Talk:Journal of Comparative Neurology 26 (1916)
THE JOURNAL
OF
COMPARATIVE NEUROLOGY
EDITORIAL liUAKl) Henrt H. Donaldson Adolf Metkr
The Wtatar Institute Johns Hopkins L nivcrslty
J. li. Johnston Olivbr S. Strong
University of Mlnneaou Columbia University
C JUDSON HkRRICK, lulvfniUy of Chlc»«o Mana«lnc Editor
VOLUME 2t) 1916
PHILADELPHIA, PA. THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
Mf 33
COMPOSED AND PRINTED AT THE
WAVERLY PRESS
By the Williams & Wii.kins Company
Baltimore, Md., U. S- A.
CONTENTS
1916
No. 1 FEBRUARY
W. J. Crozier. Regarding the existence of the 'conunon chemical sense' in vertebrates 1 William F. Allen. Studies on the spinal cord and inedula of cyclostoraes with special reference to the formation and expansion of the roof plate and the flattening of the
spinal cord. Eightj'-seven figures 9
Percival Bailry. Morphology of the roof plate of the foro-hruin and the lateral choroid plexuses in the human embryo. Thirty-one figures 79
No. 2 APRIJ.
Leslie B. Arey. The movements in the visual cells and ri'tinal pigment of tlie lower vertebrates. Thirty-seven figures IJl
11. Saxton Burr. Regeneration in the brain of .\mbIystoma. I. 'I'Im' foro-l)r:iin. Four figures 203
No. 3 JLXK
Leslie B. Arey. The function of the efferent fibers of the iii)ti<' nerve of fishes. Twelve figures (two plates) 213
(!. E. CoGHiLL. Correlated anatomical and physiological studies of the growth of the nervous system of Amphibia. IF The afferent system of the head of Amblystoma. Seventy-nine figures 247
H. .\. KocuKij. The effect of activity on the histological structure of nerve cells ^ 241
No. 4 AUGUST
Leslie B. Arey. The influence of light and temperature upon the migration of the retinal pigment of Planorbis trivolvis. Nine figures 359
A. T. R.'VSMussen and J. A. Myers. Absence of chromatolytic change in the central nervous system of the woodchuck (Marmota monax) during hibernation. Six figures
(two plates) 391
J. J. Keegan. a study of a Plains Indian brain. Eight figures 403
Martin R. Chase. An experimental study of the vagus nerve. Four figures 421
Leslie B. Arey'. Changes in the rod-visual cells of the frog due to the action of light.
Two figures ■^29
iii
IV CONTENTS
11. 11. DoNALD.soN. .\ preliminary (Ictcriiiiiiatioii of tlie part played by myelin in reducing the water content of the mammalian nervous system (albino fat). One chart.. 443 \V. J. Ckozikh. The taste of acids. Two figures 453
No. 5 OCTOBER
William A. Hilton. The nervous system of pycnogonids. Twenty-one figures 463
J. B. Johnston. Evidence of a motor pallium in the fore-brain of reptiles. One figure. 475 J. B. Johnston. The development of the dorsal ventricular ridge in turtles. Twentyseven figures 481
Perciv.\l Bailey. The morphology and morphogenesis of the choroid plexuses with especial reference to the development of the lateral telencephalic plexus in Chry semys marginata. Twenty-seven figures 507
Sumner L. Koch. The structure of the third, fourth, fifth, sixth, ninth, eleventh and
twelfth cranial nerves. Five figures 541
Caroline Burling Thompson. The brain and the frontal gland of the castes of the
'white ant,' Leucotermes flavipes, Kollar. Twenty-six figures 553
CONTRIBUTIONS FROM THE ZOQLOGICAL I.ABOR\TORr OF THE MOSEUM OF CO.\rPARATIVE ZOOLOGT AT HARVARD COLLEGE. NO. 2fi8.
REGARDING THE EXISTENCE OF THE 'COMMON CHEMICAL SENSE' IN VERTEBRATES'
W. J. CKOZIKR
I. In a recent paper by Coghill ('14) exception is taken to the view held by Plerrick f'OS), Sheldon ('09), Cole ('10), and Parker ('12), that there is in vertebrates a set of receptors (free nerve terminations) which are responsible for reactions to rather high concentrations of chemicals when applied to moist peripheral surfaces. The theoretical significance attached to this 'common chemical sense' (Parker, '12) makes it appropriate to bring forward certain facts which may to some extent serve to clear up the situation.
The evidence adduced by ( "ojihill is tliis: that in Aiiil)lystoiua larvae, before the estalilishment of the definitive nervous system, tactile and chemical irritability appear concomitantly and, so far as studied, remain completely parallel in development; and that if larvae be placed in irritating solutions of hydrochloric acid (as dilute as lo.wo)- ^^^^ epithelial cells become visibly disrupted, the reaction of the larvae being correlated with the disintegration of the skin and a grndual disappearance of reaction to tactile stimuli.
Plis conclusion, that tliere is not present in the skin any normal irritability to acid, is probably correct for the larvae experimented upon; but it by no means follows that the same conditions obtain in the adult. Coghill argues, however, that the permanently embryonic cells of the germinative layer in the skin of amjaliibians and fishes when exposed to the high concentrations of acid (40) used by Parker and Sheldon act as do the superficial ciliated cells of the Amblystoma larval skin. Because they are bound down by a "thick, less sensitive, and
^ Contributions from the Hernuida Biological Station for Kesearrh. Xo. 40.
1
THK JOl'RNAt. OK COMPARATIVE NEUROLOGY, \ OI.. 26, SO. 1 FEBntARY, 1916
J. (niOZIEK
more iinjKM'vious hiyer of cells," the disintejj;ratioii of the sensitive cells would cause exceedingly violent disturbances, presumably affecting tactile and pain terminals; and further, that it has not been satisfactorily demonstrated that it is possible (as claimed by Sheldon and by Cole) to effect a separation of tactile and 'common chemical' irritability in fishes and amphibians.
This general argument is somewhat strengthened by the fact (to which Coghill does not refer) that aquatic vertebrates possessing a soft slimy skin — cyclostomes, eels, catfish, Necturus — are known to react to local irritation by chloroform and other substances by the expulsion of mucus and even of entire gland cells. The reaction time of this response is not known accurately, though it is judged to be much greater than that, for example, of the catfish as a whole when stimulated by hydrochloric acid applied to its trunk region; still, it is not inconceivable that mechanical deformations brought about in this way might provide stimuli for tactile nerve endings, provided the mucus response were sufficiently sharp and prompt.
II. The exact manner in which solutions are to produce the hypothecated effects upon the cells of the germinative layer is left entirely untouched by Coghill. The rapidity of the responses given by the catfish and Necturus immediately negatives the idea that osmotic transfer of water is the agency of stimulation. In the case of the spinal frog, as studied by Braeuning ('04), Loeb ('05), and Cole ('10), though the reaction times are rather long, there is abundant evidence that other than osmotic factors are at work. The most important point which arises for consideration is the extent to which chemical stimulants actually penetrate the skin, i.e., the degree to which the cells of the germinative layer are exposed to the action of the stimulant. In addition to the rapidity of the reactions under discussion, it is to be remembered that they are excited by acids, salts, alcohols, and a variety of other-substances.
That the skin of aquatic animals is not to any appreciable degree damaged by the agents responsible for 'common-chemicalsense' reactions, is clearly indicated by such facts as the following:
COMMON CHEMICAL SENSE 6
1. High concentrations of acids (i^), salts (2n), alkalies {^) and alkaloids (loo). which evoke prompt and vigorous reactions from the earthworm, Eisenia foetida, cease to cause any disturbance the moment the stimulant is washed off by immersing the animal in water. If any serious disintegration were produced by these solutions, it would be reasonable to expect the continuance of activity after the external supply of the stimulant had been removed. 2. Holothurians, such as H. surinamensis (Crozier, "15 c) and Stichopus moebii (Crozier, '15 b), possess skin pigments whose loss is a fairly good indicator of changes in permeability. Yet in no case on the application, by the pipette method, of the stimuU used by Parker ('12) was there any indication of pigment loss accompanying reaction.-^ This is true also of the nudibranch, C'hromodoris zebra.
III. The value of the intracellular indicator of ("hromodoris in the study of cell penetration by acids has been pointed out elsewhere (Crozier, 'lob). When small volumes of acids, even in rs solution, are used to stimulate ( "hromodoris, there is no visible evidence that they penetrate the skin. In fact the time required for the penetration of the acids is quite high (see also Harvey, '14), the most rapid rate observed being with ^0 iso-valeric acid, where penetration recjuires 1.5 minutes.
- Experiments with earthworms also disclose certain important deficieni-ie.s in'^thc method wherol)y the animals to be tested arc entirely immersed in the stimulating solution. If the reactions are to be studied quantitatively, there are features undoubtedly obscured by this procedure. For example, the time elapsing between the complete immersion of an earthworm (P^iscnia foetida i in a tV lithium chloride solution and the instant the first writhing movement apjjcared, was found to be at least ten times greater than the reaction time of the same worm when part of it (the anterior end) was stimulated. The speed of reaction is in part conditioned by the number of receptors afTected, i)ut this does not analyze the situation completely. It is quite probable that the responses ol)served by Coghill are not at all due directly to .sensory stimulation by acid.
•^ I have observed that under certain circumstances Ptychodera sp. — a Bermudan enteropneust — reacts to chemical irritation by extruding a yellowish pigment. There is ground, ho\vever, for believing that this is an instance comparable to that of Lumbriconereis (Kschischkowski, '12), in which K salts have apparently a more or less specific action in producing the response.
4 W. J. ('JU)ZIEH
•
For -/"o HXO3 the penetration time found (Chroniodoris tissue) was 3.5 minutes; for ^, 4.3 minutes; whereas Xeoturus, under water, was found to react to I'o HNO3 (0.5 cc. of the solution being applied with a pipette) in 1 .5 seconds when stimulated on the dorsal surface of the head, 5 seconds on the lateral mid-body surface, and 2 seconds on the tail; with w HNO3 the reaction times for corresponding locations were 5, 10, and (3 seconds respectively.-* Even here, though the pipette tip was held within approximately a centimeter of the animal's surface, the concentration of the solution in the pipette does not represent the concentration which actually reaches the stimulated surface, as already noted by Parker ('12, p. 222). The conclusion must therefore be, that acids do not penetrate the skin w^ith sufficient rapidity to affect the cells of the germinative layer. TKis conclusion must also be extended to alkalies, since Harvey ('10) and others have shown the high impermeabilit}- of cells to strong hydroxides.
IV. In the light of the evidence just discussed, it seems improbable that the high concentrations of irritants employed by Parker and others in studying reactions attributed to the common chemical sense produce any violently disruptive effects when applied to the skin of aquatic animals. Indeed, so far as concerns the skin as a whole, they do not penetrate at all,^ and the cells of the germinative layer cannot be held to be exposed to their action.
This conclusion was verified, in the case of the spinal frog, by experiments of the following type:
1. The reaction time for the withdrawal of the frog's foot from w CUSO4, is about 7 seconds (at 24 ?9). After being withdrawn from the solution, the feet continue for some time to be spasmodically contracted. These subsequent contractions are entirely inhibited the instant a foot, just retracted fromCuS04,
The reaction times were measured at 20°, while the penetration of the acid was studied at 27°; the speed of penetration decreases markedly with falling temperature.
5 The mode of action of the stimulating agent on the individual receptor is entirely another question.
COMMON CHEMICAL SENSE 5
is dipped into a weak solution of K4Fe(CN)6, which precipitates the copper held by the mucus of the foot. A similar result was obtained with copper acetate. Washing the foot with distilled water does not lead to a cessation of the contractions, because, after exposure of the foot to certain solutions (Loeb, '05), water stimulates.
2. After two to four stimulations of the frog's foot by relatively strong solutions of either copper acetate or ferric sulphate, the stimulated area was sectioned and tested microchemically for the penetration of the copper or iron. The metals were found in the mucus of the surface of the foot, and in several instances doubtful traces were discovered between cells of the extreme outer portion of the epidermis. No evidence was found of any disruption of the germinative layer.
3. As a stimulating agent whose penetration would rcadil} be visible, the action of picric acid was studied. The outcome of experiments with this sul)stance at several concentrations may ho illustrated l)v the records here copied:
Kxpcrimrttts ii unit L. Picric acid, f^. K. T. = Successive tests at .5 min. intervals.
Reaction time in seconds. X. R. = \o reaction.
R.T.
H
L
1
7.8
2.1
No staining.
•)
12.8
6.6
Slight staining.
- i ...
16.8
7.8
4 .
30.2
11.8
Staining progressively
40.0*
24.6
j brighter.
G
45.0
43.2*
J
7
X.R.
X.R.
No reaction to ? formic acid.
- Not reactive to pinching beyond this point.
From these and similar tests, it was concluded that the penetration of the stimulant rapidily renders the frog's foot less re
6 W. J. CROZIER
active by killing tlie superficial cells. In the case of picric acid it is probably the II ion which is mainly concerned in stimulation, since 50 annnonium picrate is entirely ineffective, neither does it easily stain the frog's foot. This does not, however, signify that other acids behave as does picric, since as many as 20 successive reactions may be obtained from w HCl. Moreover, the staining is not directly correlated with the stimulating effect, since the skin of the frog's foot was stained by immersion for 3 minutes in g^o picric acid without producing any reaction. The skin of Necturus and the catfish may be stimulated with fQ picric acid without producing any visible stain.
V. I have repeated on the frog's foot the experiment of Sheldon ('09) and Cole ('10) regarding the separation of tactile irritability and responsiveness to irritating solution, by treatment with cocaine. A 0.5 per cent solution of cocaine hydrochloride was used, and the tests were made by comparing at intervals the reactions of the cocainized foot with those of the untreated one. After about 20 minutes' immersion, the reaction time of the cocainized leg to ^ formic acid (chosen as a powerful stimulant) was usually twice that of the normal foot; after about an hour, varying in some tests to an hour and a half, the cocainized foot no longer reacted to pinching, but gave good responses to the acid with reaction times of 10-15 seconds, still about twice the reaction time of the non-narcotized leg.
It is therefore possible, I believe, to effect experimentally a separation of sensitivity to mechanical and to chemical stimulation in the frog's foot.
There can be no question of the distinctness of the human sensation attributed to the common chemical sense (Parker, '12; Parker and Stabler, '13) as compared with any tactile sensation; and from tests made upon cocainized areas of my own mouth, ^ I am certain that the two sets of receptors are not only qualitatively distinct as regards the sensations with which they are connected, but also may be separated by the use of cocaine.
• These tests concerned mostly the inner surface of the cheek.
COMMON CHEMICAL SENSE 7
SUMMARY
1. There is positive evidence, in the case of alkalies, acids, and certain salts, that solutions supplying stimuli for the common chemical sense do not penetrate the skin of aquatic animals, nor when applied from a pipette do they damage the skin to any extent.
2. There is consequently no ground for Coghill's assumption that the cells of the germinative layer of the epithelium of fishes and amphibians are exposed to the action of the stimulating agent and thereby disrupted; and there is no histological evidence of disruption.
3. The reactions attributed to the common chemical sense depend upon a group of sense organs distinct from those sensitive to mechanical stiimilation.
UKKi;ui:xcEs
BuAEUNiNG, H. 1904 Zur Kenntnis dcr W irkunj; chi'misclicii Hcizc. Arch, gcs. Physiol., lid. 102, p. 163 1S4.
C'ocaiiLL, G. E. 1914 Correlated anatomical and physiological studies of the growth of the nervous system of Amphibia. I. The afferent system of the trunk of Amblystoma. Jour. ("omp. Xeur., vol. 24, p. 161-233.
Cole, h. W. 1910 Reactions of frogs to chlorides of ammonium, potassium, sodium, and lithium. Jour. Comp. Xeur., vol. 19, p. 273-311.
Crozikh, W. J. 1915 a The behavior of an enteropneust. Science, X. S., vol. 41, p. 471-472.
(Note — Lines 5 and 6 [p. 472] have i)een accidentally interchanged by the printer.)
1915 b The rhythmic pulsation of the cloaca of holothurians. Ibid., p. 474.
1915 c The sensory reactions of Holothuria surinamensis. Zool. Jahrb., Abt. Physiol. [Xot yet published].
1915 d On cell penetration by acids. Science, X. S., vol. 42, p. 735736.
Hakvey, E. X^. 1910 Studies on the permeability of cells. Jour. Exp. Zool., vol. 10, p. 507-556.
1914 Cell permeability for acids. Science, N. S., vol. 39, p. 947 949.
Herrick, C. J. 1908 On the phylogenetic differentiation of the organs of taste and smell. Jour. Conip. Xour., vol. 28, p. 157-166.
8 W. J. CROZIEU
Ks(nisciiu\\>Ki, K. 1912 Neue Beitriige zur Pigmentab.sondorunf^ bci Annelitk'ii. Zentnilbl. f. Physiol., lid. 26, p. 528-.532.
LoKH, J. 11)05 Production of hypersensitivity of the .skill by electrolytes. In: Studies in (ieneral Physiology, pt. II, Chicajio. pp. 748-765.
Parkku, G. 11. 1912 The relation of smell, taste, and the common chemical sense in vertebrates. Jour. Acad. Nat. Sci. Phila., Ser. 2, vol. 15, p. 221-234.
Parker, G. H., and Stabler, E. M. 1913 On certain distinctions between taste and smell. Amer. Jour. Physiol., vol. 32, p. 230-240.
Sheldon, R. E. 1909 The reactions of the dogfish to chemical stimuli. Jour. Comp. Xeur., vol. 19, p. 273-311.
STUDIES ON THE SPINAL CORD AND :MEDULLA OF CYCLOSTOMES WITH SPECIAL REFERENCE TO THE FORMATION AND EXPANSION OF THE ROOF PLATE AND THE FLATTENING OF THE SPINAL CORD'
WILLIAM F. ALLEN
Deparlinent of Anatomy, University of Minnesota
EIGHTY-SEVEN FIGURES
CONTENT?
I. Introduction it
Material and its preparation 10
11. Hereditary and mechanical causes underlying the formation of the
fourth ventricle and the tela chorioidea 12
1. Roof plate of the medulla oblongata 12
A. Amphioxus 12
B. Polistotrema (Bdellostoiun 18
C'. Petromyzon la
D. Selachians and Amphibia 23
K. Pig embryos 25
V. Human embryos 27
2. Descriptions of three roof plate expansions of the spinal cord in the
20 cm. Poli.stotrema series 28
III. Causes underlying the flattening of the spinal cord in cyclostomes. ... 32
IV. General considerations and sunmiary 35
V. Literature cited 40
INTRoDK'riOX
This paper has grown out of the stud}' of the caudal heart and the spinal cord and nerves related to it in Polistotrema. Parts of that study which are not yet completed will be published later and will deal with the origin, distribution, and phylogeny of the spinal nerves; the origin of muscle sense organs in connection with the specialized muscles of the caudal heart; and
1 A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Department of Anatomy. The University of Minnesota.
10 WILLIAM F. ALLEN
certain ganglion cells, possibly sjonpathetic, which are found along the course of the spinal and vagus nerves. The present paper includes a study of the mode of origin of the fourth ventricle in several groups, an account of a structure similar to the fourth ventricle found in the caudal part of the spinal cord of an adult Polistotrema, and a study of the development of the central canal, and the causes underlying the flattening and the ventral indenting of the spinal cord in Cyclostomes.
Material and its preparation
Since the material for this paper came from such diverse sources, and such different modes of technique were employed, too much time and space would be consumed if a detailed description were given of each method employed. With the exception of the Petromyzon material, which was fixed in Flemming, corrosive-acetic, and Perenyi's fluid, my own material was fixed either in Tellyesnicky's or Bouin's fluid. It was sectioned after paraffin or the combined celloidin-paraflfin method of imbedding. The latter method gave by far the better results, since it appears to have all of the advantages of celloidin in causing almost no shrinkage and its ability to hold yolk granules and blood corpuscles intact; besides allowing the sections to be cut as thin as paraffin alone, and causing no more difficulty in manipulation. For the most part the sections were stained in Heidenhain's iron hsematoxylin and counterstained in an alcoholic solution of orange G plus a little acid fuchsin. In a few instances, in very young Petromyzon embryos, where all the tissues were filled with yolk, carmine and hsemalum were used to advantage.
Acknowledgments are due to Prof. T. G. Lee for the gift of a very complete series of Petromyzon embryos, which were obtained from the Connecticut River. Also to Prof. R. E. Scammon for the loan of his very complete serial collection of Squalus embryos, and to Prof. J. B. Johnston for the use of a similar serial collection of Amblystoma embryos. The splendid series of human and pig embryos belonging to the Institute of
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 11
Anatomy of which Prof. C. M. Jackson is director, were especially valuable.
It is a great pleasure to the writer to have this opportunity of expressing his obligations to Prof. J. B. Johnston for his valuable suggestions and friendly criticism of this work.
A model of the caudal end of the spinal cord from a region a little in front of the caudal hearts to its extreme tip was prepared in four sections from the 20 cm. Polistotrema series. Also a model in two pieces was prepared of the cavity of the socalled first roof plate expansion and the enlarged central canal of the above mentioned series of Polistotrema. These models were constructed out of blotting paper after a modification of the Born method. Tracings of each section were made on ordinary writing paper with the aid of an Edinger-Leitz drawing apparatus, using a magnification of 100 diameters, and afterward each tracing was carefully checked up for accuracy with a higher magnification. At the very outset two base or projection lines wore drawn on the first tracing, one following the median longitudinal plane, and the other a horizontal line drawn at right angles to the first line, passing along the ventral border of the notochord. These projection lines were added by pencil to all of the succeeding tracings after the following manner: The second tracing was carefully fitted over the first, after i)lacing both over a plate glass covered box, containing an electric light reflected upward, and in like manner these lines were added to the third tracing from the second, and so on to the end of the series. Then with the aid of carbon paper these tracings and projection lines were transferred to sheets of blotting paper, having a definite thickness, previously determined after the following method. When thoroughly cooled after an immersion in melted paraffin, the blotting paper selected should have a thickness, equal to the thickness of the section multiplied by the magnification used, which in this case totalled 1.5 mm. The sheets of blotting paper containing the transposed tracings were then inmiersed in melted paraffin, drained and cooled. After which the outlines of the tracings were cut out with a sharp scalpel, and the sections were built up in regular order. In
12 WILLIAM F. ALLEN
l)iirt to maintain this regular order and in part to add strength to the model, a copper wire was inserted through each section, passing through the point of intersection of the two projection lines. After a certain number of sections had been strung on this wire the}' were securely fastened to each other by pins.
HEREDITARY AND MECHANICAL CAUSES UNDERLYING THE
FORMATION OF THE FOURTH VENTRICLE AND THE
TELA CHORIOIDEA
To the writer this problem appears to be primarily phylogenetic rather than ontogenetic; consequently this study begins with the lowest vertebrates, and is approached as a problem of evolution.
1 . Roof plate of the medulla oblongata
A. Amphioxus. Unfortunately 1 have not had access to any embryological Amphioxus material so that my inferences will have to be drawn entirely from adult material.- At the outset it can be maintained with a considerable degree of safety that the adult Amphioxus brain contains nothing which can be homologized with the fourth ventricle of higher vertebrates. Figure 67, which pasess through the highest point of the anterior ventricle (F.i) demonstrates clearly that there is nothing here comparable with the fourth ventricle. For the dorsal portion of the cavity is much narrower than the ventral portion, and there is absolutely nothing in the way of a thin and expanded roof plate. In fact, the dorsal portion of the cavity is fairly filled up with processes from ependymal cells.
A few sections behind the first ventricle there appears a small isolated cavity in the roof plate region of this series, designated as the posterior ventricle (fig. 68, V.2), and a considerable distance behind this cavity, there is a second dorsal cavity, also designated as the posterior ventricle (fig. 69, V.2). This is somewhat larger than the previous cavity, and can be readily located
- In order to eliminate minor details of description from the text, very complete and detailed descriptions of the figures have been given at the end of the paper, to which the reader's attention is directed.
SPIXAL CORD AXD MEDULLA OF CYCLOSTOMES 13
from its position immediately above a dorsal group of large nerve cells (M'.C). From the figures by Hatschek, Willey, Sterzi. and others it is evident that these isolated roof cavities described above, were once a part of a common central cavity of the embryonic brain, which later in development, became isolated 'through an invasion of ependjaiia, and it is entirely possible that this region of the central cavity in the embryo was much more suggestive of the fourth ventricle. Judging from the adult alone they may he looked upon merely as vestiges of the embryonic central canal.
B. Folistotrema i Bdellostoma) 'Sly embryonic material <>f Polistotrema confirms the statements of Price, von KuptTcr. and Dean that Polistotrema possesses well-developed ventricles in the embryo; the expansion being fully as great as in a similar stage of Petromyzon. As development proceeds the lateral plates increase in thickness from additions of fibers and cells until the fourth ventricle becomes reduced to a canal, but little larger than the central canal of the spinal cord.
Sanders, Holm, Miss Worthington, Sterzi, Cole, and Xicholl> describe and figure the fourth ventricle about as it is shown in figure 63. There is some little discrepancy in the terminolog>used, due largely to the differences (if opinion as to whether oinot Polistotrema has a cerebellum. If the jiosterior lobes of the mesencephalon should in the light of future investigation turn out to be a cerebellum, then the boundaries of the metencephalon will have to be carried further forward than we have indicated. and the so-called sinus mesoca?licus of XichoUs iS.M.) will have to be called the anterior dilation of the fourth \entricle of Miss Worthington's description.
My transverse series through the brain of Polistotrema show the condition of the ventricles to be almost identically the same as Sterzi and Nicholls found them. Until the embryological and functional areas of the brain have been better worked out it seems advisable to the writer to let the posterior border of Nicholls' sinus mesocoelicus (fig. 63, S.M.) mark the boundry line between the mesencephalon and the metencephalon, and to regard his isthmic and ventricular canals (figs. 63 and (io. A. 4V.)
14 WILLIAM V. ALLEN
as dorsal and ventral portions of the anterior end of the fourth ventricle. For the reason that in Polistotrema they extend some distance behind the posterior tip of the mesencephalon, and the dorsal or posterior canal is shown in transverse section (fig. 65) to lie close to the dorsal surface, which is then the only part of the fourth ventricle to retain the characteristic dorsal position of the higher vertebrates. These two canals appear in this series about as Nicholls has described them, the dorsal (isthmic) is the larger and contains Reissner's fiber. Although probably subject to a considerable variation, these canals apparently extend further caudad in this series than Nicholls represents them. Also in this series the dorsal canal (isthmic) gives off one or two branches at the level of the posterior tip of the mesencephalon (cerebellum of Miss Worthington) , which run parallel to the main canal, but a little to one side and below (fig. 65, A.iV.). After travelling side by side for some little distance in a mass of spongy ependymal tissue close to the roof plate, they reunite in the dorsal canal, and soon afterward both the dorsal and ventral (isthmic and ventricular) canals unite in a common canal, which is little if any. larger than the central canal of the spinal cord. This constricted portion of the fourth ventricle (fig. 63) continues caudad, rather deep-seated in a mass of loose vascular ependyma, until the posterior end of the medulla is reached, where it expands into a much larger vesicle or sinus, designated as the posterior dilation of the fourth ventricle (figs. 63 and 66, P.aV). Behind this it soon tapers down into the ordinary central canal of the spinal cord.
A glance at figures 63 to 66 suffices to show that the fourth ventricle of Polistotrema is greatly reduced as compared with that of Petromyzon. This is due probably to the rapid increase of fibers and cells in the lateral plates. Notwithstanding this reduction in size and general alteration in appearance and structure, the walls of the fourth ventricle in Polistotrema, although representing a greatly modified chorioid plexus, are unquestionably capable of producing cerebro-spinal fluid. The posterior dilation of the fourth ventricle (fig. 66, P.iV.) contains cerebrospinal fluid (S.C.F.) in the form of a deeply staining feltwork,
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 15
which is not ependjinal cilia or a tangled Reissner's fiber. Also throughout its entire length, as was noted by Sterzi, the fourth ventricle is enveloped by a rather thick layer of spongy and very vascular ependyma, which would be distinctly favorable for infiltration and possibly for secretion into the ventricle. This rich blood supply is from the blood vessels and sinuses traversing the meningeal membranes, and especially from two large arteries (A. rhombencephalica of Sterzi), one of which appears in figure 64 (M.A.). Also it would be quite possible in the anterior part of the fourth (A.:iV.) for the various canals, which run close to the dorsal, to receive infiltration direct from the outer meningeal blood and lymph sinuses.
Coagulated cerebro-spinal fluid is also to be seen in reduced amounts in the mesencephalic ventricles designated as the posterior mesocoele and the sinus mesocoelicus. They are also surrounded by a layer of vascular ependyma, which, while much thinner than the corresponding layer of the fourth ventricle, doubtless functions as a cerebro-spinal fluid forming organ.
A careful examination of this peculiar modification of the chorioid plexus of the fourth \entricle in Polistotrema leads one to believe that this is not as efficient an organ for the production of cerebro-spinal fluid as the more expanded tela chorioidea of Petromyzon and higher vertebrates.
C. Petromyzon. Petromyzon is apparently the best type that could be selected for obtaining definite information concerning the early history of the formation of the fourth ventricle and its expanded roof plate. 1) It is the lowest living vertebrate that possesses a well-de\eloped fourth ventricle and expanded tela chorioidea in the adult. 2) At no times does the medulla have a pontine flexure. 3) Its central nervous system remains a solid cord of ectoderm until after the cranial and spinal ganglia are well-differentiated. For these and other reasons the early history of the fourth ventricle in Petromyzon has been studied in the effort to determine the essential factors involved in its appearance and growth.
In order to arrive at the fundamental factors involved in the anlage and development of the fourth ventricle in Petromyzon
K) WILLIAM F. ALLEN
it is necessary to go back in the ontogeny of the central nervous system to the time when it was a solid cord. Of my embryos which represent stages killed at 5, 7, 9, 10, 11, 12, 14, 16, 18, 20, and 26 days after fertilization, all of the 5 day series revealed the central nervous system as a solid cord or keel of ectoderm cells, formed according to Calberla by a process of delamination. In places neural crest cells can apparently be seen budding off from the dorso-lateral surface of the brain and spinal cord almost identically as described and figured by von Kupffer ('90). It is obvious that the preponderance of yolk granules in all tissues makes accurate observation in these stages very difficult. The majority of the 7 day embryos disclosed a central canal either formed or in the process of formation. Out of a great number of series of 10 day embryos, three were found in which the opening of the central canal had been considerably retarded. In one series the central nervous system was still a solid cord of cells, and in the other two the central canal was in the early stages of formation. These series were selected in preference to similar stages of 7 day embryos for the reason that less yolk was present to obscure the various structures.
To facilitate comparison of similar sections of successive stages, figures 33 to 38 and figures 40 to 53 have been so arranged as to bring several regions of series in a horizontal row, while successive stages of the same region are placed in a vertical row. The conditions at the level of the trigeminal, auditory and vagal ganglia are thus readily compared.
In figure 32 we have a section through the medulla in the region of the VIII ganglion, which portrays very well an extremely early stage in the formation of the central canal. Here the medulla will be seen to be composed of a mass of nuclei imbedded in a syncytium of protoplasm. Excepting in the roof and floor plates, the nuclei have migrated some little distance to either side of the median sagittal plane, presenting the appearance of a rather broad light line surrounded by nuclei. The center of this light streak of protoplasm discloses a very conspicuous furrow or seam {C.C.S.), which appears in every section of the central nervous system of this series, very much as it looks in
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 17
this section. This furrow or seam represents the position of the future central canal. The protoplasm adjacent to this seam is sufficiently granular to suggest a secretory function at this early stage.
Figures 33 to 35 also taken from a 10 daj' Petromyzon series exhibit a slightly later stage in the formation of the central canal. Conspicuous cavities (C.C.) have appeared in the dorsal and ventral portions of this furrow, which are not only visible throughout the entire medulla, but are continuous throughout the spinal cord. It should be recorded that a few sections through the medulla possessed slightly larger dorsal and ventral cavities than were represented by these figures, but in no case had they approached each other close enough to unite. In other respects, excepting possibly for a few more fibers in the marginal layer, the structure of the medulla has remained about the same as in the previous series. Emphasis should be made of the fact that each of these cavities, when examined with a higher magnification, reveals a certain amount of fine granules, which in some cases may have completely filled the cavity, while in others they are confined to the outer edge, leaving a clear space in the center. The presence of these granules here suggests two processes: 1) disintegration of the central protoplasm, and 2) products of secretion. The lateral migration of the nuclei could i)e utilized to support either inference. That such a migration of nuclei would be favorable for disintegration is self evident, and secretory cells are usually characterized by having their nuclei somewhat remote from their lumina.
In the 11 day series (figs. 3(3 to 38) we find that the two isolated dorsal and ventral cavities of an earlier stage have not only united and formed a cleft-like cavitj^ which may now be designated as the typical embryonic central canal, but that the original dorsal and ventral cavities of this canal, especially the dorsal, have increased notably in size. The narrow central portion (figs. 37 and 38) indicates that this is the place where the protoplasm was last to separate. It should not be confused with a similar condition that occurs later, when the central and ventral portions of the lateral plates migrate inward, fuse, and
THK JOUKNAL OK COMHAHATIVE NM;l:ROL,OOY, VOL. 2b, NO. 1
18 WILLI AM F. AIJ.KN'
completely obliterate that j^ortion of the embryonic central canal. iVside from the change in the central canal the size of the medulla has increased in all directions, but especially laterally, due doubtless to an increase in the number of nerve fibers and cells in the lateral plates. Both roof and floor plates are very thin, comprised of about one layer of nuclei each. The ventral plate may be slightly thicker, due to the addition of a few nerve fibers to the outer layer. Absolutely no stretching of the roof plate has occurred, indicating that it is not under any marked internal pressure. In figure 37 (B.V.) branches of the intersegmental blood vessels have stretched out toward the roof of the brain, but at this stage they are too remote from the roof plate to be very active in infiltration. Figure 39 makes clear that the spinal cord has made equal progress with the medulla in developing a cleft-like or typical embryonic central canal. In the cord the dorsal and ventral enlargements of the central canal are shown to be of about equal size.
An additional day (figs. 40 and 41) discloses considerable expansion of the central canal throughout, with the exception of the extreme ventral portion. Very noticeable is the increase in size of the dorsal portion, the future fourth ventricle (cavita della tela coroidea of Sterzi). The roof plate exhibits no signs of stretching. Up to this stage the expansion appears to be due to the migration of the ependymal cells upward and outward, rather than to pressure within. The floor plate will be seen to have increased considerably in thickness through an addition of fibers to its marginal layer, which would obviously tend to make the floor plate more resistant than the roof plate to pressure from within from this time on. Both lateral plates disclose a remarkable growth in thickness due to an addition of both fibers and cells, some of which, however, must be attributed to the fact that the head was sectioned somewhat obliquely' (note in fig. 40 that the V and VIII ganglia appear in the same section). But little progress has occurred in the development of the intersegmental blood vessels, so that Sterzi's conjecture, that the embryonic cerebro-spinal fluid does not differ in any way from the ordinar}^ intercellular fluids, would probably hold, if it could be
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 19
established that the central protoplasm in disintegration contributed nothing to it. The spinal cord was found to be in about the same condition as the 11 da}^ series.
Moreover an 18 day series (figs. 42 to 44), some 6 daj's older than the last stage compared, shows pVacticallj' no change in the shape and size of the central canal or expansion of the roof plate, despite the fact that a notable increase of fibers had occurred in the ventral and median portions of the lateral plates. It should also be recorded that the intermediate stages, as repi-esented by the 14 and 16 day series, exhibited a like state of the central canal. A further interesting condition is revealed from a section of the spinal cord of this series (fig. 45). Here the lateral plates have expanded to such an extent from the formation of fibers in the marginal layer, that the inner surfaces of their central areas have nearly formed a complete concrescence at the center of the original central canal. In other words, the inner surfaces of the lateral plates have met, and are about to fuse, leaving dorsal and ventral cavities (CO. The ventral one of these cavities will persist as a pemianent central canal. In Petromyzon this partial closure of the original embryonic central canal of the spinal cord may have considerable bearing on the increase of internal pressure of cerebro-spinal fluid on the walls of the medulla, during this and later stages. We may regard the period from aiiout the 12th to about the ISth day as a period of rest in the formation of the fourth ventricle in Petromyzon. During this time the processes which would tend to expand the central canal have been met with equal counter forces, which would make for closing it up.
Transverse sections through the medulla of a 20 day Petromyzon series (figs. 46 to 48) demonstrate a marked change in the medulla and its central cavity, which cavity has now assumed the form of a fourth \entricle. A marked increase has taken place in the number of fibers in the ventral plate and in the ventral portion of the lateral plates. This together with pressure from the growing auditory vesicles (Aud.V.) and the notochord, has produced a nearly complete concrescence of the corresponding middle and \entral portions of the inner surfaces
20 WILLIAM F. ALLEN
of the lateral plates. Had a similar increase of nerve fibers occurred in the dorsal plate and in the dorsal portion of the lateral plates, the central canal of the medulla might have been completely obliterated. On the contrary, the dorsal cavity and the roof plate ha^e been slightly expanded, apparently through an increased internal pressure from the embryonic cerebro-spinal fluid. One evidence that increased pressure may exist was found in the marked decrease in the size of the central canal of the cord and of the ventral part of the canal in the medulla region.
A second evidence is found in the changes that the walls of the fourth ventricle are undergoing preparatory to becoming a functional organ for the production of cerebro-spinal fluid. In connection with the expansion of the roof plate it will be seen that the dorsal mesenchyme has become decidedly vascular (fig. 46, B.V.), making easy an infiltration process into the ventricle. Also ependymal cells, surrounding the ventricle, are taking on form and are probably assuming a secretory function, if they have not acquired one previously. From the recent work of Dandy and Blickfan based on the action of certain drugs and on the chemistry of cerebro-spinal fluid, it is evidence that cerebro-spinal fluid must be a product of secretion as well as of infiltration and diffusion. It is a well-established physiological fact that certain secretory cells, as for example the salivary glands, may assume a definite polarity, and produce a secretion against a very strong pressure, even stronger than that of the blood. Some investigators hold that lumina in glands are the result of pressure from secretion.
in the light of these facts, it is fair to assume that the marked lateral expansion of the fourth ventricle and its roof plate exhibited in the 26 day series (figs. 50 to 52) are the direct result of internal pressure caused by the marked increase in cerebrospinal fluid. The general increase in size of the ventricle together with the marked convexity of the roof plate and the concavity of the internal surface of the lateral plates certainly suggest internal pressure from the cerebro-spinal fluid. It should be noted in figures 50 to 52 that this expansion of the
SPIXAL CORD AND MEDULLA OF CYCLOSTOMES 21
fourth ventricle has produced a considerable secondary splitting in the concrescence of the lateral plates recorded for the previous series. Apparently this fissure did not penetrate so deeply in the region of the auditory vesicles (fig. 51, .4?^c/.T'.) as it did in front of them (fig. 50) or behind them (fig. 52), which probably indicates that the growth of the auditory vesicles in some way operated against the further splitting of medulla. It is significant that the expansion of the roof plate and the development of its mechanisms for infiltration and secretion occur at a time prior to the entrance of blood vessels into the wall of the brain and cord. The large production of cerebro-spinal fluid at this time is evidence that it serves some nutritive function.
In figure 54 we have a median sagittal section through the brain of a 26 day Petromyzon, representing a stage similar to that of figures 50 to 53. Especial attention is called to the fact that figure 54 demonstrates conclusively that the pronounced roof expansion displayed in figures 50 to 52 ha^ occurred ivithoui the aid of a pontine flexure. The slight convexity of the floor of the medulla can be attributed to the increase of fibers. Also earlier and later series revealed that Petromyzon possesses no pontine flexure. Attention should be called to the fact that the marked convexity of the cephalic end of the thin roof plate of the fourth ventricle (C.C.) in figure 54 gives every appearance of being under internal pressure from cerebro-spinal fluid. This section also makes clear, as Sterzi has previously shown, that the fourth \'entricle is formed from the dorsal portion of the embryonic central canal; while the central canal of the cord is formed from the ventral portion.
The following observations were noted in connection with the appearance of the fourth ventricle in Petromyzon, beginning at a stage when the medulla was a solid cord of undifferentiated nuclei in a syncytium of protoplasm: 1) The ependymal nuclei migrated a short distance to either side of the median sagittal plane leaving a narrow strip of granular protoplasm in the center.
2) A median sagittal furrow or seam appeared in the central protoplasm, extending from the roof plate to the floor plate.
3) An isolated cavity appeared at the dorsal and ventral ends
'11 WILLIAM F. ALLEN
of this furrow. 4) The two cavities became connected, forming a cleft-like canal, designated as the typical embryonic central canal. This canal seems to be formed by a disintegration of the central ends of the ependymal cells, now in the form of a syncytium. 5) A considerable increase in the size of the dorsal portion of the central canal occurred through the upward and outward migration of some of the roof plate nuclei and a disintegration of the inner protoplasm. No similar expansion of the ventral portion of the central canal of the spinal cord took place because its marginal layer became reinforced very early by the addition of nerve fibers. 6) Following the formation of the central canal of the medulla there was an increase of cells and fibers in the lateral walls, but for the space of about six days there was little change in the size of the central canal. 7) Next a very pronounced increase of fibers occurred in the median and ventral portions of the lateral plates, which brought about a complete concrescence of the corresponding inner margins of the lateral plates. 8) A sufficient amount of cerebro-spinal fluid was formed by infiltration and secretion to produce a marked expansion of the fourth ventricle. This expansion pushed apart the dorsal portion of the lateral plates, which had not been thickened by an addition of fibers, and stretched the roof plate to a much greater width. 9) Along with this expansion a secondary splitting of the concrescence noted in (7) took place. This fissure did not penetrate so deeply in the region of the auditory vesicles on account of the mechanical obstacle offered by these vesicles. 10) This expanded roof plate apparently assumed the function of producing cerebro-spinal fluid at a time previous to the entrance of blood vessels into the central nervous system, when its nutritive function would be of importance.
From a review of the main points in the development of the fourth ventricle in Petromyzon we are warranted in concluding : 1) That a well-developed fourth ventricle and tela chorioidea were formed in one of the lowest living vertebrates, Petromyzon, without the aid of a pontine flexure. 2) The best suggestion that has been given for the appearance of such an organ in the medulla rather than elsewhere in the central nervous system is
SPINAL CORD AXD MEDULLA OF CYCLOSTOMES 2'.^
that the roof plate has been weakened more in the medulla region through a greater migration of neural crest cells. This explanation, however, is not entirely satisfactory; since it was shown for Petromy^on (fig. 32) that the cranial ganglia were well-difYerentiated while the medulla was still a solid cord of cells. If then this were the determining factor it must have cast its shadow a long way ahead. Also it is apparent when the difference in size is considered between the medulla and the spinal cord that there are relatively no more neural crest cells extruded from the medulla. 3j The roof expansion appeared at the same time in the region of the X ganglion as in the region of the VIII and \ , and developed at a uniform rate throughout the medulla. 4) Two factqrs are evident in the formation of the fourth ventricle: first, an upward and outward migration of the roof plate nuclei followed by a disintegration of the inner protoplasm: second, internal ])ressure exerted by the rapidly increasing cerebro-spiiial fluid, infiltrated and secreted by the roof plate itself.
D. Selachians and Amphibia. As types of these clas.ses I had access to very complete sets of serial sections of Squalus and Amblystoma embryos, and to one transverse series of a 15 nun. Necturus. A careful examination of this material contributed nothing new to the ontogeny of tiio fourth ventricle. It was possible, however, to confirm in these forms many of the conclusions arrived at in Petromyzon. In both Squalus and Amblystoma the nmch earlier appearance of the central canal brings about a much earlier and more extensive expansion of the fourth ventricle. The sections shown in figures 72 and 73, while possessing enormous expansions of the roof plate, exhibit less progress in the differentiation of the structure of the medulla than was revealed in a 12 day Petromyzon (fig. 41), in which very little or no expansion had taken place in the roof plate.
In Squalus and Amblystoma the typical embryonic central canal, in the form of a vertical cleft, is a comparatively late production. The canal appears soon after the neural folds coalesce as a horizontal cleft, its presence often being indicated only by a layer of pigment. This cleft becomes elliptical, then more or less circular, and finally changes to a vertical or dorso
24 WILLIAM V. ALLEN
ventral cleft. These changes in the shape of the central canal suggest a migration of cells, rather than a disintegration process or a splitting apart of the walls as a result of pressure from the secretion of cerebro-spinal fluid. The earliest Squalus series showed an elliptical canal with its longest axis horizontal, this changed to circular, and finally to the so-called typical embryonic central canal (dorso-ventral cleft canal). For a time in the medulla of Amblystoma the roof plate is much thicker than the floor plate, a condition which can be attributed to the presence of a number of neural crest cells in the roof plate, easily distinguishable from the other cells by their large size and spherical form. After the neural crest cells had been entirely separated from the roof, the roof and floor plates are found to.be about equally thick. The same thinning out of the roof plate occurs in Squalus in part through the giving off of neural crest cells, which in this species are indistinguishable from the other cells of the medulla. It is evident in Amblystoma (figs. 74 and 83, R.P.) and also in Squalus that the roof plate of the medulla has become no thinner than the roof plate of the spinal cord through the throwing off of the neural crest cells. There is no evidence that the roof plate in the medulla is rendered any weaker or any more susceptible to expansion than is that of the spinal cord, through the migration of gangUon cells.
Apparently the first expansion of the roof plate in Amblystoma (fig. 74, R.P.) and in Squalus is produced as in Petromyzon by an outward and upward migration of the roof plate cells. The later, more pronounced expansion and stretching of the roof plate can also be ascribed, as in Petromyzon, to internal pressure due to a decided increase of cerebro-spinal fluid (see fig. 72 for Squalus and 73 for Necturus). In both species the dorsal and middle portions of the embryonic central canal of the spinal cord are obliterated by an inward growth of the lateral plates through an addition of fibers. Pressure on the cerebrospinal fluid might be increased from that source. Also a similar effect would be produced through a marked proliferation of fibers in the medial and ventral portions of the lateral plates of the medulla, which brings about a coalescence of the ventral por
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 25
tions of the lateral plates in Squalus (fig. 72, C.C.C.) similar to the condition in Petromyzon. In Neeturus (fig. 73) and in Amblystoma the roof plates are expanded to such an extent that no fusion of the lateral plates takes place; nevertheless, the thickening of the walls tends to bring about a reduction in the size of the fourth ventricle and consequent pressure on the cerebro-spinal fluid. The probability of a decided increase of internal pressure from the cerebro-spinal fluid resulting from the reduction in caliber of the embrj'onic central canal of the spinal cord by at least two-thirds, is more e\'ident, if attention is directed to the fact that the length of the central canal of the spinal cord is fully twenty times that of the fourth ventricle. In both forms the above changes took place before the blood vessels had reached the dorsal surface of the roof plate or entered the medulla. A median sagittal section through a Squalus embryo of the same stage as figure 72 shows that no pontine flexure has appeared.
E. Pig embryos. For making observations on the development of the tela chorioidea of the fourth ventricle in manunals the writer had access to a very complete set of frontal sections of pig embryos from a stage of 4 or 5 mm. up to 14 mm. embryos.
The earliest section (fig. 75), which is from a 4 or 5 mm. pig. discloses that the fourth ventricle has only begun to expand. A large part of this expansion could be attributed to an outward and upward migration of the roof plate cells, and a part to internal pressure from cerebro-spinal fluid, of which traces are beginning to appear as a coagulum (S.C.F.). It should be noted that the first blood vessels are appearing above the basal portions of the roof plate, while none have at this stage entered the medulla. Also the ependymal cytoplasm is sufficiently granular to suggest a secretory process, and finally the roof plate has begun to expand before any nerve fibers have appeared in the marginal layer.
A transverse section of the medulla of a (3 mm. pig (fig. 7(3) revealed a considerable expansion of the roof plate without the aid of a pontine flexure. It is apparent that an increase in the amoimt of cerebro-spinal fluid is the main factor in bringing
2(i AVILLIAM V. ALLEN
about this pronounced expansion of the roof plate. Since the closure of the dorsal portion of the embryonic central canal of the spinal cord occurs much later in embryonic life, no increase in pressure from cerebro-spinal fluid could take place from that source. Also no increase was shown in the number of blood vessels outside the roof plate, and no blood vessels had entered the medulla. Consequently the only means of an increase of cerebro-spinal fluid would be through secretion and a slight infiltration from the blood vessels. This section demonstrates yery strikingly, even more so than figure 75, that the roof expansion begins very early in the development of the pig's medulla, as is evident from the fact that nerve fibers have only begun to appear in the marginal layer.
Between the 6 mm. and 7 mm. stages there occurs a marked increase in the blood vessels in the mesenchyme above the roof plate and a few blood vessels are entering the substance of the medulla. There can be no question that the roof plate is now an efficient organ for the production of cerebro-spinal fluid, and there is a noteworthy increase in the amount of coagulum in the cavity (figs. 75, 76, and 77). The first embryonic cerebro-spinal fluid, probably a mere intercellular fluid, showed little or no coagulum, from the method of fixation and staining used. This may indicate a relative increase of the elements formed by secretion. Figure 77 shows that a marked expansion of the roof plate has occurred in the 7 mm. pig. Since the 7.5 and 8 mm. pig embryos have very small pontine flexures, it is evident that this pig embryo has developed a well-expanded roof plate and chorioid plexus without the influence of a pontine flexure.
The roof plate in the 10 mm. pig (fig. 78, R.Ex.) and in the 14 mm. pig (figs. 79 and 80, R.Ex.) has undergone a decided expansion, especially in a dorsal direction. This final expansion of the roof plate is unquestionably due to the action of the pontine flexure upon a fourth ventricle filled with cerebro-spinal fluid already under moderate pressure. It would have been impossible for a pontine flexure acting alone on an empty fourth ventricle, as would be implied from His' experiments with bending an empty rubber tube slit dorsally, to have brought about
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 2i
the dorsal expansion of the roof plate exhibited in figures 79 and 80. Apparently this expansion of the roof plate in the pig has been gradual, for absolutely no stretching of the roof plate has occurred, except in one place, namely in its central anterior portion (fig. 79).
A study of these sections has disclosed a direct relationshiji between the expansion of the roof plate and the amount of visible coagulum in the ventricle. Since coagulum does not appear in sections of the early fourth ventricle, but does appear after the tela chorioidea has attained the function of producing cerebrospinal fluid (as is indicated by its vascularity and the granular appearance of the cells) it is fair to assume that the non-coagulable cerebro-spinal fluid found in sections of the early embryos is an embryonic cerebro-spinal fluid, which differs in no way from the ordinary intercellular fluid of other tissues. On the other hand, the coagulum seen in sections after the roof plate has reached the stage of a functional chorioid plexus is evidence of a chemical change in the fluid, which, if a product of secretion, is capable of exerting considerable internal pressure and consequent expansion of the roof plate.
It is apparent that the greater expansion of the roof plate in the pig is produced by the same factors as were recorded for Petromyzon, namely, an early migration outward of the roof plate cells followed by an expansion from within due to the formation of cereliro-spinal fluid, plus the action of a conspicuous pontine flexure on a fourth ventricle filled with cerebro-spinal fluid alread}^ under moderate pressure.
F. Human embryos. For this studj' an 8 and a 15 nun. transverse series and a 23 mm. frontal series were available. These embryos were too far advanced to show the earliest stages of the roof expansion of the fourth ventricle. If, however, the extreme posterior end of the roof plate of the fourth ventricle is examined in the 23 mm. embryo (figs. 26 and 27, R.Ex.), in the 15 mm. embryo (figs. 28 and 29, R.Ex.), and in the 8 mm. embryo (fig. 31, R.Ex.), which represents a region of the medulla little affected by the pontine flexure, it is apparent that the roof expansion was caused by identically the same factors as was
28 WILLIAM F. ALLEN
recorded for tlie pig embryos. In view of the facts, this explanation of the formation of the roof expansion seems more tenable to the writer than to attribute all, as His has done, to the action of a pontine flexure. For beyond question, a considerable expansion of the roof plate in the human medulla takes place before the pontine flexure appears.
2. Description of three roof plate expansions of the spinal cord in
the 20 cm. Polistotrema series
An interesting variation (abnormality) was found in the spinal cord of a single specimen of Polistotrema (Bdellostoma), otherwise unusual. Certain structures appeared in the roof of the cord that were very similar to the tela chorioidea of the fourth ventricle, and will be described because of the light that they may throw on the origin of the tela chorioidea of the fourth ventricle.
What has been designated as the first roof plate expansion of the 20 cm. Polistotrema series is shown in the photographs of models 1 and 2 (figs. 1 and 2, 4 and 5, R.Ex.) to be an immense outcropping of the roof plate ependjana. Most unfortunately the anterior portion of this specimen, from which the series through the tail was taken, has been lost, so that it is impossible to state how much further cephalad this expansion of the roof plate extended, or whether there were other outcroppings of the roof plate in front of it as there are behind. It appears in the first model (fig. 2, R.Ex.) and in the first transverse section (fig. 10, R.P.Ex.) as a median mass, covering about one-half of the dorsal surface of the spinal cord; it then shifts gradually over to the right side (figs. 2 and 11); then gradually attains a median position. In this position it continues as far as the middle of model 2 (fig. 5), covering a large part of the dorsal surface of the spinal cord.
For the most part the roof plate expansion contains a cavity of considerable size, which is shown anteriorly in the cast (fig.
3, C.C.Ex.) and in transverse section (figs. 10 to 12) to be in direct communication below with the central canal. Posteriorly
SPINAL CORD AXD MEDULLA OF CYCLOSTOMES 29
such a connection is wanting (fig, 13). The fact that in various places (fig. 13) there is more or less of a string of ependymal cells between the walls of the central canal and the roof plate expansion suggests that in a more embryonic state an open communication existed between the roof plate cavity and the central canal, which became closed in the region of the posterior end of the roof expansion, and persisted in the anterior end. Another variation to be noted in the cavity of the posterior end of the roof plate expansion is that it contains numerous islands and promontories of ependymal cells and connective tissue, shown as white spaces in figure 6. These probably represent portions of the roof plate that have not been completely excavated to form a continuous cavity.
Transverse sections 11 to 13 show the cavity of tliis first roof plate expansion to be larger than the fourth ventricle in Polistotrema (figs. 64 to 66), and the whole structure more nearly resembles a typical fourth ventricle than does the fourth ventricle itself in this animal. For the most part the walls of the roof plate consist of true ependymal cells, ditTering in no way from those surrounding the central canal, except for their shorter peripheral processes. Posteriorly connective tissue takes the place of many of these cells. One of these cells is shown in figure 13A to be sufficiently granular to suggest a secretory function. Everywhere the walls of both the roof plate and the central canal are very vascular, suggesting a modified choroid plexus. Figure 13A will demonstrate the ease witli which inliltration and diffusion could take place between the blood vessels of the roof plate and its cavity. In figure 11a fold of the roof expansion, containing a blood vessel, will be seen extending into the cavity, and about it there is collected a mass of coagulated cerebro-spinal fluid {S.C.F.).
Of still greater interest are the two posterioi- outcroppings of the roof plates, designated as roof plate expansions 2 and 3 (figs. 4 and 5, R.Ex. 2 and 3), Since these two roof plate expansions are considerably smaller than the first, they can be compared directly with the roof plate of the rhombencephalon of anv embrvo.
80 . WILLIAM F. ALLEN
The so-called second roof plate expansion appears in model 2 (figs. 4 and 5, R.Ex. 2) immediately behind the first. This is a much smaller outcropping of the roof plate. It contains a cavity (figs. 16 and 17, C.C.Ex.), which spreads out a little laterally and caudally, and communicates below with the central canal. This cavity is filled with a fine fibrillar feltwork that stains deeply with orange G, and which for the most part is coagulated cerebro-spinal fluid. The ependeymal walls of both the roof expansion and the central canal are sufficiently vascular to suggest that we have here as in the previous roof expansion, a modified chorioid plexus which is producing cerebro-spinal fluid.
In model 2 (figs. 4 and 5, R.Ex. 3) the third outcropping of the roof plate is some little distance behind the second, about equaling it in size. This roof expansion has not been figured in transverse section, but from an examination of a graphic reconstruction of the central canal, three small isolated cavities were seen extending in a cephalo-caudal direction. The middle cavity was found to be in communication below with the central canal. It held cerebro-spinal fluid, and its walls apparently functioned in the production of the same.
It is evident that these three expansions of the roof plate are independent of one another. The arrangement of the three small isolated cavities in the third expansion seems to be an embryonic condition, and suggests that the larger cavities may have been produced by the union of several smaller roof expansions. It may be supposed that these expansions were formed by the multiplication of roof plate cells, which were pushed up in solid masses, in which vacuolization and confluence of adjacent cavities produced the larger cavities seen in the adult.
It is of interest to note that in this individual a posterior sinus (fig. 20, S.T.), probably representing the sinus terminalis of a normal individual, was isolated by the complete occlusion of the central canal by ependymal tissue. The ependyma surrounding this cavity is very vascular, and the cavity is distended with cerebro-spinal fluid. This sinus is much larger than the fourth ventricle in a normal individual.
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 31
When the roof expansions in the spinal cord of this series of Polistotrema are compared with the fourth ventricle of the higher vertebrate embryos, it is evident that the similarity is onlj- superficial, for the later stages in the medulla oblongata (action of pontine flexure on a thin-roofed neural tube full of cerebro-spinal fluid under moderate pressure), are dependent on factors which are not present in the spinal cord of Polistotrema. The extreme caudal end of the roof plate of the fourth ventricle in the higher vertebrates, however, has not been affected by these later factors, and presents a condition where comparison is made po.ssible. From a compari.son of the roof plate expansion in the 23 nmi. human embryo (figs. 26 and 27, R.Ex.), in the 15 nmi. human embryo (figs. 28 and 29, R.Ex.), and in the 8 mm. human embryo (fig. 31, R.Ex.) with the second roof plate expansion of the spinal cord in the 20 cm. Polistotrema series (figs. 4 and 5. 7?. Ex. 2, and figs. 15 to 17, R.P.Ex. 2) and with the third roof plate expansion of the same .series (figs. 4 and 5, R.Ex. 3), it is evident that the same main factor is present in all, namely, a migration outward and upward of certain roof plate ependymal cells to fonn an enlarged dorsal cavity. In each case the purpose of this structure is to form an organ for the production and storing of cerebro-spinal fluid. As soon as these structures as.sumed the function of infiltrating and secretory organs their walls l)ecame further expanded from internal pressure of the cerebrospinal fluid.
From a re\iew of the early stages of the formation of the roof expansion in man, the pig, Amblystoma, Squalus, and Petromyzon, it is fair to assume that the source and early development of the roof expansion of the medulla are identical to the three similar roof plate expansions, described for the spinal cord of a 20 cm. Polistotrema. It is possible that both had their plujlogenetic anlaqe as mutations, the former from some primitive vertebrate, and the latter from a normal Polistotrema, that both were useful and dominant characters, and in case of the medulla, where the animal was allowed to reproduce, this character became preserved for the race.
82 WILLIAM F. ALLEN
CAUSES UNDKRLYIiNG THE FLATTENING OF THE SPINAL CORD IN CYCLOSTOMES
If our attention is first directed to a transverse section through an adult Polistotrema (figs. 10 and 71) it might be inferred, since there is ample room in the membranous neural canal for a wellrounded spinal cord, that the flattening of the spinal cord might be attributed entirely to internal factors. A glance at a transverse section through a developing Polistotrema spinal cord (fig. 57) suffices to show that there is proportionately much less room within the membranous neural canal, and that a mechanical force in the form of a rapidly growing notochord is at work immediately below the spinal cord.
At the outset it seems advisable to establish arbitrarily a typical state of an embryonic spinal cord, by which a direct comparison of one form can be made with another. The examination of very early stages of the spinal cord in a large number of embryos of Squalus, Amblystoma, the chick, and the pig, in all of which the neural tube is formed by the rolling up of the neural plate, shows that the neural tube passes through three stages: a) A depressed tube with the central canal in the form of a horizontal cleft; b) a cylindrical tube with the canal circular in cross section; and c) a laterally compressed tube with the canal in the form of a vertical cleft (figs. 81 to 86). The existence of this series of changes in Squalus has been shown in a table of developmental stages compiled by Scammon. The third stage may be selected as the typical embryonic spinal cord. This stage is reached at about the time of the first appearance of nerve fibers in the marginal layer.
As a result of a comparison of the typical embryonic stages of the spinal cord in the following transverse sections^ (fig. 39 for Petromyzon, fig. 55 for Polistotrema, fig. 81 for Squalus, fig.
83 for Amblystoma, fig. 84 for the turtle, fig. 85 for the chick, and fig. 86 for the pig) it is clear that we have all gradations from
- It is obvious that this comparison would have no value unless the sections
were truly transverse sections. To avoid selecting oblique transverse sections, these figures were always drawn from anterior trunk sections if the embryo showed any flexures.
SPINAL CORD AND MEDILLA OF CYCLOSTOMES 33
the nearly cylindrical spinal cords of the Cyclostomes (Petromyzon and Polistotrema) to the very much compressed (flattened laterally) cords of the chick and pig. Were it not for the intermediate stages of Amblystoma and the turtle we might bo justified in establishing two distinct types of the embryonic spinal cord: t\i)o 1 cylindrical, and type 2 compressed. We could even go fuithor and classify the Cyclostome embryonic cord under type 1 and the Gnathostome cord under type 2.
While the equal expansion of the spinal cord through the addition of nerve cells and fibers to the typical embryonic stage in the pig would tend to produce a cylindrical spinal cord, and in the C'3'clostomo would tend to produce a depressed cord, the internal structure shows that the origin of the differences between the spinal cord of a Cyclostome and a mammal is not so simple. The neural axis of Pctiomyzon in an early stage, corresponding to stage fa) above, is decidedly c()mi)ressed instead of depressed. The problem then is to explain the change from a laterally compressed cord in the early IVtromyzon embryos to the gradunllx depressed, ribbon-like. si)inal cord of the adult.
A careful examination of figures of) to .')". wliicli are taken from practically the same region from three dilYerent Polistotrema embryos, shows \-ery elcMi-ly that the growing notoeliord is bringing about the marked flattening (depression) and ventral indenting of the spinal cord. Tn connection with figure 5") it should be noted that tlie spinal cord is in the so-called typical embryonic stage, and amid siuToundings peculiarly favorable for undergoing a depression from a rapidly growing notochord. It will be seen that the spinal cord is closely enveloped by a meningeal membrane (P.M.), approximating a layer of connective tissue, the future^ membranous neural arch, which is firmlj^ attached below to the growing notochord. Directly above, the mesenchyme is proliferating and migrating toward the center to form a median dorsal cartilage (M.D.C.) ; while there is apparently but little lateral resistance in the way of massing of mesenchyme and the formation of myotomes. In a somewhat later stage (fig. 56) some growth has taken place in the notochord. producing a slight indentation on the spinal cord. In a much
THE JOrRNAl. OK CO.MI'AH\TI\ K NElROI.UtiY, VOL. 26, NO. 1
M WILLIAM F. ALLEN
Inter sta^e (fig. 57) a nieciiaii dorsal cartilage has been formed. The inuoh stronger membranous neural arches are firmly attached above to this dorsal cartilage and l)elow to the notocliord. The soft plastic spinal cord is thus closely confined by the dorsal cartilage above and less closely by the neural arches laterally. The notochord beneath it has gi'own very rapidly and its enormous increase in size has brought about a decided flattening (depression) of the spinal cord and indentation of its ventral surface.
It is equally clear in Petromyzon also that the growing notochord is to be looked upon as a direct cause for bringing about the flattening of the spinal cord. The external conditions surrounding the spinal cord are shown in figure 58 to be equally favorable for assisting the notochord in this process, with the possible exception that the membranous neural arch is attached abo\'e to a membranous neural spine instead of a cartilage, which, however, may be compensated for by an increased dorsal growth of the myotomes.
The hypothesis that the flattening of the spinal cord in Cyclostomes is largely brought about by the upward growth of the notochord after the manner set forth in the previous paragraph is considerabl}^ strengthened by the fact that a certain relationship exists between the size of the notochord and the amount of flattening of the spinal cord.
This is clear in the 20 cm. Polistotrema series from sections of the posterior end of the spinal cord (figs. 19, 21, and 22) and from the photographs of the model of the same region (figs. 7 to 9) ; the flattening (depression) of the spinal cord becomingless evident as the notochord decreases in size. More striking is a similar relationship shown in the tail region of the 70 mm. Polistotrema (figs. 59 and 60) for the reason that these two sections were only one-fourth of a millimeter apart. The above relationship between the size of the notochord and the depression of the spinal cord can be demonstrated fully as conclusively in the medulla region (see figs. 61 and 62). It should l)e recorded for these two sections that their structure is the same as that of the spinal cord and that they are less than one-half
SPINAL CORD AXD MEDULLA OF CYCLOSTOMES 35
a millimeter apart. Also the same relationship could be shown in sections anterior to figure 62, and from similar sections of a Petromyzon larva. A po.ssible objection to applying this argument in the tail region might arise from the fact that the extreme posterior end of the spinal cord is non-nervous, consisting entirely of ependyma and undifferentiated embryonic cells. In reply to this we would invite comj)arison of figures 21 and 22, where the structure is non-nervous in both cases, and where the effect of the notochord is obvious.
That the spinal cord of the higher vertel)rat('s has not been depressed by pressure from the notochord is due ()i)viously to the fact that the notochord is an embryonic structure, which never attains sufficient size to have any influence on the spinal cord. Tliis is clear from figures 85 and 86. In Cyclostomes, liowcNcr, Ihc notochord is a very important structure, develops early and grows foi- a long period of time, and serves as the skeletal axis of tli(> :i(lult. in fishes. Aiiii)liil)ia, and reptiles th(> growing notochord iiia> have some slight (effect in flattening of the adult spinal conl. hi Ami)hioxus the ventral surface of the spinal cord clearlx shows the indenting effect of the growing notochord ' lig. 70 ). and in the trunk region where the diameter of \hr noto('h()i-(l is gicatest. th(> s|)inal cord is most depressed.
(;i;\i;k \i. coxsidi;!} a rioxs and si .\imai;v
l'"roiii th(> foi'cgoing tacts the following conclusions .seem full\warranted :
1) In the dc\ clopinciU of the loot' ])lale ('\|)aiisioii itela chorioideai in the medulla oblongata of most vertebrates three separate stages or (>pochs of expansion should be recognized: a) A first enlargement of the dorsal portion of the embryonic central canal took place from a migration outward and upward of certain of the roof plate cells, or as was the case in Petromyzon from the migration of the nuclei and jirobable disintegration of the cytoplasm, b) The second stage in the expansion of the roof plate was the direct result of an increase of pressure from the cerebro-spinal fluid, produced from at least two possible
8() WILLIAM V. ALLEN
st)ur('(\^. First in the lower \ei-tei)rjites, as a conseciuence of considerable embryonic cerel:)r()-s|)inal fluid being forced into the fourth ventricle from the closure of the tlorsal and central portions of the embryonic central canal in the spinal cord and the ventral portion of the embryonic central canal in the medulla, through the union and fusion of the corresponding portions of the lateral plates. Second, in all vertebrates, through the production of cerebro-spinal fluid by the walls of the fourth ventricle assuming the role of infiltration, diffusion and secretion. That the ventral portion of the embryonic central canal of the spinal cord was not expanded by the same cause is explained by the fact that it was reinforced at a very early stage by nerve fibers and supported by a growing notochord. c) A third and final stage in the expansion of the roof plate in the higher vertebrates was brought about by the appearance of a pontine flexure acting on a thin-roofed medulla filled with cerebro-spinal fluid, itself under moderate pressure.
2) It is the opinion of the writer that the most important factors in bringing about the expansion of the roof plate of the medulla are those concerned in the second stage described above. For a considerable period of time these factors apparently work in conjunction with the forces of the first stage in resisting counter ingrowths of the lateral plates which would tend to close up the central canal. Since it was shown that no pontine flexure occured in Petromyzon, it can be concluded that the rather extensive expansion of the roof plate in the medulla of this genus was accomplished solely through the factors entering into the first and second stages. For the second stage it was recorded in most cases, especially well shown in the pig, that the size of the fourth ventricle and the expansion of its roof plate bear a close relationship to the amount of coagulum seen in sections. Since this coagulum did not appear in sections until after the roof plate had attained the function of producing cerebro-spinal fluid, it probably indicates a chemical change in the cerebro-spinal fluid, the products of secretion now being added to the tissue fluids which entered the ventricle b\" infiltration. As a product of secretion, the cerebro-spinal fluid would be capable of exerting
SPINAL CORD AXD MEDULLA OF CYCLOSTOMES 37
considerable pressure on the thin and phistic roof plate. The tela chorioidea is differentiated as an organ for the production of cerebro-spinal fluid before blood vessels have entered the central nervous system, at a time when the nutritive function of this fluid is important.
3) The appearance of a third stage in the roof expansion of the medulla, due to a pontine flexure, is of little significance save in the higher vertebrates, where it was held that without cerebro-spinal fluid confined in the ventricle there would be no reason for maintaining that a further expansion of the roof plate would take place from the action of a pontine flexure: more than likely, the roof plate would have been folded up within the ventricle. In His' experiment with the bending upward of a dorsally-slit piece of rubber tubing, the elasticity of the rubber tubing, which forced apart the cut surfaces, would be com])arable to the action of the cerebro-spinal fluid under moderate pressure within the ventricle, which factor His has ajiparently disregarded.
4) In the adult Amj^ihioxus there is nothing which for a certainty could be homologized to the fourth ventricle and its expanded roof plate. Two isolated cavities in the region of the roof plate, which might be taken for the anlage of the fourth ventricle, ai;)pear to the writer to be nothing more than vestiges of a much larger emliryonic central canal. If Amphioxus possesses no fourth ventricle in the adult we may saf(>ly conjecture that more primitive vertebrates had a central nervous system in which there was no distinction between medulla and spinal cord.
5) In an attempt to trace the phylogenetic history of the roof expansion of the fourth ventricle in living vertel)rates, the peculiar modification of the fourth ventricle in the adult Polistotrema (Bdellostoma) should be recorded here even though it has been accurateh' described b}- Sanders, Holm, Miss Worthington, Sterzi, Cole, and Nicholls. From the adult it is evident that the well-formed fourth ventricle of the embryo has become transformed through a process of centralization to a deep-seated canal, for the most part no larger than the central canal of the spinal cord. Of especial interest is the fact that its anterior and pos
3S WILLIAM F. ALLEN
terior i)()rti()ns has developed into specialized organs for the production of cerebro-spinal fluid. Notwithstanding this specialization, the fourth ventricle is thought to be decidedly inferior to the tela chorioidea of Petromyzon as an organ for the production of cerebro-spinal fhiid.
6) In the spinal cord of one individual of Polistotrema there occurred at least three expansions of the roof plate which resemble the roof of the fourth ventricle in other vertebrates. From the fact that these expansions were very vascular and their cells granular it is inferred that they functioned as choroid plexuses for the formation of cerebro-spinal fluid. The writer presents the hypothesis that the fourth ventricle in ancestral vertebrates may have originated as a mutation, similar to this sport plexus in the spinal cord of Polistotrema; that such sport expansions may have occurred at various places such as the diencephalic segment, the roof of the mesencephalon where a choroid plexus still exists in Petromyzon, and in the hind brain and spinal cord. Such mutations, proving to be useful have been preserved in the vertebrate race.
Concerning the flattening of the spinal cord in Cyclostomes
7) A great variation in the shape of the so-called typical embryonic spinal cord is to be recorded. In Petromyzon it was found to be nearly cylindrical, to be moderately compressed in Squalus, Amblystoma, and in the turtle, and decidedly compressed in the chick and the pig.
8) To obtain this typical stage the original compressed spinal cord of Petromyzon must have undergone a marked depression, and the early depressed neural tubes of Squalus, Amblystoma, turtle, chick, and pig must have undergone a decided compression. The main factor causing this depression in the former was thought to be ventral pressure from a growing notochord, and the compression of the latter was attributed to lateral pressure from the growing myotomes.
9) Transverse sections immediately before and during the time that the greatest depression of the spinal cord is taking place
SPINAL CORD AND MEDULLA OF CYCLOSTOMES 39
in Polistotrema and Petronwzon show conclusively that the main factor involved is the pronounced growth of the notochord. It was further established that the embryonic spinal cord was not only in a very plastic condition, but that the general environment was decidedh' favorable for bringing about a depression of the spinal cord through this agency.
lOj The conclusions outlined in (9) were cousideral)ly strengthened by the fact that a direct relationship was established, in both the medulla and tail region, between the size of the noto(^hord and the amount of depression exhibited in the spinal cord. This was shown in late embryos in both Polistotroina and Potromyzon, and in adult Polistotrema.
11) That a similar depression did not take place in the higher vertebrates from a growing notochord was explained by the fact that the notochord is relatively a transitory and insignificant structure; while in the Cyclostomes it is not only formed earl>in embryonic life, but grows rapidly and continuously.
40 WIIjLIAM F. ALLEN
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I'LATIO 1
KXri.ANATION OF FIGURES
1 to 9 rc'picsciit pliolographs from models of the extreme posterior end of the spinal cord tioiii a 20 cm. Polistotrcma (Bdellostoma), illustrating a series of three extensions of the central canal into roof plate expansions. These models were prepared at a magnification of 100 diameters, and were reduced one-half in i)li()t()graphing. In a few cases where certain outlines were somewhat indistinct in the photographs they were strengthened with pen and ink. The roof plate expansions in those models were painted lighter and appear the same in the photograi)hs. Kxccpt at the posterior end of the last model, all of the models of the spinal cord exhibit a marked depression and in ventral views they show a pronounced indentation at the center immediately above the notochord. As indicated by an arrow, the caudal direction in figures 1 to 3 is toward the left; while in figures 4 to 9 it is toward the right.
1 The most anterior of the four models seen from the right side. It includes a distance of about two segments. Observe that the roof plate expansion, which covers a large portion of the dorsal surface of the spinal cord, is rather low. It is not known where it begins, or whether there are other outcroppings in front of it as there are behind. It extends caudad some little distance on this model. On the dorsal surface of the cord one dorsal or sensory root is shown, and on the ventral side one ventral root is seen in entirety, being composed of several rootlets. X 50.
2 Dorsal vievvof the same modelshown in figure 1, seen from above. Note that the roof expansion covers a large portion of the spinal cord. At the anterior end it occupies a large central portion, then becomes gradually smaller, and at the same time is confined largely to the left side, after which it gradually increases in size until the posterior end of the model is reached, where it is decidedly wider than at the anterior end, and extends farther over to the right side than to the left. X 50.
3 Ventral view of the cast of the central canal and extension of the same up and out into the roof expansion of the same region of the cord as is shown in figures 1 and 2. It will be seen that the central canal and cavity of the roof plate expansion are connected throughout. In certain places on the right side, shown in white, the roof plate is solid, consisting of ependymal and connective tissue in place of a cavity. For some distance posteriorly on the right side, where the roof expansion is widest, there is no cavity in the roof e.xpansion. The knob-like projections from the right side of the cast of the central canal represent diverticula, and in sections through such a region there would appear to be two central canals. X 50.
ABBREVIATIONS
C.C, central canal or cast of the same M.R., motor or ventral spinal nerve
C.C.Ex., central canal extension into root
roof plate expansion or cast of the R.Ex., roof plate expansion same S.R., sensory or dorsal spinal nerve
root
MXAL CURD AND .VEDLLLA < F CYC LOsiM )MJ:
WII.I.rAM F. AII.KN
-^^
aEx,
M. R.
S.R.
R.E
C.C.
acEx.
- c.a
FLAri'
EXPLANATION OF FIGURES
4 I.aliMal vii'w of the left side of the second model of the Polistotreina spinal curd, which should follow figure 1. Observe the continuation of the first roof plate expansion noted in hgure 1 and two additional outcroppings of the roof plate (/?. ^.r. 2 and 3). Between these outcroppings the spinal cord is perfectly iionnal. Three motor and sensory roots are shown in the figure. X 50.
.') Dorsal photograph of the same model as figure 4. The extent and positions of the three roof plate outcroppings previously mentioned in the description of figure 4 are well portrayed here, as are also the sensory roots. X 50.
6 Dor.sal view of a cast of the more caudal portion of the central canal and cavity of the first roof plate expansion. This cast differs from the more anterior cast in that there are no connections between the central canal and the cavity of the roof expansion. Evidence from transverse sections favors the view that a connection once existed, which has been cut ofY in later development. In the two posterior outcroppings of the roof plate there is a connection between the central canal and the cavity of the roof expansion. The light places in the photograph are indicative of places in the roof expansion where it is solid and contains no cavity. They are more numerous and decidedly larger than was shown in the more anterior model (fig. 3). X 50.
Between models 2 and 4 (figs. 4 and 7) comes model 3, the photograph of which has not been included as a figure. It is about equal in length to models 2 and 4. simply eoimecting the two, without presenting any peculiarities in roof plate expansion, etc.
ABBREVIATIONS
M.R.. motor or v(Mitral spinal nerve R.Ex.{Z), third roof plate expansion
root in the Polistotrema cord
R.Ex., roof plate expansion S.H., sensory or dorsal spinal nerve
R.Ex.i^Z), second roof plate expansion root in the Polistotrema cord
46
SPINAL CURD AND MEDULLA OF CYCLOSTUMES
WILLIAM F. ALLEX
R.E
M.R,
R.Ex,
-R.Ex,
47
I'LATK 3
lOXri-ANATION OK FKiUKIOS
7, 8, and 'J rc|ucsciit laterul, dorsal, and ventral photographs of the, lourMi model, which includes the extreme posterior end of the spinal cord. Note (especially the swelling above and below (S.T.) caused probably by an abnormal sinus terminalis. From this point caudad two factors occur, which may greatly modify the shape of the spinal cord. First, the notochord gradually decreases in caliber and ends at {Nc. 1); second, the spinal cord has not developed a nervous structure, consisting solely of ependjTiia and round undifferentiated embryonic cells. As a result the spinal cord will be seen to gradually become rounded, and after extending past the notochord it ends in dorsal and ventral processes that become lost in the surrounding connective tissue. The posterior motor roots exhibit a reduction in the number of rootlets, and they approximate each other more closely. The posterior sensory roots become greatly reduced in size, and the last left one has no corres]:)onding roots on the opposite side. X 50.
ABBREVIATIONS
M.R.{1), last motor or ventral spinal S.R. (1), last sensory or dorsal spinal
nerve root nerve root
Nc. (1), posterior end of Polistotrema S.T.. sinus terminalis
notochord shown in model
platp: 4
EXPLANATION OF FIGURES
10 to 23, show 14 transverse sections through the posterior spinal cord region of a 20 cm. Polistotrema (Bdellostoma), the same as was modelled and shown in figures 1 to 9. The outlines were all drawn with the aid of a Leitz-Edinger drawing apparatus, using a magnification of 140 diameters and were reduced onehalf in reproduction. They are numbered consecutively from anterior to posterior. Figures 10 to 14 pass through what has been designated as the first roof plate expansion; figures 15 to 17 through the second roof plate expansion; figure 20 through the abnormal sinus terminalis; and figures 21 to 23 at various intervals through the extreme posterior end of the spinal cord, which has failed to develop any nervous structures, and has not to any extent been flattened in its development by the growth of the notochord. The enormous concavity seen on the ventral surface of spinal cord in figures 11 to 14 is to a large extent an artifact due to the preparation of the series. It should be noted that the cavities of the roof plate expansions are full of a fibrillar feltwork, for the most part coagulated cerebro-spinal fluid, that their walls are moderately expanded by it, and their cells are sufficiently granular to suggest a secretary function.
10 is from the most anterior section of this series. The entire neural arch, median dorsal cartilaginous bar, and a portion of the notochord are included in this figure. The so-called first roof plate expansion covers a large area of the central portion of the spinal cord; its cavity is in communication with the cen tral canal. The ependymal walls of both the cavity and central canal are composed of several layers of cells. Motor roots, motor cells, substantia gelatinosa cells, and blood vessels are to be seen in transverse section. X 70. {Continued on pnge, 50) 48
SPINAL CORD AND MEDULLA OF CY(L08T0MES
WI[.I lAM F. VLLEX
S.T,
V S.R.
THK .lOllRN'Vt, OF rOMI'Mi\TI\
I!)
- y. vol. 26, xr
{C'otitiii lied from jkkjc 4^)
11 A transverse section taken 840 microns behind figure 10. It would pass ( hrou-;!! al)()ut the center of thefirst model (figs. 1 and 2) . Note that the roof expansion, while containing an enormous cavity, is confined almost entirely to the right side. It is decidedly suggestive of a chorioid plexus forming cerebrospinal fluid. A blood vessel lies in a fold in its wall and about this fold there is a great mass of coagulum. Throughout the first roof plate expansion, the cavity, and in many places the central canal itself, is larger than either the third or the fourth ventricle. X 70.
12 From a section 645 microns behind figure 11, and not far from the caudal end of the first model (figs. 1 and 2). At this point the first roof plate expansion and its enclosed cavity attain their greatest width, which cavity is broadly connected with the central canal. X 70.
13 Transverse section 80 microns behind figure 12 and at the very beginning of model 2 (figs. 4 and 5). The dorsal wall of the first roof expansion is very wide and extremely vascular, and the convexity of its walls and the arch of the cavity bear evidence of moderate internal pressure from cerebro-spinal fluid. The wall, while still quite thick, contains a lesser number of ependymal cells, but more connective tissue. Note especially the absence of any direct connection between the cavity of the roof expansion and the central canal. A chain of ependymal cells still connects the two, and may be indicative of a former embryonic connection that has been lost. From this region, caudad, there is no communication between the cavity of the roof expansion and the central canal. That a former connection occurred may be indicated by the fact that at various intervals ependymal cells are scattered between the two. A sensory root can be seen sending its fibers inward toward what is believed to be the substantia gelatinosa. X 70.
13 A, represents a small portion of the first roof plate expansion and its inclosed cavity from a section taken 410 microns behind figure 13. Observe especially the rich blood supply for the dorsal wall and the ease by which diffusion could take place between a blood vessel and the cavity. A large roof plate cell is drawn separately, highly magnified, directly to the left of 13 A. Note the fine granules in cytoplasm, which gives evidence of being secretory. X 125.
ABBREVIATION.^
B.V., blood vessel M.R., motor or ventral spinal nerve
C.C, central canal root
C.C.Ex., central canal extension into .V.A., membranous neural arch
roof plate expansion N.C., notochord
Ep.N., layer of ependymal nuclei R.P.Ex., roof plate expansion
M.C., motor or effective cells S.C.F., cerebro-spinal fluid
M.D.C., median dorsal cartilaginous S.G., substantia gelatinosa
bar S.R., sensory or dorsal spinal nerve
M.F., MixUcrian or giant fiber root
50
.SPINAL COHD AND .MEDLLLA OF CYCLOSTOMES
WILUAM F. ALLEN
PLATE 4
C.CJ7;,
■ \:',.\
1 1, rcpre
>\ ili(
■ first roof
'""■'•■
A motor
.stcri
or throuf2;li
uf pi
ate, whic-li
roof
expansion
I'LA'i'i'; .-)
KXl'I.ANATIONT OK KICUIUCS
II I'loiii a Iraiisvcrsc section lakcn I!) microns hcliiTid limine seiits a condition i'oiuul tluouKliout the entire posterior end ol expansion, where a series of cavities are connected with one ano root is seen issuing from the cord. X 70.
15 to 17 Three transverse sections, anterior, middle, and po what has been designated as the second outcroi)i)ing of the riK in model 2 (figs. 4 and 5) appears immediately behind the first already figured. In figure 15, which is through the anterior end of this roof plate expansion, the outcropping is one of cells only. In figure 16 a distinct cavity containing coagulum is visible, connected below with the central canal. Observe the vascularity of its walls, of the ependyma surrounding the central canai and the diverticula of the same, which doubtless serve as a modified chorioid plexus for producing cerebro-spinal fluid. In figure 17, which is through the posterior end of ths roof expansion, a cavity is still present. Its walls, which are mainly connective tissue, are rich in blood vessels. A few scattered ependymal cells and fibers represent a possible connection with the central canal. A third outcropping of the roof plate has been described, but not figured in transverse section. It is located behind the second expansion, and the fact that no ependymal cells are encountered between the first and the second, and the second and third roof expansions, except immediately surrounding the central canal, favors the view that these separate outcroppings of the roof plate in the adult were never connected in an embryonic state. X 70.
18 and 19 Two sections some distance caudad of figure 17 and some little distance apart. They show the spinal cord to l)e jjcrfectl}^ normal, l)ut to be gradually tapering down in size. Figure 18 is from that part of the cord represented by model 3. The ependymal area in the center has maintained its original size, the reduction that has occurred in size is to be found in the nervous portion. Not only does the ependymal area broadly divide the spinal cord into two halves, but it has here as elsewhere in a number of places, entirely obliterated the central canal for a space of a few microns. Figure 19 is some distance caudad of figure 18, passing through the sensory nerve roots one segment anterior to the abnormal sinus terminalis (model 4, fig. 9, S.T.). The spinal cord will be seen to be fast losing its nervous structure, for no motor cells will be seen in this section or in any further caudad. X 70.
ABBKEVIATIO.N'S
B.V., blood vessel M.V.C, median ventral cartilaginous
C.C., central canal bar
C.C.Ex., central canal extension into A^^l., membranous neural arcli
roof plate expansion or cast of the Nc, notochord
same R.P.Ex., roof plate expansion
Ep.C, ependymal cells R.P.Ex. (2), second roof plate expun Ep.N., layer of ependymal nuclei sion
M.D.C., median dorsal cartilaginous S.G., substantia geUitinosa
bar S.R., sensory or dorsal spinal nerve
M.R., motor or ventral spinal nerve root
root
52
SPINAL CORD AND MEDULLA OF CYCLOSTOMEK
WILLIAM F. ALLEN
C.(;.Ex.
CC.
Ep.M
R.PEx(^.
C,C.
^^f^M
BX
CC.
15
CC
R.P.Kk^iu
,.^'
%Al^-}
Ef>.N.
16
S.R
M.R
M.R.
18
^ 7JR.
M.V.C.
53
I'LA'I'I'; c.
20 'I'hi'ough tlu' ceiitcT of tlu' ubnoniial sinus tcnniiuilis, showiiifj; llu' l;ist. pair of sensory roots (not last sensory root) in section. Observe especially the large al)norinal sinus together with the rich vascular supply for the tissue immediately surrounding it. Note also the dorsal and ventral swelling caused by this sinus and compare with photograph of the model (figs. 7 and 9) . They show every evidence of being expanded by the cerebro-spinal fluid. From this point caudad no nervous structures have developed in the spinal cord. .Vlso from this point caudad the notochord gradually decreases in caliber. X 70.
21 to 23 represent three transverse sections taken through the extreme posterior, non-nervous, end of the spinal cord, which is composed solely of supporting tissue and undifferentiated embryonic cells. Figure 21 is the most cephalic, and passes through the spinal cord a short distance behind the abnormal sinus terminalis (figs. 9 and 20, S.T.). The spinal cord is still flattened here, but not indented ventrad, and contains a normal central canal. In figure 22 there has occurred a marked reduction in the size of the notochord. Observe that the spinal cord has not become flattened as it has more anteriorly, where the notochord is massive. With figures 21 and 22 compare figures 59 and 60, which are taken from a similar region of the spinal cord from a 70 mm. Polistotrema embryo. Figure 23 is the last of this series of drawings. It passes through the extreme posterior end of the spinal cord, some 45 microns caudad of the last trace of the notochord. Note the presence of a central canal, and that the cord has separated into several processes, which further on become lost in the surrounding connective tissue. X 70.
PLATE 7
EXPLANATION OF FIGURES
24 to 31 From transverse frontal sections through the medulla and spinal cord of several human embryos, drawn with the aid of an Edinger-Leitz drawing apparatus, and reduced 3 diameters in reproduction.
24 and 25 From transverse sections through the rhombic brain (frontal through the embryo) of a 23 mm. human embryo; figure 25 passes through the V root and posterior end of the cerebellar rudiments (lateral lobes), while figure 24 is from a more anterior section. The roof expansion (chorioid plexus) is shown as a conspicuous black line in these figures. Everywhere within the boundaries of the roof expansion, the cavity is filled not only with coagulated cerebro-spinal fluid, but with embryonic red corpuscles. Whether these entered through venolymphatic openings (C) or are the result of extravasations was not determined. Wherever mesenchyme borders the roof expansion it is very vascular. It is apparent that the roof expansion is under moderate internal pressure. At first glance the roof expansion will show resemblance to the so-called first roof plate expansion of the spinal cord of the 20 cm. Polistotrema, already figured, but its later mode of development was shown to be very different. X 10.
{Continued on -page 56)
54
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILIIAM F. AI.LEN
-M.D.Q.
C.C.
M.B.C.
B.V.
C.C.
V^ -,^*«\r^'
C.//.A.
Nc.
M.V.C.
22
\ ' V ^\ 1/
■ M.V.C.
23
AltllKKVIATIONS
/^. r., I)lood vessel
r.(\, central canal
(\X..\., cartilaginous neural arch
C.T., white fibrous connective tissue
Ep., epcndynia
M.D.C, median dorsal cartihiuinous l)ar
M.V.C, median ventral cart ihifiinous liar
Myii., myotonies Xc, notochord S.C.F., cerebro-spinal Sp.M., spinal cord S./?., sensory or dorsn
root S.T., sinus terminalis
1 spinal nerve
55
( Coniihucd fraiii ikkji ').', )
L't) imd L'7 licpicsciil I wo ( rniisvcrsc seel ions tliroiiuli I lie nir(|iill;i, piissiiiK tliroUKli til |-.isl(M-i(.r cii.l of llic luiirlli vculiiclr ..f llic s;iiiic scries iron: \vlii<-li tiglllTs -Jl and L':> were drawn. Xolc csi.ccially liic cxiiansion of the rool' plalc and coniparc willi the so-called second roof phitc; expansion of tlic 20 cm. Polistotrcnia spinal cord i li)j;. Itii. ll is (pii'st ional)le vvhothor tlio opcninsR (C) arc: artifacts or not. As was noted previously, the inesencliynie outside the roof phite is very vascular and tiie loof pla I e has t he apjx'arnce of heino; under anioderate (letiree of intci-nal pi-essure. :< 10.
L'S .V ratheidl.li(|ue rr(udal section tliroujih t he medulla of a i:. mm. human end)ryo (Inst, of .\nat.. Iians, seiies, II L'o), In (his region the contour of the rhomhic Kr.ain is such that t he i)osterior part of the roof expansion of the fourtli ventricle is cut t ransversely w Idle t he more antericji- portion, seen below, appears more or less in fiontal section. More anterior sections would show the roof plate to 1)1' continuous. The [josterior end of the fourth ventricle will admit of direct comparison with the second roof expansion of the 20 cm. Polistotrema spinal cord (fig. 10). Had the fourth ventricle been empty, as was the case of the rubber tubing in His' experiments, there would be absolutely no grounds for believing that the anterior portion of the roof plate would be expanded as it is by the appearance of a pontine flexure. It might on the contrary have been folded up within the ventricle. X 16.6.
29 A transverse section through the extreme posterior end of the fourth ventricle of the same series as figure 28. There is here a slight roof plate expansion containing no cavity. Compare with figure 15. X 16.6.
30 Transverse section through the thoracic spinal cord, taken from the same series as figure 28. Note that the roof plate consists of ependyma only, while the floor is reinforced by white matter, and even at this late stage if any marked increase in pressure occurred from the cerebro-spinal fluid of this region, an expansion of the roof plate would have been entirely possible. X 16.6.
31 Similar to figure 28, but from an 8 mm. human embryo ("Inst, of Anat., series H4). In this plane the posterior end of the fourth ventricle is cut nearly transversely, and is directly comparable with the second roof expansion of the 20 cm. Polistotrema spinal cord (fig. 16). The cavity was full of coagulum, its walls have the appearance of being under moderate internal pressure, and the adjacent mesenchyme is very vascular. X 46.6.
ABBREVIATIONS .
B.V., blood vessel 01., inferior olive
C, an apparent communication be- R.Ex., roof plate expansion
tween the veno-lymphatics and the R.L., rhombic lip
fourth ventricle R.P., roof plate of the central nervous
C.C., central canal system
Ch n., chondrocranium S.C., semicircular canals
C.P., choroid plexus of the fourth S.R., sensory or dorsal spinal nerve
ventricle root
Crh.L., lateral lol)es of the cerebellum T.S., tractus solitarius
C.T., white fibrous connective tissue VIII. G., auditory ganglion
E])., ependyma V.L.S., veno-lymphatic sinus
Ex.Ar., external arcuate fibers V.R., trigeminal root
/.L., inner or ependj'mallayer of nuclei W.R.F., white reticular formation
Mar.L., marginal layer XI. R., accessory nerve root M.L., mantle layer
56
SPINAS COKD AND .\iEDl LLA (»1' CYCLOSTUME. WIL I AM F. AI.LEX
c C.C.
/Ex. At.
01.
V.L.S.
s.C.::f;- \fjynit^-^ 26
-R.Ex. ^- CC.
- BV
^^ A
31 '" >^'^-^
..H.E..
. R£x
c
/■■■
1
"f^- ^ Marl
cc
\^-^
Ex.A.
H
01
29
•Vv,
R.R
S.R.
•€.C
30
57
i;\ri,A.\.\TI()N OK I'KidKKS
',i2 to M A .st'iics of triUisvtT.sc scclioiis througli tlic region of llic V, VIII,
- ui(l X Hiinglia in embryos of Pctromyzon of ages varying from 10 to 2G days. It
will hv seen from these sections that Pctromyzon develops an extensive roof expansion willioiit the aid of a pontine flexure, and the cranial and spinal ganglia aic wcll-fornicd while the (•ciitral nervous system is a solid cord. All of the figures were ilrawn with the aid of an Edinger-Leitz drawing apparatus. With figure 54 a magnification of 76.6 diameters was used, while 250 diameters was used for the others. In reproduction they were all reduced one-half.
32 Transverse section through the medulla in the region of the auditory vesicle from a 10 day Pctromyzon. This is my oldest embryo in which the central nervous system has remained a solid cord. Ordinarily it becomes tubular during the seventh day. This section shows the medulla to consist of a syncytium of protoplasm, consisting of a mass of round nuclei, much yolk, and a few fibers in the marginal layer. The nuclei have migrated a short distance to either side of the median dorso-ventral line. A seam (CCS.) has appeared here, which marks the position and beginning of the embryonic central canal. The protoplasm bordering the central canal seam is finely granular and may be assuming a secretory function. The acustic ganglia and fibers are shown to be welldififerentiated on both sides. X 125.
33 to 35 Three transverse sections passing through the medulla region of another 10 day Pctromyzon embryo, in which the central canal has been somewhat retarded in development. These sections pass through the V, VIII, and X ganglia respectively, and with the exception that the central canal furrow or seam (CCS.) has expanded into small dorsal and ventral cavities (no dorsal cavity has appeared in fig. 35) the general structure of the medulla is about the same as in figure 32. Later these cavities will become the dorsal and ventral expansions of the embryonic central canal of the medulla. The protoplasm in the region of this seam is granular and may be secreting an embryonic cerebrospinal fluid. From these figures it will be seen that the beginning of the central canal occurs at the same time throughout the entire rhombic brain. The anterior portion of the spinal cord, while not figured, contains a central canal furrow in the same stage. X 125.
{Continued on page 60)
ABBREVIATIONS
And. v., auditory vesicle or otocyst Mes., mesencephalon
Br. A., branchial arch M.L., mantle layer
B.V., blood vessel My:)., myotomes
CC, central canal Nc. notochord
CCC, central canal closure, caused P.B., pineal body
by fusion of lateral plates R.P., roof plate of the central nerCCS., central canal seam or furrow, vous system
in Petromyzon Sp.M., spinal cord
Ep.N., layer of ependymal nuclei Sy.P., syncytium of protoplasm
F.L.P., fused lateral plates of the Tel., telencephalon
spinal cord V.G., Gasserian or semilunar ganglion
F.P., floor plate of the central nervous VIII.VIL, acustico-fascialis ganglion
system; W.M., white matter
G.C., germinal cell X.G., vagus ganglion (nodosum). Mar.L., marginal layer ' Y ., yolk granules
58
SPINAL CORD AXD ifEDULLA OF CYCLOSTOMES
WILLIA^f F. ALLEN
^
o
Q, o
tyT
>
CO
U
3
d
O
A
^^
a: <^
>^ U
-■'^>TC CD
'--•■w;:V CO
s s
d^d ^
a. 1
dd^
^^^-<r7
^
O)
03
- CO
O,
59
{Cnilnnn.lfrnm i>a,,, .5S)
- >(; toiiS Ivoiu a I 1 .lay I'd roiii.v/.on crnhiyo IIhoiikIi Mic saiiic icnic.iis as I lio.so
shown in liLimfs 'M\ lo .'!,"), and to racililale coinparisun were placed dircu^tly under lliein. Consideralde proiiress iuis occurred everywhere. Note Ij that I he (•(•ntrai canal scam with its small dorsal :ind ventral cavities in the 10 nun. sci'ics has ^^ivcn place to a 1\ pical cmUryonic central canal, whicii is much wider al the top and hottom than at the center. The constricted jiortion of coui'se rei)resents the last place for tlu; protoplasm to give way or to be disintegrated. ■J) 'i1ie floor |)late is slijilitl>- thicker and less expanded than the roof plate, being riMnl'orced on the outside by white matter and by a rapidly growing notochord lielow .')) A r(>w more nerve fibers and nuclei have appeared in the lateral plates. I) 'I'he number of dividing germinal cells has increased while the number of yolk graiudes remains about the same. 5) The first blood vessels have put in appearance directly outside the meningeal membrane (fig. 37, B.V. and nearer the roof plate on the opposite side). X 125.
.39 Transverse section through the spinal cord of the same 11 day Petromyzon series as figures 36 to 38, showing the so-called typical embryonic si)inal cord. X 125.
40 and 41 Somewhat obliciue transverse sections from a 12 day Petromyzon, passing in figure 40, through the V ganglion on one side and the VIII ganglion on the opi>osite side, and in figure 41 through the X ganglion on one side and a region l)ehind the X ganglion on the opposite side. They can readily be compared with the 11 day series above (figs. 36-38). The lateral plates have apparently increased notably in the number of nerve fibers and nuclei, some of which, however, will have to be attributed to the fact that the sections are cut quite obliquely. Also the numbers of nerve fibers have increased in the floor plate. Throughout, the central canal has increased in width. Of especial interest is a small central mass of protoplasm (Sy.P.) in figure 40, which for a space of 50 microns persists as the last remnant of a once solid mass of protoplasm in the center of the medulla. It is obvious at this stage that some factor must have produced sufficient internal pressure to i)revent the closing up of the ventricle on account of the rapid increase of cells and fibers in the lateral plates. It is fair to assume that this factor is internal pressure from cerebro-spinal fluid. X 125.
54 of this plate will be described in its proper place, opposite the next plate.
60
PLATE 9
EXPLANATION- OF FIGURES
42 to 44 Three tran.sv(M-sc sections through the iiKMliiUa of an Is day Petromyzon embryo. i)a.ssinp; through the V. VIII. and X ganglia, and for the sake of comparison preserving the same order or arrangement as was used for the earlier embryos. A slight increase in the white matter is to I.e noted for the lateral and ventral plates over the \2 day series; but little, if any change has taken place in Ihe central canal, unless possibly the central jmrtion has increa.sed slightly in width. Ab.solutely no further expansion of the roof plate has occurred. Since sections through the medulla of a 14 and 10 flay series jiresented about the same appearance as figures 42 to 44 none were figured. X 12.").
45 Tran.sverse section through the ce|)halic portion of the spinal coni fn.ni the same series as figures 42 to 44. Dbserve especially tiie begiiming of the dorsal closure of the central canal (CCC). showing the central canal to consist of dorsal and ventral cavities and a central scam, strongly resembling the stage wiieii it first api)eared in the spinal cord. This <lorsal closure of the embryonic central (•anal begins in the anterior portion of the spinal cord much earlier than the corresponding ventral closure of the embryonic central canal in th«- nu'dulla, occuring in my .series of 14 and In days. It is obviously caused by the ingrowth of the lateral plates due to the great increase in the ninnb(>r of nerve fibers. X 12.'). 4(1 to 4.S Three tran.sver.se sections through the same region of the medulla of a 20 day Petromyzon as is shown above in figures 42 to 44 for the l.S day embryo. It will be .seen that many noticeable changes have taken i)lace. du«' primarily !.• a marked increa.se of nerve fibers in the lateral and ventral plates, and to a slight increa.se in the number of cells in the lateral plates. The shape of the medulla has become more compressed (flattened out in a dorso-ventral plane. Unquestic.nably the increase of fibers in the lateral plates has occurred largely in the median and ventral portions. Note the result on the embryonic central canal, which has been completely closed, except for a small dor.sal triangular cavity (C.C) the early fourth ventricle. Its roof plate, however, has expanded somewhat. Ob.serve that true ependymal cells are l)eginning M take on form .ibout the ventricl(>, and those in the roof j)latemay soon assume a secretory funclioii, if tlirv .nc not already active. .Vlso the blood vessels have become more .ilniiidaiii outside of the medulla, especially in the region of the roof plate, which would make infiltration and diffusion thniugh the mof plate into the ventricle easy. X 12."..
lit From ;i transverse section through the anterior portion of the s|)inal cord. Obs.MVe tile great increase in fibers in the lateral and ventral plates, and n<ite that tile (left-like embryonic central canal has become entirely closed, but for a small portion, which will remain as the adult central canal. It is obvious that the two ])()rtions of the emliryonic central canal which persist in the medulla and t lie spinal cord are the opposite, being the dorsal in the medulla :ind the ventral in the cord. X 12.").
.')0 to 53 Four transver.se sections through the medulla of a 2(j day Petromyzon embryo, taken through the V. VIII, and X ganglia, and through a region liehind the fourth ventricle where the central canal is j^assing ventral to assume its characteristic position in the spinal cord. A comiiari.son with the 20 day series above (figs. 4(5 to 4S) will demonstrate a marked increase in the size of the medulla and the fourth ventricle, and a greater expansion and convexity of the (Continued on page 62) 61
{( oiilnumlj'nnn piuji HI)
roof l)l!iti-. in (■..iisrciucncc of which I lie (h)r,sal tii)s of the Lateral ph'ilcs arc widely scpai-iitoil. 'IMhtc is a marked increase in (he nuinl >er uf nerve lil.ersin I he hateral l)lates, especially in I he median and \-eidral portions. Of prime inii)ortance is the great expansion of the lonrth \-entricle and the roof plate, which apparently in Petroinyzoii can he explained only from inlcrnal factors, the mosl obvious of wliich is the meidianical expansion due to an increase in the cercsbro-spinai Huid. It will he seen that these forees were sufficiently strong to more than offset the thickeninfi; of the hvteral plates which would tend to obliterate the dorsal i)ortion of the embryonic central canal as it has the ventral portion. It is apparent that this internal pressure has pushed the lateral wall apart in the dorsal region, where the lateral plates are thinnest and weakest. As was pointed out in the 20 day series the ependymal cells are becoming difTerentiated and probably ha\-e assumed a secretory function. Likewise the increase in the numi)er of blood vessels above the roof plate favors filtration and diiTusion into the fourth ventricle. X 125.
.54 (See preceding plate.) Median longitudinal section through the head region of a 20 day Petromyzon embryo introduced for a comparison w^ith the transverse sections in figures 50 to 53. Note especially that the marked convexity of the roof plate of the fourth ventricle is suggestive of expansion from an increase of cerebro-spinal fluid. Absolutely no pontine fiexure is to be seen, the little convexity that occurs in the floor plate can easily be attributed to an increase in the number of nerve fibers. Observe that the fourth ventricle (CC.) is the remains of the dorsal portion of the original embryonic central canal, while the central canal of the spinal cord is the remains of the ventral portion. The ventral portion of the embryonic central canal of the medulla has been obliterated through the fusion of the ventral portions of the lateral plates. X 38.3.
ABBREVIATIONS
Aud.W, auditory vesicle or otocyst Myo-, myotomes
B.V., blood vessel N.C., nerve cell
C.C., central canal R.Ex., roof plate expansion
C.C.C, central canal closure, caused R.P., roof plate of the central nervous
by fusion of lateral plates system
Ep., ependyma V.G., Gasserian or semilunar ganglion
Ep.N., layer of ependymal nuclei VIII. VII., acustico-fascialis ganglion
G.C., germinal cell W.M., white matter
G.M., white matter X.G.. vagus ganglion (nodosum). M.L., mantle layer
PLATE 10
EXPLANATION OF FIGURES
55 to 62 Taken from various transverse sections through embryonic and larval Polistotrema and Entosphenus (Pacific coast lamprey). Introduced to show the effect of the developing notochord on the spinal cord in Cyclostomes. They were drawn with the aid of an Edinger-Leitz drawing apparatus and reduced one-half in reproduction.
55 Transverse section through the caudal region of a 20 mm. Polistotrema embryo. It will be seen at this stage that the notochord has produced very little visible effect on the spinal cord. Cyclostome embryos of this stage (compare fig. 39 for Petromyzon) present a nearly cylindrical spinal cord; while that of all other vertebrates is more or less elliptical in cross section, the greater (Continued on page 64) 62
SPINAL CORD AND MEDULLA oK CYCLOSTOMES
WILIJAM F. ALI.EN
liaiiiclcr Im'Iiio .loisu-vrni ral. Il sli..nl<l l.c .idled llial I (ic spin.-.l c.r.l is criv.'l..|).'<l lin;|,tly hy a iiiciii nsica I iiiciiil.iaiic, iim.ic or less fiiscil with comicctivc lissur nutsi.l.'that will Iniiii I ii.r n.'ural arcli. \\lii<-li is iiir.ily alla.'licd to the iiolochonl licli.w. Iiniiicilialcly al.ovc, I lie iiicsciicliy iiic is prolirci aliiifi; rapidly
and mij>i-al iiii; In I lie ccnlcr wlicic il will luiin I he i iiati doisal cartilaninous
i)ar. Lilllc pro-rcss has Mcciincd in I lie loriiial ion of I he .nyoloincs al liic side, and clsrw Ihmv llinv is ,,iily loos.' incscn.diynic. ■- 70.
.')('. Similar Iransx'crsc section to li>iiiic :)."). I)iil IVoin a '-'7 iiiiii. I'oiislol rciiia rnil.ivo. -nils sliiilitly lat.T sta-c shows coiisideralilc -rowt h of the tiofochord and a niedian indentation on I he ventral surface of the spina! cord as tlu; result. Xolc ( liat lli(>coii(litions siinoiindin^ I he dex'elopinent of t ho notocliord previously enmneraled undei' I he descii|)l ion of lijiiire .").') are insi ruriieiiial in assisting the notochord in |.ro(lucinji the Ki'ulii.il ilatteninu' (depression ) of tlie spinal cord seen in the nexl liiinre. X 70.
.")7 'i^-ansveise section of the spinal cord of a 'iO inin Tolist ot rema embryo from the same re-iion as ii.iiure .")(). It will be seen tliat the spinal cord is enclosed in a membranous canal of dense connective tissue, attached below to the notochord and above to the median dorsal cartilaginous bar. .\bove this there ai'e developing cartilaginous rays surrounded l)y (l(>nse connect i\c tissue. The d(>veloping myotonies rest against the neural arches l)oth laterall\ and dorsally. 'rii(> notochord has increased greatly in size and, pushing up against the soft spinal ciu'd. produces the depression and ventral indent.alion of the spinal cord exhibited in this figure. It should be noted that the roof plate is still ependynaa and an e\|);iiision of the roof jihate could take iilaci' even in this late stage if the
mechanical factors enumerated for the i liilla of Pet romyzon u('r(> operative
here, 'bhe thickening of the lateral plates has about obliterated t h(> central portion of the embryonic central canal, leaving only the dorsal and ventral |)ortioiis. in which I luu-e is a fibrillar feltv.ork, jjiobably representing both cerebid-spinal lliiid and ependyma cilia. Reissner's fiber is visible in the ventral or |iermanent central canal. X 70.
.")S Transverse" section through the tail region of a '_'() mm. I'^ntosphenus larva. It will b(» observed that the si)inal cord is fuither developed than in the 27 mm. l%jlistotr(>nia einlnyo (tig. .Ki). It is apparent that the same factors are involved in flattening the spinal coi-d as were enumerated for Polistotrema. The notochord has made fully as much growth and the structures surrounding the spinal cord arc the same as in Polistotrema, with the except ion t hat instead of a median dorsal cartilage for the attachment of the membranous neural arch there is a membranous neural si)iiie. To some extent this may reduce the dorsal resistance, but on the othei- hand it may be compensated for by a greater development of the myotomes above the neural arch. X 12o.
A)iMI{KVI.VTIONS
r..l., caudal artery M.V.C, median ventral cartil.aginous
('.('., central canal bar
r.//., caudal heart Mjni., myotomes
('.v., caudal vein A'..l., membianoiis neural arch
D.R.. dorsal cartilaginous rays .Vr.. notochord
Ep.N., layer of e{)endymal nuclei P.M.. pia mater or meningeal mem L.S., lateral veno-lymphatic sinus or brane of the young(!r stages
anlage of the same A'./'., loof ])Iale of the central nerxous
Mar.L.. marginal layer s\stem M.D.C, median dorsal carl ilaginoiis Sj).(;., si)inal ganglion
bar r.7'., ventral veno-lymphatic tiiink
ir..l/., white matter
64
SPINAL COHl) AXD MEDULLA OF CYCLOSTO.\rES
WILLIAM F. ALLEN
PLATE 10
THE .lOL.RNAL OK COMPARATrVE NErROLOGY. VOL. 26. NO. 1
PLATK 11
KXl'LANATION OK FIGUHI>
51) ;uul t>() Two I laiisvciisc sections only 290 nii(;rons ;i[)urt through the extreme posterior end of the spinal cord of a 70 mm. Polistotrema embryo. In the more anterior section (fig. 59) the spinal cord is depressed, contains developing nervous elements, and the notochord is large proportionately. In the posterior section (fig. (50) the diameter of the notochord is much reduced and only supporting elements appear in the spinal cord. As a result no flattening of the spinal cord has taken place in this region. Compare with figures 21 and 22, which are similar sections through an adult Polistotrema. X 70.
61 and 62 Taken from two transverse sections 480 microns apart, through the medulla oblongata of an adult Polistotrema. In both sections no nervous structures have appealed that were not present in the spinal cord. Note as you pass caudad (figs. 62 to 61) that the medulla becomes flattened ventrally and dorsally in direct proportion to the increase in size of the notochord. This relationship can be shown fully as marked in more anterior sections, and in sections taken from a similar region of larval Petromyzon. X 25.
ABBREVIATIONS
Aud.V., auditory vesicle or otocyst M.V.C., median ventral cartilaginous
B.V., blood vessel bar
C.C., central canal Myo., myotomes
E.N., undifferentiated embryonic nu- N.A., membranous neural arch
clei N.C., nerve cell
Ep., ependyma P.M., pia mater or meningeal memEp.N., layer of ependymal nuclei brane of the younger stages
L.S., lateral veno-lymphatic sinus or P.P., parachordal plate
anlage of the same Sp.G., spinal ganglion
M.D.C., median dorsal cartilaginous W.M., white matter
bar X.G. in figure 62 should be Sp.G.
M.F., Miillerian or giant fiber X.N., vagus nerve M.L., mantle layer
PLATE 12
EXPLANATION OF FIGURES
63, represents a diagrammatic reconstruction of the fourth ventricle from an adult Polistotrema series, and the planes from which the transverse sections were drawn for figures 64 to 66 are indicated by lines bearing those figures. Observe how the large fourth ventricle of the embryo has been reduced to a small central canal, having a posterior dilation (P4F), and how the anterior end breaks up into two or more small longitudinal canals that soon terminate in the sinus mesocoelicus.
64 and 65, represent transverse sections, taken at different levels of the fourth ventricle of Polistotrema. These sections were drawn with the aid of an Edinger-Leitz drawing apparatus and were reduced one half.
64 From a transverse section through the caudal portion of the mesencephalic lobes (cerebellum of Miss Worthington). Exact plane indicated by line 64 in figure 63. The section passes through the posterior mesoccele or cerebellar ventricle {M" .) and the sinus mesocoelicus (anterior dilation of the fourth ventricle of Miss Worthington), a short distance behind the branching off of the posterior mesocoele. The cavity contains a fibrillar feltwork, wliich is in part coagulated cerebro-spinal fluid and in part ependymal cilia. The ependyma surrounding the fourth ventricle is rich in blood vessels, which derives its arterial sui)ply from {Continued on page 68)
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILLIAM F. ALLEN
M&O
c^:i^
P ^
§^6l^ ^
1 >
iCiinli iiiicil J'rtiiii /"/(/< 'I luo iiii'diilla aiicrii's [M.A.). Here as clsowlicrc, the cpciKlyina siinouiidiii^ the rourtli ventricle doubt less functions as a inodilieil chorioid plexus, discharging eorebro-spinal fluid into the fourth ventricle. It will be seen that the cavity of the fourth ventricle is smaller than the i)e(!uliarly modified central canal and roof exi)ansion cavity of the Polistotrema spinal cord portra\cd in h^ures 10 to 13. X 25.
65 A more caudal section through the extreme tip of the posterior lobes of the mesencephalon (cerebellum of Miss Worthington), its exact plane being indicated by line 65 in figure 63. It will be seen that the fourth ventricle of the embryo has in this region of the adult become reduced to three small longitudinal canals (.l.i V.), which are nnbedded in a rather large, dense, and vascular ependymal mass. The most dorsal of these canals contains Reissner's fiber. Here again the ependymal walls are probably functional as a modified chorioiod plexus. Very shortly these canals reunite and continue some little distance caudad as a small central canal, no larger than the central canal of the spinal cord. X 25.
66 Transverse section through the posterior end of the medulla of the same series as figure 64. The exact plane of the section is indicated by line 66 in figure 63. It passes through what has been designated as the posterior dilation of the fourth ventricle {P4V), which is nothing more than a fair-sized centrally located cavity, the remains of a much larger embryonic fourth ventricle, surrounded by a great mass of vascular ependyma. The center of this cavity contains a fibrillar feltwork (S.C.F.) composed largely of coagulated cerebro-spinal fluid and some ependymal cilia. Here as more anteriorly we probably have a modified chorioid plexus, the ependymal walls and their blood vessels secreting and filtering eereliro-spinal fluid into the fourth ventricle. X 25.
ABBREVIATIONS
AtV., anterior fourth ventricle M.A., medulla artery
B.V., blood vessel Mes'., posterior lobes of the mesen Ep., ependyma cephalon, cerebellum of Miss Worth Hab. B., habenular body ington
Inf., infundibulum P4V., posterior fourth ventricle
M., mesocoele or mesencephalic ventri- S.C.F. , cerebro-spinal fluid
cle S.M., sinus mesocalicus
M'., anterior portion of the mesoca?le V.M.R., motor V root
or sub-commissural canal of Nicholls 4V., fourth ventricle
M"., dorsal portion of the mesocoile VIII. G., auditors ganglion
or optocoel and posterior portion of X.R., vagus root
the optocoel of Nicholls
PLATE 13
EXPLANATION OF FIGURES
67 to 69 Tliree transverse sections through the brain region of a 37 mm. Amphioxus. No blood vessels were seen in any of these sections, but the membranous neural canal is surrounded on three sides by enormous veno-lymphatic sinuses, and the structure of the central nervous system is to a considerable extent made up of rather coarse supporting tissue, making infiltration an easy method for nourishing the brain. Drawn with an Kdinger-Leitz drawing apparatus and reduced one-half in reproduction.
{Con tinned on page 70) 6S
INAL CORD AND MEDULLA OF CYCLOSTOMES
WII.l.I.VM F. ALLEN
PLATE 12
iCoiilnnnil frniii piiii, US)
I'lif iii.ot ;iiilirii)i- si'ctidii, li-iuri' (17, piisscs tliroiijili t lie iinU^rior vcjil.ricdi; at its lii^ilu'sl i)()iiit, wliicli is a sliort (lislaiicc hcliiiid tli(^ nciiroporc. This ventricle has no (h)isal dilation suggestive of the fourth ventricle. What dilation occurs, is niedian and ventral. Cilia-like processes from the border of the cells enter the cavity. I f (lie cpciidyiual cells are not secretory it is possible that the cerebro-spinal lliiid ul' Atnphioxu.s does not differ from the serum of the adjacent veno-lymphatic siiuiscs. If nerve cells occur in this region they are small, and in ordinary i)reparat ions indistinguishable from ependymal cells. X 125.
OS (50 microns behind figure 67. The large central canal of the eml)ryonic brain has evidently become reduced in this region to a ventral central canal {C.C.) and a small dorsal isolated cavity {V 2.). This isolated dorsal cavity can not be comi)ared with the fourth ventricle of higher vertebrates. It is rather to be looked upon as a vestigeal structure, which may aid in the infiltration of lymph from the outer veno-lymphatic sinuses. X 125.
69 Taken from a section 530 microns behind figure 68. It passes through that part of the brain in which there are accumulated a great number of giant cells {M' .C .) in the region of the roof plate. As in figure 68, there is an isolated cavity near the dorsal surface, which was probably a portion of the large embryofiic central canal, but which in the adult is separated from the central canql and from the more anterior isolated dorsal cavity by ependyma. It seems best to the writer to regard this and the preceding dorsal cavity as vestigeal structures. X 125.
70 (See next plate.) Transverse section through the anterior spinal cord from the same series as the three previous figures. Observe that the Amphioxus spinal cord is not depressed as is the Cyclostome spinal cord, but is indented ventrally by the notochord. The central canal, which in some places exists as a dorso-ventral cleft, is almost obliterated here by the ingrowth of ependymal tissue. X 125.
71 Cephalic transverse section through a portion of the spinal cord, meningeal membranes, neural arch, notochord, spinal ganglion, and sensory root of an adult Polistotrema. Observe the depression of the spinal cord, its ventral indentation, the ventral or permanent central canal {C .C .), which contains Reissner's fiber, and immediately above, the dorsal portion of the embryonic central canal, which is here more or less filled with ependymal cells and their processes. It will be seen that the gray matter is as much flattened out as is the cord itself, and the ventral horn and motor cells are crowded laterad, while the dorsal horn, substantia gelatinosa {S.G.), is apparently median and dorsal. Within the neural arch there is abundant room for a spherical spinal cord. The cord is held in place by the usual meningeal membranes. X 70.
ABBREVIATIONS
Ar., Arachnoidea Nc, notochord
C.C, central canal P.M., pia mater or meningeal mem C.T., white fibrous connective tissue bi-ane of the younger stages
D.M., dura mater S.G., substantia gelatinosa
D.S., dorsal veno-lymphatic sinus Sp.G., spinal ganglion
Ep., ependyma S.R., sensory or dorsal spinal nerve
L.S., lateral veno-lymphatic sinus or root
anlage of the same Suba.S., subarachnoid cavities
M'.C, Miillerian or giant cells Subd.S., subdural spaces
M.F., Miillerian or giant fiber W.M., white matter
Myo., myotomes V.L, anterior ventricle Amphioxus
A^.^., membranous neural arch V.2., vestiges of the embryonic central N.C., nerve cell canal in Amphioxus
70
SPINAL CORD AND MEDULLA OF CYCLOSTOME,
WILLIAM F. ALLEN
PLATE 13
71
I'LAri'i II
KXl'I.WATION OK I'KIKKKS'
7'J III SO ic|)ics('rit M nuiiilxTor I r;msvcrs(> so(^t ions through the medulla of shark, iunphihian, and pi-^ cndtryos lor tlic purpose of demonstrating various stages of roof plate expansion.
72 leather ohliciue transverse seel ion tlirout^h tlic medulla of a 19 mm. Squalus emhryo in the region of the \ni ganglion (from series No. 2 of Professor Seainmon's colleet ion) . Note the well-formed fourth ventricle, and the Ijroadly expanded and very mueh stretched roof plate. Its collapsed appearance in this section is doubtless due to fixation or preparation. On account of a great proliferation of cells and nerve fibers the lateral plates have fused ventrally, as in Petromyzon, obliterating that part of the embryonic central canal. Attention should be called to the fact that the medulla roof plate in sharks begins to expand much earlier than it does in Petromyzon. This figure shows a well-expanded roof plate, while the cells in the mantle layer are no more differentiated and there are no more nerve fibers in the marginal layer than appear in a 12 day Petromyzon embryo (fig. 40), where there is no fourth ventricle and no expansion of the roof plate. X 70.
73 Transverse section through the medulla of a 15 mm. Necturus taken through the VIII ganglion. Observe the wide fourth ventricle and the broadly expanded and greatly stretched roof plate, and the coagulated appearance of the cerebro-spinal fluid in the ventricle. Like Squalus, the fourth ventricle begins relatively much earlier than in Petromyzon. It should be noted that no blood vessels have reached the level of the roof plate or entered the medulla; hence the coagulum in the ventricle must be largely a product of secretion. X 39.
74 From a transverse section of a very young Amblystoma embryo in the region of the auditory vesicle (Professor Johnston's series No. 50). Here there has occurred a dorsal and a smaller ventral excavation of the cleft-like central canal. It will be seen that the larger dorsal cavity, the beginning of the fourth ventricle, possesses no thinner roof plate than does the spinal cord (fig. 83). Also in this section (fig. 74) the roof and floor plates are about equally thick. What has taken place dorsally and ventrally throughout the embryonic central canal has been a migration of the cells outward. The fact that in this section of the medulla the roof plate is no thinner than in the section of the spinal cord (fig. 83), taking note that the spinal and cranial ganglia are well-formed, is evidence against the hypothesis, that the greater migration of the neural crest cells of the medulla was the prime cause of the thinning out of the roof plate of the rhombic brain. X 70.
(Continued on page llf)
ABBREVIATIONS
Aud.V., auditory vesicle or otocyst M.L., mantle layer
B.V., blood vessel Myo., myotomes
C.C, central canal or cast of the same N.A., membranous neural arch
C.C.C., central canal closure, caused N.C., nerve cell
by fusion of lateral plates A^c, notochord
Ep., ependyma P.O., pigmented or eye cells of Amphi Ep.N., layer of ependymal nuclei oxus
P.P., floor plate of the central nervous R.Ex., roof plate expansion
system R.P., roof plate of the central nervous
G.C., germinal cell system
L.S., lateral veno-lymphatic sinus or S.C.F., cerebro-spinal fluid
anlage of the same V.G., Gasserian or semilunar ganglion
Mar.L., marginal layer VIII.G., Auditory ganglion
M'.C, Miillerian or giant cells W.M., white matter
M.F., Miillerian or giant fiber 4 V., fourth ventricle
72
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILUAM F. ALLEN
?■■/
vnic^.
c.c.
72
WIG.
73
-S.C.F.
R.Ek.
73
(( mill ini( (I. J idiu jxii/i 72}
7;") to SO rcprcscMl livt- t r.'iiisvcrsc sections throiitih I he il(vcloi)iiif; lourtli vcntricli" .•mil roof phitr cxpaiisioii iti |)iti cinlnvos from '> nirii. up (o 14 mm. With till" ("\ccpt ion of lijiuic 7."), w hicli is I'loin my collccf ion, the rcunaininfj; figures are from frontal series helonging to the Institute of Anatomy. That there is a dirert rolationsliip between the amount of visible coagulum in the form of a fibrillar feltwork and the expansion of the roof plate is evidenced by the fact that this ct)agulum does not ajipear in the early embryos l)efore the roof plate has assumed the ai)pearance of an organ capable of the production of cerebrospinal fluid (as indicated by vascular supply and granular appearance of the cells). It may l)e inferred that the earliest non-coagulablc cerebro-spinal found in the earliest stages is an cmbi yonir fluid which differs in no way from the ordinary intercellular juices, but that the appearance of coagulum at the time when the roof plate has attained the appearance of a functional chorioid plexus is indicative of a chemical change in the fluid, which if a product of secretion is capable of producing a marked increase of internal pressure in the corebro-spinal fluid and consequent expansion of the roof plate.
75 From a transverse section through the medulla of a 5 mm. (or less) pig embryo, in the region of the auditory vesicle. It will be seen that the peripheral branches of the intersegmental blood vessels have about reached the roof plate, but no blood vessels have entered the medulla. The protoplasm of the inner margin of the ependymal cells is sufficiently granular to suggest a secretory function. The small amount of coagulum in ventricle is probably the result of secretion, but the cerebro-spinal fluid has probably not exerted much internal pressure. X 70.
76 Transverse section of a 6 mm. pig medulla through the widest portion of the fourth ventricle, namely, at the level of the V ganglion. This is the only portion of the roof plate to have undergone any stretching of its cells, and this is confined solely to the most centrally located cells. This section shows a considerable increase in the size of the fourth ventricle and expansion of the roof plate, together with some increase in the amount of coagulable cerebro-spinal fluid (S.C.F.) and an increase in the number of blood vessels above the roof plate; but no blood vessels have entered the substance of the medulla. At this stage the pontine flexure could not have been a factor in producing the roof expansion. The collapsed appearance of the roof plate at its center is not natural, but rather a result of the preparation of the material. X 39.
PLATE 15
EXPLANATION OF FIGURES
77 Taken from a transverse section through the V ganglion of a 7 mm. pig embryo. Note the increase in the number of blood vessels above the roof plate, which together with the increase in the coagulable cerebro-spinal fluid suggests a functional chorioid plexus. The pontine flexure in this stage is too slight to have any effect on the expansion of the roof plate. It should also be recorded that a few blood vessels have entered the outer surface of the medulla. As in the previous series the sections have suffered a collapse of the roof plate from fixation or later preparation of the material. X 39.
78 From a transverse section of a 10 mm. pig embryo through the region of the V ganglion. The increased vascularity of the mesenchyme above the roof plate together with the enormous amount of coagulated cerebro-spinal fluid (S.C.F.) in the ventricle are evidences of the factors which have produced the increased expansion noticed in the roof of the ventricle. Also at this stage the pontine flexure has increased to such an extent that its action on a fourth ventricle full of cerebro-spinal fluid, itself under a moderate pressure, would produce a further expansion of the roof plate. As in the preceding sections the roof plate has suffered a collapse in the preparation of the material. X 39.
74
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILLIAM F. .VLLEN
C^.F
71) and SO I'wo transvi'isi- sections tlinmnh tlu- anti-riur ami |iii>ici mr i-iiils
of the fourth ventricle from a 14 mm. frontal series of a pig. .\ more advanced
stage in the development of the chorioid plexus together with a more pronounced
pontine flexure has produced a much larger fourth ventricle and expanded roof
plate than is shown in the previous series (fig. 78). The thickening of the lateral
walls of the medulla is taking place as it did in Petromyzon and Squalus, but the
greater expansion of the ventricle in the pig (fig. 79) has prevented the walls
from fusing ventrally. Nevertheless, the thickening of the ventral portion of
the lateral plates would increase the pressure of the cerebro-spinal fluid. X 25
and 30.
ABBREVI.^TIONS
R.Ex., roof plate expansion
S.C.F., cerebro-spinal fluid
V.G., Gasserian or semilunar ganglion
4 v., fourth ventricle
X.R., vagus root
B.V., blood vessel
C.S.F., cerebro-spinal fluid
Ep.N., layer of ependymal nuclei
Mar.L., marginal layer
M.L., mantle layer
R.C., embryonic red corpuscle
75
PLATE 16
EXPLANATION OF FIGURES
81 to 87 Represent true transverse sections through what has been termed in the text, the typical embryonic spinal cord, from a number of different vertebrates, all of which have developed a tubular nervous system after the neural fold method. They were drawn with the aid of an Edinger-Leitz drawing apparatus and reduced one half in reproduction.
81 From a transverse section through the anterior portion of the spinal cord of a 10 mm. Squalus embryo (Professor Scammon's series No. 16). This socalled typical embryonic spinal cord is decidedly compressed. An earlier stage possessed an elliptical cord with its greatest diameter from side to side. The floor plate is slightly thicker than the roof plate. Each contains a single layer of nuclei. The ventral portion of the cleft-like central canal is expanded into a cavity, which persists as the permanent central canal. A well-formed spinal ganglion is seen to the left. X 125.
82 Transverse section of the spinal cord of a 19 mm. Squalus embryo (from Professor Scammon's series No. 2). Note that the dorsal closure of the lateral plates, due to fiber and cell proliferation, is the same as was figured for Petromyzon. They meet in a seam, leaving dorsal and ventral cavities, of which only the ventral one persists. As in Cyclostomes this method of closure would tend to throw a large part of the embryonic cerebro-spinal fluid into the brain cavities. X 70.
H'6 and 84 Transverse section through the anterior portion of the spinal cord of an Amblystoma and a turtle embryo. The former (taken from Professor Johnston's series No. 50) is a rather early representative of the so-called typical embryonic stage; while the latter is a rather late representative of this stage. Both cords may be said to be compressed (elliptical, having its greatest diameter dorso-ventral), but only slightly so, when compared with birds and mammals (figs. 85 and 86). As a result, granting an equal proliferation of fibers and cells in the lateral plates, it would be expected that the adult cord in Amblystoma and the turtle would be more depressed, which is found to be the case. X 70 and 125.
85 and 86 From anterior transverse sections of the spinal cord of a 93 hour chick and a 5 mm. pig. Both are good illustrations of the so-called typical embryonic stage, the pig being in a slightly more embryonic state. In these we have the most compressed of all embryonic cords examined, while the adults cord are nearly cylindrical. X 125.
87 Transverse section through the caudal end of the same spinal cord shown in figure 86. Observe spherical appearance which is indicative of an earlier phase in its development. X 125.
76
SPINAL CORD AND MKDULLa'uK CVCLTiSTOMES
•WILLIAM F. ALI.EK
C.C.C.
MX.
Mo^.L. C.C.
PLATE Ki
==^-Rf^
r
4^- -C.C.
S)-Nc.
R.P.
..^•s.'?So-j MX.
^' C.C. M.R.
Nc.
R7
C.C, ccMitral canal
ABBREVIATIONS
M.L.. inantk' lavfi
C.C.C, central canal closure, causod M.R., motor or ventral spinal nerve
by fusion of lateral plates Ep.N., layer of ependymal nuclei F.P., floor plate of the central nervou.s
system G.C., germinal cell Mar.L., marginal layer
root Nc, notochord R.P.. roof plate of the central nervous
system Sp.G., spinal ganglion Sy.P., syncytium of protoplasm
77
MORPHOLOGY OF THE ROOF PLATE OF THE FOREBRAIN AND THE LATERAL CHOROID PLEXUSES IN THE HUMAN EMBRYO
PERCIVAL HAILEY
From the AtuUortu'cnl Lnhornloni of the University of Chicago
THIKTV-ONE FIGURES
rOXTKXTS
Introduction TO
History SO
Material and iiictliods S4
Description S6
1 . The 19 mm . embryo . S6
2. The 28 mm. embryo 91
3. The 32 mm. embryo 96
Discussion 09
1. Telencephalon '.lO
2. Diencephalon 108
Summary 110
IXTKODI (IK IN
The researches of Minot, von Ku))ffer, Buickhaidt, G. Elliot Smith, C. Judson Herrick, J. B. Johnston and others, have succeeded in honiologizing with considerable certainty the structures in the roof of the prosencephalon of the lower vertebrates, and of some of the lower ^Mammalia. The results of these comparative studies Johnston extended to the human embryo and it seemed desirable to examine some other human embryos of different ages in the hope that additional light might be shed on the method of development of these structures.
79
80 PEK(;iVAL BAILEY
UISTOHV
Concerning the structures with which this study is concerned, the writings of such authors as Faivre ('54), Luschka ('85), and Haeckel ('60), previous to the work of Wilhehn His, contain very Httle of vakie.
Of the choroid plexus of the lateral ventricle, His writes ('04) :
Soin deni Thalamus angehefteter Randstreifen bleibt ependymal und in ihm bildet sich die Fissura chorioidea, von der aus die Epithelfaltungen des Corpus chorioideum in den Seitenvontrikel sich einstiilpen.
Minot ('01) considers the lateral plexus to be developed from the velum transversum.
In both birds and mammals the lateral portions of the velum, i.e., the choroid plexus of the lateral ventricle is highly developed. It thus appears that as we ascend the vertebrate series there is first a broadening of the velum, and an increase in its lateral development, then occurs a further reduction and flattcnino- out of the velum, and a much greater growth of the lateral plexus.
G. Elliot Smith ('03) attempted another explanation for the formation of the lateral plexus.
Now, although in the whole of its extent the epithelial layer of the choroid plexus presents uniform features, it is difficult to admit a common origin for the whole structure; with regard to that part of the plexus which is found in the region of the foramen of Monro, there can be little doubt of its origin from the primitive roof of the forebrain But the case is very different with that portion of the
plexus which is not directly connected with the roof of theforebrain, but is attached to the stria terminalis. There is no evidence to show that this portion is derived from the roof, and all the facts of development point to the conclusion that its proximal attachment to the optic thalamus is a primitive and not a secondarily acquired relation. Such lieing the case, the caudal extension of the epithelial choroidal fold in the mammalian hemisphere would appear to be derived from a stretching of the attachment of the labium caudale of the cerebral hemisphere to the optic thalamus. As a result of this, the connecting band becomes reduced to an epithelial lamina, which becomes invaginated and folded by an extension backward of the choroidal folding which begins farther forward in the i-egion of the foramen of Monro.
Johnston ('09) after showing that the velum transversum is continued down the side-wall of the prosencephalon as the ditelencephahc groove, returns to a modification of Minot's original idea.
DEVELOPMENT OF THE CHOROID PLEXUS 81
In the angle between the [cerebral] vesicle and the diencephalon appears the choroid plexus pushing into the lateral ventricle. It appears as a folding of the anterior limb or wall of the velum transversum and its lateral prolongation [di-telencephaUc groove].
Concerning the mesodermal portion of the lateral plexus, Aleek ('07) maizes the following statement.
The choroid plexuses of the lateral ventricles are due to an ingrowth of the pia mater pushing the mesial wa I of the hemispheres into the ventricles.
This is the current notion.
The arachnoid is not supposed to be prcsent, although the plexus is but a fringe of the velum interpositum. into the structure of which the arachnoid docs enter. The neural wall is, of course, iireserved, but consists only of a simple epithelium. The plexuses are then thin laminae coverecl with an epithelium, beiKvith which is a connective tissue stroma containing an extraordinarily rich network of bloodvessels.
Findlay's ('99) idea of a single membrane, the pia-arachnoid. removes the difficulty concerning the involvement of the arachnoid.
Innally, Hochstetter ('13) has written a purely descriptive account of the development of the lateral choroid plexus with no attempt to analyze its parts. He summarizes his work as follows :
Fassen wir rlas bisher Mitgeteilte zusammen. so koinien wir sagen, dass sich die Plexus chorioidei der Seitenventrikel ungefahr in dersel•ben Richtung entwickeln wie die Hemisph:irenl)lasen selbst. Zuerst angelegt, wenn auch nicht gleich als Anlage der Plexus chorioidei kenntlich, ist ihr vorderster, im Hereiche der Decke des C'avum Monroi beftndlicher Abschnitt. Er ersteht hier, wie wir gesehen haben, aus den die Sulci hemisphaerici [di-telencephalic grooves] bildenden Hirnwandfalten, sowie aus dem diese beiden, in der Forts(>tzung des Zwischenhirndaches verbindenden. vorerst kielformig vorspruigentlen Wandteile des Endhirns. Ein zweiter Abschnitt erscheint wesentlich spiiter in Form einer jcderseits zuniichst einfachen gegen den Hohlraum der Seitenkanuner zu vorspringenden Falte, der als Area chorioidea bezeichneten Wandplatte der Hemisphare. Diese Falte geht vorn in die Wandfalte des Sulcus hemisphaericus liber, withrend sie sich nach riickwiirts etwas von ihr entfernt (fig. 4), noch weiter riach riickwiirts aber l)ald verstreicht. So erscheint bei dem iiltesten von den drei
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 20, NO. 1
82 PERCIVAL BAILEY
hishcr hosprochencn JOiubryonen (H. Sch. 2) dcr hintersto deiu Zwischeiihirn auliost^ndo Abschnitt der Area chorioidca nach vollkoninKsn glatt iiTui uiist'faltet.
All of his figures, with the exception of figure 6, appear to be through the diencephalon, back of the velum transversum.
The development of the paraphysis in the human embryo has never been followed. In fact, its identification is very much in doubt. Francotte ('94) claims to have found it in an embryo of twelve weeks. It is said to be an organ characteristic of all vertebrates, but becomes very rudimentary in birds and mammals. Its development has been followed by Dexter ('02) in the common fowl, and it has been described by Selenka ('91) in the opossum. A good review of the literature is given by Warren ('05).
Of the human embryo, Streeter in Keibel and Mall's Human Embryology says,
Orally this choroid roof [of the third ventricle] is continued into the telencephalon where it forms a pointed pouch overlapping the lamina terminalis and the contained commissures. . . . The anterior choroidal pouch has been homologized with the paraphysis of the lower vertel^rates.
It should be borne in mind, that in all vertebrates, the paraphysis, if present, arises from the roof of the telencephalon just cephalad to the velum transversum. In view of this fact, the structure labelled paraphysis by Goldstein ('03) is obviously not so, since it lies behind a structure called the .velum transversum and at the posterior end of the diencephalic roof.
The epiphysis is a constant organ in the vertebrate series^ (except in the alligator) but probably concerning no other organ has there been so much confusion and misinterpretation. For a review of the literature, reference may be had to Gaupp ('98). The development of the epiphysis "has not been followed completely in the human embryo, and so far as I know, an indication of the division into the two parts, epiphyseal stalk and pineal vesicle, which seems to be so characteristic of many vertebrates, has never been recorded.
With the recognition of the velum transversum in Cyclostomes by Sterzi (07), the velum has been established as a constant
DEVELOPMENT OF THE CHOROID PLEXUS 83
morphological feature in the roof of the prosencephalon of vertebrates.
The tela chorioidea diencephali has been the subject of an extensive anatomical, embryological and comparative study by Lachi ('88). It presents few features of interest. A number of names have been applied to parts of it (Zirbelpolster, dorsalsac, post-paraphysis, post-velar arch, etc.), but it has generally been recognized as extending from the superior commissure to the velum transversum.
With regard to the roof of the telencephalon, however, there is no such unanimity of opinion. I shall not attempt to review the observations on the lower vertebrates. Johnston ('13) reviews the literature fully and on the basis of this and his own extensive observations presents the following scheme as covering all the forms of the roof of the telencephalon, beginning with the preoptic recess in wliich the sulcus limitans ends:
Lamina tenninalis (containing the anterior eoinniissurei Recessus ncuroporicus
Lamina supraneuroporica (containing tlie pallial commissures^ Recessus superior Tela chorioidea telencephali • Paraphysis
Vehim transversum (anterior leafi
Instead of tela chorioidea telencephali, the more definite term, tela chorioidea telencephali medii, will be used in this paper. This plan may be completed for the diencephalon as follows:
Velum transversum (posterior leaf' Tela chorioidea diencephali Commissura superior Epiphysis
The posterior commissure belongs to the mesencephalon. The neuropore itself has never been followed through the successive stages of its development in the human embryo.
The evidence for transferring the above scheme to the human embryo is not wholly conclusive, and the present work was undertaken primarily to determine in how far the morphology in this region in certain human embryos was compatable with the above scheme.
84 PERCIVAL BAILEY
1 wish here to express my indebtedness to Ur. C. J. Herrick, whose broad knowledge and mature judgment has been of invakiable assistance, and especially to Dr. Geo. W. Bartlemez, at whose suggestion the work was undertaken and without whose kindly interest the work would have been impossible. Thanks are also due to Mr. A. B. Streedain for the care he has taken with the illustrative work.
MATERIAL AND METHODS
The material upon which this study was chiefly based consists of three very well preserved human embryos, cut in transverse series, stained in bulk with, borax carmine, and counterstained on the slide with orange G. Wax-plate reconstructions were made, the plates being stacked from a side view of the embryo drawn from a photograph, taken after fixation. The shrinkage after embedding was calculated and the outline reduced accordingly. Although the primary object of this study is the morphology and relations of the roof plate, in two cases the entire forebrain has been modeled. This was done because the embryos happened to fall in at opportune intervals between His' embryo CR (13.6 mm.) and the embryo of 50 mm. also modeled by him, and also in order that the relations of the choroid plexuses might be seen more clearly.
Embryo H 173 was obtained from an aborted ovum of 42 x 32 X 19 mm., presented by Dr. N. R. Engels of Chicago. The only available history w^as that the patient had missed two menses. The intact ovum was placed in physiological salt solution and kept at about 0°C. for 11 hours. It was then opened and fixed in formalin-Zenker for 24 hours, stained in bulk with borax carmine, imbedded by the celloidin-paraffln method and cut 10m thick in a plane parallel to the hindbrain. The embryo measi;red 19.1 mm., crown-rump length after fixation. The sections were counterstained on the slides with orange G. The total shrinkage was about 20 per cent. There are frequent mitoses in embryo and chorion. The brain was modeled at a magnification of 50 diameters with the aid of the Edinger pro
DEVELOPMENT OF THE CHOROID PLEXUS 85
jection apparatus. One millimeter plates were used and ever}other section drawn except in the region of the foramen interventriculare, where half millimeter plates were used and every section drawn. The epiphyseal region was modeled at a magnification'of 100 diameters. Millimeter plates were used and every section was drawn.
Embryo H 91 was obtained from an aborted ovum 50 x ;U X 30 mm. presented by Dr. G. C. Dittmann of Chicago, whose data indicate a clinical pregnancy of 60 days. The ovum was left unopened in physiological salt .solution for 10 hours, then opened and fixed in an S per cent .solution of formaldehyde, neutralized with magnesium carbonate. It measured 27.8 mm. crown-rump length after fixation in formalin and showed a shrinkage of 13. () per cent after imbedding in paraffin. It was stained in bulk in borax carmine and on the slide with orange G. It was cut in 20m sections, and modeled at a magnification of 40 diameters. Millimeter plates were used and every fifth section was omitted.
Embryo H 41 was obtained fiom an u\uni of 71 x 39 x 32 nun., presented by Dr. L. A. Beaton of Chicago. The chorion was opened and the entire ovum fixed in formalin. The crownrump measurement of the embryo after fixation was 32.1 mm. and it showed a shrinkage of 10 per cent after imbedding in paraffin. The staining was the .same as that of H91. Tliis embryo was sectioned 20m in paraffin, and modeled at a magnification of 25 diameters. ^Millimeter plates were used and every other section was drawn. The region around the foramen of Monro was modeled at a magnification of 100 diameters; 2 mm. plates were used and every section was drawn.
Two points in the technical procedure are to be emphasized because they are in large measure responsible for the exceptionally good preservation of the form relations of the delicate roof plate of the brain. Both of the older embryos (H 91 and H 41) had the cranial cavity opened by an incision in the line of the sagittal suture. Distortions due to unequal shrinkage of the brain and overlying structures were thereby in great measure avoided. All three were passed from 95 per cent alcohol to
86 PERCIVAL BAILEY
ether-alcohol, then through 0.5, 1, 2 and 3 per cent celloidin, hardened in chloroform-alcohol, cleared in benzol and imbedded in paraffin under the air pump.
The plane of section in each case is shown in figures 29; 30 and 31.
Several other human embryos were studied, the most helpful being embryo H 44 of the Chicago collection, which measured 60.4 mm. after fixation in formalin. A transverse series from a 25 mm. pig in the collection of Dr. F. R. Lillie was also used.
DESCRIPTION
1. The 19 mm. embnjo {H. 173)
The recessus preopticus is well marked (fig. 18, r.pre.). The roof plate stretches dorsad from this recess as a thickened lamina (fig. 18, l.t.) to about the level of the sulcus separating the medial and intermediate roots of the corpus striatum. Above this point the roof plate narrows and extends cephalad and dorsad (fig. 18, Z.s.?), forms a broad arch (fig. 18, r.s.), and then passes caudad and dorsad (fig. 18, tx.t.m.) as a still thinner membrane toward the velum transversum. Just in front of the velum transversum, the roof plate forms a small narrow arch (fig. 18, p.a. and fig. 2, p. a.), from the sides of which arise the lateral choroid plexuses.
(Throughout these descriptions, narrow and wide are used of dimensions tangential to the ventricular surface, and thick and thin of dimensions perpendicular to the ventricular surface. For example, in figure 12, the tela chorioidea diencephali is thin and wide.)
The velum transversum is well marked (fig. 18, v.L), indicating the boundary in the roof plate between the diencephalon and telencephalon.
The roof of the diencephalon (fig. 18, tx.d.) is still narrow throughout most of its extent. It is also relatively thick, with several rows of nuclei in cross-section. It is narrowest at its posterior end and remains narrow almost to the anterior end of the thalamus, where it suddenly widens (fig. 1, t.c.d.). The
DEVELOPMENT OF THE CHOROID PLEXUS 87
entire structure, when viewed from above, is somewhat trumpetshaped, with the bell at the anterior end. There is no indication of plexus formation. The entire roof plate of the diencephalon is perfectly smooth. The commissura superior is clearly indicated (fig. 18, com.s.).
The epiphyseal evagination is a hollow outgrowth (figs.* IS and 3, e.e.). The top of the evagination is cupped and in this cup lies a ball of cells (figs. 3, 10 and 18, e.v.). This ball of cells has an irregular lumen in its center (fig. 10, e.v.). The cells of the ball stain more lightly than the cells of the cup, and the line of separation is fairly distinct. It will be seen that this ball of cells, while not in actual contact with the epidermis, approaches it very closely (fig. 10, e.v.). The epiphyseal evagination lies some distance cephalad of the commissura posterior (fig. 18, c.p.).
The extent and morphology of the lateral choroitl plexus is shown in figure 19. It is clearly divisible into two parts, an anterior part (figs. 2 and 19, p.c.v.L, p.o.) attached to the lateral margin of the paraphysal arch along its entire length, and by the taenia 'fornicis (fig. 2, /./.) to the medial hemisphere wall immediately above and lateral to it; and a posterior part (figs. 1 and 19, p.c.v.L, p:p.) attached by the taenia chorioidea (fig. 1. t.c.) to the lateral thalamic wall and by the taenia fornicis (fig. 1, t.J.) to the medial hemisphere wall immediately below the hippocampus. The fissura chorioidea is very wide throughout most of the extent of the posterior part of the lateral choroid plexus.
If the angle between the taenia chorioidea and the thalamic wall be followed anteriorly, it is found to be continuous with the velum transversum; if the angle is followed posteriorly, it continues backward between the lateral thalamic wall and the medial hemisphere wall, turns downward between them and passes anteriorly and downward behind the optic nerve on the lateral wall of the hypothalamus (fig. 19, d-t. gr.). This groove is more clearly marked on the lateral wall of the 32 mm. embryo.
The ependymal portion of the plexus is still relatively thick (fig. 27). The mesenchymal tissue of the plexus is typical em
8S
PERCIVAL TJAILRY
hryonal connective tissue. The blood capillaries are particularly numerous near the ependyma.
If one examines now the ventricular surface, the corpus striatum (fig. 19, c.s.) appears at the posterior point of attachment of the lateral plexus as a single ridge in the floor of the lateral ventricle. As it is followed anteriorly, this ridge is soon divided by a groove into two portions. The lateral portion is
REFERENCE LETTERS
(I.e. (I., area chorioidea anterior.
a.c.p., area chorioidea posterior.
n.p., anterior pouch (of the tela chorioidea diencephali)
nq.S., aqueduct of Sylvius
cb., cerebellum
CO., chiasma opticum
com.s., commissura superior
c.p., commissura posterior.
C.S., corpus striatum
c.s.i.r., corpus striatum, intermediate root
c.s.i.r., corpus striatum, lateral root
c.s.m.r., corpus striatum, medial root
d.p., deep pit (in telencephalic roof plate)
d-t.gr., di-telencephalic groove
e.e., epiphyseal evagination
ep., epidermis
c.r., epiphyseal ridge
e.v., epiphyseal vesicle
f.a., fissura arcuata
f.c, fissura chorioidea
f.int., foramen interventriculare
f.r.; fasciculus retroflexus (Meynerti)
h.a., and hip., hippocampal area
hem., hemisphere
h.s-t.r., habenulo-subthalamic ridge
hy., hypothalamus
inf., infundibulum
I.S., lamina supraneuroporica
Id., lamina terminalis
mt., metathalamus
n.h., nucleus habenulae
n-p.a., neopallial area
n.post., recessus postopticus
o.n., optic nerve
p.a., paraphysal arch
p.c.v.L, plexus chorioideus ventriculi lateralis
p.c.v.L, p.a., plexus chorioideus ventriculi lateralis, pars anterior
p.c.v.L, p.p., plexus chorioideus ventriculi lateralis, pars posterior
p.o.hy., pars optica hypothalami
r.m., recessus mamillaris
r.n., recessus neuroporicus
r.posL, recessus postopticus
r.pre., recessus preopticus
r.s., recessus superior
s.a., striatal area
.s.L, sulcus limitans
.s.?«., stria meduUaris
S.M., sulcus Monroi
s-L, subthalamus
t.c, taenia chorioidea
Led., tela chorioidea diencephali
t.c.t.m., tela chorioidea telencephali medii
t.f., taenia fornicis
tg., tegmentum
th., thalamus
th.l, th.2., parts of thalamus
th., e.s., thalamus, ependymal surface
th., p.s., thalamus, pial surface
t.i-c, taenia infrachorioidea
t.r-p., telencephalic roof plate
t.s-c, taenia suprachorioidea
<.<., taenia thalami
r.t., velum transversum
DEVELOPMENT OF THE CHOROID PLEXUS 89
at first in the floor of the lateral ventricle but anteriorly comes to lie in the lateral wall (figs. 2 and 23, c.s.l.r.). The medial portion
pcv t,p.p.
1 2
Fig. 1 Section through thediencephalon of the 19mm. embryo, H 173. X 13J. Slide 21, Sect. 11. Compare the photograph of this section, figure 27.
Fig. 2 Section through paraphysi.i and hypothalanuis of the 10 mm. embryo, H 173. X13J. SIide23. Sect. 13."
Fig. 3 Section through epiphysis of the 19 mm. embryo, n 173. X 15. Slide
12, Sect. 4.
Fig. 4 Section through epiphysis of the 28 mm. embryo, H 91. X 10. Slide 9, Sect. 148. Ependyma solid black.
90 PERCIVAL BAILEY
continues to the foramen interventriculare where it divides into two parts, the lateral part passing forward in the floor of the lateral ventricle (the intermediate root of the corpus striatum, figs. 2 and 23, c.s.i.r.), and the medial part passing through the foramen interventriculare, forming its floor, and extending in the lateral wall of the third ventricle behind the lamina terminalis as far as the recessus preopticus (fig. 18, c.s.m.r.). It is very likely that this elevation which I have just described as the medial root of the corpus striatum contains in its lower end other things besides striatal tissue. I shall continue to describe the entire elevation as the medial root of the corpus striatum, following the usage of His, and shall not enter into a discussion of its internal structure.
Above the taenia fornicis on the ventricular surface of the medial hemisphere wall may be seen an elevation (fig. 19, hip. ) extending from the posterior pole of the hemisphere just above the choroid plexus, and beyond it over the foramen interventriculare. This is the anlage of the hippocampus, at least in part. There is corresponding to it on the outer pial side of the hemisphere wall, a shallow groove (figs. 1 and 2,f.a.). This groove is not due to the folding in of a thin weak place in the wall, for the wall at this point is very definitely thicker (fig. 27).
Turning now to the wall of the third ventricle, we find just below the tela chorioidea diencephali a low ridge (fig. 18, n.h.) extending from the epiphyseal evagination almost to the velum transversum. This is the habenula. From its anterior end a sharp ridge runs backward and downward to the subthalamus (fig. 18, h.s-t.r.). Above this ridge, the wall is shrunken and thin up to the habenular thickening. Below the ridge, is an elevation (fig. 18, th.l) which extends upward and forward in front of the habenula toward the velum transversum.
The sulcus limitans is indicated on figure 18 by a dotted line running above the tegmentum and subthalamus, below the elevation last mentioned (fig. 18, th.l) and behind the corpus striatum to the preoptic recess. Just back of the corpus striatum and between it and the hypothalamus, the sulcus limitans runs into a very deep recess (fig. 2) .
DEVELOPMENT OF THE CHOROID PLEXUS 91
Below the sulcus limitans lie the subthalamus (fig. 18, s-t.) and hypothalamus (fig. 18, hy.). The elevation which marks this region is very long. The pars optica is only indistinctly marked off (fig. 18, p.o.hy.). At its posterior end, the elevation divides into two portions, one of which, subthalamus, continues upward into the tegmentum, the other, hypothalanms, backward into the mammillary recess.
The floor plate is thin and somewhat widened (fig. 3). The infundibulum (fig. 18, inf.) lies a considerable distance back of the optic chiasm. The postoptic recess is but jioorly marked (fig. 18, r. post.).
2. The 28 mm. embryo (H. 91)
The entire telencephalon is not modeled in this embryo. The model was made primarily to show the lateral choroid plexus. Those portions not modeled differ in no essential respect from the corresponding j^ortions of the 32 mm. embryo.
Immediately ccphalad of the velum transversum (fig. 20, v.t.), which is clearly indicated, the roof plate forms a small arch (fig. 20, p.a.) to the sides of which are attached the lateral choroid plexuses. In front of the arch, the roof plate becomes very thin for a few sections (fig. 5, t.c.t.m.). Then as we pass cephalad of this thin lamina, we come to a region where the roof plate thickens in a peculiar manner (fig. 0, t.r-p.).
The hemisphere wall on each side of the roof plate is thin and in it two distinct zones are discernable, a broader zone next the ependymal surface, where the nuclei are very numerous, mantle layer, and a narrower zone next the pial surface which is relatively free from nuclei, marginal layer. In the roof plate, however, these two. zones are not discernable, the nuclei are equally numerous throughout from the pial to the ependymal surface, and at the ependymal surface are loosely arranged, so that the outHne is indefinite and irregular. This is the characteristic arrangement, and such an arrangement I have found in this region in five human embryos of about this age in the collection of the Department of Anatomy and in a 25 mm. pig embryo in the
92
PEUCIVAL BAILEY
collection of Dr. ¥. \\. Lillie. In the embryo under consideration toward the anterior end of this region (fig. 7, t.r-p.) the morphology is somewhat different from that shown in figure 6. In the mid-line again, the nuclei are evenly distributed from ependymal to pial surface and are very numerous. Immediately on either side, however, the wall is greatly thickened in such manner that,
Fig. 5 Sectionthrough tela chonoideatelencephalimedii of the 28 mm. embryo,
H91. X 50. Slide 28, Sect. 362.
Fig. 6 Section through roof plate of telencephalon medium of the 28 mm. embryo, H 91. X 50. Slide 29, Sect. 367.
although the pial surface still forms a regular curve, the ependymal surface shows a deep notch in the mid-line. The outline of the ependymal surface is again indefinite owing to the loosely arranged cells. The marginal layer approaches almost to the midline. At the extreme anterior end of this region the notch disappears; the ependymal outline becomes definite; and the roof plate thickens markedly in an undoubted lamina terminalis ffig. 20, l.L).
DEVELOPMENT OF THE CHOROID PLEXUS
93
The tela chorioidea diencephali is very broad and very thin (fig. 11, Led.). It does not exhibit any folding except at the extreme anterior end. Toward the posterior end of the tela, a narrow strip of the alar plate is curved lateralward, resembling the rhomboidal lip of the rhombencephalon, and which we may term the thalamic lip (fig. 9, /./.).
Fig. 7 Sec-tion through the roof plate of (elcn(ei)li:il()ii moiliiiiii i>f the "JN in
embryo, H 91. X 50. Slide, 29, Sect. 372.
Fig. 8 Section through the roof plate of te]cn(ei)h:il()n inediiiin in the 32 in cinbrvo, H 41 . X 50. Slide 32. Sect. 2.
The thalamic lip carries with it lateralward the taenia thahiini (fig. 9, t.t.) and the roof plate. Toward the anterior end of the tela, the thalamic lip bends laterally more and more until its pial surface comes into contact with the pial surface of the thalamic wall (fig. 11, t.l). The ependymal surface of the roof plate is hereby brought into contact with the ependymal surface of the thalamic lip (fig. 11). At the apex of the angle between the thalamic lip and the thalamic wall lies the stria medullaris (fig. 11, S.771.). The entire tela when viewed from above is wedge
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PERCIVAL BAILEY
shaped, the anterior end being very broad. Heuser ('13) has noted a similar condition in the pig. The commissura superior is well marked (fig. 20, com. s.).
It will be seen that the roof of the epiphyseal evagination (fig. 20, e.e.) becomes epithelial for a short space in its uppermost portion. Just in front of this epithelial region there is a small aggre^'ation of cells (fig. 4, e.v.) which recalls the ball of cells described in the epiphysis of the 19 mm. embryo. The nuclei
Fig. 9 Section through the diencephalon of the 28 mm. embryo, H 91. X
6§. Slide 15, Sect. 228.
Fig. 10 Section through the epiphysis of the 19 mm. embryo, H 173. X 150. Slide 12, Sect. 4.
of these cells stain densely like the nuclei of the ependymal cells, and lie very close together. The cytoplasm is stained a deep yellow like the cytoplasm of the ependymal cells. They are surrounded by more Hghtly staining cells, and are nowhere in connection with the ependymal cells. The lateral wail of the epiphyseal evagination is very massive (fig. 4, e.r.). A ridge arises from the postero-superior portion of the lateral wall of the diencephalon and extends upward and backward to the epiphyseal evagination (fig. 21, e.r.).
DEVELOPMENT OF THE CHOROID PLEXUS
95
The lateral choroid plexus (fig. 21, p.c.v.l.) is of considerable size, but does not nearly fill the ventricle (fig. 11, p.c.v.l.). In antero-posterior extent the plexus measures 1.55 mm., the ventricle measuring 2.97 mm. The anterior end of the plexus is much the larger and is less folded. Thompson ('09) has described a similar condition in the cat. The attachment of the plexus to the roof plate is now much narrowed, owing to the relatively small size of the paraphysal arch (fig. 20, p.c.v.l., p.a.). The taenia fornicis is now closely approximated to the taenia chorioidea, the fissura chorioidea being reduced to a narrow
11
12
Fig. 11 Section throuuli tlif iliciKophaloii of the L'S nun. embryo, H'.)l. X61. Slide 24. Sect. 318.
Fig. 12 Section througli the (licnccphalon of the 32 mm. emliryo. 1141. X OL Slide 24, Sect. 2.
slit (fig. 11, /.r.). In its growtii, the j)lexns has extended some 0.25 mm. anterior to its point of attachment to the mid-line of the telencephalon. The connective tissure resembles that of the 19 mm. embryo closely, except that the cells are farther apart, but the ependymal layer has become much thinner and consists clearly of a single layer of columnar cells, with the nuclei in the ends of the cells next the ventricular cavity (fig. 28). The epend\^ma of the posterior j^art of the plexus resembles more closeh' that of the 19 mm. embryo, there being several layers of irregularly arranged nuclei. The posterior part of the plexus is much attenuated and much more folded than the anterior end.
96 PEIICIVAL HAILEV
111 the wall of the third ventricle, the habeiiula is well marked (fig. 20, n.h.) and from its posterior end a ridge extends downward to the tegmentum (fig. 20, f.r.). Just below the habenula, the wall of the diencephalon is much thicker than in the 19 mm. embryo, and extends anterior to the velum transversum to form the posterior wall of the foramen interventriculare.
3. The 32 mm. embryo (H. 4I)
Just in front of the velum transversum, as in the 28 mm. embryo, the roof plate forms a low arch (figs. 22 and 25, p.a.) to the sides of which are attached the lateral choroid plexuses. Immediateh^ anterior to this arch the roof plate becomes very thin (fig. 25, tx.t.m.). Anterior to this thin lamina, the roof plate is thickened again in the manner noted m the case of the 28 mm. embryo (see fig. 6, t.r-p.). At the anterior end of this region (fig. 25, l.sJ), there is an indication of the median notch on the ependymal surface (fig. 8, t.r-p). It is, however, not nearly so well marked as the similar notch in the 28 mm. embryo (fig. 7, t.r-p.) At the extreme anterior end of this region is a structure not found in any of the other embryos. A deep narrow pit extends into the roof plate from the pial surface, which causes the roof plate to project into the ventricle (figs. 13, 14, and 25, d.p). This pit is found only in one section, with indications of it in the two adjacent sections. Immediately anterior to the pit, the roof plate thickens markedly and extends as a thickened lamina (fig. 22, l.t.) to the recessus preopticus. There is, however, a short distance anterior to the pit a shallow notch on the ventricular surface of the lamina terminalis (fig. 25, r.n.f).
The tela chorioidea diencephali shows only slight indications of longitudinal folding. The outwardly curved and very prominent thalamic lip (fig. 12, t.l.) is not in contact with the lateral wall of the thalamus. At the anterior end, the tela chorioidea diencephali is very broad, and a pouch (fig. 25, a.p.) arises which extends forward over the velum transversum. The whole tela resembles very closely the same structure in the 28 mm. embryo.
DEVELOPMENT OF THE CHOROID PLEXUS
9:
The commissura superior is very readily identified (fig. 22, com.s.).
.The epiphyseal evagination (fig. 22, e.e.) is, in all essential respects, identical with that of the 28 mm. embrj'o.
The lateral choroid plexus (fig. 12, p.c.v.l.) also resembles that of the 28 mm. embryo. The posterior portion is more swollen, resembhng more nearly the anterior part. The ependyma
13
14
•pl.al.
(liurii of the
Fin. 13 Section tliiouKli tin- roof i)latc of tlic tele 32 mm. embryo, H 41. X 50. Slide 32, Sect. 4.
Fig. 14 Section through the roof plate of the telenrephalon medium of tiie 32 mm. embryo, H 41. X Ti. Slide 32, Sect. 4.
is almost entirely a single layer of cells except at the extreme caudal end of the plexus. The plexus extends through 4.44 mm.; the ventricle through 7.2 mm. The plexus extends 1.6 ♦mm. anterior to its most anterior point of attachment.
On the ventricular surface we find again that the corpus striatum exhibits three roots at its anterior end (fig. 24, c.s.). The most medial root, in the lateral wall of the third ventricle, is narrow, lies immediately back of the lamina terminalis, and
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, N<
98 PERCIVAL BAILEY
forms more the anterior boundary than the floor of the foramen interventricuhire (fig. 22, c.s.m.r.). The intermediate root is also small, extending beyond the foramen interventriculare in the floor of the lateral ventricle. The intermediate root unites at its posterior end with the medial root, the two being separated from the lateral root by a groove. The lateral root is the largest, and lies more in the floor than in the lateral wall of the ventricle (fig. 24, c.s.l.r.). The two ridges, one formed by the lateral root and the other by the union of the medial and intermediate roots, extend backward some distance and then turn sharply downward as a single elevation (fig. 24).
The hippocampus (fig. 12, hip.) is a broad, slightly thickened portion of the medial hemisphere wall, just above the taenia fornicis. The external sulcus is very shallow and broad.
The habenular ridge is prominent, extending on the ventricular surface of the diencephalon just below the tela chorioidea diencephah from the region of the velum transversum to the epiphysis (fig. 22, n.h.). The ridge extending from the posterior end of the habenula to the tegmentum is also prominent (fig. 22, /.r.).
The most striking feature, probably, is the great thickening of the posterior extremity of the thalamus. The anterior extremity of the thalamus extends well into the foramen interventriculare, some distance anterior to the velum transversum.
The sulcus limitans is indicated on figure 22 by a dotted line. It is deepest below the posterior pole of the thalamus. Behind the lamina terminalis it is relatively shallow.
The hypothalamic region is marked mainly by its great length. The mammillary recess is indicated (fig. 22, r.m.) ; the floor plate is wide and thin. The infundibular recess (fig. 22, inf.) is near the optic chiasm but is separated from it by an unmistakable postoptic recess (fig. 22, r.post.). There is no clear external division between hypothalamus and sub thalamus. ♦
DEVELOPMENT OF THE CHOROID PLEXUS 99
DISCUSSION
1. Telencephalon
a. Recessus preopticus. In all three embryos there is no doubt about the identity of this recess.
b. Velum iransversum. Just as obvious is the location of the velum transversum. It is marked by the groove running across the roof plate joining the anterior ends of attachment of the tela chorioidea diencephali to the thalamus. At the lateral end of the velum transversum the taenia thalami meets the taenia chorioidea ffig. 26) and at this point the velum transversum becomes continuous with the angle between the taenia chorioidea and the lateral thalamic wall. In figure 26 an arrow lies in the angle between the taenia chorioidea and the lateral thalamic wall and continues in the velum transversum. The position of the head of the arrow in the mid-line is shown in figure 25. It was noted in the account of the 19 mm. embryo and appears more clearly in the 32 nun. embryo, that if this angle be followed backward it passes behind the attachment of the hemisphere to the thalamic wall, and then as a diagonal groove downward and forward across the lateral wall of the hypothalamus and ends at the optic chiasm. This is the di-telencephalic groove (figs. 14 and 19, d-l.gr.) of Johnston.
With the preoptic recess and the velum transversum fixed, the extent of the telencephalic roof plate is determined. Between the preoptic recess and the velum transversum should appear the lamina terminalis, recessus neuroporicus, lamina supraneuroporica, recessus superior, tela chorioidea telencephali medii, and paraphysis.
c. Paraphysis. Just in front of the velum in each embryo is a small arch (figs. IS, 20 and 25, p.a.) relatively largest in the 19 mm. embryo, and smallest in the 32 nun. embryo. In each case it lies immediately in front of the velum transversum. In each case also the lateral choroid plexuses arise from its sides. There can be no doubt that this is the paraphysal arch. No indication of the development of a glandular structure could be found. The resemblance of the paraphysal arch in
100 PERCIVAL BAILEY
the 19 mm. embryo to those of the 10 mm. cat and 20 hr. chick figured by Tilney ('15) and of a 4 mm. embryo of Platydactylus mauritanicus, figured by Tandler and Kantor C07), is rather striking.
d. Recessus neuroporicus. An identification of this point is absolutely essential to a final definition of the boundary between lamina terminalis and lamina supraneuroporica. By recessus neuroporicus is meant the most caudal point, i.e., last point, of closure of the neuropore. In many vertebrates at this point a recess appears on the ventricular surface of the roof plate (Johnston, '09).
It was found impossible to identify with certainty this point in the embryos examined. In the 19 mm. embryo, and the 28 mm. embryo, no such recess is apparent. In the 32 mm. embryo, there has been noted just in front of the pit in the roof plate, a shallow notch on the ventricular surface (fig. 25, r.n.f). But there is no evidence that this is the recessus neuroporicus, since no such notch appears in either younger embryo.
e. Lamina terminalis. The upper end of the lamina terminalis, as defined by Johnston, has not been determined because the recessus neuroporicus is not apparent. Concerning the major portion of the lamina, however, there can be no doubt. The thick lamina above the recessus preopticus is unmistakable (figs. 18, 20 and 22, l.L).
f. Tela chorioidea telencephali medii. Just in front of the paraphysal arch in the 28 mm. embryo, and the 32 mm. embryo, the roof becomes a single layer of flattened cells (fig. 5, t.c.t.m.). This is certainly tela chorioidea telencephali medii. The identification of this tela in the 19 mm. embryo is not so easy. However, I am inclined to identify the summit of the greater arch of the roof plate (fig. 18, r.s) as the recessus superior. This would make the roof between this point (fig. 18, r.s.) and the paraphysal arch, tela chorioidea telencephali medii (fig. 18, t.c.t.m.). The roof plate here is somewhat thinner than the lower limb of the greater arch (fig. 18, l.s.f) and the adjacent hemisphere wall is considerabl}^ thinner. The angulus terminafis
DEVELOPMENT OF THE CHOROID PLEXUS 101
of His probably represents all the membranous parts anterior to the velum trans versum.
g. Lamina supraneuroporica. Between what is obviously lamina terminalis and what is just as obviously tela chorioidea telencephali medii in the 28 mm. embryo and the 32 mm. embrj^o, lies the peculiar thickening of the roof plate before noticed Cfig. 6, t.r-p.) which seems by a process of elimination to be lamina supraneuroporica. Whether this interpretation is correct, I cannot say, because I have not had material with which to follow this region in its later development. There is in the 32 mm. embryo, a short portion of the roof plate between the deep pit (fig. 25, d.p.) and the shallow recess on the ventricular surface (fig. 25, r.n.?) which may be lamina supraneuroporica. In the 19 mm. embryo, the lower limb of the greater arch (fig. 18, l.s.f) seems its most likely location.
h. Plexus chorioideiis ventriculi lateralis. The problem of the formation of the lateral choroid plexuses is one of considerable difficulty, because of the complex morphological relations involved, but if a few facts of development be remembered, the problem l)ecomes relatively simple.
If the brain of a half-grown frog tadpole be examined, it will be found that "the membranous roof of the forebrain ventricle is attached to the massive wall of the hemisphere by the taenia fornicis which is directly continuous caudad with the taenia thalami" (Horrick, '10). The taenia thalami is the attachment of the roof i)late posterior to the velum transversum to the lateral wall of the thalamus. It might be added also that the taenia fornicis becomes continuous with the taenia thalami at the lateral end of the velum transversum. In the middle of the membranous roof in front of the velum transversum arises the paraphysis. There is no plexus lateralis in the larval or adult frog, but in urodele Amphibia it is between the paraphysis and the taenia fornicis that the lateral choroid plexus makes its appearance, pushing into the ventricle. Warren ('05) in describing the development of the lateral plexuses of Necturus maculatus says, "The telencephalic plexus develops from the
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PERCIVAL UAILEY
I)anii)hysal arcli. . . ." "Tlie plexuses of the hemispheres arise on either side from the origin of the telencephalic plexus and pass into the lateral ventricles. . . ."
If we turn now to the human embryo and examine, say, the 19 mm. embryo, H 173, we find that it is easy to trace the tela chorioidea telencephali medii over the paraphysal arch and velum transversum to the tela chorioidea diencephali. If, however, we attempt to follow backward the taenia fornicis, it will not be found to be continuous with the taenia thalami at
Fig. 15 Median view of a model of the forebrain in His' 6.9 mm. embryo,
Br. .3.
the lateral end of the velum transversum, but is separated from the taenia thalami by the fissura chorioidea. In order to explain the difference between the condition in the frog and in the human embryo it is necessary to analyze more closely some younger human embryos.
Figure 15 shows a medial view of the forebrain of His' embryo Br 3, 6.9 mm. in length. On it is indicated by a row of crosses a line homologous with the taenia fornicis et taenia thalami in the tadpole. The anterior limb of the di-telencephalic groove is marked by small circles and labeled area chorioidea posterior for reasons which will appear later. On the opposite side of
DEVELOPMENT OF THE CHOROID PLEXUS
10.3
the taenia fornicis from the area chorioidea posterior, that is, between the taenia fornicis and the mid-dorsal line, is placed another area of circles labeled area chorioidea anterior. It is
17
Fig. 16 Median view of a model of the forebrain in His' 13.6 mm. embryo, CR.
Fig. 17 Hypothetical view of the median wall of the cerebral hemisphere from His' 13.6 mm. embryo, CR. The hemisphere has been excised along the line b-a-d in figure 16.
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in this latter region tiiat the lateral choroid plexus makes its appearance in urodele Amphibia as we have just remarked, and here also it makes its first appearance in the human embryo. The hippocampal area is not apparent in this embryo but later developments show that its anlage must lie in some such position as indicated in figure 15. The part of the telencephalon which is evaginated to form the cerebral hemisphere is the part anterior to the line d-a-b. The division between telencephalon and diencephalon is represented by the line b-a-c.
Keeping these relations in mind, we may easily understand His' embryo CR, 13.6 mm. in length. Figure 16 shows a medial view of the forebrain. An invagination of the telencephalic roof has taken place between the taenia fornicis and the midline, forming the fissura chorioidea in the region which was marked area chorioidea anterior in figure 15. The hemisphere vesicle has enlarged mainly by enlargement and evagination of the neo-pallial area (fig. 15, 7i-p.a.), so that the hippocampal area which in figure 15 lay below the roof plate and in front of the area chorioidea posterior, now lies above the roof plate and behind the area chorioidea posterior, and its ependymal face has now come to look lateralward. So also with the area chorioidea posterior, which lies now in the evaginated cerebral hemisphere behind the di-telencephalic groove, and its ependymal face is now its lateral face. When the hemisphere evaginated, the wall bent for the most part along the line of the taenia fornicis in figure 15, the di-telencephalic groove as far as the point a, and then along the line a-d.
If now the hemisphere in embryo CR be excised, along a line homologous with the line b-a-d in figure 15, the relations of the area chorioidea posterior and area chorioidea anterior are more clearly seen. The medial surface of such a hypothetical hemisphere is represented in figure 17. The area chorioidea anterior has invaginated into the lateral ventricle. The resulting plexus has been cut off in figure 17, leaving the fissura chorioidea. Since the invagination takes place between the taenia fornicis and the mid-line of the telencephalic roof, the taenia fornicis lies along the lateral and upper margin of the fissura
DEVELOPMENT OF THE CHOROID PLEXUS 105
chorioidea, or in other words, along the hne of attachment of the plexus to the medial hemisphere wall. If, therefore, we wish to follow the taenia fornicis et thalami in the embryo CR, we must follow the Hne marked with crosses in figures 16 and 17 along the wall of the diencephalon (figs. 1() and 17, t.t.), across the velum transversum, and then between the area chorioidea posterior and area chorioidea anterior above and lateral to the fissura chorioidea (figs. 1() and 17, t.f.).
It will be seen (fig. 17) that the area chorioidea anterior hes entirely anterior to the velum transversum. The lateral choroid plexus never approaches the mid-line except along the sides of the paraphysal arch, anterior to the velum transversum. The area chorioidea posterior has relations entirely analogous with its relations in figure 15, except for its change of face. It lies between the hippocampus and the di-telencephalic groove, and adjoins the posterior end of the taenia fornicis on the upper side, which in figure 15, was the lower side of the taenia.
This stage in the process bears a close resemblance to the condition in the brain of Phitydactylus mauritanicus, as described by Tandler and Kantor ('07). Concerning the development of the structures in the region of the foramen of Monro, they write (italics mine) :
Die zwischoii don bcidcn Foramina Monroi gclegene Deckc dcs Ventriculus impar [telencephalon niediuni] ist rein epithclialer Natur and grenzf sicli laterahi'drts durch je cine dcutliche Furche Sulais tcgmenli ah.
The lateral edge of this sulcus tegmenti is the taenia fornicis.
Die Fissura chorioidea, wclche von hinten her den Rauni des Foramen Monroi einengt, entwickclt sick, wie die Durchsichi dcr Serielehrt. derart, dass sie im vordercn Ahschnitt aus dem Sulcus tegmenti selbst. hinten aber oberhalb diescr Furche entsteht, und hier die mediale Hemispharcn wand einschneidet.
Der Plexus chorioideus des Ventriculus lateralis stijlpt niimlich nur ein ganz kurzes Stiick der Hirnwand ein. Die Einstiilpungsstelle selbst, liegt wie man inn Stadium V zeigen kann, gerade dort, ico das hinfere Ende des Sulcus tegmenti die Decke des Telencephalon impar von der medialen H emisphdrenwand absetzt.
106 PERCIVAL BAILEY
Here again, as in the brain of Necturus maculatus, the half grown frog tadpole, and His' embryo CR, the taenia fornicis can be followed along the lateral margin of the sulcus tegmenti past the velum trans versum to the taenia thalami. But when the sulcus tegmenti invaginates to form the fissura chorioidea, the posterior end of the taenia fornicis and a small part of the medial hemisphere wall are drawn in also. This latter process is carried much farther in the human embryo.
In the evagination of the hemisphere in the human embryo, the point marked x in figure 15, at the junction of the hippocampal area, striatal area, and area chorioidea posterior, remains always at the posterior pole of the hemisphere. The result on the corpus striatum, as the hemisphere extends backward, is to draw out the tail of the caudate nucleus. The result upon the area chorioidea posterior is to draw it out in a thin lamina marked in figure 17 by small circles.
The area chorioidea posterior now buckles into the lateral ventricle, and we have the condition found in the 19 mm. embryo, H 173. The area chorioidea posterior is clearly shown by figure 19, being all of the plexus back of the point marked z, and has buckled inward only slightly, leaving a very wide fissura chorioidea. The point z lies opposite the velum transversum.
It thus appears that the lateral choroid plexus is composed of two parts, a pars anterior plexus chorioidei ventriculi lateralis which is formed by the invagination of the area chorioidea anterior, between the paraphysal arch and the taenia fornicis, and a pars posterior plexus chorioidei ventriculi lateralis formed by the infolding of the area chorioidea posterior in the medial wall of the hemisphere.
If the taenia fornicis be now followed in the 19 mm. embryo, it will not be found to become continuous with the taenia thalami because the area chorioidea posterior, to which it was attached toward its posterior end, has now buckled into the ventricle. If, therefore, we follow the attachment of the lateral choroid plexus to the medial hemisphere wall, we follow the taenia forni
DEVELOPMENT OF THE CHOROID PLEXUS 107
cis as far as the area chorioidea posterior, then along the upper margin of the area chorioidea posterior (anterior margin in fig. 15) called also taenia fornicis in adult anatomy, around its posterior extremity (lower extremity in fig. 15), and back along its lower margin (posterior margin in fig. 15) called also taenia chorioidea, to the lateral end of the velum transversum where we finally reach the taenia thalami.
It thus appears that the portion of the taenia fornicis of human anatomy which hes adjacent to the pars posterior plexus chorioidei ventriculi lateralis is not homologous to the taenia fornicis in Anura and would better be called taenia suprachorioidea, and the taenia chorioidea correspondingly termed taenia infrachorioidea. The illustrations have been labeled in accordance with established usage. To place them in accord with the foregoing conclusions, in figures 1, 11, 12, 18, 24 and 26 taenia fornicis should be changed to taenia suprachorioidea, and taenia chorioidea to taenia infrachorioidea.
In later stages the taeniae infra- et suprachorioiilea become approximated closely as is found in the 2S mm. embrj'o and in the 32 mm. embrj^o, and the fissura chorioidea is reduced to a narrow slit, its axis in the plexus of the 19 mm. embryo, lying probably along the dotted line in figure 19.
In the 28 mm. embryo and the 32 mm. embryo, it is impossible to distinguish the dividing lino between the pars anterior and the pars posterior of the lateral choroid plexus, and the taenia fornicis in its restricted sense is relatively of very short length.
i. Sulcus limitans. The sulcus limitans is lost in a deep recess between the corpus striatum and hypothalamus. This recess has disappeared by fusion of its walls in the 32 mm. embryo. If the sections of the 19 mm. embryo be followed, the beginning of this process can be readily seen (fig. 2).
j. Corpus striatum. There is nothing extraordinary about the corpus striatum in either embryo. The approximation of the thalamus and corpus striatum in the foramen interventriculare (fig. 22) is of interest when one remembers Goldstein's work. Of course, the entire connection between the thalamus and
108 PERCIVAL BAILEY
corpus striatum is not formed by fusion, and there is as yet no fusion here. The intermediate root of the corpus striatum probably extends into the medial hemisphere wall, but the external morphology does not suggest it.
2. Diencephalon
a. Tela chorioidea diencephali. It is to be noted that the tela chorioidea diencephali shows no indications of folding except at its anterior extremity. In the 32 mm. embryo, a pouch arises at the anterior end and extends forward over the velum transversum and the paraphysal arch (fig. 25, a.p.). Streeter, in Keibel and Mall's textbook, as was mentioned in the history, makes the statement that Orally this choroid roof [of the third ventricle] is continued into the telencephalon where it forms a pointed pouch overlapping the lamina terminalis and the contained commissures. . . . The anterior choroidal pouch has been homologized with the paraphysis of the lower vertebrates." There is not up to this stage any pointed pouch in the telencephalic roof. The pouch noted above lies in the roof of the diencephalon, just back of the velum transversum and hence cannot be the paraphysis. The true paraphysal arch has been pointed out above. In comparing Francotte's figures with this region in embryos of approximately the same age in the Chicago collection, I feel sure that it was this anterior pouch of the tela chorioidea diencephali which he described as the paraphysis.
I have not followed the thalamic lip fully in later stages, but in an embryo of 60.4 mm. greatest length, H 44 of the Chicago collection, the thalamic lip is fused with the lateral thalamic wall toward the anterior end.
b. Epiphysis. The epiphysis, in the 19 mm. embryo especially, shows marked indications of a differentiation into epiphyseal stalk and epiphyseal vesicle. Such a condition is very characteristic of lower vertebrates, especiallj^ reptiles, but has never before been noticed in the human embryo. The epiphyses of the 28 mm. and 32 mm. embryos show similar indications.
DEVELOPMENT OF THE CHOROID PLEXUS 109
In the 32 nun. embryo and in the 28 mm. embryo, the tela chorioidea continues on to the epiphyseal evagination. A similar condition has been noted in the cat by Thompson ('09).
c. Habenula. The character of the ridge running from the anterior end of the habenula to the subthalamus in the 19 mm. embryo (fig. 18, h.s-t.r.) is not apparent. It is probably only a temporary fold mechanicall}' produced by inequalities in development of the thalamic wall. It has entirely disappeared in both older embryos.
The ridge extending from the posterior end of the habenular prominence in the 28 mm. embryo and the 32 mm. embryo (figs. 20 and 22, f.r.) indicates the position of the fasciculus retroflexus (Meynerti).
d. Thalamus. In the 19 mm. embryo, only the anterior, inferior part of the thalamic wall (fig. IS, th.t.) lying between the sulcus limitans and the habenulo-subthalamic ridge is thickened. In the 28 mm. embryo, the ridge has disappeared and the anterior portion of the thalamic wall which lay above the ridge is thickened (figs. 18 and 20, Ih. 2). This region probably contains the principal nuclei of the thalamus. The posterior extremity of the thalamus (figs. 18 and 20,) is still somewhat flattened and thin in the 28 mm. embryo, but becomes very thick in the 32 mm. embryo (fig. 22).
e. Sulcus limitans. There is no difticulty in following the sulcus limitans. In the 32 mm. embryo under the posterior pole of the thalamus, it is very deep.
/. Hijpolhalamus. The hypothalamus in all these embryos is of great antero-posterior extent. It will be noticed also that the infundibulum, especially in the 19 mm. embrj'o, lies a considerable distance back of the optic chiasm. It seems quite probable that, as Johnston ('09) has remarked, in His' model of the embryo CR of 13.6 mm., the recess marked infundibular is really postoptic, and the real infundibular recess lies back of it and is labelled tuber cinereum. Only in the 19 mm. embryo is the subthalamus clearly separated externally from the hypothalamus.
1 10 PERCIVAL HAILEY
SUMMAliV
1. The plexus chorioideus ventriculi lateralis is composed of two distinct portions, of which the anterior is developed from the roof plate in the angle between the paraphysal arch and the medial wall of the hemisphere, and the posterior from that part of the medial wall of the hemisphere just anterior to the di-telencephalic groove and homologous to the anterior hmb of the velum transversum.
2. The taenia fornicis of adult human anatomy, except for its extreme anterior end, is not homologous with the taenia fornicis of Anura.
3. The position of the recessus neuroporicus could not with certainty be ascertained, and identification of the lamina supraneuroporica was therefore uncertain.
4. The tela chorioidea telencephali medii is present in the embryos of 28 and 32 mm.
5. The paraphysal arch can be followed to the embryo of 32 mm., as an arch of the roof plate of the telencephalon. It lies just anterior to the velum transversum and from its sides arise the lateral choroid plexuses. The anterior pouch of the choroid plexus of the third ventricle lies in the diencephalon and is not, therefore, homologous to the paraphysis of the lower vertebrates. No indication of the development of a glandular structure was found.
6. The velum transversum can be traced up to the 32 mm. embryo, joining the anterior extremities of attachment of the tela chorioidea diencephali to the lateral thalamic wall, and its groove is continuous laterally with the angle between the taenia chorioidea and the lateral thalamic wall, the di-telencephahc groove.
7. The tela chorioidea diencephaU is not folded nor vascularized except for its extreme anterior end in the embryo of 32 mm. It is broadened by the formation of a thalamic lip, very much resembling the rhomboidal lip of the rhombencephalon.
8. The epiphyses of all three embryos show indications of the presence of the homologue of the pineal vesicle of the lower
DEVELOPMENT OF THE CHOROID PLEXUS 111
vertebrates. In the embryos of 28 and 32 mm., the tela chorioidea diencephaU is continued on to the epiphyseal outgrowth.
9. The position of the fasciculus retroflexus (Meynerti) is indicated in the embryos of 28 and 32 mm. by a jironounced ridge.
10. There is evidence that the connection of the corpus striatum and thalamus is thickened by fusion of the medial root of the corpus striatum with the anterior extremity of the thalamus in the foramen interventriculare.
11. The sulcus Hmitans is lost in a very deep recess in the embryo of 19 mm. between the corpus striatum and hypothalamus, and this recess disappears in later stages by fusion of its walls.
12. The length of the liyj^ollialaniu.s in embrycjs from 19 to 32 mm. is relatively very great. In the embryo of 19 mm. the subthalamus is separated externally from the hypothalamus.
13. The infundibular outgrowtli is still some distance back of the optic chiasm at 19 mm. and becomes sjiifted nearer only in much later stages.
mtkkah UK ('iTi:i)
Dkxteu, 1'\ 1'JU2 Tin- (Icvcldpinciil of tlic parjipliysis in tlic ooiiimnn fowl.
Am. Jour. Anivt., vol. 2, no. 1, pp. 13-24. Faivre 1S54 Lcs plexus choroitlcs. Revuo Modicalc do I'ari.s. Hritisli ami
Foreign Mod. Chir. Review. FiNDLAY, .1. W. 1809 Tlie ohoroid i)lexu.ses of the lateral ventricles <tf tlie l)rain.
Brain, vol. 22, pp. 161-202. Francotte, p. 1S94 Xotc sur I'oeil parietal, I'epiphyse, la |)araphyse et les
plexus choroidcs du troisienie ventricule. Bull, de I'aead. royale,
des sci. d. Bclg., 3 Scric, T. 27, N. 1, pp. 84-113. Gaupp, E. 1898 Zirbel, I'arietalorgan und I'arapliysis. Krgeh. d. .\nat. u.
Entwick., Bd. 7, pp. 208-285. Goldstein, K. 1903 Beitnige zur Entw-gesch. d. Mensciil. (.Jeiiirns. Arcii.
f. Anat. u. Phy.s., Anat. Abt. Haeckel, E. 1860 Zur nonnalon und pathologischen Anatomie des Plexus
Choroideus. Schmidt'.^ Jahrbiioher. Virehow's Archiv, Bd. 16,
1859, pp. 253-289. Hkrrick, C. J. 1910 Morphology of the forebrain in Amphibia and Reptilia.
Jour. Comp. Neur., vol. 20, pp. 413-547. Heuseu, C. H. 1913 The development of the cerebral ventricles in the pig.
Am. Jour. Anat., vol. 15, pp. 215-239.
112 PERCIVAL BAILEY
His, W. 18S0 Die Forinentwickelung dcs menschlichcn Vorderhirns vom Ende
des ersten bis zum Bcginne des drittcn Monates. Abhdl. d. konigl.
siich. Akad. d. Wiss., math. phys. CI., Bd. 15.
1893 tlber das frontale Ende des Gehirnrohres. .Arch. f. Anat. u.
Entw. pp. 157-171.
1904 Die Entwicklung des menschlichen Gehirns. Leipzig. HocHSTETTEU, F. 1913 Uber die Entwickelung der Plexus Chorioidei der
Seitenkammern des menschlichen Gehirns. Anat. Anz., Bd. 45, pp.
225-238. Johnston, J. B. 1909 On the morphology of the forebrain vesicle in vertebrates.
Jour. Comp. Neur., vol. 19, pp. 457-539.
1910 The evolution of the cerebral cortex. Anat. Rec, vol. 4,
no. 4.
1913 The morphology of the septum, hippocampus, and pallial
commissures in reptiles and mammals. Jour. Comp. Neur., vol. 23,
pp. 371-478. Lachi, p. 1888 La tela corioidea superiore e i ventricoli cerebral! dell'uomo.
Atti della Soc. Tosc. d. Sc. nat., vol. 9, Fasc. 1. LuscHKA, H. VON 1885 Die Adergeflechte des menschlichen Gehirns. Bd. 6,
174 pp., 4 pi., 4°. Berlin, G. Reimer. Schmidt's Jahrblicher, G. 88,
p. 376. (Review.) Meek, W. J. 1907 A study of the choroid plexus. Jour. Comp. Neur., vol. 17,
pp. 286-306. MiNOT, C. S. 1901 On the morphology of the pineal region, based on its development in Acanthias. Am. Jour. Anat., vol. 1. Selenka, E. 1891 Das Stirnorgan derWirbelthiere. Biolog.Centralbl., Bd. 10,
pp. 323-326. Smith, G. Elliot 1903 On the morphology of the cerebral commissures.
Trans, of the Linnaen Society, London, vol. 8, part 12, July. Sterzi, G. 1907 II sistema nervoso centrale dei vertebrate. Vol. 1, Ciclostomi,
Padua. Streeter, G. L. 1912 The development of the central nervous .system. Kei bel and Mall's Human Embryology. Tandler, J. and Kantor, H. 1907 Beitriige zur Entwickelung des Vertebra tengehirnes. I. Die EntwickhmgsgeschichtedesGekohirnes. Anatom.
Hefte, Bd. 33, Heft 101. Thompson, P. 1909 Description of the brain of a foetal cat, 20 mm. in length.
Jour. Anat. Physiol., vol. 43, pp. 134-145. Tilney, Frederick 1915 The morphology of the diencephalic floor. Jour.
Comp. Neur., vol. 25, no. 3, pp. 213-282. Warren, John 1905 The development of the paraphysis and pineal region in
Necturus maculatus. Am. Jour. Anat., vol. 5, pp. 1-29.
DEVELOPMENT OF THE CHOHOll) PLEXUS
113
p«,
"lySOTlO
r pre
,^
"H
CO.
Fiji. 1^ Median view of a miHld ol ilir turcl.rMin tr..iii the I'.t inin. cniltryn,
H 173. X '221. Tho dotted lino follf)\vs the .sulrus liinitans. The limiting
Mienihrane i>ouiuling the ejiiphyseal vesicle, e.w. ventrally in this figure should
Ix' reiiresented as iiu'onij)Iete: cf. figure 10.
In this anil some of the following figures the i)lanes of the cross sections in the text-figures are indicated. The arrows at the right and left ends of the models are marked with the slide and section luuiibers of the first and last sections represented in tiie models. The reference letters for this and the following figures are tOuiul on jiage SS.
>.\1I'.\UAT1VE NEl-ROI.OGY, VOL. 26, NO.
114
l'Kl{(I\',\L HAILin
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Fig. 19 Lateral view of a model of the forebrain from the 19 mm. embryo, H 173. X 22§. The lateral wall of hemisphere has been cut away, exposing the lateral choroid plexus. Median surface of this model is shown in figure 18.
Fig. 20. Median view of a model of a portion of the forebrain from the 28 mm. embryo, H91. X 18.
Fig. 21 Lateral view of a model of a portion of the forebrain from the 28 mm. embryo, H 91. X I'S. The lateral hemisphere wall and corpus striatum have been cut away, exposing the lateral choroid plexus. Median surface shown in figure 20.
DEVELOPMENT OF THE CHOROID PLEXUS
115
20
21
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Fig. 22 Median view of a model of the forebrain from the 32 mm. embryo, H41. X 12^. The dotted line follows the sulcus limitans.
Fig. 23 View of the anterior end of the model of the forebrain from the 19mm. embryo, H 173, shown in figure 18. X 22|. View taken slightly from the medial side and above. A portion of the median hemisphere wall and lamina terminalis has been removed exposing the corpus striatum.
Fig. 24 View of the posterior extremity of the corpus striatum, in the model of the forebrain of the 32 mm. embryo, H 41, shown in figure 22. X 12^. View taken from above, behind and lateralward.
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Fig 25 Median view of a model of the region around the foramen mterventriculare from the 32 mm. embryo, H 41. X 50. (The entire forebram of this
embryo is shown in figure 22.) The model was made at a magnification of 100
diameters. Anterior end to the left.
DEVELOPMENT OF THE CHOROID PLEXUS 119
th.,p.5.
vl.pa
Fig. 26 Lateral view of a model tif the region around the foramen interventriculare from the 32 mm. embryo, H 41. X 50. Median view of the same model is shown in figure 2o. For help in orientation, the approximate position of the roof i^hitc, which lies on the opposite side of the hemisphere wall, has been projected through as a dotted line. The location of the taenia thalami is indicated by a row of dots and dashes. An arrow lies in the di-telencephalie groove and continues across in the groove of the velum transversum. The position of its head may be seen by referenceto figure 25. .Vnterior end of model totheright.
Fig. 27 Photograph of a section through the diencephalon of the 19 mm. embryo, H 173. X 16f. Slide 21, Sect. 11. The break in the hemisphere wall to
the left is an injury to this individual section. Compare figure 1, drawn from the
same section.
Fig. 28 Photograph of a section through the lateral choroid plexus of the 28 mm. embryo, H 91. X 20. Sect. 355. Section taken midway between figures 7 and 11, as shown in figure 20.
Fig. 29 Photograph of the 19 mm. embryo, H 173. X 2. Plane of section indicated.
Fig. 30 Photograph of the 28 mm. embryo, H 91. X H- Plane of section indicated.
Fig. 31 Photograph of the 32 mm, embryo, H 41. X U- Plane of section indicated.
120
CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORT OP THE BTaSEUM OF COMPARATIVE HARVARD COLLEGE, NO. 272
THE MOVEMENTS IN THE VISUAL CELLS AND
RETINAL PIGMENT OF THE LOWER
VERTEBRATES
LESLIE B. AREY Northwestern University Medical School
ONE TEXT FIGURE AND FIVE PLATES
CONTENTS
L Introduction and historical review 121
2. Material and technique 124
3. Experimental part 126
A. Determination of adaption times 126
(a.) Retinal i)i{iment 127
(b) Visual cells 129
B. Effect of temperature (normal animals) 133
(a) Retinal pigment 133
(b) Visual cells , 151
C. Experimentation upon excised eyes 163
(a) Effect of light and darkness 163
(b) Effect of temperature 168
D. Effect of anaesthetics. 171
(a) Retinal pigment 172
(b) Visual cells 175
E. Effect of oxygen 176
F. Interrelation of integumentary jihotoreceptors and retinal elements. 179
4. Discussion ■ 181
5. Summary . 184
Bibliography 1S8
1. INTRODUCTION AND HISTORICAL REVIEW
The positional changes that occur in t'he vertebrate retina chiefly through the action of hght have attracted the attention of many workers in continental Europe although, strangely enough, neither English nor American investigators have hitherto concerned themselves with this particular field of endeavor. The results acquired from these researches have proved of such interest and significance as to equal, and perhaps even to sur 121
THE JOURNAL OP COMPAR.VTIVE NEUROLOGY, VOL. 26, NO. 2 APRIL, 1916
122 LESLIE B. AIIEY
pass, those obtMiued from tho two other branches of retinal physiology, namely, the study of the visual purple and of the action currents in the optic nerve.
In order that the reader may understand the present status of this subject, it is desirable to summarize briefly the general conclusions that have been established relative to the efficiency of light in producing movements in the retinal elements. For a more extensive review of this literature reference may be made to a separate paper by the writer (Arey, '15) or to the excellent compilation of the German author, Garten ('07).
Although a variability in the position of the retinal pigment had been noted by early workers (H. Miiller, '56; Morano, '72) the cause remained unsuspected until Boll ('78) and Kiihne ('77) independently discovered that in the light the retinal pigment of the frog extends nearly to the external limiting membrane whereas in darkness it retreats, thereby forming a compact layer next to the choroid^ (figs. 1 and 4). Later observations have corroborated Kiihne 's original view that pigment migration is not due to the extension and retraction of cell processes but to the movement of pigment granules in the protoplasm of relatively fixed cells.
The most extensive pigment migration occurs in fishes (Stort '86) and in anuran amphibians (Boll, '78, and Kiihne '77, on the frog; Arcoleo, '90, on the toad), whereas the positional changes in the pigment of urodeles are relatively limited (Angelucci, '78). Well defined movements of the retinal pigment are also found among birds, including not only those that are diurnal, but also some that are nocturnal in habit. Among reptiles (Angelucci, '90) and mammals (Angelucci, '78) limited pigment changes have probably been detected, notwithstanding the contradictory evidence presented by various workers.
' Czerny ('67) found that when sunlight was concentrated by a lens on the retinas of various animals, the pigment became more highly expanded in the affected regions than in other portions which had been exposed to light but not so treated. In his experiments no comparisons were made with dark-adapted retinas. These somewhat pathological tests were not well substantiated by the later work of Deutschmann ('82).
MOVEMENTS IX THE VISUAL CELLS 123
Movements of the inner member of the vertebrate cone cell were first observed by Stort in 1884, although the earliest announcement of this discovery was pubhshed by Englemann ('85) in whose laboratory Stort worked. To the contractile portion of the cone's inner member Englemann applied the significant term 'myoid' (figs. 30, 31; my. con.). The contractility of the myoid is extraordinary, since in some fishes light produces a shortening of this part to 10 per cent of the length which it assumes in darkness (figs. 25, 27). If effective at all, fight always causes a shortening and darkness an elongation of the cone cell.
Stort ('87) extended his first discovery on the frog by experimentation upon representatives of the various other vertebrate classes, thereby showing that in fishes and birds extensive movements of the cones likewise occur. In the salamander, as a type of urodele amphibian, responses of the cones to fight have both been asserted (Angelucci, '90), and denied (Garten, '07). Among a few reptiles (Englemann, '85) and mammals (Garten, '07) slight changes have apparently been detected.
The visible response of the rod's inner member presumably is not identical throughout all the vertebrate classes. Angelucci ('84) was the first to observe a shortening of the frog's rod after exposure to light and later, in 1890, he applied the term 'myoid' to the contractile portion of the rod's inner member (figs. 30, 31 ; my. bac.) in a sense similar to that in which Englemann has previously used it for the corresponding jiortion of the cone; Arcoleo ('90) likewise reported that the rod myoid of the toad shortens in the fight. Recently, however, Lederer ('08) has asserted that the photomechanical change in the rod myoid of the frog is not a shortening but an elongation. In all fishes that possess cones, the rod myoid lengthens in the fight and shortens in darkness, as Stort ('87) first believed. The response in the rods of day-birds (Stort, '86) is similar to that in fishes, although in night-birds (Garten, '07) movements of these cells may be entirely absent. No experimentation has been performed upon the rod cells of mammals or of reptiles ; in the latter group, however, the retina usually lacks these elements.
124 LESLIE B. AREY
2. MATERIAL AND TECHNIQUE
Tlio work of Chiarini ('04a, '06), in which he systematically investigated the effect of light on the retinas of representative vertebrates, led him to the conclusion that the maximal changes in the position of the pigment and of the cone cells occur in the lower vertebrates and particularly in the fishes, whereas in the highest vertebrates these changes are extremely hmited or may even be imperceptible. For experimentation involving variable temperature poikilothermous animals are necessary, and no vertebrates are more easily available for this purpose than are fishes and amphibians. When to availability is added the advantages which they offer by the possession of exceptionally well developed visual and pigment cells, which are capable of undergoing the greatest dimensional changes found in any of the vertebrates, it will be seen that these animals are particularly favorable for such physiological studies as were conducted in the present investigation.
The following fishes were used: the common horned pout (Ameiurus nebulosus Lesueur); the common killifish (Fundulus heteroclitus Linn.) ; the shiner (Abramis crysoleucas Mitchill) ; and the goldfish (Carassius auratus Linn.). Of these, all but Fundulus are fresh water forms, while Fundulus is found in brackish water, and especially at the mouths of fresh water streams. Abramis and Carassius belong to the same family, the others to different families. Goldfish were obtained for the most part from dealers although a number of feral animals were used. Many of the streams and ponds about Cambridge are stocked with feral goldfish, liberated by accident or design. The majority of such animals have lost their gold coloration and have returned almost completel}^ to the original olivaceous type.
The amphibians used were as follows: the leopard frog (Rana pipiens Schreber) ; the green frog (Rana clamitans Latreille) ; the bull frog (Rana catesbiana Shaw) ; and the mud puppy (Necturus maculatus Raf.). Of the three species of frogs, the leopard frog was used almost exclusively for experimentation on adult animals; the larvae only of the bull frog were employed.
MOVEMENTS IN THE VISUAL CELLS 125
The technique of preparing retinas for microscopical examina tion was very simple. Eyes were removed from their orbits in two ways. In Ameiurus, where the eyes are prominent and the skin is soft, excision was performed directly. The eyes of amphibians and of fishes other than Ameiurus, particularly in experiments conducted in the dark where rapidity of operation was desirable, were not excised directly but according to the following procedure. With heavy scissors the cranium was bisected in a median sagittal plane; following this, a transverse cut just posterior to the orbit freed the two halves of the cranium, with the contained eyes, from the rest of the body. In either case the operation was performed in a few seconds and the eye, without being handled, was allowed to drop into the fixing fluid. Both Perenj'i's and Kloinenbcrg's fluids gave good fixation (Howard, '08; Palmer, '12), but of the two, Perenyi was preferred. The toughness of the sclera and the consequent slowness in the penetration of fluids demands generous allowances of time during the various steps preparatory to imbedding in paraffine. Dehydration and clearing in xylol should, however, progress as rapidly as possil)le since otherwise the sclera liecomes extremely hard.
Two methods were used in removing the lens, one of which, although longer, gave much more satisfactory results. The first somewhat tedious procedure consisted in paring away the front face of the eyeball with a razor after the eye had been previously imbedded in parafline. After removing the face of the eyeball slightl}^ l)eyond the ora serrata, the lens was pried from its paraffine matrix with a dissecting needle; following such manipulation reimbedding was of course necessary. The second and simpler method was to remove the face of the eyeball with fine curved scissors after the eye had been hardened in absolute alcohol; if, however, the eye was not sufl^ciently hardened or the
^ A method of simple decapitation used by Pcrgens has lieen criticized by various workers who contend that slight changes occur in the position of the retinal elements after the head is immersed in the killing fluid. In a series of controlled experiments, however, I could detect no post-mortem disturbances when the head was both bisected and cut from the trunk.
126 LESLIE B. AREY
greatest care was not exorcised, the retina proper easily separated from the pigmented epithelium. On the whole, the first method was ])ref erred to the second because of the wrinkling of the retina that usually accompanied the use of the latter.
Sections were cut 7 ^u to 10 /x thick, and except in a few special cases only those passing through the region of the optic nerve were retained for examination. Preparations were stained with Ehrlich-Biondi's triple stain or were double stained in Heidenhain's iron hematoxyhn and a plasma counterstain. EhrlichBiondi in some instances gave excellent differentiation of all elements, while at other times it would show the capriciousness in producing satisfactory results for which it is notorious; iron hematoxylin gave uniformly good preparations.
It often became necessary to bleach the pigment in order to study the visual cells, which would otherwise be masked by the partially or completely extended processes. The method employed was essentially that of Mayer ('81), in which nascent oxygen^ is the effective agent.
The aim of the present investigation has been to determine the influence of light, temperature, anaesthetics and oxygen on the movements of the rods, cones, and retinal pigment in the normal and excised eyes of fishes and of amphibians.
To Prof. G. H. Parker, under whose direction this research has been conducted, I wish to acknowledge my indebtedness for much inspiration and valuable suggestion. Advantage is taken of this opportunity to express appreciation for the facilities of the Zoological Laboratory placed at my disposal by the Director, Prof. E. L. Mark, and for many courtesies extended by him during my residence at Cambridge.
3. EXPERIMENTAL PART A. DETERMINATION OF ADAPTION TIMES
Before extensive experimentation can be undertaken on the retinal elements, it is necessary to determine the various lengths
3 When potassium chlorate and hydrochloric acid interact, it is commonly said that nascent chlorine is the agent causing bleaching. As a matter of fact the reaction liberates free oxygen.
MOVEMENTS IN THE VISUAL CELLS 127
of the time which they require in assuming the positions characteristic of Hght and of darkness. In most cases it is not easy to state definitely when adaption is completed, for the response becomes less vigorous as it nears the end and consequently the factor of personal equation is unavoidable.
Light intensity and temperature "(Dittler, '07) represent variables which undoubtedly play a part in the determination of adaption time. No attempt was made to discover the exact role of either of these factors, although the general experimental conditions were kept approximately uniform during successive trials.
The effect upon adaption time of a long or short prehminary subjection to light or darkness, has never been taken into account, although such influences were asserted by Gaglio ('94).
a. Retinal pigment
Pergens ('96) found that after 2 minutes' illumination the retinal pigment of I.euciscus began to expand. After 1 minute of darkness a noticeable contraction occurred, which was completed in 20 minutes. Chiarini ('04b), working on the same fish, came to somewhat different conclusions. He observed a sensible pigment expansion after direct exposure to sunlight for 1 minute, although complete light adaption necessitated a period of 1 hour. The reverse process of dark adaption was not initiated until the animal had been subjected to darkness for from 4 to 5 minutes, and a minimum of 1 hour was required to complete the contraction.
When the retinal pigment of fishes has undergone a maximum expansion, it accumulates distally' near the external limiting membrane, whereas the proximal portions of the cells are to a greater or less extent devoid of pigment (figs. 9, 10). Such a
^ The term 'proxiinal' as iisod in this jiaper will refer to movements toward the nuclei of a given cell, either pigment or visual. In like manner 'distal' will have reference to movements away from the nucleus. Since the distal movement in the pigment cells is the reverse of that in the rods and cones, an arbitrary nomenclature with reference to the eye-ball becomes confusing, while the terminology here suggested lends itself readily to descriptions of the moving parts.
128 LESLIE B. AREY
condition of distal acciunulation requires additional time after the 'front rank' of pigment granules has arrived at the position of maximal extension. In the determinations made by me, light adaption was not considered to be completed until the pigment was thus maximally expanded.
The results obtained on the retinal pigment of fishes were as follows:
Aineiunis
Diffuse daylight
30 minutes, pigment incompletely extended
40 minutes, pigment fully extended, but homogeneously distributed 1 hour, maximal expansion Darkness
45 minutes, pigment about half contracted 1 hour, maximal contraction
The movements of Abramis were more rapid, notwithstanding an apparently heavier pigmentation of the retina.
Abramis
Diffuse daylight
15 minutes, lightly pigmented processes three quarters extended. Most of
the pigment at the bases of the cells
30 minutes, processes fully extended. Distal pigment accumulation begun
45 minutes, maximal expansion Darkness
20 minutes, pigment about half contracted
30 minutes, maximal pigment contraction
The results of the dark adaption on Fundulus were less satisfactory, since the delimitation of the pigment in darkness is poorly defined.
Fundulus
Diffuse daylight
45 minutes, pigment fully extended; but little tendency exhibited towards
distal accumulation 1 hour, maximal expansion Darkness
45 minutes to 1 hour, maximal contraction
As regards the adaption time in the frog, Stort ('87) beheved that in 1 hour of bright, diffuse daylight or in 4 hours of dark
MOVEMENTS IN THE VISUAL CELLS 129
ness, maximal light- and dark-adaption of the pigment respectively was produced. Kiihne (79) stated that complete darkadaption occurred in 1 to 2 hours, this period corresponding to the time necessary for the regeneration of the visual purple. Chiarini f'04b) maintained that following an exposure of half an hour to direct sunlight one and one-half hours were needed to complete the adaption in darkness. Although no direct experimentation was made on the frog, the general experience gained from working with this animal leads me to suspect that 1 hour for light-adaption and 1 to 2 hours for dark-adaption are approximately the proper amounts.
h. Visual cells
Relative to the adaption times of the cone cells, Pergens ('96) stated that in Leuciscus the first visible shortening occurred after an exposure to light of 1 minute. According to his illustrations, the cones were very much shortened after 2 minutes while those that had been subjected to light for 5 minutes were practically in the position characteristic for light. When light adapted animals were introduced into the dark, a lengthening of the cones was evident after 1 minute and in 5 minutes the elongation was nearh^ complete, although it did not become maximal for 20 minutes. Pergens' method of allowing decapitated heads to remain in the light during fixation has been criticized by Garten ('07), and the later work of Pergens ('99) has likewise been questioned by Herzog ('05). These workers assert that it is entirely possible that the changes initiated by the action of light continue until the actual penetration of the fluid into the eye fixes the retina. Certain experiments of \\'eiss, instigated by Garten, indicate that the action of light can influence the position of cones and retinal pigment after decapitated heads have been introduced into the fixitive. These results are in opposition to the statement of Ghiarini ('04b) that light cannot be effective during fixation.
In an attempt to avoid this source of error, at the completion of an experiment the hght-adapted eyes were fixed in exceedingly
130 LESLIE B. AREY
dim li«2;lit. It is probable that this precaution was sufficient, for. as will be shown later, the movements of the cones of Abramis, the only fish worked upon, are not rapid and moreover no changes occur even when the excised eyes, immersed in water, are subjected to fight or to darkness.
Temperature is an important factor that must be considered in the adaption of cones. In anticipation of certain results that will be found in another part of this paper, it may be said that the cones of fishes are maximally extended at about 25°C. in the dark (fig. 27), and in the case of Abramis at least, they are also maximallj' shortened at 5°C. in the dark (fig. 25) . Moreover, as temperature does not affect the length of the cones when they are under the influence of light, the animals may be kept at a temperature of 25°C. during an entire experiment and the resulting movement of the cones will then be solely traceable to conditions of fight or darkness.
The results on the cones of Abramis may be summarized as follows :
Diffuse daylight
15 minutes, cones much shortened — perhaps two thirds
23 minutes, approximately the same condition as at 15 minutes
30 minutes, shortening not quite complete in most animals
45 minutes, maximal light adaption Darkness
13 minutes, cones somewhat extended — one third {'!)
20 minutes, extension practically complete
30 minutes, maximal dark adaption
The adaption times of the cones of Abramis are longer than those given by Stort for Leuciscus. This, in part, may be due to the wider range between the positions of maximal light- and dark-adaption which was produced by the aid of elevated temperature.
Englemann ('85), working on the frog, was the first to discover that the movements of the cones were not accomplished instantaneously, but required definite periods of time. He also observed that elongation in the dark was a longer process than shortening in the light. My results on Abramis do not entirely support his latter view. At first the cones of this animal do
MOVEMENTS IN THE VISUAL CELLS 131
respond more actively when stimulated by light, but a longer time is required to complete the process of light-adaption than the reverse changes in the dark.
No experimentation seems to have been performed upon any animal to determine the adaption time of the rod cells. Ameiurus was selected for these tests, for the rods differ greatly from those which are characteristic of fishes in general. Instead of the slender elements 1.5 n to 2.0 // in diameter, which, for example, are found in Abramis (fig. 25), the rods in Ameiurus (fig. 31) are robust and resemble more closely those of the frog (fig. 35). The barrel-shaped ellipsoid measures about 4 m in either dimension, and the width of the outer membrane is the same, \\ith. this can be compared the width of the outer member of the rods in the frog, which in my preparations of R. pipiens measured, for the most part, 5 fi, although Howard ('08) states the width as 6 fx, and H. Miiller ('5(3) as from G /i to 7 ^i.'• The species was not mentioned by either of these WTiters.
Unlike the cones, the rods of Ameiurus in the dark form a more or less even row close to the external limiting membrane (fig. 31), while in the light the myoid elongates carrying the ellipsoid and outer member far up' into the pigment layer (fig. 30). The extent of these positional changes may be judged from measurements of rods in darkness and in light which give extreme values of 70 fx and 7 /x respectively for the length of the myoid. I know of no fish in which rods of this size have been described, although Garten ('07) makes particular mention of the pike as possessing 'grosse Stabchen.' It is evident that the large size and the extensive positional changes which the rods of Ameiurus undergo make them especially favorable for physiological experimentation.
The effect of temperature upon the length of the rods is comparatively slight, hence the following determinations on the rods of Ameiurus were conducted at room temperature.
- Perhaps tlu- fact that my nioasuremcnts were made on dark-adapted rods
accounts for this discrepancy, for in rod cells the outer member is said to become longer and slenderer in darkness than in light.
132 LESLIE B. AREY
Diffused tlaylight
30 minutes, rods two-thirds extended
45 minutes, maximal light adaption. (Cones also light-adapted) Darkness
15 to 20 minutes, rods, in most cases, almost completely shortened
30 minutes, maximal dark adaption. (Cones still in position of light adaption)
The quicker response of the rods in darkness than in Hght is noteworthy. The rod shortens in the dark, the cone in the hght; since in both cases the process of shortening is more vigorous than the lengthening, it would appear that the contractility of either type of cell is the responsible factor and the relative efficiency of light and darkness is not primarily involved. In the last analysis, however, the situation may not be reducible to such simple terms. If these responses are merely the expression of the action of hght and darkness on the protoplasmic myoids, why should the direction of movement of the two elements be opposed? A discussion of this phase of the problem will be found in another place.
I attempted no experimentation upon the cones of the frog. Angelucci ('90) stated that after an exposure to candle-light for 5 minutes, the cones were strongly retracted, although other experiments of his do not seem to support this conclusion. Herzog ('05) found that at medium light intensity complete lightadaption occurred in 2^ minutes.
The most surprising discovery in this series of determinations, taken as a whole, was the length of time required to complete the adaption of the rod and cone cells in comparison with the retinal pigment. From the results of earlier workers, I had expected the positional changes of these cells to be completed in a few minutes, hence the actual values obtained were wholly unlooked for, and were only accepted after many repetitions of individual experiments.
MOVEMENTS IN THE VISUAL CELLS 133
B. EFFECT OF TEMPERATURE (NORMAL ANIMALS)
a. Retinal -pigment
No investigations have hitherto been made to determine the effect of temperature on the retinal pigment of fishes, whereas several workers have used the frog for experimentation of this kind. The problem considered here was to determine the response of the retinal pigment of normal fishes and amphibians to various temperatures, both in light and in darkness.
/. Fishes. Experiments in the light were performed in tlie following way. Light-adapted" fish were placed in large battery jars close to north windows where they received strong diffuse daylight; sheets of white paper were always placed under the jars to aid reflection.^ The highest temperature to which it is safe to subject fishes is about 28°C., although by gradual elevation a somewhat higher temperature can be withstood (Loeb and Wasteneys, '12). A low temperature that did not vary beyond the limits of 3° and 5°C. was produced l)y introducing small pieces of ice directly into jars with the fish. At this temperature fish are for the most part inactive, the respiration rate decreases and they remain quietly at the bottom of the jars.
At the end of an experiment, which was never less than threes hours long, the eyes were excised and immersed in fixing fluid in the light. ^
During the earliest trials retinas from the same fisli were compared, eyes being subjected to the extreme temperatures in
- The terms 'light adaption' and 'dark adaption' as used throughout tliis paper
imply that the animals had been previously subjected for a inininmm of 4 hours either to bright diffuse daylight or to total darkness.
' The possibility of a dark background influencing the (listril)ution of retinal pigment was considered. Such a visual control, if present, would correspond to the known role of the eye, as determined bj' Pouchet ('76) and others, in animals which adapt their bod)- color to the immediate environment. A series of careful comparisons, however, failed to show any recognizable differences between the pigment distribution in the retinas of fishes that had been kept over dark or light backgrounds.
- In all work which involved the use of temperature, the fixing fluid was kept
at the same temperature as that at which the experiment had been performed. Such treatment eliminated a possible source of slight error.
134 LESLIE 13. AREY
successive experiments. At first this seemed to be the correct procedure, but rigorous controls showed that such precautions were unnecessary. When interpreting the results of experiments conducted in the hght it is not the absolute amount, but rather the relative distribution of pigment that serves as a basis for decisions. In experimentation in the dark, absolute differences in the degrees of pigmentation could give rise to errors in judgment, for the pigment, gathered into compact masses in the cell 'cups' might mask or apparently reverse the effect of temperature. Fortunately, how^ever, the retinas, as judged from the expanded light condition seem, on the whole, to be very equally pigmented and the width of the contracted pigment zone, therefore, gives a fair index of the effect of temperature in the dark. When working in the light especially, it was found to be very desirable to have the experiments at contrasted temperatures conducted simultaneously in order that advantage might be taken of identical light conditions, for as will be shown, light intensity is an important factor in obtaining the maximum expansion of pigment.
Experimentation in the dark was conducted as follows. A tireless cooker, lined with black paper, was used as a dark chamber, on account of the minimal loss of heat incurred by it during the course of an experiment. If such an apparatus be previously brought to the temperature of the introduced jar of water, an experiment can be continued for several. hours without further attention. In all determinations mentioned in this paper which were conducted in the dark, precautions were taken against the possible influence of light during the few seconds necessary for excision and transference of eyes to the fixative. A long series of careful comparisons showed identical results whether the operation was carried out in total darkness or in light of just sufficient strength to permit the operator to see the animal and his instruments. Indeed, the results obtained by operating in an ordinarily lighted room showed no recognizable differences in either pigment, rods, or cones from those secured by working in darkness.
A detailed description follows of the conditions found in each of the four fishes:
MOVEMENTS IN THE VISUAL CELLS 135
1. Ameiurus. At 25°C. in the light (fig. 2), the characteristic position of the expanded pigment is in a broad band about 95 m wide, which extends nearly to the external limiting membrane. The pigment granules are evenly distributed and show no tendency to aggregate distally.
At 15°C, the condition is very .similar to that just described. The pigment, on the whole, tends to be homogeneously distributed, although in many retinas at this temperature there is a slight distal accumulation.
The disposition of pigment at 5°C. is markedly dilTerent (fig. 1) for it migrates to an extreme dstal situation and forms a dense zone, approximately 30 ii wide, close to the external limiting membrane, although the pigment of fishes and amphil)ians, under normal conditions, never actually touches this membrane. Between this heavy pigment-mass and the bases of the cells lie scattered granules, but the intervening space, nevertheless, appears relatively devoid of pigment. This extreme condition is best produced on the brightest days, and it is impossible to obtain as complete a migration on cloudy days, regardless of the temperature. On the other hand, the uniform distribution characteristic of incomplete expansion at 2o°C\, is independent of the intensity of diffuse daylight. This would suggest that a high temperature is more efficient than light in the regulation of pigment distribution, and that cold, that is, the absence of heat, merel}- allowed light to act unrestrained. Light and high temperature, then, are antagonistic in their effects.
The results at 5°, 15*", and 25°C\ are, in a way, what might have been expected. A temperature of 15° to 25°C\ probably represents the greatest average warmth to which the animal is subjected in nature; this range from 15° to 25°C., then, represents the limits of what may be called a warm environment for the animal. In the same way from 0° to 10°C'. may be called a cold environment, and from 10° to 15°C. a neutral environment, neither particularly warm nor cold. Hence it is not surprising that the results at 15°C. are more similar to those at 25°C. than at 5°C. At 10°C. the distribution of pigment approximates rather more closelv that at 5°C\ A curve, therefore, obtained
136 LESLIE B. AREY
by pl()ttiiif>; toinperatures as abscissas and the quantitative amount of distal migration as ordinates would show a gradual slope from 0° to 10°C\, from 10° to 15°C. a rapid drop and from 15° to 25°C. a nearly horizontal but slightly sloping line.
The results of experiments performed in the dark, where the pigment is highly contracted, are usually not as clear cut as those just described. The reason for this is because it is impossible to see the qualitative distribution of the pigment granules and hence decisions regarding the effect of temperature must depend largely on measurements of the width of the narrow pigmented layer. Such a criterion, as has previously been pointed out, is open to the criticism that individual eyes may vary enough in the absolute amount of contained pigment to disconcert judgments concerning the effect of temperature. After having studied a great number of preparations, I do not believe that such an unequal pigmentation is in truth a factor that warrants serious consideration. However this may be, an obvious precaution consists in the prolonged repetition of each type of experiment. It may be said that in the course of my experimentation on the effect of temperature on normal fishes alone, over 200 retinas have been examined.
The evidence obtained from Ameiurus, was more conclusive than that from any of the other fishes, with the possible exception of Carassius. At 25°C. (fig. 4) the pigment forms a densely contracted layer, the mean width of which is about 25 /u- In contrast with this is the condition at 5°C. (fig. 3), where the cells have short pigmented processes, the total extent of which is approximately 38 ix. The differences apparent at these two extremes of temperature were so slight in comparison with the much greater variation in the light, that thorough experimentation at the intermediate temperature of 15°C. was not attempted, although it was sufficiently demonstrated that the results at this temperature do not vary to any great extent from those at the two extremes, and probably more closely approximate the highly contracted condition at 25°C.
The results of some of the temperature determinations were not conclusive. Reference has already been made to the fact that
MOVEMENTS IX THE VISUAL CELLS 137
on dull days maximal expansion in the light was hard to obtain. Among fishes in general, more doubtful cases occurred in experiments conducted in the dark than in the light, yet in all such cases the uncertainty merely lay in deciding between two nearly equal conditions, while in practicalh' no instance was there evidence of a definite reversal whereby a greater distal migration occurred at 25°C. than at 5°C.
2. Fundulus. The conclusions reached from the study of Fundulus, as well as from the other fishes, are similar to those given for Ameiurus, but each fish shows individual peculiarities in the disposition of the pigment and these will be briefly described.
In the light the pigment of Fundulus tends to migrate to a great extent forming a broad zone at the distal ends of the cells, much denser and more sharply defined than in Ameiurus. Between this zone, which has a width of 30 m, and the bases of the cells there is a clearer area, almost devoid of pigment at 5°C. (fig. 5), while at 25°C\ (fig. 0) this region contains a considerable amount of evenly distributed pigment granules. In the latter case, however, the pigment still forms a closely aggregated zone distally, although it is reduced to a width of 17 mAs in Ameiurus the condition at lo°(\ more closely resembles that at 25^C. than at 5°C.
A peculiarity in preparations of the light-adapted retina of Fundulus (at least with Perenyi's fixation) is that at the higher temperatures the pigment extends in columns from base to periphery of individual cells (fig. (i), while between such columns of adjacent cells are elongated areas free of pigment but taking the plasma stain. Examination under high magnification does not show the presence of excessive shrinkage, although a casual observation might suggest that this had occurred. AMiere the pigment is aggregated at the base and periphery of the cells, cell boundaries are not distinguishable and the pigment appears as homogeneous masses. The separate columns connecting these two continuous zones give the whole an appearance not imlike a ladder with rungs set very close together.
THE JOt'RNAL OF COMPAKATIVE NEUROLOGY, VOL. 26, NO. 2
138 LESLIE B. AREY
111 th(^ (lark the contraction is very incomplete and sometimes tends to give rise to a distal accmnulation of pigment which faintly resembles the distribution in the light, although such a condition is not constantly present as a characteristic of dark adaption. It is impossible to state the cause of this pigment massing. It may be due to a greater activity at the distal ends of the cells in producing a contraction, or what would bring about a similar end result, a contraction of pigment en masse.
Measurements were taken of preparations at the extreme temperatures in the dark, the degree of variation being greater than in any of the other fishes studied. Thirteen retinas at 5°C. (fig. 7) showed the pigment to be extended a mean distance of 20.4 divisions of the ocular micrometer, whereas fifteen retinas at 25°C. (fig. 8) had a corresponding value of 14.4.
3. Abramis. In the hght the condition in the eye of this fish is somewhat similar to that in Fundulus. The pigmentation is very heavy, forming a broad zone near the external limiting membrane; between this distal zone and the base of the pigment cells pigment granules are also present, the density of pigmentation depending on the temperature.
At 5°C. (fig. 9) the distal zone is wider than at 25°C. (fig. 10) in the ratio of 50 n to 38 /z, while the proximal area is much less heavily pigmented than at 25°C. Although each area is sharply defined, the one appears to grow at the expense of the other. It was not possible to get so complete a distal accumulation of the pigment as in Fundulus and, as will be presently shown, the extent of migration in Fundulus is still less complete than that in Carassius.
The dark phase is one of great contraction, and although some retinas show evident differences at the extreme temperatures, yet throughout the whole set judgment of the eye has to be supplemented by actual measurements. Such measurements show that the mean expansion at 5°C. (fig. 11) exceeds that at 25°C. (fig. 12), the values being 30 /x and 20 ^ respectively.
4. Carassius. The eye of the goldfish is more heavily pigmented than those of the three other fishes. At 5°C. in the
MOVEMENTS IN THE VISUAL CELLS 139
light (fig. 13) all the pigment is located distalh" forming a broad, dense zone. Between this band and the base of the cells is a narrow clear area in which scattered pigment granules are visible only with the aid of high magnifications — for practical purposes it may be said to be free of pigment.
The situation at 2o°C. in the Hght (fig. 14) is variable. In some cases it closely simulates that at 5°C'., although the clearer space is always relatively more heavily pigmented; in other instances, the pigment is uniformly distributed from the proximal to the distal extent of the cells.
The contraction that usually occurs in the dark was less pronounced in C'arassius than in any fish heretofore described; in fact in some eyes that had been subjected to a low temperature, the actual l)readth of the pigment layer nearly equalknl that of a light-adapted eye. The relative expansion at the extreme temperatures in the dark, however, leaves no doubt that at 5°C. (fig. 15) the contraction is less than at 25°C. (fig. 16). Measurements (in terms of the divisions of an ocular micrometer) taken from eight eyes at 25°('. and ten eyes at 5°C\ gave respective mean values of S.O and 12.1.
From observations on these four genera certain generalizations are suggested. The degree of pigmentation in the eye of Ameiurus (figs. 1, 2) is much less extensive than in the other fishes, as a comparison with Carassius (figs. 13, 14) shows in a striking manner. The three other fishes, however, offer better opportunities for comparison since their pigmentation is more nearly equal. At o°C. in the light the pigment of both Carassius and Fundulus (figs. 13, 5) migrates distally to such an extent that a proximal zone, devoid of pigment, is created. Moreover, the dark phase of the pigment, in both these animals (figs. 16, 8), is one b}^ no means extreme when compared with the highly compact layer in .Vbramis and Ameiurus (figs. 12, 4). In the last named fishes, on the contrary, it is impossible under the most favorable conditions of light and temperature to obtain the proximal clearer area entirely free from pigment; correlated with this absence of complete expansion is the high degree of contraction which is evoked in the dark.
140 LESLIE B. AREY
It (loos not seem probable that quantitative differences in the degree of pigmentation can be the cause of such relations, for if the relative amounts and the distribution of pigment in Fundulus and Abramis be compared at 5°C. in the Ught (figs. 5, 1) and at either temperature in the dark (figs. 7, 3) it must be admitted that quantitative differences do not adequately explain the conditions that exist.
A clearer insight is gained if these responses are viewed from the standpoint of cell organization. We can think of two general types of pigment cells in which the pigment distribution is correlated with the behavior outhned above. Thus, in one type, the pigment would tend to remain at the distal end; such a cell would show maximal expansion but relatively incomplete contraction. In a second type of cell, in which the pigment aggregates more proximally, maximal contraction but incomplete expansion would be accompHshed. Although other species of fishes should be studied before a final conclusion is reached, this set of relations may be general. The correlation can be stated, at least tentatively, as follows — the highest degrees of expansion and contraction in the retinal pigment of fishes are mutually exclusive in the same retina.
2. Frog {adult and larva) . As in many other lines of physiological work, the frog has been used to a large extent by investigators of retinal physiology.
Ewald und Kiihne ('78) performed the first experiment in which the position of the retinal pigment of the frog was shown to be dependent upon temperature. According to their account, after 2 hours' immersion in ice water in the dark, the pigment showed a distribution similar to that obtained at 17°C., which may be called a state of contraction.^ When, however, frogs were subjected to 30°C. for 2 hours, an expansion occurred in which lightly pigmented processes were said to extend even to
^ This, however, does not coincide with an earlier statement ('77, p. 250) of the same authors, "Vor allem ist die Temperatur von ausserordenlichem Einflusse .... Frosche, welche 1-2 Stunden in Eiswasser gehalten wurden, liefern schwarze Netzhiiute, indem das ganze Epithel mit ausschliipft, und nicht viel besser verhalten sich die Priiparate von solchen, die bei 5°-10°C. im Dunkeln verweilten."
MOVEMENTS IN THE VISUAL CELLS 141
the external limiting membrane. These workers were primarily interested in discovering mider what conditions of temperature an accumulation of pigment in the rod area could be avoided, for their investigation dealt with the visual purple. Since the frogs used for these determinations were treated with curare in order to produce pigment relaxation, and, moreover, since these animals were in an oedematous condition as a result of this poisoning, it is evident, as Herzog ('05) pointed out, that much weight can not be given to their conclusions alone.
To Gradenigro ('85) belongs the credit of having performed the first temperature experiment upon the retinal pigment of normal animals. He introduced a dark-adapted frog into a dry, darkened chamber and removed the whole to a dark room. A temperature of 30°C. was maintained until heat rigor set in, when, on examination, the pigment was found to be in a condition of maximal light expansion (fig. 19). Gradcnigro's results were confirmed by Angelucci ('90) and by Fujita ('11).
Herzog ('05), without knowledge of Gradenigro's contribution, undertook a detailed study of the relation between temperature and pigment distribution. His results not only corroborated those of Gradenigro, but also estabhshed the additional facts that at low temperatures (0°-14'C\) in the dark the distribution of pigment is identical with that at high temperatures (fig. 17), while only between 14° and 18°C. in the dark (fig. 18) is maximal pigment contraction obtained.
His experiments were performed in the following manner. Dark-adapted frogs (R. temporaria and H. esculenta?) were placed in a heating chamber at an initial temperature of 20°C'., the introduction of the animals cooling the apparatus to 17°-18°C'. Progressive heating raised the temperature in 15 minutes to 24°C., in 30 minutes to 32°C., in 45 minutes to 37°C., and in one hour to 39-40°C. At each fifteen minute interval frogs were removed and their eyes prepared for microscopical examination.
At 24°C. the position of the pigment was not essentially different from the normal state of maximal contraction, although delicate fringed processes did extend towards the external Umiting membrane. It is probable that such an experiment did
142 LESLIE B. AREY
not fairly test the effect of this temperature on pigment migration. One could hardly expect the body temperature of the animals to become adjusted to that of the apparatus in such a short time, especially since the heating was progressive and the final temperature was not realized until the end of the experiment.
At 32°C. the pigment had extended to the maximal distance but the distribution was nearly homogeneous.
A condition of extreme expansion occurred at 37°C. A dense massing of pigment near the external limiting membrane masked the rod ellipsoids completely, while the outer members of the rods were nearly free from pigment. When the temperature was raised to 39° or 40°C. clonic spasms occurred which ended in death; the pigment, nevertheless, retained the same position as at 37°C.
A second series of experiments, in which frogs were cooled in a refrigerating apparatus, showed that a subjection to 0°C. in the dark for 2 hours produced incomplete expansion, while after 3 hours the pigment was distributed in a zone of maxim_um breadth, but with only a slight tendency toward distal massing. This discovery, which was Herzog's most interesting contribution, is not only in disagreement with the commonly quoted result of Ewald und Kiihne's earlier work, but also has not been substantiated by the recent investigation of Fujita ('11), who, however, states that high temperature does induce pigment expansion, as the'other investigators have all maintained. Fujita tried the effect of ^ow temperature on only four animals, the duration of his experiments ranging from 30 minutes to 6 hours, yet he drew the following positive conclusion (p. 170) : "Das Resultat war in alien Fallen das gleiche: ich konnte keine Hellstellung konstatieren. Die Zapfen waren nicht kontrahiert, das Pigment nicht vorgewandert."
Since all these results on the frog's retinal pigment are not only fundamentally different from those found by me in fishes, but also have no parallel in the movements of vertebrate and invertebrate melanophores, and since, moreover, there is no general agreement concerning the effect of low temperature, a thorough reinvestigation of the problem seemed to be needed.
MOVEMENTS IN THE VISUAL CELLS 143
My first effort, therefore, was to repeat Herzog's work using apparatus and methods that essentially agreed with his, in order to ascertain whether identical results would be reahzed. If the movements of the frog's retinal pigment are really exceptional among other lower vertebrates, such responses have considerable theoretical as well as incidental interest.
The following apparatus was de\nsed. A cubical cage, made of fine wire netting with a cover of the same material, was supported by uprights inside a large battery jar which was fittcul with a glass cover perforated by a small hole to allow slow diffusion of air. The wire cage, made to receive the frog, did not come in contact with any part of the surrounding glass receiver which was to serve as a constant temperature chamber. The glass receiver sat upon a platform in a large cyhndrical metal tank. A thermometer passed through the cover of the tank and also through the cover of the temperature chamber into that chamber itself. A funnel connected with a rubber tube to exclude light was fitted into the cover of the tank and served for introducing water into the tank.
Tests were conducted in the following way. A dark-adapted frog was wiped dry and plac(>d within the wire cage^" inside the constant temperature chamber. Previously the chambei- had been brought to the desired temperature by one of two simple methods. If the efTect of heating was to be studied, the metal tank was partially filled with watei- at the appropriate temperature and the whole system allowi^l to adjust itself until the thermometer ins'de the constant temjierature chamber registered the required degree of warmth. After the frog had been introduced, the temperature could be regulated without opening the apparatus by admitting warmer water through the funnc^l and drawing off an equivalent amount from the bottom of the tank. If, on the other hand, a temperature near the freezing
'" Herzog lays much stress on the facts that the animaTs body was never in contact with solids other than the wires of the cage, and that the body was always wiped free from secretions or excretions at the commencement of an experiment. He seems to fear lest there should be a chemical stimulation due to heated secretions or excretions that would affect the results in case these precautions were not followed.
144 LESLIE B. AREY
point was desired, the outer chamber of the tank was filled with a mixture of ice and water; if a sufficient amount of ice was provided a temperature of from 3° to 5°C, could be maintained, without further attention, throughout the whole experiment. At the expiration of the time allowed for temperature adaption (usually 3 hours), the apparatus was placed in the dark or in weak red light and the frog's eyes were removed and fixed in darkness at the same temperature as that at which the experiment had been conducted.
In the course of these tests nearly 100 eyes were examined, yet the general results obtained were identical with those described by Herzog. At 3°C. and 33°C. (figs. 17, 19) the pigment was expanded approximating the condition characteristic of light; between the temperatures of 14°C. and 19°C. (fig. 18), however, the pigment was contracted to a narrow compact layer. It should be noted that 14°C. and 19°C. may not represent the limiting temperature at which pigment contraction occurs, although Herzog states this to be the case; no attempt was made by me to determine these intermediate temperature-limits. At low temperatures the expansion of pigment was generally not as complete as at a high temperature or in the light, and there was often considerable variation in different parts of the same retina, yet the general result was one of unmistakable expansion.
In some cases, however, there was little or no evidence of a pigment expansion at the lower temperature, yet such instances were comparatively rare. Although the cause of discrepant results of this kind is not evident, they perhaps furnish additional proof for the nervous control of the frog's retinal pigment, as many workers maintain {vide infra). It seems probable that Fujita was unfortunate enough, in the few experiments which he performed at a low temperature, to obtain nothing but this lack of typical results, although in my own work the occurrence of such anomalous cases was always sporadic.
A careful comparison was made of the results obtained at 3°C. and 33°C. No constant difference in the amount of migration could be detected, although Herzog states that at 37°C. the distal migration is greater than that obtained at low temperature.
MOVEMENTS IN THE VISUAL CELLS 145
Hence we may conclude that the condition found in the frog is unhke that found in the fishes. It should be noted, however, that between certain limits the two animals show similar tendencies in their pigment responses. These hmits are approximately 0° to 19°C. in the dark for the frog, and 0° to 28°C. in either darkness or light, for the fishes.
Since the tendency of pigment migration under the influence of temperature agrees between the limits of 0° to 19°C. for the dark adapted frog and 0° to 28°C. for the fishes either in light or in darkness, the query may be raised — is it not possible that if the fishes were subjected to higher temperatures a reversal of the temperature effect would occur in which an expansion of the pigment would again be found as in the frog? The fact that Herzog found but slight differences at 18°C. and 24°r. increases this suspicion. I am convinced, however, that a tendency toward such a response does not exist in the retinal pigment of fishes even to the slightest extent. In the first place, as stated before, prolonged heating of the frog's retina at 24*^C. would be likely to produce more striking changes than Herzog obtained. Furthermore, a few experiments in which the temperature of fishes was raised to 30°C. and over failed to show, in either light or darkness, anything beyond the characteristic response of less complete expansion than at the lower temperature.
The extent to which the retinal pigment of the frog and of fishes moves under the influence of temperature differs in a high degree. Among the fishes the differences are small and amount to little more than a redistribution of pigment in the dark and light phases respectively, whereas in the dark-adapted frog varying temperature induces the whole range of pigment response usually occasioned by light or darkness. This further indicates that the nature of the response in the two kinds of pigment cells differs fundamentally. In the fishes probably the response is through the direct action of temperature on the cell protoplasm, while in the frog the pigment migration may be produced indirectly through the intervention of the nervous system.
146 LESLIE B. AREY
All the (Xperinieiits of i)revious workers on the effect of temperature have been performed in the dark and I, therefore, set about to discover what results would be obtained in the light. The only change in the apparatus from that previously described was that a 20 litre jar replaced the metal tank; this, when filled with water at the desired temperature, performed the necessary heating function while its transparency did not interfere with the entrance of light into the inner chamber. Experiments were made at the same temperatures as in the dark — 3° to 5°C., 16°C. and 33°C., but the results were, for the most part, conditions of uniform expansion independent of temperature.
It was observed that when frogs were subjected to a low temperature they became quiescent and tended to keep their eyehds closed. Although the lower lid (the only one which is movable to any extent) is more or less transparent, the possibility of its influencing the results led to its removal in a number of instances; no difference, however, was obtained by the observance of this precaution. From these results, therefore, we conclude that in darkness, temperature is the controUing factor, while in the light temperature is subordinate to the stronger stimulus, light.
At this juncture a doubt arose as to the exact temperature the frog's retina was experiencing while in the apparatus. It is well known that at the surface of the frog's body rapid evaporation can take place; hence it is perfectly conceivable that the rate of evaporation in the temperature chamber might be such as to keep the body temperature for some time considerably below that of the surrounding air. This possibility was first checked by taking the oesophageal and rectal temperatures of animals that had been subjected to various temperatures in the apparatus for several hours. The recorded temperature, however, was nevei found to vary more than a fraction of a degree from that of the air in the containing chamber.
To be absolutely certain on this point, a prolonged set of experiments was made in which the frogs were immersed in water media of appropriate temperatures. It is certain that after a short time the animal, as a whole, must assume the temperature
MOVEMENTS IN THE VISUAL CELLS 147
of the medium irrespective of acti\ities at the surface of the body.
The apparatus for this verification consisted merely of a large battery jar, a sheet of coarse wdre gauze and suitable weights. The jars were filled with water to within a quarter of a centimeter of the top, and the gauze, held in place by weights, served as a cover. This device worked in the following manner. Animals coming to the surface to breathe could only get their nostiils above water, the rest of the head and bod}' remaining submerged, hence, in a short time the body temperature of the frog necessarily approximated that of the surrounding medium.
At 3°C\ the body temperature of the animal quickly fell, th(> respiration rate was reduced until it practically ceased and the bodily activities diminished until the frogs, for the most part, remained quietly at the bottom of the jar. although some animals would occasionally swim to the surface to breathe. At33°('., on the contrary, the frogs were very active and had to return at shoi't intervals to the surface, where they would sometimes remain for several minutes clinging to the netting.
A series of experiments was performed both in darkness and in light. In the dark nothing new was learned beyond the conditions already described. In the light, the first experimeni showed a state of extreme pigment expansion at 3°C., which was comparable to that of Ameiurus under similar conditions. The other trials, at 16°C. and 33°C\, on the contrary, showed the pigment uniformly distributed. Another experiment, a short time after, gave the same result at 3°C\ but not so decisively. The possibility of discovering a similarity in the pigment responses of frogs and fishes iii the Hght, led me to repeat these experiments many times without, howe^^er, again obtaining similar results.
If extreme jiigment expansion occurred at 3°C. it would be interesting from another standpoint since Herzog reported a similar condition, in darkness, at the highest temperatures which these animals can withstand.
Occasionally during experiments both in light and in darkness, an anomalous condition arose whereby the distribution
148 LESLIE B. AREY
of pigment in one part of the retina was markedly different from that in the remaining portions. i^ Such conditions may have been due to a variety of disturbing factors, Angelucci ('90) has recorded noises, unilateral pressure on the eyeball and 'mechanical or electrical stimulation of the body as causing the migration of pigment in dark-adapted animals. Herzog ('05) likewise states that a frog tied up for 24 hours in the dark showed the pigment in the light position. A whole series of experiments and observations are on record to show motor control of some sort not well understood. It certainly is evident from a comparative study of pigment in other forms, that is, in the retinas of fishes as well as in vertebrate and invertebrate melanophores, that the situation in the frog is entirely unlike that in any other animal concerning which we have data.
Herzog explained the temperature responses of the frog's pigment in the following way. If the effect of temperature is purely physical, its action presumably consists in accelerating or retarding chemical processes in the protoplasm of the pigment cell. Since, however, the movements of the dark-adapted pigment are not directly correlated with the temperature gradient, a physical action of temperature is probably not responsible for the observed phenomena. If, on the other hand, it is assumed that the response of the pigment involves the principle of 'specific energies,' then any positive stimulus, acting through the nervous system, will cause a pigment expansion, and thus a satisfactory explanation for the known facts is furnished.
In connection with the special case offered by the frog an interesting speculation arises as to the kind of pigment responses shown by the frog larva. The larva" in a general way, is comparable to a fish; at least it may be said that the larval stage recapitulates certain conditions persistent in the adult fish. Is it not possible, therefore, that under the action of temperature the pigment of the larval frog will show a distribution similar to
11 A somewhat similar lack of consistfency was also noted by Fick ('90), in his attempts to obtain maximal contraction in dark-adapted eyes. He concluded, however, that inequality in the pigment distribution was characteristic of darkadapted retinas.
MOVEMENTS IN THE VISUAL CELLS 149
that in fishes? The material, on which an answer to this question was sought, was the larva of the bullfrog (R. catesbiana). Animals were obtained during the month of April, 1914; these, of course, represented larvae hibernated from the previous season, since two or even three years are required to complete the metamorphosis of this species. A few experiments were also made on animals obtained in November, 1914.
From the larvae procured, two size limits were selected for experimentation. The smallest larvae had a total body length of 4.5 cm., the hind legs of such animals not being visible; the largest larvae had a body length of 7.0 cm., and the hind legs were developed as two small buds with the digits just differentiated.
In neither the 4.5 cm., nor the 7.0 cm. animals were the eyes as deeply pigmented as in the adult of R. pipiens. Of the two sizes, the 7.0 cm. larvae had the pigment more highly developed, and consequently better differentiation was obtained at the various temperatures with these, than with the smaller animals.
The 4.5 cm. larvae at 3°, 26°, and 32°C. in the dark showed the pigment in an expanded state (cf. figs. 20, 22) but not in a firm zone of uniformly distributed granules. On the contrary, the cells displayed pigmented processes that seemed to be more or less independent of each other; the appearance of the zone being that of a more uniform base with a fringe of pigment extending distally from it. The degree of expansion at 2(3°C. was distinctly less than at either 3°C. or 32°r., although at neither of the extreme temperatures was the pigment as fully extended as in the light. At 16°C\, however, a striking difTerence was found (cf. fig. 21), for the pigment lay contracted in a narrow compact layer near the choroid.
The 7.0 cm. animals gave results (figs. 20, 21, 22) quite similar to those just described, although the contrasts at the various temperatures were considerabh' sharper due to the heavier pigmentation of the eyes at this stage.
In these lar\'ae, therefore, the behavior of the retinal pigment to temperature is identical wdth that characteristic of adult frogs. Since these animals were always immersed in water, there is no
150 LESLIE B. AREY
question coiict>rniii^' tlio correspoiulenco of their body temperature and that of the surrounding^ medium.
What would be discovered in a study of earher stages I can not say, but I suspect great difhculty would be encountered in interpreting the results due to incomplete pigmentation, for the differences at various temperatures in the 4.5 cm. tadpole, although fairly well marked, showed much less contrast than those exhibited by the 7.0 cm. animals.
3. Necturus. A comparison between the frog and some urodele suggested another interesting problem. Is the condition exhibited in the frog restricted to anurans or is it common to the whole group of amphibians? This query becomes all the more pertinent when it is recalled that urodeles are not in the direct line of ascent to the anurans; that is, they are not amphibians which have never gone beyond the water inhabiting stage, but are more probably a group that were once land animals and have again returned to the water as a secondary adaption.
The common mud puppy, Necturus maculatus, was chosen because of the ease with which it is procured and kept in captivity. These animals were treated according to the technique used for fishes, hence a temperature higher than 28°C\ was not attempted.
The first experiments performed were to secure typical lightand dark-adapted retinas in order that some basis of comparison might be had. The results were by no means as striking as one might wish. In both cases the pigment was extended, not in a band with even contours, but generally in large conical processes from the individual cells, which, hke other cells of Necturus, appear to be very large; these conical processes surround the distal ends of the outer segments of the rods.
In the Hght, the pigment was usually somewhat more extended than in darkness. The processes were not of uniform length but mean measurements may be expressed by the values of about 38 n in the light and 30 /x in the dark, — ^a condition of slight contrast when compared with fishes or the frog. The presence of a certain amount of migration in Necturus has been previously noted by Howard ('08), who took advantage of
MOVEMENTS IN THE VISUAL CELLS 151
the contraction in the dark in hi.s study of visual cells. This situation is comparable to that found in Triton where movements of the pigment through the influence of light were discovered by Angelucci (78), Stort ('87) and Garten ('07). The extent of pigment migration in this animal is described as being very limited by Garten (p. 70) who says: ". . . . diesclbe ist aber hier unvergleichlich viel schwacher als bei vielen andereii niederen Wirbelthieren."
In an examination of the effects of various temperatures on dark-adapted eyes, however, no constant differences were noted. In some instances the processes of the cells seemed to be less heavily pigmented and the bases more heavily pigmented at 15°C, than at the two extreme temperatures, but this was by no means constant. At least, it can be said that there is no marked contraction of the pigment in the dark at any temperature.
The evidence from Xecturus and the limited pigment migration in Triton conclusively prove that the pigment responses typical of the frog are not common to all amphibians. It is probable, therefore, that such peculiarities as were described for the frog have been developed solely within the anuran group.
b. Vi^sual cells
Although the myoids of both th(^ rod and cone cells of fishes are capable of a high degree of contractility (a 90 per cent retraction occurs in some instances) , the effect of various temperatures on these cells remains untried up to the present time; in fact, the frog is the only animal upon which such work has been attempted. CJradenigro ('85), Angelucci ('S4b), Herzog ('05), and P'ujita ('11) found that warming produces the same effect on the cones of the frog as does light. Herzog also stated that coohng to 0°C. Hkewise caused the cones to shorten, although Fujita denies that this occurs.
There is no record of any attempt to determine the effect of temperature upon the rods of vertebrates beyond the statement of Gradenigro ('85) that at 30°C. the rod of the frog shortens as in light.
152 LESLIE B. AREY
The object of the work upon visual cells to be described in this section parallels that stated for pigment, that is, to determine the effects of various temperatures on the myoid of rod cells and of cone cells in normal fishes and amphibians.
The apparatus and technique employed were similar to those which were used in the experimentation upon pigment. Particular care was exercised at the termination of experiments conducted in the dark to guard against the action of light on the highly sensitive visual cells. In the fishes, measurements of the cone myoid were made from the external limiting membrane to the proximal edge of the ellipsoid, and in the frog, from the external Umiting membrane to the proximal side of the oil drop which is situated at the distal end of the ellipsoid. The lengths of the rods were measured from the junction of the inner and outer members to the external limiting membrane. Each value given in the tables represents the mean measurement, in micra, of many (12 to 24) individual elements.
1. Fishes. (1) Ameiurus. Of the four fishes studied, the cones of Ameiurus, in many ways, gave the least satisfactory results. These elements are not located at uniform levels and the differences between the elongated and shortened condition, when stimulated by extremes of temperature, are not striking to the eye. The additional fact that the cones, when maxi-. mally shortened under the influence of temperature, never closely approach the external limiting membrane makes these animals rather unsatisfactory for certain kinds of experimental work.
Tables 1 and 2 present data for both rods and cones from typical retinas at 5°C. and 25°C. in the dark.
It will be seen that at the higher temperature the myoids of both cones and rods lengthen (figs. 32, 33). Especially in the cones is this response unmistakable. The lengths of the rod's inner member, after subjection to the extreme temperatures, varies within only a few micra, yet the relative change may be 25 per cent or more; moreover, since the mean ranges at the extreme temperatures do not overlap, these differences are presumably significant. If the length of the rod ellipsoid, 4 /x,
MOVEMENTS IN THE VISUAL CELLS
153
TABLE 1
Measurements from the retinas of four Atneiurus which had been kept at 5°C. in Iht dark. The values are in micra and represent measuretnents taken along axe-coinciding loitK radii of the eyeball
NUMBER OP ANIMAI,
NERVE FIBER
LAYER TO
EXTERNAL LIMITING
MEMBRANE
CHOROID TO
EXTERNAL LIMITING
MEMBRANE
CONE MYOID
ROD INNER
MEMBER
1
2 3 4
35
35
50 43
95
95
95 100
19 32
13-20 19-31 13-24
7-9
7-9
9
9-10
Mean
41
96
16-27
8-9
Measurements from the retinas of four Ameiurus which had been kept at 25°C. iit the dark. The values are in micra and represent measurements taken along axex coinciding ivith radii of the eyeball
NUMBER OF ANIMAI.
NERVE FIBER
LAYER TO
EXTERN AI MMITINO
MEMBRANE
CHOROID TO
EXTERNAL I.IUiriNa
MEMBR.\NE
CONE MYOID
ROD INNER
MEMBER
1
2 3 4
50
43
42
50
105
100
S7
93
32-38
32-38
25-31
31-42
9-13
12-15
10
9-12
Mean
46
96
30-37
10-13
is subtracted from the iiieaii values, the relation existing between the corresponchng vahies at either end of the tenii)erature range will be expressed by the following proportion: 25°C: 5°C. =3:2. The variabiUty in the length of adjacent rods in any section is relatively so great that this disparity in the length of the myoids is not apparent until actual nieasuTements are made.
In a few observations upon the effect of temperature on the extended rods in the light, the range at 25°C. seemed to run higher than at 5°C. by 15 to 20 per cent. The lengths of the extended inner members were approximately 50 m at 5°C. and 60 p. at 25°r. Similar tendencies will be noted among certain other fishes.
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NC
154
LESLIE B. AREY
(2) Abramis. The cones, with their large oval eUipsoids 10 M in length, are very conspicuous in stained preparations. The fine rods, on the contrary, are not always, demonstrable when treated with the Ehrlich-Biondi stain, which acts in an unusually capricious fashion in respect to these elements.
The elongated cone cells are more or less uniformly extended, although the variabiHty in length is greater under these conditions than when in the retracted state. The rods, however, are arranged in the dark at very irregular levels so that retinas present a fairly even distribution of them from 8 ^ to sometimes as far as 50 ^ from the external limiting membrane. In the light the condition is one of more uniform elongation.
In tables 3, 4, and 5, are presented the data obtained from measurements at 5°, 15°, and 25°C. in the dark.
Measurements from the retinas of four Abramis which had been kept at 5°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball
NUMBER OP ANIMAL
NERVE FIBER
LAYER TO
EXTERNAL LIMITING
MEMBRANE
CHOROID TO
EXTERNAL IIMITING
MEMBRANE
CONE MYOID
ROD INNER
MEMBER (MAXIMUM)'
1
2 3
4
90
72 85 82
90
85 91
88
7-12
5-12
5- 9
6-10
40
Mean
82
89
6-11
40
TABLE 4
Measurements from the retinas of three Abramis which had been kept at 15°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball
NUMBER OF ANIMAL
NERVE FIBER
LAYER TO
EXTERNAL LIMITING
MEMBRANE
CHOROID TO
EXTERNAL LIMITING MEMBRANE
CONE MYOID
ROD INNER
MEMBER
(maximum)
1
2 3
95
100
88
94
105
69
16-29
15-24
24-34
59 ,
60
25
Mean
94
89
18-29
48
MOVEMENTS IN THE VISUAL CELLS
155
TABLE 5 Measurements from retinas of four Abramis which had been kept at 25°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball
NUMBER OF ANIMAL
NEHVE FIBER
LATER TO
EXTERNAL LIMITING
MEMBRANE
CHOHOIP TO
EXTERNAL LIMITING
MEMBRANE
CONE MYOID
ROD INNER
MEMBER
(maximum)
1
2 3 4
80
72
105
76
89
84
84
65
21-49
2^-40
30-40
28-40
44
44
Mean . . .
83
81
27-42
44
The effect of temperature, therefore, upon the cones of Abramis is very marked, the length of the myoid at 5°C. (fig. 25) averaging only 25 per cent of that at 25°C. (fig. 27), while in extreme cases this ratio is as low as 10 per cent. If the mean limits of myoid extension are averaged, values of 9, 24, and 35 micra are obtained for the temperatures of 5°, 15° and 25°C. respectively. These values are in the ratio of 1.0: 2.5 : 4.0, or in other words, the 'coefficient of expansion' for the myoid of Abramis is 2 =*= for 10°C.
As a matter of fact, in the great majority of these temperature experiments, 5°C\ represents a value too high and similarly 25°C\ a value too low for the actual temperatures maintained. 3°C\ and 26°C. are more nearly the actual values. If temperature is plotted as abscissas and the myoid length in micra as ordinates, the resulting curve (fig. A) is a straight line showing that the temperature effect is uniform between these limits.
The straight fine obtained in the plot may indicate that the temperature response is the result of two or more opposed chemical reactions which operate in a compensatory manner. Since the response of the nn-oid in elongating is directly correlated with the temperature gradient, it seems feasible that the effect of temperature is physical (in the sense of Herzog), and through its action chemical processes in the protoplasm are uniformly accelerated. If the length of the myoid is a fair index of the chemical activity that causes elongation, and if the effect of
156
LESLIE B. AREY
teinponituiv is ])urely physical, the coefficient of 2± for 10°(\ is of interest, on account of its agreement with the value found for the temperature coefficient of various vital processes as well as of ordinary chemical reactions.
The incomplete data concerning the maximum lengths of the inner members of rods are hardly significant, although both of the mean values at 15°C. and 25°C. are slightly above the one measurement at 5°C. The EhrHch-Biondi stain was used on most of these preparations and it was only rarely that the rod ellipsoid took the stain sufficiently to render its identification certain.
—
3
^
2 B
_
^^
h
'
z 8
- ^^
§
-_ '^^
H
C
- ^
1 1 1 i 1 1 1 1 1 i..J_L_L
1 1 1 1 1 1 1 1 1 1 1 1 J IJ
TEMPERATURE IN DEGREES CENT.
Fig. A. Plot showing the relation between temperature and niyoid length in the cone cells of Abramis.
Table 6 gives measurements from two retinas which had been subjected to extreme temperatures in the light. The measurements for the cone myoids are identical, and in no one of the four fishes was there a demonstrable change ascribable to temperature under these conditions. It should be noted that the cone measurements at 5°C. in the dark (table 3) are either equal to or, as in this case, are actually smaller than those representing the highly retracted light condition. This dependence upon temperature was taken advantage of in all experimentation to be described later where elongation of the cones was
MOVEMENTS IN THE VISUAL CELLS
157
desired. Although the disparity between the rod lengths given in the table is probably extreme, such values have the same purport as the corresponding measurements made on the rods of Ameiurus.
TABLE 6 Measurements from the retinas of two Abramis, one of which had been kept at 5°C., the other at 25°C. in the light; the values are in micro and represent measurements taken along axes coinciding vilh radii of the eyeball
NUMBER OF ANIMAL
TEMPERATURE
"C.
NERVE FIBER
LAYER
TO EXTERNAL
LIMITING
MEMBRANE
CHOROID
TO EXTERNAL
LIMITING
MEMBBANE
CONE MVOID
ROD INNER
MEMBER
(MAXIMUM)
1
5
25
54
88
78
100
S-14
8-14
50
75
(3) Fundulus. The retina of this fish is interesting because of the presence of prominent 'double cones.' Such elements are found in representatives of all the vertebrate classes, with the exception of mammals ((Jreeff, '00). They consist of two cones with fused inner members, although close examination is necessary to demonstrate this union. One component is usually larger and is known as the 'chief cone' (fig. 29; ell. con.), whereas the smaller is the 'accessory cone' (fig. 29; con. ace.) The chief cone alters its position independently of its accessory cone, which remains close to the external limiting membrane and is not moved to any gi'eat extent by the action of light or other stimulating agents.
The rods, although nitlu-r small, are (luite in i>vitlence and differ from those of Abramis in maintaining fairly uniform degrees of elongation.
In the tables (7 and 8) which show the characteristic results of experimentation in the dark, the mean myoid length of the chief cones at 5°C. (fig. 28) is only about one-third that at the higher temperature (fig. 29). The differences in the accessory cones are not striking, although the lower values are somewhat increased at 25°C. Since the movements of the accessory cones are very Hmited, this probably represents a significant elongation.
The contrast between the extension of the rod at 5° and 25°C. is striking and, added to the evidence gained from other fishes,
158
LESLIE B. AREY
Measurements from the retinas of five Fundulus which had been kept at 6°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball
NUMBER OP ANlMAf.
KERVE FIBER
LAYER
TO EXTERNAL
LIMITING
MEMBRANE
CHOROID
TO EXTERNAL
LIMITING
MEMBRANE
CHIEF CONE
MYOID
ACCESSORY
CONE MYOID
ROD INNER
MEMBER
1
3 4
5
100
106
88 105
75
69
68
69
68
52
5-11
5
5
3
5-9
1-5
1-5
1-5
1-4 1-5
16-19
11-19 16-23
Mean
95
65
5-7
1-5
14-20
Measurements from the retinas of five Fundulus which had been kept at25°C. in the dark; the values are in inicra and represent measurements taken along axes coinciding with radii of the eyeball
NUMBER OF ANIMAL
NERVE FIBRE
LAYER
TO EXTERNAL
LIMITING
MEMBRANE
CHOROID
TO EXTERNAL
LIMITING
MEMBRANE
CHIEF CONE
MYOID
ACCESSORY
CONE MYOID
ROD INNER
MEMBER
1
2 3 4 5
120
120
130
135
135
87
87
100 94
110
14-22
15-22
17-26
17-27
17-26
2-5
2-6
6
6 1-3
24
31
21
19
38
Mean . .
128
96
16-25
3-5
27
indicates that in these animals a lengthening of the inner members is favored by a high temperature.
A series of measurements of chief cones in the light failed to show any differences at the extreme temperatures.
(4) Carassius. The retina of Carassius, as well as that of Fundulus, has prominent double cones.
In the few eyes measured, the chief cone elongated with in creased temperature (figs. 23, 24) but the accessory cone did not change its position, at least to any extent.
Table 9 summarizes the results from typical retinas.
MOVEMENTS IN THE VISUAL CELLS
159
TABLE 9 Measurements from the retinas of four Cara^sius, two of which had been kept atS°C., and two at 25°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball
NUMBER OF
TEMPERATURE
NERVE FIBER
LATER
TO EXTERNAL
LIMITING
MEMBRANE
CHOROID
TO EXTERNAL
CHIEF CONE
ACCESSORT
ANIMAL
"C.
LIMITINO
MEMBRANE
MYOID
CONE MYOID
1
25
75
88
21
3-5
2
25
69
69
21
2-3
3
5
56
63
1-3
2
4
5
69
94
3-4
2-3
2. Frog. (1) Rana pipiens (adult). Gradenigro ('85), Angelucci ('90), Herzog ('05) and Fujita ('11) have all stated that at a temperature of 30°C. or more, the cones of the frog shorten until they assume the position characteristic of light. Thus, Herzog (p. 419) says: "Aufenthalt im Briitschrank 1/2 Stunde lang, Temperatur von 21° bis 30° C. ansteigend: . . . dieselbe zeigt auch, dass die Zapfen maximal contrahiert sind. Die Lange der Zapfen betriigt mit ganz vereinzelten Ausnahmen 0.0091 mm. (v. d. Lim. (^xtern. — Oelkugel excl.)."
The action of low temperature was first tried by Herzog ('05), who makes the following statement (p. 424). concerning the result of two hom's" cooling to (f(\ in the dark: Dagegen sind die Zapfen bereits nahezu hochstgradig verkiirzt. Ihre Lange betragt fast durchweg 0.0078 bis 0.0091 mm."
Fujita experimented upon six frogs, which were kept at a low temperature in an 'P^isgefass' for periods of 30 minutes to 6 hours, after which the decapitated heads were fixed in ice-cold fluid. His conclusion (p. 170) is diametrically opposed to that of Herzog. "Das Resultat war in alien Fallen das gleiche : ich konnte keine Hellstellung konstatieren. Die Zapfen waren nicht kontrahiert "
Measurements showing the elongation of the cone myoid at intermediate temperatures (14° to 19°C.) are not given by Herzog, who, however, states (p. 418) concerning retinas that had been raised in the course of 15 minutes from 18° to 24°C.: Ein wesentlicher Einfluss is nicht zu erkennen Die
160
LESLIE B. AREY
ZMpfenliinge (von der Limitans externa bis zur Oelkugol im Ellipsoid exclusive) betriigt im Durehschnitt maximal 0.034 mm., im Mininuim 0.0169 mm."
With apparatus and methods similar to those described in connection with the experimentation upon frog's retinal pigment, an attempt was made to discover the exact responses of the cone myoid to high, medium, and especially to low temperatures.
In table 10, which gives the measurements of visual cells from a few typical preparations, it will be observed that the cones are greatly shortened at 33°C. (fig. 36), but that at the other temperatures they retain the elongated condition typical of darkness (figs. 34, 35). ^^ Measurements of the cones gave two modal lengths, which are approximately expressed by the figures in the table representing the extreme values. The maintenance of elongation at a low temperature is in agreement with Fujita's ril) experiments, 1'" but is opposed to Herzog's conclusion, which was apparently based upon exhaustive investigation. It is true that the temperature of 0°C. which the latter worker used was a few degrees less than the lower limit (3° to 5°C.) employed
TABLE 10 Measurements from the retinas of eight Rana pipiens which had been kept at 3°, 14°, 19°, and 33°C., respectively, in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball
NUMBER OF ANIMAL
TEMPERATURE
°C.
NERVE FIBER
LAYER
TO EXTERNAL
LIMITING
MEMBRANE
CHOROID
TO EXTERNAL
LIMITING MEMBRANE
CONE INNER
MEMBER
ROD INNER
MEMBER
1
3
140
66
12-20
10±
2
3
160
75
13-22
9±
3
14
103
56
11-20
11±
4
14
88
50
11-16
10 ±
5
19
103
61
13-19
13 ±
6
19
94
63
14-23
10±
7
33
163
63
8-12
10±
8
33
126
63
7-10
11±
1* The actual values at medium and high temperatures vary somewhat from those given by Herzog for R. temporaria (and R. esculenta?).
13 This experimentation upon the retinal elements of the frog was practically completed before Fujita's paper was known to me.
MOVEMENTS IN THE VISUAL CELLS 161
by me, yet it seems improbable that such a small difference would cause the cones to shorten maximally. Any error that could be introduced during these determinations would tend to shorten the cones, hence Herzog's results are at a disadvantage in this respect. I have continued experiments for hours, yet the results were always the same; in no case was there found a general shortening of the cones that in any way resembled the extreme condition at 33°C.
From these results, which, in a general way, are the reverse of those found in fishes, it is evident that the responses of the cones are not comparable to those of the retinal pigment. The pigment may indeed be under nervous control, so that stimulating agents such as heat, cold and light produce a migi-ation according to the principle of specific energies, j-et if the cone cells are influenced by the nervous system, these experiments can not be said to furnish proof of such a relation.
In order that there should be no doubt concerning the etlect of low temperature upon the cone myoid, a further determination was made long after the results which are tabulated above were obtained. In this later experiment rigorous precautions were observed to eliminate possible errors. After frogs (R. pipiens of various sizes), kept at a temperature of 18°C., had been subjected to a ])reliminary treatment of darkness for 72 hours, they were introduced into a vessel cooled to 1°C, where they remained for a period of 4 hours. The eyes were then quickly excised in dim, red light, the operation not requiring more than 15 seconds, after which they were returned into darkness where fixation at approximately the freezing temperature ensued. The average measurements of the cone myoids in these preparations were as follows: 12 to 14 m; 10 to 15.4 n; 10 to 14 m; 10 to 11 m; 13 to 15.4 n;9 n. These values, although somewhat smaller than those given in table 10, can not be said to prove that low temperature shortens the myoids as does high temperature.
Gradenigro ('85) found that elevated temperature induced a shortening of the rod in the dark. After measuring the myoid length in a considerable number of preparations from retinas subjected to various temperatures both in light and in darkness,
162 LESLIE B. AREY
I was unable to discover constant differences in length that could be correlated with definite temperatures. The agreement of mean values obtained from various retinas under identical temperature conditions was not good, and since the rod myoid measures only 6 yu to 12 /i in length, even small variations furnish serious obstacles in determinations of this kind. In any one preparation, moreover, variability in the length of adjacent myoids tended somewhat to mask a possible temperature effect. If anything, my measurements showed the reverse of what Gradenigro maintained, the elongation at 33°C. in the dark being greater than at 5°C., but, as stated before, these results are by no means trustworthy.
A shortening of the rod through the action of high temperature, as claimed by Gradenigro, is of interest because, according to most investigators, light produces the same result. With this can be compared an analogous correlation in fishes, where light causes an elongation of the rod myoid and, as I have shown, elevated temperature does likewise. It is certain, on the contrary, that although the cones of both frogs and fishes shorten in the light, heating produces unlike responses in the dark,
(2) Rana catesbiana (larvae). Although the cones in both the 4.5 cm. and the 7.0 cm. larva of this frog are of large size, clearly defined temperature responses were not observed; indeed, the difference between the positions assumed even in light and darkness is not striking, the cone myoids in the light remaining well elongated in comparison to those of the adult R. pipiens. The variability in length in different preparations is considerable, yet if anything, the cones appeared more shortened at 3°C. than at higher temperatures. There is no marked shortening at 33°C., for the cones under these conditions were as long as at lower temperatures, and in some cases longer. It is probable that the responses of the cones in adult R. catesbiana will be found to agree with those in other species which have been studied, although no experimentation was performed to determine this point.
3. Necturus. The rods, and the single and double cones of Necturus are very large, yet positional changes with varying tempera
MOVEMENTS IN THE VISUAL CELLS 163
ture (3° to 28°C.) were not observed. Individual cells vary more or less in the height at which they are situated above the external limiting membrane, yet no constant differences of significant amount could be correlated with definite temperatures. With these results should be compared Garten's ('07) denial of a change in the position of the cones of the 'salamander' through the influence of light, such as Angelucci ('90) had previously claimed. Stort ('87), however, described movements in both the rods and the cones of Triton.
C. EXPERIMENTATION UPON EXCISED EYES
a. Effect of light and darkness
The results of a number of investigators since the first work of Englemann ('85) have indicated that the retinal elements of some vertebrates, and especially the frog, are subject to a nervous control, the action of which is not well understood.
The role which the nervous system plays either in producing or in assisting the movements of the various retinal elements is hard to demonstrate. Experimentation involving the direct action of light on excised eyes can not be expected to solve the problem decisively, for if no movements result, autoanaesthetization, or some similar disturbance due to the interrupted blood supply, may be the real cause. If, on the other hand, responses are called forth by direct stimulation, it by no means follows that a similar phenomenon necessarily occurs in the living animal, any more than a demonstration of the direct stimulation of muscle fibers proves that this rather than a nervous impulse is the normal method of muscle stimulation. The limitations which restrict a wide interpretation of results, however, do not lessen the interest involved in determining the extent to which the retinal elements can be direct!}' stimulated.
Hamburger ('89) maintained that the cones and retinal pigment in excised eyes of the frog assumed the positions characteristic of light or darkness according to the conditions of the experiment. Dittler ('07) working on isolated frog's retinas obtained
104 LESLIE B. AREY
a shortening of the cones in localized areas through the action of Hght but found darkness to be ineffectual. The spread of the response to portions of the retina unstimulated by light, led Dittlcr to investigate further the cause of cone retraction. He was able to furnish experimental proof that weak acids, resulting from catabolic processes in the retina, caused the cone myoid to shorten; hence he concluded that the cone myoid was not of itself 'UchtempfindHch,' as Englemann ('85) had believed, but was stimulated to movement through chemical agents resulting from the action of light on the retina. Fujita ('11), as a result of very limited experimentation, stated that the pigment of the excised eye of a frog expanded in the light but did not contract in the dark.
Ringer's solution, normal saline solution, and tap water were used by me for the immersion of excised eyes. When the first two media were emploj^ed the movements of the rods and cones of Ameiurus, through the action of light, were never clearly demonstrated; possibly such results are to be interpreted as evidence of a chemical control somewhat comparable to that described by Spaeth ('13) for the melanophores of Fundulus. Tap water did not inhibit the movements of any of the retinal elements of Ameiurus and consequently it was used in all subsequent experimentation.
The pigment of Ameiurus, Abramis and Fundulus did not contract when excised eyes from light-adapted fishes were subjected to darkness for periods of 4 hours or less. At most there was only evidence of a retraction of the distal accumulation of pigment, which is characteristic of hght-adapted eyes, to form a more homogeneously pigmented zone (figs. 2, 10, 6). When the reverse experiment (subjection to light) was performed, the pigment of Ameiurus became maximally expanded in 2 hours (figs. 1, 3). Only the sHghtest tendency toward expansion, however, could be found after similar experimentation on the two other fishes. It thus appears that light acts directly on the pigment of Ameiurus only, while darkness is totally ineffective on all three animals.
MOVEMENTS IN THE VISUAL CELLS 165
WTien the rods and cones of Ameiurus and the cones only of Abramis and Fundulus were tested, the following results were obtained. The rods of Ameiurus moved both in light and in darkness, whereas the cones were stimulated only by light, no elongation occurring in the dark even when the experiment continued for 4 hours. The cones of Abramis and Fundulus did not change their positions to any extent either in hght or in darkness. In the light the cones of Abramis, which were more carefully investigated than those of Fundulus, at most showed only slight retraction and never closely approached the external hmiting membrane. If light did exert a direct influence on the cones or retinal pigment of this fish, the changes would be extremely easy to distinguish due to the wide difference between the light- and dark-adapted phases.
Since neither the cone cells nor retinal pigment of Abramis underwent movements under these conditions, it is possible that the accumulation of catabolic products, occasioned by the interruption of the blood supply, was responsible. Experimentation of the following kind shows the importance of maintaining the vascular circulation. If the optic nerve only of Abramis is cut, the retinal elements undergo their normal movements in darkness and in light. If, however, all the blood vessels and muscles are cut and the eye ball is attached to the body by the optic nerve only, no movements result. The objection may be made that some nervous mechanism is deranged by cutting these muscles and blood vessels, but this is hardly probable, as further experimentation, to be presented in a subsequent paper, on this and other fishes has shown.
In cases Uke the movements of the pigment or cones in excised eyes of Ameiurus through the action of light only, it is probable that an inhibitory tendency is also present, but the response to the stimulus furnished by light is sufficiently vigorous to overcome it. Either a less vigorous stimulus or response may explain why no movements of the cones and pigment occur in darkness.
An inhibition due to the presence of unremoved catabolic products, as postulated here, would be merely a form of auto
166 LESLIE B. AREY
anaesthesia. As will be shown, carbon dioxide and other anaesthetics do, in fact, arrest the movements of all the retinal elements of fishes.
Dittler ('07) accounted differently for the absences of elongation in the cones of isolated frog's retinas which were introduced from light into darkness. In order to appreciate his way of viewing this situation, it is necessary to understand the general theory advanced by him to explain the movements of the cones. In darkness an equilibrium was supposed to exist in the metabolism of the retina, the elongated cone myoid representing an unstimulated condition. Through the action of light, however, catabolic processes preponderate, and the accumulated acid wastes chemically stimulate the myoid to shorten. These conclusions were based upon experimental evidence by which it was shown that weak, free acids could be detected if isolated retinas were subjected to light in Hmited amounts of Ringer's solution, and further that such an acid solution was capable of causing other dark-adapted cones to shorten while still in the. dark. To return to the case under consideration, Dittler believed that the accumulation of the catabolic products formed in the light merely continued its contractile influence after the isolated retina was removed into the dark, and since these products were not removed, the metabolic equiUbrium could never be restored and consequently elongation failed to occur.
This theory of chemical stimulation is not supported by the condition in Fundulus and especially in Abramis, where the cones of excised eyes do not shorten even when exposed to light, for under these favorable conditions the tendency toward the production of a catabolic excess should be maximum. In still another way Dittler's theory does not explain a typical response of the cones of fishes. The cone myoid in isolated retinas of the frog shortens when the temperature is raised to 30°C. or more in the dark (fig. 36), and this fact Dittler used to support his view in the following logical manner. It is well known that most chemical reactions are accelerated by raising the temperature; hence in this case the autonomic equilibrium normally existing in the dark would become disturbed, the re
MOVEMENTS IN THE VISUAL CELLS 167
suiting increase of catabolic products causing the cone myoid to shorten." Although Dittler's statement is not altogether clear, it seems evident that low temperature was supposed not only to reduce the metabohsm of the retina to a low level, but also to render the cone myoid less 'empfindhch' to chemical stimulation.
The conditions in the dark-adapted cones of fishes, however, are entirely different, for here not only do the cone myoids elongate when the temperature is increased, but also elongated cones can be made to shorten by the use of low temperatures. If the shortening of the cones of fishes in the fight were due to a chemical stimulation, how can the elongation of these elements in the dark through the action of heat be explained, since manifestly in this case the metabolic equilibrium tends to become destroyed, the result being the formation of an excess of catabolic wastes, which by analogy with the conditions in the frog, should cause the cone myoids to shorten? Moreover, the efficiency of low temperature in retracting elongated cones, and the correlation between the uniform degree of myoid elongation and the temperature gradient (p. 155) finds no explanation through Dittler's hypothesis.
It should be said, however, that Dittler strictly limited his conclusions, the experimental evidence for which appears to be well established, to the material upon which he worked, and was even reserved in suggesting the occurrence of a similar method of stimulation in living animals.
The reason why the rods of Ameiurus move in darkness, while the cones do not, may be as follows. The rod myoid normally shortens in the dark, whereas the cone myoid elongates. It is probable that the contractile function of the mj'oid is more vigorous than the reverse process of elongation. This is not only substantiated by the fact that dark adaption of the rod takes less time than light adaption, but also by experiments
'* Dittler did not formulate this conception in extenso as I have expressed it. yet several statements (pp. 317-318) show that this was his belief. A concluding quotation reads: .... "der Einfluss der Temperatur uberhaupt ganz nach physikalischen Modus zu Wirken scheint, und bereciitigt uns, seine Wirkung rein in diesem Sinne zu fasscn."
168 LESLIE B. AREY
in which the optic nerve was cut. In such cases the rod never elongated in the Hght although it often showed a tendency to shorten when introduced into the dark, whereas the behavior of the cone cells in light and darkness was the exact opposite to that of the rod, since they tended to shorten in the hght but remained unchanged in the dark. On these grounds, therefore, an explanation is offered to show why it is that through the strong stimulation produced by the direct action of light, both types of cells show characteristic responses, while in the dark only the more vigorous contractihty of the rod myoid becomes effective. It must be remembered, however, that although the direction of the movements of the rod and cone cells are opposed, the real response of the protoplasmic myoid may be similar in both cases. If this were true, the apparent inconsistency in the movements of these elements would be due to a difference in the axis of contractihty in the two kinds of myoids, and the explanation just advanced would not stand. Reference will be made to these possibilities in another place.
b. Effect of temperature
Previous attempts to determine the direct influence of temperature upon the retinal elements have been confined to the frog. Gradenigro ('85) found that if excised eyes of dark-adapted animals were subjected to a temperature of 30° to 36°C. the rods and cones shortened and the pigment expanded, both end-results b^ng characteristic of light-adaption. Dittler ('07) was able to confirm Gradenigro's discovery concerning the cone cells. When the isolated retina was heated to 35° to 37°C. in the dark for 50 to 60 minutes, the cone myoids shortened. After retinas had been subjected to a temperature of 1° to 2°C, for many hours, on the contrary, no shortening of the cone myoid was observed.
The apparatus and methods used by me were similar to those described in connection with the experiments upon living fishes. The excised eyes were contained in test tubes which were suspended in jars of water kept at appropriate temperatures. Eyes
MOVEMENTS IN THE VISUAL CELLS 169
from the same animal were used simultaneously, one at each temperature extreme.
1. Effect of temperature upon retinal pigment. After many trials it was found that sharp differentiation of the retinal pigment of fishes was hard to secure at the extreme temperatures when the initial temperature had been intermediate. Accordingly, the expedient was employed of subjecting the Uving animals to a preliminary treatment either at 3°C. or at 25°C., and as a result uniformly satisfactory differentiation was obtained.
The retinal pigment of Ameiurus behaved precisely as in hving animals. At a low temperature, both in darkness and in light (figs. 3, 1), the degree of distal migration was greater than that at a high temperature (figs. 4, 2). Particularly in experiments conducted in the light was this strikingly apparent, since in many preparations at 3°r. the pigment migrated so far distally that the more proximal portions of the cells were free of granules, while a sharp line of demarcation existed between the pigmented and non-pigmented zones.
A few experiments were made upon the dark-adapted eyes of Abramis. In this case also, a greater pigment expansion was found at 3°C\ than at 25°(\ (figs. 11, 12).
Reference has been made to the contention of many investigators that there is an apparent nervous control over the movements of the frog's retinal elements. It has been shown that at a medium temperature in the dark the pigment is maximally contracted (fig. 18), whereas at higher and lower temperatures (figs. 17, 19) a considerable degree of expansion is effected. Not only is the amount of migration occasioned by temperature much more extensive than that in fishes, but also the similaritj^ between the effects of the two temperature extremes as contrasted with an intermediate temperature, has no parallel among other pigment cells or even in melanophores.
Since it is at least agreed that the pigment of excised eyes of the frog expands in the light, it ought to be possible to observe the effect of temperature, if this agent acts directly upon the pigment cells. Excised eyes from animals that previously had been at an intermediate temperature in the dark, were subjected
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 2
170 LESLIE B. AREY
to temperatures of 3°, 16°, and 33°C., but no changes were observed in the position of the retinal pigment. These results, which do not agree with Gradenigro's statement, by no means furnish conclusive proof that in the Hving frog temperature operates through the nervous system, yet when supported by a comparative study of the retinal pigment and melanophores of other animals, the conclusion reached by Herzog ('05), that the expansion of the frog's retinal pigment under these circumstances is of nervous origin, involving the principle of specific energies, becomes highly probable.
According to Fujita ('11), when excised eyes from dark-adapted frogs are retained in the dark for 20 minutes, the pigment assumes a partial light position. My observations do not confirm this result, for no migration of any consequence occurred.
2. Effect of temperature upon visual cells. When a study of the visual cells of Ameiurus was made, the effect of temperature was found to be essentially similar to that upon normal animals.
WTien eyes from dark-adapted individuals of Abramis that had previousl}^ been kept at a temperature of 25°C. were likewise excised and subjected to 5°C. and 25°C. in the dark, those at 25°C. (fig. 27) retained the elongated position while those at 5°C. (fig. 25) shortened to a considerable extent although somewhat less than in living animals. Mention has already been made of the significance of these results in connection with the applicability of Dittler's theory of chemical stimulation to the cones of fishes. Although the rods of Abramis at both temperatures showed a distribution extending over wide limits, yet the shortest measured 12 /x at 5°C. as compared with 20 n at 25°C., and the modal elongation at the same temperatures, as judged by the eye, was 18 m and 25 ^ respectively. It is interesting to note that in the excised eyes of dark-adapted Abramis, temperature is able to produce changes in both the retinal pigment and visual cells, notwithstanding the fact that light and darkness are wholly ineffectual in this respect.
Table 11 summarizes these results from typical retinas.
A few experiments were performed upon the cone cells of the frog. The results obtained were identical with those stated
MOVEMENTS IN THE VISUAL CELLS
171
TABLE 11 Measurements of the visual cells from the retinas of two Ameiurus and tico Abramis, of which one of each had been kept at 5°C. and the other at 25°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball
FISH
TEMPERATURE "C.
CO.N-E MYOID
ROD INNER MEMBER
Ameiurus No. 1
o
4-16
5-6
Ameiurus No. 2:
25
19-32
8-12
.\bramis No. 1
5
10-30
18
Abramis No. 2
25
35-50
25
by Dittler ('07), who used isolated retinas. In dark-adapted eyes which were placed in water at a temperature of 33°C. the cone myoids shortened (fig. 36), while at 3°C. or HJ°C. (fgs. 34, 35) the myoids remained for the most part unchanged. These results are identical with those found by Fujita ('11) and myself on the cones of living animals, and indicate that, unlike the pigment cells, the movements of the cones are not dependent upon nervous control. If an influence of the nervous system over these elements exists in the normal animal, it is at least not manifested as is the control over the retinal pigment, in which changes at both high and low temperatures can be interpreted according to the principle of specific energies.
D. EFFECT OF ANAESTHETICS
Various instances have been noted throughout this paper in which the behavior of the retinal pigment and the visual cells, when deprived of their blood supph', cast suspicion upon autoanaesthetization as being the factor causing suspension of movement. Certain conditions discovered in the responses of melanophores in the web of the frog's foot had previously suggested such a possibility; indeed, it was this difficulty which led to the abandonment of the frog's melanophore as material for an investigation somewhat similar to the present one. In this way my interest was aroused to determine the effect of anaesthetics on the movements of the retinal elements, both in normal animals and through the more direct action upon excised eyes.
172 LESLIE B. AREY
The effect of certain drugs, as quinine and strychnine, upon the retinal pigment ('protoplasmagifte') is in dispute. It is clear, however, from the work of Ovio ('95) and of Lodato ('95) that cocaine can arrest pigment migration.
As a precaution against a possible source of error, animals were never introduced from one condition of light or darkness to the other without having been previously subjected to a brief preliminary treatment of the anaesthetic which was to be tested.
a. Retinal pigment
1. Carbon dioxide. The carbon dioxide used in these experiments was a commercial soda-water product sold under the trade name of Tureoxia.' Quantitative determinations of the concentrations used were made by titration with fo sodium carbonate, using phenolphthalein as an indicator.
In the first experiments made on Ameiurus the movement of the pigment was arrested by a strong solution of carbon dioxide, but since none of the animals survived such treatment the obvious objection exists that the pigment cells also may have been killed.
A slight refinement in method consisted of revivifying the fishes at intervals, by temporary removal to running water, until opercular movements were restored. By this method fishes were kept alive for 2 hours, during which time four or five revivifying treatments were necessary. The migration of retinal pigment was shown to be checked both in light and in darkness, yet controls proved that the cells were not permanently injured.
A method which gave more satisfactory results was devised after repeated trials had given a mixture of tap water and carbonated water of sufficient strength to anaesthetize an Ameiurus but not to prohibit opercular movements of greatly reduced amplitude. The record of an experiment will well illustrate both the method and the results.
Experiment 4-i-6- A dark-adapted Ameiurus was placed in a mixture of 1 part of Tureoxia' to 4 parts of tap water, and after re
MOVEMENTS IN THE VISUAL CELLS 173
maining 10 minutes in the dark the jar was removed into strong diffuse dayhght for Ij hours. During this time, the fish was practically motionless except for a very weak but rhythmical pulsation of the opercular rims. At the end of the experiment one eye was removed and fixed. The Ameiurus was allowed to recover until the next day when the other eye was removed. The pigment in the eye which had been subjected to carbon dioxide was in the typical dark position (cf. fig. 4) while the pigment of the control eye was maximally expanded (cf. fig. 2). Titration of the anaesthetizing solution showed that the concentration of carbon dioxide had been in the ratio of 60.14 cc. per litre of water.
In the converse experiment from light to darkness an Ameiurus lived 3 hours in a similar .solution (53.07 cc. of carbon dioxide per litre) during which time the pigment retained its light distribution, whereas the control eye removed on the next day, showed maximal contraction.
These results prove conclusively that in the presence of certain concentrations of carbon dioxide the pigment cells are not injured but are in a condition of anaesthetization whereb}' there is a failure to respond to the normal stimulus causing contraction and expansion. Such experimentation, however, does not show whether this failure is due to a direct effect upon the pigment cells or to an inhibition through the central nervous system.
To demonstrate which alternative is true, the effect of carbon dioxide was tested on excised eyes of Ameiurus. If, under these conditions, a migration occurs a direct influence of the anaesthetic on the cell itself will be disproven, while on the other hand, if no migration ensues one can only infer that a similar direct action on the pigment cell is responsible for the whole course of events in the living fish, whereas an inhibition through the central nervous system may be involved as well.
For such an experiment the excised eye of Ameiurus is well adapted, since its pigment has been shown to migrate from the dark to the light position, although the reverse process does not occur. Excised eyes of dark-adapted fish were exposed to light in a solution of carbon dioxide having a strength of about 60 cc. per litre. The pigment in each case was arrested in the contracted position.
174 LESLIE B. AREY
The work described for living Amciurus has been repeated on both Abramis and Funduhis with identical results. In every case the pigment maintained the position it occupied previous to the application of the anaesthetic.
The results obtained in this study, as a whole, are very different from those of Fick ('90), who concluded that the retinal pigment of the frog expanded when the animals were subjected to an atmosphere of carbon dioxide gas. Fick attributed this result to asphyxiation and it is certain that the experimental conditions in his work differed greatly from those in my tests. In order to make the experiments more comparable, frogs should be treated with a mixture of oxygen and carbon dioxide gases in which they could live.
2. Ether. The anaesthetic effect of ether on the retinal pigment was demonstrated by a series of tests that duplicate those described with carbon dioxide. Care must be observed against using an excess of ether since otherwise a partial or complete disintegration of the pigment cells results.
Both dark and light trials were made on Ameiurus, Abramis, and Fundulus. In each animal the pigment was found to be completely arrested in whatever position it occupied at the beginning of the experiment. Controls proved that ether, if used in small amounts, does not permanently injure the pigment cells.
Ether also checked the migration of pigment in excised eyes of dark-adapted Ameiurus when such eyes were subjected to light.
3. Chloretone and ur ethane. These substances are such satisfactory narcotizing agents that their effect was tested upon the retinal pigment of Ameiurus. Individuals lived in 0.1 per cent chloretone or in 1.0 per cent urethane, but the pigment was not arrested in its movement from the dark to the light phase. In concentrations of 0.5 per cent chloretone and 2.5 per cent urethane, the pigment migrated when fish were brought from darkness to light although the animals died in both cases.
The results from all the foregoing experimentation are of interest in showing the difference in the effect upon pigment cells of four powerful anaesthetics, of which only two were
MOVEMENTS IN THE VISUAL CELLS
175
efficient. The experiments with chloretone and urethane also prove that even though the animal as an organism dies, the pigment, nevertheless, can expand independently.
b. Visual cells
Experiments similar to those just described were repeated in order to determine the action of anaesthetics on both rod and cone cells. Since the cone myoid is maximally elongated at about 25°C. in the dark (figs. 25, 27), this condition was taken advantage of in producing sharp contrasts between dark and light phases. The cones of Abramis and Fundulus, on account of their great contractility, were particularly favorable for observation, as were the rods of Ameiurus becau.se of their large size.
The results of these experiments are shown in table 12.
TABLE 12 A tabulation of the effects of carbon dioxide and ether upon the movements of the visual cells of Ameiurus, Abramis, and Fundulus; X indicates that the movements of the elements were completely arrested; conditions corrcspotidittg to the blankspaces were not investigated
CONE
ROD
Dark to Light
Light to Dark
Dark to Light
Light to Dark
Ameiurus
Abramis
Fundulus
X
X
X
X
X
X
X
X
The conclusion is, therefore, that both ether and carbon dioxide anaesthetize the visual cells of normal fishes to such an extent that neither light nor temperature is efTecti\e in causing positional changes.
A few experiments upon the excised eyes of Ameiurus showed that both carbon dioxide and ether have the same anaesthetic effect on the rods and cones as that described for normal animals.
Whether or not autoanaesthetization prevented movements of the retinal elements, as was suspected in previously described experiments when the normal blood supply was interrupted, it is at least demonstrable that certain anaesthetics do act in a
176 LESLIE B. AREY
similar way. The effect of carbon dioxide is especially interesting for, as it is the commonest catabolic product, it may have been the agent that prevented movements in those cases. This conception is opposed to Dittler's ('07) view, which assumes the existence of a balance in the metabolism of the unstimulated cone cells which is disturbed by the increased catabolism through the action of light. The movement of the cone cells in the isolated retina of the frog was stated by Dittler to be due to the action of a weak free acid, the product of increased catabolism. This conclusion, v/hich was supported by experimental evidence, is opposed to that postulated by me; nevertheless, it must be pointed out that the responses of the retinal elements differ considerably in fishes and in the frog, and while evidence for autoanaesthetization is indirect yet the results obtained from experhnentation upon fishes can be consistently interpreted in this way, whereas Dittler's hypothesis does not meet all the known facts. A discussion of these points was given in another section of this paper.
E. EFFECT OF OXYGEN
Spaeth ('13) showed that the isolated melanophores of Fundulus contract in the absence of oxygen, but contracted melanophores do not expand when oxygen is the only stimulating agent present. Fick ('90) deprived dark-adapted frogs of oxygen by submergence in water or by introducing them into an atmosphere of hydrogen or carbon dioxide. As a result of this treatment he asserts that the retinal pigment underwent expansion. Dittler ('07) states that. in frogs which are about to hibernate the cones are never as fully elongated as in active animals; but after subjection to an atmosphere of pure oxygen the cones can again be obtained in the maximal dark position. It seems probable that in this case the effect of oxygen was indirect, and the increased activity of the cone cells accompanied pari passu the return of other body activities.
In order to test whether or not the amount of oxygen available to a fish in any way controls the distribution of its retinal
MOVEMENTS IN THE VISUAL CELLS 177
pigment, a series of experiments, chiefly upon Ameiurus, were performed.
In experiments involving a reduced oxygen supph-, the apparatus was simple. A 3| litre flask was filled with boiling water. The flask was then closed with a three-hole rubber stopper through which passed, (1) a glass tube extending to the bottom of the flask, which served to introduce gas from a hydrogen generator, (2) a glass overflow tube extending about three-quarters of the way do^v^l the flask, which served chiefly as an outlet for the hydrogen gas, (3) a mercurj' pressure regulator. As soon as the flask of boiling water was stoppered, the hydrogen supply was turned on and as a result, water was forced to escape through the overflow tube, its place being taken by hydrogen gas. When the water level reached the bottom of the overflow tube no more escaped, l:)ut the gas after bubbling through the water did do so and was conductetl through a water trap to the outside air. As the water cooled do\\Ti to room temperature it could not take up oxj'gen since none was present , and furthermore, the bubbhng hydrogen gas tended to expel mechanically any residual oxygen present in the boiled water.
Water containing an excess of oxygen was j^repared by bubbling oxygen gas through water in a flask similar to that described in the former ex])eriment, whence it escaped by means of an overflow tube loading into a water trap. The wat^r used had previously been boiled and reoxygenated by an aquarium aerating device.
Quantitative determinations of the oxygen content were made at the expiration of all experiments by the method of Winkler (Trcadwcll and Hall, '05).
Ameiurus was used in most of the experimentation, although Abramis served in a few cases. The description which follows applies particularly to Ameiurus.
It was possible to reduce the oxygen supply to an amount in which the fish could not live.^' This, for example, happened
'5 The normal oxygen saturation of water at 20°C. is 6.356 cc. per liter (Treadwell and Hall, '05). Boiled water which had been cooled rapidly was found to contain about 0.93 cc. per liter.
178 LESLIE B. A KEY
when only 0.8 cv.. of oxygen per litre was present. On the other hand, in water containing an excess of oxygen (7.5 cc. per litre) respiratory movements of the operculum ceased, the fins appeared reddish in color and respiration may have been largely cutaneous.
In parallel experiments conducted both in the dark and at various light intensities, no difference could be detected in the positions of the pigment or visual cells under the extreme conditions of oxygen supply. This is not surprising, for presumably but little oxygen is needed to permit the cells to function, and since for the success of the experiment, the animal must have enough oxygen with which to keep itself alive, a crucial test involving a complete elimination of oxygen is not possible. Since the pigment can not be made to contract in excised eyes, but only to expand, a decisive experiment in which all oxygen might in this way be eliminated (similar to Spaeth's work on isolated chromatophores) was impossible.
Pigment, rods, and cones respond in a normal fashion when brought from darkness to light or vice versa in water containing the minimum oxygen content, about 0.9-1.0 cc. per litre, in which the Ameiurus can live.
Since no indication was observed of a tendency toward expansion in the retinal pigment cells of fishes which w^ere deprived of oxygen, it is evident that the expansion described by Fick ('90) in dark-adapted frogs whose respiration rate had been reduced by covering the head with a velvet hood, is exceptional. In view of the well known respiratory function of the frog's skin it is possible that Fick's results are open to other interpretations, especially since his experiment, a repetition of the earlier work of Englemann ('85), is not in agreement w^ith the latter's conclusion relative to the absence of movement in the retinal elements when frogs provided with velvet hoods were retained in the dark as controls to other experiments.
The chief value, therefore, of the work done by me is to showthat within normal experimental limits the retinal pigment and visual cells of fishes are not affected by an increased or diminished oxygen supply.
MOVEMENTS IN THE VISUAL CELLS 179
F. INTERRELATION OF INTEGUMENTARY PHOTO-RECEPTORS ANT) RETINAL ELEMENTS
The skin of several lower vertebrates has been sho^^^l to be sensitive to hght. Among the fishes, Eigenmann ('00) stated that certain bhnd forms hving in caves gave motor responses when stimulated by light, the photo-receptors presumably being located in the skin. Parker ('05) followed up some negative results obtained by one of his students on Fundulus by an investigation on ammocoetes, and proved that the integumentary nerves were sensitive to light, causing movements of the animal that were both 'phototropic and photodynamic' A photoreceptivity of the skin of certain other vertebrates was first demonstrated by the following workers: Graber ('84) on Triton: Dubois ('90) on Proteus; Kordnyi ('92) on the frog; Carleton ('03) on Anolis; and Eycleshymer ('08) on Necturus.
Englemann ('85) co\'ercd the heads of dark-adapted frogs with a velvet cap and exposed the bodies to sunlight. Under these conditions, he asserted that in 15 minutes the pigment and cone cells assumed the maximal light position, whereas the same elements in control experiments conducted in the dark remained unchanged. Illumination of the skin for longer periods was said to result in a falling off ('herabsteigen') in the exjxmsion of the retinal pigment and to a weakened response on the part of the cones. From these results he concluded (p. 507) : ". . dass Zapfen und Pigment des Auges von entfeniten Korpergegenden aus reflectorisch in Bewegung gebracht werden konnen."
Fick ('90), in repeating this experiment of Englemann. found the pigment in an expanded condition while the frogs, ready for the test, were still in the dark, and after supplementary experimentation of various kinds he decided that pigment expansion accompanied disturbed respiration. In the case under consideration the velvet hood was supposed to have caused partial asphyxiation.
Koranyi ('92) refers to the similarity in the responses of the retinal pigment resulting either from the illumination of the retina or of the skin only, yet he does not state that he actually observed this condition himself.
180 LESLIE B. AREY
More recently Fujita ('11) asserted that after the head and forward extremities were bound with wet black cloth and the rest of the body was exposed to sunlight for 15 to 20 minutes, the 'eyes' remained in the dark-adapted condition.
I, myself, before learning of Fujita's work, had performed several experiments of the same kind. The head and fore body of dark-adapted frogs were bandaged with many thicknesses of black velvet and the remainder of the body and the hind legs were exposed to direct or diffuse sunlight for periods of 15 minutes to 1 hour. Care was taken to keep the skin moist and to guard against the heating tendency of direct sunlight by using a heat filter. Although the animals were in an active condition at the end of the experiment, there was in no case a distinct change in the position of the cone cells or retinal pigment as Englemann maintained.
In an unpublished investigation by Mr. S. G. Wright, no direct responses were observed when Ameiurus, from which the eyes had been removed, were illuminated with the light of an electric arc. The normal fish, as is well known, is a night feeder, yet it also frequented equally the light and dark halves of an aquarium jar.
It is, conceivable, however, that the soft skin of this animal contains a photoreceptive mechanism, even though the motor responses to light fail to indicate its presence. One of the possible ways in which an integumentary photosensitivity could be manifested is through an influence on the position of the retinal elements, similar to the relation which Englemann believed to exist in the frog. In view of such a possibility several experiments were performed in which dark-adapted fish with bandaged heads were exposed to daylight and to the light of an electric arc for periods of 45 minutes to 1 hour. In no case, however, was there the sHghtest tendency toward movement on the part of the rods, cones, or retinal pigment.
These results indicate that neither in the frog nor in Ameiurus are movements of the retinal pigment or visual cells evoked by a cooperation of dermal photosensitivity and 'retino-motor' nerve fibers. Consequently, Englemann's experiment does not
MOVEMENTS IN THE VISUAL CELLS 181
have the significance that he beheved it possessed, when he attempted to furnish phj^siological proof that in the frog: "Jedenfalls aber laufen .... auch retinomotorische Fasern von den grossen Nervencentren aus durch den Sehnerv zuvi Auge" ('85, p. 506).
4. DISCUSSION
We have so accustomed ourselves to view the phenomena exhibited by living organisms from the evolutionary standpoint that an 'explanation' which will reveal the adaptiveness of an organism to its environment is demanded whenever a system of relations involving constant responses to definite stimulating agents is discovered.
To make dogmatic assertions regarding the presence or absence of adaptation in a set of responses is, obviously, a matter of exceeding danger, j-et if the phenomena exhibited by a series of representati\e animals to definite stimulating agents are shown to be variable, it is at least evident that a single inclusive explanation will not be forthcoming.
The writer has attempted to show elsewhere (Arey '15) that the discontinuous occurrence of photomechanical responses in the visual cells and retinal pigment, both in the various vertebrate classes and among different representatives of certain individual classes, renders it extremely difficult from the adaptational standpoint to devise a satisfactory ox])lanation for the meaning of these movements. The majority of theories which attempt to link the known responses of the retinal elements with the mechanism of light perception are, without doubt, highly speculative, and since for the most part they lack an experiment^al basis of any kind, they must remain of interest only as ingenious and interesting possibilities. In the case of the retinal pigment at least, it is possible to compare the responses to light and darkness with those exhibited by melanophores in general (Parker '06, p. 413), and from our present knowledge we are unable to see either in the reactions of the retinal pigment or in those of the rods and cones anything more
182 LESLIE B. AREY
than the presence of constant protoplasmic responses to a definite stimulating agent.
When the effect of temperature is considered, a lack of uniformity at once becomes apparent.
Among the fishes the efifect of temperature upon the retinal pigment is in agreement with Parker's ('06) general conclusion that in all melanophores, low temperature has the same effect as light and high temperature the same effect as darkness. ^^ In the retina of the living frog where this statement holds true only between the temperatures of 0° and 19°C. in the dark, notwithstanding the identical photic responses of the retinal pigment in fishes and amphibians, it is probable that a nervous control is responsible for the expanded condition both at low and at high temperatures; hence the responses in this animal are not comparable to those in fishes. Since only a limited temperature reaction occurs among fishes, and this in darkness as well as in light, it probably has no immediate adaptive significance, but merely represents the survival of a tendency shown in the responses of melanophores in general. ^^ In homothermous animals temperature, of course, can play no part in the normal activity of the retinal pigment, and even if the temperature responses in poikilothermous animals have an adaptive significance, it is evident that this particular advantage must be unavailable to warm blooded vertebrates, in which keenness of sight is best developed.
Moreover, the action of temperature upon the cone cells in the dark is variable. In the fishes, the cone myoids greatly elongate when warmed and shorten when cooled, while in the frog the cones are maximally shortened at high temperatures only and at all other temperatures remain unchanged.
The inconsistent action of temperature in producing movements of the visual cells apparently has no common adaptive
IS It should be pointed out, however, that in the case of the melanophores of the frog's skin, the reverse of this statement is true, since high temperature produces similar effects to light, and low temperature to darkness.
1^ Below 38°C. temperature has practically no effect on the rate of decomposition of the visual purple, as Kiihne ('79) showed, hence the changes of the pigment through the action of temperature are not related to this phenomenon.
MOVEMENTS IN THE VISUAL CELLS 183
value, and, moreover, as in the case of retinal pigment, variable temperature can play no part in the normal movements of these cells in homothermous animals.
It maj' be asked how it happens that high temperature in the dark produces diametrically opposed results upon the cone myoids of fishes and of the frog, when the responses to light are identical. The nervous system, presumably, is not involved in these reactions since temperature has a similar effect upon excised eyes. Between the minimal and optimal limits the movement of undifferentiated protoplasm is uniformly accelerated with increased temperature, and as has been shown in another place (p. 135) the responses in length of the cones of Abramis are markedly similar in this respect. Whatever the details of the process may be, it seems evident that among the fishes tem])erature acts purely as a physical agent in controlling the velocity of the reactions leading to positional changes of the cones. In frogs temperature apparently acts in an entirely different manner. The fact that only high temperature is effective in producing a change, a shortening of the myoid, is best explained on the basis of Dittler's theory of chemical stimulation, whereby increased tem])erature can be conceived of as favoring the formation of catabolic wastes which chemically stimulate the cones to shorten, while low temperature probably acts both in retarding catabolism, and by reducing the sensitivity of the myoid toward such products as are fonned even under these conditions.
A comparative study of the responses of the visual cells, throughout the various classes, to light and temperature reveals difficulties in explaining the mechanism by which their positional changes are accomplished. Why do the rod cells of some animals shorten in the light whereas others lengthen? Why do the rods of fishes and of birds lengthen through the action of light, whereas the cones shorten?
From the physiology of simple protoplasm, two alternatives are open. Either the myoids of the visual cells have become specialized to respond to certain stimulating agents in different ways, or, in various retinas the morphological structure of these
184 LESLIE B. AREY
contractile regions is such, that although the protoplasm responds similarly in all cases, the visible result upon the position of the entire cell is variable. The latter alternative would be realized if the active protoplasm were differentiated into analogues of myofibrillae which were arranged in some cases axially in the myoid and in other cases transversely or spirally. To become effective in elongating the myoid through contraction, a simple spiral 'myofibrir would have to make an angle greater than 45 degrees with the long axis of the myoid. ^^
In attempting to interpret the adaptiveness of the movements of the vertebrate retinal elements, it is evident from the foregoing discussion that neither with respect to the action of temperature nor of light has a satisfactory and constructive conclusion been reached. From the present state of our knowledge, therefore, the situation may be summarized in the following way. Although the movements of the visual cells and retinal pigment, when present, may have a certain unknown significance in connection with the mechanism of light perception, such movements can be interpreted at present only in terms of protoplasmic responses to definite stimulating agents.
5. SUMMARY
1. The retinal pigment of the fishes studied requires 45 minutes to 1 hour for light adaption and from 30 minutes to 1 hour for dark adaption. The cones of Abramis assume the light (shortened) position in 45 minutes, the dark (elongated) position in 30 minutes. Maximal elongation of the rods of Ameiurus in the light occurs in 45 minutes, maximal shortening in darkness in 30 minutes.
2. Both in light and in darkness, the retinal pigment of fishes shows greater expansion at a low than at a high temperature. High temperature is apparently more efficient in causing this
1* In connection with these possibilities mention will be made of only the fibrils found by Hesse ('04) in both the inner and outer members of the rods and cones of several vertebrates, and of the longitudinal fibrils which Howard ('08) was able to trace throughout both the rods and cones of Necturus. Both workers, however, considered such structures as neuroid in character.
MOVEMENTS IN THE VISUAL CELLS 185
redistribution of pigment than is low temperature. The results obtained from the four fishes studied indicate that extreme conditions both of expansion and of contraction are not to be found in the retinal pigment of any one fish.
3. In darkness, the retinal pigment of the frog undergoes striking expansion l)etween the temperatures of 0° to 14°C. and 19° to 33°C., whereas at the intermediate temperatures of 14° to 19°C. it is highly contracted. Temperature is without effect upon light-adapted retinas. Shice the retinal pigment of the larvae of Ran a catesbiana shows temperature responses identical with those characteristic of adults, there is no temporary- lar^'al recapitulation of the responses characteristic of fishes.
4. Light and darkness protluce but limited changes in the distribution of the retinal pigment of Necturus. No definite effect of temperature could be detected. It is ])robable that the peculiar res])onses found in the frog have been developed within the anuran grou]).
5. The cone inyoids ot' fishes sjiortcn at low temperatures in the dark: at liigh temperatures they lengtlieu. Not only is elongation i)rogressive, extreme conditions Ix'ing found at 0° and 30° ± C, but also the rate of change is directly proportional to the tem])evature gradient. Tem])erature is iiieffectual in the light.
(). The myoid of the rods of fishes also elongates at high tem])eratures and shortens at low temjieratures, but the extent of change is mucli less than that of cone cells.
7. The cone myoiil of adult frogs in the dark shortens when subjected to a temperature of 19° to 33°C., but remains elongated at all lower temperatures. Xo definite temperature responses of the cone myoids were found in the larwae of Rana catesbiana.
8. A correlation between the length of the rotl myoid and temperature was not detected in the frog either in darkness or in light.
9. The positions of the visual cells of Necturus are not affected by temperatures l)etween the limits of 0° and 28°C.
THE JOIRNAL OF CO.MP A HATI \ E NEUKOI.OOY. \ OI.. 2<i, NO. 2
186 LESLIE M. AREY
10. Ill excised eyes of \\w four fishes studied, light causes a migration of the retinal pigment in Ameiurus only, whereas the pigment of none of the fishes moves in darkness. The rods of excised eyes of Ameiurus undergo movements both in the light and in the dark, the cones move in the light only. Neither exposure to darkness nor to light produces positional changes in the cone cells of excised eyes of Abramis orFundulus. Where tested, temperature was found to cause movements in the retinal pigment and cone cells in the excised eyes of fishes.
11. It is probable that the absence of responses in the excised eyes of fishes is due to an autoanaesthetization caused by the accumulation of catabolic products.
12. Dittler's theory of the chemical stimulation of the cone myoid, propounded to explain the movements of the cone cells in isolated frog's retinas, does not satisfactorily meet many conditions found in the responses of the cones of fishes.
13. The effects of temperature upon the rods, cones, and retinal pigment of the excised eyes of fishes are identical with those found in living animals, hence it is probable that temperature has a direct action upon these elements, its effect being physical in the sense that the chemical activity of the protoplasm is thereby accelerated to varying degrees.
14. Temperature has no effect upon the retinal pigment of the excised eye of the frog, therefore it is plausible that the action of temperature in living animals is physiological, whereby any adequate stimulus acting through the central nervous system can produce a striking pigment expansion according to the principle of specific energies. As in living animals the cone cell of the excised frog's eye responds by a shortening at an elevated temperature only ; it is probable that temperature acts directly upon the cone myoid, for this response, unlike that of the pigment, can not be interpreted by the principle of specific energies.
15. Neither in the frog nor in Ameiurus are movements of the retinal elements evoked by exposure of the skin only to light. Hence the existence of an interrelation between dermal photosensitivity and the responses of the retinal elements by
MOVEMENTS IX THE VISUAL CELLS 187
means of 'retino-motor' nerve fibers, as maintained by Englemann, is not substantiated.
16. Within the experimental limits at which fishes can be kept alive, the retinal pigment and visual cells are not affected by an increased or diminished oxygen supply.
17. Both in darkness and in light, and in excised as well as in normal eyes, carbon dioxide and ether completely check the movements of all the rethial elements of fishes. Chloretone and urethane, on the contrary, are inefficient hi this respect. The action of carbon dioxide suggests that this may be the catabolic ])roduct that in many cases restrains the movements of the retinal elements when the circulation of the blood is interrujited.
IS. Although the movements of the visual cells and rethial pigment, when ])resent, may have a certain unknown significance in connection with the mechanism of light perception, such movements can be inteipreted at present only in terms of ]irotoplasmic responses to definite stimulating agents.
(\nfibri(l(/c, Mass., April 10, 1915.
I'OSTSrUII'T
A study of the inllucncc of Hght on the in()\enients of the frog's rod has just been completed by the writer. Careful measurements prove that these elements are extended in the light and are retracted in darkness. Hence the results of the older workers (p. 123), who believed that the jihotic responses of the frog's rodmyoid are the re\-erse of those occurring in fishes and birds, are not subslaiitiated.
188 LKSLIE B. AREY
Paperf! innrkcd irith <ni <if<trrisl: hare mil hccii itcccf<sihle in the original.
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ITaf.
MOVEMENTS IX THE VISUAL CELLS 189
Dubois, K. 1890 Sur la perception des radiations lumineuses par la peau,
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2. Bd. 3. Kail. 12, Anhang, .30 pp.. 5 Taf., 49 Textfig. (iuAHKH. \". 1S84 tJruntllinien zur Erforschung desHelligkeits- und l'arl>ensin ncs der Tiere. Prag u. Leipzig. 8 vo, viii + 322 jtp. (lUADENicuo, (1. 1885 Cber den Einfluss des Lichtes und der Wiirme auf die
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- Hambuuc;eu, D. J. 1889 Dorsnijiling van der rervus opticus bij Kikvorschen,
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KXI'F.AXATIOX OF PLATES
The figures of Plates 1 to 4 are photomicroKraiilis; tho fiRurcs of Plate 5 were drawn with the aid of a camera luc-ida.
AHBl{i;\ lATIONS
bac, rod con., cone
con. ace, accessory cone ell.hac, rod ellipsoid ell.con., cone ellijisoid glt.ol., oil globule mb.lim.e.v., external liniil my. bac, rod niyoid
ineinl)
my. con., cone niyoid
pd.cl.pig., base of pigment cell
pr.'^.d.'tl.bac., rod outer member
pr.'^.chl.con., cone outer member
rln., retina
.■*(•/., sclera
.<it.til.cx., external nuclear layer
Kt.pig., pigment layer
191
PLATE 1
EXPLANATION OF FIGURES
The photographs in this plate, which show the influence of temperature on the distribution of the retinal pigment of fishes, are all at a magnification of 170 diameters.
1. Ameiurus, 5°C. in the light.
2. Ameiurus, 25°C. in the light.
3. Ameiurus, 5°C. in the dark.
4. Ameiurus, 25°C. in the dark.
5. Fundulus, 5°C. in the light.
6. Fundulus, 25°C. in the light.
7. Fundulus, 5°C. in the dark.
8. Fundulus, 25°C. in the dark.
192
PLATE 2
EXPLANATION OF FIGURES
The photographs in this plate, which show the influence of temperature on the distribution of the retinal pigment of fishes, are all at a magnification of 170 diameters.
9 Abramis, 5°C. in the light.
10 Abramis, 25°C. in the light.
11 Abramis, 5°C. in the dark.
12 Abramis, 25°C. in the dark.
13 Carassius, 5°C. in the light.
14 Carassius, 25°C. in the light.
15 Carassius, 5°C. in the dark.
16 Carassius, 25°C. in the dark.
194
MOVEMENTS IN' THE VISUAL CELLS
I KSME B. AREY
9
10
-St i>i'j.-
^t.hac.corii:
lib. I ill I. a,
rlnr---
12
13
14
15
16
195
PLATF] 3
EXPLANATION OF FIGURES
Figures 17 tu '22, wliich are photographs showing the iriHucuce of temperature on the distribution of the retinal pigment of the frog (adults and larvae), are all at a magnification of 170 diameters. The larval Rana catesbiana, from which figures 20 to 22 were made, had a total body length of 7.0 cm. A Leitz /o homogeneous immersion objective was used in making figures 23 and 24, which are magnified 715 diameters.
17 R. pipiens (adult), 3°C. in the dark.
18 R. pipiens (adult), 18°C. in the dark. 1!) R. pipiens (adult), 33°C. in the dark.
20 R. catesbiana (larva), 3°C. in the dark.
21 R. catesbiana (larva), 16°C. in the dark.
22 R. catesbiana (larva). 32°C. in the dark.
23 Carassius, 27°C. in the dark.
24 Carassius, 3°C in the dark.
196
PLATE 4
EXPLANATION OF FIGURES
These photographs, taken with a Leitz -^2 homogeneous immersion objective, are at a magnification of 715 diameters and show the responses of the myoids of the cone cells of fishes when under the influence of temperature.
25 Abramis, 3°C. in the dark.
26 Abramis, 15°C. in the dark.
27 Abramis, 27°C. in the dark.
28 Fundulus, 3°C. in the dark.
29 Fundulus, 27°C. in the dark.
198
MOVEMKNTS IN THE VISUAL CELLS
LESLIE B. AREY
St. piy.
1
^>« - • f II con;
"m%\ » • r"^^ mh.ltm.cz
^^:, :::.'■'■■
25 cUcon:
r-f»*i '-^i*! Jf' J 1 '"^ "'" "•
^m ■'
^L k ell. con.
ell. bac•C iffj,/^ . /nb./.m.eo:.
27
199
)>■
- f^^ . ^
^:»i!ij
26
1 i . 1
29
PLATE 5
EXPLANATION OF FIGURES
All drawing;s in this plate are at a magnification of 930 diameters, a Leitz -jV homogeneous immersion objective being used.
30 Showing the positions of the rods and cones in a typical light-adapted retina of Ameiurus.
31 Showing the positions of the rods and cones in a typical dark-adapted retina of Ameiurus.
32 Showing the effect of low temperature (3°C. ) on the position of the dnrkadapted rod- and cone-cells of Ameiurus.
33 Showing the efTect of high temperature (27°C.) on the i)osition of the darkadapted rod- and cone-cells of Ameiurus.
34 From the retina of a Rana pipiens that had ])een previously kept at a temperature of 3°C. in the dark. The cone myoids remain elongated as at an intermediate temperature.
35 From the retina of a Rana pipiens that had previously l)een kept at 18°C. in the dark. The cone myoids are elongated.
36 From the retina of a Rana pipiens that had been kept at 33°C. in the dark, showing the resulting shortening of the cone myoids.
200
-MOVT^MENTS I.N THE VISUAL CELLS
I.ESr.IE B. AREY
^.'Prs.dst.bac.
prs.dst.bac.
36
prs.dst.con^
prs.dst.con.
32
33
201
THE JOUBNAI, OF COMPAn
VTIX E XElKOI.Oin
rfx;exeratiox tx the braix of amrlystoma
I. THE REGENERATION OF THE FOREBRAIN
H. SAXTOX BURR
The Audtinnicdl Lnhoratori/ of The School of Medicine, Yale Cniversity
KOIH FIOl'RES
Up to the present time the data deahng with regeneration in the central nervous system have been exceedingly conflicting. The early workers reported the regeneration of \ari()iis definitive parts after they were extir])ated. As far hack as 1890 Danielewsky found that the removal of the cerebral hemispheres of the frog residtcd in the formation of a 'cerebral mass' which he believed contained embiyonic nerve cells, though this mass was in no way a new licinisphci-c. More recently Bell, in 1007, removed the "lateral half" of the brain of the frog and found that the brain nearly always is rcfornuMl though never does it reach the normal size.
On the other hand. Schaper ('98) found tiiat the icinoval of ])ortions or of the entire brain of Rana esculenta was never followed by regeneration. Rubin in 190;^ working with Rana fusca larvae found that no regeneration occurred aftei- the removal of the brain or of ])arts (^f it.
The results of experiments published elsewhere (Burr '10) show conclusively that the nasal placode of Ambly stoma and also of the frog does not regenerate when it is com})letely removed. In addition, the large amount of work by Lewis and others has shown that the eye ^^^ll not regenerate when all of the anlage is remo\ed. It was deemed probable, therefore, that complete extirpation of the cerebral hemisphere would not be followed by even a partial regeneration of the part removed. The discrejiancy in the results previously reported might be due to incomplete operations. The regeneration of parts of
203
204 H. SAXTON BURR
the brain would then be due to the fact that the entire substance of the hemisphere was not removed, enough being left to carry the regeneration through to a considerable degree of completeness. A condition similar to this has been shown by Harrison to exist in the extirpation of limb buds in Amblystoma (Harrison '15).
So far as it has been possible to ascertain, no attempt has previously been made to control the stimulus afforded by the functional activity of the end organ normally connected with the part of the central nervous system removed. Extirpation of a portion of the brain has invariably carried with it the removal of the end organ. It is a little difficult then to see why a part should regenerate when its activity has ceased owing to the removal of the end organ.
In order to obtain some answer to the above question the following experiments were performed on Amblystoma larvae. Two series of operations were undertaken on embryos possessing neither peripheral nerves nor a circulatory system. In the first of these the right cerebral hemisphere and the right nasal placode were extirpated, the cut that severed the hemisphere from its connections passing directly in front of the optic stalk. In the second series the right telencephalon was removed but the right nasal placode was left in position. This was accomplished by turning back the flap of skin containing the placode and removing the underlying forebrain. The flap was then returned to its former position and held in place until the wound had healed. Great care was taken to remove all of the cells of the cerebral hemisphere in all the operations.
The above experiments subject the brain tissue left by the extirpation of the hemisphere to two conditions. In the first series of operations the nervous tissue is left to regenerate without the possibility of any stimulus from the end organ that is normally connected with it. In the second series the end organ, the nasal placode, is left in its normal position and may therefore act as a stimulus to the nervous tissue (Burr '16).
In the first series of experiments in which the right hemisphere and the right nasal placode were removed the wound usually
BRAIN REGENERATION IN AMBLYSTOMA
205
healed within the first twenty-four hours. Five days after the operation a new wall had formed connecting the wall of the right diencephalon with the wall of the left telencephalon. At first this wall consists of a nari'ow band of new cells bridging the interventricular foramen. As growth proceeds the narrow
Fifl. 1 Transverse section of embryo five days after operation, hy which
the cerebral hemisphere was removed, leaving the nasal placode in place, .-^howinp: curtain of cells across the interventricular foramen. X 50.
band of cells is drawn (nit into a thin plate, never more than two or three cells thick, stretching across the foramen (figs. 1 and 3). A careful inspection of a series of operated larvae makes it (juite clear that the new tissue thus formed is derived from the primary ependymal cells that line the neural tube. Figure 2 dra^^^l from a section of an operated larva of the second series shows the origin of this new tissue from the margin of the dien
2()()
H. SAXTON liVlUi
('(>))lial()ii. It is ('()inj)()S('(l (Mitirclx' of tlic ty])ic;il colutuiiai' cells thai make u]) the opeiulyiiui. As j»;r()vvth ])r()ceeds the cells of the lunv nieiiil)nine lose their coluiiniar shape and become metainor])hose(l into flattened quadrilateral cells. In the three months old larva, a section of whose brain is shown in figure 3, it is evident that this metamorphosis is accompanied by a thinning of the tissue so that at this time the membrane is
Fig
the same operation „
diencephalon. X 50.
'U^-^ ^ ^^
Portion of the regenerated curtain of an embryo seven days aiier in figure 1, showing regeneration from the margin of the
after
composed of only a single layer of cells whose somewhat elongated nuclei are parallel to the surface.
It is evident from the above that the regenerated portion is not made up of nerve cells but rather of the primitive germinal epithelium which lines the neural tube, the only regeneration that takes place being that necessary to close the wound made by the removal of the hemisphere. We have then, forming as a result of the operation, a curtain of primary ependyma across the interventricular foramen. This is not nervous and hence constitutes no true regeneration of the telencephalon.
BRAIN REGENERATIOX IX AMBLYSTUMA
207
Hardesty ('04) has showii that the dividmg elements of the primary ependyma, the germmal cells, give rise to a nuclear layer just outside of the epend>ina from which later develop the neuroblasts and the spongioblasts. It is c(mcei\able that the differentiation of the nuclear layer into ner\e cells results from the stimulus derived from the ingrowth of nerve fibers
Fig. 3 TransvcM-so section of embryo without the right heiiiispluTc or nasal
placode three months after tlie operation, showing the thin ( urtain across tlie
interventricular foramcii. X 50.
from othei- definiti\e ])arts of the nervous system. Evidence of this is seen in the fact that the fiber tracts of the telencephalon do not develop until the olfactory nerve fibers have grown into the peripheral part of the hemis])here from the nasal placode. The possibility of such a stimulus has been used by Kappers ('14) in his neuro-biotaxis theory of the phylogenetic migration of nuclear centers in the central nervous system.
208 H. SAXTON BITRK
On thv other hand Harrison ('10) has shown that embryonicnerve cells taken from the medullary cord of frog larvae will produce protoplasmic nerve processes when cultivated in vitro, but it is manifestly impossible to determine by such methods the extent to which development will proceed without the intervention of functional activity. As has been shown elsewhere (Burr '16), development and differentiation of the central nervous system will progress to a certain point without the presence of a functioning end organ. Beyond this point functional activity is essential for continued growth. In the experiments just referred to, the telencephalon was quite completely organized without the ingrowth of the olfactory fibers, but this nerve is not the only one whose fibers penetrate the hemisphere, for Herrick ('10) has shown that in the posterior third of the telencephalon of Amblystoma there are at least two centers that are directly connected with the hypothalamus and the pars dorsalis thalami by ascending projection fibers. Hence it seems probable that the apparent self-differentiation of the telencephalon may be due in some degree to the presence of the forward growing nerve fibers which establish this connection, the functional stimulus imparted by them being sufficient to carry differentiation some distance. Experimental evidence that is in favor of this view is seen in the fact that in the posterior margin of the curtain which develops across the foramen of Monro on the removal of the telencephalon and nasal placode, there appears in the older larvae a rounded mass of nerve cells and fibers. This mass is in direct communication with the nucleus habenulae and the hypothalamus, a tract of fibers reaching it from each of these regions. Evidently the neuroblasts at the posterior margin of the interventricular foramen have been stimulated to further development by the ingrowth of axones from lower centers. This mass is, then, in all probability the rudiment of the primitive pallium.
The second series of experiments involved the removal of the right hemisphere without the removal of the nasal placode. The results here are definite and conclusive. A curtain of ependymal cells is formed across the foramen of Monro as in the
BRAIN REGENERATION IN AMBLYSTOMA 209
previous exiDerinient. The healing of the wound brings the nasal placode back to approximately its normal position though a slight shifting anteriorly has been noted. Five days after the operation the curtain is composed of a single layer of cells (fig. 1). Two days later this single layer of cells has become converted into a thickened mass of colmnnar cells that are evidently of ependymal origin (fg. 2). By the time the twelfth day is reached the olfactory- nerve has established a connection with the curtain, which has in the mean time increased considerably in size. The establishment of the nervous connection betw(^en the nasal placode and the telencephalon takes ]ilace iu the noi-mal larva about the tenth day, only a slight delay in this union occumng as a result of the operation. In the unoperated larvae the nasal placode lies in close apposition to the thick wall of the telencei)halon, the olfactory fibers growhig into the hemisphere at the ])ohit of contact. In the oi)erated foil lis the nasal anlage lies close to the newly formed curtahi. which as nulicated al)ove shows a slight thickening not seen in the operated larvae possessing no nasal placode. This thickening anticipates the ingrowth of the olfactoiy nerve by several days. It is not possible at lliis tiuu^ to give any explanation of this thickening, though it is conceivable that the presence of the olfactory anlage stimulates the ependymal cells to grow and divide through some mechanical or chemical factor.
Two days after the ingrowth of the olfactory ner\e the new telencephalon, as it must now be called, has reached quite an advanced stage of de\'eloi)ment. During these two days the thickened curtain has l)een differentiated into a small but nevertheless typical telencei^halon. Neuroblasts are present and a ventricle has ap]ieared. Outwardly the form has changed. A forward growth has converted the thick flat plate of cells into an ovoid mass of neuroblasts and spongioblasts. The ingrowth of the olfactory nerve on the lateral aspect of the mass is accompanied by the appearance of the large lateral forebrain tract (fig. 4). Almost sinuiltaneously there appears from the dorsal aspect of the olfactory bulb the tractus olfactorius dorsolateralis and from the ventral aspect the tractus olfactorius
210
H. SAXTON BURR
medialis. At tliis time the new telen('0])halon is similar in organization to its follow but it is considerably smaller in size. From tliis tiiiu^ on •growth and differentiation continue in both hemispheres hi an entirely normal fashion, though apparently there is to some extent a compensating increase in the rate of growth of the ncnv t(4encephal(m foi- in tlu^ oldest larva of the
Fig. 4 Transverse section of an embryo one and one-half months after the
operation showing the regenerating telencephalon as a result of the presence of
the nasal placode. X 44.
series, a larva some three and a half months old, the new hemisphere had completely regenerated and could not be in any way differentiated from its fellow.
SUMMARY
It is evident from the above experiments that the forebrain of Amblystoma will not regenerate when it and its functional end organ are completely extirpated. The healing of the wound
BRAIN REGENERATION IN AMBLYSTO.MA 211
results in the formation of a curtain across the interventricular foramen derived from the epend\nna lining the neural tube. If, on the other hand, the forebrain is removed without removing the end organ, the nasal placode, the presence of the latter acts as a stimulus to the regeneration of a new telencephalon through the ingrowth of the olfactory nerve. The pallial region of the telencephalon is regenerated in all cases owing to the stimulus afforded by the forward growth of axonos from lower centers in the brain and cord.
LITKHATURK CITED
Bell, Iv '1'. l'.K)7 Some (•xixTiincnts on the dovflopmont ami rcnonoratioii of
the eye and nasal orsan in frof; onihrvos. Arch. f. ICntw.-Moch..
B(l. 23. S. 457. ]U HH. II. S. 1910 Tho offoct of Iho removal of the nasal placocle.s on Anit)ly stoma enihiyos. .Jour. Kxi). Zool.. vol. 20. p. 27. l).\Mi:i.i:\vsKV. H. IWO (M)er die Hepeneration der (Jrosshirn Henii.-^ijharcn
luini Fro^icli Veih. X. inter, med. Kongr., Bd. 2. IIaudkstv. I. I'.IOl On th(> developin(Mit and nature of ncuroplia. .\ni. Jour.
.Vnat., vol. \A. j). 22'.t. IIai{HIs()n, 1{. C. I'.tio The o\itgro\vlii of liie nerve fit)er a.s a mode of proto jihisniif movement. .lour. Exp. Zcx)!.. vol. i). p. 7S7. IIkuuk K. (". .1. lini) The morphology of the forehrain in Ampliil)ia and Hej) tilia. .lour. Comi). Xeur., vol. 20, p. 413. K.MM'KKs, ('. U. AaiENs. 1914 Weitcre .Mitteihmpen ui>er Neurol>iotaxi.-<.
\lll. Folia-Xeurol)io.. Bd. S. S. .3S3 Lkwi.-^. W. II. 1901 llxiMM-imental studies on the development of tlie eye in
.\mi)hil)ia. .Vm. .Jour. .\nat.. vol. 3. p. .50.5. RrniN. H. 1903 Beziehungdes Xcrvensystems zur Hepeneration hei .\mi)hil)ien.
Inaug. Dissert. Hostoek. Scu.M'KU, .\. 1898 Experimentelle Stmlien an Amphil.ienlarven. .\reh. f.
i;nt\v.-Meeh., Bd. (i. S. l.-)l.
CONTRIBUTIONS FROM THE ZOai.OGICAL LABOBATOBY OF THE MTI8EDM OF COMP.VBATIVE ZOOLOGY AT HABVABD COLLEGE. NO. 276.
THE FUNCTION OF THE EFFERENT FIBERS OF THE OPTIC NERVE OF FISHES
LESLIE B. AREY
Northwestern University Medical School
TWELVE FIGURES (tWO PLATES
CONTENTS
Preliminary 213
Statement of the problem 215
Material and teciinical methods 217
Experimental part 218
a. Experimentation upon Ameiurus 218
1. Retinal pigment 21fl
2. Visual cells 230
b. Experimentation upon Abramis and Kundulus 233
Theorot cal considerations 234
Summary 239
Bibliography 240
PRELIM IN ARY
A number of observations have been recorded, chiefly upon the frog, which indicate that the retinal pigment and i^isual cells are at least partially under the control of the central nervous system, although the manner and extent of this influence are not well luiderstood. An interrelation between the retinal elements of the two eyes has also been maintained by which stimulating agents, such as light or salt crystals, appUed to one retina induce changes^ in the other. It has not only been declared by
' Extensive experimentation has demonstrated the existence of photomechanical responses in the retinal pigment of most of the lower vertebrates. In all cases in which movements of the retinal pigment are demonstrable, light causes an expansion (i.e. a migration toward the external limiting membrane), and darkness a contraction of the pigment (figs. 1 and 4). A portion of the cone's inner member, to which the appropriate term 'myoid' has been applied, is also capable of actively shortening when the retina is stimulated by light, a compensatory
213
THE JOURNAL OF COMPARATIVE NEUROLOOY, VOL. 26, NO. .3, JUNE, 1916
214 LESLIE B. AREY
some workers that tliis interrelation is independent of the brain, but also tliat it continues after the optic nerve is cut distal to the chiasjua. The latter assertion, however, has been the subject of much controversy.
Observations on the frog relative to these conditions have • been presented by the following workers: Englemann ('85), Grijns ('91) ; Nahmmacher ('93) ; Angelucci ('90; '05); Lodato e Pirrone ('01); Chiarini ('04) and Herzog ('05). Similar statements were made by Pergens ('96) for fishes, and by Birch-Hirschfeld ('06) for the pigeon. The diversity of stimuli which have been reported as being effective in producing positional^ changes in the cones and pigment has caused Pick ('89; '90; '91) to doubt the reflex nature of the process, although some of his own experiments by no means disprove many of the contentions of the other investigators. More recently Fujita ('11) has shown wherein the older experimentation on the interdependence in the responses of the elements of the two eyes through the action of light, is not trustworthy.
Englemann ('85), who was the pioneer in asserting the presence of 'retino-motor' nerve fibers, further supported his view with results obtained by illuminating the skin only of darkadapted frogs, whence changes in the cone cells and retinal pigment were said to occur. This observation, however, is not in agreement with those of Fujita ('11) and myself (Arey, '16).
A number of other statements are on record which ascribe a control over the movements of these elements to the central nervous system. An enumeration of stimulating agents supposed to act in this way would include such as the following: noises, unilateral pressure on the eyeball, mechanical irritation (Angelucci, '90), and trussing up a frog for 24 hours (Herzog, '05).
The first direct experimentation in which the relation of the optic nerve to movements of the retinal elements was tested
elongation taking place when the retina is again subjected to darkness (figs. 10 and 11). In fishes and birds, at least, the rod myoid is likewise contractile (figs. 10 and 11), although the direction of movement in light and in darkness is the exact reverse of that executed by the cones. For a review of the literature on this question, reference may be made to an earlier paper by the writer (Arey, '15).
EFFERENT FIBERS OF THE OPTIC NERVE 215
was performed by Hamburger ('89), whose results were corroborated by Fick ('91). After the optic chiasma of the frog was severed, light- and dark-adaption occurred as in normal animals, hence the pigment and cone cells were viewed as independent structures, responding to direct stimulation, the movements of which are not dependent upon the mtegrity of the optic ner\'e. Arcoleo ('90) likewise foiuid that the retinal elements of the pithed toad exhibited ]ihotomechanical changes. On the other hand,* Nahnunacher ('93) showed that stimulation of the frog's optic chiasma with salt crystals induced clianges in the cones, only if the optic nerve was intact.
Hence it seems probable that, in the frog, the retinal elements are capable of more or less intle])endent n\ovemeiit, but over this is superhnposed a nervous (efferent) control, the nature of which is not altogether evident.
Apart from the work of Pergens ('96), who l)elieved that the illumination of one eye of a fish induced cone contraction m the other, practically no attempt has been made to detennine the conditions under which movements of the retinal elements of fishes are accomj^lished. The negative results of Ciertz ('11) concerning the etfeet of electrical stuuulation of the eyes of Abramis brama differ only sliglitly from those of Fujita ('11), who, however, maintained that intluction shocks caused an insignificant pigment contraction in the light-adapted eye of the 'Weissfisch.'
STATEMENT OF THE PROBLEM
It has been shown that the retinal pigment of most fishes expands to a marked degree when the normal animal is brought from a situation oi darkness to one of light, and that tlie pigment again contracts when the procedure is reversed (figs. 1 and 4).
The writer (Arey, '16) has also found that the pigment in the excised eyes of Ameiurus, at least, is capable of expanding when subjected to light, which, in this case, presumably acts as a direct stunulus on the pigment cells; a contraction in darkness, how
216 LESLIE B. AREY
ever, does not occur. In several other fishes (Abramis ; Fundulus ; Carasshis) a general pigment migration could not be demonstrated under these conditions even in the light, and such a lack of response was accounted for by postulating the occurrence of an autoanaesthetization of the pigment cells, presumably through the accumulation of catabolic wastes. Furthermore, the rods of the excised eyes of Ameiurus undergo movements both in the Hght and in the dark, whereas the cones move in the hght only; yet neither darkness nor light induces positional changes of the cones in the excised eyes of certain other fishes (Abramis and Fundulus). As will be seen farther on, the total absence of .response in the excised eyes of these fishes throws important light upon the question of a probable autoanaesthetization of the retinal elements.
If the optic nerve transmits afferent impulses exclusively, as hitherto has been believed, no disturbance in the movement of the retinal pigment should be introduced when the optic nerve only is severed, and the retina is thereby freed from this source of cranial innervation. The present paper will be devoted to the description of experiments devised to test the validity of this hypothesis.
In order that the reader may better interpret the various experiments about to be described, as well as the significance of their progressive sequence, it is advisable to state in advance the thesis which this paper aims to establish. Experimental evidence will be presented which offers physiological proof for the existence in Ameiurus of two distinct components of a mechanism, through the balanced action of which are movements of the visual cells and retinal pigment alone possible. One component involves the efferent nerve fibers of the optic nerve, whereas the second component (possibly the ciliary, autonomic, nerves) is closely associated with the eye muscles. This latter set of nerve fibers exerts a constant inhibition upon the movements of the retinal elements, while the impulses in the efferent optic nerve fibers, on the other hand, serve only as a block to this tonic inhibition, thus allowing photomechanical responses to occur.
EFFERENT FIBERS OF THE OPTIC NERVE 217
The following paper presents one phase of an investigation upon the visual cells and retinal pigment, pursued at Harvard University under the super\-ision of Prof. G. H. Parker. To Professor Parker I am greatly indebted for the continued interest and kindly criticism that characterizes his instruction.
MATERIAL AND TECHNICAL METHODS
The fishes used in this in\'ostigation were as follows : the common horned pout, Ameiurus nebulosus Lesueur; the shiner, Abramis crysoleucas Mitchill; and the coinmon kilUfish, Fundulus heterocUtus Linn. A greater part of the experimentation was done upon .Ameiurus, since in this animal the activities of the retinal pigment and visual cells proved to be especially favorable imder the experimental conditions imposed upon thorn. Furthennore, the occurrence of responses in the excised eyes of Ameiurus was a happy concomitant circumstance, since the results were thereby controlled at several important points.
The technical methods used in preparhig retinas for microscopical examiniition were sun]:)lo. The eyes of Ameiurus were excised directly, for the skiji of this annual is soft and the eyes are prominent. In the two other fishes, es])ecially when rapidity of o]:)eration was desirable, the following procedure was obser\'ed. With hea\'y scissors the cranium was bisected in the sagittal plane; following this, a transverse cut just posterior to the orbit freed the two halves of the cranium, with the contained eyes, from the rest of the body. In either case the operation was performed in a few seconds, and the o>-e, without being handled, was allowed to drop into the fixhig fluitl.
Perenyi's fluid gave good fixation and was used exclusively. Fixatives containing nitric acid have long been recognized as favorable in the obtainment of faithful presentation of the retina. The preparatoiy steps prior to embedding in paraffine demand generous allowances of time, yet the processes of dehydration and clearing should progress as rapidly as possible, since otherwise the sclera becomes extremely hard.
Two methods were used in removing the lens, one of which, although longer, gave much more satisfactory results. The first,
218 LESLIE B. AREY
somewhat tedious, ])roccdure consisted in j^aring away the front face of the eyeball with a razor after the eye had previously been imbedded in paraffine. After removing the face of the eyeball slightly beyond the ora serrata, the lens was pried from its paraffine matrix with a dissecting needle; following such manipulation reimbedding was of course necessary. The second and simpler method was to remove the face of the eyeball with fine curved scissors after the eye had been hardened in absolute alcohol; unless, however, the eye was sufficiently hardened and the greatest care exercised, the retina proper easily separated from the pigmented epithelium. On the whole, the first method was preferred to the second because of the wrinkling of the retina that usually accompanied the use of the latter.
Sections were cut 7^t to lOju thick, and only those passing through the region of the optic nerve were retained for examination. Preparations were stained with Ehrlich-Biondi's triple stain or were double stained in Heidenhain's iron haematoxylin and a plasma counterstain. Ehrlich-Biondi in some instances gave excellent differentiation of all elements, while at other times it would show the capriciousness for which it is notorious ; iron haematoxylin gave uniformly good preparations.
When it became necessary to bleach the pigment, in order to study the visual cells, which would otherwise be masked by the partially or completely extended processes, the method employed was essentially that of Mayer, in which nascent oxygen- is the effective agent.
EXPERIMENTAL PART
a. Experimentation upon Ameiurus
Ameiurus is well adapted for operations involving the optic nerve. The soft skin and the relatively small eye allow easy access to the orbit without causing the serious disturbance and shock that is almost unavoidable when operating on fishes which have eyes set in prominent bony sockets.
2 When potassium chlorate and hydrochloric acid interact, it is commonly said that nascent chlorine is the agent causing bleaching. As a matter of fact the reaction libei'ates free oxygen.
EFFERENT FIBERS OF THE OPTIC NERVE 219
The method of operation was as follows. With curved scissors an incision was made in the skin ventral to the eye and the cut carried upward around both sides of the ball until a semicircular (or greater) incision resulted. The eyeball could now be rolled back, and when thus displaced, no excessive strain was exerted on the conjoined tissues. By dissecting through the aperture thus exposed, the optic nerve was separated from the overlying muscles and severed. If a delicate razor-edge scalpel, such as ophthalmologists use in cataract operations, is employed, it is comparatively easy to cut the ner\'-e without injury to the surrounding parts. Ameiurus has no large central arter>' and vein in the optic nerve, as is the case in mammals. A blood-vessel, however, runs beside the optic ner\'e, but if care is taken it need not be injured by the operation.
1. Retinal piqmenl. My first effort was to discover the efTects upon pigment migration of the severance of the optic wqtyq only, A typical cx})oriment will illustrate the response of the pigment when a previously dark-adapted fish was operated on and exi)osed to light.
Experiment 8.1.12. Tiie optic nerve of a dark-adapt od fish was severed and the animal allowed to recover from any shock cfToct until the next day, when it was exposed to diffuse daylight for 2\ hours. At the expiration of this time both the operated ancl the normal eye were excised. Subsequent examination showed that the retinal pigment in the operated eye had retained the position typical of tlarkness (fig. 4), while the pi^nnenl in the control evo had nuf>;rato(l norniallv (%. 1).
Thus it is seen that the integrity of the otitic ner\'e must be maintained in order that the retinal pigment of an otherwise intact animal may undergo a positional change when stimulated by light.
In successful ex|)erinients of this type the length of exposure ^^aried between 45 minutes and 3 hours, yet uniform results were obtained. ^ A possible influence of operative shock, on animals that were subjected to experimentation directly after the
' Under normal conditions the pigment is fully extended by light in about 45 minutes (Arey, '16).
220 LESLIE B. AREY
optic nerve was severed, was shown to be non-existent. It was also sliown that roughly performed operations, in which various mutilations of the eye muscles and the adjacent blood vessels were intentionally made, did not interfere with the obtainment of essentially typical results.
This type of experiment has been repeated very many times. Among animals used during both the spring and the fall of 1913, no deviation was found from the conditions recorded above. When work was begun again on a new supply of Ameiurus in the spring of 1914, the results secured from operated animals brought from darkness into the light, were not always as decisive as those of the preceding year. Some experiments showed a complete retention of the pigment, while others resulted in a partial migration, which, in some cases, was quite extensive although never as complete as in normal light adaption. Further experimentation on other animals procured in the fall of 1914 also gave inconsistent results, varying from complete to very incomplete control.
I am unable to state the cause of these discrepancies. That the results of the work done in the year 1913, which embraced the larger part of this experimentation, are beyond question, I feel confident; the remarkable consistency of a long series of experiments about to be described substantiates this conviction. On the other hand, the persistent occurrence of more or less incomplete, mingled with complete control in the work of the year 1914, was also undeniable.
It is possible that individual fish vary considerably in their, ability to inhibit the migration of pigment after sectioning of the optic nerve, or what amounts to the same thing, the activity of the pigment in producing expansion may be variable. I shall attempt to present evidence, based on experimentation to be described later, that demonstrates an inhibiting mechanism which prevents the pigment from migrating when the optic nerve is severed. Since it has been proven (Arey, '16) that the action of light on completely excised eyes can directly stimulate the retinal pigment to movement, it is evident that there must be a competition between light stimulation and this inhibiting mech
EFFERENT FIBERS OF THE OPTIC NERVE 221
anism for the control of pigment migration. If the inhibiting mechanism is weak or becomes exhausted, or if the response of the pigment cells to the direct action of light is especially strong, the common result will be the production of a more or less extensive movement of the pigment.^
This, nevertheless, does not explain how different lots of animals, obtained in the spring and fall of one year, differed as a whole from other lots, procured in corresponding seasons of the following year. All the fish used were su])i)lied by one collector, who secured them from se\'eral small ponds, according to their var^ang abundance from year to year. It is possible that the stocks of different ponds may show more or less consistency, due to common inheritance, in the behavior of their retinal pigment under the previously mentioned exjDerimental conditions. The writer, however, wishes to present this suggestion merely as a possibility that may or may not be true.
A second type of experiment, in which light-adapted animals were subjected to diirkness, is illustrated by the following record.
Experiment 8.1 .49. One optic nerve only of a light-adapted Ameiurus was severed. The animal was subjected to darkness for 18 hours, after which both eyes were excised. No pigment contraction was found to have occurred in the operated eye (fig. 2), whereas the pigment of the control eye showed the extreme contraction of darkadapted retinas (fig. 4).
The greatest aanount of contraction e^'er obser\'ed was a withdrawal of the distal accumulation of pigment, which even at room temperature is often characteristic of light adaption, to form a uniform zone (fig. 2). Sometimes a shght thinning out of ]:)igment was seen, whereby the expanded mass appeared less dense than normally. In no case, however, was there observed a withdrawal of pigment, as a whole, from the zone usually
- A few determinations of the rapidity of pigment adaption seemed to indicate
that the migration occurred more slowly in the fishes used during the first season, which gave constant results, than in the animals used during the following j-ear. This fact not onlj' tends to support the hypothesis that a variability in the activit}' of pigment migration was responsible for the lack of consistency' in the later results, but also indicates that the response of the pigment in the animals of the first year, taken as a whole, may have been less vigorous.
222 LESLIE B. AREY
occupied (hiriiij;- light adaption. In this type of experiment, therefore, it is also evident that severance of the optic nerve restrains the normal pigment migration. The fact that the pigment of excised eyes does not contract in darkness, and that pigment contraction in general seems to involve a less vigorous response than does expansion, explains why consistent results were obtained in all these experiments, for the competition with the inhibiting mechanism presumably was less keen.
To remove any doubt that the integrit}^ of the optic nerve is necessary for the migration of pigment in light and in darkness, the nerve was cut inside the cranium near the chiasma. In order to do this, a small aperture was made in the cranium over the region of the optic nerve and the severance of the nerve was accomplished by introducing a cataract scalpel through the opening thus made. The removal of bone from the thick cranium had to be done with care or the fish failed to recover from the resulting shock effect. In successful experiments the results obtained were similar to those previously recorded. Since the eye and the surrounding parts were left intact during the whole procedure, it is safe to conclude that in Ameiurus the optic nerve is intimately related to the phenomenon of pigment migration.
A microscopical study of preparations demonstrating the relation of the optic nerve to the nerve-fiber layer of the retina shows that the arrangement of nerve fibers is approximately of a radial nature, and that the fibers from any sector of the retina are extended into the adjacent side of the optic nerve. These relations probably become disturbed a short distance from the emergence of the optic nerve from the eyeball. The optic nerve of Ameiurus joins the retina by several roots (fig. 7, rdx. n. opt.), hence this condition is not shown as diagrammatically as in preparations of the eye of Abramis which cut the optic nerve in a radial plane. Here the nerve fibers form a distinct V-shaped ^parting' as they pass to the two sides of the retina. On the periphery of the nerve this is especially evident although at the center there is undoubtedly more decussation, as w^as shown by Kohl ('92) for several of the lower vertebrates.
EFFERENT FIBERS OF THE OPTIC NERVE 223
This relation should stand the physiological test imposed by partial section of the optic nerve. In view of the results already established, one would expect to find the pigment situated in a sector adjacent to the cut to be unaffected by varying conditions of light and darkness, while the pigment adjacent to the intact portion of the nerve should expand or contract in a normal manner. The description of an actual experiment will make this clear.
Experiment S.1.3S. The (Ji)tic nerve of a lij^ht-iulapted Aineiurus was two-thirds severed close to the eyeball, and the fish was transferred to darkness. After 2 hours the eye was excised, a pointed Hap of skin bein}< left attached to the eyeball to insure proper orientation. In a preparation, sectioned to include portions of the retina adjacent to both the cut and the uncut fibers, a striking contrast was evident (fig. 7). On the uncut side the pigment was contracted maximally, while on the cut side it still remained in the expanded position characteristic of light adaption.
I can think of no experiment that could be devised to support better the view concerning the role which the optic nerve plays in the movement of retinal pigment, than the one just described.
The similar treatment of dark-adapted fi.sh which were subjected to light did not give as decisive results as those of the reciprocal set. The pigment on the 'cut' side of the eye was not retained in a contracted state, but migrated to a considerable extent (fig. 6). In no case, liowever, was the expansion maximal, that is, involving an accunudation near the external limiting membrane, but at most only a broad evenly pigmented zone was formed, which was markedly in contrast with the maximal expansion of pigment in that half of the retina adjacent to the intact portion of the nerve.
By the evidence of previous experimentation, a reason for this difference in behavior is suggested. It has already been shown that light is an efficient stimulus in producing expansion of the pigment in excised eyes, whereas darkness does not contract expanded pigment. Furthermore, after complete section of the optic nerve, the pigment in certain cases still tended to migrate when the animal was exposed to light. In the experiment last described, it is probable that there was a more or less extensive
224 LESLIE B. AREY
decussation of fibers distal to the cut, and that these fibers, although few in number, since they come from the intact portion of the nerve and distribute themselves through the retina of the opposite side are capable of allowing the pigment to migrate when the eye is subjected to the action of light. In producing this result, these fibers are doubtless servient to the direct action of light on the pigment cells themselves. In darkness, without the aid of some independent and efficient factor, such as the direct stimulus of light, it may well be that these straj^ fibers are not sufficiently potent to overcome the inertia of the pigment.
If the cause of the apparently inconsistent behavior described in these two types of experiment is something of the nature of that just outlined, then the evidence for the control of pigment migration through the optic nerve thereby receives additional support.
It was thought that if a small cut were made through the eyeball and retina, near the entrance of the optic nerve, the portion of the retina peripheral to the cut would be freed from all connections with the optic nerve, and consequently some decision could be reached concerning the role of the decussating fibers just considered. This method, if successful, would also corroborate the general conclusions which were drawn as to the control of pigment migration by the optic nerve fibers.
On the whole, the results showed quite conclusively that in the earlier experiments the presence of decussating fibers had caused the pigment expansion on the side of the retina adjacent to the cut optic nerve, when the eye was stimulated with light. Cuts about 1.0 mm. in length were made with a cataract scalpel. Although the blade of the scalpel was very thin, tapering to a needle-like point and the edge was of great keenness, nevertheless, in many cases the retina was torn away from the contracted pigment layer. The ease with which a* separation of the retina and the pigment layer is effected, especially when the pigment is not expanded to strengthen the union, is well known.
In successful operations (fig. 5), the pigment lying peripheral to the cut showed but little expansion, although it was often the case that toward the extreme periphery of the retina, pigment
EFFERENT FIBERS OF THE OPTIC NERVE 225
expansion was again found. Since it is probable that the fibers of the nerve-fiber layer do not have a precise radial distribution, the latter condition is readily explained by assuming the presence in this peripheral expanded region of intact nerve fibers, which, near the fundus, bordered on the incision.
The decrease in intraocular pressure necessitated by cutting the eyeball has no effect on the activity of the pigment, for normal animals whose corneas have been punctured show typical responses.
One is driven to the conclusion, by all the experiments heretofore described, that there must be some mechanism, either in the eye muscles or in the blood vessels of the eye, that exerts an inhibition on the movements of the retinal pigment, for proper control experiments have eliminated the skin as a possible factor.
If this supposition is true, the retinal pigment should undergo expansion when all the eye muscles and blood vessels of a darkadapted fish are cut, and the eye, connected to the bodj^ by the optic nerve only, is subjected to light. This condition was indeed realized, the pigment distribution being essentially like that in totally excised eyes. The same experiment under reversed light conditions did not result in a contraction of the pigment. As in the similar failure of excised eyes to show contracted pigment when subjected to darkness, I believe there is a strong probability of an anaesthetic action on the pigment cells due to the accumulation of catabolic waste. If the vascular circulation could be preserved, it is probable that the pigment cells would contract in an experiment of this kind. Possibly with artificial circulation the pigment of an excised eye would also contract in the dark.
The next step was to discover whether the presence of certain eye muscles could be correlated with the inhibition of the pigment response. \Mien dark-adajDted Ameiurus, having the optic nerves cut, were brought into the light and the dorsal oblique and posterior rectus muscles (those innervated by trochlear and abducens nerves respectively) were severed, no pigment migration occurred. It is evident, therefore, that the inhibiting mechanism does not involve these muscles alone. Reciprocally, the dorsal, ventral
22() LESLIE B. AREY
and antorior rectus and the ventral oblique muscles (those innervated by the oculomotor nerve) were cut, leaving the eyeball attached to the body by the two remaining muscles. Under these conditions, the pigment migrated much as in a normal animal.
Hence it appears that the inhibitory mechanism has been located as existing in association with the muscles (or possibly the blood vessels of the muscles) which are innervated by the oculomotor nerve. The objection may be raised, however, that there is a possibility of the dorsal oblique and posterior rectus muscles possessing an inhibitory function also, but that the inhibition produced when only two muscles are left in connection with the eye is not sufficient to prevent the pigment response. The evident check experiment which answers this criticism consists in severing all the muscles except two which are innervated by the oculomotor nerve. When this was done (the dorsal rectus and the inferior oblique being left intact) no movement of the pigment was observed. This result indicates that the inhibitory mechanism is found associated with those muscles innervated by the oculomotor nerve and is not demonstrably associated with the other eye muscles.
Since the dorsal oblique and posterior rectus muscles are not potent in restraining pigment migration, it was possible to make the following experiment. All the muscles excepting these two were cut and light-adapted fish with the optic nerves either intact or severed were placed in a dark situation. If the retinal pigment contracted under these conditions, it could be reasonably assumed that the presence of a partial vascular circulation had been responsible for the change. However, no movement of the pigment was detected, yet as the blood vessels in these muscles are small in number and size, the results neither support nor detract from the general view that absence of blood supply prevents pigment contraction in the dark.
Although the relation of the autonomic fibers to the oculomotor nerve of teleosts has not been worked out as completely as in some other forms, it is at least evident that from the ciliary ganglion the so-called ciliary nerves pass to the eyeball, probably
EFFERENT FIBERS OF THE OPTIC NERVE 227
following the same general courses as do the branches of the oculomotor nerve. In man, where the ciliary nerves have been traced rather carefully, autonomic fibers supply the sclerotic and choroid coats, ciliary muscle, iris, and cornea of the eyeball (Carpenter, '06). The ciliary ganglion of fishes according to Onodi ('01) also receives fibers from the trigeminal nerve, the relation being more intimate than Schwalbe (79) believed.
The action of atropine upon sympathetic nerves is to paralyze the endings of the postgangUonic fibers. If the mechanism that inhibits pigment migration involves sympathetic nerves, it was thought that it might be possible to eliminate its action by the use of this drug.
By supplying fishes with water through a tube, they could be retained indefinitely in the air. Dark-ada])ted fish with severed optic nerves were brought into the light, and occasionaly during the course of an hour small amounts of a 0.5 per cent solution of atropine sulphate were introduced into the orbit. Subsequent examination of the retina showed that a migration of pigment to the light phase had occurred.
The i)ossibility of a direct stimulation of the pigment cells presented a difficulty, however, that had to be tested, for Spaeth ('13) found that a 1.0 per cent solution of atropine caused a rapid expansion of the melanophores of Fundulus. When dark-adapted eyes were placed in a 0.5 or 1.0 per cent solution of the dnig and were returned to darkness for an hour or more, the pigment did expand to an extent which nearly equalled that caused l)y incomplete light adaption.
It is certain, therefore, that any evidence gained from experimentation of the kind described is valueless, and it is doubtful if much more dependence can be placed on the significance of a pigment migration when atropine was painted on to the eye muscles in such limited amounts that a direct stimulation of the pigment cells seemed improbable, or when small quantities of atro]:)ine were injected into the cranial cavit3\^
^ The use of nicotine, which paralyzes the synapse of the preganglionic fiber with the sympathetic nerve cell, might furnish more interesting results, provided it did not have a toxic effect on the pigment cells. Unfortunately, no experiments of this kind were performed.
228 LESLIE B. AREY
An ;itt(»inj)t was made to cut the oculomotor nerve and thus to separate the sym])athetic fibers which arise with it from the brain. To make a large opening through the cranium leads to operative shock from which Ameiurus does not recover. Accordingly, a relatively small aperture was made, through which a cataract scalpel was introduced, all the nerves presumably being cut on one side of the brain between the optic and the trigeminal. A dark-adapted Ameiurus with severed optic nerve, when exposed to the light after this treatment, showed pigment expansion. This experiment, although properly controlled, was not of a refined type. It is possible that disturbances other than the mere section of the oculomotor nerve may have led to the observed results.
It is not intended that any of the experimentation in which an attempt was made to locate the fibers of the inhibiting mechanism, should be received as conclusive. The results obtained from cutting muscles and from the severance of the oculomotor nerve suggest that autonomic fibers are involved, and that these enter with the oculomotor nerve. A strong suspicion is therefore cast upon the sympathetic fibers in connection with the ciliary ganglion as the causal agents in preventing movement of the retinal pigment when the optic nerve is cut. .
No statement has previously been made concerning the nature of the mechanism in the optic nerve, the integrity of which is necessary for allowing positional changes of the pigment to occur. If the inhibitory fibers associated with the eye muscles are conceived as acting after the manner of a brake, it follows that the optic nerve must contain active components, which in some way, directly or indirectly, permit expansion and contraction of the pigment.
If the optic nerve contains fibers of an efferent nature, it is of interest to discover whether these components can be made to function by electrical stimulation. The description of a typical experiment will best illustrate this point.
Experiment 8.1 .23. A previously dark-adapted Ameiurus, in which both optic nerves had been cut, was retained in the air, respiratory water being supplied through a tube. The peripheral end of the cut
EFFERENT FIBERS OF THE OPTIC XERVE 229
optic nerve of one eye was exposed and stimulated for 1\ hours, in the light, with a weak current from an induct orium. Inspection of the two retinas showed that the pigment in the eye that had licen electrically stimulated was uniformly expanded (fig. 2), while in the control eye the pigment still remained contracted as is characteristic of dark adaption (fig. 3).
Since repeated trials confinned these results, the conclusion follows that there must be nen'ous elements of an efferent nature in the optic nerve of this fish which nonnally receive unpulses from the central nervous system, and can be made to function experimentally by electrical stimulation. The result in either case is a release from an inhibition exerted upon the pigment cells by a mechanism possibly involving the autonomic fibers from the ciliar\' ganglion.
Previous oxperijuentation (Arey, '16) has sliown tlial botli in light and in darkness the retinal pigment of fishes is more highly expanded at low temperature (0°(\ +) than at high temperatures (25°C. ±), hence a series of experiments was next made to discover whether after the optic nerve was cut temperature woiUd still be efficient in producing the characteristic temjierature res]K)nses. In detenninations jnade l)oth in light and in darkness, the results were identical with those found in normal animals, for at r>°Q\, the expansion of ])igiiK'iit was greater tlian at 2')"(\ (figs. 1 and 2; 3 and 4).
In order to ])r()du{'e slunp contrasts (in the light at least), before the optic nerA'e was cut, a preUminaiy treatment at a tem])eratiu'e of o°C. was necessaiy. If the i:)relijninan' treatment was at 25°C. the pigment remained in the ])osition characteristic of that temperature, regardless of the temperature that followed. From this result additional evidence is obtained, over that already advanced (Arey, '16), to show that a high temperature is more efficient in causing positional changes of the retinal pigment than is a low temi^eratiu'e, and that temperatiu'e is more efficient than either light or darkness."
- It shoulil be reinLMnl:)ercHl, however, that temperature merely produces a
quantitative redistribution of the already expanded or contracted pigment, the extent of its influence always being limited in this way.
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 3
230 LESLIE U. AREY
2. ]'isu(tl cells, 'llw luoro important oxpei'huonts, which show the rohition of the optic nerve to the pigment migration of Ameiiiriis, were repeated in order to discover whether a similar set of relations exists between the optic nerve and the visual cells.
It will be remembered that the myoid of the cone cells of fishes elongates in the dark and shortens in the light (figs. 10 and 11), whereas the behavior of the rod myoid is the converse of this. Moreover, in excised eyes both types of cells are stimulated to movement through the direct action of light, whereas only the rods show a response in darkness.
In general it may be said that these experiments, which extended discontinuously throughout a period of two years, showed that whenever the migration of pigment was inhibited the movements of the rods and cones likewise failed to occur. In those cases, discussed previously (p. 220), where cutting of the optic nerve only partially inhibited the pigment response, the movements of the visual cells usually were not prevented. Practically all of the experiments to be described were performed in the spring and autumn of 1913 before cases of incomplete control were discovered.
When the optic nerve of a dark-adapted fish was severed, and the animal was subjected to light (fig. 12), the rods remained as in the condition of dark adaption. The cones, on the contrary, usually became more or less shortened, although they did not approach the external limiting membrane as closely as when exposed to light under normal conditions.
In the converse experiment, in which operated fish were transferred from light to darkness, the cones showed no tendency to elongate, but in some cases remained maximally shortened for several hours. The rods, however, did not always retain the highly elongated position characteristic of darkness, for they commonly exhibited partial retraction, although the final position was unmistakably that of semi-elongation.
From these two types of experiment one striking correlation is evident. The cones have a greater tendency to undergo positional changes in the light, while the rods behave similarly in
EFFERENT FIBERS OF THE OPTIC NERVE 231
darkness. The end result of this tendency towards movement, nevertheless, is identical in both kinds of \'isual cells, for a shortening of the myoid results in either case. This would suggest that the process of retraction, as in simple contractile tissue in general, is more vigorous than is elongation. This behavior of the cones is comparable to that observed in excised ej'esof Ameiurus, where these cells shortened in the light but remained unchanged in darkness; the rods of excised eyes, on the contrarj^ moved both in light and in darkness. The posulation of a more vigorous I'esponse in retraction offers an explanation for the obser\'ed facts, for under such conditions the inhibitoiy mechanism would be less likely to check com])lotely the positional changes of these elements.
Reference has been made (p. 223) to experiiuonts in wliicli the optic nerves of light -ada])tod fish were ])artly severed. \\'hen these annuals were transferred into the dark, the retinal ]:)igment showed distinct areas of expansion and contraction, corresponding respecti\'ely to the cut and uncut sides of the ojitic nerve. In one such jireparation which had been stained, the position of the cone cells was also obser\(Hl to \'ar>' with respect to the same areas. Where the pigment lia<l undergone movement, the cones were elongated, whereas in th(> half of the retina adjacent to the cut fibers the cones remained inaxunally shortened.
A control experunent, in which the light-ada])ted eye, connected to the bod}' by the optic ner\'e only, was subjected to darkness, showed that the rod cells had shortened although not always completely. The cones, as in excised eyes, did not change their positions in the least. There is no evidence that the optic nerve of itself ]irohil)its the inoveinents of the rod and cone cells.
The results which ha\'e thus far been outlined indicate that, just as with the retinal pigment, there is a mechanism associated with the muscles or blood vessels of the eye which tends to inhibit mo\-ements of the visual cells, ^^'^len the dorsal oblique and posterior rectus muscles are cut, and after section of the optic nerve the dark-adapted Aineiurus is exposed to light, no changes in the position of the rods and only incomplete
232 LESLIE B. AREY
changes of tlio cones occur. The .muscles innervated by the trochlear and abducens nerves evidently are not essential in causing an inhibition of movement.
A few reciprocal experiments were performed by cutting all the eye muscles except the dorsal oblique and the posterior rectus. Although the extent of positional change was not as striking as in excised eyes, it seems probable that the same mechanism that controls the migration of pigment, and is presumably associated with the muscles innervated by the oculomotor nerve, also acts on the visual cells.
The evidence, therefore, suggests that normally there are impulses which travel in the optic nerve and render ineffective the inhibition produced by a second set of fibers. Hence it becomes a matter of interest to discover whether it is possible, by artificial stimulation of the severed optic nerve, to cause the visual cells to move more freely than they would otherwise do. The details of these experiments were similar to those previously described when the migration of pigment was tested. , The cut optic nerves of dark-adapted fish were stimulated in the light by a weak faradic current. Both rod and cone cells assumed their characteristic light positions, while in control eyes of the same animals, the optic nerves of which had been cut but not stimulated, the visual cells remained for the most part as in darkness.
These results on the visual cells of Ameiurus, taken as a whole, closely agree with those obtained from the study of retinal pigment, and the general conclusion concerning their significance is so identical with one previously stated (p. 229) that it hardly needs to be repeated here.
It was shown in a former paper (Arey, '16) that in fishes an elevated temperature (25°C. ±) causes an elongation of the myoids of dark-adapted cone-visual cells, whereas a low temperature (0°C. ±) induces a shortening of the myoids. The rod myoid exhibits a similar behavior but in a less marked degree.
The effect of temperature was tested on the visual cells of darkadapted fish the optic nerves of which had been cut. At 5°C. in the dark the cone myoid measured 18 m, the rod myoid 8 /x ± (fig. 8). At 25°C. in the dark the cone myoid elongated to 25 fi, the rod to 10 ^u ± (fig. 9). These results are in agreement with
EFFERENT FIBERS OF THE OPTIC NERVE 233
those obtained with normal animals. The effective action of high temperature in causing an elongation of the cone cells is noteworthy, since without the aid of temperature no change in the position of the cones occurred under these conditions.
b. Experimentation upon Abratnis and Fundulus
Having presented evidence which indicates that the optic nerve fibers of .\meiurus are not all afferent in function, the question arises as to the occurrence of this condition among other fishes as well as among other classes of vertebrates. A series of exiieriments was carried out upon Abramis and Fundulus in which the effect produced on the retinal oh^nents liy the severance of the optic nerve was ti-ied.
Both in light and in darkness the retinal ])igment and the cone cells of Abramis underwent movements which were essentially normal, and hence independent of the cut nerve. Both in the cone myoid, which is capable of a 90 per cent retraction, and in the pigment, which exhil)its extreme conditions of contraction and expansion, was an inde])endent movement strikingly (exhibited. The stain used in' niiiknig these preparations did not demonstrate the rods to advantage, except in one instance where a light-adapted fish had been subjected to darkness. In this case the rods were shortened, occui>ying the characteristic dark position.
A\'hen all the eye muscles and blood vessels of dark-adapted Abramis were cut, the eye remaining attached to the body by the optic nerve only, no marked change in the position of the pigment or cones accompanied a removal into the light. Since the pigment and cones show no mo\'ements when the eyes are com])letely excised and exposed to light or darkness, these experiments fa\'or the view that the absence of mo^'ement in such cases is not due to an inhil)ition tlirough the optic ner\'e but to an int(M-i-u])tion in tlie vascular supply."
' 111 several instances a large blood vessel that lies near the optic nerve was accidentally cut in experiments where the optic nerve only was being severed, yet the changes in the retinal elements occurred as before. This further supports the view that the blood supply to the retina by means of vessels in connection with the eye muscles is important in allowing the positional changes to occur.
234 LESLIE B. AREY
'I'hc (>lYo('t of se\'(M'iiif2; the optic norvo of Fiin(hilus was investigiited in a simihir nuimior. When brought from darkness to light the pigment migrated normally after the usual operation. The converse experiment also showed an independent movement of the pigment, although the poorly defined contraction which characterizes dark adaption rendered this type of experiment less decisive than the clear cut results on Abramis.
It is evident, then, that unlike Ameiurus the movements of the retinal elements of Abramis and Fundulus are not dependent upon the integrity of the optic nerve. From the results of Hamburger ('89) and Fick ('91) upon the frog, who showed that the retinal pigment underwent movements after the optic nerve was cut at the chiasma, it is safe to conclude that in this animal also the optic nerve does not control pigment migration. The experiments of these latter workers are all the more interesting since in many observations which have been recorded showing the retinal elements of the frog to be under nervous control, it has generally been directly stated, or at least implied, that the optic nerve was involved. It would be unprofitable to speculate concerning the further occurrence among the vertebrates, or even among the fishes, of individuals possessing efferent optic nerve fibers which act similarly to those in Ameiurus.
THEORETICAL CONSIDERATIONS
Although serious doubt has beezi cast on many of the earlier results upon the frog which were supposed to demonstrate the presence of efferent fibers, both between the brain and the retina and between the two retinas by way of the optic chiasma, yet it is probable that there is a residue of truth in the general proposition of a nervous control of the retinal elements in this animal.
Fick ('90, p. 84), in a paper attacking Englemann's assertion of the existence of efferent nerve fibers, takes the following position :
Wenn dieser Schliiss richtig ist, so kann man niir ruhig durch die ganze bisherige Sinnesphysiologie einen Strich machen und ihre Erforschung von Neuem beginnen ; denn die rein centripetale und specifische
EFFERENT FIBERS OF THE OPTIC NERVE 235
Leitung in den Sinnesnerven gilt als die eigentliclie Grundlage dieses Abschnittes der Physiologie.
This statement may have been partially justified in view of the diversity of stunuli reported to be potent in causing; changes in the retinal elements of the frog, but even if the presence of efferent fibers of the kind physiologicall}' demonstrated in Ameiurus were found in all the vertebrate classes, it by no means follows that the fundamental princii^Ies of sensor\' pliysiologAwould be seriously endangered. In tlie normal Ameiurus the efferent function does not in the least interfere witli the moA-ements of tlic retinal elements, and stbnulathig agents which do not act directly upon these cells presmnably are ineffectual in producing changes. Since, moreover, there is no e\-idence of any intrusion on the part of these elements upon any of the sensoiy processes, our ideas of retinal physiology- scarcely re(iuire modification and certainly do not demand reorganization.
From the study of Ameiums exj^erimental jiroof has been advanced sliowing the existence of an hihibitoiy mechanism, not associated with the optic nei-xc. wliicli tends to ]^revent the movements of pigment cells and retinal pigment. Moreo\'ei-, a second mechanism associated with the optic ner\'e was dcjuonstrated, the hitegrity of which is necessaiy for the accom])lishment of ty])ical movements on the part of the retinal elements.
It ma}^ now be fairly asked wlietlier there is any e^•idence indicating the possible modus oi^ei'andi of these two systems. Several schemes are readily suggested by which the facts hitherto presented could be explained. If, however, one conceives of the efferent fibers in the optic nerve as acti\'ely causing moA'cments of the retinal elements, both hi darkness and in light, either two kinds of efferent fibers must be postulated, or one movement of e^ch of the elements is passive — a return to the unstimidated condition — and in some way is interfered with by the mhibitoiy mechanism wlien the optic nerve is cut. Nevertheless^ either one of these ex]ilanations becomes discrepant wlien ai)])lied to the total behavior of the retinal elements.
Since electrical stimulation of the cut optic nerve in the light induced changes in the retinal elements of an otherwise intact
236 LESLIE R. AUEY
fisli, it would seem reasonable to expect that the same would occur in darkness, provided efferent impulses in the optic nerve directly stimidate the i-etijial elements to undergo positional changes.
CJertz ('11) tested the effect of electrical stimulation for one to two minutes in dai'kness and in light upon the eyes of Abramis brama, both excised and in vivo, but only negative results were obtained. Englemann ('85) had previously asserted that the cones and retinal pigment of the excised or normal eyes of darkadapted frogs responded to induction shocks in a similar way as to light, and Arcoleo ('90) also claimed to have observed pigment migration under these conditions in a pithed toad and frog, as well as upon dark adapted excised eyes. The more recent work of Lederer ('08) and of Fujita ('11) upon normal frogs nevertheless, has not supported this view.
When the cut stump of the optic nerve of a dark-adapted Ameiurus was stimulated with a faradic current in total darkness, no distinct changes occurred in the retinal pigment or rod cells, even though the stimulation continued for 45 minutes. Similar treatment of excised eyes also gave negative results. These observations indicate that a satisfactory explanation of the action of the efferent nerve fibers must be sought by viewing the situation in a different way.
It is instructive to adopt the point of view suggested by a study of the vasomotor nerves. In this case a tonic condition is presupposed either through the action of vasoconstrictor nerve fibers or possibly by the intrinsic properties of the muscles themselves. Dilation is believed to be accomplished by the action of dilator nerve fibers, whose impulses inhibit the tonic contraction of the musculature, thus indirectly causing relaxation. To work out a detailed application for the condition found in Ameiurus would be both unprofitable and unwarranted. The simplest conception is that impulses^ from the efferent compo
- This view of tonicity differs greatly from that which Herzog ('05) believed
to exist in the frog. His statement that after destruction of the central nervous system the pigment and cones gave abnormally vigorous responses in the dark, as if released from an inhibition, does not agree with the observations of Hamburger ('89), Arcoleo ('90), Dittler ('07) and Garten ('07).
EFFERENT FIBERS OF THE OPTIC NERVE 237
nents of the optic nerve block, i.e., counteract, the tonic inhibition produced by the second nervous mechanism, thereby allowing conditions of light and darkness to act directly upon the retinal elements.^ This would explain why electrical stunulation of the cut optic nerves of dark-adapted fish, in the light only, led to the usual changes in the retina. Although no determinations were made, it follows that if this hypothesis is tnie, the converse experiment with light-adapted fish in the dark should I'esult in the assum])ti()n of the dark phase on the part of the retinal elements.
Assuming that the balanced action of a system lik(> the one suggested is indeed a reality, it is eWdent, nevertheless, that questions relative to its adaptive significance are not easily answered. It is certainh' diflicidt to ex]:>lain the rationale of a situation whereby an animal possesses a mechanism the components of which act antagonistically, thus allowing photomechanical influence to be exerted un(hsturbed.
Since stnicture and function go hand in hand, the value of i:)hysiol()gical evidence always liecojues much enhanced by the coexistence of a correlated structiu'al basis. The possibility of double conduction in one set of nerve fibers is hardly to be considered, hence there arises a pertinent quer>' relative to the anatomical proof for the existence of efferent components of the optic nerve.
Englemann ('85) first postulated 'retino-motor' nerve fibers to explain certahi conditions which he asserted occurred in the frog, and fiu-ther suggested that the anterior arcuate conunissure of Hannover in the chiasma, tlu^ ])hysiological significance of which had pre\'iously been unknown, served as an association tract through which the movements of the retinal elements of the two e\'es were interrelated.
" It w ill lie remembered in the experiments on Abramis and Fundulus (p. 233) that after severing the optic nerves the usual responses to light and darkness were maintained. Hence it seems probable (since the retinal elements undergo no changes in excised eyes) that the direct action of darkness as well as light is effective, provided normal circulatory conditions are maintained.
23S LESLIE B. AREY
By tlie use of (lolgi methods niul by juethods of primary and secondary degeneration, centrifugal fil)(u-s oi-iginating in the vertebrate brain and extending to the retina can be demonstrated. The following quotation from Johnston ('06, p. 265) summarizes the general results gained from studies of this kind:
In fishes in which one eye has long l)een lost the optic tract of the opposite side degenerates with the exception of these efferent fibers, which persist and are stained by the Weigert method. In mammals, following section of the optic tract there occurs secondary degeneration of cells in the anterior quadrigeminum, and in the dorsal part of the geniculatnm laterale and pulvinar. These fi.ndings in mammals agree with those in fishes by the Golgi and degeneration methods, where the centrifugal filiers arise from the tectum opticum and geniculatum (Catois, '02) . The significance of these fibers is not understood but their presence in all vertebrates seems to show that they have some constant function.
A further anatomical difficulty is presented by the fact that a connection between optic nerve fibers and the pigment cells has never been demonstrated, for even the erfetont fibers shown by Cajal ('94) and others have their endiii.;s near the internal nuclear layer. Garten ('07, p. 85) suggColcJ that an actual connection might not be necessary: Natiirfich liesse sich behaupten, die centrifugalen Opticusfasern rufen in der Stabchenzapfenschicht einen Erreginigsvorgang hervor, der sich 'per contiguitatem' dem Pigmentepithel mitteilt." In view of the tentative conclusion reached by me concerning the balanced action of the 'retino-motor' and the inhibitory nerve fibers, it is difficult to state what anatomical conditions would be imposed by such a system. Such matters of detail need not stand in the way of the fact of fundamental importance, which is the existence of demonstrably functional efferent optic nerve fibers.
The significance of these efferent nervous elements in the light of the theory of nerve components may appear to demand no serious consideration, since the majority of neurologists prefer not to homologize the optic 'nerve' with true cranial nerves. Neither the afferent nor the efferent fibers of the optic nerve are comparable to the components of a true peripheral nerve, for both lie within the primary optic apparatus, which is itself a differ
EFFERENT FIBERS OF THE OPTIC NERVE 239
entiated portion of the brain. Johnston ('06) and Herrick ('15) have assigned the optic nerve proper to the central system of tracts of the somatic afferent division. From the conchision previously drawn concerning the function of the efferent o])tic nerv-e fibers, it is evident that they may, at least with as much propriety, tentatively be called visceral efferent elements.
SUMMARY
When the optic ner\'e only of Ameiurus is severed, the rods, cones and retinal pigment fail to execute their characteri^tic photf)mechanical responses. In other words, the movements of the retbial elements dojiend upon the maintenance of the integrity of this nen-e. After hemisection of the nen-e, movements of the elements occur only in tliiit region of the retina adjacent to its intact side. It can not only be shown (since essentially normal responses occur m excised eyes and in eyes attached to the body by the o])tic nerve alone) that a second mech;inism exists in association with the muscles innen'ated by the oculomotor nerve whidi inhil)its these movements when the o]Mic nerve is cut, but also that electrical stimulation of the ])(Mi])h(Mai stump of the optic nerve can o\'ercome this inhibition.
Hence experunental evidence has been advanced which offers physiological ])roof for the existence of functional efferent nerve fibers in the optic nerve of Ameiurus. Only by the balanced interaction of these elements with a second extrinsic set of nerve fibers (possibly the ciliaiy ner\'es), which independently exert an inhibitory effect upon the retinal elements, are normal photomechanical movements of the rods, cones, and retinal pigment accomplished. Although light is ineffectual after section of the optic ner\'e, temi^eratui-e ])roduces essentially nonnal res]ionses in both the pigment and ^'isual cells. It is probable that efferent ijui)ulses in the optic nerve fibers do not directly stimulate the retinal elements to motion, but rather such impulses have an indirect action, possibly by counteracting, that is, blocking, the tonic inhibition exerted by the second system of nerve fibers. If these efferent optic nerve fibers fit at all into the scheme of
240 LESLIE B. AUEY
'iiervc (■()jii])()ii(Mils,' Ihcv luay l)c <lcsi^ji;itod as visceral efferent eleineiits.
Severance of the ()i)tic nerve of certain other hshes (Abramis and Fiin(hihis) lias no inhibitory effect upon the movements of the retinal i)i^inent or of the rod- and cone-cells, and a similar relation has been reported for the frog by other workers. Hence it is impossible to state the extent to which the mechanism discovered in Ameiurus may be distributed throughout the vertebrate group, if, indeed, it is not peculiar to Ameiurus alone.
BIBLIOGRAPHY Papers marked with an asterisk have not been accessible in the original
Angelucci, a. 1890 Untersuchungen liber die Sehthatigkeit der Netzhaut und
des Gehirns. Untersuch. zur. Naturlehre d. Menschen u. d. Thiere
(Moleschott), Bd. 14, Heft 3, pp. 231-357, 2 Taf.
1905 Physiologie generale de I'oeil. Encyclope.die frangaise d'oph thalmologie, tome 2, pp. 1-141.
- Arcoleo, E. 1890 Osservazioni sperimentali sugli elementi contrattili della
retina negli animali a sangue freddo. Annali d'Ottalmologia, Anno
19, Fasc. 3 e 4, pp. 253-262. Arey, L. B. 1915 The occurrence and the significance of photomechanical
changes in the vertebrate retina — An historical survey. Jour. Comp.
Neur., vol. 25, no. 6, pp. 535-554.
1916 The movements in the visual cells and retinal pigments of the
lower vertebrates. Jour. Comp. Neu-., vol. -6, no. 1, pp. 121-202. BiRCH-HiRSCHFELD, A. 1906 Dcr Einfluss der Helladaptation auf die Struktur
der Nervenzellen der Netzhaut, nach Untersuchung an der Taube.
Arch. f. Ophthal., Bd. 63, Heft 1, pp. 85-111. Cajal, S. R. 1894 Die Retina der Wirbelthiere. Bergmann, Wiesbaden, 4to,
8 + 179 pp. Carpenter, F. W. 1906 The development of the oculomotor nerve, the ciliary
ganglion and the abducent nerve of the chick. Bull. Mus. Comp.
ZooL, Harvard Coll., vol. 48, no. 2, pp. 141-229. Catois, E. H. 1902 Recherches sur I'histologie et I'anatomie microscopique
de I'encephale chez les Poissons. Bull. Sci. France et Belgique, Tom.
36, pp. 1-166.
- Chiarini, p. 1904 Cambiamenti morphologici, che si verificano nella re ina
dei vertebrati per azione della uce e dell' oscurita. Parte 1. Retina
dei pesci e degli anfibi. Boll, della R. Accad. Med. di Roma, Anno, 30,
pp. 75-110. DiTTLER, R. 1907 tJber die Zapfenkontraktion an der isolierten Froschnetz haut. Arch. f. ges. Physiol., Bd. 117, Heft, 5 u. 6, pp. 295-328.
EFFERENT FIBERS OF THE OPTIC NERVE 241
Englemann, T. W. 1885 t'ber Bewegungen tier Zapfen- unci Pigmentzcilen der Netzhaut unter dem Einfluss des Lichtes und des Xervensystems. (Xach einem 1884 gehaltenen Vortrag.) Arch. f. ges. Phj-siol., Bd. 35, Heft 10, 11, u. 12, pp. 498-508.
FiCK, A. E. 1889 tJber die Lichtwirkungen auf die Xetzhaut des Frosches. Bericht iiber d. 18. Versammliing d. Ophthalmol. Gesell. zu Heidelberg 1889, pp. 177-183.
1890 Uber die Ursachen der Pignientwanderung in der Xetzhaut. Vierteljahrs-schrift d. naturf. Gesell. Zurich, Jahrg. 35, pp. 83-86.
1891 Untersu hungen iiber die Pignientwanderung in der Xetzhaut des Frosches. Arch. f. Opththal., Bd. 37, Aht. 2, pp. 1-20.
FujiT.\, H. 1911 Pigmenthewegung und Zapfen kontraction im Dunkelauge des
Frosches bei Einwirkung verschiedener Reize. .\rch. . vergl. Oph thal., Jahrg. 2, Heft 2, Xo. 6, pp. 164-179. G.\RTEN, 8. 1907 Die Veriinderungen der Xetzhaut durch Licht. Graefe Saeinisch Handbuch ges. Augcnheilkunde, Leipzig. Aufl. 2, Bd. 3, Kap.
12, Anhang, 130 pp. Gehtz, H. 1911 Gibt elektrische Reitzung phototrope Xetzhautreaktion bei
Al)ranus brania? Arch. f. Ophthal., Bd. 78, Hoft 1, pp. 224-226.
- Ghijn.s, G. 1891 Bijdrago tot do i)hysiologie van don norvu.s opticus. Utrecht
8vo.. 3 + 7a pp.
- H.'\MnunRER, D. J. 1889 Dorsnijding van der nervus opticus bij Kikvorschen,
in verl)and met de Beweging van Pigment en Kegels in het Xetvlies,
onder den Invloed van Licht en Duister Onderzoekingen d. Ttrecht sche Hoogeschol, Reeks 3, vol. 11, pp. 58-67. Hekhh'k, ('. J. 1915 An introduction to ncurologv. Saund(M-.s, Phila.. Svo,
335 pp. Ukhzoc, H. 1'.K)5 llxpcrimcnti'llc rntcrsuchung zur Physiologie der Bewegungs vorgiinge in dor Xetzhaut. Arch. f. Anat. u. Physiol., Physiol. Abt.,
Jahrg. 1905. Heft 5 u. 6. pp. 413-464. Johnston, J. B. 1906 The iicrvou.s system of vcrt(>l)nitcs, Blakiston, Phila.,
Svo., XX + 370 i)p. Kohl, C. 1892 Rudimentiire Wirbelthieraugen. Bii)liotheca Zoologica. Bd. 4,
Heft 13. pp. 1-140. Lederer, R. 1908 Wirken elektrische Reize auf das Pigmentepithel des^Frosch auges? Centralbl. f. Physiol., Bd. 22, Xo. 24, i)p. 76.5 766.
- LoD.\TO, G.,E PiRRONE, D. 1901 Sulle vie associative fra le due retine, studio
sperimentale. Arch, di Ottalmol., vol. 8. pp. 465-489. X.\HMM.\CHER, W. 1893 t^ber den Einfluss reflectorisehe und centrale Opticus reizung auf die Stellung der Zapfen in der Froschnetzhaut. Arch. f.
ges. Physiol., Bd. 35, Heft 9 u. 10, pp. 375-387. (5nodi, a. 1901 Das Ganglion ciliare. .Vnat. .Vnzeiger, Bd. 19. Xo. 5 u. 6, jip.
118-124.
- Pergens, E. 1896 Action de la lumiere sur la retine. Ann. Soc. Roy. Sc.
Med. Xat., Bruxelles, tome 5, pp. 389-421. ScHWALBE, G. 1879 Das Ganglion oculomotorii. .lena. Zeitschr. f. Xatur wissensch. Bd. 13, pp. 173-208. Spaeth, R. A. 1913 The physiology of the chromatoiihoros of fishes. Jour.
K\p. Zo(')l., vol. 15, no. 4, pp. 527-585.
AHUHKVIATIONS
cll. bac, roil cUipsuiil ///-.s. dtit. bac, outer incmljcr of rod
ell. con., cone ellipsoid /)/-.s-. dst. con., outer member of cone 7nb. Urn. e.i-., extornal liini1iii, a single cell {VI 1 1) represents the distal end of the ganglion. Towards the brain from this position the ganglion can be followed distinctly (fig. 2, G.VIII). The auditory vesicle has detached itself entirely from the skin and has made very perceptible progress in dilTcrentiation since the non-motile stage, as may be seen by comparing figures 73 and 75. Mitotic figures are still very abundant in the inner portion of the epithelium. The root fibers of the nerve are seen in figure 27 (R.VIII) entering the brain on the ventral aspect of the root of the geniculate ganglion. In figure 28, taken from the next section caudad of 27 (10 m) no descending auditory root fibers can be definitely recognized.
C. THE COILED-REACTION STAGE
As in the case of the trigeminal ganglia, the ganglia of the facial complex have become more consolidated towards the brain and around the antero-ventral aspect of the auditory vesicle.
1. The lateral line component. The lateral fine primordia have become greatly extended since the early flexure stage (Paper I,
THE JOURNAL OP COMPARATIVE NEUROLOGY, VOL. 26, NO. 3
262 G. E. COGHILL
fig. 58). In addition to the priniordia shown in the latter figure there is now a priniordium which runs mesad from the base of the balancer on the ventral surface of the head. In this epithelial thickening there is a lowly differentiated lateral line organ at the mesial end and possibly another at the base of the balancer. This primordium is not connected with the other preauditory priniordia, unless it is by a very slight ectodermal thickening of an indifferent nature.
The lateral line ganglia a and h are in close contact with each other proximally but project distally into distinct gangha (fig. 3, G.L.L.VII,a,b). Ganglion a still reaches almost to the skin. In its distal portion it still has two parts, from each of which arises a nerve. These nerves follow the priniordia closely attached to the skin for a considerable distance. The finer divisions have no sheath cells and their exact length is therefore difficult to determine. Ganglion b projects ventrad behind the spiracular pouch and from it nerve fibers pass to the skin at or near the lateral line primordium which has been -described as occurring in this stage at the base of the balancer.
The roots of the lateral line ganglia enter the brain as illustrated in figures 38 to 41, figure 38 being the most rostral of the series. Here the root fibers of ganglion b {R.L.L.VII,b) are entering the brain and collecting into a bundle immediately beneath the external limiting membrance. In figures 39, 40, and 41, the next successive sections caudad, the root fibers of ganglion a are entering in a more dorsal position {R.L.L.VII,a). Within the brain these fibers appear to bifurcate. At least they form short ascending and descending tracts as shown in figure 5 {L.L.Asc.,Des.,a and b). Figure 38 shows the ascending tracts, and figure 41, the descending tracts in cross section, under high magnification.
2. The visceral sensory component. The proximal portion of the geniculate ganglion has not changed its position noticeably (fig. 3, G. gen.). Distally it projects to the spiracular pouch, where its appearance has materially changed. Along the caudal aspect of the entodermal wall of the pouch a fiber or two may be traced along the bases of the entodermal cells. Their course, however.
THE NERVOUS SYSTEM OF AMPHIBIA 263
is very short, for they can not be traced into the hj'omandibular trunk. They take a course in conformity with this trunk, and probably represent an incipient r. alveolaris. This part of the ganglion apparently corresponds to the geniculate ganglion which has been described in the earlier stages. In addition to this there is now a very massive connection of the ganglion with the ectoderm running forward over the pouch and at the same time in close relation with the entoderm. The structure here gives one the impression that the ectoderm is contributing large numbers of cells to the ganglion. This is probably the epibranchial placode, although my specimens have not been selected at short intervals and are not adequate for the deciding of this question. No fibers emerge from this portion of the ganglion.
The root of the geniculate ganglion is shown entering the brain in figure 39 (R.VII,vis.). Within the brain they turn caudad and form a distinct fasciculus solitarius (figs., 39 to 41, Fas. Sol), whi<'h lies against the limiting membrane and wedged in between the lateral line tracts dorsally and the auditory tract ventrally. This tract can now be followed to the level of the glossopharyngeal rooti, from which it receives an increment, and then on to the level of the root of the vagus nerve (fig. 5).
S. The auditory organ atid nerve. The wall of the auditory vesicle has become greatly reduced in thickness and its lumen much enlarged since the eady flexure stage (fig. 77). In the inner portion of the ventral wall mitosis is still going on rapidly. The auditory ganglion is closely applied to the vesicle ventromesially, while the endolymphatic appendage is closely pressed against the brain. This contact is ob\'iously a secondary thing, brought about by the enlargement of the vesicle (compare figs. 73, 75, 77, 79). The auditory nerve can be traced to the caudal extremity of the vesicle, closely applied to the vesicle in about the same relative position as it appears in figure 77. The root fibers (R.VIII) enter the brain as shown in figures 40 and 41 ventrally of the root fibers of the geniculate ganglion. Their course caudad on the ventral aspect of the fasciculus solitarius is indicated in figure 5 {VIII,des.).
264 G. E. COGHILL
D. THE EARLY SWIMMING STAGE
The facial and auditory ganglia have in this stage become still more consolidated about the auditory vesicle and the roots enter the brain farther caudad relative to the vesicle.
1. The lateralline component. The preauditory lateral line primordia have advanced greatly in distribution and differentiation (Paper I, fig. 59). The supraorbital primordium can now be traced far down in front of the eye and the olfactory organ and spreads out at the end, in anticipation apparently of the line of organs that develops later around the rim of the snout. The suborbital primordium extends beneath the eye into close relation with the olfactory organ, while the proximal portion of this primordium is now continuous with the primordium of the hyomandibular region. The latter primordium now passes ventrad behind the balancer, then mesad, where it appears on the ventral surface behind the oral plate in two distinct ridges, in which the characteristic organ structure .is well pronounced.
Silver impregnations of the lateral line fibers now show them extending practically the full length of the primordia, and following rigidly the course of the primordia as in the earlier stages. The nerve trunks are everywhere closely adhering to the epithelium and here and there fine fibrils can be followed in among the cells.
The root fibers of the lateral line ganglia impregnate much more deeply with the silver methods than do the peripheral fibers. They enter the brain as illustrated in figures 55 to 60, in two roots, R.L.L.VII, a and b). From a study of silver impregnations of these fibers it is impossible to be absolutely sure that all fibers from ganglion a enter root a, or that root b is exclusively made up of fibers from ganglion b. But if there is an intermingling of fibers from the different ganglia in the individual roots this must be very slight. The roots, upon entering the brain, form ascending and descending tracts, as shown in figure 6. The descending tracts reach the level of the middle of the auditory vesicle, and the ascending tracts are of about the same length or a little longer. The relation of the descending tracts
THE NERVOUS SYSTEM OF AMPHIBIA 265
of the facial to the ascending tracts of the postauditory lateral line roots is shown in figure 61, where the tract VII a is most dorsal of the series. Between this and tract VII b is the dorsal division of the postauditory roots. Most ventral in the set is the ventral division of the latter. Good silver impregnations" in longitudinal section make it appear that these tracts may all be considerably longer than figure 6 represents them.
2. The visceral sensory component. The geniculate ganglion is now in contact with the ectoderm by only a slender column of cells which rests also upon the entoderm. This ectodermal contact of the ganglion lies about midway between two other projections of the ganglion: the one contributing fibers to the r. hyomandibularis as the latter passes ventrad in very close relation with the caudal wall of the spiracular pouch (fig. 4, e); the other, directed rostrad on the dorsal surface of the entoderm, giving off a well defined r. palatinus (fig. 4, d). This nerve passes mesad and a little rostrad over the pharyngeal cavity almost to the middle line and forward to within abougt 50 n of the rostral end of the foregut.
The root fibers of the geniculate ganglion enter the brain ventrally of the lateral line roots, as seen in figure 56 {R.VII,Vis.) and then turn caudad as the fasciculus solitarius (fig. 6, Fas.SoL). The tract still lies immediately against the limiting membrane. It is distinctly larger than in the coiled-reaction stage.
3. The auditory organ and nerve. The endolymphatic appendage is more sharply differentiated from the body of the auditory vesicle than it was. in the earlier stage, and is still closely pressed against the brain (fig. 79). The auditory ganglion and nerve extend to the caudal border of the vesicle in the position shown in figure 79. In silver impregnations the fibers of the nerve now appear as exceedingly fine filaments which become closely merged with the basal ends of the epithelial cells or with a basement membrane which is also slightly impregnated. Nerve ending upon the epithelial cells of the vesicle, however, have not been observed.
The root fibers of the auditory ganglion, entering the brain as shown in figures 57 to 60, form a descending tract which extends
266 G. E. COGHILL
along the ventral aspect of the fasciculus solitarius to the level of about the middle of the auditory vesicle (fig. 6).
3. The glossopharyngeal and vagus nerves
This complex includes lateral line, general cutaneous and visceral components.
A. THE NON-MOTILE STAGE
1. The lateral line component. There are two postauditory lateral line primordia (Paper I, fig. 56, Po.LL.l and 2). The one of these is closely associated with the ectodermal thickening over the first branchial pouch; the other lies along dorsally of the thickening of the ectoderm in the vicinity of the more caudal pouches. From the primordium over the first branchial pouch a slender strand of cells reaches to the brain (fig. 1, G.L.L.IX, R.L.LJX,X). These cells are laden with yolk spherules and are mostly of an indifferent nature, although some of them have the appearance of neuroblasts. This is the lateral line ganglion of the glossopharyngeus. While the root reaches the brain, nothing can be determined concerning its fibers within the brain.
The main lateral Une ganglion of the vagus is a spindle-form body, situated opposite the first and second myotomes. It has a broad attachment with the lateral line primordium of that region. Its root is so slightly developed that it can be recognized only as it lies in the plane of section. A very few root fibers reach the brain in connection with the lateral line root of the ninth nerve (fig. 1, G.L.L.X).
2. The general cutaneous component. On the lateral aspect of the lateral line ganglion of the vagus is a loose aggregation of cells which extends caudad into very close relation if not into contact with an ectodermal thickening which is practically continuous with the lateral line primordium associated with the vagus nerve. This cluster of cells, representing the jugular ganglion of the vagus, is slightly more condensed in its most rostral portion, which bends mesad ventrally of the lateral line
THE NERVOUS SYSTEM OF AMPHIBIA 267
ganglion. This incipient root, however, does not reach the brain (fig. 1, G. Jug.).
3. The visceral sensory component. The visceral ganglion of the glossopharyngeus fuses with the ectodermal thickening which rests upon the entoderm of the first branchial pouch (Paper I, fig. 56, Ec.Th.). This structure is obviously the epibranchial placode of the glossopharyngeus. From it a strand of ganglion cells follows along the lateral line root to the brain. No definite relation of root fibers, however, can be made out (fig. 1, G. Vis. IX).
The visceral ganghon of the vagus nerve is in a still more embryonic condition. It consists for the most part of a loose collection of cells situated on the ventral side of the lateral line and general cutaneous gangha. The most highly different iated part of it reaches out to the ectodermal thickening over the second branchial pouch, with which it fuses. Farther caudad there is loose connection of the ganglion with the ectodermal thickening over the third and fourth visceral pouches. The ganglion at this time has no root connection with the brain (fig. 1, G.Vis.X).
B. THE EARLY FLEXURE STAGE
1. The lateral line component. The lateral line primordium, which is situated over the first branchial pouch, has become elongated dorso-vent rally, and two other primordia appear, the one situated over the second branchial pouch ojiposite the first myotome and the other opposite the second to fifth myotomes (Paper I, fig. 57).
The lateral line ganglion on the ninth nerve has now become spindle form, but it still reaches to the primordium (fig. 2, G.L.L.IX). Its root fibers reach the brain in close contact with the root fibers from the lateral line ganghon on the vagus. The latter ganglion now sends out a projection of cells which reaches to the primordium which is situated over the second branchial pouch, and extends caudad to the primordium of the trunk (fig. 2).
The roots of these postauditory lateral line ganglia approach the brain together (figs. 29 and 30). In figure 30 this root
268 G. E. COGHILL
appears in two parts, a dorsal, from the ganglion on the vagus, and a ventral, from the ganglion on the ninth nerve. There are no perceptible ascending and descending divisions of these root fibers within the brain at this stage.
2. The general cutaneous component. The jugular gangHon holds about the same position relative to the other ganglia as in the younger stage, although there is now no apparent connection with the skin in its more caudal portion. It does' connect with the skin, however, at a point near the second post-auditory lateral line primordium. This strand of cells is in very close association with the ganglionic projection to this primordium. It forms an incipient r. auricularis vagi.
3. The visceral sensory component. The epibranchial placode over the first branchial pouch is now massive and has a broad connection with the visceral ganglion of the ninth nerve (fig. 2, G.Vis.IX). It is now contributing cells, apparently, to the ganglion. The latter extends along the lateral line division of the nerve to the brain. Its attachment to the brain is shown in figures 30 and 31 {R.Vis.IX).
A projection of the visceral ganglion of the vagus still connects with a placode-like mass of cells situated over the second branchial pouch. The larger portion of the ganglion projects caudad and ventrad towards the other visceral pouches, near which it ends in indefinite clusters of cells. A slender strand of indifferent cells reaches from the ganglion to the brain, in which there may be a few root fibers (fig. 32, R.X). In figure 32 the strand of cells which represents the root (G.X), extends caudad between the brain and the myotome (M). Here a single root fiber appears. This may, however, be a motor fiber. No root fibers can be positively recognized as arising from cells in the ganglion.
C. THE COILED-REACTION STAGE
1 . The lateral line component. The lateral fine ganglion of the ninth nerve still reaches to the skin, but a relatively large strand of fibers without sheath cells now spreads across the primordium. This is an incipient r. supratemporalis IX. The ganglion has
THE NERVOUS SYSTEM OF AMPHIBIA 269
enlarged greatly and stretches along the root till it comes into close relation with the lateral line root of the vagus (fig. 3). There is, then, a considerable advance made towards the consoUdation of all the postauditory lateral line ganglia into a common mass (compare Goghill, '02, figs. 1 and 2). The ganglionic projection, however, which reaches out on the r. auricularis vagi now amounts practically to a distinct ganglion with its own root connection with the main lateral line root of the vagus. In the terminal portion of this smaller ganglion fibers connect with the primordium, but this nerve does not seem to be quite as well developed as the r. supratemporaHs. It is a question whether these two nerves innervate a common primordium (Paper I, fig. 58) or whether there may not be two primordia here separated by an ectodermal thickening to which the jugular ganglion connects.
The primorida of the trunk now apj)ear in two distinct patches (Paper I, fig. 58, LL.). One of these lies opposite the tliird and fourth myotomes and the other opposite the seventh to the tenth myotomes. To both of these the main lateral line ganglion of the vagus sends nerves, which separate from each other a short distance from the ganglion.
Figures 42 to 46 rej)resent five successive serial section which show the lateral line roots entering the brain. There are still two divisions of the roots, a dorsal and ventral, marked, respectively, a and h. Each division forms an ascending and a descending tract, the longitudinal extent of which is shown in figure 5.
2. The general cutaneous component. The jugular ganghon still lies on the lateral aspect of the proximal portion of the lateral line ganglion of the vagus as an ill-defined group of cells. It reaches the skin as a ganglion rather than as a nerve, in close relation with the small lateral line ganglion already mentioned. Its point of contact with the skin seems to be between the lateral line primordium innervated by the latter ganglion and that innervated by the r. supratemporaHs. It projects also caudad in loose relation with the visceral ganglion, but its relations in this region are ver}' indefinite. A root-like projection of the ganglion
270 G. E. COGHILL
passes ventrally of the lateralis ganglion and through the Vshaped cleft of the first myotome, but no fibers can be traced from this region into the brain.
3. The visceral sensory component. The visceral ganglion of the ninth nerve has enlarged greatly since the last stage, in which it appeared to be receiving cells from the epibranchial placode. It now touches the auditory vesicle (fig. 3, G.Vis.IX). Distally, however, it still reaches to the entoderm, where it has a very indefinite border. Throughout its distal portion it shows two rather distinct portions, the more dorsal of which projects the farther over the entoderm. No nerve trunks are yet to be found arising from this ganglion. It has a root, however, which enters the brain as a relatively strong bundle of fibers on the ventral aspect of the lateral line roots. This relation is shown in figures 44 and 45 (R.Vis.IX). The fasciculus solitarius {Fas. Sol.) is seen in figure 42 rostrally of the root. Caudally of the root it appears in figures 45 and 46, where it is larger. The most of the visceral sensory root fibers must, therefore, turn caudad in the fasciculus (fig. 5).
The visceral ganglion of the vagus still reaches out to the branchial pouches. No peripheral nerves yet arise from it. Its root enters the brain as shown in figures 47 to 49. Figure 47 shows ascending vagus fibers just ventrally of the glossopharyngeal portion of the fasciculus solitarius (X). Figure 49 shows that the fasciculus solitarius {Fas. Sol.) is smaller on the caudal side of the root. It is apparent from this that the visceral sensory fibers are directed chiefly cephalad in the fasciculus at this time. The fasciculus solitarius extends only a short distance caudad of the root (fig. 5, R.Vis.X).
D. THE EARLY SWIMMING STAGE
1. The lateral line component. In addition to- the lateral Une primordia of the trunk there are now two distinct postauditory primordia, the one innervated by the r. supratemporalis, the other by the lateralis component of the r. auricularis vagi (fig. 4; Paper I, fig. 59) . The lateral line can be traced clearly as far
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caudad as the seventeenth myotome, almost the full extent of the primordia. Fibers reach the primordium of the inferior Hne, also. The small lateral hne ganglion which is associated with the r. auricularis vagi still stands out distinctly. The root fibers from the gangha on the vagus pass to the brain on the dorsal surface of the root of the ninth nerve. The root fibers have extended their tracts as indicated in figure 6 {L.L.IX,X Asc.,Des.). The relation of the root fibers to the tracts is shown in figures 62 to 65. The roots here are very compact bundles of fibers. There is no apparent interchange of fibers between the roots from the different ganglia. The dorsal tract seems to represent the vagus ganglion and the ventral tract the glossopharyngeal ganglion. The ventral tract runs rostrad in very close relation to the fasciculus sohtarius and at the level of the middle of the auditory vesicle takes a position ventrally of the lateral line tracts of the facial nerve (fig. 61, L.L.IX,XJ)).
2. The general cutaneous component. The jugular ganghon holds about the same position relative to the other ganglia as in the earUer stages. Only in favorable preparations can it be distinguished from the visceral ganglion, over which it lies in the form of a rather tliin cap. It gives rise to the r. auricularis vagi, which is still a very short nerve. The exact distribution of the fibers can not be made out. General cutaneous fibers join also the projections of the visceral ganglion which reach out to the second and third branchial pouches. It has been impossible to demonstrate fibers from the jugular ganghon to the external gills, to the skin around the base of the gills or to the ventral surfaces of this part of the head. The cutaneous innervation, so far as the ganglionic system is concerned, is in a much lower state of development in the post auditory region than it is in the preauditory.
The root of the jugular ganghon now reaches the brain, passing across the dorsal surface of the visceral root from the rostral to the caudal side (fig. 4, R.GJug.). It enters the brain at a slightly more ventral level than does the visceral root, as shown in figures 67 to 69 {R.GJug., fig. 69). This root is continuous from the ganghon to the brain surface with the spinal accessory
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root (fig. 4, R,XI), which in this stage extends caudad to the level of the third myotome (compare Coghill '02, fig. 1, root X4,ci,h,c,d). The latter root is probably largely motor in the early swimming stage, although cells that look like neuroblasts occasionally occur in it. The root of the jugular ganglion enters the descending trigeminal tract, as shown in figure 6. The diffuse nature of the tract in this region is illustrated in figures 66 to 70 {Tr.Des.V).
3. The visceral sensory component. The end of the glossopharyngeal ganglion still rests upon the entoderm, but there is now a postbranchial nerve arising from it which extends ventrad behind the pouch and runs a considerable distance rostrad in the primordium of the first branchial bar (fig. 4, G.Vis.IX,g). This is the ramus lingualis.
No fibers can be traced from this nerve to the skin. Neither do fibers pass from it into the entoderm. Nevertheless it must be primarily visceral sensory, although it may at this time contain motor fibers also. The nerve adheres in its course very closely to muscle primordia, just as do the r. mandibularis V, and the r. hyomandibularis VII of this age.
The root of this nerve still enters the brain in close contact with the ventral surface of the lateral line component (figs. 4 63, 64). Figures 62 and 65, respectively, show the condition just rostrally and caudally of the entrance of the root fibers, which join the fasciculus solitarius {Fas. Sol.).
The projections of the vagus ganglion have not yet completely lost their connection with the entoderm of the first and second branchial pouches, while the caudal portion of the ganglion frays out into loose cells over the branchial region. The first n. branchialis vagi passes some distance into the primordium of the second gill arch behind the corresponding pouch, but it is not as well developed as the r. lingualis of the glossopharyngeus (fig. 4, G.Vis.X,h). No other nerves can be found arising from the ganglion at this time. There appear to be no pharyngeal or prebranchial divisions of any of the postauditory branchial nerves in the early swimming stage.
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The root of the vagus ganglion is distinctly more developed than in the earUer stage. As it approaches the brain it turns distinctly dorsad from beneath the general cutaneous root (fig. 3, 67 to 69, R.Vis.X). These root fibers must have a very short course within the brain, for the fasciculus sohtarius (Fas. Sol.) diminishes considerably in thickness within the space of 20 m from the root entrance, as shown in figure 66. A few fibers of the fasciculus, however, extend caudad for some distance (fig. 6). Dorsally of the fasciculus sohtarius are found a few fibers of the sensory ascending tract from the spinal cord (figs. 66, 67, 68, 70, DT); and ventrally of it is the descending sensory tract of the trigeminus. At no place has the fasciculus sohtarius become ciisplaced from its earliest position beneath the external limiting membrane to the deeper position which characterizes the adult.
4- Tlie eye and optic nerve
"WTiile the retina is recognized as essentially a part of the brain and the optic nerve as morphologically a tract of the central nervous system, the function of the eye as an exteroceptor requires that it be treated here with the afferent system. The structure of the eye at the four successive stages of development under consideration has been illustrated by figures 72 (non-motile), 74 (early flexure), 76 (coiled-reaction), and 78 (early swimming). These are all taken from transverse sections of embryos at the level which shows the greatest extent of connection of the lens with the skin, or as nearly as could be determined, through corresponding points in the eye.
In the non-motile stage (fig. 72) the pigmented layer is thick at the margins but soon tapers off into a relatively thin, simple epithelium, which has not yet acquired a high degree of pigmentation. It fits down closely upon the retina, excepting in the ventral portion where clefts may occur here and there beneath it. The central ends of the retinal cells have well defined boundaries, and have a considerable pigment deposited about them. Mitosis is going on rapidly throughout the entire peripheral (ventricular, with reference to the brain) border next to the pigment laj-er. The lens at this stage is simply a discoid thickening
274 G. E. COGHILL
of the epithelium, which fits into the optic cup. Its margin conforms closely to that of the retina but there is a narrow space between them. In the ventral portion of this space mesenchyme cells occur.
In the early flexure stage (fig. 74) the pigment layer has become thinner, particularly at the margins, and its pigmentation has increased considerably. The optic cup has greatly deepened. The cell boundaries along the central border of the retina have become less distinct and cells occur there which suggest the nature of neuroblasts. Mitotic figures are still abundant in the central portion of the ventricular margin of the retina. The lens has acquired the form of a hemisphere, or shghtly in advance of this, since the line of contact with the skin does not equal the diameter of the lens. Mitosis is going on rapidly in the central portion of the lens and there is a faint suggestion of a cavity in it. The space between the lens and the retina has greatly increased, owing, apparently, to the increased curvature of the retina, and mesenchyme cells have pushed in almost to the center of this space (fig. 74, Mes.). Mesenchyma is invading the space, also, from dorsad.
The retina of the coiled-reaction stage (fig. 76) has rounded up into more nearly a spherical form. Cells which appear to be neuroblasts are now conspicuous in the middle region of the inner margin. The cells along the whole outer border (ventricular surface) are still proliferating rapidly by mitosis. While the. lens is almost free from the skin, there is still a small area of close contact with it. A distinct cavity has appeared in the lens, on the outer side of which the epithelium is much the thicker. The skin over the lens is still thick, and mitosis continues in the lens.
In embryos of the early swmming stage (fig. 78) the skin over the lens has become reduced to a thin epitheUum, which, however, is still pigmented in a considerable degree. The lens, though in contact with the skin, is perfectly detached from it. The cavity in it has increased greatly in size, and the inner epithelium is now much the thicker. The cells here are long and columnar and their borders well defined. Mesenchyma extends
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into the space between the skin and the retina, practically into contact with the lens both dorsally and ventrally. The pigment layer of the retina has now become highly pigmented, so that most of its nuclei are obscured from view. Mitosis is still going on in the middle of the outer layer of the retina, but it appears to be more abundant towards the margins. In the inner layer of the retina there now occur many ganglion cells, particularly in the middle region. Good silver impregnations show that the axones of these cells form an optic nerve which extends along the optic stalk into the brain, where they enter the most caudal portion of the chiasma ridge. The form and position of these cells are illustrated in figure 71. The axone of this cell passes into very close relation with the internal limiting membrane of the retina, where it mingles with other axones. Nothing can be made out concerning dendritic processes of these cells. They appear still to be unii)olar.
According to favorable siher impregnations of both A. punctatum and A. microstomum Cope, these optic fibers form a chiasma which is in intmiate relation with postoptic commissure at this time. The optic fibers, being more deply impregnated than the other fibers of the commissure, may be followed clearly across the middle line of the brain. They appear to be a constant feature of the brain of the early swimming embryo, though they do not stand out clearly in stained preparation as tho\- do in silver impregnations.
In the same preparations which demonstrate the ganglion cells and their fibers clearly nothing of a nervous nature can be seen deeper in the retina. There are some round nuclei which suggest neuroblasts destined to become bipolar cells; but the layer of rods and cones seems veiy embryonic, since, as mentioned above, mitosis is still going on in its central region and no well defined cellular organization appears there.
5. The olfactory organ and nerve
In the non-motile stage the olfactory organ consists of a greatly thickened patch of ectoderm which has externally a very slight concavity. It touches the brain only lightly in its dorso-caudal
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region but not in the region from which the olfactory nerve of later stages arises. In the early flexure stage the external concavity has deepened slightly. The cells of the olfactory epithelium have become highly columnar and heavily pigmented peripherally. The organ has come into rather close contact with the brain in the rostral region, but no direct connection can be made out between them. In the coiled-reaction stage the external concavity has become a shallow pit with well defined margins, but the depth is not equal to the diameter of the aperture. There is now very intimate contact between the olfactory epithelium and the brain at the point where the olfactory nerve is about to appear, but no fibers have been observed entering the brain in this stage. In the transition from this to the early swimming stage the olfactory pit has deepened considerably but has not yet dilated perceptibly within the nares. The olfactory nerve has now become well established. It arises from near the rostral margin of the olfactory epithelium, where the latter is in actual contact with the brain. Silver impregnations indicate that the fibers arise from neurones situated near the base of the nerve, though the impregnations of the cell bodies has been very imperfect and unsatisfactory. In frontal sections (parallel with the longitudinal axis of the embryo) the fibers appear running both caudad and rostrad within the brain into a zone of highly differentiated neurones which, presumably, are the mitral cells. There is a cell-free area here which obviously represents the glomerular zone.
6. Summary
A. THE NON-MOTILE STAGE
The ophthalmic and Gasserian ganglia are both connected with the skin at their distal ends (figs. 1, 7 to 12). No fibers can be clearly traced from them to the skin. Their root fibers, however, reach the brain. Large cells, having the characteristics of the Rohon-Beard cells of the spinal cord, situated near the entrance of the trigeminal root, send processes out into this nerve (figs. 20, 21). The general cutaneous ganglion of the vagus (jugular)
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is very indefinite in outline and has neither peripheral nor root fibers arising from it. The lateral Une ganglia all have root fibers connecting with the brain. Their peripheral ends reach to the corresponding primordia of the lateral lines, excepting in the case of the ganglion of the hj'omandibular system, the relation of which to the skin is very indefinite. The auditory vesicle (fig. 73) is continuous with the skin and with the auditory ganglion. A barely perceptible auditory root connects with the brain. The visceral sensory system is represented b}' the geniculate ganghon, the glossopharyngeal and vagus. All of these connect with thickened patches of ectoderm which are associated with the visceral pouches. The geniculate has a very small root which reaches the brain. A strand of cells connects the glossopharyngeal ganglion with the brain but no fibrillar elements appear in this. The vagus ganglion has no root. The structure of the eye at this stage is illustrated in figure 72. The olfactory epithelium has barely begun to invaginate and does not yet touch the brain at the point of future origin of the olfactory nerve.
B. THE EARLY FLEXURE STAGE
The ganglia of the trigeminal nerve have severed their connection with the skin and now have peripheral fibers which connect obscurely with the skin (fig. 2). The root of the nerve forms a very short ascending tract and a descending tract which, augmented by neurones of the second order, extends to the level of the auditory vesicle (fig. 22). The jugular ganglion of the vagus has a very indefinite outline. It connects directly with the overlying skin but has no perceptible peripheral or root fibers. The ganglia of the acustico-lateral system have acquired greater definiteness of outline and compactness of structure. They now send fibers to the various prunordia, with which they are still in contact. Their root fibers enter the brain distinctly. The auditory vesicle has become detached from the skin and from the auditory ganglion (fig. 75). The visceral sensory ganglia are still connected with the epibranchial placodes and no peripheral fibers arise from them. The root fibers of the geniculate
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278 G. E. COGHILL
and glossopharyngeal ganglia enter the brain, but the vagus ganglion has no root. The optic cup has deepened greatly and the lens projects deeply into its cavity. The invagination of the olfactory epithelium is more marked but there is not yet an olfactory nerve.
C. THE COILED-REACTION STAGE
The disti-ibution of the trigeminal fibers to the skin is much more obvious than in the earlier stage, the mesial terminal branch of the r. ophthahnicus profundus being well defined. A descending trigeminal tract of a composite nature extends to the spinal levels (figs. 3, 5). The giant ganglion cell component of the trigeminus is now particularly clear (figs. 34 to 37). The outline of the jugular ganglion is still indefinite and no perceptible fibers leave the ganglion. The lateral line primordia have become much extended and a mandibular line makes its appearance. Lateral line nerves follow along the primordia closely attached to the skin, although no fibers have been traced in this stage to the mandibular line. All of the lateral line roots form ascending and descending tracts in the brain, and the auditory root forms a short descending tract (fig. 5). The auditory vesicle of this stage is illustrated in figure 77. The geniculate ganglion is still united with its epibranchial placode, but from it emerge several fibers which project ventrad behind the spiracular pouch as an incipient r. alveolaris. The root fibers from this ganglion form the fasciculus solitarius which runs caudad immediately beneath the external limiting membrane to the level of the glossopharyngeal root which enters it. Peripherally the glossopharyngeal ganglion connects with its epibranchial placode. The vagus ganglion now has a small root which joins the fasciculus solitarius. Distally this ganglion connects with the ej^ibranchial placodes, but gives rise to no peripheral fibers. The condition of the eye is shown in figure 76. The olfactory epithelium is now in close contact with the brain, but olfactor}^ root fibers do not certainly enter the brain in this stage.
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D. THE EARLY SWIMMING STAGE
Of the general cutaneous system the lateral and mesial terminal branches of the r. ophthalmicus profundus, the r. maxillaris, r. mandibularis with a nerve to the balancer, and a nerve from the ophthalmic ganglion directly to the overlying skin are well defined (fig. 4). The r. auricularis vagi is perceptible but its distribution is uncertain. The root fibers of the trigeminus now extend in the descending tract to about the level of the root of the vagus nerve, and this tract is joined by the root of the vagus ganglion (fig. 6). The giant ganglion cell component of the trigeminal nerve is still present, but its exact distribution through the nerve can not be determined. The hyomandibular lateral line primordium is now continuous with the other preauditory primordia and is innervated by the r. hyomandibularis VII. The other lateral line primordia are now innervated practically throughout their entire extent, the lateral Une of the trunk extending into the level of the seventeenth myotome. The extent of the ascending and descending tracts of the lateral Hne roots is shown in figure 0, where the extent of the descending auditory fibers is also figured. The visceral sensory component is now represented by a well developed r. alveolaris VII, r. lingualis IX, and a less developed first r. branchiahs vagi. There are no prebranchial or pharyngeal branches of the postauditory branchial nerves. The vagus root now connects with the brain and contributes fibers to the fasciculus solitarius. In the retina mitosis is still going on rapidh' in the middle of the external layer, while in the internal layer ganglion cells are already developed which send their axones along the optic stalk into the brain. They decussate in the most rostral portion of the postoptic commissure. Only a suggestion appears of neuroblasts in the region of the bipolar cell la^'er. The olfactory nerve is well established and its fibers distribute themselves in a very definite glomerular zone of the olfactory lobe.
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II. THE PHYSIOLOGICAL PAirr 1. Response to tactile stimulation
Early in my study of the responses of these embryos to tactile stimulation it became apparent to. me that the skin of the preauditory region was less sensitive than was that of the postauditory region. For conclusive evidence upon this point, however, I am indebted to one of my graduate students, Miss Ruth Orcutt, who in March, 1911 carried out a series of experiments, two of which I introduce here as typical of her results.
Ten specimens were selected which were approaching very near to the typical coiled-reaction stage, their movements being not quite extensive enough to be typical. These were touched with a hair one after the other in regular order in the following manner: At each trial an embryo was at first touched in the region of the eye. If this stimulus produced a response, the next embryo of the series was touched; if it did not produce response, the same embryo was, after an interval, touched over the postauditory region in the territory of innervation by the vagus nerve. If response occurred, the next embryo was tested; if not, the same embryo was then touched over the myotomes of the cervical region, the territory of the Rohon-Beard cells of the spinal cord. The ten embryos were worked over in regular order in this manner till one hundred forty-three responses occurred. Of these five were in response to touch on the preauditory region; 66, to stimulation of the territory of the vagus nerve; and 72, to stimulation of spinal territory strictly.
Another group of embryos of a distinctly more advanced stage, being in the advanced coiled-reaction stage, were tested in the same manner. These gave 94 responses, of which 12 were in response to stimulation of the trigeminal region; 41, to stimulation of the territory of the vagus; 41, to stimulation of the spinal region.
These and other experiments upon this question give unequivocal evidence that stimuli from the spinal field reach the motor centers with much greater facility and certainty than do stimuli
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from the territory of the vagus nerve; and that, as compared with stimuU from these areas, stimuU very rarely reach the motor centers from the trigeminal field in embryos of the tj^jical coiled reaction stage. At the time these experiments upon this subject were made we had no exact information upon the nature of the innervation of the regions under consideration. The anatomical part of this paper, however, shows that the trigeminal nerve, throughout the whole period of development up to the early swimming stage, runs far ahead of the general cutaneous component of the vagus in the development of both peripheral and root fibers. The relative accessibihty of motor centers, therefore, to stimuh from trigeminal and vagus territories can not depend upon the relative development of these nerves. The certainty of responses, when the stimuli are applied to the spinal region, obviously depends upon the perfection of the giant ganglion cell system of afferent neurones and their direct access to motor centers (Paper I). The neurones of this system occur as far rostrad as the second myotome, which is essentially within the limits of the medulla oblongata. They may, indeed, occur here and there farther cephalad as shown in this paper. Their peripheral fibers, therefore, probably invade the region which was regarded in the above mentioned experiments as belonging to the vagus, but in a less efficient manner than in the trunk, so that response is not as certain from stimulation here as it is from stimulation farther caudad. The response to stimulation of the whole postauditory territory and trunk is in all probability effected through the giant ganghon cells system in the coiledreaction stage. And since we now recognize cells of this type in the trigeminal nerve, such responses as occur to stimulation of this region in the earlier periods may take place through this system also. Granting, however, that all the giant ganglion cells in the trigeminus are distributed to the skin rather than to the muscle primordia, the innervation of the preauditory region by this system must be very sparse as compared with that of the postauditory and trunk regions, for there are only a few giant ganglion cells in the vicinity of the trigeminal root. Such sparseness of inner\'ation, upon the hypothesis that the gan
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glionic portion of the trigeminus is not functionally connected with the motor centers, might explain the low degree of responsiveness to stimuli in this region. On the other hand, if the responses are effected through the ganglionic system of the trigeminus their infrequency in the coiled-reaction stage must depend upon the limitations of the zone of influence of the root fibers within the brain, for the peripheral distribution of the nerve at this time is very well developed.
As a factor in this problem the descending trigeminal tract naturally suggests itself. This tract has been described in the first part of the paper as being of a composite nature — made up of processes of neurones of the first and second, and possibly higher orders, in varying proportions according to the region. The root fibers of the trigeminal ganglia run some distance caudad in the coiled-reaction stage — probably almost to the level of the auditory vesicle. Beyond this the tract, now extending into the spinal region, consists of processes from central neurones along the course. If, then, stimuli reach the motor centers in the lower regions of the medulla oblongata through the descending trigeminal tract they would have to traverse a series of relatively short neurones with intercalated synapses after leaving the trigeminal conductors. Such a conduction path would certainly introduce relatively great resistance if it did not block the stimuli altogether. This is, in all probability, the really significant factor in determining the behavior of embryos of the coiled-reaction stage to tactile stimulation of the preauditory region.
In the early swimming stage the condition is quite different. The trigeminal fibers now reach almost or quite to the level of the vagus root, that is, into the immediate vicinity of the centers that dominate the whole motor mechanism. Accordingly, response to stimulation of the trigeminal region has become prompt and regular. The data at hand, therefore, afford very good evidence that such a descending trigeminal tract as occurs in the early flexure stage (fig. 22) is at best a very inefficient conductor; and that before response can become prompt and constant the root fibers of the nerve must reach directly into the approximate levels of the motor centers. We have in this con
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ditioii, then, the significance of the rapid growth of the descending trigeminal tract between the coiled-reaction and early swimming stages, for there is very close correlation between the development of the power of locomotion and the introduction of the most anterior sensory field of the organism into direct connection with the motor centers so that it becomes effective in determining the direction of locomotion.
The significance of this correlation in the growth of the motor and sensory systems finds a striking demonstration in the reactions of embryos of advanced swimming stage which have been transected in the upper portion of the trunk (Paper I, p. 200). This operation cuts out both the exteroceptive and proprioceptive stimuU from the greater portion of the trunk and leaves the motor centers more exclusively to the influence of the cutaneous field of the head. In head pieces of this kind, which have just enough of the trunk musculature attached to give unequivocal responses, the movements are almost uni\'ersally away from the side touched. It is obvious, therefore, that this avoiding form of response to stimuli about the anterior end of the embryo is a basic thing in the orientation of the swimming animal to its environment; and, judged by the anatomical results of this paper, its perfection and eflSciency depend upon the growth of the trigeminal root fibers into proximity with the motor centers.
In the above experiments upon the relative irritability of different areas it was observed that the regions untlcr consideration differed materially with reference to the nature of the response they evoke. In the first set, three of the five responses to stimulation of the trigeminal area were away from the side touched, whereas 62 of the 66 responses to stimulation of the vagus field were away from the side touched, as were all of the 72 responses to stimulation of the spinal field. In the second experiment mentioned the embryos were considerably older and all of the 94 responses were away from the side touched excepting two of the twelve responses to stimulation of the trigeminal area. These data are in accord with my general experience with these embryos, namely, that with increased responsiveness there is increased purity of type in response. In other words, embryos
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that are not readily excited to motor response are much more likely to move towards the side touched than are embryos that are easily excited to movement.
The conclusion of the last paragraph raises a question which has been mentioned in an earlier paper ('09, p. 252) as to whether movement away from the side touched is a fundamentally primary thing in behavior or a secondary thing, derived by a process of selection out of a diffuse or irregular type of response. From the conclusion reached concerning the structural basis for prompt and certain response to stimulation of the trigeminal field, it might be inferred that this problem also hangs directly and solely upon the degree of development of the peripheral neurones in their relation to the motor centers. In other words, if a stimulus must pass through such a descending trigeminal tract as is shown in figure 22, representing the early flexure stage, there is a larger element of chance or accident in the direction of the response than there would be if the stimulus traveled into the immediate vicinity of the motor nuclei through the peripheral neurones. This would be an attractive hypothesis, but, to my mind, it is only of secondary consideration. When, for instance, there is a high degree of responsiveness (a low threshold of stimulation for the entire reflex arc concerned) a stimulus such as is employed experimentally would act in a much more localized or restricted area than it would when there is a low degree of responsiveness. Under the latter condition the stimulus would probably be transmitted to relatively distant receptors, possibly even to the opposite side of the embryo and, finding here access to an arc of lower threshold, it would excite a response that apparently violates the regular law of behavior. This explanation gains credence from Hooker's experiments ('11) which show that amphibian embryos of the ages under consideration are very sensitive to stretching of the skin by pressure upon relatively distant points. In fact his experiments show conclusively that in order that a tactile stimulus act locally in any appreciable degree in these embryos there must be a very low threshold of stimulation for the reflex arc as a whole. Undue resistance at any point in the arc would certainly allow the
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stimulus to act on more distant receptors of arcs with, possibl3^ a lower resistance. The cause of irregularity in the direction of response when responsiveness is low is, therefore, in all probability to be found in the receptor sj'stem and adequate stimulation of an arc as a whole rather than in an hypothetical diffuse form of conduction through the central nervous sj'stem.
2, Respofise to chemical sti?nulatwn
The question of the existence of a true irritabihty to substances in solution as opposed to their destructive action on the skin has been discussed in Paper I. Bj- spraying small jets of acid upon restricted regions of the embr\'o and by immersing head pieces of transected specimens in acid it has been shown that the skin of the head is like that of the trunk in its sensitiveness to the action of acid. It appears, therefore, that the definitive, ganglionic nerves of the ages under consideration do not differ from the giant ganglion cell system in respect to chemical stimulation.
3. Response to light
During the season of 1910 I performed a series of experiments upon the reaction of larvae and embryos of A. opacum and A. punctatum to light. Sunlight, thrown from a mirror through a water jacket and condenser, was employed in such a way as to bring to bear very high illumination upon sharply restricted regions of the animals. As a check on the adequacy of the stimulus a larva of A. opacum was used which was about ten days beyond the age when swimming begins. When intense illumination was brought to bear over the head and eyes of this larva responses were fairly prompt, as the following figures show, the reaction times being indicated in seconds, with the interval between one response and the next stimulus indicated in parentheses: 5, (15); 10, (15); 8, (20); 8, (30); 8, (20); 3, (30); 8, (30); 7, (30) ; 8, (30) ; 8. The responses were always quick, total body movements and swimming away from the stimulus, slightly to one side rather than straight forward from the position when stimu
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lated. Immediately following this series of responses the larva lay with its trunk and tail only in the highly illuminated field for five minutes without movement, excepting one small movement of the head. In a fainter light, but with illumination sufficiently strong to cast a distinct shadow upon the dark surface on which the larva rested, it lay for ten minutes longer motionless. The responses recorded above, therefore, must have been to stimulation of the eyes and not to an action of the light upon the skin; and the irritability of the skin to light in this relatively late stage must be negligible.
The methods of the above experiment were applied to five embryos of A, punctatum which had almost but not quite reached the swdmming stage, but the results gave no evidence of irritability to light. Four specimens which had reached the early swimming stage, however, gave the following results (the time of application of the stimulus being indicated in seconds, the plus sign indicating a reaction after the time indicated, the minus sign indicating no reaction) :
Specimen a. 20 +, 20 +, 10 +, 60 -, 30 +, 30 +, 10 +.
Specimen b. 60 -, 20 +, 120 +, 10 +, 60 +, 10 +, 60 -.
Specimen c. 60 -, 70 +, 60 -, 60 -, 20 +, 15 +.
Specimen d. 60 -, 45 +, 50 +, 60 -, 60 -, 60 -, 60 -.
The specimens were stimulated one after the other so that there was a relatively long interval between successive stimuli applied to the same specimen. The illumination during this period may not have been absolutely constant but it was at all times very brilliant. As embryos of this age only rarely move without some form of external stimulation, it appears that the responses observed in this experiment were due to the stimulation of the retina by light, but the threshold of stimulation is exceedingly high. The shortest reaction time was ten seconds, as compared with three seconds in the older larva; whereas one specimen lay in one trial for two minutes before responding and in two other trials gave the shortest reaction time.
Another set of specimens of the same age were tested in the same manner with the following results:
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Specimen e. 60 -, 60 -, 60 -, 60 -, 60 -, 60 -, 60 -, 60 -, 60 -.
Specimen f. 60 -, 60 -, 35 +, 25 +, 60 -, 55 +, 60 -, 60 -, 25 +, 30 +.
Specimen g. 60 -, 60 -, 30 +, 60 -, 60 -, 45 -, 50 +, 60 +, 60 +, 35 +.
Specimen h. 55 +, 60 -, 60 -, 60 -, 60 +, 60 -, 60 -, 60 -, 53 +, 60 -.
In brief, specimen e gives no evidence of irritability to light, / and g show noteworthy evidence of it, while the other three show slight evidence of it. It may be said, therefore, that at the time swimming begins the retina is just beginning to be irritable to high illumination.
•The anatomical explanation for these results will not be fully before us till the central nervous system is better understood, but it is important to note here that it is just at this stage that the optic nerve makes its connection with the brain and that the retina is in a very embryonic condition in its outer layers. In the light of these reactions interest attaches to this order of development in the retina. The ganglion cells, it has been noted, are well differentiated and their fibers decussate in the incipicMit chiasma. These neurones have as high a degree of difTcrcMitiation as have other neurones that are known to be in the functional condition. The inference, therefore, is that the high and xariable threshold of the retina at this stage is due to the embryonic condition of the more peripheral elements in the reflex circuit. On the other hand, it is interesting to know that with the layer of rods and cones and the bipolar cell layer in such an embryonic condition there could be any optic reflexes stimulated. One almost questions whether the ganglion cells themselves ma>' not possess at this time a certain degree of irritability to light. In the early swimming stage, as described in the anatomical part of the paper, mitosis is till going on in the central zone of the layer of rods and cones and only suggestions of neuroblasts can be detected in the region of the layer of bipolar cells. It seems incredible that speciahzed photic receptors and conduction paths can be already- established in such embryonic structures. Upon the basis of studies which are now in progress on the central paths of the optic reflexes it will be necessary to return to more exhaustive cytological study of the retina and to an amplification of
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the experimental evidence of the development of function in the organ.
4. Response to olfactory stimulation
In the anatomical part of the paper it was noted that the olfactory nerve is well established in the early swimming stage. Preliminary experiments were made in 1912 to discover whether olfactory stimuli in any way stimulate movement. Pellets of various substances were enclosed in gauze and placed in the center of flat dishes about 9 by 13 cm. In these dishes embryos somewhat older than the early swimming stage were distributed in cistern water. Pellets of pure gauze were used as checks. In other pellets were placed, egg masses, fresh algae and debris from aquaria which emitted a strong odor. The embryos in all the dishes were frequently agitated to discover whether the olfactory organ had any influence in directing movements stimulated from other causes. Over thirty embryos in the different test dishes and the same number in the check dish were kept under observation for three hours without the manifestation of any tendency on the part of the specimens to collect in the vicinity of the odiferous substance. A number of experiments of this kind gave no further suggestion of olfactory reflexes. Experiments of this kind, however, should be carried further to determine whether other substances may not be found which would stimulate olfactory reactions, either directly or by reinforcing or inhibiting other responses. It has been noted however, that, although olfactory neurones have grown into the brain in considerable numbers, the olfactory epithelium is very poorly differentiated in the ages under consideration.
5. The auditory and lateral line organs
The relative development of the lateral line system of organs and nerves during this period arouses inquiry concerning their function. In the postauditory region this system develops distinctly in advance of the general cutaneous component in both its central and its peripheral relations. Even in the preauditory
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region, where the general cutaneous system is more precocious, the connection of the lateral line nerves with the skin becomes readily recognizable earlier than in the case of the general cutaneous nerves, and throughout the period under consideration the lateral line nerves have the more intimate and the more extensive contact with the skin. Judged upon the anatomical basis, therefore, the lateral line system would certainly be held as playing a larger role in the life of the organism at this time than do the general cutaneous nerves. I have been unable, however, to discover what this function may be and have further experiments planned.
The auditory organ, being a simple dilated vesicle, gives no evidence of being a mechanism that would be adapted as a receptor to the function of equilibration, so that the auditory nerve, which has established quite extensive relations within the brain and intimate contact with the vesicular epithelium in the early swimming stage probably does not enter into the reactions of the organism till a later period. As is well known, the embryos of this stage lie on the side while at rest and hold the upright position only during locomotion. The embryos which I select as representing the early swimming stage move upon the substratum, and their ujoright position during locomotion is probably the result of the lateral thrust of the head, which would tend to prevent them from settling down on either side till motion ceases.
6. Summary
a. The j^reauditory portion of the ganglionic general cutaneous system develops far in advance of the postauditor}' portion. The former becomes a factor in quick and certain response only about the time the embryo begins to swim, although response to preauditory stmiulation occurs irregularly and infrequently for a considerable time before this.
b. The entrance of the receptor field of the trigeminal nerve into function as an important factor in the behavior is closely correlated with the extension of the root fibers of the trigeminal
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nerve into the ininiediate vicinity of the motor centers in the lower portion of the medulla oblongata.
c. Such irregularity as appears early in development in the direction of movement relative to the side touched depends upon the condition that the point of adequate stimulus is not the point actually touched, by reason of the variability in the threshold of stimulation of reflex arcs as a whole, and not upon diffuse conduction through the nervous system. Reflex arcs are from the first definite and fixed during the period under consideration.
d. The afferent system of the head is like that of the trunk with reference to chemical stimulation.
e. A slight responsiveness to high illumination of the retina occurs about the time the animal begins to swim, and this is in close correlation with the development of the first fibers of the optic nerve into the brain. Owing to the exceedingly embryonic condition of the retina at this time this topic demands further investigation.
f. Although the olfactory nerve is well established at the end of the period under investigation there are no perceptible reactions to olfactory stimulation.
g. There is no available evidence that the auditory organ or lateral line nerves have any part in reactions, although the latter, judged upon the basis of its anatomical relations, would be regarded as more efficient than the ganglionic general cutaneous system.
h. The physiology of the eye, olfactory organ, auditory organ and lateral line organs require further investigation in connection with anatomical studies of the brain, which are now in progress.
III. DISCUSSION
It is the purpose to deal here with the work of other investigators which seems to have direct bearing upon the results of this paper.
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1. Lateral line primordia, placodes and ectodermal thickenings
In the use of the terms 'lateral Hne primordia,' 'placodes' and 'ectodermal thickenings,' I am following Landacre and Conger in their work upon Ameiurus and Lepidosteus (Landacre, '10, '12; Landacre and Conger, '13). I wish, however, at the outset of this discussion, to emphasize the fact that mj- series of Amblystoma embryos is not adapted to a critical study of the histogenesis of these structures, for my specimens have been selected at relatively long intervals according to physiological stages and with reference to a problem that is not primarily one of histogenesis. It seems obvious, nevertheless, that there is a striking resemblance between Amblystoma and Lepidosteus (Landacre and Conger, '13) in the early processes of differentiation of sense organs and nerves.
While I have not undertaken to determine to what extent epibranchial placodes contribute to the formation of the various cranial ganglia, it seems certain that the visceral ganglia of the facial, glossopharyngeal and vagus nerves all receive masses of cells from this source, particularly (hu'ing the middle and latter part of the period covered by my studies. This tardy differentiation of the visceral as compared with the somatic sensory ganglia is correlated with a late entrance of the visceral sensory field into the behavior, and is particularlj' noteworthy in connection with Landacre's observation that the relatively late differentiation of the ejiibranchial i^lacodes in Lepidosteus as compared with Ameiurus is correlated with a late development of the taste buds in the former as compared with the latter, accoitling to particular growth periods.
No lateral line placodes have been recognized in Amblystoma of m}' series, unless the ectodermal thickening which joins the auditory vesicle to the preauditory lateral line primordium be regarded as a vestige of a dorso-lateral placode. In section this thickening of the skin has the appearance of that which is formed in the early flexure stage over the ophthalmic ganglion where the latter has just become detached from the ectoderm. The significance of this attachment of the ophthalmic ganglion to
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the skin docs not seem to be understood. Landacre ('12) observed the same rehitions in Lepidosteus, but, apparently, did not satisfy himself concerning the meaning of it.
The ectodermal thickenings which Landacre and Conger have differentiated from the epibranchial placodes and the lateral Hne primordia certainly will be found also in Amblystoma, although my studies have not extended to the details of their relations. This subject deserves exhaustive study from material selected for the purpose. My object in touching upon this topic has been merely to bring the salient features in the differentiation of the nervous system which these structures represent into correlation with physiological processes in the organism as a whole. Limited as this study has been, however, I have been increasingly impressed with the results of Landacre's studies on this subject as fundamental to a broadly biological view of the development of the nervous system of the vertebrate head. These ectodermal thickenings and proliferations impress me not simply as vestiges of an ancestral metamerism or branchiomerism, but as centers of physiological processes which may be essential factors in the regulation of the growth of the nervous system. There is a field here for experimental studies, it seems to me, which might prove as illuminating as the well known observations upon the correlative development of the lens and the retina, and it is to be hoped that the purely embryological studies of these structures may be extended to other types of vertebrates.
2. The junction of the auditory vesicle
Streeter ('06) has studied the development of the ear as correlated with the development of the function of equilibration in the tadpole of the frog. He found that shortly before tadpoles acquire the ability to maintain an upright position the labyrinth consists of a closed epithelial sac incompletely subdivided into compartments and possessing differentiated nerve endings which are connected with the brain by the acoustic nerve and ganghon." The semicircular canals are "in the process of development, but are not completely pocketed off until after equilibration is already
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established. Consequently the semicircular canals as such ai'e not an essential factor in equilibration." He observed, further, that tadpoles from which he removed one auditory vesicle in the 3 mm. stage began, when 9 to 9.5 mm. long, to differ from normal tadpoles in the manner of swimming — the operated specimens having a tendency "to swim with the operated side under, and in the rolling movements around the long axis of the body it is from the operated side under to the opposite." Streeter concludes that it is at about this time that "functional union is normally established between the ear vesicle and the spinal cord reflex centers, upon which the individual is dependent for maintaining its position in free water." Estimated by Street er's notes, this is about two days after the animals begin to swim, and, therefore, at a stage much beyond that which I call the early swimming stage in Amblystoma. This difference in age is expressed in the structure of the ear as well as in the efficiency of the motor system in locomotion, for the ear of the Amblystoma early swimming stage is a perfectly simple sac, except for the endolymphatic appendage — a much simpler organ than the ear of the frog tadpole when it acquires the power to swim upright in free water. Streeter notes that the auditory nerve is developed at this time, but does not describe its central relations in detail. While it would be interesting to know these central relations from the point of view of functional mechanism as a whole, Streeter's results seem to be conclusive on the question of the relation of the auditory organ as such to the function of equilibration, and they do not lead us to expect any functional union of the ear with the motor centers in embryos of Amblystoma of the early swimming stage.
3. The sensory nerve roots in relation to later development
The medulla oblongata of Amblystoma of lengths from 17 to 38 mm. has been recently described by Herrick ('14). Such larvae are practically adult so far as reactions are concerned and in the structure of the medulla oblongata Herrick considers that all the fundamental relations of the adult have been acquired.
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A comparison of his descriptions with the anatomical results of this paper will show that the general plan of the sensory centers of the medulla oblongata is laid down by the growth inward of the root fibers of the peripheral nerves. The trigeminal and lateral line fibers have formed extensive ascending and descending tracts, and the auditory and geniculate ganglia have formed descending tracts of considerable length by the time the animal begins to swim. These tracts are all closely grouped and all lie immediately beneath the external limiting membranes, and constitute the substantia alba of the dorsal portion of the medulla oblongata. They lie embedded in the marginal zone of the substantia grisea, in which are neurones directed dorso-ventrad across the whole series of tracts of the various sensory modes. In the neurones of this group there can scarcely be any specificity of function with reference to the different sensory nerves (compare Herrick and Coghill '15). To this elementary plan the adult has added the longitudinal association tracts a and h of Kingsbury and Herrick, and an additional lateral line tract, while the fasciculus solitarius has become detached from the perip'hery and with its displacement inward the lateral line centers have developed greatly ventrad as well as dorsad.
Without knowledge of intermediate stages between the early swimming stage and the younger of Herrick's descriptions it is impossible to say just how the three root bundles of the lateral line VII of Herrick are derived from the two bundles of the younger stage. Since, however, the root bundles of the younger stage have the same relation to each other and to the root bundles of the postauditory system as do the two more ventral bundles of Herrick, and since the most dorsal bundle of Herrick has, in younger stages, smaller fibers than the other bundles, it is reasonable to infer that the most dorsal bundle of Herrick is a distinct and newly acquired tract, and not a detached portion of one or the other, or of both of the tracts of the younger stage.
In the relation of the several root bundles to particular ganglia Herrick finds that, in the case of the facial division, each of the tracts receives fibers from both the preauditory ganglia. This is a very difficult relation to determine in the young embryos,
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for they must be studied under very high magnifications, and the roots come into very close relations with each other as they approach the brain. It seems unquestionable, however, that the more dorsal tract is derived chiefl}' from the dorsal ganglion (a) and that the ventral tract comes chiefly from the ventral ganglion (b). In the case of the postauditory group, the dorsal tract is derived chiefly from the vagus and the ventral, chiefly from the glossopharyngeal ganglion. It is impossible to say that there is no interchange of fibers between the roots of either set, but if such anastomosis does occur it nuist iinolve only a very few fibers.
If these lateral line tracts were each composed of fibers from one ganglion exclusively there would be grounds for assuming that they effect a certain degree of localization in the brain with reference to the particular areas with which they are severally connected. Herrick's observation of the mingling of the fibers of the different roots, however, makes it necessary to seek some other explanation for this early differentiation of the lateral line system within the brain. There is nothing in the sense organs to suggest that this has to do with different sensory modes. Its significance, therefore, must in some way have to do with the directness with which stinuili may i)ass to the motor centers. But that this mechanism as it is found in early swimming embryos could serve such a function seems incredible, particularly in view of the nature and arrangement of the neurones of the second order in the circuit. It is hoped that the study which is in ]')rogress on the association paths of the brain nia>' offer something towards the solution of this problem.
In view of Herrick's observations of the bifurcation of the root fibers, it is of interest to note, further, that in the case of the trigeminal and lateral line nerves this takes place very early in development. In the case of the visceral sensory and auditory roots, however, there is little or no evidence of bifurcation of the fibers in the early swimming stage.
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4. The development of function in the neurone and in the reflex arc
It has already been pointed out (Paper I) that the giant ganghon cells of the spinal cord on the one hand and the motor neurones on the other have their connections made with their respective end-organs for a considerable time before they become integral parts of a functional reflex arc, and that, for this reason, these neurones can not be used as a standard by which to judge the structural changes in the neurone that mark the beginning of nervous function. Now we find that the cranial afferent nerves also are in the earlier stages developed in a degree that seems out of proportion to the part that they can be demonstrated to have in reflexes. We have noted, for example, that, in the coiled reaction stage, the trigeminal nerve has a very general distribution to the skin and that it has root connection with the brain from a much earlier period, whereas it becomes an important factor in behavior only about the time the embryo begins to swim. The neurones of this nerve must for a considerable time receive stimuli and conduct impulses that are never discharged through any reflex arc to the effectors. The root fibers of the visceral sensory system also are well developed and the fasciculus solitarius is extensively developed for many days before the neurones of this system are subjected to physiological stimulation by the development of the mouth. None of these neurones, therefore, can give us a clue as to when they take on nervous functions.
Our observations upon the development of the optic nerve in relation to the function of the eye show that the same uncertainty is attached to the neurones of the second or higher order in the arc. In this case the retinal ganglion cells are highly differentiated and can, under very strong stimulation of the retina, become an integral part of a reflex arc at a time when the more peripheral sensory elements of the arc are in an exceedingly embryonic condition. The high threshold of the optic reflex must then be due to the undeveloped condition of the retina whereas the ganglion cells, optic nerve fibers, and the central neurones reaching caudad from the diencephalon to the lower
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portion of the medulla oblongata must be capable of conducting stimuli for a considerable period before they can be excited by normal stimulation of the retina. The development of the olfactory nerve and centers suggests that the same principle applies to much of the central nervous system in early development. We have, then, within our knowledge of the development of reflexes and reflex arcs no rational basis for cytological studies of the difi"erentiation of the neurone as correlated with the development of nervous function. It is hoped that the studies which are now in progress upon the central conduction paths will furnish such a basis for cytological investigations; and that the api)lication of cytological methods to nerve cells of known physiological capacity may reveal the essential structural basis of conduction in the neurone.
5. The cranial nerves of Amphibia
Some points of interest concerning the morphology and development of the cranial nerves of Amphibia deserve mention here.
In my paper on the cranial nerves of Amblystoma ('02, p. 215, and figure 1) the lateralis ganglion which corresponds to Vila of this paper is described as closely fused with the ganglion of the ophthalmicus profundus, and the latter as very intimately related with the Gasserian ganglion. In the younger embryos of this study these ganglia are widely separated, but in the latter part of the period under in\Tstigation these ganglia, particularly the Gasserian and profundus, approach each other rapidly, and finally the Gasserian and profundus ganglia estabhsh a wide contact, the lateralis ganglion VII a stiU standing apart from them. A similar change, though of less degree, occurs in these ganglia in Rana pipiens, according to Landacre and McClellan ('12), who describe the ganglion of the ophthalmicus profundus in the 8 mm. embryo as standing out "rather distinctly, indicating its definite character," and as being much more isolated in younger stages. In the early development, therefore, of both Anura and Urodela there is a process of consolidation going on between the various ganglia of the V -|- VII complex, a process
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which goes farther in Anura than in Urodela, that is to say, till all the g;anglia become fused into a common mass, whereas they become two masses in the Urodela.
This migration and fusion of ganglia involves not only a shifting of position relative to each other but also a general movement of the ganglia towards the brain along the path of their respective roots. There is also a shifting of the otocyst rostrad with reference to the point of entrance of the facial and auditory roots into the brain. Such a movement of the vesicle together with its expansion in growth, if carried far enough before chondrification of the auditory capsule sets in, might explain the fusion of all these preauditory ganglia into a common mass in Urodela; but this could not explain the migration caudad of the ganglion of the opthalmicus profundus, for instance, as illustrated in figures 1 to 4. There is apparent no purely mechanical cause for such movement on the part of these ganglia.
A very similar migration and a consolidation of ganglia occur in the postauditory ganglionic complex as well. In adult Amblystoma there is a single postauditory ganglionic mass which can be resolved into its component ganglia only with difficulty (Coghill, '02, p. 231 and fig. 1). In the ages studied here (figs. 1 to 4), however, there are two widely separated ganglionic masses, one upon the glossopharyngeal and the other upon the vagus nerve. Changes very like this occur in Anura according to Landacre and McClellan, for they describe a rather marked distinctness of the different ganglia of the postauditory complex in 8 mm. embryos of Rana pipiens. The fusion of these ganglia may be accounted for by the dilation of the auditory vesicle in later development, and the consequent pushing caudad of the glossopharyngeal ganglia upon those of the vagus. Before this takes place, however, there is a perceptible migration of some of these ganglia along their respective roots towards the brain, although this is not as clear as the migration of the trigeminal ganglia. The chief differences, therefore, between the cranial ganglia of Anura and those of Urodela depend, apparently, upon two factors — an active migration of the various ganglia towards
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the brain and pressure exerted by the dilation of the auditory vesicle in its growth.
In my discussion of the hyomandibular nerve of Amphibia ('02, pp. 264-271) I pointed out differences between the Anura and Urodela which then seemed to me to warrant the hypothesis that the ramus alveolaris VII of the latter was a pretrematic nerve. The essential basis for that hypothesis was the apparent prebranchial position of the nerve in relatively advanced larvae "anteriorly of the deep pharyngeal evagination which represents the embryonic spiracular cleft" ('02, p. 228). This position, however, must be secondary, for, as described in this paper, the first nerve that grows out from the geniculate ganglion enters the truncus hyomandibularis and passes behind the ectodermal-entodermal contact of the spiracular pouch. This position must be primary, as Emmel found it to be for the chorda tympani in the development of ^licrotus ('04), and the nerve must undergo very much the same kind of a shifting in position from an actual postbranchial position to an apparent prebranchial position just as the chorda tympani does in mammals, according to Emmel's descriptions. Contrary to my earlier interpretation, therefore, the r. alveolaris MI of Urodela should be regarded as homologous with the chorda tympani of manunals.
6. Xeurobiotaxis
In numerous contributions (cited in his report which is mentioned in the bibliography appended to the present paper) Kappers has presented an interesting array of data to show that the definitive position of the perikarya of motor neurones in the brain of vertebrates is determined by "a process of taxis or tropism occurring under normal conditions of nervous action, that is, under the influence of reception and propagation of stimuU." This process he designates neurobiotaxis. It involves the conception of a bodily migration of the motor neurones towards the chief source of stunulation by the afferent neurones or through the tracts of the brain. A discussion of this conception as regards motor neurones does not belong in this paper on
300 G. E. COGHILL
the afferent system, but it is important to observe here that this paper describes specific instances of migration of cells masses in the form of several of the cranial ganglia. The clearest cases of this are the Gasserian and profundus ganglia, particularly the latter. This movement of the ganglia along the root towards the brain deserves more exhaustive and specific study, but some factors in the problem may be definitely stated. The ganglion of the ophthalmicus profundus V, for instance, in the earlier period is anchored to the skin far out over the eye. While this anchorage is intact root fibers make their connection with the brain. In embryos of the early flexure stage these fibers have established a firm anchorage to the brain by the bifurcation of the root beneath the external limiting membrane, and now their hold upon the skin is lost. Immediately a movement of the ganglion begins which finally carries it snugly up against the brain immediately around the entrance of the root (figs. 51 to 54).. During this migration there are no apparent extrinsic mechanical factors introduced. A comparison of figures 1 and 2 shows that the migration has begun before the ganglion comes into contact with the eye, and comparison of figures 76 and 78 shows that after the ganglion comes in contact with the most dorsal portion of the eye it moves caudad over the surface of the eye — a median section through the eye in figure 78 showing only the tip of the ganglion while a similar section in figure 76 shows the massive portion of the ganglion. The ganglion moves caudad, therefore, with reference to the eye as well as with reference to the brain and auditory vesicle. There must be, then, some intrinsic factor involved in this movement.
As such a factor the root fibers with their firm achorage within the brain immediately suggest themselves (fig. 22). As has been described above, these fibers are growing very rapidly caudad towards the chief motor centers in the lower portion of the medulla oblongata and the upper portion of the spinal cord. This growth may be regarded not simply as an increase in mass but as an active stretching out of the whole fiber in an amoeboid fashion after the manner of the movements of nerve cells growing in vitro as Harrison ('10, '11) has described them. In other
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words, the entire axone may be thought of as creeping inward past its point of anchorage at the surface of the brain, as an amoeba would creep between obstacles, and as di-agging the perikaryon after it. What the stimulus for such an action may be I shall not 'discuss here. I only wish to emphasize that this is a concrete case of migration of a group of neurones and that the rapid advance caudad of the root fibers within the brain and the concomitant shortening (not wrinkling) of the extracerebral root fibers convinces me that the axones are active factors in this process.
Another feature of the development of the sensorj^ cranial nerves deserves mention in connection with the question of neurobiotaxis, namely, the relative rates of development of the nerve trunks and their respective roots. In case of the trigeminal nerve it has been noted in the anatomical part of this paper that the root connections with the brain are well established at a time when no peripheral fibers can be traced to the skin The lateral line primordia, also, are very small and are reached directly by the corresponding ganglia at a time when the latter have relatively long roots connecting with the brain. The most conspicuous case of this kind, however, is the visceral sensory system, the ganglia of which are connected with the placodes and are without peripheral nerves, excepting a few fibers of indefinite distribution from the geniculate ganglion, at a time when the root fibers have already formed a well defined fasciculus solitarius which reaches from the facial root to that of the vagus.
It is apparent, then, that in the development of the sensory cranial nerves the axone runs far ahead of the dendrite. This suggests the inference that, from the point of view of taxes or tropisms, normal peripheral excitation does not stimulate the growth of the axone inward. This is not meant to imply that there can be no nervous processes in these neurones before the dendritic terminals are estabhshed, but that, if there are such processes, they can have no reference to such stimuli as normally act upon the corresponding definitive nerves and have significance in the reactions of the animal. Similar conditions
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prevail in the development of the reflex mechanism of the trunk, where the Rohon-Beard cells have their typical connection with the skin and the motor cells have their terminals established upon the muscles for a considerable time before they become incorporated into a functional reflex arc and are, therefore, of any value to the animal as regards behavior. From these facts it appears, then, that the behavior-value of nervous processes, if there be such in these nerve cells of early stages of development, has no regulating influence in establishing the primary plan of the integrating mechanism of the organism. WTiat this regulating agency may be is one of the chief topics of interest in my study, but it can be best discussed upon the basis of an exhaustive anatomical and physiological analysis of the development of the various elements of the central nervous system during this early period. Such a study I already have well under way.
Since this paper was submitted for publication new evidence of a 'general chemical sense' in the skin of animals has been published by Crozier ('16) with particular reference to my suggestion ('14, pp. 205-207) that the stimulating effect of substances in solution may be accounted for by their destructive action upon the epithelial cells. He concludes that there is "no ground for Coghill's assumption that the cells of the germinative layer of the epithelium of fishes and amphibians are exposed to the action of the stimulating agent and thereby disrupted; and there is no histological evidence of disruption."
Without entering into further discussion or expression of opinion I wish here merely to raise the following questions, which seem to me to require settling before the main question at issue can be regarded as closed:
1. Since the skin of the invertebrate is very different anatomically from that of the vertebrate (the earthworm, for instance, having sensory nerve cells in the skin with endings free on the surface) may the reactions of the one be legitimately judged upon the basis of the structure and function of the other?
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2. Since, according to Crozier the "concentrations of irritants employed b}^ Parker and others" "do not penetrate at all" are there free nerve endings at the surface of the skin of vertebrates to function as receptors for these chemical stimuli (see HerrickCoghill, '98)?
3. Since theepidermis of fishes and amphibians may be thought of as a system of three colloidal agglomerations, normally in equilibrium with each other — (1) the cellular protoplasm, and (2) surface films of mucus-like substance, which bathe opposite surfaces of (3) the cell membrane — Jiiay not very great disturbances of equilibrium with consequent violent effect upon neighboring parts result from the addition of a foreign solvent or solute to the surface film actually without the added solvent or solute penetrating the cell membrane (Nelson, '13, particularly table 5)?
4. In order to stinuilate the tactile nerve endings mechanically through the reaction of the epithelial cells nmst the action of the reagent be so violent as to produce histologically or chemically demonstrable effect upon these cells? May not relatively slight increase or decrease in the turgor of the epithelial cells be sufficient to stimulate?
5. Why should the stinuilating effect of water when applied after the application of a chemical stinnilus be evidence of destruction of tissue in one case ("If any serious disintegration were produced by these solutions, it would be reasonable to expect the continuance of activity after external supply of the stimulant had been removed." Crozier, '16, p. 3) and in another case be evidence of a 'general chemical sense' ("Washing the foot with distilled water does not lead to cessation of the contraction, because, after exposure of the foot to certain solutions (Loeb '05), water stimulates" Crozier, '16, p. 5)?
6. How can this last mentioned irritability to water after chemical stimulation be explained upon the hypothesis of a "general chemical sense," and if it can be so explained why should it appear so conspicuoush^ in the behavior of amphibian embryos (Coghill, '14. ])p. 197, 198) the responses of which, Crozier con
304 G. E. COGHILL
cedes, are probably not at all due to sensory stimulation by acid?"
7. Granting that the so-called chemical stimuli act indirectly through the epithelial cells upon the nerve endings mechanically, may not the api)arent physiological separation of the tactile and chemical receptors by use of cocaine still be accounted for upon the basis of the comparative strength of stimuli (Coghill, '14, pp. 206, 207)?
8. Regarding psychological differentiation of the two forms of stimuli as applied to the inner surface of the cheek of man, are the components of the cranial nerves of man sufficiently known to warrant the inference that morphologically equivalent receptors are concerned in the oral cavity of man and in the skin of the fish or frog? May not the visceral sensory system be a factor in the irritability in the oral cavity?
Some of these questions I have under investigation and I shall probably discuss them further in a later paper. Meantime, in the interest of the leading problem of my investigations, I shall hope to see Crozier's methods of inquiry projected further into the question concerning the existence of a 'general chemical sense' in the skin of vertebrates.
THE NERVOUS SYSTEM OF AMPHIBIA 305
BIBLIOGRAPHY
CoGHiLL, G. E. 1914 Correlated anatomical and physiological studies of the
growth of the nervous system of Amphibia. I. The afferent system
of the trunk. Jour. Comp. Neur., vol. 24, no. 2. Crozier, W. J. 1916 Regarding the existence of the 'common chemical sense'
in vertebrates. Jour. Comp. Neur., vol. 26, no. 1. Emmel, V. E. 1904 The relation of the chorda tj'mpani to the visceral arches
in Microtus. Jour. Comp. Neur., vol. 14. Harrison, Ross Granville 1910 The outgrowth of the nerve fiber as a mode
of protoplasmic movement. Jour. E.xp. Zool., vol. 9, no. 4.
1911 On the stereotropism of embryonic cells. Science, N. P., vol. 34, no. 870.
Herrick, C. Judson 1914 The medulla oblongata of larval Amblystoma.
Jour. Comp. Neur., vol. 24, no. 4. Herrick, C. Judson, and Coghill, G. E. 1915 The development of the reflex
mechanism in Amblj'stoma. Jour. Comp. Neur., vol. 25, no. 1. Herhick, C. L., and Coghill, G. E. 1898 The somatic equilibrium and the
nerve endings in "the skin. Jour. Comp. Neur., vol. 8. Hooker, Davenport 1911 The development and function of voluntary and
cardiac muscle in embryos without nerves. Jour. E.xp. Zool., vol. 11,
no. 2. Kappers, C. U. Ariens, 1914 Phenomena of neurobiotaxis in the central
nervous system. XVHth International Congress of Medicine, London :
1913, Anatomy and Embryology. Landacre, F. L. 1907 On the place of origin and method of distribution of
taste buds in Ameiurus nielas. Jour. Comp. Neur., vol. 17.
1910 The origin of the cranial ganglia in Ameiurus. Jour. Comp.
Neur., vol. 20, no. 4.
1912 The epibranchial placodes of Lepidosteus osseus and their relation to the cerebral ganglia. Jour. Comp. Neur., vol. 22, no. 1.
Landacre, F. L., and Conger, A. C. 1913 The origin of the lateral line pri mordia in Lepidosteus osseus. Jour. Comp. Neur., vol. 23, no. 6. Landacre, F. L.. and McClell.vn, Marie F. 1912 The cerebral ganglia of the
embryo of Rana pipiens. Jour. Comp. Neur., vol. 22, no. 5. LoEB, J. 1905 Production of hypersensitivity of the skin by electrolytes. In:
Studies in General Pliysiology, pt. II, Chicago, pp. 748-765. Nelson, C. Ferdinand 1913 Studies in osmosis. Jour. Amer. Chem. Soc,
vol. 35, No. 6. |^(^ Streeter, George L. 190^ Some experiments on the developing ear vesicle
of the tadpole with relation to equilibration. Jour. Exp. Zool., vol. 3. i^.
306 G. E. COGHILL
EXPLANATION OF FIGUUES
All of the figures are taken from Amblystoma puuctatum Cope, excepting figure 71, which is from A. microstomum Cope, and figures 11 and 12, which are from another species of Amblystoma, probably jeffersonianum.
Methods. The methods followed in the preparation of Nos. 444, 449, 467 and 473 were described in Paper I. In Nos. 543, 54G, 545, 550, 557 and 593 the fixation was with Van Gehuchten's fluid, and staining with erythrosin and toluidin blue. For No. 455 fixation was with formol-Zenker solution; staining with Bomer's haematoxylin and slightly acidulated orange G. For Nos. 561, 562 and 567 fixation was with Zenker's solution and staining with iron haematoxylin and orange G., excepting that the latter was omitted in No. 567. For 447 and 496 the fixation was with corrosive sublimate-acetic acid mixture and staining with alum cochineal (447) and alum carmine (496) in tof.o with Lyon's blue in 95 per cent alcohol on the slide. No. 635 was prepared with Paton's silver method: neutral 10 per cent formalin about three months [formalin (40 per cent formaldehyde) is saturated with sodium carbonate or lithium carbonate and the fixing fluid is made by mixing one part of this stock solution with nine parts of distilled water]; washed in running water over night; f per cent silver nitrate five days and four hours; f per cent nitrate of silver to which four drops of sodium hydroxide have been added for every 20 cc. of the solution, and then about twelve drops of ammonia, two hours; bath in distilled water to which one drop of glacial acetic acid has been added for every 2 cc, twenty minutes; 1 per cent freshly dissolved hydrochinone, sixteen hours; washed in distilled water; dehydrated through graded and absolute alcohol in about 6f hours; cleared and embedded through cedar oil, cedar oil one part and paraffin one part, cedar oil one part and paraffin two parts, then pure paraffin, in two hours or a little less. My experience is that this method of clearing and embedding is very satisfactory in use upon amphibian embryos, in which the yolk is a very refractory element. The silver-impregnated material does not suffer from treatment with cedar oil for a day or more.
ABBREVIATIONS
Ad., area of adhesion between the skin Ent., the point of entrance of the nerve
and ganglion or otocyst as indicated indicated into the brain
Aud.V., the auditory vesicle or otocyst Fas. sol., the fasciculus solitarius
c, the branch of the n. mandibularis V g, ramus lingualis of the glossopharynto the balancer geal nerve
d, ramus palatinus VII G.G., the Gasserian ganglion DC, the Rohon-Beard cells of the spinal G.gen., the geniculate ganglion
cord and cells of like character in the G. Jug., the jugular ganglion of the
brain vagus
DT, the dorsal sensory tract of the G.L.L.VII, the lateral line ganglia of
spinal cord and its extension into the the facial nerve
brain G.L.L.IX, the lateral line ganglion of
e,n. hyomandibularis VII the glossopharyngeal nerve
Ec.Th., thickened regions of ectoderm G.L.L.X, the lateral line ganglion of
End., the endolymphatic appendage the vagus
of the otocyst
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307
G.oph., the ganglion of the ophthalmicus profundus
G.VIII, the ganglion of the auditory nerve
G.Vis.IX, the visceral ganglion of the glossopharyngeus
G.Vis.X, the visceral ganglion of the vagus
h, the postbranchial division of the first ramus branchialis vagi
L.o.p.V, the lateral terminal branch of the ophthalmicus profundus
L.L.VII, lateral line fibers of the facial nerve within the brain
L.L.VII, Asc, the ascending divisions of the lateral line root fibers of the facialis
L.L.VII,Des., the descending divisions of the lateral line root fibers of the facialis
L.L.IX,X, the lateral line root fibers of the glossopharyngeus and vagus within the brain
L.L.IX,X,Asc., the ascending divisions of the lateral line root fibers of the glossopharyngeus and vagus
L.L.IX,XJ)c-'^., the descending divisions of the lateral line root fibers of the vagus
M, myotome, the third in figures 1 to 4
Mdb.V, ramus mandibularis trigemini
Mes., mesenchyma
M.o.p.V, the mesial terminal branch of the ophthalamicus profundus
Mx., ramus maxillaris trigemini
Neu. II, neurones of the second order in the reflex circuits
Nuc.vis.ni., the visceral motor nucleus
Olf., the olfactory organ
Op.St., the optic stalk
o.p.V., the ramus ophthalamicus profundus
P.L., the pigment layer of the retina
R.G.G., root of the Gasserian ganglion
R.G.Jug., root of the jugular ganglion, or the somatic sensory root of the vagus
R. L.L.VII, the lateral line root of the facialis
R.L.L.IX,X, the lateral line root of the glossopharyngeus and vagus
R.M., the root mass, a collection of chiefly indifferent cells about the entrance of the nerve roots into the brain
R.oph., the root of the ophthalmicus profundus
R.V., root of the trigeminus
R.V,ni., the motor root of the trigeminus
R.VII,m., the motor root of the facialis
R. VII, vis., the visceral sensory root of the facialis
R.VIII, the root of the auditory nerve
R. Vis. IX, the visceral sensory root of the glossopharyngeus
R.Vis.X, the visceral sensory root of the vagus
R.X,nt., the motor root of the vagus
R.XI, the spinal accessory root, or the most caudal motor root of the vagus
t. a dorsal liranch of the ramus ophthalmicus profundus
Tr.Asc.V, the ascending trigeminal tract
Tr.Dcs.V, the descending trigeminal tract
Tr.S, a tract in the motor zone, probably bulbo-spinal
r, ncrvus trigeminus
VC, the somatic motor neurones of the ventral column
]'//, nervus facialis
VIII, nervus acusticus
VT, the somatic motor tract
X, nervus vagus
30S G. E. COGHILL
Figs. 1 to 4 Graphic projections from serial, transverse sections upon the median plane to show the ganglia and sensory nerves of the head in the four physiological stages:
Fig. 1 (No. 467), the non-motile.
Fig. 2 (No. 473), the early flexure.
Fig. 3 (No. 449), the coiled-reaction.
Fig. 4 (No. 444), the early swimming stage. X 50.
The brain is drawn in heavy lines; the myotomes and sense organs, in light lines; the general somatic sensory ganglia, in stipple; the lateral line and auditory ganglia, in diagonal lines; the visceral sensory ganglia, in rectangular crosshatching. The lateral line nerves are not fully represented here. They follow the course of the lateral line primordia, the distribution of which is shown in Paper I, figures 56 to 59. These, figures with the descriptions in the text of this paper will give the important features of the lateral line nerves in the stages described. Silver impregnations of the early swimming stage show that these nerves are practically coextensive with the primordia of the organs. This is probably true of the earlier stages also, although satisfactory impregnations of the fibers have not yet been secured. The ganglia and sense organs have been projected mechanically with great care. The nerve trunks, which are too small to project distinctly at the magnification which has been used, have been sketched free-hand.
THE NERVOUS SYSTEM OF AMPHIBIA 309
PLLVll R'1-I-IX.X G.LL.X G.Jug.
G.ophA G'OP'^
Ec.T^Ent■R.VR.M.^, — ,-p p.y
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.V.L OF COMPARATIVE NEUROLOGV. VOL. 26, NO. 3
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G. E. COGHILL
Ent.V ^ R:L.LVI1 ^^^
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311
Tr.Des.V\ L.L.IX.X.Asc , , ,^ ^
Ent.V XL.L.VIIDes. Ent.L.L.IX.X
Ent VII / / L.L.IX.X.Des
Motor Column
L.L.VII Des. L L. VII Asc TrAsc.V
lEntVII
R.VII.Vi
Ent VIM VIII Des Tr Des.V
Figs. 5 and 6 Projections from serial, transverse sections upon the sagittal plane, of the sensory root fibers after they have entered the medulla oblongata, in the coiled-reaction stage (fig. 5, No. 449) and in the early swimming stage (fig. fi. No. 444). X 50. The most rostrally located Rohon-Beard cell of (he spinal cord is represented in each case, and the ascending fibers of these cells are shown as far cephalad as the root of the vagus nerve. The approximate extent of the trigeminal root fibers caudad is indicated in solid black, while the whole fiber system which I have called in the text the descending trigeminal tract but which is made up in large part of neurones of the second order is indicated in light lines about and beyond the root fibers proper. These figures are in a measure schematic, as will be seen by comparing them with figures 38 to 70, with wliich thev should be studied.
312 G. E. COGHILL
Figs. 7, 8, 9 Non-motile stage (No. 467). X 50.
Fig. 7 (section 1-3-12) illustrates the adhesion of the ophthalmic ganglion to the skin (Ad.G.oph.) dorsally of the eye, and the relation of the eye to the olfactory epithelium (0//.).
Fig. 8 (section 1-4-3) is 120 fx caudad of figure 7 and shows the constricted nature of the root of the ophthalmic ganglion, although at this level it is still ganglionic, also the relations of the retina, optic stalk and brain.
Fig. 9 (section 1-4-8) shows the adhesion of the Gasserian ganglion {G.G.) to the skin at Ad., just ventrally of the lateral line primordium iSup.L.L.). These drawings are made from the same embryo as is figure 1, with which they should be studied.
Fig. 10 Non-motile stage (No. 546, section 4-3-1). X 50. The section- lies approximately in the frontal plane, and shows favorably the two divisions of the ophthalmic ganglion {G.oph.) and the adhesion of one of these divisions with the skin (Ad.). Farther caudad the Gasserian ganglion appears in section (G.G.). A little farther caudad is the ectodermal thickening that is associated with the spiracular pouch (Ec. Th.).
Figs. 11 and 12 Non-motile stage (No. 455, sections 2-1-10 and 13). X 50.' The plane of section is longitudinal and latero-ventral, favorable to show the relations of the ophthalmic and Gasserian ganglia and their roots.
Fig. 11 shows the two divisions of the ophthalmic ganglion, a and h. Fig. 12 is 30 M farther ventrad and shows the root connections of these nerves with the" brain [R.V). Around the base of these roots is amass of cells iR.M.) which does not seem to be a part of the ganglia at this stage. I have called this the root mass. The preauditory lateral line ganglion and the visceral sensory ganglion of the facial nerve (G.VII) are represented as a single mass. Its rostral end is in contact with the entoderm of the spiracular pouch.
Fig. 13, 14 and 15 Early flexure stage (No 473, sections 1-3-14, 1-4-6 and 1-4-12 respectively.) X 50. These sections are selected from the series from which figure 2 was made, and should be compared with it.
Fig. 13 illustrates the ophthalmic ganglion after it has detached itself from the ectoderm, leaving an ectodermal thickening at Ec.Th.
Fig. 14 shows the attenuation of the root of the ophthalmic ganglion (R.oph.) 160 fi caudad of the last figure and at the level of the caudal portion of the eye.
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313
up.LL
314 G. E. COGHILL
Fig. 15 is 60 fi still farther caudad, showing the Gasserian ganglion {G.G.) connected by a slender root with the root mass (R.M.) at the surface of ther brain, and the relation of this ganglion to the lateral line primordium {Siip.L.L.).
Fig. 16 Early flexure stage (No. 550, section 1-2-14). X 50. This figure is introduced to show the plane of section of figure 22, which is taken from the same section.
Fig. 17 Coiled-reaction stage (No. 561, section 1-3-14). X 50. This figure is introduced to show the plane of section of figure 33, which was taken from the region indicated between the lines at c.
Fig. 18 Early flexure stage I'No. 545, section 5-2-14). X 500. A ganglion cell with its peripheral, dendritic process, in the distal portion of the ophthalmic ganglion. The spherules in the cytoplasm are yolk.
Fig. 19 Early flexure stage (No. 593, section 2-5-7). X 500. Ganglion cells of the Gasserian ganglion, with axones reaching towards the brain. Other indifferent cells appear around the ganglion cells. The yolk is indicated in the cytoplasm as in the last figure.
Figs. 20 and 21 Non-motile stage transverse section (No. 543, sections 3-3-16 and 13, respectively). X 500.
Fig. 20 is 15 M caudad of figure 21, and shows root fibers of the trigeminal nerve within the brain {Tr.Des.V). Both figures show cells of the root mass (R.M.) Fig. 21 a large cell [D.C.) which has the anatomical features of RohonBeard cells of the spinal cord sends a process out of the brain into the trigeminal root. A similar cell [D.C.) appears in figure 20, in which figure the motor nucleus of the trigeminus (Nuc. vis.m.) is seen to be in a much more ventral position.
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315
m\^ .*^^ j i
^-^-^l^fisa^^^
^m/G ^ ^ :j^ DC
R.M
316
G. E. COGHILL
R.M
Aud.V
R.VII'
Fig. 22 Early flexure stage (No. 550, section 1-2-14). X 200 The plane of section of this figure is illustrated in figure 16. It illustrates a longitudinal section of the medulla oblongata, in which the entrance of the trigeminal root appears (R.V). There is a short ascending trigeminal tract {Tr.asc.V) and a descending trigeminal tract {Tr.des.V) which reaches to the level of the middle of the auditory vesicle (Aud.V) The ventricular pits opposite the entrance of the roots are conspicuous features of such a section.
Figs. 23 to 28 Early flexure state (No. 473, sections 1-5-14 to 19). X 500. These figures are from six successive sections of the series from which figure 2 was made, and show the relations of the root bundles of the seventh and eighth
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317
R.L.LVII
w I
R.L.LVII
R.VIII
lUMVcs as tliov enter tlie l)rain. Figures 25 and 26 show the root fibers of the
lateral line component {R.L.L.VII, a and 6) and figures 24 and 23 show the ascending divisions of these root fibers {L.L.VII,Asc.). P'igure 25 shows also the root
fibers of the visceral sensory component (R. VII. vis.) of the facial nerve approaching the brain amid cells of the root mass, and figure 27 shows this component
entering the brain. Immediately ventrally of this is the root of the eighth nerve.
In the next section caiidad, figure 28, descending divisions of the lateral line
root fibers (L.L.VIIJJcs.) appear, but nothing can be seen of the other components of this complex. The entire cephalocaudal range through which the
lateral line component can be recognized in this series within the brain is only
60 ju.
318 a. E. COGHILL
Figs. 29 to 31 Early flexure stage (No! 473, sections 1-6-16 to 18). X 500. three successive transverse sections through the entrance of the root of the glossopharyngeal nerve into the brain. The lateral line component {R.L.L.IX,X) enters in figures 29 and 30, and the visceral sensory component (R.Vis.IX) enters in figures 30 and 31 at a slightly more ventral and caudal level than the entrance of the lateral line component. No ascending or descending fibers of this complex can be made out beyond the limits of these three sections, a range of 30 m Fig. 32 Early flexure stage (No. 473, section 2-1-12). X 500. This figure shows the only perceptible connection of the vagus nerve other than the lateral line root with the brain at this time, a single nerve fiber (R.X.), which is of doubtful nature. It may be motor.
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319
320 G. E. COGHILL
Fig. 33 Coiled-reaction stage (No. 561, section 1-3-14). X 500. The plane • of section is shown in figure 17, in which the region of this figure is included between the lines and indicated by a. The caudal end of the figure is to the left, and is a great deal farther dorsad than is the rostral or right end of the figure. The root of the trigeminal nerve {R.V) here approaches the brain in two divisions, the root from the Gasserian ganglion (R.G.G.) and that of the ophthalmic ganglion (R.oph.). The latter is entering the brain and bifurcating into a short ascending tract (Tr.Asc.V) and a massive descending tract {Tr.Des.V). In the angle of this bifurcation is a large neurone {D.C.), the very large nucleus of which is in sharp contrast with the surrounding nuclei. This cell lies in the dorsoventral level of the sensory root and is far removed from the motor nucleus, several cells of which can be recognized in a more ventral position {Nuc. vis. m.).
Fig. 34 Coiled-reaction stage (No. 562, section 1-4-2). X 500. This is from' a transverse section located 56 fx rostrad of the entrance of the trigeminal root. It shows the presence at this level of ascending root fibers of this nerve {Tr.Asc.V). In a more dorsal position there is a cell of the Rohon-Beard type {D.C.) which has a long process extending into the immediate vicinity of or actually into the ascending trigeminal tract.
Figs. 35 to 37 Coiled-reaction stage (No. 562, sections 1-4-9, 10 and 11). X 500. Three successive transverse sections through the brain at the entrance of the trigeminal root {R.V).
Figure 35 is the most rostral in position, and shows two or three cells of the type described in the last two figures {D.C). Another cell of this type appears in figures 36 and 37 {D.C), two sections through the same cell. In both figures the peripheral process of the cell is shown entering the trigeminal root. In figure 37 there is a stump of a dorsally directed process, and in figure 36, evidence of a ventrally directed process. Cells of the root mass {R.M.) appear in all these fioures.
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Tr.Des.V ■>■ -^-V;i-,-;^. .^«:iS:~ Tr.AscV
322 G. E. COGHILL
Figs. 38 to 41 Coiled-reaction stage (No. 447, sections 1-5-10 to 13). X 500. Four successive, serial, transverse sections through the entrance of the seventh and eighth nerves into the brain. These figures are from the same specimen as are figures 3 and 5, with which they should be studied. Figure 38 is the most rostral of the series. In this figure the root of the lateral line ganglion on the hyomandibular division of the nerve {R.L.L.VII,b) is entering, and the ascending fibers of the other division {L.L.VII,Asc.a) appear in a slightly more dorsal position. These fibers are seen entering the brain in the three following figures {R.L.L.VII,a). The visceral sensory component of the facial nerve is entering the brain in figure 39, and its fibers appear as the fasciculus solitarius (Fas. Sol.) in the two following figures. The auditory root {R.VIII) enters in figures 40 and 41. All of these root systems lie dorsally of the descending trigeminal tract (Tr.Des.V), which consists here of scattered fibers through a rather extensive
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323
^;:^P^^^^
Nuc.vis
324
G. E. COGHILL
R L L Vll^a
RVIIl
Figs. 42 to 46 Coiled-reaction stage (No. 447, sections 2-1-6 to 10) X 500.
Five successive serial, transverse sections through the entrance of the glossopharyngeal nerve into the brain. Figure 42 is the most rostral. In it can be
readily recognized the ascending fibers of the lateral line component of the ninth
and tenth nerves {L.L.IX,X,Asc.a and b), and ventrally of these the visceral
sensory component of the facial nerve as the fasciculus soUtarius (Fas. Sol.).
The lateral line components are entering the brain in the three successive figures,
while the visceral sensory (R.Vis.X) enters in figure 44. In the two following
figures this component appears with the facial fibers as the fasciculus solitarius,
and in figure 46 the descending divisions of the lateral line roots occur (L.L.
IX,X,Des.a,b). The descending trigeminal tract (Tr.Des.V) appears as scattered
fibers immediately beneath the external limiting membrance throughout this
region. Neurones of the second order (Neu. II) stretch across the whole mesial
face of this system of root fibers.
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325
Neu.ll
R.LLIX.X
Nuc.vis.m
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326 G. E. COGHILL
Figs. 47, 48, 49 Coiled-reaction stage (No 449, sections 1-6-17, 18, 19). X 500 Three successive serial transverse sections through the brain at the level of the entrance of the root of the vagus nerve. Figure 47 is the most rostral of this set: In it the visceral sensory root of the vagus (R.Vis.X) is approaching the brain surrounded by cells of the root mass {R.M.), and the fasciculus solitarius appears in two clusters of fibers {Fas. Sol.), a more dorsal representing the facial and glossopharyngeal portion and a ventral consisting of ascending fibers of the vagus root. This figure, as well as the following, extends ventrad to show the relation of the sensory centers to the motor column (FT), and the visceral motor nucleus {Nuc.vis.vi.). The root fibers of the vagus enter in figure 48, and in the following figure a few descending root fibers can be recognized {Fas. Sol.).
Fig. 50 Early swimming stage (No. 567, section 2-1-19). X 500. From a transverse section through the medulla oblongata 35 m caudad of the entrance of the trigeminal nerve. Around the dorsal margin of the descending trigeminal tract {Tr.Des.V) is a cluster of three or more large neurones {DG) of the giant ganglion cell type. Their processes run ventrad towards the motor nucleus of the trigeminus {Nuc.vis.vi.).
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327
32S G. E. COGHILL
Figs. 51 to 54 Early swimming stage (No. 557, sections 2-1-1,5,7,11). X 200. Four sections selected from a transverse series through the brain in the vicinity of the entrance of the root of the trigeminal nerve. The massing of the ganglia against the brain is shown here, a relation which should be noted in the study of figures 1-4. Figure 51 is the most rostral in this set, and figure 54 the most caudal. In figure 51 the ophthalmic ganglion (G.oph.) appears with a crescentic piece of the Gasserian ganglion (G.G.) on its ventral surface. The fibers from this ganglion enter in figure 52 (R.oph.)., while the root of the Gasserian ganglion enters in figure 53 {R.G.G.) more dorsally. This section is 28 m caudad of the last. In figure 54 the motor root of the nerve appears.
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329
Fourth Ventricle
'R.Vm
330 G. E. COGHILL
Figs. 55 to 60 Early swimming stage (No. 496, sections 1-5-4 to 9). X 500. Six successive serial transverse sections through the entrance of the roots of the seventh and eighth nerves into the brain. Figure 55 is the most rostral of the set. In this figure the ascending divisions of the lateral line component {L.L. VII,Asc.a,b) and the roots of the visceral sensory component iR.Vn,Vis.) and the auditory root R.VIII) are beginning to enter. The lateral line roots (R.L. L.VII,a,b) enter in figures 56 to 59. In figure 58, 59 and 60 the fibers of the visceral sensory component have become the fasciculus solitarius {Fas. Sol.), and the descending auditory root fibers (yiII,Des.) appear along the dorsal margin of the descending trigeminal tract (Tr.Des.V). Neurones of the second order (Neu. II) here lie across the entire sensory zone. These figures should be compared with figure 6.
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331
LLVIIAsc
Neu.ll
Nuc.vis.
L.LVII,
R. LL. VII :::^^hSi^H-^^^^.' E'^c^
Fas.Sol.
R.VIII
332 G, E. COGHILL
Fig. 61 Early swimming stage (No. 496, section 1-5-15). X 500. From the same series as figures 55 to 60, at the level of the middle of the auditory vesicle and endolymphatic appendage (End). This should be compared with figure 6. It shows the relations of the descending facial and ascending postauditory lateral line components in this early stage; also the fasciculus solitarius {Fas. Sol.) and the descending auditory fibers {VIII Des.). Mesenchymal cells are now pressing in between the brain and the otocyst.
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333
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Figs. 62 to 65. Earh^ swimming stage (No. 496, sections 1-5-24 to 1-6-2). X 500. Four successive transverse serial sections through the entrance of the glossopharyngeal nerve into the brain. Figure 62 is the most rostral. The lateral line roots {R.L.L.IX,X) enter in figures 63 to 65, and on their ventral surface the visceral sensory root (R.Vis.IX) enters in figures 63 and 64. These figures should be compared with figure 6.
THE NERVOUS SYSTEM OF AMPHIBIA
335
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336 G. E. COGHILL
Figs. 66 to 70 Early swimming stage (No. 496, sections 1-6-19 to 23). X 500. Five successive serial transverse sections through the entrance of the root of the vagus nerve into the brain. Figure 66 is the most rostral in position. In this figure the fasciculus solitarius {Fas. Sol.) can be recognized between the descending trigeminal tract (Tr.Des.V) ventrally and the ascending fibers of the spinal Rohon-Beard cells dorsally (DT). The visceral sensory root of the nerve (R.Vis.X) enters in figures 67 and 68 and the somatic sensory root {R.G.Jug.) enters in figure 69 ; the visceral sensory root entering more dorsally. Figure 70 shows fasciculus solitarius fibers caudally of the entrance in a relation like that in figure 66, which is on the rostral side of the root. The somatic motor column is shown in 67, 69 and 70 {VT). The visceral motor nucleus appears in all the figures of this set, and lying between it and the external limiting membrane is a tract {Tr.S) which can not be described in detail in this paper. It is probably a bulbo-spinal tract. The figures of this set should be compared with figure 6.
THE NERVOUS SYSTEM OF .\:MPHIBIA
337
RLL.IX
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338
G. E. COGHILL
R.Vis.X
Fig. 71 Early swimming stage of Amblystoma microstomum Cope (No. 635,
section- 1-3-7). X 500. A transverse section of the embryo through the eye,
to show the nature of the ganglion cells of the retina and their fibers, which at
this time decussate in the most rostral portion of the postoptic commissure.
Silver impregnation after fixation in neutral formalin.
THE NERVOUS SYSTEM OF AMPHIBIA
339
74
Figs. 72 to 79 From transverse sections through the eye and ear of the four stages.
Figs. 72, 73 non-motile (No. 467). Figs. 74. 75 early flexure (No. 473).
340
G. E, COGHILL
Brain
Figs. 76, 77 coiled-reaction (No. 449); early swimming, figures 78, 79 No.
444). The sections through the eye were selected at levels which show the most
extensive area of the lens, and those of the ear were. selected at levels which
show the clearest differentiation of the endolymphatic appendage.
THE EFFECT OF ACTIVITY ON THE HISTOLOGICAL STRUCTURE OF NERVE CELLS
R. A. KOCHER
From the Henry Phipps Psychiatric Clinic, Baltimore,^ and the George WiUinms
Hooper Fouiidalion for Medical Research, University of
California, San Francisco
INTRODUCTION
The study of the effect of functional activity on the nior])ht)li)»;y of nerve cells has been the subject of numerous investigations in the past. These researches liave covered a considerable range as to kinds of cells studied, degrees of fatigue, and as to methods of treatment of the material. There is almost complete agreement on one point, namely, fatigue results in appreciable changes in cell structure. There is, however, the utmost divergence of opinion as to the nature of the clianges, and one who tries to correlate the findings of the different workers in this field is utterly confused. The chief findings relate to" (a) size of cell body and nucleus and (b) amount and distribution of chroniiitic material. While one group of workers concludes that fatigue results in decrease in the size of cell and nucleus (Hodge (1), Van Durme (2), Luxemburg (3), Legendre and Pi^ron (4), Lugaro (5), and Nissl (0) ), another grouj) finds an increase in the size or a change in the nucleus-plasma relation (Crileand Dolley (7), Mann (8), Vas (9) ). Lambert (10) noted no change in the size of cell or nucleus. Increase in chi'omatic substance (hyperchromatism) as the chief result of fatigue has been noted by Nissl. Alost workers (Van Durme (2), Vas (9), Lugaro (5), Mann (8) ) have described a decrease in chromatic material as
' These experiments were carried out at the suggestion and under tlie direction of Prof. Adolf Meyer, and completed in 1912. The computations were in part made after leaving Baltimore. It g ves me great pleasure to express here my thanks and appreciation to Doctor Meyer for his keen interest in the experiments and for many helpful suggestions.
341
THE JOURNAL OF COSfPAR.VTIVK NEVROLOGY, VOL. 2S, NO. 3
342 R. A. KOCHER
the result of exercise. Luxemburg (3) described a breaking up of the chromatic bodies into granules, and Dolley tried to reconcile these contradictions by regarding the hyperchromatism as a result of moderate activity, and the hypochromatism the result of excessive activity or exhaustion. They find that both stages may be present in the same animal simultaneously. Other changes, such as wandering of the nucleus toward the periphery of the cell (Magini (11), Lambert (10), Vas (9) ), rapture of the nuclear and cell membranes (karyolysis and karyorhexis) have been described. Dolley noted thirteen stages of cell change from hyperchromatism to disintegration or death of the cell, corresponding to the degrees of moderate activity up to complete exhaustion. Eve (12), in a study of sympathetic nerve cells, concludes that "there are usually some small differences before and after stimulation, but these are nearly all inconstant and generally reversible. Such divergence in the results is not so surprising when we stop to consider the complicating factors necessarily attending these experiments, such as, (1) difficulty of separating the effects of normal activity from unavoidable shock or injury to the nervous system in killing the animal (the nervous system 'does not 'die' as soon as the heart stops beating); (2) postmortem changes ensuing between the time of death and complete penetration of the tissue by the fixing agent due to the action of autolytic enzymes present in all tissue; (3) varying chemical action of fixing agents; for example, formaldehyde coagulates protein by combination w'th the amino groups, alcohol by dehydration, sublimate by formation of salts, etc.; (4) the solvent action of materials used in fixation and in imbedding, for example, alcohol, xylol, paraffine ; (5) varying effects of chemical reaction between basic or acid dyes used in staining and the different cell structures; (6) effect of subjecting tissue to temperatures of 50° to 54° in the paraffine oven for a period of several hours.
In the present series of experiments I attempted as much as possible to minimize the formation of artefacts, having in mind the above mentioned considerations. It was hoped that by using special care in the handhng of material, the use of im
EFFECT OF ACTIVITY OX XERVE CELLS 343
proved technic in fixation and .staining, and by varying the kind of degree of activity in a long series of experiments, using different animals and studying various kinds of nerve cells, that some degree of uniformity of results might be obtained.
METHODS
A resting control animal was used in each experiment. The animals were killed in most cases by bleeding after ether anaesthesia, the nerve material removed as quickly as possible, cut into small pieces, and the control and fatigue specimens placed in the same fixing solution, imbedded side by side in the same block of paraffinc, cut with the same stroke of the knife, mounted and stained together on the same slide. The detailed data of experiments showing animals used, kind of stimulation, length of time, microscopic technic, etc., are sho\\ii in table 1. In all, fifteen experiments were performed. Examination of the table reveals the fact that almost every foi-mof activity was used; normal activity, forced activity, activ-ity resulting from electrical stimulation, both faradic and galvanic, chemical stimulation, and shock being ai)plied in tlie exi)criments. The kinds of animals used were dogs, cats, rats, sparrows, jjigeons, and frogs. The microscopic technic used was varied considerably, not only as to fixative but also as to staining fluid. The stain most frequently used was Held's modification of Nissl's method.
The examination of the nxaterial for comparison was facilitated by having both control and fatigue specimens mounted on the same slide. Changes such as have been previously described as resulting from fatigue were carefully examined for; namely, comparative amounts and distribution of chromatic substance, size of granules, nucleus-plasma relation, relative size of cells and nuclei, etc. In order to determine whether any change in the size of the cell had resulted from activity, a large series of camera lucida drawings were made. These drawings were made of cells without selection. A field was taken, and every cell showing a nucleolus was included. This precaution was necessary in order to be assured that the cell was cut through a com
344
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])arablo ])laiie. Colls were first drawn using a low power for orientation, each cell numbered on the paper, then the one-sixth objective or the one-twelfth objective was used to project the cell, care being taken to draw always at the same distance from the microscope. The area of the cells was then computed with the use of the polar planimeter. The data are tabulated in table 2, and will be referred to later in connection with the individual experiments.
Normal Activity
Experiment I. Dogs. The animals which served for this experiment were two fox tei-rier puppies from the same litter, three months old. A female was used for activity while the male served for the control. The latter remained quiet in a cage, where he had been kept for several weeks previously. The activity animal was led by a chain on a fast walk into the country; the distance covered was fifteen miles in three and a half hours. This was a considerable feat for a puppy of this size, as the pace meant running all the way for her. At the end of three and a half hours, she was so fatigued that she refused to go any further, and had to be carried home. She was then killed less than one hour after exercise had ceased, the brain and cord at once removed, and sections taken from the lumbar and cervical enlargements, from the cerebellum, and from the cruciate gyrus, and the sections placed in 10 per cent formalin and in Held's fluid. The control dog was killed at the same time, in the same way, and corresponding sections taken from the brain and cord, and placed in the same fixing fluid with those from the 'fatigue' animal.
MICROSCOPIC STUDY OF THE NERVE CELLS Cervical eyilargement of the cord
The cells are uniformly stained, the Nissl bodies standing out clear and distinct. In both control and fatigue specimens, there is an occasional cell showing sHghtly clear areas about the nucleus, but this is no more marked in either section, and these cells are as numerous in the control as in the other. Drawings were made with the Leitz camera lucida, using the one-twelfth objective and no. 2 ocular. The camera lucida outlines of the cells and nuclei were traced with a planimeter with the following result :
Control cells, 43 measured, average area 0.44r square inches.
EFFECT OF ACTIVITY ON NERV^E CELLS
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Fatigue cells, 36 measured, average area 0.420 square inches. These results are tabulated along with the measurements of cells from other regions and experiments in table 2.
LUMBAR ENLARGEMENT OF THE CORD
Examination of sections stained according to Nissl's original method, e.g. seifen methylen blue," after fixation with 95 per cent alcohol, and according to Held's modification of this stain. As regards amount and distribution of chromatic material, there is no difference between the control and fatigue sections. Several sections stained by Held's method showed grouping of cells near the dorsal part of the anterior horns where the chromatolysis was slightly more marked in the fatigue specimen than in the control. This paucity of chromatic substance was not only around the nucleus but also extended to the dendritic trunks. This was not constant in all sections nor throughout the same section. Cells in the extreme anterior horn show no difference from the control. It seems probable that the particular section cut through a nucleus of the cord where the character of the cells was slightly different, since the similar variation in favor of the control cells was observed in some of the subsequent experiments. The nuclei of the control and fatigue animals show no difference whatever in the amount and nature of the staining of the chromatic material.
CRUCIATE GYRUS
These cells are uniformly stained, the nuclei and nuclear membranes and chroniatic bodies being distinct. No difference in the morphology of these cells can be made out with the highest power of the microscope.
CEREBELLUM
The cells of the cerebellar cortex stained with Held's method are well defined, and show a distinct architecture. Numerous cells were examined, but without any discoverable difference in the staining reaction between control and fatigue specimens.
EFFECT OF ACTIVITY ON NERVE CELLS 349
Sparrows
Experiment 2. A male sparrow was shot and instantly killed with a rifle ball at 6 a.m. The brain, cord, and brachial and dorsal ganglia were removed one-half hour later. These were placed in Held's fluid and in 10 per cent formalin.
Fatigue bird. A male sparrow that had been flying about all day was shot, and the brain, cord, brachial and dorsal ganglia removed one-half hour later, and placed in Held's fluid and 10 per cent formalin.
Microscopic study. Brachial ganglia — one-twelfth objective, no. 2 ocular, Leitz. The cell architecture is distinct in both the fatigue and control specimens. Sections stained with Held's method show the cell bodies stained diffusely pink with distinct Xissl gi-anules stained tlark blue. There is no difference as regards depth of stain of either cell bodies or the amount and size or depth in staining of the Xissl h)odies. No cells in either section showed any crenation of the nuclear membrane such as described by Hodge. Some cells show excentric nuclei and nucleoli, but by actual count this occurrence is just as common in the control specimen. Examination of the anterior horn cells of the spinal cord in the region of the cervical enlargement as well as the examination of the Purkinje cells of the cerebellum show no variation from the control morning specimens with respect to size of cells and nucleus' or in the morphological markings.
Pigcous
Experiment 3. For this exjieriment pigeons were selected which were in the daily hal)it of making long flights, sometunes remaining on the wing for over an hour at a time. One of these pigeons was killed just at dusk, and as a control, one approximately of the same age was killed from the same flock at six o'clock the following morning. For study the entire brain was removed as well as the brachial ganglia and sections from the spinal cord in the region of the brachial and lumbar enlargements.
Microscopic study. Brachial ganglia. The evening (fatigue cells) show no crenation, no central chromatolysis, and there is no apparent difference in the size or distrilnition of the granules from the controls. ]\Ieasurement of a large number of cells show that the differences in size of the cell bodies or nuclei in the fatigue and control specimens fall within the hmit of "variation."
Anterior horn cells. Brachial cord. No difference in any respect could be detected between control and fatigue specimens.
Cerebellum. A large number of the cells were studied from both the evening and morning pigeons, but no constant variation in morphological marldngs could be detected.
350 E. A. KOCHER
Rats
Experiment 4- This and the following experiments differ from the preceding in that here the activity was forced to the point of exhaustion. In the previous experiments exercise was voluntary. Two half grown white rats from the same litter served for this experiment. They had previously been kept in a cage and well fed. The fatigue rat was kept running in a revolving wheel for one-half hour, having become tired, he refused to run, and clung to the wall of the wheel. The exercise was then changed from running to swimming. The rat was placed in a tank of lukewarm water, where he kept up constant swimming in an attempt to escape. At the end of one hour he was quite exhausted, was taken out, and allowed to rest for an hour. He was then made to swim a half hour again, followed by a half hour of rest. This was continued until the total time of swimming was three hours. He was then killed at the same time as the control. The total brain and portions from the cervical and lumbar cords were removed. The brains were cut sagittally, and placed in 10 per cent formalin; the other portions were fixed in Held's fluid.
Microscopic examination. In this experiment a large number of sections were cut in series, and a thorough search made for constant differences in staining reaction, amount of chromatic material, size of cells and nuclei, etc. No such constant differences appeared as would go beyond the limits of simple variation. In some slides one might be quite sure of a preponderance of cells of a certain type; for example, showing central chromatolysis ; but on actual counting and comparison, the number of such cells will be balanced by an equal number of the same type in the control.
Forced activity
Experiment 5. Dogs. Four young fox terrier dogs of approximately the same size and age were used for this experunent; one served for a control, the other three were subjected to continuous running for periods of one, two and a half, and five hours respectively. They were killed immediately after the exercise, and the nerve tissue from the four animals given identical treatment as to fixation, imbedding, staining, and cutting, the four pieces being mounted side by side in the same block of parafhne, and cut with the same stroke of the microtome knife. In this way the effect of exercise of various grades of intensity could be studied in the cells of the anterior horn of the cord, of the posterior ganglia, and of the cerebellum.
Microscopic examination. Thorough study of all the sections numbering over a hundred, most of which were made in series, was made in this experiment. The various types of cells described by Dolley were particularly kept in mind, and an attempt made to correlate them with various grades of fatigue. Dolley (7) describes thirteen different stages of fatigue corresponding to different grades of work and over-work.
EFFECT OF ACTIVITY ON NER-VT. CELLS 351
Representatives of practically all these types of cells were found in my specimens, from the resting control animal, as well as from those animals exercised for one, two and a half, and five hours. In order to determine as accurately as possible the relative proportion of these t\'pes of cells in the different specimens, a table was made out listing each of these cell tj-pes, and then beginning with a section under the microscope, every cell showing a complete nuclear membrane was taken in order, and checked in the proper colunm. In this way over three thousand cells were counted with the result listed in table 3.
As wall be seen in the table, the number of a particular type of cell varies considerably, but this variation is the same for the different animals. There are neither progressive clianges in the juorphology of the cells from rest to exliaustion nor are there any qualitative or quantitative differences in t>'pe of cells from resting and fatigued or even exhausted animals. The animals used in Dolley's experiment exercised at most up to three to four hours altogether. Dolley also describes these t^TDes of cells as persisting in the effort to recu])erate for from two weeks to several months after exercise no more severe than tliat of two or three hours running in a treadmill. All these types of cells are admittedly present at the same time (Dolley, .American Journal of Physiology, vol. 28, p. 151). Dolley describes thirteen stages of fatigue in one aniniid where the animal exercised one hour in a tread mill. Also, the cells selected by him for illustration of these stages are taken from a single preparation of three sections in the same experiment. Obviously the observations were not over a large enough range of sections nor sufficiently controlled by actual counts of the various tj-pes of cells. In the Journal of Medical Research, vol. 21, p. 104, Dolley says, "Measurements were made of five cells of each type in five anaemia exjieriments, one a fatal resuscitation, the other a repeated hemoj-rhage ;" a little farther on, ""^Measurements were made of ten cells in each of three groups." A great many cells were skipped (those not entire) in Dolley's method of counting, giving a large leeway for a personal factor in the selection of types. Ibid, volume 20, page 291, " ^Measurements were made
352
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of camera lucida outlines of cells and nuclei. This was done in
four experiments Five cells of each type and after
each fixative seemed to give a fair average." Dolley's miterial in which consecutive stages were studied did not receive the same treatment as to fixation, staining, mounting, and cutting. The control and fatigue material was handled entirely separately (Amer. Jour, of Physiol., vol. 25, p. 155). Slight unavoidable variations in the exposure of the tissue to the various agents and different thickness of the cut sections would make such material worthless for comparative study. As pointed out above, this objection cannot bo applied to my o^^^l experiments, as the handling of material was identical in all cases. Dolley's method of measuring size of cells and luiclei is o])eii to serious objection. In the Journal of Medical Research, volume 21, he .states, "While not adapted for exact measurement on account of their shape, the largest diameters (cells and nuclei) were always taken." In my own experiments the actual relative areas of the nuclei and cells was computed with the use of the polar planimeter, which traces the entire outline of the cell and gives very accurate results. As seen in the table, I found no difference in the size of the cells of the control and exercise aninuds.
Electrical stimulation {gdlvanic)
Experiment 6. Pigeons. In this experiment a i)if;eon was fastened by the feet in a standing position, while a wire from four 50 amperes galvanic i)atteries was wrapped around the legs. A current was allowed to i)ass through the wire once a second hy a clock make and hreak arrangement. With the entrance of the stimulus there was a strong contraction of the leji; and iIukIi nuisclcs. After four hours the contractions became feei)le. owing to fatigue, and the pifjeon was killed. Rigor mortis of the leg and thijih muscles set in innnediately. A control pigeon was killed at the same time.
^Microscopic study revealed absolutely no constant morphological differences in the anterior horn cells and in the dorsal gansHon cells from the two birds.
Faradic stimulation
Experiment 7. Faradic stimulation of the sciatic nerve in a cat was appHed in this experiment. A cat weighing 2 kilos was anaesthetized with ether, and decapitated according to the method of Sherrington by
A'A R. A. KOCHEIl
lyiii^- off tlie carotids, etc. After a rest of about three-quarters of an hour to give the animal a chance to recover somewhat from the shock, a canuhi was inserted into the carotid artery, and connected with a manometer for record. The left sciatic nerve was then exposed and stimulated with a faradic current from two dry cells. The secondary coil was placed at eight, later at six. Stimulation was applied at intervals, fifteen seconds stimulation was followed by forty-five seconds rest. Stimulation began at twelve o'clock and continued until five P.M. The heart was still beating strongly at the end of the expeiiment, the blood pressure remained fair, and reflexes were obtained throughout b}^ stimulation of the sciatic, both in the right and left leg. The contractions on the right were spasmodic, those on the left — the stimulated side, were tetanic. The latter were feeble, and gave all signs of fatigue. The three pairs of dorsal ganglia of the sciatic nerve as well as the lumbar and sacral cord, were removed and placed in the fixing solution. Microscopic examination of the cells and measurements of cells and nuclei from the fatigued and unstimulated side of the cord of the' same animal failed to disclose any difference in morphology.
Experiment 8. Faradic stimulation of the sciatic nerve of the frog was used in this experiment, the unstimulated dorsal ganglia cells and anterior horn cells of the unstimulated side of the same animal as well as corresponding material from a second resting frog killed at the same time served as control. The two frogs were pithed and placed in a moist chamber. One-half minute stimulation of the sciatic nerve was followed by one minute rest. The electrodes were applied just above the knee at the back. This interrupted stimulation was continued six hours. The muscles showed response by tetanic contractions. At the end of the period the muscles were still irritable. Three pairs of dorsal ganglia corresponding to the sciatic nerve as well as the spina' cord of the same region were taken for study.
The stimulation produced no changes in the cell morphology that could be detected by a measurement of the size of the cells and nuclei or by study with various powers of the Leitz compound microscope.
Drug stimulation (strychnine)
Experiments 9 to I4. Verworn (13) attributed fatigue in nerve cells in part at least to a local asphyxia of the cells due to an accumulation of fatigue substances and an insufficient supply of oxygen. By perfusing fatigued frogs with oxygenated salt solution, inserting the canula into the aorta, he was al^le to restore irritability of the nerve cells and corresponding response of the muscles in contraction after they had ceased to respond from fatigue. He succeeded in keeping the muscles and nerve responsive to stimulation for mariy hours longer than would ordinarily be the case. In experiments 9 to 14 this method was applied in order to continue stimulation and corresponding nerve exhaustion to a degree not possible by the usual methods. Strychnine in doses of ^ to I of a grain was given in each case by injecting into the subcu
EFFECT OF ACTIVITY ON NERVE CELLS 355
taneoiis area of the lower back just after beginning perfusion with normal salt solution and by adding to the perfusion solution. The heart continued beating throughout the experiment. The strychnine caused violent tetanic contractions at first, which later gave place to continued fibrillary contraction. This served as an indication of the condition of irritability of the spinal cord cells. To prevent direct action of the strychnine on the nerve endings in the gastrocnemius muscle, the femoral arterj- was tied off. This muscle continued in a state of irritability for several hours. The experiments carried out by this method were continued for a length of time varying from two to ten hours. Controls were used, treated in the same manner, except for the strychnine injections. The subsequent treatment of the nerve material (sections of the spinal cord and from the thoracic and lumbar regions) was identical. There is a .striking change in both the control and treated cells, which had been perfused for a long period. Many cells as well as nuclei are swollen beyontl the normal size, owing, doubtless, to a .simple turgescence caused by a flooding of the circulatory system with the salt solution, and the staining reaction is diffuse. There are, however, no differences in morphological characters between the cells of the strj-chnine stinmlated and the control cells of the spinal cord.
Shock {anaemia)
Experiment 15. A four kilogram dog was given morphine and bled from the jugular vein; 20() cc. were removed, following which the dog remained semi-comatose for five hours. At the end of this time lie was killed along with a control dog of four and a half kilograms weight. ]\Iaterial was taken from the cruciate gyrus and cerebellar cortex, fixed in 10 per cent formalin, and stained with polychrome methylene l)lue. Examination with the microscope revealed no tlifference in the character of the nerve cells in the shock animal as compared with the control.
DlSCrSSIOX OF RESULTS
It luxs long; been known that prolonged activity of mucous gland cells results in cliaracteristic liistological changes in these cells, owing to the disappearance from the cell of certain granules (zymogen granules). This granular material is evidently used to make organic material of the secretion. It was doubtless on the basis of such observations that certain physiologists were led to seek for similar alterations in tlie highly specialized nerve cell following functional acti\-ity. ]Many of the investigations of these physiologists were confined to one or two experiments, the material was often not sufficiently controlled by normal tissue
356 11. A. KOCHER
for comparison, and frequently the histological technic was faulty. In explanation of the diverse changes described by these workers as resulting from fatigue, it has been assumed that certain materials present in the nerve cells have been katabolized to form energy for the nerve impulse, and that the using up of this material during activity can be detected histologically by the depletion of the granules (chromatic substance), changes in size of cell and nucleus. In a long series of carefully controlled experiments, I could find no evidence of any analogy between the effect of activity in certain glands and activity in nerve cells. In no experiment did the histological structure of the nerve cell following activity show any constant deviation from that of the corresponding resting cells of the controls. Some very sweeping generalizations have been drawn from the conclusions of previous workers; namely, that fatigue, fear, shock and exhaustion may lead to permanent damage and even disintegration of nerve cells. Crile's present theory of surgical shock and of certain aspects of Graves' disease, based essentially on these assunptions, may be cited to show to what extremes these deduct ons based on insufficiently controlled experiments of this kind have led.
SUMMARY
The effect of various grades of activity on nerve cells was studied in a series of fifteen separate experiments. The animals used were dogs, cats, pigeons, sparrows, frogs, and rats. ii]very experiment was carefully controlled by a resting animal of the same species, of the same approximate age and size, and the material from both given identical treatment, except for the activity. The nerve cells studied were from the cruciate gyrus, from the cerebellum, from the anterior horn of the spinal cord, and from the dorsal ganglia. In one of the experiments over thirty-five hundred nerve cells classified into thirteen types according to histological characters were counted to determine the relative frequency of characteristics which might be correlated with grades of activity. There was no deviation from the normal in even the most advanced fatigue. Over a thousand cells
EFFECT OF ACTIVITY OX NERVE CELLS 357
and nuclei were measured by computing the areas of the projected outlines with the planimeter. There was found to be no constant difference in size of cells or nuclei resulting from activity. Furthermore, no quahtative differences in histological characters could be found between fatigue and resting nerve cells.
BIBLIOGRAPHY
(1) Hodge, C. F. 1892 Journal of Morphology, vol. 7, p. O.j. 1S94 vol. 0.
18S7 American Jour, o"" Psychiatry, vol. I, ]). 171.
(2) V.\N DuRME, Pau . 1901 Xevraxe. p. 11.5.
(3) Luxemburg, J. 1899 Neurol. Zentralblatt, vol. 8, p. 629.
(4) Legendre, RenIo, et Pi6rox, H. 1908 Coniptes Rendus dc la Soc. do liiol.
p. 1102. (o) LuGARO, E. 1895 Archives Ilalicnnes dc Biologic, Xr. 24, p. 2.5S.
(6) Ni.ssL 189) Neurol. Zcntralhlatt, Xr. 1.5, p. .39. 189G Zcitschr. f. Psy<hi atrie, vol. 48.
(7) DOLLEY D. H. 1909 American Jour, uf Physiology, vol. 2.'). p. \rA: Ciui.i;,
1911 Jour, of Med. Research, p. 309; 1909, p. 95.
(8) Mann, G. 1895 Jour, of .Vnatomy and Physiology, vol. 29. p. KM).
(9) Vas, F., Archiv f. mikr. Anat., 1892. Bd. 40, p. 375. (10^ La.mbert. M. M. 1893 Comptcs Rendus.
(11 *• Magini, G. 1895 Archives Italiennes do Biologic, Nr. 22.
(12i Eve, F. C.
(13) Verworn, .Max 1900 .\rchiv f. Anatomic u. Plnsiologie, supp'. p. 1.52.
THE JOUBNAI, OF CO^^PAR\TIVE NEUROLOGY, VOL.
CONTRIBUTIONS FKOM THE ZOOLOGIC.VL LAJBORATORT OF THE MUSEUM OF COMPAKATIVE ZOOLOQT AT
HABVARD COLLEGE. NO. 278, AND FROM THE ANATOMICAL LABORATORY OP THE NORTHWESTERN
UNIVERSITY MEDICAL SCHOOL, NO. 36.
THE INFLUENCE OF LIGHT AND TE:\IPERATURE
UPON THE MIGRATION OF THE RETINAL
PIGMENT OF PLANORBIS TRIVOLVIS
LESLIE B. AREY
NINE FIGURES
CONTENTS
Introduction and historical survey 359
Material and methods 362
Description of the eye of Planorbis 365
Experimental part 369
a. Effect of light and darkness 369
1. Effect on normal animals 370
2. Effect on excised eyes 371
b. Determination of adaption times 374
c. Effect of temperature 376
1. Effect on normal animals 376
2. Effect on excised eyes 377
d. Effect of anaesthetics 378
Discussion 379
Summary 385
Bibliography aS6
INTRODUCTION AND HISTORICAL SURVEY
The pigment granules of many animal cells, wliich in general are termed melanophores, are capable of undergoing positional changes when adequately stimulated. This property has made possible the development of an interesting field of research, which has had for its goal, the correlation of migratory movements of the pigment with the presence of definite stimulatmg agents. At first, only the influence of such important environmental factors, as light and temperature, as well as the effects of more artificial stijuulating agents like pressure and electricity were sought, and but Uttlc attention was given to the role played .by chemical agents. Recently, however, the possibility of
359
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4, AUGUST, 1916
360 LESLIE B. AREY
chemically stiinulating certain types of isolated pigment cells has been shown in a striking manner (Spaeth, '13^; 'IS**).
All investigations relating to the physiology of pigment cells may be divided into two general categories, depending on whether body chromatophores or retinal pigment cells serve as the material for experimentation. The general tendency, however, to keep these fields of research sharply separated is wrong, inasmuch as the behavior of the two types of cells has much in common, and it is only by summating our knowledge, after the comparative method, that a thorough understanding of either may be gained.
Light is the commonest and most potent of the natural stimulating agents concerned in pigment migration. In general, it may be said that light induces an expansion, and the absence of light a contraction, of pigment cells.
Photomechanical changes have been demonstrated in the body chromatophores of crustaceans, cephalopods, fishes, amphibians, and lizards. The earliest experimentation showing the effect of light upon the retinal pigment of vertebrates was performed by Kiihne (77) and by Boll (78), who worked upon the frog. Their results have since been extended upon representatives of the remaining vertebrate classes, although striking movements of the retinal pigment of reptiles and mammals have not as yet been demonstrated. Among invertebrates, photomechanical changes have been found in the compound eyes of insects (Exner, '89), crustaceans (Exner, '91), arachnids (Szczawinska, '91), and in the eye of cephalopods (Rawitz, '91).
Of the molluscs, the cephalopods constitute the only group in which a response of the retinal pigment to photic stimulation has hitherto been demonstrated. Hensen ('65), upon theoretical grounds, hazarded the guess that the pigment in these animals possessed a certain mobility, for Babuchin ('64) had previously referred to finding the visual rods entirely free from pigment in some specimens of Sepia and Octopus. It was left for Rawitz ('91) to show that in the light the visual rods of Sepia officinalis are pigmented along their whole length, with an especial accumulation at the lens border, whereas in the
RETINAL PIGMENT OF PLANORBIS 361
dark the pigment is limited to the base of the. rod. Chun ('03) confirmed these general conclusions on deep-sea cephalopods, as did Hess ('05) by making a comparison of the pigment responses in pelagic and httoral forms.
Temperature is likewise a controlling factor in determining the ultimate distribution of pigment in many bodj^ melanophores. In general (Parker, '06), low temperature has an influence similar to that of light, inasmuch as it favors pigment expansion; high temperature, on the contrary, like darkness, induces pigment contraction.
A similar condition liolds for the retinal pigment of certain animals. Congdon ('07, p. 547) found that: "In both Palaemonetes and Cambarus the proximal retinal pigment migrates distally when the temperature is lowered and proximally when it is raised." The writer (Arey, 16") found an identical tendency in the retinal ])igment of fishes. Working with the frog's retina, Gradenigro ('85) first showed that at a temperature of 30°C. the pigment expands, thereby closely simulating the distribution characteristic of light. Later Herzog ('05) confirmed this discovery, and further stated that maximal expansion likewise ensues when the temperature api^roaches the freezing point, the contracted condition typical of darkness being obtainable only between the temperatures of 14° antl 18°C. A reinvestigation of this matter recently made by the writer (Arey, 16") has convinced him that Herzog's results are substantially correct and that the behavior of the retinal pigment of the frog to temperature must indeed be considered exceptional; the explanation for this is presumably to be found in the existence of a superimposed nervous control, resulting in the obliteration of the more primitive response.
Positional changes of the retinal pigment of gasteropods in response to definite physiological stimuli have never been recorded. Smith ('06), in his work on the structure of the eyes of pulmonate gasteropods, noticed that the retinal pigment of Planorbis trivolvis Say showed processes of variable length, and accordingly he performed a few experiments to determine whether the pigment distribution could be correlated with
362 LESLIE B. AREY
corresponding conditions of light and darkness. His results concerning a photomechanical influence were entirely negative, although he states his behef (p. 255) in the existence of an appreciable pigment migration caused by certain unknown factors. Assuming that the retinal pigment of these animals does undergo migratory movements, it is reasonable to expect that the behavior of pigment cells, having no demonstrable nervous connections (Smith, '06), is dependent upon definite environmental stimuli, hence I decided to reinvestigate the influences that simple stimulating- agents such as light and temperature might have upon the retinal pigment of these snails.
MATERIAL AND METHODS
Planorbis was obtained in abundance at certain localities on the bank of the Charles River, Cambridge, Mass. During warm weather it is easily procurable within arm's reach of the shore, but as the water grows colder a gradual withdrawal of animals occurs toward the deeper bed of the river. My experience in collecting during the fall of 1914 will illustrate this behavior. In the second week of October a great wealth of material was discovered at a location where the previous June no specimens had been seen; at the time of this collection the snails were distributed as far up as the water's edge. The next collecting trip was made on November 21, and, in the same spot, where several weeks previously hundreds of animals were obtainable, there now remained a few stragglers only, and these were withdrawn from the bank almost beyond reach. Ten days later scarcely a specimen was left within sight.
I was interested to discover whether this withdrawal was due to an active migration of individuals, or to the effect of the low temperature which might cause the snails to become inactive, whence they would be washed by the current down the sloping bank into deeper water. Mr. W. F. Clapp of the Museum of Comparative Zoology informs me that the former of the alternatives is undoubtedly correct. Moreover, low temperature does not necessarily cause these animals to become inactive, since he reports having often broken through the ice
RETINAL PIGMENT OF PLANORBIS 363
in mid-winter and dredged Planorbis from the deeper water to which they resort; under these conditions he has always found them to be in an exceedingly active condition.
This interesting migratory behavior to what is presumably the stimulus of low temperature has a definite 'protective' value to the snail, since an animal thereby removes itself to a depth at which ice is not formed. It is probable that the situation is not one in which an annual rhythm, independent of temperature, has been impressed upon the species, for when Planorbis, during its retreat to deeper water, is removed to laboratory aquaria and kept for some time at room temperature it neither manifests a tendency to seek the deepest water, nor does it exhibit negative geotropism. On the other hand, I do not recall that anijnals introduced into jars of ice water showed any marked tendency to seek the bottom of the dish. It is possible that there is, in fact, a temperature response which, however, comes on but slowly, as evinced by the gradual withdrawal under natural conditions.
Except for minor changes, the following is quoted directly from an enlightening statement made by Mr. Clapp during a correspondence in which I sought his aid in solving the migratory behavior of this animal :
Wliat you write in regard to the migration of Plaiiorl)is is my idea also. There is little doubt that Planorbis lias a rest period comparable to that of nearly all other gasteropods — the effeet of inactive pha-ses in the life cycle is often clearly marked on the shell. In many species of land shells there are two annual rest periods, the shorter occurring in the summer, the longer in the winter. I have never observed any general inactivity or burrowing during the summer on the part of our New England species of Planorbis, and therefore have thought it probable that their rest period occurs during the winter months.
The winter rest period of the land shells in this section of the country is governed entirely by the temperature. When spring arrives earlj' the shells appear early — the hardier species first, the more delicate species later — but all appear earher than in other years when the warm weather comes on tardily. It seems probable that Planorbis, Physa, L>nnnaea and similar shells should follow the same principle, in other words, become inactive and bury themselves when the temperature of the water falls below a definite point. To a certain extent this may be true, but if so, the rest period must be much shorter than in land shells, and, I think, not compulsory as with the latter
364 ' LESLIE B. AREY
forms. My reason for believing this is that I have frequently obtained Planorbis, Lymnaea and Physa, in mid-winter, by breaking through the ice and using a small dredge or net.
Some believe that the fresh water pulmonates do not hibernate at all. That appears to me to be based on negative evidence, since they have not l)een observed under natural conditions, or rather have been observed under unnatural conditions (vivaria).
At the conclusion of all experiments, to be described subsequently, the animals were beheaded, fixation of the head and the contained eyes occurring under conditions of light and temperature identical with those at which each experiment had been conducted. Perenyi's fixing fluid gave very satisfactory preservation. Sections of 8 m thickness were cut parallel to the long axis of the eye and were stained with Heidenhain's iron-haematoxylin and with orange-G.
Whenever the experimentation involved the employment of light, strongly diffused daylight, such as is obtainable at north windows, was used.
The microscopical preparations were studied and measurements were made at a magnification of 1400 diameters. In expressing the extent of pigment distribution in nimierical terms, two measurements were used. One, which I shall call the 'zonal measurement,' represents the breadth of the band of dense pigment, exclusive of the finger-like processes extending toward the bases of the constituent cells (figs. A, 4 and 5). The second, or 'process measurement,' consists of the zonal measurement plus the average length of the pigmented processes just referred to.
The sense in which a few descriptive terms will be consistently employed needs explanation. 'Peripheral' and 'central' will designate those parts of the eye which are respectively farthest from and nearest to its center (fig. B). 'Distal' and 'proximal,' used in describing retinal cells, are retained in a sense similar to that employed in descriptions of uninvaginated ectodermal cells — hence distal indicates the portion nearest the lens or the lumen of the optic sac, while proximal refers to that part of a cell nearer the connective-tissue sheath at the periphery of the eye (figs. B and C).
RETINAL PIGMENT OF PLANORBIS
365
DESCRIPTION OF THE EYE OF PLANORBIS
Planorbis is a representative of the group of pulmonate gasteropods which is characterized by the possession of but one pair of non-retractile tentacles, at the bases of which the eyes are located. Although the tentacles are not retractile after the manner of an introvert, they nevertheless are capable of exhibiting a high degree of muscular shortening whereby their surface becomes thrown into transverse ridges (fig. A, ta.).
fbr.mus.
oc. n.opt.
Fig. A An axial section of both the eye and the tentacle of Phinorbis, showing the position and mutual relation of these structures (X 60). fbr. mus., muscle fibers; n. opt., optic nerve; oc, eye; ta., tentacle.
The eye (fig. A, oc), which lies just bene<ath the surface epithelium, has the shai:)e of a cone or pear whose base, corresponding to the corneal portion, points outward. The base has a diameter of about 150 m, whereas the axial measurement is 200 M An axial section of the eye (fig. B) shows the following parts : (1) optic capsule; (2) cornea; (3) retina; (4) optic nerve; (5) lens ; (6) vitreous humor.
The optic capsule (fig. 3, cps. opt.) is a thin, connectivetissue sheath surrounding the eye, to which the retinal and corneal elements are attached. It is continuous with the sheath of the optic nerve.
366
LESLIE B. AREY
The cornea and retina represent differentiated portions of an original sac-like epithelial invagination (figs. A and B). The wall of this closed 'optic sac/ as it is called, never becomes more than one cell thick. The pigment-free corneal cells are only slightly columnar in shape, hence this portion of the optic sac remains as a thin membrane, which is strikingly in con
Fig. B An axial section (semi-diagrammatic) of an entire eye of Planorbis (X 275). cm., cornea; hu. vit., vitreous humor; Ins., lens; nl. cl. rtn., nucleus of retinal cell; n. opt., optic nerve; pr^., pigment; rtn., retina.
trast with the thick cornea of some other pulmonates. Its extent is limited to the broad base of the conical eye (fig. B).
The retinal portion of the optic sac is easily distinguishable from the cornea by the presence, in the former, of pigmented cells and by the great elongation of all its elements (figs. B and C). In general, the constituent cells maj^ be said to have a radial arrangement with respect to the optic sac. Notwithstanding the fact that the retina is only one cell thick, it is con
RETINAL PIGMENT OF PLANORBIS 367
venient to follow the suggestion of Smith ('06) and distinguish three concentric zones, which, although of an arbitrary nature, are on the whole rather clearly defined. The peripheral zone (fig. C, rtn. ex.) is in contact with the optic capsule and is quite free from pigment; it contams the cell nuclei. The pigment of the non-sensory cells is aggregated in a middle or intermediate zone (fig. C, rtn. m.); peripherally its limits are ill defined because of irregular processes, which extend down into the cells, whereas centrally a sharp line of demarcation separates the pigment from a third or central zone {rln. i). The position of the boundary conmion to the peripheral and intermediate zones is somewhat dependent on the degree of pigment migration under various envii'onmental conditions, which will be discussed in the main part of this paper. The internal or central zone (fig. C, rtn. i.) is of constant width and comprises the portion of the retina between the pigmented zone and the lens. It contains the rods (bac.) — the photoreceptive elements.
Two kinds of cells form the retinal epithelium, the non-sensory- pigmented cells (fig. C\ nl. cl. pig.) and the unpigmented sensory cells {nl. cl. sm.). The pigmented cells, which are the more numerous of the two, are grouped about the sensor>' cells thereby isolating the latter from each other. These pigmented cells are of two kinds. One set has its nuclei situated close to the optic capsule, while the nuclei of the other set are near the center of the cells and slightly distal to those of the sensory elements. Both types of pigment cells are exceedingly slender, although those with more distally placed nuclei generally have an enlargement just distal to the nucleus; according to Smith ('06, p. 255) : "When this part of the cell is free from pigment it appears to contain a \'acuole."
The distribution of pigment is variable under different circumstances; the limit of proximal migration in either tj-pe of cell, however, is conditioned by the position of the nucleus (fig. C), for pigment is never found proximal to it. The length of the pigment cells, and therefore the thickness of the retina as a whole, varies at different levels in the eye. The thickest portion of the retina is on the sides at a distance of about 60 m
368
LESLIE B. AREY
from the apex of the optic sac. At this thickest region, the distance from the optic capsule to the central zone, that is, the length of the pigment cells, is approximately 35 /x.
The unpigmented or sensory cells (fig. C, nl. cl. sns.) are much more robust and have larger nuclei than either kind of pigment cell just described. These cells are of an elongated spindle shape, thickest in the region of the nucleus. The cell body
fbrlo.
fbrl.
)rtn.ex^
pre. n't'.
Fig. C A portion of an axial section of a Planorbis eye, showing the component retinal elements (X 1000). ax. bac, axis of rod; cps. opt., optic capsule; fbrl'., fibrillae of rod-axis; /6rZ"., fibrillae of rod-mantle; ivlr. bac, mantle (involucrum) of rod; nl. cl. pig., nucleus of pigment cell; 7il. cl. sns., nucleus of sensory cell; pig., pigment; pre. n't., neurite-process of sensory cell; rtn. ex., peripheral zone of retina; rtn. i., central zone of retina; rtn. m., middle or pigmented zone of retina.
continues through the pigment zone and ends, in the so-called central zone, in a structure known as the 'rod' — this being the photoreceptive portion of the cell. Each rod consists of two parts, centrally a core or 'axis' (ax. bac), and peripherally a radially striate "mantle' (ivlr. bac). Proximal to the nucleus, each sensory cell gives off a neurite {pre. nH.), which courses along the inner face of the capsule and is ultimately gathered up with similar neurites to form the optic nerve (figs. A and B,
RETINAL PIGMENT OF PLANORBIS 369
n. opt.). The optic nerve is covered by a sheath of connective tissue continuous with that of the optic sac. The work of Babuchin ('65), Henchman ('97), and particularly that of Smith ('06) has proved that the sensoiy cells of pubnonate gasteropods are of fibrillar composition, the terminal brushlike neurofibrils of the rod mantle (fig. CJhrl.") being continuous with the fibers of the rod axis {fhrl.') of the cell body, and of the neurite. The length of the rod is 6 )u or 7 m The lens (fig. B, Ins.) is a large pear-shaped body occupying almost the whole interior of the optic sac. It is a non-cellular secreted mass, the outer portion of which forms a denser capsule or rind.
The vitreous humor (fig. B, hu. vit.) fills whatever spaces intervene between the central zone of the retina and the lens, as well as the spaces between the indi\-idual rods.
EXPERIMENTAL PART
a. Effect of light and darkness
The initial experimentation consisted in determining whether or not the retinal pigment of Planorbis undergoes positional changes, whereby a characteristic distribution is assumed in light and in darkness.
The citation of Smith's statement ('06, p. 255) relative to the tests performed by hun on this animal will serve to show both his methods and his results :
In spite of the fact that the rods, lying, as they do, distal to the pigment zone, cannot be protected by pigment migration, as can the rods in cephalopods, the variable position of the proximal region of the pigment suggests the possibility of pigment migration in the eye of Planorbis. I therefore attempted to determine b}- a few experiments whether differing light conditions would produce corresponding changes in the position of the pigment. Several specimens of Planorbis were placed for an hour or more in water in a white, porcelain dish which was set in a sunny window. Their heads were then cut off with scissors and fixed in Perenyi's fluid. Sections were made in the ordinary way and stained in Heidenhain's iron-haematoxylin, followed by orange-G. Similar preparations were made from specimens which had been kept in darkness for an hour or more before killing. Comparisons were
370 LESLIE B. AREY
then made between the two kinds of preparations in order to learn whether the position of the pigment was different in the two cases. More cases in the 'hght' eyes had the pigment reaching quite to the nuclei than in the 'dark' eyes; but there was such a lack of uniformity in different animals, and even in the same retina, that the evidence was not at all conclusive. Repeated experiments did not lead to more definite results. I am satisfied that the pigment does travel up and down in the pigment cells under the influence of some stimulus, but just what is the exciting factor I have not determined. I have not been able to get any evidence of pigment migration in the eyes of either Helix or Limax.
In another part of the same paper Smith again returns to a consideration of these results and becomes more bold in his inferences. Selected excerpts (pp. 268-269) make plain his final attitude toward the situation in Planorbis.
It is probable that pigment movement is a direct response to lightstimulation in Planorbis .... Not having been able as yet to determine the exact conditions under which it occurs, I can only suppose from analogy that the migration is a response to light. The shape of the cells, the position of the pigment in some cells as compared with that in others, and the apparent need of pigment migration in the" eye of Planorbis, all point to a probable responsiveness of its pigment cells to light; ....
A personal experience (Arey, 16^) in determining the lengths of time needed to complete light and dark adaption of the highly mobile retinal pigment of fishes led me to suspect that the 'hour or more' which Smith allowed for the execution of his experiments might be wholly insufficient to permit the photomechanical response to proceed to completion. Hence in my experimentation ample time allowances were made for both light and dark adaption.
1. Effect on normal animals. Five animals were placed in a battery jar containing water and were kept in a dark-chamber for 24 hours. In the same manner, an equal number of animals were subjected to daylight for 10 hours. A microscopical examination of the resulting preparations gave data as in table 1.^
1 The final measurements recorded for each eye represent the mean of the values obtained from several measurements on each side of the retina at a distance of about 50 ij. from the entrance of the optic nerve. Experience showed that
RETINAL PIGMENT OF PLANORBIS
371
Measurements shovoing the relative distribution of the retinal pigment of Planorbis in darkness and in light at room temperature. The vahies are mean values expressed in micra, and indicate: (1) the thickness of the main pigment mass {zonal measurement) ; (2) the zonal measurement plus the length of the pigmented processes {process measurement); (3) the length of the pigment processes obtained by subtracting {1) from {2). The percentage change in each mean measurement in darkness as compared with that in light is also computed
CONDITION OF ILLUMINATION
NUMBER
OF RETINAS
MEASURED
MEAN
ZONAL
MEASUREMENT
PERCENTAGE
CHANGE
IN ZONAL
MEASUREMENT
MEAN
PROCESS
ME.VSUKE MENT
PERCENTAGE
CHANGE
IN PROCESS MEASUREMENT
MEAN
LENGTH OF PIGMENT PROCESSES
PERCENTAGE
CHANGE
IN LENGTH
OF PIGMENT
PROCESSES
Light
10
10
11.0
18.0
64
16.5
25.0
52
5.5
7.0
Darkness
27
Since the distal margin of the pigment zone — the boundary between central zone (fig. C, rtn. i.) and iniddle zone {rtn. m.) — is fixed in relation to each pigment cell, these values show quite decisively that in the light (fig. 2) the pigment migrates distally in the cell, that is, toward the source of illumination, whereas in the dark (fig. 5) the reverse is true. An appreciable thinning out of the pigment u\ darkness due to its bemg spread over a greater area, is not demonstrable. It may be that the pigment involved in migration is only that which lies most proximally in the middle zone, or there may occur a general proximal movement, that is, a readjustment of the relative quantitative amounts, throughout the whole mass. AMiichever alternative is true, it is certain that the edge of the pigment zone which forms the boundary between the middle and central zones (fig. C) maintains a constant position in relation to the retinal cells.
2. Effect on excised eyes. Since light exercises a photomechanical influence on the retinal pigment, the interesting query arises as to whether this effect is direct, upon the cell itself,
restricting the measurements to this most favorable region afforded the fairest numerical representation of the pigment distribution. In these, as well as all experiments to be described subsequently, individuals of Planorbis were selected of a more or less uniform size — this precaution will, I think, serve to obviate criticism, if not actual error.
372 LESLIE B. AREY
or whether it is accompHshed through some nervous mechanism related to the cerebral ganglion. It is true that the probability is against the existence of nervous connections with the pigment cells, since all previous researches have failed to demonstrate structures of this kind, yet it is entirely conceivable that such neuro-fibrils may exist and have hitherto escaped detection; indeed, the extreme difficulty with which the neuro-fibrils of the sensory cells are demonstrated, makes this possibility all the more real. Even if a direct influence of light can be proved, the existence of such neuro-fibrils is not precluded, nor yet their cooperation in effecting the migration of pigment; but it does render this event less likely, and definitely shows, moreover, that whatever may happen in the normal animal, a direct action of light on the pigment cell is a demonstrable phenomenon. If the direct action of light can not be shown to induce migratory movements of the pigment in excised eyes, three possibilities exist: (1) fibers in connection with the pigment cell perform double conduction, transferring afferent impulses to the cerebral ganglion and efferent impulses back to the retina; (2) afferent impulses travel in the optic nerve to the cerebral ganglion while efferent impulses return by hypothetical nerves to the pigment cells, or (3) the optic nerve is of a mixed nature, possessing both afferent and efferent components. Since, however, the existence of double conduction in nerves is a mere postulation, the first of the three possibilities may be safely eliminated. There is, nevertheless, still another way of viewing the situation, and one that demands serious consideration. The metabolism of an organ isolated from the body to which it belongs is, of necessity, fundamentally altered. Not only is its supply of nutriment cut off, but what is more serious in a short experiment, the elimination of waste is not adequately provided for. If isolated pigment cells do not respond to the direct action of light, the reason may easily be ascribable to an autoanaesthetization caused by the accumulation of the cell's own catabolic products. The probability of this course of events was suggested in some experiments upon the eyes of fishes (Arey, 16^; IG*"). Moreover, it will be shown in another part of this paper (p. 378)
RETINAL PIGMENT OF PLANORBIS 373
that anaesthetics are probably capable of exerting an inhibition on the migration of pigment, even in normal snails.
Spaeth ('13^) found the melanophores on the isolated scale of Fundulus to be responsive to ultra-violet rays but not to those of the \'isible spectrum. Parker ('97), working upon Palaemonetes, obtained responses from all three types of pigment cells when excised eyes were brought from light into darkness, or the reverse. This result was observed equally well when stalks containing ganglia were used or when merely retinas, exclusive of ganglia were employed. Hamburger ('89) reported that the pigment of enucleated frog's eyes exhibits migratory movements both in darkness and in light. In another paper (Arey, '16") I have described the occurrence of a pigment migration when the previously dark-adapted excised eye of the common horned pout, Ameiurus, is brought into the light; in the reverse exposure (hght to dark), however, no response ensues; furthermore, in several other fishes which were studied, even the direct influence of light could not be shown.
Experiments on Planorbis were niiide upon animals that had been adapted to light or to darkness for 6 and 24 hours, respectively. Such animals were beheaded and small pieces of tissue, bearing a tentacle, were then placed in a watch glass containing an abundance of Ringer's solution and subjected to light or to darkness according to desire. The exposure to light lasted 4 hours, whereas in the dark, eyes were left for 5 hours. Table 2 sununarizes the results of these determinations.
The results of both these sets agree very closely with the values given previously for nornml light-adapted eyes (11.0 /x and 16.5 n for zonal and process measurement respectively). The obvious conclusion to be drawn from these experiments is that light has a direct influence on a previously dark-adapted pigment cell, but that light-adapted retinas do not change in darkness. The direct action of light on the retinal pigment of this animal, therefore, is identical \\'ith that of Ameiurus.
I am inclined to interpret these results in the following way. The conditions under which an isolated eye is placed undoubtedly favor the accumulation of catabolic products. This unremoved
374
LESLIE B. AREY
Measurements showing the relative distribution of pigment in the excised eyes of Planorbis in darkness and in light. The values are mean values expressed in micra and indicate: {!) the thickness of the main pigment mass {zonal measurement); (;S) the zonal measurement plus the mean length of the pigmented processes {process measurement); {3) the length of the pigmented processes obtained by subtracting (/) from {2). The percentage change in the mean zonal measurement in darkness as compared with that in light is also computed
CONDITION OP ILLUMINATION
NUMBER
OF RETINAS
MEASURED
MEAN ZONAL
MEASUREMENT
PERCENTAGE
CHANGE IN
ZONAL MEASUREMENT
MEAN
PROCESS
MEASUREMENT
MEAN
LENGTH OF
PIGMENT PROCESSES
Dark-adapted eyes sub
6
11.0
12.5
14
14
14
3.0
Light-adapted eyes subjected to darkness
1.5
waste exerts an inhibitory or anaesthetic influence upon the normal activity of the protoplasm of the pigment cells. Since in the case of the retina it is even probable that light favors catabolism, it follows that the movement of the retinal pigment in the light only, must be due to the greater efficiency of light as a stimulating agent. The strong stimulatory effect of light, therefore, is able to break through the protoplasmic inhibition caused by accumulated wastes, whereas darkness is ineffective in this respect. If this reasoning is tenable, one may infer that movements of the pigment would also be realized in darkness, provided artificial circulation could be maintained. That light actually is a more efficient stimulus than darkness, is further suggested from the determinations of adaption times {vide infra).
(b) Determination of adaption times
It is evident from the foregoing observations that photomechanical changes do occur in the pigment of the Planorbis retina, and furthermore, one is led to suspect that the indecisive results obtained by Smith ('06) are assignable to the short duration of his experiments, which, in his own words, lasted but "an hour or more." Hence the pertinent inquiry as to
RETINAL PIGMENT OF PLANORBIS 375
the exact periods of time necessary to complete light- and darkadaption becomes the next object of consideration.
Snails which had xjreviously been thoroughly adapted to light (fig. 2) or to darkness (fig. 5), were subjected to opposite conditions of illumination. Indi^'iduals were removed and killed at half-hour intervals until the experiments had continued for 5 hours. Microscopical examination of the retinas gave the following results:
Dark-adapted Planorbis subjected to light:
Incoinplctc light-adaption occurred in 3 hours
Complete light-adaption occurred in 4 hours Light-adapted Planorbis subjected to darkness:
Incomplete dark-adaption occurred in 4 hours
("omi)lete dark-a<Iaption occurred in 5 hours
Our suspicion as to the cause of Smith's failure to procure satisfactory and consistent results is corroborated, since the time alloted foi- his ox]ierimentation was quite uiadeciuate.
From the comjiarative standpoint, it is interesting to see how the rai)idi1y of pigment migration in Planorbis agi'ees with corres]ionding movements in the eyes of other animals.
In the compound eyes of the prawn, Palaemonetes, Parker ('97) gives the following data for the time consujued in tlu^ adaption of the proxnual, or retuial, pigment:
Darkness to ligiit :iO to 4.^ minutes
Light to darkness 45 to (U) minutes
The writer (Arey, 16"), working upon several fishes, has shown that the comi)lete adaption of the retinal pigment re(iuires longer periods of time than have generally been siq^posed. These determinations may be combined in the following summarization:
Darkness to light 45 to 60 minutes
Light to darkness 30 to 60 minutes
An intoi-esting comparison with the cephalopod type of eye is made possible through the values determuied by Hess ('05), from whose work it appears that many hours (48 or more) are
THK .lornVM Ol- C<>\ir \HATIVE NEUROLOGY, VOL. 26, NO. 4
376
LESLIE B. AREY
necessary for dark-adaption, whereas a much shorter time (approximately I5 hours) suffices for light-adaption. Hess's results upon Octopus defilippi (one of the several species studied by him) are especially astonishing, since he states that darkadaption occurs in 48 hours, whereas light-adaption may be practically c()]n])lotod in 15 minutes.
(c) Effect of temperature
Next to light, temperature is the commonest environmental stimulating agent that controls the movement of retinal pigment. Accordingly, a series of tests were made, both on normal and excised eyes, to discover whether a correlative temperature response is demonstrable.
Measurements showing the relative distribution of pigment in the eyes of Planorbis at 3° and 32°C. both in darkness and in light. The values are mean values expressed in micra and indicate both the thickness of the main pigment mass (zonal measurement), and the zonal measurement plus the length of the pigmented processes (process measurement). Corresponding values at room temperature, copied from Table 1, are given in parenthesis
CONDITION OF ILLUMINATION
NUMBER
OF RETINAS
TEMPERATURE
MEASURED
deg. C.
6
3
6
30
4
3
6
32
MEAN
PROCESS MEASUREMENT
Light.... Light.... Darkness Darkness
10.0
14.0
(11.0)
(16.5)
12.5
19.0
13.0
19.0
(18.0)
(25.0)
14.0
24.0
1. Effect on normal animals. Fully light- and dark-adapted snails, retained in jars of water, were each subjected to temperatures of approximately 3° and 32° C. At the end of 4 hours the eyes were fixed at temperatures identical with those at which the respective experiments had been conducted. The data from these determinations are presented in table 3.
These data show that temperature is extremely efficacious in evoking positional changes in the retinal pigment. Since,
RETINAL PIGMENT OF PLANORBIS
377
both in the Hght and the dark, the pigment shows greater distal migration at low (figs. 1 and 4) than at high temperatures (figs. 3 and 6), it follows that low temperature and light on the one hand, and high temperature and darkness on the other, tend to influence i)igment migration similarly. As the data show (this being a logical corollary of the proposition just stated), extreme distal migration is favored by the cooperation of light and low temperature (fig. 1), whereas an extreme proximal migration is best obtained at high temperatures in the dark (fig. 6). It is pertinent to mention in this connection that the pigmented processes at the higher temperatures in the dark (figs. 5 and 6) were very thick as well as long.
It will be noticed that in the light the values given in ])arenthesis (representing the results at room tem])erature co])ied from table 1) are intermediate between the measurements at the extreme temperatures; in the dark, on the contraiy, this progressive relation does not hold.
T.VBLE 4 Mcasiireiiicnls showing Ific nldliic distribulion of pigment in the excised eyes of Planorbis at 2° and 30°C. in the light. The values are mean values expressed in 7nicra and indicate both the thickness of the inain pigment mass {zonal measurement), and the zonal measurement plus the length of the pigmented processes {process measurement)
CONDITION OF ILLUMINATION
NUMBER
OF RETINAS
MEASURED
TEMPERATURE
MEAN ZONAL
MEASVREMENT
MEAN
PROCESS
MEASUREMENT
Light
Liglit
3
3
deg. C.
2
30
9.6
10.3
10.2
17.0
2. EiJect on excised eyes. A few experiments were performed to determine whether the temperature response is obtainable in light-adapted eyes wliich are isolated from the body. In all respects the method of experimentation was identical with that employed when normal animals were used.
Table 4 gi^-es the results from the small number of retinas measured, an accident having destroyed the remainder of the material.
378 LESLIE K. AREY
Tlio ivsiilts tlnis tabulated arc so similar to those just discussed in the case of normal animals that comment is scarcely necessary. The specimens used were rather undersized and this probably accounts for the low values obtained in each set.
Unfortunately no material was available when temperature experiments upon dark-adapted excised eyes might have been performed. Had an influence of temperature been proven under these conditions it would have been of considerable interest, inasmuch as darkness was found (p. 371) to be ineffective upon excised eyes which had previously been subjected to light. This result was indeed realized in my work (Arey, '16^) upon the isolated eyes of fishes, thereby suggesting that temperature is a more efficient stimulating agent than is light.
(d) Effect of anaesthetics
In a few instances only, have clear cut observations been recorded as to the anaesthetic action of definite substances upon retinal pigment cells. Ovio ('95) and Lodato ('95) agreed that cocaine arrested pigment migration in the frog. The writer (Arey, '16^) has found that carbon dioxide or ether, without permanently injuring the retinal pigment cells of fishes, is capable of arresting completely the movements of the pigment in light as well as in darkness. Chloretone and urethane, on the contrary, although of sufficient strength to kill the animals as organisms, did not prohibit pigment migration. Since 5 per cent solutions^ of carbon dioxide effectively controlled these migrations, it was suggested that this, the commonest of catabolic products, might well be the effective agent in preventing migratory movements of the pigment in excised eyes.^ It is possible that the results, already presented, in which it was found that the pigment of the isolated eyes of Planorbis migrated in light but not in darkness, may be interpreted in a similar manner.
- No attempt was made to ascertain the minimal concentrations which would accomplish this end.
3 Of four fishes used, Ameiurus, Abramis, Carassius, and Fundulus, the retinal pigment of the excised eyes of Ameiurus changed its position in tlie light, whereas in none of these species did a change occur in darkness.
RETINAL PIGMENT OF PLANORBIS 379
A few experiments seemed to indicate that both ether and carbon dioxide did completely check the pigment movements when Planorbis was introduced from dar! ness into light, j^et the snails were very susceptible to the anaesthetics and in no case survived. By careful experimentation it might be possible to find concentrations at which movements of the pigment would be prevented, yet the pigment cells and the animals (as determined by controls) survive. After several trials, in all of which the snails did not outlive the 3 to 4 hours subjection to the anaesthetics necessary for light adaption, the work was discontinued.
It is evident that the cliicf vnhw of the few determinations made lies in their suggest i\'eness and not in the actual results gained, which, because of the absence of a])propriate controls, are properly open to criticism. Hence one may be allowed merely to suggest that the ])igment in the excised eyes of Planorbis, similarly to that in Ameiurus, is able to overcome the probable anaesthetic effect of accunmlated wastes in the light only, whereas the weaker response in the dark fails to appear at all because of the presence of the same catabolic products.
DISCUSSIOxN
In 190(3 Parker analyzed the results of many workers concerning the influence of light and temperatiu'e ujDon melanophores and stated his conclusions in the following generalization (p. 413) : *'It is probable that in all melanophores in which there is a migration of ]ngment, light or low tem])erature will induce a migration toward the source of illumination and the absence of light or a high temperature a migration in the reverse direction." Certain cases, how(»\ei-. may be cited which do not conform to Parker's dictiun. tSuch reversed behavior to light is exhibited by the melanophores of the frog (Harless, '54), the eel (Steinach, '91) and Triton (Hertel, '07). A lack of conformity is likewise shown in the temperature responses of the frog's retinal pigment (Herzog, '05; Arey, '16='). It is not improbable that, in these animals, a more or less active nervous control
380 LKSLIH n. AllKV
Ims liocii sii])(M'uni)()S(Ml ii])()n tlio more pi-iiuitive direct melaiiophore response, thereby making the behavior appear anomalous.
It is evident, however, that the results enumerated in this paper relative to the infhience of light and temperature upon the retinal pigment of Planorbis, are conformable with the previously quoted generalization of Parker. The existence of a similar agreement in the behavior of the retinal pigment to temperature was found by Congdon ('07) upon the prawn, Palaemonetes, and by myself (Arey, 'lO"*) upon several fishes.
The role played by body chromatophores in the economy of an animal is supposedly adaptational with respect to its environmental coloration, and perhaps the regulation of its body temperature as well. The significanace of movements of the retinal pigment, on the contrary, are by no means patent, and the agreement of its responses to light and temperature with those of melanophores in general is of considerable speculative interest. Is the reason for this unaminity of response a phylogenetic one, retinal pigment cells retaining a primitive behavior? Or is it merely the similar but discontinuous expression of common physiological needs? Or is the agreement purely fortuitous, not involving the fulfilment of any common need? Or is the influence of light and temperature upon the pigment-containing protoplasm necessarily similar wherever mobile pigment cells are found, the pigment migration under any condition (when not controlled by the nervous system), therefore, being always predictable? By allowing one's fancy free rein, numerous other possibilities may be conceived.
Since w^e know many more instances of immobile than of mobile pigment cells in the tissues of animals, it is reasonable to suppose, and, moreover, evolutionary doctrines compel us to assume, that the existence and perpetuation of the latter type of cells is not without significance. Thus, ignoring all questions as to the reason for this or that behavior of pigment cells, we at least can safely assume with Parker ('06, p. 411) : " . . . it might be supposed that if a case arose in which a reversed migration of pigment would be of service to the organism, such form of migration would be evolved and a set of pigment cells
RETINAL PIGMENT OF PLANORBIS 381
in which the pigment granules under illumination would migrate away from the source of light instead of toward it would be produced."
It has already been pointed out that the exceptional behavior of the bod}' chromatophores of the frog, eel, and Triton probably is the result of a nervous control. As far as direct responses are concerned, Parker's further assertion ('OG, pp. 411-412), for all we know to the contrary, is true: "Hence it seems probable that the melanophores, retmal pigment cells, and other like structures in which dark pigment granules exhibit migratory movements, are restricted as to these possibilities, and that in light they always transport their ])igment toward the source and never in the reverse direction."
Since the pigmented cells of the gasteropod eye have no demonstrable nervous connections, this condition, if true, renders these cells wholly indifferent in the process of light i)erception. It follows, therefore, that such cells are not comparable to the pigment-bearing retinular cells of certain arthropods in which the ])igment granules, contained within the sensory cells themselves, change ])()siti(ni in light and in darkness. The situation in the gasteropod, howe\'er, is quite similar to that in the vertebrate eye in tliis respect.
In several crustaceans (e.g., Parker, '99, upon the am])hii)od, Gammarus) it has been shown that in darkness the pigment of the retinulai- cells mo^es proximally. thereby leaving part of the rhabdome devoid of pigment, whereas in light the rhabdome again receives a pigment sheath. These responses were interpreted as having the following significance. In the light, the rhabdome, surrounded by pigment, is protected from overstimulation by light reflected internally from the white pigment; in dim light, on the contrary, the efficiency of the visual apparatus is increased by the withdrawal of pigment, whereby the reflecting mechanism enables the eye to make the best use of the available diffuse light. Theories involving the principles of oA-er-stimulation as well as of optical isolation have also found many supporters among those who attempt to explain the phenomenon of pigment migration in the vertebrate eye (Arey, '15).
382 LESLIE B. AREY
Tlio iiitcrpictatioJi of iuij>;nit()ry movcinents in the retinal pigjnent of giusteropods, or to go further, the interpretation of the presence of such pigment at all, from the standpoint of preventing overstimulation or of securing optical isolation, involves greater difficulties than is the case in arthropods. In the snail, the brush-like rods, which have been described as the photo-receptive portions of the sensory cells, are entirely distal to the pigmeirt zone, hence these elements are at all times exposed to the full strength of light. It has been pointed out, however, that the pigment may serve as a background to prevent reflection and thus it indirectly produces a limited 'optical isolation.'
The accumulation of pigment far from the cell bases, and the slight extent to which it can be induced to move proximally, suggests that its chief utility may exist in a relationship with that portion of the sensory cell which it immediately surrounds. In this connection, a statement by Smith ('06, p. 270) is suggestive :
Aside from preventing internal reflections within the rod zone, it is possible that the pigment is directly protective to that part of the visual cell which is surrounded by it. We do not know that the middle part of the sensory cell is not sensitive to light. Neither do we know how or where light vibrations are transformed into nervous impulse. If the transformation takes place in the middle zone, the pigment may serve some purpose there.
Having thus seen that the presence of retinal pigment of gasteropods is only to be interpreted with difficulty, how much greater is the task of assigning explanations to the feeble movements exhibited by this pigment. It may be said that explanations of the presence of retinal pigment and of its movements are self-inclusive, an explanation of one necessarily involving the other. That presence and mobility represent two more or less distinct factors, follows, however, from the fact that in some, and perhaps in most gasteropods (e.g., in Helix and Limax, Smith, '06) the retinal pigment is non-motile.
Theorists who have viewed the migratory movements of retinal pigment in those eyes where striking changes occur as indicative of the prevention of overstimulation or the procural
RETINAL PIGMENT OF PLANORBIS 383
of optical isolation, have likewise associated a retreat of the pigment in dim light with the presence of a more diffuse and weaker photic stimulation, but, withal, a stimulation which, under the circumstances, thereby rises to its highest efficiency. An assumption, however, is involved in this reasoning, for it must be shown that the pigment really does retreat in dim light, as, in truth, it does in darkness. This essential point, however, has too often been ignored.
Furthermore, if the distrilnition of pigment in twilight were essentially smiilar to that in bright light, it follows that explanations which attempt to solve the meaning of the position assumed by the pigment in light, only answer half of the questions involved, for it may well be asked — Why should there be an extensive movement in total darkness, or why, since a . permanently expanded condition would seem to be all that is required, any mo\'ement at all? These queries merely show that easily devised ex])lanations, as for examj^le those based upon the theory of optical isolation, may not touch the ]irimaiy reason at all — such oln-ious relations may have a secondary ini])ortance or may even be ])urely fortuitous.
Returning to the case under ct)nsiderati()ii, the movements of the retinal pigment of Planorbis, through the influence of light, are very limited in comparison with those in many animals. The dispersion in the conqiacted ]iigment mass, and the formation of spai'se gramUar ]irocesses, do not visibly alter the density of the main pigmented zone. It is difficult to see (assuming that a proxunal migration does occur in dim light as well as in darkness) how these changes would be of any great value to the animal in the ways in which the pigment commonly has been supposed to act. Even the vague assumptions of a nutritional relation of the pigment to adjacent sensory cells is discre]iant with certain ]ihases of the ]ihoto- and thermo-mechanical changes.
In the body chromatojihores of certain animals, e.g., lizards, the response of the pigment to light and temperature may be of use in regulating the body temperature of the animal. Thus (quoting from Parker, '06, p. 411): "The dark color of the lizard's skin in modei'ate illumination at a moderate temperature
- ^S4 LESLIE B. AREY
insur(>s, ])()ssil)ly, iunoii*;- otluM- tilings, a (-vriii'm dc^roo of waniith wliich would be superfiuous, if not dangerous, at a higher teiu])erature, and in consequence the skin })ecomes light-colored in hot sunlight."
The significance of temperature responses of the retinal pignuMit of Planorbis is even more obscure than the meaning of photic influences. It is easy to construct several groundless hypotheses to explain why low temperature, acting similar to light, and high temperature, acting similar to darkness, could be of advantage to the snail in the regulation of its retinal economy. Such interpretations, how^ever, possess even less value than the speculations concerning the role played by light.
To devise an explanation from the adaptional standpoint that will account for the temperature responses occurring in total darkness would seem a hard task. It may be, however, that the cell thus responds to temperature stimulation because the change is of use in the light-adapted eye only, whereas in darkness the similar response to similar stimulation is gone through with perfunctorily, as it were, even though it is of no use to the organism.
The cautiousness of the following position, with the formulation of which this discussion w411 be closed, may be its only commendation; yet when our absolute ignorance of fundamental facts is considered, caution may well qualify as a cardinal virtue. An analysis of the conditions of pigment migration in the various vertebrate classes has led the writer (Arey, '15; '16*^) to an identical conclusion for these animals as well.
Recognizing, therefore, that the movements of the retinal pigment of Planorbis, as well as of other animals, to light and to temperature may have an adaptive significance, and, furthermore, realizing that (with the possible exception of arthropods) we at present are quite unaware of the meaning of these movements, it would seem that we are permitted to indulge only in interpretations formulated in terms of protoplasmic responses to definite stimulating agents and that the questions of utility thereby involved must await the establishment of a more thorough knowledge of the co-existing factors.
RETINAL PIGMENT OF PLANORBIS 385
SUMMARY
1. Light causes the retinal pigment of nornuil Planorbis to migrate distally (toward the source of ilhmiination). Darkness causes a proximal migration.
2. About 4 hours is necessary for the pigment to assume the distribution characteristic of light-adaption, whereas about 5 hours is demanded in accomplishing the reverse process of dark-adaption.
3. The pigment of dark-adapted, excised eyes exhibits migratory movements when exposed to light, and is, therefore, capable of direct stinmlation. In the converse treatment (light to dark) of excised eyes, on the contrary, no change in the position of the pigjuont occm-s. It is possible that the absence of positional change iji darkness is indicative of an anaesthetic effect, produced by the imremoved products of catabolism, the more vigorous influence of light, however, being able to o\ercome this inhibitory tendency.
4. High tem])rrature (30°C.) induces proximal migration of the normal retinal ]Mgment both in darkness and in light. Low temperature (3°C\) hiduces distal migration both in dai-kness and hi light.
5. Upon light-adapted excised eyes, low temperature likewise favors proximal migration and high temperatm-e favors distal pigment migration.
G, Planorbis afifords the first case among gastero])()ds in which the occurrence of positional changes in the retmal pigment has been correlated with the presence and absence of light. For the first time among molluscs, a thenno-mechanical influence upon the retinal pigment has been demonstrated.
7. It is probable that low concentrations of anaesthetics, such as carbon dioxide or ether, are capable of completely arresting migratory mo\'ements of the Planorbis retinal jjigment. This suggests that carbon dioxide, as a product of catabolism, may be the inhibiting factor that prevents migratory movements of the pigment when excised eyes are brought from light into darkness.
386 LESLIE B. AKEY
8. The jidaptional significance of the existeiico of mobile pigment in the retina of Planorbis is at present quite obscure. These movements can only be correlated with the presence of known environmental conditions, and interpreted in terms of protoplasmic responses to definite stimulating agents.
Anatomical Laboratory, Nortluvestern
University Medical School
December 6, 1915
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Heidelberg, Bd. 1, pp. 15-104.
- LoDATO, G. 1895 Riccrchc sulla fisiologia dello strato neuroepitheliale della
retina. Arch, di Ottalmologia, vol. 3, pp. 141-148. Ovio, C!. 1895 Di un speciale azione della cocain sulla funzione visiva. Annali
di Ottalmologia, Anno 24, Suppl. al I'asc. 4. p. 23. Paukeh, (1. H. 1897 Photomechanical changes in the retinal pigment cells
of Palaemonetes. and their relation to the central nervous system.
Bull. Mus. Comp. Zool., Harvard Coll., vol. 30, no. 6. pp. 273-300.
1899 The photomechanical changes in the retinal pigment of Gam niarus. Bull. Mus. Comp. Zool., Harvard Coll., vol. 35, no. 6, pp.
141-148.
1906 The influence of light and heat on tiie movement of the nielan ophore iiigment, esi)ecially in lizards. .lour. K\p. Zool., vol. 3, no.
3, pp. 401-414. Hawh/., H. ISDl Zur Piiysiologie der Cepiialoi)odenretina. .\rch. f. .\nat.
u. Pliysiol., Physiol. Ai)t., Jahrgang 1891, Heft 5 u. 6, pp. 367-372. Smith, (I. 1906 The eyes of certain pulmonale gasteropods. with special
reference to the neurofibrillae of Limax maximus. Bull. Mus. Comp.
Zool., Harvard Coll., vol. 48, no. 3, pp. 233-283. Spaeth, H. A. 1913" The mechanism of the contraction in the niclanophores
of fishes. Anat. Anz., Bd. 44, No. 20-21, pp. 520-524.
1913 The physiology of the chromotai)hores of tislies. .Jour. Exp.
Zool., vol. 15, no. 4, pp. 527-585. Stki.nacm, K. 1891 Ueber den Farbenwechsel bei niederen Wirbelthieren,
bedingt durch direkte Wirkung des Lichtes auf die Pigmentzellen.
Centralbl. f. Physiol., Btl. 5, Xo. 12, pp. 326-330. SzczAWiNSKA, W. 1891 Contribution k I'etude des yeux de quelques Crustaces
et recherches e.xperimentales sur les mouvements du pigment granu leux et des cellules pigmentaires sous I'influence de la lumiere et I'ob scurite dans les yeux des Crustaces et des Arachnides. Arch, de
Biol., Tome 10, pp. 523-566.
388 LESLIE B. AKEY
PLATE 1
EXPLANATION OF FIGURES
eps.opt., optic capsule rtn.ex., peripheral zone of retina
pig., pigment rtn.i., central zone of retina
The figures of this plate, taken from axial sections of the Planorbis eye, are photographs at a uniform magnification of 540 diameters and show the distribution of retinal pigment at various temperatures in light and in darkness.
1 At 3°C. in the light.
2 At room temperature (21°C. ±) in the light.
3 At 30°C. in the light.
4 At 30°C. in the dark.
5 At room temperature (21°C. ±) in the dark.
6 At 32°C. in the dark.
L. n. A. Photo.
ABSENCE OF CHROIMATOLYTIC CHANGE IN THE CENTRAL NERVOUS SYSTEM OF THE WOODCHUCK (MARMOTA MONAX) DURING HIBERNATION
A. T. RASMUSSEX AND J. A. MYERS
Frimi llic I'hi/Hialogical Lahoralorij, Medical College, Cornell University, Ithaca,
New York
SIX FICIUKS (two plates)
IN TRODUCTIOX
Since the discovery hy Xissl in ISS.") that \\w clironioi^hilous granules in the cytophisni of nerve cells — grannies whicli had already been described by Flennning ('S2) and which von Lenhossek later tenned tigroides, though now more generally spoken of as Nissl gi'anules are iiitens(>ly stained by basic aniline dyes, especially by nietliyleiic bhic tlic (lis])()siti()n of these granides, under numerous ])hysi()l(»ui('al and ])ath()logical conditions, has been the subj(>('1 of umcli >tiid>-. From the extensive functional alterations that numerous authors (\'alentin, Dubois, Merzbacher, etc.) have reported to occur in the nen'ous system during hibernation, and from the morphological variations which are said to take i)lace in the nerve cells in certain otheiconditions, such as sleep and star\'ation, one would natm-ally expect to find marked changes, especially in the Nissl granules, during hibernation. This state, as is well known, is attended in some animals by nlmost continuous sleep and profound torpor for four months and even longer, during which time no food whatever may have been eaten and the body temperature has been reduced to but a few degrees above the freezing point.
HISTORICAL
An examination of the Uterature revealed the fact that some observers have reported marked structural changes in the nerve cells during hibernation. Levi ('98) in the toad (Bufo vulgaris)
391
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4
392 A. T. RASMUSSEN AND J. A. MYERS
found tliiit the \issl bodies had greatly diminished in size and the acido])lul p-aiiules had become basophiUc during wintersleep; but in liibernating mammals no changes were observed. A more detailed study by Legge ('99), however, led this author to conclude that the cells of the cerebro-spinal axis of hibernating bats do undergo visible changes. In the cells of the cerebral cortex he found the Nissl granules to have elongated, being more fusiform in shape, and to be displaced towards the periphery, forming a sort of envelope to the cytoplasm. Baroncini and Beretta ('00) also reported morphological changes in the spinal cord and especially in the cerebral cortex of mammals (muscardin and bat) during winter-sleep. They found that the Nissl granules had greatly decreased and stained more diffusely. The chromatic substance of the nucleus was also more diffuse than in active specimens and the nucleolus seemed to have disappeared in many cases. Essentially the same changes in the Nissl granules in the cells of the cerebro-spinal axis and in the Purkinje cells of the cerebellum of hibernating hedgehogs, were reported by Marinesco ('05) w^ho found a distinct decrease in these granules during the torpid state. Those that remained were reduced to fine granules or were diffused in the cytoplasm. A more recent study by Zalla ('10) agrees with the older results obtained by Levi in regard to mammals. Zalla found no appreciable morphological difference in the Nissl substance in the dormouse (Myoxus glis) during hibernation as compared with the active state. His results in amphibia were not constant, but in reptiles he found a distinct decrease in this substance in the motor cells of the cord and pons during winter-sleep.
The question of changes in the chromophilous substance of the nerve cells during hibernation in mammals thus seems to be unsettled. Legge, Baroncini and Beretta, and Marinesco apparently have observed marked changes, while Levi and Zalla could not establish any such chromatolysis. However, these authors did not all work on the same species of mammal. In amphibia there is also some disagreement. Levi found a distinct decrease in the Nissl granules during hibernation, while Zalla, whose results were not very constant, believes that there
CHROMATOLYTIC CHANGES DURI^XI HIBERNATION 393
are no changes. In reptiles, however, Zalla did find a marked decrease during torpoi-.
Some other morphological changes in the nerve cells during hibernation may be briefly mentioned. Querton ('98) bj' means of the Golgi method on cerebral neurones found that the protoplasmic prolongations retract and assume a moniliform appearance during the dormant period. This, however, could not be substantiated b}' Baroncini and Beretta ('00). Some work has been done on the neurofibrillae. Tello ('03) reported that in the motor cells of the spinal cord of lizards the neurofil:)rillae are much thicker than usually found. Tello and Cajal ('04) found that this was true only during the cold season when the animal is dormant, and that when the animal is warmed up and made active the fibrillae become more numerous and much finer. Cajal thus believes that the hy]KMti-()])hy of the neurofibrillae is due to the cold and resulting diminished s])inal reflexes, bcc.Musc exposure (»r llic nniiiial t<. a low temperature brought about this gi'-ntisni of the ii('Ui<.lii)rillae while warming it up caused a i-ctui-n 1(» llic iK.inial. and l)ec;'use this change is not seen in the telence])hal()n and mesencei)halon whose cells retain their activity to a nuich greater extent during the lethargA'. jNIarinesco CO')) has re]ieatetl these ex])erhnents on young cats and dogs and Dustin ('()()) has done the same on young rabbits with essentially shnilar results. A temjierature below 10°C., however, was found to be less effecti\e than 10°C. in bringing about these changes in the neurofibrillae, according to iMarinesco. This may be related to the fact that a temj^erature too low excites hibernating mannnals and Anally wakes them up since the body temperature rises as a result of increased actiA'ity. The latter author found no such modification of the fibrillae in hibernating hedgehogs, which fact he interprets as indicating that the activity of the nervous system of hibernating mammals is not reduced to the extent that it is in the lizard and other cold blooded animals. Zalla ('10), on the other hand, found that the neurofibrillae were fewer and farther apart in the dormouse during hibernation.
- >!M A. T. UASMUSSEN AND J.
I'RKSKNT INVESTKIATION
In view of the conflicting reports as just reviewed and the more recent observations by Crile on changes in the brain cells under various emotional and other conditions, much less striking than the phenomenon of hibernation, the work we have done on the woodchuck in this regard, seems worthy of a brief note. Woodchucks, or ground hogs, which represent the American marmots, are some of the best examples of hibernating mammals in this country. All s]:»ecies remain dormant for four to six months each year, and hence constitute good material for a study of hibernation. This work was commenced early in January 1913 by J. A. Myers, who fixed and imbedded the central nervous system of six woodchucks, four of which were killed at various intervals while hibernating (January 18, February 6, March 15 and April). One was killed shortly after waking up (March 15) and another, during the following summer.
In addition to studying the above series by means of the Nissl stain, the other co-author prepared another series consisting of the brain and spinal cord of fifteen woodchucks killed during the autumn, winter and spring of 1913-1914. This series includes one animal killed about a month before hibernation (October 25), one just before hibernation (November 22), five during hibernation (February and March) — one of these was partly awake when killed on February 16, but was sluggish and had a rectal temperature of 19°C. — one within two days after waking up (March 16) and seven others which had been awake from three days to more than a month. Three of this last group had been fed for one, two and three weeks respectively. These animals were kept in the artificial burrows which were designed by Professor Simpson of this laboratory and which have already been described elsewhere.^ The rectal temperature of the dormant animals varied from 8°C. to 12°C., whereas, the temperature of the active animals ranged from 32°C. to 38°C.
' Rasmussen, A. T., Amer, Jour. Physiol.. 1915, vol. 39, p. 20.
CHROMATOLYTK^ CHANGES DURING HIBERNATION 395
All the animals of both series were killed quickly by transfixing the heart through the chest wall. No anaesthetic was used, exce])t in five cases where only suflficient ethei- was given to keep the animal quiet. These five animals were all killed after hibernation while awake and active. The amount of ether given did not seem to have any noticeable effect on the Nissl granules. If, however, these five cases are excluded from consideration because of the introduction of this additional factor, the two series involve as strictly comjjarable cases two before hibernation, nhie during hibernation and five after hiliernation. The blood was washed out immediateh' after deatli with normal saline solution by injection through the aorta. The saline was followed by a satui'ated acjueous solution of bichloride of moicury to which had been added 10 ]ier cent of formalin. Tluis the central nei'vous system was fixed \-ery (juickly in situ. The whole brain and cord were then removed and cut into transverse secti(ms a few millimeters thick. The desired levels were further fixed in a saturated aciueous solution of bichloride of mercuiy for 48 hours, washed in running water 36 hours, and dehydrated in graded alcohols containing iodine in the usual manner. The tissue was cleared in xylol and imbedded in ])araffin melting at 54°C. The levels thus imlx'ddcd were: olfactory bulb, motor cortex, mid-thalamiis, midbrain at the level of the superior colliculus, mid-cerebelhmi and ])()ns. nu'dulln oblongata at extreme inferior border of fouith Ncntiicle. lijst cei'vical, sixth cervical, sixth thoracic, second lumbar and lowei- Imnbai- segments of the spinal cord.
Sections were cut five microns in thickness, except in the case of the spinal cord where the sections were six mici'ons thick, and stainetl on the slide for ten minutes in a large quantity of hot (70°C.) solution of 1 per cent methylene blue in water saturated with aniline oil. The excess stain was washed off rapidly in water and decolorization carried on by transferring the slides directly to 95 per cent alcohol for two to ten minutes. Dehydration was completed in absolute alcohol and clearing in cajuput oil followed by xylol. The sections were mounted permanently in Canada balsam dissolved in xylol. Where the size
39() A. T. KASMUSSEN AND J. A. MYEllS
])('rmitt(Hl. ('()iT('S])()ii(lin<>; sections IVom ull aiiiinnls of a series were fixed on the same slide to insure ec^iud staining. In other cases several sections from the same block were placed on one slide and all the corresponding slides stained together by carrying tliem through the reagents by means of a basket, or rack, which would contain the entire lot.
RESULTS
The nerve cells of the woodchuck are essentially typical, containing the usual large round nucleus, with one nucleolus. The chromophilous substance in the cytoplasm has the usual appearance and arrangement so well known that no description will be necessary here. In spite of all precautions there are noticeable variations in the size, distinctness and arrangement of the Nissl bodies in homologous cells of animals in the same state and even in the cells of the same group in a particular section. These variations are found in both dormant and active animals. We can detect no modification in the Nissl granules characteristic of the hibernating as compared with the non-hibernating state. Certainly in these woodchucks there is not the difference indicated by the figures given by Marinesco in the case of the hedgehog. The chromophilous substance is present during hibernation in at least as great a quantity as at other times and presents the usual appearance when stained with methylene blue. The arrangement of the granules varies somewhat even in cells of the same group, being more abundant in the periphery of the cells in some cases and in others being grouped more densely around the nucleus. The size and shape of the bodies vary from fine irregular granules to larger elongated ones; but when a large number of cells are examined the extreme variations may be found in animals in the same state and often in the same section. A predominance of a particular variation in either state can not be established. The accompanying figures and explanations will suffice to indicate the general cell picture before, during and after hibernation. The larger types of nerve cells were selected as illustrations because in them the Nissl bodies are more distinct and make better photographs.
CHROMATOLYTIC CHANGES DURIXG HIBERNATION 397
We wish to thank Prof. Sutherland Simpson for his helpful guidance in this research. We are also indebted to Prof. B. F. Kingsbury for assistance in taking the photoniicrographs.'
LITERATURE CITED
Baroncini, L., and Beretta, A. 1900 Ricerche istologiche sulle modif.
degli organi nei mammiferi ibernanti. Riforma med., vol. 16, p. 206. Cajal, Ram6x y 1904 Variaciones morphologicas, normales et pathologicas
del rctic'ulo neurofibrillar. Trabajos del lab. de invest, biolog. de la
Univ. de Madrid, vol. 3, p. 9. Conipt. Rend. Soc. Biol., vol. 56, p. 372. DusTiN, A. P. 1906 Contribution k I'etude de I'influencede I'age et de I'activ it6 fonctionnellc sur le neurone. .\nn. Soc. Roy. d. Sc. mod. et
natur. de Bruxelles, vol. 1.). Legge, F. 1899 Sulle variazioni della fina .struttura ehe presantano iluranto
I'hibernazione le cellule cerebrali dei pipi.'^trelli. .Moiiit. zool., vol. 10,
p. 152. Levi, G. 1898 Sulle niodificazioni inorfologiche delle cellule nervose di animali
a .sangue frcddo durante rhibernazione. Riv. d. patol. nervos. e.
med., p. 443. Marinesco, G. 1905 La sensibilite de la cellule nerveusc aux variations de
temperature. Revue Neurolog., No. 14 (July 30, 1905), p. 7S4. Re cherches sur les changements de structure que les variations de teni p('>rature impriment h la cellule nerveusc. Revista Stiintelor Medicale,
No. 3, Bucarcst.
1906 Recherches sur le.-^ changements dcs ncurofibrilles consecutifs
aux diffcrents troubles de nutrition. Le Nevraxe, vol. S, j). 149. (^rEKToN 1S98 Le sommeil hiliernal et les modifications des neurones ct'rei)raux.
.\nn. Roy. d. Sc. med. et natur. de Bruxelles, vol. 7, p. 147. Teli.o 1903 Sombre la existencia de neurofibrillas gigantes en la medula espinal
de los reptiles. Trabajos del lab. de invest, biolog. de la I'niv. de
Madrid, vol. 2, p. 223. Zalla, M. 1910 Recherches experimcntales sur les modifications morpholo giques des cellules nerveuses chez les animaux hibernants. (Resume).
Arch. ital. de biol., vol. 54, p. 116. Riv. d. patol. nervos. c. ment., ann.
15. p. 211.
-In regard to tlie general problem of the correlation of structural changes to activity in nerve cells, attention should be called to the experiments carried out on six different species of animals by R. A. Kocher, reported in this journal (vol. 26, No. 3, p 341) since this article was submitted for publication. This author could find no correspondence between the size or structural characteristics of nerve cells ami various grades of activity.
PLATE 1
EXPLANATION OF FIGURES
The three photographs in this plate were taken from the nucleus hypoglossus. The sections from the various animals were mounted on the same slide and hence stained together. X 320.
1 Before hibernation. Woodchuck killed October 25, 1913, without any anaesthetic. Animal active. Rectal temperature 37.6°C.
2 During hibernation. Woodchuck killed March 7, 1914, without any anaesthetic. Animal very dormant and had been so nearly all the time for at least three months. Rectal temperature 9°C.
3 After hibernation. Woodchuck killed April 11, 1914, without any anaesthetic. Animal active. Had been fed for two weeks. Rectal temperature 37°C.
CHROMATOLYTIC CHANGES DURING HIBERNATION
A. T. RASMUSSEN AND J. A. MTEBS
' ^
^in»*
8^ ^
.%
' " ■ . «3
^
.n
■t.f
3
399
PLATE 2
EXPLANATION OF FIGURES
Photographs of motor cells from the antero-lateral group in the sixth cervical segment of the spinal cord. The sections were mounted on the same slide and hence stained together. X 320.
4 Before hibernation. Same animal as in figure 1.
5 During hibernation. Same animal as in figure 2.
6 After hibernation. Same animal as in figure 3.
400
CHROMATOLYTrC CHANGES DURING HIBERNATION
A. T. RASMCSSEN AND J. A. MYERS
•* ^
- 1^
J ■■
- 4
-M^'
^
401
A STUDY OF A PLAINS INDIAN BRAIN
J. J. KEEGAX
From the Anatomical Department, University of Nebraska. Omaha, Nebraska
EIGHT FIGURES
IXTRODUCTIOX
The priniaiy object of this paper is to furnish an account of the morphology of the cerebral hemispheres of a North American Indian brain, thereby filUng a gap in the literature of racial cerebral anatomy. Although a large amount of work has been done upon the Indian tribes from an anthropological standpoint, no description of an Indian brain has, to my knowledge, ever been given. The study of cerebral morphologA- has not yet furnished characters distinctive of race, but the studies of I^lli()t Smith ('04), Duckworth ('07), and others have revived ail iiitcn'st ill this work and advanced groally tli(> inteq^retation of sulci and gyri.
The secondaiy but more im])ortant aim of tliis study. fi-oiii which the conclusions are largely tleduced, is the application of the principles of fissure relation to cortical areas, both in the interpretation of sulci and in the comparison of cortical areas and sulci of the two hemispheres and of other cerebra. This is especially \'aluable in this case on account of the completeness of the history and the exceptional mental abilities evidenced.
The study of cortical localization has been conducted by two methods, cortical stinudation and histological differentiation. The most important names connected with the former method are those of Sherrington and Griinbaum ('01) in their experimental work upon the ape brain, supplemented by a few similar observations upon the human cortex and numerous clinical observations proved by operation or autopsy. This work necessarily gave results in only a few regions of the cortex, mainly
403
404 J. J. KKEGAN
tlic somalic motor ajid sensory areas of the central region. The histological field was first entered by Flechsig ('96) who delimited cortical areas by the order of the myelination of their fibers. Campbell ('05) and Brodmann ('07) made piu-ely histological surveys of the cortex, but were not able by this tedious method to examine enough material to establish the relation of many cortical areas to fissures. E. Smith ('07) was the first to make such a topographical survey of the human cortex and, while in general the areas plotted agreed with the areas of Campbell and Brodmann, the added value of their interpretation in relation to fissures facilitated greatly the study of the fissures of the cerebrum.
The value of E. Smith's interpretation is evidenced by its general acceptance for the occipital region by most of the recent writers, as Duckworth ('07), Appleton ('10), Cole ('11), Schuster ('08), and many others. The application of this method to other regions, however, has been neglected, chiefly on account of the difficulty in obtaining fresh material and the inexpertness in distinguishing the lamination in macroscopic sections. The boundaries of the area striata are so easily identified that this area can be plotted even in imperfectly preserved material, which accounts for the general application in this region.
In this study an attempt has been made to apply by comparison the cortical area plan of E. Smith ('14) to a valuable cerebrum in which the preservation prevented a knife-section analysis of the cortical areas and their relation to sulci. The accuracy of this method may well be questioned for undoubtedly many small errors are present, but it was found that such a plotting aided greatly in the interpretation of fissures and perhaps made possible a more intelligent comparison of similar cortical areas of the two hemispheres and of other cerebra. It at least furnishes the most expressive figures of the sulci and gyri that present knowledge makes possible and might be applied profitably for comparison to the figures accompanying studies of other brains.
AN AMERICAN INDIAN BRAIN 405
HISTORY
The brain which is the subject of this account was obtained at autopsy from the body of a female Indian of the Omaha tribe, and thanks are due Dr. A. A. Johnson of the Department of Pathology for the opportunity of the use of the specimen for study.
The individual was fifty years of age, about 12.") pounds in weight and possessed rather typical Indian features although not extreme. There was united in her person the blood of the Indian, the French, and the English settlers of Nebraska, her grandfathers carrjdng the half French and the half English admixture and the grandmothers of pure Indian descent. It is thus seen that the Indian l^lood was carried down entirely on the female side of the family, which might give rise to s]:)eculation upon the formerly nuich discussed question of the relation of sex to cerebral morphology'.
The intellectual status was far above the average as su])erficially judged of Indian peo])le, and above the average of the white race as judged from the high cjuality of her attainments. The educational training consisted of mission school and government Indian school as a child, five years of general education in preparatory schools, and a three >eai- medical course which she completed in two years, graduating at the head of her class. A year's hospital training comjileted the medical work. Her life was spent in medical service with her own peoi)le. She was generally recognized by the profession and hmnen alike as a woman of veiy superior personality whose intellectual endowments and qualities of character placed her quite above the ordinary \qyq\ of humanity.
DESCRIPTION
The brain, after standing several weeks in a formaldehyde solution, weighed 1353 grams with the dura mater removed. A comparison of the weights of the two hemispheres showed the right considerably heavier than the left, 605 grams and 575 grams.
406 J. J. KEEGAN
respectively, but these figures are unreliable on account of the pathological condition of the left hemisphere, which had destroyed a part of the temporal lobe. This hemisphere also was distorted in preservation, due to the softened condition of the cerebral tissue, consequently no measurements of any value of lengths or indices could be made. Such measurements were established by Cunningham ('92) but have not proven of comparative value even in perfectly preserved brains.
The general type of fissuration presented nothing very unusual, gi^'ing the impression of a fairly well and evenly fissured cerebrum. There was no undue prominence of any region, perhaps a greater tendency to a vertical course of the sulci of the central region of the right hemisphere and a tendency to irregular fissuration in the posterior parietal region of the left hemisphere. There was no indication of an exposure of the insula.
The following brief account of the sulci and gyri is not intended as a description of all points observed, for many of these of no recognized significance or variation can be determined equally as well by an examination of the accompanying figures. These illustrations were made by tracing from photographs and subsequently inking in the fissures by careful examination of course, depth and bridging gyri. The depth of the more important fissures is entered in the drawing. In general the heavier lines indicate the deeper fissures. A bridging gyrus is indicated by an interruption of the line and an intervening dot. The cortical areas are filled in with different symbols, the plan being to place no two of similar type adjoining.
These areas were determined by placing those first in which there could be little doubt of their boundaries. The area striata was delimited by macroscopic examination of knife sections. The remaining areas generally could be filled in by a process of elimination, but in some cases they were determined by arbitrary judgment. This latter is very evident in cases where the boundary does not correspond to a fissure, but it is surprising how little is left to the judgment and how easily the area conformation can be made to agree with the plan of E. Smith without violating the rule of the deeper fissures being in the main bounding fis
AN AMERICAN INDI.\N BRAIN 407
sures. With this scheme it is hardh' necessary' to label the different fissures, for a comparison with E. Smith's figures in Cunningham's Text-book of Anatomy will show the interpretation.
The fissura cerebri lateralis is shar]3ly upturned and bifurcated in the left hemisphere at its posterior extremity, but horizontal and single in the right. Since this bifurcation is caused by an overgrowth of the posterior inferior parietal region, as has been determined in the Negro brain by Poynter and Keegan ('15), the two hemispheres are peculiarly contrasted in their centers of greatest growth in the inferior parietal lobule, the left being in the posterior region and the right in the anterior region. This is further evidenced in the conformation of other sulci in these areas, and will be discussed at a later point. In both hemispheres there is a distinct operculation by the superior lip in the region of the areii subcentralis. The anterior rami of the fissure are completely separate on both sides, enclosing a prominent pars triangularis.
The sulcus centralis is unusual in its upi^er third in the left hemisphere. It lacks several millimeters of reaching the mesial border and in the superior third is displaced anteriorly in an arcuate form, apparently due to an overgrowth of the gyrus centralis posterior opposite the motor are^ of the lower limb. According to Cole ('10), interniption of the sulcus by a submerged or bridging g>'rus occurs at the posterior arch of the supeiior genu in poorly convoluted brains and just above in well developed brains. This brain shows the gyrus exactly at the apex of the superior genu in both hemispheres, narrower and more prominent in the left. An examination of a large number of Negro and Caucasian brains failed to corroborate Cole's conclusion. The gA'rus is always at the same point, varying only in depth and width. The maximum depth of the sulcus centralis is about the same as the sulcus interparietalis. The greater depth of the latter is stated by Appleton ('10) to be a distinctive feature of Australian and simian brains. It is a question whether this would not simply indicate an overgrowth of the parietal lobe in a human brain and not an undergrowth of any region.
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4
408 J. J. KEEGAN
The sulci precentrales are typical of Cunningham's ('92) description in the right hemisphere, but in the left hemisphere the superficial interrupting gyrus occurs lower, apparently between the two rami of the sulcus precentralis inferior. The sulcus diagonalis is incorporated in the ramus verticalis in both hemispheres, thus reducing the posterior area of the gyrus frontalis inferior to a small area in the floor of the sulcus precentralis inferior. The factor which would account for this might be an overgrowth of the motor area of the mouth region in the gyrus precentralis inferior or of the motor speech center in the gyrus frontalis inferior. Cole ('10) has attempted to relate the submergence of this area or gyrus to a simian condition and quotes Kohlbriigge and Bolk as supporting his contention that the sulcus diagonalis represents a detached portion of the simian sulcus precentralis inferior.
The sulcus frontalis superior is well defined in both hemispheres and superficially continuous from the s. precentralis superior to the s. fronto-marginalis, maintaining about an even distance from the mesial border throughout. A prominent bridging gyrus near the posterior extremity permits the interpretation of the continuity of the posterior middle frontal region with the anterior superior frontal region as illustrated in E. Smith's chart. Appleton ('10) considers the tendency towards extra segmentation of the s. frontalis superior as a lowly character.
The sulcus fronto-marginalis is better defined in the left hemisphere than in the right but is too irregular in both to permit of much comparison.
The sulcus frontahs inferior is a high arcuate fissure on both sides in full communication with the s. precentralis inferior. Its length and its distance from the fissura cerebri lateralis are each about a centimeter greater in the right hemisphere. This gives rise to a larger gyrus frontalis inferior on the right side which would be contrary to expectation if the speech center had any relation to the size of this gyrus.
The sulcus frontalis medius is a very subordinate system of shallow sulci, according to the interpretation of the s. frontalis superior. The greater width of the gyrus frontalis medius of
AN AMERICAN INDIAN BRAIN 409
the left hemisphere does not seem to increase the regularity of these elements. The greater tendency is towards a transverse direction.
The sulcus frontaUs mesiaUs is seen as an irregular system of alternating transverse and longitudinal elements extending to the frontal pole. The more posterior of these were chosen arbitrarily as a guide between the superior frontal area antl the anterior frontal area.
The orbital surface has the usual U-yye of fissuration, the only unusual feature being the extension of the mesial limb of the s. orbitalis transversus into the fossa Sylvii of both hemis])heres.
The sulcus cinguli corresponding to E. Smith's chart is ver>' poorly developed in its anterior two-thirds,, the more prominent sulcus being the s. paracinguh. This might indicate a disappearance of the s. cinguli due to an increased growth of the mesial region, but was called to attention by Cole ('10) in a microcephalic brain in which he suggested that the supracingulate sulcus was the older.
The sulcus postcentralis is a ver>' prominent fissure in both hemispheres, extending from the mesial border to within a few millimeters of the fissura cerebri lateralis. In the right hemisphere there is ho communication with the s. interparietalis, thus giving rise to a fissure veiy similar to the s. centralis. In the left hemis])hore the posterior reflection of the two extremities forms an arcuate sulcus and a wide gjTUs centralis posterior in the corresponding regions.
The sulcus inter]mrietahs is contrasted on the two sides. In the right hemisphere it appears as a fourth vertical sulcus in series with the precentral, central and postcentral sulci. It communicates across an almost superficial bridging g>Tus with the s. paroccipitalis. The tendency in the left hemisphere is towards a sagittal and more posterior arrangement. The sulcus is represented by two horizontal elements in intercommunication with the s. postcentralis and the body of the s. paroccipitalis across bridging g>Ti.
The sulcus paroccipitalis is more lateral and more prominent in the right hemisphere. The independence of this sulcus is
410 J. J. KEEGAN
well represented in both hemispheres. Appleton ('10) has classified this as a fetal tendency. The narrow antero-posterior width of the gyrus arcuatus posterior is a noticeable feature and might be indicative of an unusual growth in the superior parietal area.
The sulcus parietalis superior is poorly represented, perhaps due to its partial incorporation in surrounding sulci.
The lobulus parietalis inferior gives evidence of a prominent growth. This is more noticeable in the left hemisphere where the prominent sulcus interparietalis extends into the anterior region as an accessory fissure. The s. angularis, which to some extent is an index to the growth of the inferior part of this lobule, is not independent from the s. temporalis superior as found in the majority of Negro brains. It appears as the sharply upturned extremity of this fissure, more anterior in the left hemisphere.
The sulcus temporalis superior of the right hemisphere was injured by the tumor growth. The absence of the anterior transverse element may be associated with an increased growth of the acoustic area of the superior temporal gyrus. This was further evidenced by very prominent transverse temporal gyri of Heschl and the extension to the lateral surface of the fissure separating the two larger of these gyri.
The sulcus temporalis medius and the sulcus temporalis inferior have never been interpreted well enough to permit morphological comparison. The ascending ramus of the former, sulcus occipitalis anterior of some authors, is typical in the right hemisphere of an arcuate communication with the s. occipitalis inferior, classified by Appleton ('10) as a simian character.
The fissura rhinalis is present in both hemispheres as a shallow groove connecting the Sylvian fossa with the s. collateralis. The interpretation of these two fissures, applied as in the Negro brain, indicates an earlier or less developed condition in the right hemisphere but in neither is the simian or fetal type approached, regardless of the greater depth than u.sual.
The sulcus collateralis is superficially continuous to the occipital region in both hemispheres. The posterior portion lies considerably more lateral in the right, which, as a limiting sulcus for the area peristriata, would indicate a greater extent of this
AN AMERICAN INDIAN BRAIN 411
area on this side. The rhinal element of the sulcus is separated in both hemispheres by a bridging gATus near the anterior extremity.
The sulcus lunatus can be very- plainly identified in the left hemisphere. It is 35 millimeters in length and lies nearer the lateral border about 3 centimeters from the occipital pole. It has an arcuate or rather angular form and a slightly operculated posterior lip. The area striata, delimited by knife sections, lacked about 2 millimeters of reaching this lip but followed the course of the fissure quite regularh\ In the right hemisphere the tendency is towards a longitudinal disposition of the fissures in this region, to which the boundary- of the area striata does not bear any definite relation. The most prominent sulcus is interpreted as a modified s. prelunatus or s. occipitalis laterahs of some authors. Comparison of the extent of.the area striata in the two hemispheres shows \c\y iilainly a greater lateral extent in the left hemis]iliere.
The sulcus occipitalis paramesialis hes more upon the lateral surface in the left hemisphere but is verj' prominent in both hemispheres. This lateral position indicates that it bears a relation to the lateral extension of the area striata.
The sulcus occipitalis inferior courses on the tentorial surface along the lateral border. It is verj' similar on the two sitles, a deep, slightly operculated fissure about 40 millimeters in length, communicating at the two extremities with the sulci of the corresponding regions.
The sulcus calcarinus in both hemispheres is separated from the fossa parieto-occipitalis by a small bridging gyms cuneus and from the sulcus retrocalcarinus by a larger bridging g^'rus.
Tlie sulcus retrocalcarinus temiinates in both hemispheres in a deep operculated polar element but separated by a prominent bridging g>Tus. Landau ('15) interprets this polar element (s. extrenms, s. occipitalis triradiatus, s. calcarinus externus of E. Smith, s. occipitalis polaris), as the true posterior bifurcation as described by Cunningham ('92), separated bj- the g>Tus cuneolingualis posterior and homologous to the sulcus triradiatus of ape brains. The prominence of this sulcus was noted by the writer ('15) in the Negro brain, in which it was described as an
412 J. J. KEEGAN
independent yulciis occipitalis pokiris. If the interpretation of Landau is correct, its prominence would be an indication of a greater proportionate development of the area striata in the same manner as the sulcus lunatus.
The fossa parieto-occipitalis is quite typical of E. Smith's description in the left hemisphere. The gyrus intercuneatus is about 5 mm. in height and separates widely the s. paracalcarinus and the s, limitans praecunei. It is traversed by an independent sulcus incisura which appears superficially on the lateral surface, separate from the termination of the sulcus limitans praecunei in the gyrus arcuatus posterior. The s. paracalcarinus undermines the posterior wall of the fossa, becoming superficial along the border of the hemisphere posterior to the gyrus arcuatus. The fossa of the right hemisphere is less typical. The gyrus intercuneatus is faintly indicated, the deepest part of the incisura being formed by the sulcus paracalcarinus. The s. limitans praecunei incises the lobulus praecuenus.
The sulci limitantes areae striatae are easily identified by the delimitation of the area striata.
The insula, as far as could be determined, presented nothing unusual in its fissuration. The operculation was complete in both hemispheres.
SUMMARY
The detailed study of the fissures and convolutions brings a number of points to attention. First is noted the striking difference between the two hemispheres in almost every fissure or region examined. This variation is so great that a comparison of hemispheres is of very little value in the interpretation of sulci, which method proved so valuable in the interpretation in the Negro brain. A similar fact of asymmetry is stated by Appleton ('10) to represent an agreement with European cerebra in general and a contrast with supposedly lower types of cerebra. Many writers have called attention to the asymmetry of the cranium, brain, and intracranial venous sinuses separately, but comparatively little work has yet been done to study the co-relation of these asymmetrical conditions the one to the other or to explain their origin. E. Smith ('07), in a note upon
AN AMERICAN INDIAN BRAIN 413
the asymmetry of the brain and skull, contrasts the asjTnmetry of the cranium of the higher races of man with the symmetry of the apes and to a less degree of the black races. This lack of symmetiy of the cranium in man is attributed to the unequal development of homologous parts of the two cerebral hemispheres, especially the great parietal and frontal association areas. The greater size of the right parietal association area is given as an explanation of the greater prominence of the corresponding parietal bone and a relative shifting backward of the right parietal boss, of the usually greater extent of the lateral part of the left visual cortex and the retention of a more pithecoid form in the left hemisphere.
This as}Tmnetr>' is suggestive of the predominance of finictional areas upon one side or the other of the brain. This has been generally accepted in the predominance of the speech center upon the left side in the inferior frontal g>'rus, Broca's convolution, and of the visual cortex in the more extensive area striata and more prominent sulcus lunatus in the left hemisphere of the majority of brains. Cumiingham ('02) attrilnited right-handedness to a functional pre-eminence of the left hemisphere, al-though not supported by any constant observation of greater weight or greater prominence of the so-called motor ann center, of appreciable histological difference or of any regular plan of asymmetrj\ The asymmetr>', although noticeable also in the lower animals, "never attains the same degree as in man." A deficient growth of the ascending parietal convolution (gyrus post-centralis) has been found associated with the congenital absence of the arm of the opposite side (Moorhead, '02). The fact that this is not in the so-called motor center of the arm is interesting in that it shows that the functional center does not necessarily correspond to the motor center.
The importance of this aspect of cerebral mor]iholog\' would tend to compUcate the comparison of sulci and gyri, not only between the two hemispheres of the one cerebrum but also between hemispheres of different cerebra, of individuals or of races. It would be necessary, before a rational comparison could be made, to establish as many points as possible of functional similarity. At the present time there are few such points for
414 J. J. KEEGAN
comparison, but it is not unreasonable to expect that a closer clinical or functional analysis may disclose more features that can be translated as indications of the predominance of functions upon one side or the other of the brain. An interesting observation would be to determine if a predominant vision with the right eye is related to a more prominent area striata and sulcus Kmatus of the left hemisphere.
The most striking differences between the two hemispheres of this brain are found in the occipital, parietal and central regions. The sulcus lunatus can be identified only in the left hemisphere where it is very prominent and associated with a more lateral extent of the area striata, a larger, more lateral and more independent sulcus occipitalis triradiatus, a sulcus occipitalis paramesialis more upon the lateral surface, a sulcus occipitalis inferior nearer the lateral surface and in arcuate communication with the sulcus occipitalis anterior.
The inferior parietal area is more extensive in the right hemisphere and the increased growth appears to be in the anterior portion. This is evidenced by the extension of the anterior extremity of the sulcus interparietalis into this region and the more posteriorly situated sulcus angularis. The conformation of the sulcus iriterparietalis of the left hemisphere would seem to indicate a predominance of growth in the superior parietal lobule. This has resulted in a low position of the sulcus interparietalis, a posterior deflection of the sulcus post-centrahs and a striking anterior arching of the sulcus centralis above its superior genu. The lower part of the gyrus post-centralis is also widened by the posterior deflection of the inferior extremity of the sulcus post-centralis. The entire gyrus is noticeably wider than in the right hemisphere.
The value of these observations of the dissimilarity of the two hemispheres is difficult to judge on account of the lack of knowledge of the extent of unilateral functional predominance and of clinical tests to corroborate the morphological findings. The study is interesting from an anthropological standpoint, demonstrating that in a race of inferior status all of the elements necessary for a higher individual development are present. The same condition was concluded in the Negro brain (Poynter
AN AMERICAN INDIAN BRAIN 415
and Keegan, '15). No single morphological point could be selected which would represent inferiority. AVhile in the Negro the mental characteristics may be in part explained by the great predominance of the parietal lobe over the frontal lobe, in this brain the characteristic feature is not a disproportionate growth of any large area, nor any striking complexity or simplicity of fissuration, but a marked asymmetrv' of the fissures and convolutions.
COXCLUSIOXS
1. This Indian bruin represents in practically all features a high type of cerebrum, the only possible exceptions being the shallow communication of the incisura rhinalis with the sulcus collateralis and the presence of a typical sulcus lunatus in the left hemisphere.
2. The great asymmetry' of the two hemispheres in fissuration is the most convincing evidence of a highly specialized cerebnun. Future combination of physiological observation with cerebral morphology will undoubtedly lead to a better interpretation of such variations.
3. The comparison of cerebra of different hidivitluals should take into consideration tins relation of asymmetrj' to functional localization and should be confined to hemispheres of the same side in individuals of as near similar mental traits as possible.
4. The method of plotting the cortical areas estabUshed by E. Smith, although inaccurate in many details, aids greatly in the interpretation of fissures and m the comparison of the development of different areas. It serves as the most expressive manner in which the morphologA' of cerebra can be presented for comparison.
LITERATURE CITED
Appleton, a. B. 1911 Descriptions of two brains of natives of India. Jour.
Anat. and Physiol., vol. 45. Brodm.\nn, K. 1907 Beitriige zur histologischen Lokalization der Grosshirn rinde. Journ. fiir Psychol, und Neurol., Bd. 6. Campbell, A. W. 1905 Histological studies on the localization of cerebral
function. Cambridge. Cole, S. J. 1910 On some morphological aspects of microcephalic idiocy.
Jour. Anat. and Physiol., vol. 44.
1911 Remarks on some points in the fissuration of the cerebrum.
Jour. Anat. and Phvsiol., vol. 46.
416 J. J. KEEGAN
Cunningham, D. J. 1892 Contribution to the surf;u;o anatomy of the cerebral
hemispheres. Cunningham Memoirs of the Roy. Irish Acad., no. 7,
Dublin.
1902 Right-handedness and loft-braincdness. Jour. Anthropol. Inst.,
vol. 32. Duckworth, W. L. II. 1907 The brains of aboriginal natives of Australia.
Jour. Anat. and Physiol., vol. 42. Flechsig, p. 1896 Gehirn und Seele. Leipzig. Landau, E. 1915 Zur vergleichenden Anatomie des Hinterhauptlappens.
Folia Neuro-Biol., Bd. 9. MooRHEAD, T. G. 1902 A study of the cerebral cortex in a case of congenital
absence of the left upper limb. Jour. Anat. and Physiol., vol. 37. PoYNTER, C. W. M. AND Keegan, J. J. 1915 A study of the American Negro
brain. Jour. Comp. Neur., vol. 25. Schuster, E. H. J. 1908 Descriptions of three Chinese brains. Jour. Anat.
and Physiol., vol. 42. Sherrington, C. S. and GRtJNBAUM, O. 1901 Observations on the physiology
of the cerebral cortex in some of the higher apes. Proc. Roy. Soc,
vo'.69. Smith, G. E. 1904 Studies in the morphology of the brain, with special reference to the Egyptians. Recs. Egyptian Gov't. School of Med., Cairo,
vol. 2.
1907 Asymmetry of brain and skull. Jour. Anat. and Physiol., vol. 41.
1907 A new topographical survey of the human cerebral cortex.
Jour. Anat. and Physiol., vol. 41.
1914 The nervous system. Cunningham's Text-book of Anatomy,
fourth edition.
ABBREVIATIONS
A, Sulcus angularis PL, Sulcus prelunatus
C, Sulcus centralis PO, Fossa parieto-occ'pita is CA, Sulcus calcarinus POC Sulcus paroccipitalis
CL, Fissura cerebri lateralis PRI, Sulcus precentralis inferior
CO, Sulcus collateralis PRS, Sulcus precentralis superior
D, Sulcus diagonalis R, Sulcus radiatus
FI, Sulcus frontalis inferior RA, Ramus ascendens of fissura cerebri FM, Sulcus frontalis medius lateralis
FMA, Sulcus fronto-marginalis RC, Sulcus retrocalcarinus
FS, Sulcus frontalis superior RH, Ramus horizontalis of fissura IP, Sulcus interparietalis cerebri lateralis
L, Sulcus lunatus RI, Fissura rhinalis
OA, Sulcus occipitalis anterior RO, Sulcus rostralis
01, Sulcus occipitalis inferior SA, Sulcus subcentralis anterior
OL, Sulcus olfactorius SC, Sulcus cinguli
OP, Sulcus occipitalis paramesialis SP, Sulcus subcentralis posterior
OR, Sulcus orbitalis transversus TA, Sulcus temporalis anterior P, Sulcus occipitalis polaris (retro- TI, Sulcus temporalis inferior
calcarine bifurcation) TM, Sulcus temporalis medius
PC, Sulcus postcentralis TS, Sulcus temporalis superior PCI, Sulcus paracinguli
AN AMERICAN INDIAN BRAIN
417
Fig. 1 Right hemisphere, lateral view Fig. 2 Left hemisphere, lateral view
418
J. J. KEEGAN
Fig. 3 Right hemisphere, mesial view Fig. 4 Left hemisphere, mesial view
AN AMERICAN INDIAN BRAIN
419
Fig. 5 Both hemispheres, basal view
Fig. 6 Both hemispheres, superior view
420
J. J. KEEGAN
Fig. 7 Both hemispheres, occipital view Fig. 8 Copy of E. Smith's ('14) figure
AN EXPERLMENTAL STUDY OF THE VAGUS NERVE
MARTIN R. CHASE From the Anatomical Laboratory of the Northwestern University Medical School^
FOUR FIGURES
In a previous paper a study was made of the structure of the roots, trunk and branches of the vagus nerve (Chase and Ranson '14).
It was shown, in conformity with the work of Molhant ('10) and Van Gehuchten and Molhant ('11) that the vagus nerve in the dog contains myelinated fibers which can be classified as large, medium and small. In addition there were found enormous, numbers of unmyelinated fibers. These are present in certain of the vagus rootlets, and throughout the tmnk of the vagus. In the cervical and thoracic tiimk they constantly increase in proportion to the myelinated fibers, so that at the level of the diaphragm the vagus is almost a pure unmyelinated nerve.
The most of the large myelinated fibers, and many of medium size, are given off in the cervical branches of the vagu-. The bronchial and esophageal rami receive nearly all of the remaining myelinated fibers. Relatively few unmyelinated fibers are found in the cervical branches. The increased proportion of unmyelinated fibers in the lower vagus is due in part at least to the withdrawal from the trunk of the myelinated fibers by the upper branches. Essentially the same histological picture was seen in sections from the vagus of man (Ranson '14), the rat and the rabbit.
Gaskell '86 observed unmyelinated fibers in the vagus nerve, and found the lower thoracic vagus to contain few myelinated fibers. He interpreted the unmyeUnated fibers in the vagus
1 Contribution No. 37.
421
422 MARTIN R. CHASE
as being post-gaiiglioiiic fibers arising from sympathetic cells in the vagus ganglia.
Molhant '10 working with the Cajal reduced silver method, saw masses of unmyelinated fibers in the vagus nerve and thought they were of sympathetic origin, being presumably post-ganglionic fibers arising from cells in the sympathetic ganglia. Neither Gaskell nor Molhant demonstrated unmyelinated fibers in the roots of the vagus.
Langley has suggested the possibility of j)reganglionic visceral efferent fibers losing their myelin sheaths during their course down the vagus.
Ranson ('15) has recently studied the sti-ucture of the vagus nerve in the turtle, and has made observations of importance in clearing up the origin of the unmyehnated fibers in the vagus nerve. The vagus nerve in the turtle divides high in the neck into a cervical and a thoraco-abdominal ramus. The cervical ramus is composed almost entirely of myelinated fibers and the cells of the cervical ganglion of the vagus are associated only with the fibers of this ramus.
The thoraco-abdominal ramus presents the same structure as the thoracic vagus in mammals. It has a ganglion in the upper part of the thoraco-abdominal cavity. It consists largely of unmyelinated fibers, with scattered myelinated fibers, and maintains the same structure from its origin to the origin of the recurrent nerve. Ranson shows that the unmyelinated fibers are not post-ganglionic fibers arising from cells in the cervical ganglion, since the thoraco-abdominal trunk is not associated with this ganglion. Neither do preganglionic visceral efferent fibers lose their myelin sheaths, since the structure of the thoraco-abdominal ramus does not change. There is no association with sympathetic ganglia, and the proportion of unmyelinated fibers does not change below the thoraco-abdominal ganglion, so there are probably no sympathetic elements in this ganglion.
In the dog there is a very close association of the vagus and sympathetic trunks in the neck, and it seemed possible that some of the unmyehnated fibers of the thoracic vagus in this animal
THE VAGUS NERVE 423
might be of sympathetic origin. The present paper presents the results of some experiments performed on dogs to determine by degeneration methods to what extent sjniipathetic fibers entered into the composition of the vagus.
Figure 1 is a diagram of the right ^'agus nerve in the dog, showing the relations with the sympathetic, and the origin of the branches. Note that the two trunks are closely united from high in the neck to the upyier thoracic region. Note also that just below the bronchial rami, r.b., each \-agus gives off a branch which goes to unite with the nerve of the opposite side. In addition each nerve is also joined by smaller twigs from the contra-lateral vagus. Sections of either vagus below the level of the bronchial rami may include fibers which originate in the opposite vagus, a fact of importance in interpreting the histolog>^ of the degenerated nerves.
It would be possible for post-ganglionic libers to enter the vagus from communications with the superior cervical ganglion of the sympathetic. In the ])re]iaration of the former paper (Chase and Ranson) we were unable, in a study of serial sections, to trace any considerable mnnber of such fibers into the vagus. Throughout their course in the neck, although the two ner\-es are contained in a conunon sheath, no interchange of fibers could be traced. At the level of tlic inferior cervical (g.c.i.) ganglion of the sympathetic, however, the association of the vagus and sympathetic is veiy close, and numerous large bundles of sympathetic unmyelinated fibers can be seen in serial sections entering and leaving the vagus tiimk. If, then, any considerable proportion of the unmyelinated fibers in the thoracic \'agus of the dog have their origin in the s^^upathetic trunk, they nuist arise from cells in the inferior cervical ganglion g.c.i., or ganglion stellatum, g.s.
METHODS
The right vagus ner\'e was se\-ered in a number of dogs. In some cases it was separated from the sympathetic tnmk high in the neck and about an inch of the vagus nerve was removed (fig. 1, A). There would then be no interference with fibers
THE JOLKXAL OF COMTAR-VTIVE XEUROLOGV, VOL. 26, XO. 4
424
MARTIN 11. CHASE
4 b.
THE VAGUS XERVE 425
from the superior cervical ganglion, the sympathetic trunk or inferior cervical ganghon.
In other cases about an inch of combined vagus and sympathetic trunk was removed in the midcervical region (fig. I, B). Such a procedure would cut off any fibers to the vagus from the superior cervical ganglion, but would in no way interfere with fibers from the inferior ganglion.
The dogs were autopsied after 2 to 8 weeks and jiortions of the nerves studied after pre])aration by the pyridine silver method fRanson '11. '12) and the osmic acid method.
CHANGES AKTEIl .SIXJTIOX OF THE VACU'S ALONE HKUI IN THE NEf'K
Sections fi-oiii llie right \agus of a dog autoi)sietl five weeks after removal of a ])oition of \]\o upper cervical vagus, the .sympathetic l)eing left intact, show definite degenerative changes. A cross section stained l)y the pyridine silver method, taken
Via. 1 Diagram of tlic ri<ili( vami.s iiorvo in the (I<ir
J'in. L' From a .soction of riKlit vajius norvc at level indicated (fig. 1, f, 5), following removal of a jiortion of the vao;us and sympathetic trunks at level B, figure 1. Pyridine silver. X 1140.
Fig. 3 From a section of the same nerve at same level as figure 2. Osmic acid. X 1140.
Fig. 4a From a section of a normal vagus nerve just l>elo\v pulmonary ]ilexuses (fig. 1, 4«)- Pyridine silver. X 1140.
Fig. 4b From a section of the same nerve as figure 2. helow the pulmonary plexuses (fig. 1, 4b)- Pyridine silver. X 1140.
a, vagus rootlets n.r.i.. iiervus caroticus internus A,B., indicate levels at whicli portions n.l.s., nervus laryngeus superior
of the vagus trunk were removed n.r., nervus recurrens
art.s., arteria suhclavia n.r., nervus vagus
a.s., ansa subclavia ^^.r.^.s., nervus vagus and truncus sym b, bulbar rootlets of the accessory nerve pathicus
c, spinal root of the accessory nerve p.o., plexus csophageus g.c.i., ganglion cervicale inferior r.b., rami bronchiales
g.c.s., ganglion cervicale superius r.e., ramus externus n. accessorii
g^.j., ganglion jugulare r./j., ramus pharyngeus
g.n., ganglion nodosum t.s., trunkus sympathicus
g..s., ganglion steliatum 2, 3, 4a, 4b, indicate levels at which the
I.V., left vagus corresponding figures were taken
426 MARTIN U. CHASE
throup;li the thoracic vagus just above the bronchial rami, sliows no normal myelinated fibers and only a few scattered unmyelinated axones. There are present, however, a few bundles of unmyelinated fibers which are clearly of sympathetic origin. Th(y cling in groups and stain in a characteristic manner. Similar groups of unmyelinated fibers, present at this level in the normal vagus nerve, were readily identified as of sympathetic origin by their contrast with the vagus fibers. They occupy only a small part of the total cross section area.
Sections taken below the pulmonary plexuses show the right vagus to have retained its degenerated character. It is joined early by twigs from the normal left vagus, whose deeply stained fibers contrast sharply with the degenerated areas. It is impossible to locate in these sections the sympathetic fibers seen at a higher level. They are not present in bundles, and it would be impossible to differentiate individual sympathetic fibers from normal fibers from the left vagus.
CHANGES PRODUCED BY REMOVAL OF A PORTION OF THE CERVICAL VAGO-SYMPATHETIC TRUNK
In the animal from which the illustrations are taken, a portion of the right sympathetic vagus trunk was removed as indicated (fig. 1, B) and the dog was allowed to live four weeks.
Figure 2 is a drawing from a cross section of the right vagus trunk just proximal to the bronchial rami (fig. 1, 2, 3) stained by the pyridine silver method. There are seen no normal myelinated or unmyelinated nerve fibers. The aggregations of granules, surrounded by definite zones, are nuclei of neurilemma cells, as is proven by longitudinal section. The entire cross section shows the same structure, there being only an occasional naked axone. There are no bundles of unmyelinated sympathetic fibers as was seen at the same level in the specimen previously described.
Figin-e 3 is a drawing of a small part of a cross section of the same nerve at about the same level, stained with osmic acid. Note the presence of a number of deeply stained globules, which are the remains of degenerated myelin sheaths. There is pres
THE VAGUS NERVE 427
ent in the entire cross section only an occasional myelin ring, and most of these clearly represent degenerating fibers.
Figure 4 is a composite drawing. Figure 4a, the upper half of the figure, is taken from a cross section of a normal right vagus nerve just below the origin of the bronchial rami (fig. 1, 4a). There are present in this field nine small myelinated axones. The remainder of the field is packed with unmyelinated axones. Figiu'e 4a may be taken to represent the condition in the normal thoracic vagus, it being remembered that above the bronchial rami there are more myelinated axones, while near the diaphragm there are even fewer than in the figure. Figure 4b is a drawing from a cross section of the same nerve as figures 2 and 3, taken at a level just proximal to the union with the branch from the left vagus nerve (fig. 1, 4^). There are in the field no normal unmyelinated or myelinated axones, and the entire cross section shows the same structure, there being only an occasional naked axone.
In both pyridine silver and osmic acid preparations of the nerve below the level described sections, both of the right nerve after its union with the branch from the left vagus, and of the left nerve after \mion with the brancli from the right, show a mingling of the degenerated nerve with the normal, but the degeneration can be clearly traced to the diaphragm.
In the second case, wliere both tlie \agus and sym])athetic were cut in the mid-cervical region, no bundles of fibers of sympathetic origin coukl be found, the entire nerve being degenerated.
We conclude, then, that following section of the cervical vagus ner\-e in the dog, with or without section of the sympathetic trunk, all fibers of the vagus, myelinated and unmyelinated alike, imdergo complete degeneration. There are to be found in th(^ degenerated thoracic tiimk, above the level of the pulmonary plexuses, in .some individuals bundles of unmyelinated nerve fibers having their origin in the cells of the .symi)athetic, presumably in the inferior cervical ganglion. In some individuals no bundles of this sort are found. In no case does the total number of fibers of sympathetic origin present amount to more than an insignificant fraction of the total mnuber of unmyelinated
428 MARTIN K. CHASE
fibers normally present. In the nuiterial studied it was possible to demonstrate more groups of unmyelinated fibers of sympathetic origin in the thoracic vagus in the animals in which only the vagtis was severed high in the neck, than in those operated by division of both vagus and sympathetic trunks. It is thought that this finding is the result of individual ^'ariation in the animals, but it is possible that the difference in operative procedure has something to do with it.
The unmyelinated character of the thoracic vagus nerve in the dog is not due to the presence of fibers derived from the sympathetic trunk, during the close association of the vagus and sympathetic nerves in the neck.
BIBLIOGRAPHY
Chase and Ranson 1914 The structure of the roots, trunk and branches of
the vagus nerve. Jour. Comp. Neur., vol., 24, no. 1, pp. 31. Gaskell, W. H. 1886 On the structure, distribution and function of the nerves
which innervate the visceral and vascular systems. Jour. Phys.,
London, vol. 7, p. 19.
1889 On the relation between the structure, function and origin of
the cranial nerves. Jour. Phj^s., London, vol. 10, p. 153. Langley, J. N. 1900 The sympathetic and other related systems of nerves.
Schiifer's text-book of Physiology, vol. 2, p. 665.
1903 Die Kranialen autonomen Nerven und ihre Ganglien. Ergeb.
der. Phys., Bd. 2, p. 852. MoLHANT, M. 1910 Le nerf vague. Le Nevraxe, vol. 11, p. 137. Ranson, S. W. 1911 Non-medullated fibers in the spinal nerves. Am. Jour.
Anat., vol. 12, p. 67.
1912 The structure of the spinal ganglia and of the spinal nerves.
Jour. Comp. Neur., vol. 22, p. 159.
1914 The structure of the vagus nerve of man as demonstrated b}^ a difTerential axon stain. Anat. Anz., Bd. 46, S. 522.
1915 The vagus nerve of the snapping turtle (Chelydra serpentina). Jour. Comp. Neur., vol. 25, p. 301.
CHANGES IN THE ROD-VISUAL CELLS OF THE FROG DUE TO THE ACTION OF LIGHT
LESLIE B. AREY
From (he Anntomicol Lalxiratonj of the Northwes em L'tiiversily Medical School^
TWO FIGURES
CONTEXTS
L rrcliiiiiiKiry 420
2. Experimentation 434
A. Influence of light on the position of the red rod nucleus 435
B. Influence of light on the length of the rod myoid. Relative changes
at center and periphery of retina 43(5
a. Red rods 436
b. Green rods 439
C. Influence of light on the diameter of the red rod 440
3. Summary 441
4. References 442
I'ln'.Ll.MlNAKV
Whenov(M- tlic fcsiilts of cxiMM-iinontatioii u])()ii any ]);(iti('iilar anhiial, or groiij) of animals, are at \'ariance with a well cstabHsIkhI canon, it is ilosiniblo that such results be sul^jectod to the closest scnitiny possible before the existence of a deviation from the generally accepted norm is admitted. The present paper, which attempts to answer the following; quer>', is concerned chiefly wdth the reexamination of a case of this kind — Query: Are the photoviechanical responses of the insual rods of the frog similar to those occurring in the retinas of other inrestigated vertebrates?
I^otli the rods and the cones of many vertebrates undergo positional changes when subjected to photic or thermal stinnilation (Arey '15, '16).
1 Contrilnition Xo. 3S. April 20, 1916.
429
430 LESLIE B. ATIEY
Wherever iiioveinents of the cones have been detected, hght causes a shortening, and darkness an elongation, of the contractile portion of tlie inner member known as the myoid (figs. 1 and 2, my. con.).
Stort ('86), working on the crow, first showed that the rod myoid of birds exhibits photomechanical changes which are the exact reverse of those found in the cone — that is, elongation in the hght and retraction in the dark. The following year ('87) he extended his observations upon certain fishes. Although this striking response in fishes can be demonstrated with comparative ease (especially in fishes possessing large rods; Arey, '15, '16), it was not until long after that these forgotten observations were corroborated and their correctness admitted.
With respect to the frog's rod,^ however, Angelucci ('84, '90) and Gradenigro ('85) came to a different conclusion. These workers recorded that the myoid of the inner member (figs. 1 and 2, my.bac.rb.) of the frog's rod shortens when exposed to light and elongates in darkness, Gradenigro (p. 343) states this conviction most emphatically: "Zuerst habe ich mit positiver Bestimmtheit ersehen dass an der Froschretina unter der Einwirkung des Lichtes die Stabcheninnenglieder kiirzer und dicker werden, in ahnUcher, wenn auch nicht so ausgedehnter und rascher Weise, wie Englemann bei den Zapfeninnengliedern die Beobachtung gemacht hat."
Angelucci ('90, p. 245) makes a similar straightforward assertion, which applies to both the red and green visual rod:
- There are two kinds of visual rods in the frog's retina. The more numerous form (figs. 1 and 2) has a short inner member {my.hac.rh. + ell.bac.rb.)
and a long outer member (prs.dst.bac.rb.). Since the first imperfect observation by Midler ('51), it has been shown repeatedly that, in the fresh retina of an animal which has previously been retained in darkness, the outer member of the rod appears reddish due to the presence of unbleached visual purple (rhodopsin). Schwalbe ('74) saw a second type of rod (figs. 1 and 2) characterized by an elongated inner member {my.bac vr. + ell.bac vr.) and an abbreviated outer member (prs.dst.bac.vr.); Boll ('77) also described this element and interpreted correctly its green appearance, in fresh dark-adapted retinas, as due to a specific photo-sensitive material, which, from its color has been called visual green (chloanopsin). Hence red and green rods differ both anatomically and as to the nature of their photo-sensitive contents. Unless otherwise stated reference to the red visual rod is usually understood.
CHANGES IN THE VISUAL CELLS OF THE FROG 431
"Die Stiibchen sind dick iind sowohl in ihreni iiusseren Glied als in ihrem myoiden Theil zusamnicngezogen. . . . Auch die griinen Schiralbe' i<chen Stiibchen sind sowohl in ihreni iiusseren als in ihrem inneren Cllicde zusanimengezogon."
Arcoleo ('90), using pithed toads, likewise reported similar responses.
More recently, Lederer ('08), in a brief communication, has challenged the results of the previously named workers on the frog's rod. From the study of fixed material, which had been subjected to teasing, he concludes (p. 7()4): " Die Hellstabchen vvaren im allgemeinen liinger, schlanker und hat ten gleich breites Innen- und Aussenglied, wiihrend bei den Dunkelstilbchen, die kiirzer und plumper erschienen, das Innenglied dort, wo es an das Aussenglied grenzt dicker wird." It should be remarked in passing tliat Lederer's two schematic figures of isolated frog's rods have ])resumably been interchanged — at least, it is obvious that they illustrate conditions exactly the reverse of those which his text descriptions maintain. His experience with stained celloidin sections is summarized in the following statement (p. 764) :
man iiach den Zupfpriiparatoii am gefiirbton Scimitte cbcnfalls ahnliclu' N'criuiitnissc' liiit tc erwartcn sollcn. Indcssen waren hier die Venindorimgou der Stahchonschichi schr wenig markant. In ungofiiiir der Hiilfte der geschnittonen Licht-Bull)i zoigten die Stiibchcn liingere, gcstrechtcrc Form, grosseren Al)stand ilircs Aussongliodos von der Alembrana limitans externa, die Dunkelstiibchen gedrungenere gestalt. kleinere Dislanz von der iiusseren (Jrenzmembran. In der anderen Ilalfle der Sehnitte al)er war eine Veriinderung der Stellimg der Stiibchen sehr wenig ausgesproehen, und die Hellund Dukelsliihchen das Aussenglied ungefiihr gleich weit von der ]\Ienil)rana limitans externa entfernl.
The rods of certain amphibians exhibit photomechanical movements which should be clearly distinguished from those produced by the contractilty of the myoid. Thus Stort ('87) first asserted, that, in the dark, the nuclei of the rods in Triton (figs. 1 and 2, st.nJ.cx.) migrate partially through the external limiting membrane, thereby causing the whole rod to become extended, whereas, in the light, these nuclei lie wholly within the outer nuclear layer. The contractility of that portion of the
432
LESLIE B. AllET
rod-visual coll between the rod nucleus and the external reticular layer was believed to cause these changes. Angelucci ('90) made shnilar observations on the salamander, as did Garten ('07) on Triton.
Changes in the cylindrical outer member (figs. 1 and 2, prs.dst. bac.rb.) have also been reported. Ewald und Kiihne ('78) first observed a swelling of the outer member of the frog's rod as the result of strong illumination. That Lederer ('08) obtained
prs. dst. bac. vr
prs. dst. con
-prs. dst. bac. rb.
mb. Iim. ex<
Fig. 1 From a dark-adapted retina of tlie frog, Rana pipiens, showing the positions assumed by the rods and cones. X 1130. ell. bac.rb., ellipsoid of red rod; ell.bac.vr., ellipsoid of green rod; ell.con., cone ellipsoid; gtt.ol., oil globule; mb.lim.ex., externa limiting membrane; my.bac.rb., myoid of red rod; my.bac.vr., myoid of green rod; my.con., cone myoid; prs. dst. bac.rb., outer member of red rod; prs.dst.bac.vr., outer member of green rod; prs.dst.con., outer member of cone; ^t.nl.ex. external nuclear layer.
results, the exact opposite of those reported by Ewald and Kiihne, is assumed from the context of the previously cited quotation (p. 431), and from his illustrations.
Angelucci ('84) measured the length of the outer member of the frog's rod and found it shortens in the hght; later ('90) he confirmed this result by measurements of the large rods of the
CHANGES IN THE VISUAL CELLS OF THE FROG
433
salamander where the differences in length were more striking. Arcoleo ('90) and Garten ('07) reported similar conditions for the toad and frog respectively.
In most accounts oif the changes occurring in the anuran rod, due to the action of light, reference is not made to the number of individuals experimented upon, and in no case are definite measurements of length given, judgment of the eye apparently being the only critorion adopted.
prs. dst. bac. rb.
Fifj;. 2 Fri)in a li^lit-:ulai)t(Ml retina of the frojr, Hana pipicns, showiiiK
positions assunicil Ky tlie rods ami eoncs. X 1130.
In the experimentation which forms the basis of this paper, an attempt has been made to correlate an influence of light with the following possible changes in the frog's visual rod: 1) changes in the position of the red rod nucleus with respect to the external hmiting membrane; 2) changes in the length of the red and green rod myoids; 3) relati\'e changes in the length of the red rod myoid at the center and at the perij^hery of the retina; 4) changes in the diameter of the various comi)()nent parts of the red rod.
434 LESLIE B. AREY
KXPKIUMKNTATION
Individuals of Hiina pipiens, ai)])r()xiinately uniform in size, were exposed to ])right diffuse daylight for eight hours, or to total darkness for a minimum length of twenty-eight hours. At the expiration of these periods of light and dark-adaption, the cranium of each animal was first split sagit tally, and then cut transversely just caudad to the eyes. The resulting moieties of the cranium, with the contained eyes, were dropped into Perenyi's chromo-nitric fixative; in this solution they underwent fixation, the condition of illumination being identical with that to which they had been experimentally subjected. The operation on dark-adapted animals was accomplished by the light from a photographic red lamp and demanded but a few seconds time.
After dehydration and the removal of the lens, the eyes were imbedded in paraffine, sectioned 8m thick, and stained with Ehrlich-Biondi's acid fuch^in-orange G-methyl green mixture. In light-adapted retinas the expanded pigment masked the visual cells to a greater or less degree, hence, nascent oxygen was used as a bleaching agent in these preparations. The results obtained with the Ehrlich-Biondi stain were very satisfactory, yet it is of interest to note that the staining reaction of several definite structures was often variable. For example, the outer member of the red rod in most cases stained red with the acid fuchsin, yet in some retinas from the same series they were colored an intense orange from the orange G, although the treatment in both cases had been, so far as is known, identical, the series having been carried along simultaneously step by step. Moreover, in one preparation, at least, it was observed that the outer members in one half of the retina were stained orange, whereas in the other half they were stained red. This selective stainability is doubtless indicative of a variable physiological cytoplasmic state. Preparations in which the outer members of red rods selected the orange G, showed the green rods stained red with the acid fuchsin. In retinas that had been bleached (potassium chlorate and hydrochloric acid being the reagents
CHANGES IX THE VISUAL CELLS OF THE FROG 435
used), the rod outer member varied in color from blue-violet to red-violet. Cases involving a somewhat similar variability might be cited with respect to several other structures in the retina.
Experimentation was made upon sixty retinas, from which twenty-three light-adapted and twenty-three dark-adapted preparations were selected for measurement. Preparations, in which the external limiting membrane was not apparent, or in which wrinkles caused oblique sections, were rejected. All measurements were made with a Leitz 1 12 homogeneous hnmersion ol)jective and a Zeiss Xo. 2 micrometer eyepiece.
A. Influence of ligJit on (lie position of the red rod nucleus
'riic object of this series of measiu'cments was to determine the effect of light and darkness u])on the ]iosition of the imclei of the red rods with respect to the external limiting membrane.
The nuclei of the rod- and cone-visual cells comprise the external nuclear layer (figs. 1 and 2, st.nl.ex.) of the retina. According to Greeff ('00, p. 133), these two kinds of nuclei in am]ihibians can be distinguished, morphologically, only with great difficulty; hence it is essential to inciuire whether any criterion exists whereby the rod nuclei can be identified witli a tolerable degi'ee of certainty. The illustrations of the frog's retina given by Clreeff ('00, pp. 96, 102) represent the nuclei of the red rod-visual cells as lying considerably nearer the external hmiting membrane than the nuclei of either the green rod-visual cells oi- of the conevisual cells. If. therefore, in any preparation attention be directed to the nuclei which protrude farthest beyond the external limiting membrane, one may be reasonably sure that the nuclei of the red rods only are under consideration. Ten light-adapted and ten dark-adapted retinas were selected at random for measurement. Not only was considerable regional ^'ariability in the position of nuclei found in indi\idual retinas, but also the variability in any limited area was extensive — ranging from nuclei whose edges were at the same level as the external limiting membrane to nuclei which were 3.0 ij. above that membrane. Partly for this reason, and partly to be certain that only the
436 LESI.IK 15. AliKV
lUR'loi of I'od roils were under observation, attention was directed solely to the mean distance to which the maximally extended niielci protruded beyond the external limiting membrane.
The following values (grand means) were obtained: in light (10 retinas), 2.9m; in darkness (10 retinas), 2.2fx.
On account of the degree of variability observed, I do not regard this slight difference, which, incidentally, is not in agreement with the results of Stort ('87) and Garten ('07) on Triton, or of Angelucci ('90) on the salamander, as indicative of a photic influence; Since the condition recorded in Triton and the salamander is not clearly demonstrable in my preparations of the frog, it follows that whatever changes are found in the rod myoid will be due chiefly to the activity of the myoid itself.
B. Influence of light on the length of the rod myoid. Relative changes at center and periphery of retina
a. Red Rods. In every retina, at least ten measurements were made, on each side of, and not far from, the optic nerve. The averages of these twenty or more values are given in tables 1 and 2 as central measurements. Similarly the means of at least twenty measurements (ten on each side), made well toward the periphery of the retina, are recorded as peripheral measurements. In order to avoid unconscious selection all measurements were made on consecutively-placed rods. The myoid length in the tables constitutes the distance from the external hmiting membrane to the nearer edge of the rod ellipsoid (figs. 1 and 2, 7ny. hac.rh.).
The tables show that there is considerable variation in individual retinas, yet the rod myoid unmistakably elongates in the hght and shortens in darkness (figs. 1 and 2). Although the mean values, at the center and periphery of individual retinas, may also vary greatly in either set, the grand means are practically identical.
The measurements of individual light- and dark-adapted rods overlap in many instances; furthermore, the mean for certain groups of ten deviates considerably from the average for both groups of ten, which constitutes the central or peripheral values
CHANGES IN THE VISUAL CELLS OF THE FROG
437
of the tables, as the case may be. In such instances the process of a\'eraging two groups of ten serves to mask this condition, hence the tables, for the sake of compactness, are faulty in this respect.
The conclusion follows, therefore, that the photomechanical responses of the frog's red visual-rod myoid arc in agreement with those of other vertebrates in which changes have been demonstrated.
The older workers (Angelucci '84: '90; CIradenigro, 'So: and Arcoleo '90) perhajis erred, either in not making actual measure
Meafsureinenl.s from lircidii-thrce dark-adapted nliiias of liann }ti/)icn.-^. The
values are in micra and represent measurements taken along axes coinciding
with radii of the eyeball. Each value for the length of the red rod myoid is the
mean obtained from twenty consecutively-placed elements
NITMDER OF ANIMAL
NERVE FIBER
LAYER TO
EXTERNAL LIMITINi;
MEMBRANE
CHORIOID TO
EXTERNAL LIMITING
MEMBRANE
LENGTH OF
RED ROD MYOID
AT CENTER OP
RETINA
LENGTH OF
RED ROD MYOID
AT PERIPHERY
OF RETINA
1
107
04
5.0
4.9
2
\:u\
70
4.0
5.7
3
114
72
4.6
3.3
4
114
04
4.4
4.9
5
122
m
6.6
7
6
110
C!)
5.3
6.7
7
100
(•>4
4.0
6.4
8
10(i
(')4
6.7
6.6
9
12<»
07
6.4
6 4
10
100
()9
9.3
7.6
11
114
69
6.b
6.9
12
12!t
09
5.9
5.7
V.i
m
73
6.2
5.7
14
',17
()2
3.6
4.3
15
07
03
4.7
5.0
16
111
69
4.6
5.1
17
107
63
5.4
6.0
IS
107
64
6.4
6.2
19
114
63
6.6
6.4
20
103
60
7.2
6.9
21
89
57
7.2
6.3
22
93
62
5.7
7.2
23
93
60
6.4
6.2
Mean
109
66
5.8
6.0
438
LESLIE B. AREY
iiHMits, or wcic influenced by their observations on the more strikingly mobile cone (figs. 1 and 2; my. con.), the movements of wliich are the reverse of those exhibited (such is my beUef) by mobile rods in general.
In recent papers ('15; '16) the writer favored the anomalous photomechanical responses of the frog's rod, reported by the several older workers, as being more trustworthy than the somewhat confusing account of Lederer ('08). The writer ('15),
TABLE 2 Measuremefds from Lwenly-three light-adapted retinas of Raiia pipiens. The values are in micra and represent measurements taken along axes coinciding with radii of the eyeball. Each value for the length of the rod rod myoid is the mean obtained from twenty consecutively-placed elements
NUMBER OF ANIMAL
NERVE FIBER
LAVER TO
EXTERNAL LIMITING
MEMBRANE
CHORIOID TO
EXTERNAL LIMITING
MEMBRANE
LENGTH OF
RED ROD MYOID
AT CENTER OF
RETINA
LENGTH OF
RED ROD MYOID
AT PERIPHERY
OF RETINA
1
100
67
9.9
10.2
2
111
73
13.6
14.9
3
93
74
11.9
12.0
4
129
62
11.7
12.6
5
117
72
11.9
12.6
6
114
72
12.6
11.9
7
114
64
13.3
12.2
8
127
64
9.2
12.2
9
127
69
8.6
10.3
10
119
72
16.9
16.2
11
114
72
13.9
14.7
12
120
72
14.3
14.7
13
92
62
9.9
9.2
14
102
69
13.4
13.0
15
107
60
13.0
11.9
16
125
63
9.6
9.2
17
100
62
11.4
12.4
18
106
62
9.9
9.6
19
93
61
8.6
7.9
20
96
69
12 7
9.6
21
107
64
7.9
7.2
22
97
64
8.6
9.7
23
129
69
11.9
12.9
Mean
100
67
11.5
11.6
3 Lederer s figures of isolated rods do not necessarily show how long he rod myoid really was, either in darkness or in light. Presumably, the myoid, as the result of teasing, broke approximately at the level of the external limiting
CHANGES IN THE VISUAL CELLS OF THE FROG
439
furthermore, made use of these data in an argument against the feasibihty of attempting to advance (in the hght of our present knowledge)' a single rational explanation for the diverse photomechanical movements of the \dsual rods. It is evident, however, that the conclusion reached in the present investigation renders this particular objection invalid.
It is reasonable to expect, although material is not available at present to put the matter to an experimental test, that the photomechanical behavior of the toad's visual rod will be found to vary in no essential detail from that herein described for the frog.
b. Green rods. From the sixty retinas experimented upon, the ten light-adapted and ten dark-adapted preparations which showed the most perfect histological preservation were selected for measurement. In each retina, measurements were made of the myoid length (figs. 1 and 2, my.bac.vr.) of ten consecutivelyplaced green rods; the results are recorded in tables 3 and 4.
TABLE 3 Measurements from ten dark-adapted retinas of Rana pipiens. The values arc in micra and represent measurements taken along axes eoinciding tcith radii of the eyeball. Each value for the length of the green rod myoid is the mean obtained from ten consecutively-placed elements
NUMBER OF A.NIMA.L
NERVE FIBER LAYER
TO EXTERNAL LIMITING
MEMBRANE
CBORIOID TO
EXTERNAL LIMITING
MEMBRANE
LENGTH OF
GREEN ROD MYOID
1
2
3
4 5 6 7 8 9 10
95
94
98 90 112 135 102 109 105 115
66
65
75 69 68 82 63 70 70 72
21.4
23.0
28.6
26.2
22.5
27.7
24.4
24.0
24.1
22.5
106
70
24.4
membrane, although nothing to this effect is stated Furthermore, the varicose and atypical appearing rods naturally increases one's caution in accepting his conclusions. Although Loderer gave but little emphasis to his observations on sectioned material (p. 431). I believe that those observations constitute the strongest evidence in support of his thesis.
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL.
440
LESLIE B. AREY
TABLE 4
Measurements from ten Uq hi -adapted retinas of Rana pipiens. The values are in micrn and represent measurements taken along axes coinciding with radii of the eyeball. Each value for the length of the green rod myoid is the mean obtained from ten consecutively-placed elements
NUMBER OF ANIMAL
NERVE FIBER LAYER
TO EXTERNAL LIMITING
MEMBRANE
CHORIOID TO
EXTERNAL LIMITIN-J
MEMBRANE
LENGTH OP
GREEN ROD MYOID
1
2
3
4 5 6
7
8
9
10
105
102
127
120
97
112
127
120
127
123
68
72
75
69
68
63
87
75
72
66
25.0
26.1
28.2 28.2 31.0 28.2 25.5 27.7 32.4 24.9
Mean
116
72
27.7
The tables show that although the difference in the length of the rod myoid, in darkness and in light, is small, the length of the light-adapted element is quite consistently the greater (figs. 1 and 2); hence it seems probable that this difference is significant and the photomechanical responses of the red and green rods are analogous.
This conclusion is not in accord with the results of Angelucci ('94). Angelucci believed that the red and green rod myoids responded similarly to photic stimulation, yet in both cases a shortening was said to take place.
C. Influence of light on the diameter of the red rod
From my preparations many rods were measured, yet no constant or significant differences were found in the diameters of the outer members (figs. 1 and 2, prs.dst.bac.rb.), or of the ellipsoids (figs. 1 and 2, ell.bac.rb.) of the inner members, which could be attributed to the influence of light and darkness. The myoid of the inner member (figs. 1 and 2, my.bac.rb.) naturally becomes tenuous when elongated by the action of light (p. 436).
One is inclined to question the morphological normaUty of the teased rods figured by Lederer ('08), especially since the appear
CHANGES IN THE VISUAL CELLS OF THE FROG 441
ance of these elements in his own sectioned material was dissimilar. In the figures corresponding to the text descriptions of dark-adapted rods (the figures and their appended descriptions are apparently interchanged in his paper), the varicose outer members are t\\dce, and the ellipsoids three to four times, as broad as the corresponding parts of the light-adapted rods.
My observations, therefore, are opposed both to those of Lederer ('08) who believed that darkness causes the rod to swell, and to those of Ewald and Kiihne ('78), who recorded that light acts in this manner.
SUMMARY
1. Distinct movements of the nuclei of the red rod-visual cells, due to photic stimulation, are not demonstrable. Hence movements of the rods are not produced indirectly in this way.
2. The myoid of the rod-visual cell elongates in light and shortens in darkness. Therefore, contrary to the conclusions of the older workers, the photomechanical response of the frog's rod myoid is found to be similar to that occurring in the retinas of all other investigated vertebrates.
The mean length of aj^proximately 1000 myoids from 23 light-adapted retinas is ll.G m The mean length of approximately 1000 myoids from 23 darkadapted retinas is 5.9 fx.
3. A significant regional difference in the length of the redvisual rod myoid is not api^arent from comparisons of approximately 2000 measurements made at the center and the periphery of light- and dark-adapted retinas.
4. The myoid of the green visual rod probably elongates slightly in the light and shortens in darkness.
The mean length of 100 myoids from 10 light-adapted retinas is 27.7 fji.
The mean length of 100 myoids from 10 dark-adapted retinas is 24.4 fjL.
5. Definite changes in the diameter of the outer member, or of the ellipsoid, of the red visual-rod can not be correlated with photic influences. The rod mj^oid, however, does become tenuous in the light.
442 LESLIE B. AREY
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und der Glasskorpers. Graefe-Saemisch, Handbuch der gesam. Augenheilkunde, Leipzig, Aufl. 1, Bd. 1, Teil 1, pp. 321-456. Stort, a. G. H. VAN Genderen 1886 tjber Form und Ortsveranderungen
der Elemente in der Sehzellenschicht nach Beleuchtung. Bericht
liber d. 18. Versamm. d. Ophthal. Gesell. zu Heidelberg., pp. 43-49.
1887 Mouvements des elements de la retine sous 1' influence de la
lumiere. Arch, neerlandaises des Sciences exact et naturelles, publ.
p. 1. Soc. hoUand. des Sciences, Tom. 21, Livr. 4, pp. 316-386.
A PRELLAIINARY DETERMIXATIOX OF THE PART PLAYED BY MYELIN IX REDUCIXG THE WATER COXTEXT OF THE MAMMALIAN NERVOUS SYSTEIVI (ALBINO RAT)
H. H. DONALDSON From The Wislar Institute of Anatomy and Biology
ONE CHART
It is a familiiir fact that there is a progressive loss of water in the brain and spinal cord with advanchig age. This is illustrated for the albino rat in Chart 1.
f^SiBSii^
rrn^-t:
T tr^iiu:::.'!:: v. :; ir :tr:jirn trr::: : it:
-n^
JX
Chart 1 Showing the percentage of water on age in the central nervous system of the alljino rat. The upper graph gives the values for the water in the brain as determined by the formuhis (Hatai. in 'The Rat,' Donaldson, '15). The lower graph gives the corresponding values for the spinal cord, determined in the same way.
The small black dots indicate the observed values for the several age groupa for the brain, and form the data for the formulas. The small black triangles have a like value in relation to the spinal cord.
443
444 H. H. DONALDSON
It is also well known that in some mammals all the axons in the central nervous system are unmyelinated at birth, while in other species a greater or smaller number of them may already have their sheaths. In all cases, however, an active formation of myelin occurs during the period of rapid growth (Koch and Koch '13).
In a previous study on the percentage of water in the albino rat I was misled by certain graphs (Donaldson '10) to the conclusion that probably both the axons and their myelin sheaths changed in their water content so as to produce the well known reduction of water which is observed, but further study has shown that this is an incorrect view, and it is the object of this paper to present the evidence of my revised conclusion.
Since it has already been shown that the loss of water in the human brain follows the same course as in the brain of the albino rat — and has similar limits (Donaldson '10) — it is permissible to use in the argument certain observations on the human brain.
From the data for the human brain already in the literature I have selected those published by de Regibus ('84), because this author evidently examined only the outer layers of the cortex when making his determinations for the water in the gray substance and because he was able also to obtain remarkably uniform results for all of his determinations.
De Regibus tested four male brains, 25 to 76 years of age and three female brains, 30 to 60 years of age.
In Table 1 there appear also determinations of the water content in the human cortex and in the fibers at birth. These are based on the records of Weisbach ('68).
This enables us to contrast in Table 1 the conditions at birth with those at maturity.
TABLE 1
Percentage of water in the gray and white substance of the human brain at birth and
at maturity
CORTEX (gray)
At birth (Weisbach)
At matoirity (de Regibus)
per cent
88
per cent
88 70.4
WATER CONTENT — MAMMALIAN NERVOUS SYSTEM 445
According to this table the (gray) cortex has lost 2 points and the (white) callosum 17.6 points in the process of maturing.
It is never possible at maturity to obtain the cortex or any other gray mass without some admixture of myelinated fibers and I have therefore, provisionally, credited one point of the loss, noted by de Regibus in the water content of the cortex, to the presence of myelin. This implies but a small proportion of myelin since if we assume that myelin has 48 per cent of water the reduction of 1 per cent would mean that about one thirtyninth of the mass was represented by myelin. According to this assumption the mature gray substance (cortex) — when the myelin is excluded — contains 87 per cent of water, and in the computations which follow the neurons without myelin are assumed to have 87 per cent of water, except at ten days, when they are credited with 88 per cent.
The fact that the fibers without myelin have at birth a high percentage of water (88%) while at maturity, after myolination, they have lost 17.6 points is the sort of evidence which furnishes the basis for the current, but unsupported view, that the loss of water is to be associated with the formation of the myelin. It is our object to present more precise information bearing on this point.
To obtain a notion of the apjiroxunato distribution of the water between the myelin and neiu'ons proper, it is necessary to have data on the relative abundance of these two constituents of the brain.
In 1913 W. Koch and ]\I. L. Koch made a study of the chemical composition of the brain of the albino rat at six ages between birth and maturity, and of the spinal cord at one age. The data thus obtained are those which will be utilized here. The authors determined seven fractions: Proteins, organic extractives and inorganic constituents, which three taken together, we shall designate protein (or non-lipoid), and phosphatides, cerebrosides, sulphatides and cholesterol, which four taken together, we shall designate lipoid.
These data give us at each age, therefore, the protein and the lipoid present ui the brain, or to be a little more exact, we should
446
H. H. DONALDSON
say tlio lii)oicl uiul the non-lipoid fractions. The lipoid (in part) represents the myeUn sheaths, while the protein, with the remainder of the lipoid, represents the cell bodies and their unsheathed axons.
With the exception of the one day group, the ages for which analyses were made, are given in table 2.
TABLE 2 To show, fur Ihc brain of the albino rat at five ages and for the spinal cord at one age the percentage of water in the myelin as computed according to the method described. The protein and lipoid are given in percentages of the total dry substance. (Based on table 2. Koch and Koch '13)
Brain
(1)
(2)
CORRECTED PROTEIN
(3)
CORRECTED LIPOID
ii)
PROPORTION
OF LIPOID FOR
DIFFERENT
AGES.
LIPOID AT 20
DATS = 1
(5) (6)
PERCENTAGE OF WATER
(7)
WATER.
DAYS
Entire
brain
observed
In Neurons
= protein
(C) (assumed)
LIPOIDS (C)
(COMPUTED)
(See p. 447)
10
per cent
93.80
per cent
6.2
86.5
88
63.81
20
40
120
210
88.88
82.86
75.11
76.36
11.12
17.14
24.84
23.63
1.0
1.5
2.2 2.1
82.5
79.4
78.4 78.1
87
87
87
87
46.542.7
52.4
47.4
Average of 20 to 210
47.8
Spinal cord
120
52.92
47.08
4.2
70.4
87
51.0
1 First traces of myelin. - Myelin well shown.
At one day — or practically birth — it is found that the lipoid is present to the extent of 0,31 or nearly one-third of the weight of the protein. There is, however, no visible myelin at this age, so it is concluded that this proportion of the total lipoid is normally associated with the protein and is not to be included in the lipoid which forms the myelin sheaths. We have treated the data for the later age groups in accordance with this relation, and in each case have taken from the total lipoid found an
WATER CONTENT — MAMMALIAN NERVOUS SYSTEM 447
amount equal to 0.31 of the protein found. The remammg amount of hpoid is assumed to be that used for the sheaths.
In table 2, the column (2) headed Corrected Protein gives the observed protein (non-lipoid) plus 0.31 of itself and the column (3) headed Corrected Lipoid gives the observ^ed lipoid less the amount of lipoid added to the protein.
In table 2 the data are given in five age groups for the brain and in one age group for the spinal cord. It is to be noted that the 10 day brain group — which stands just at the beginning of the myelin formation — is for the moment excluded from the discussion and we begin the comparisons which are to be made, with the 20 day brain group.
In the brain series {with, one cxco])tion) the corrected protein dhninishes and the corrected lipoid increases with advancing age. Between 20 and 210 days the proportion of the lipoid doubles — column (4). We have in column (5) the observed percentage of water in the brain as a whole. It is assumed, as previously noted, that the corrected ])rotein (neurons in the strict sense = both cell bodies and axons) have 87 per cent of water. From those several data we can cominite the ]iercentage of water to be assigned to tlie corrected lipoid, which re])resonts the myelin.^
The method of computation may be illustrated by the data for the 20 day group. Reference to table 2 shows that at this age there is 1 part of lipoid (11.12<;^o) to 8 parts of protein (SS.SS^c^. This gives 9 parts, representing the entire brain and having S2.5 per cent of water. The product, 9 X 82.5 = 742.5. We assume that the 8 parts of protein have 87 per cent of water. The product, 8 X 87 = 696. The 1 part of lipoid, representing the myelin, will then have a percentage of water equal to the difference of these products = 742.5 - 696 = 46.5 per cent.
'It is hardly necessary to point out that a division of the brain into mj'elin on the one hand and neurons on the other fails to enumerate several structural elements which are also present though representing a relatively small mass. There are, in addition to the neurons, glia and ependjnnal cells; blood and lymph vessels; blood and Ij-niph and a slight amount of connective tissue. Over against the neurons plus this group of elements we put the myelin, but for the purposes of the present discussion it is convenient to speak of the rest — the non-myelin portion — of the brain as if represented by the neurons alone.
448 H. H. DONALDSON
As table 2 shows, similar computations give percentages of water for the myelin in the braiii, which range from 42.7 per cent to 52.4 per cent and which yield a mean value of 47.8 per cent. The computation for the spinal cord gives 51 per cent. The significance of these results lies not in the particular percentage of water here determined for the myelin — as that depends somewhat on the percentage of water assumed for the protein — but on the similarity of the values found in all the five cases examined.
However, it is found on trial that one cannot depart far from the value of 87 per cent for the proteins without obtaining rather improbable percentages for the myelin, so that this value is probably nearly right.
DISCUSSION
Those familiar with the pubhshed data for the percentage of water in the cortex will at once perceive that the value given by de Regibus (86%) seems high. The various water records for the cortex run down as low as 83.5 per cent. The differences between the various determinations are, how^ever, almost certainly due to the varying amounts of white substance included in the sample, and as has already been stated the value chosen is probably close to the true value.
In connection with the computations there are, however, two conditions which have been assumed to be constant but which in all probability, are subject to variation. I refer to the density of the myelin and to the fraction of the hpoid to be assigned to the protein. As to this last condition, it would be plausible to think of a larger fraction of lipoid in the axons than in the cell bodies. If this were true it would be necessary to increase this fraction in the case of the spinal cord or the callosum.
It is also possible that aside from this method of distribution the fraction of lipoid in the neuron may increase with age. The slight loss of water during the first ten days of (rat) life is possibl}^ due to such an increase. Finally, the density of the myelin may change with age, as its chemical composition certainly does, and it is conceivable that it has a higher water content when first formed, as is suggested by the 63.8 per cent given for the ten day record in table 2.
WATER CONTENT — MAMMALIAN NERVOUS SYSTEM 449
If we compare the drawings of Watson ('03), showing the increase of the visible m^-eUn in the cerebral hemispheres and in the spinal cord of the rat, with the chemical results here used, we see that the histological pictures show a more gradual appearance of myelin than the chemical results, or the water determinations, would suggest. This probably depends on the fact that it is only a fraction of the lipoids forming the sheaths, which takes the haematoxylin stain, and -this stainable fraction forms at first a smaller, but later a larger portion of the entire sheath (Koch and Koch '13; Smith and Mair '08).
There is still one more modification in the formation of myelin. Tribot ('05) has contrasted in terms of diy substance the relati^'e amounts of albuminoides and the fats in the nerv-e tissue of the guinea-pig at tlifferent ages. The percentage value of the fats increases from 11 days (his first observation) up to 120 days — after which it begins to fall. The fats of Tribot are the lipoids of Koch's analysis and it is of interest to note that the 120 day record for the brain in table 2, column (4), also shows the highest proportion of corrected lipoids. The observations of Dunn ('12) which show in the myelinated fibers of the second cer\'ical nerve of the rat the highest relati\e areas for the myelin sheaths at 75 days and 132 daj's — seem to fit with these other observations and to suggest that the fonnation of lipoids with advancing age fluctuates in such a way as to show a maximum about the end of the active growing period of the central nervous system.
This discussion of possible factors modifying these determinations has been introduced to clear the way for further work on the main question, but so far as one can foresee the effect of taking them into consideration, it would tiMul to make more uniform the values thus far obtained.
CONCLUSIONS
We conclude from these results that there is no evidence that the cell bodies and their unsheathed axons suffer more than a slight loss of water between birth and maturity, and that the progressive diminution in the water content of the entire brain and spinal cord is mainly due to the accumulation of myelin.
450 H. H. DONALDSON
Avith a water content of about 48 per cent. Moreover, the myelin must be regarded as a more or less extraneous substance, having but little significance for the characteristic activities of the neurons.
If we compare the loss of water in the case of the nervous system with that in the muscular system, which also contains a large proportion of fat (Tribot '05), we find that while the two systems lose about the same percentage of water between birth and maturity (Lowrey '13), yet in the case of the nervous system alone is this lipoid (or non-protein substance) accumulated outside of the cell. From this it is seen that the neuron is peculiarly able to maintain its early water-solids composition and that it accomplishes this by throwing out the material, which in the muscles is retained within the cells.
As the diminution in the percentage of water in the central nervous system is preeminently a function of age, and as it appears to be due almost entirely to the formation of the myelin, it follows that the myelin formation is also a function of age (Donaldson '11). A glance at the graphs in Chart 1 shows that the most active production of myelin, as indicated by the rapid loss in the percentage of water, occurs early, i.e., during the first forty days of rat life, in the brain, and during the first hundred days, in the spinal cord.
WATER CONTENT — MAMMALIAN NERVOUS SYSTEM 451
LITERATURE CITED
Donaldson, H. H. 1910 On the percentage of water in the brain and in the
spinal cord of the albino rat. Jour. Comp. Neur., vol. 20, no. 2, pp.
119-144.
1911 The effect of underfeeding on the percentage of water, on the
ether-alcohol extract, and on meduUation in the central nervous
system of the albino rat. Jour. Comp. Neur., vol. 21, no. 2, pp. 139-145.
1915 The Rat. Memoirs of the Wistar Institute of Anatomy and
Biology, no. 6, pp. 1-278. Dunn, E. H. 1912 The influence of age, sex, weight and relationship upon the
number of medullated nerve fibers and on the^size of the largest fibers
in the ventral root of the second cervical nerve of the albino rat.
Jour. Comp. Neur., vol. 22, no. 2, pp. 131-157. Koch, W. and Koch, M. L. 1913 Contributions to the chemical differentiation
of the central nervous system. III. The chemical differentiation of
the brain of the albino rat during growth. J. of Biol. Chemistry, vol.
15, no. 3, pp. 423-448. LowREY, L. G. 1913 The growth of dry substance in the albino rat. .\nat.
Rec, vol. 7, pp. 143-168. DE Regibu-s, C. 1884 Determinazione dell' acqua contcnuata nellc sostanze
grigia e bianca del cervello umano. Atti. d. reale Accad. di Med.
di Torino, vol. 6, pp. 323-32S. Smith, J. L. and Mair, W. 1908 An investigation of the principles underlying
Weigert's method of staining medullated nerve. J. of Pathology and
Bacteriology, vol. 13, pp. 14-27. Tribot, J. 1905 Sur les chaleurs de combustion et composition chimiqiio dcs
tissus nerveux et musculairc chez le cobaye, en fonction de I'age.
C. R. Acad. Sc. Paris, T. 140, pp. 1565-1566. Watson, J. B. 1903 Animal education. Contributions from the Psychol.
Lab. of the University of Chicago, vol. 4, no. 2. Univ. of Chicago
Press, Chicago, 111. Weisbach, a. 1868 Der Wasscrgchalt des Gehirns nach Alter, Geschlecht und
Krankheiten. Med. Jahrbiicher, vol. 16, nos. 4 and 5, pp. 1-76.
THE TASTE OF ACIDS^
W. J. CROZIER
TWO FIGURES
I. The problem of irritability is essentially a matter concerning sense organs; in these structures we find cells "as nearly as possible unifunctional" fLucas, '09, p. 328) with respect to the capacity for being stimulated. Hence the analysis of irritability should properly be founded u]i()n the study of receptor physiology in higher animals, rather than upon an examination of the properties of protoplasm in deceptively simple protozoans.
Two contrasted methods have in the past been used in the study of stimulation. The first of these deals with the responses of unicellular organisms, together with the (usually vague) application of principles derived from cells of more or less generahzed type. The second method has consisted in the analysis of sensations. Yet, if we consider such questions as those involved in taste stimulation, we find that no definite conclusions have been reached. The very complexity of organization in 'simple' forms is probably in the main responsible for the results obtained in the first field, results which we find stated in such weak generalizations as occur in Verworn's book ('13, pp. 41, 86, 94). The second procedure, the study of human sense organs, has ine\itably come to be clouded by psychological interpretations.
It appears that the possibility of arriving at some understanding of what occurs in stimulation has not yet been sufficiently tested out from the standpoint of sense organ irritability as revealed in animal reactions. This paper is a contribution toward that end; it deals with the problem presented by the taste of acids. I am indebted to Prof. G. H. Parker for his criticism of the manuscript.
^ Contributions from the Bermudii Biological Station for Research, Xo. 46.
453
454 W. J. CROZIER
II. Practically the whole of the difficulty is contained in the often considered case of acetic and hydrochloric acids. A comparative study of the reactions to these and other acids as given by the earthworm (Hurwitz, '10; Crozier, '16), leads to a preliminary simplification of the matter, namely to the formulation of two separate problems: these acids stimulate, they also result in a sour taste. These two problems must be considered independently; we must distinguish between (a) that property of an acid which causes it to be efficient in stimulation and (b) that which determines the sourness of an acid solution.
The relative efficiency of an acid as an agent of stimulation may be measured by the liminal effective dilution. The point I wish to make is that the property of hydrochloric acid which causes it to be tasted in solutions about five times as dilute as that limiting the tastability of acetic acid may have no immediate connection with the common cause of the sourness perceived when these acids act upon the tongue.
This point of view contains the possibility of an explanation for (and is, reciprocally, in part justified by) the fact that acids exhibit a characteristic astringency in solutions so dilute that they are no longer sour. There is some evidence, also, that the sourness and astringency may be separated experimentally, as by the use of cocaine. There is therefore ground for the opinion that in taste excitation by acids two processes occur, and that the production of a sour taste is the secondary one.
The points which require solution with respect to human taste excitation by acetic and hydrochloric acids are:
(a) Both these substances stimulate.
(b) They stimulate in different degrees, in dilute solutions the stimulus from hydrochloric acid being the more intense.
(c) They both result in a sour taste.
(d) They fail to stimulate when too greatly diluted.
(e) The dilutions which limit the capacity of the two acids to stimulate are very different (HCl n/900 ± ; acetic, n/200 ±).
Each of these points — except (c) — is closely paralleled by the details of earthworm reactions to acids (Crozier, '16), and also
THE TASTE OF ACIDS 455
by the reactions of ^olosoma, a freshwater oligochaet (Kribs, '10).
It adds somewhat to the clearness of the discussion if, as I have previously proposed (Crozier, '14, p. 16), we restrict the word 'stimulus' to mean the change induced in a receptor by the action of a stimulating agent. The explanation of the acid taste is made easier by the fact that one is not called upon to account for a sour taste resulting from heterologous stimulation; there is no good evidence for the existence of a sour taste not directly produced by acid. The extreme specialization of the acid taste makes it a very favorable case for analysis.
III. It is sufficiently obvious that only the surface of the receptor is immediately concerned in stimulation. This view is reasonable upon purely morphological grounds, such as the modifications of the exposed ends of sensory cells- and the relations of nerve fibrils to the surface of secondary sense cells. The way in which this conception of the cell surface as the organ of irritability has been elaborated by R, S. Lillie and others need not be discussed here. But it may be pointed out that the method of interpreting mechanical excitation has been indicated by Osterhout ('15) and by such observations as that of Evans and Winternitz (Evans and Schulemann, '14, p. 453). Unequivocal evidence in this direction is afforded by Harvey's work on the penetration of cells by alkalies, (Harvey, '14 c), by his experiments with acids (Harvey, '14 c), and by my own stud}' of this latter subject (Crozier, '15, '16); the evidence referred to is, in brief, to the effect that penetration of the body of the cell substance is altogether too slow a process. to account for the rapidity of taste stinuilation. The slowness of acid penetration is tlue to the resistance offered by the cell surface, a resistance which practically disappears with the death of the cell. An examination of cell penetration by various acids, employing a wide range of dilutions, should therefore yield valuable information regarding the composition and behavior of the resistant cell surface. In several cases a study of this
2 The propable chemoreceptors of the earthworm have been described by Miss Langdon ('95) and Bovard ('04).
THE JOURNAL OF COMPARATIVE NEIROLOGY, VOL. 26, NO. 4
456
W. J. CKOZIER
kind has been made possible by the discovery of intracellular indicators sensitive toward acids (Harvey, '14 b, Crozier, '15). Measurements of cell p.enetration by acids show, so far as concerns the present problem: 1) that both acetic and hydrochloric acids are able to penetrate the cell; 2) other things being
100
ZOO 300 400 500 600
Dilution (Liters containing one equivalent)
Fig. 1 The average time of penetration of cells by hydrochloric, butyric, and acetic acids.
1, Acetic acid ]
3, Butyric acid \ integument of Chromodoris zebra, a nudibranch. 5, Hydrochloric acid
2, Butyric acid
4. Hydrocloric acid
1, 3, 5, from Crozier ('16), at 27°.
2, 4, from Harvey ('14 c), at 28°.
Butyric acid is introduced for comparison, since Harvey (loc. cit.) gives no data for dilutions of acetic; the principle is the same for both these acids. It should be noted that in Harvey's experiments the acid was dissolved in a balanced salt solution, in mine in distilled water.
testis epithelium of Stichopus ananus, a holothurij
THE TASTE OF ACIDS 457
equal, the speed of this penetration varies with the acid and its concentration; 3) the curves relating penetration time to concentration are different for the two acids, dilute solutions of hydrochloric penetrating much more easily than acetic. Points of resemblance, in fact of identity, are found among the characteristics of stimulation bj' these acids, and have been enumerated as a, b, e, in section 11. The closeness of the parallehsm indicates that the stimulus is due to the penetration of the surface layer of the cell. In stimulation by these acids the essential step is the union of the stimulating agent with some constituent of the ceptor surface.
According to Becker und Ilcrzog ('07), the decreasing order of intensity of taste sensation due to the acids they tested was HCl, HNO3, trichloroacetic, formic, lactic, acetic, butyric. These acids penetrate the indicator-containing cells of Chromodoris zebra in the order HCl, HXO3, formic, monochloroacetic, lactic, butyric, acetic (consiilering a range of concentrations; C'rozier, '16). There is reason to believe that trichloroacetic penetrates more speedily than monochloroacetic; and with this in mind, the parallelism between these two series becomes surprisingly close. This particular case is quoted with the purpose of pointing out that a small series of acids, such as that just cited, may to all ai^pearances support the view that the intensity of taste excitation is proportional to the ionization strength of the acids; similarly, a penetration series may be chosen which seems to prove a corresponding relation. Yet, further examination of a large number of acids shows that ionization is but one of the factors determining cell penetrating power, and the existence of influences in addition to ionization has also been suspected with reference to acid stimulation; direct evidence upon this point has hitherto been lacking.
IV. Analysis of penetration data obtained with eighteen acids has shown (Crozier, '16) that in any given case abihty to penetrate the cell appears to depend upon two (or more) factors: one is the ionization strength of the acid; the second depends upon some other aspect of the acid molecule. To account for these results it is further necessary to assume that the cell surface is
458
W. J. CROZIER
of heterogeneous constitution. This conclusion may be extended to include the surface of chemoreceptors, since it enables us to account not only for the reactions of the earthworm to dilutions of acids (Crozier, '16), but also clears up a well known anomaly in regard to human taste.
It was shown by Richards ('98) and by Kahlenberg ('98) that the hydrogen ion muSt in some way be responsible for the sour taste. Yet acetic acid, which ceases to be tasted at dilutions
eo
70
So
g -90
tS 30
so
.0
(?>-J
5 10 90 95 100
Per cent ionization
Fig. 2 The relation between percentage ionization and time of cell penetration from solutions of hydrochloric, butyric, and acetic acids.
1, Acetic acid
3, Butyric acid \ integument of Chromodoris zebra. 5, Hydrochloric acid
2, Butyric acid
4, Hydrochloric acid /, 3, 5, from Crozier ('16). 2. A. from Harvey ('14c).
testis epithelium of Stichopus ananus.
THE TASTE OF ACIDS
459
below about n/200 is only 6 per cent dissociated at that concentration, whereas hydrochloric acid, fully dissociated (99 per cent), is tasted down to about n;900. The actual hydrogen ion concentrations in these solutions limiting tastability are: HCl (n 900), C„ = 0.00119 N; acetic (n '200), C, = 0.00035 N. Acetic acid is more stimulating than would be calculated from its dissociation.
Exactly similar conditions are fountl in studying the penetration of cells by acids, as will be clear from an inspection of the two sets of curves in figure 2. In table 1 acid solutions giving
The ionization of hydrochloric, acetic, and butyric acids, in solutions which penetrate cells within equal titnes. For sources of data see corresponding reference numbers in figure 2.
IONIZATION, PER CENT
OF PENETRATION,
MINUTES
Hydrochloric
Acetic
Butyric
15)
(41
111
131
12]
5
10
15
20
50
95.0
98.5
98. 8
99.0
94.5
96.7
97.4
97.5
2.1
3.0
4.0
2.0
3.3
4.0
5.5
2.0
2.1
2.5
2.6
3.5
the same penetration time appear in the same horizontal row; it is seen that the penetration of cells by acetic acid is much more efficient than if its dissociation were the deciding influence.
It follows that the stimulus produced by acetic acid is due, in the first place, to its union with the cell surface; this penetration of the plasma membrane is more efficient than if dissociation strength were the determining factor. The indication is that this acid dissolves in a fatty substance located at the cell surface (for further evidence, see Crozier, '16). The stimulus due to hydrochloric acid also depends upon its penetration of the cell surface, but there is some evidence that it involves proteins.
It is not clear to what extent stimulation by acetic and hydrochloric acids may be interpreted in terms of the effects which
460 W. J. CROZIER
these substances have upon surface colloidal conditions, and thus upon cell permeabiUty (Spaeth, '16). Herlitzka ('10) has discussed this question with reference to the taste of salts, but measurements of the effect of acid on permeability (Osterhout, '14) have not considered the possibiUty that characteristically different results may follow from the action of diverse acids; Osterhout's ('14) measurements deal only with hydrochloric acid.
V. There remains to be accounted for the production of a unitary taste quaUty, the sourness of acid solutions. This must be related to ionizable hydrogen (Kahlenberg, '98, Richards, '98). But it has just been shown that hydrogen ions outside the cell surface cannot be the effective agents, since stimulation by these acids parallels so closely the peculiarities of their penetration of the cell. The hydrogen ion must therefore act after the acid has united with the ceptor surface. At least two possibilities are obvious: (1) the presence of potentially ionizable hydrogen within sufficiently concentrated undissociated molecules is enough to produce the sour taste, or (2) the acids ionize secondarily after entering. The first suggestion is not so far fetched as it may at first seem, though it is not in accord with current teaching. Numerous parallels could be drawn from the taste of organic substances (Francis and Fortescue-Brickdale, '08, pp. 331 et seq.). I believe that this view is possibly correct, although something could also be said for the hypothesis of secondary ionization following some reaction involved in gaining admission to the outer layer of the cell. It is in this latter direction that the explanation (Beutner, '14) must be sought for the source of electromotive effects accompanying stimulation in general, which presumably occur also in taste excitation.' This interpretation disposes of the difficulty (very conspicuous in the case of taste) which is encountered by those who would make the process of stimulation and the production of electromotive effects completely identical in every case.
^ A taste cell bathed by a stimulating solution is in a condition very similar indeed to that of the various tissues experimented upon by Loeb and Beutner (Loeb. '15).
THE TASTE OF ACIDS 461
SUMMARY
An attempt has been made to account for the action of acetic and hydrochloric acids upon the sense of taste. It is shown that the stimulus due to these substances probably depends upon their union with diverse constituents of the ceptor surface. It is thus possible to explain, by comparison with the penetration of cells by these acids: how they stimulate at all, why they stimulate to different degrees, and why acetic acid gives a more powerful stimulus than if it acted by dissociation alone. WTiy these acids produce a unitary (sour) taste quahty is a problem of a different order; it is possible that this depends upon the presence of potentially ionizable hydrogen within undissociated acid molecules, though secondary ionization may also play a part.
agar's island
BERMUDA.
LITERATURE CITED
Becker, C. T. und Herzog, R. O. 1907 Zur Konntnis dcs Geschmackes. I. Mit teilung. Zcits. f. physiol. Chein., Bd. 52, p. 406-505. Beutner, R. 1914 Studies on a new kind of E. M. F. Jour. Amer. Chem.
Soc, vol. 36, p. 2040-2045. Bovard, J. F. 1904 The distribution of the sense organs in Microscolex elegans.
Univ. Calif. Publ., Zocil., vol. 1, p. 269-2S6. Crozier, \V. J. 1914 The orientation of a holothurian by light. Amer. Jour.
Physiol., vol. 36, p. 8-20.
1915 On cell penetration by acids. Science, X. S., vol. 42, p. 735-736.
1916 Cell penetration by acids. Jour. Biol. Chem., vol. 24, p. 2.55-279. Evans, H. M., and Schulemann, \V. 1914 The action of vital stains belonging to the benzidine group. Science, N. S., vol. 39, p. 443-454.
Francis, F., and Fortescue-Brickdale, J. M. 1908 The chemical basis
of pharmacology. London, 8°, xii + 372 pp. Harvey, E. N. 1914 a The relation between the rate of penetration of marine
tissues by alkali and the change in functional activity induced by the
alkali. Publ. Carnegie Instn. Wash., No. 183, p. 131-146.
1914 b Cell permeability for acids. Science, N. S., vol. 39, p. 947-949.
1914 c The permeability of cells for acids. Intern. Zeits. Physik. chem. Biol., Bd. 1, p. 463^78. Herlitzka, a. 1910 Contribute all'analisi fisico-chimica del sapore dei sali.
Archivo d. fisol., vol. 7, p. 557-578. HuRWiTZ, S. H. 1910 The reactions of earthworms to acids. Proc. Amer. Acad.
Arts and Sci., vol. 44, p. 67-81.
4()2 W. J. CROZIER
Kahlenberg, L. 1898 The action of solutions on the sense of taste. Bull.
Univ. Wisconsin, Sci. Ser., vol. 2, p. 1-31. Kribs, H. G. 1910 The reactions of iEolosoma to chemical stimuli. Jour.
Exp. Zool., vol. 8, p. 43-74. L.vNGDON, Fanny 1895 The sense-organs of Lumhricus agricola, Hoffm.,
Jour. Morph., vol. 11, p. 193-234, pi. 13. Larguier des Bancels, J. 1912 Le gout et I'odorat. Paris, 8°, vii + 94 pp. LoEB, J. 1915 Electromotive phenomena and membrane permeability.
Science, N. S., vol. 42, p. 643-646. Lucas, K. 1909 The evolution of animal function, II. Science Progress,
vol. 3, p. 321-331. Osterhout, W. J. V. 1914 The effect of acid on permeability. Jour. Biol.
Chem., vol. 19, p. 493-501.
1915 The nature of mechanical stimulation. Science, N. S., vol. 41,
p. 175. Richards, T. W. 1898 The relation of the taste of acids to their degree of
dissociation. Amer. Chem. Jour., vol. 20, p. 121-126. Spaeth, R. A. 1916 The vital equilibrium. Science, N. S., vol. 43, p. 502 509. Verworn, M. 1913 Irritability. New Haven, 8°, xii + 264 pp.
THE NERVOUS SYSTEM OF PYCNOGOXIDS
WILLIAM A. HILTON Zoological Laboratory of Pomona College, Clarciuont, Cal.
TWENTY-ONE FIGURES
The noi'vou.s system of pycnosoiiids presents many ])eculiarities. It is rather difficult to find the counterpart of this system in other arthro])ods. The nervous system of some Crustacea suggests it, especiallj' in those forms with an elongatetl thoracic region and refhiced alxlomen. The general arrangement of the gangha is totally unlike the central nervous system of arachnids although the general form of the body of 'sea spiders' strongly suggests arachnid relationships. The rather small supraesoi)hageal ganglion and the well develoi)od chain of ventral ganglia suggest a rather iirimitive type of nervous systrni. but the imier\ation of the i^harynx and jiroboscis presents complex and apparently unicjue conditions.
Although there is an extensive.literature on the classilicaiiou, structine and development of pj'cnogonids, there is littU^ or nothing on the structure of the nervous system.
The general form of the ganglia with their chief branches is quite well known, for nearly every pajier on the classification of the groui) contains a more or less detailed sketch of the animals described with the nervous system shown in place.
The supraesophageal ganglion seems to contain but two i)airs of ganglia recognized by early authors in other arthropods as the i^rotocerebriun and deutocerebrum, the tritocerebrum found in some arthropods being absent. This is but one of several structures that point to a closer relationship with arachnids than with Crustacea. However, without going into further reasons at this time, I am inclined to side with Dohrn and consider Pycnogonida a separate class.
4G3
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 OCTOBER, 1916
4()4 WILLIAM A. HILTON
As the tendency has been to regard these animals as arachnids, it may be worth while to glance through the neurological literatiu-e on this group.
Among the earliest work on the nervous system of arachnids was that of Treviranus in 1812. No hint of pycnogonids is given in this paper, nor is there any mention of these animals in the work of B. Haller just a century later. There is no reference to pycnogonids in the extensive work of Saint Remy, '90. Dahl, in 1913, gives a brief summary of the work of Dohrn in connection with various types of arachnids. If we go through the extensive literature on the pycnogonids as a group we find, it is true, little of the structure of the nervous system, but much about the arrangement of the ganglia composing it.
From' the works of Hoek, 78, '81, Dohrn '70, '81, Sars '91, Meinert '98, and a number of others, as well as from the study of Pacific coast forms, we learn that the central nervous system consists of a supraesophageal ganglion and a ventral chain of from four to five chief ganglia. The smaller number of ganglia we find when the body is less elongate. The supraesophageal ganglion has a ventral median nerve to the proboscis, nerves to the eyes and a pair to the chelifori. Each ventral ganglion has at least one main branch. Three branches from the first ventral ganglion are as follows: 1) A small pair or two pairs to the proboscis; 2) a pair to the palps; 3) a pair to the ovigers; 4) if the first ganglion is fused with the second as it is in those with four ganglia, then there is also a pair to the first pair of walking legs.
Figures 1 to 7 show different types of nervous systems from Pacific species of pycnogonids. The method by which the nervous system was studied by some observers was simply to determine the position of the ganglia through the transparent body-wall. This was tried with a number of specimens after the animals had been fixed in mercuric fluids. In some cases the whole animal was stained and mounted in such a way as to show the internal ganglia. In some cases the animals to be studied were placed for a short time in caustic or acid and by one or the other of these methods the internal parts were
NERVOUS SYSTZM OF PYCNOGONIDS 465
cleared so that the gangUa might be seen. Serial sections of the whole animals were also made for study, but the chitin often makes }:)erfect series impossible. Hoek and possibly
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Figs. 1 to 7 Drawings from the adult nervous systems of a number of species of California in'cuogonids. The supraesophageal ganglion is shown at the upper end of the figures in everj' case. The nerves are not all shown in every figure. All are shown from the ventral side, the ganglia were exposed by various methods and all arc not drawn to quite the same scale.
Fig. 1 Eurycyde spinosa, Hilton.
Fig. 2 Halosoma viridintestinalis, Cole.
Fig. 3 Tantystylum intermedium, Cole.
Fig. 4 Ammothella tubercuata, Cole.
Fig. 5 Pycnogonium stearnsi, Ives.
Fig. 6 Palene californiensis, Hall.
Fig. 7 Anophlodactylus orectus. Cole.
4()0 WILLIAM A. HILTON
some others have used gross dissection with the hii'ger species. I jilso tried this method and found that it was not difficult to expose and remove the whole nervous system from even the smallest specimens. For the structure of the ganglia serial sections were made from these removed ganglia.
There seems to be some difference of opinion as to number and position of abdominal ganglia. There are without doubt ganglia in the adult that may be called abdominal, but they are often not evident or indicated by slight knobs on the last ganglion. Probably in no case are these little ganglia in the abdomen (figs. 1, 2, 6, 7, 19).
The special nerve supply to the proboscis has been described by Hoek, Dohrn and others. I was able to dissect it out in a number of species where I have found essentially the same features already described. In the genus Pycnogonum I found a similar condition as shown by Hoek. Practically the same condition was found in two other genera not before described. There are three main branches which run to the three divisions of the proboscis: a dorsal branch running from the mid-ventral line of the supraesophageal ganglion, and two lateral branches springing from the forward part of the first ventral ganglion. Each of these branches has numerous small ganglia along its course and near the end of each branch there is a much larger ganglion. Branches connect the three trunks with each other and fine nerves run from each ganglion to adjacent parts of the proboscis. Lateral to these three ganglionated branches is a more external nerve which sometimes has a separate origin from the larger ganglia or from the ganglionated trunks. These three more superficial branches appear to fuse in places with the deeper branches, but they do not bear ganglia.
This whole complicated structure seems quite unusual and some have seen in this proboscis region the representations of other segments of the animals. However I prefer the assumption of Dohrn that the proboscis represents only a secondary growth of the lips of the stomodoeum. I believe the special nerves of the proboscis represent the system of frontal nerves and ganglia which we find in Insecta and other arthropods.
NERVOUS SYSTEM OF PYCXOGONIDS 467
The small ganglia of the proboscis are rather new structures, but the large, represent the frontal and lateral head ganglia of other forms.
The develo]:>ment of the nervous system of pycnogonids has been especially studied by ^Morgan. '91, and Meisenheimer, '03, although a number of others have studied the general life histories, or special stages. According to ]\Ieisenheimer, in the embryos of Ammothea the early development of the ganglia is much as in other arthropods, a longitudinal strip or germ band enwTaps the yolk and paired thickenings of the ectoderm occur which rei:)iesent cerebial and jiost-oral ganglia. I have not followed these earliest stages in any of my material. At the time that the free larva is liberated, there is seen a supraesophageal ganglion and thiee i^airs of sub-intestinal centers such as shown in figure 13 of a California foiin of the same genus. The second of these two ganglia is comjiosed of two jiarts and represents the second and thiid ])arts fused. This t>pe of larval nervous system seems rather ty]ucal of this sort of larval form. What the changes are from this to the adult are not exactly known, but suggestions may l)e obtained fi'om tlie study of other species. Morgan in Tanystylum gives some idea of the gradual development of additional ganglia in the caudal region as the larva with three i)airs of ajipendages add a fourth and a fifth ])aii" successively and later a sixth pair. At this last period \ho maximum lumibci' of ganglia is attained, this number becomes re(lu('(Hl with the growth of the seventh pair of appendages and llie adult condition is reached. During the early stages the aildition of extra gangha is probably not so much from the backward growth of nervous tissue as from later de^•elopments from the surface. In Palene the type of development is different because of the large yolk mass. Separate ganglia for the segments are developed, each of these has at an early period an invagination or Central organ.'
The species whose development I have especially studied seems intermediate between the free living larval form and such a continuous type of development as shown by Palene. This genus Ano]ihlodactylus is more parasiMc during larval stages
468
WILLIAM A. HILTON
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c
Fig. 8 Central nervous system of A. erectus during first larval stage. X 350.
Fig. 9 Nervous system of A. erectus during the second larval period. X 350.
Fig. 10 Section through whole larva of A. erectus during the third larval period. X 350.
Fig. 11 Central nervous system of A. erectus at about the third larval stage. Drawn from a whole mount which did not show as much as some others. X 300.
Fig. 12 Longitudinal section through the central nervous system of A. erectus during the last larval stage. X 350.
Fig. 13 The central nervous system of the first larval stage of Ammothella. X 350.
NERVOUS SYSTEM OF PYCXOGOXIDS 469
than the others mentioned. The first larval stage soon attaches itself to, and enters hydroids. It has three appendages in the first larval stage, one pair is chelate, the last two have long tendril-like extensions. At such a period the nervous system is not easily made out from surface views, but it is much like that of Ammothella. Figure 8 shows three parts, a larger thicker portion which has nerves to the larger first appendages, and on each side back of this a group of cells corresponding to the other appendages. A moult within the hydroid gives rise to a small larval form without the long appendages and it is at
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Fig. 14 Outline of ventral view of larva and ganglia from below A. crectus, third larval stage. X So.
Fig. 15 Outline of a ventral view of a later stage larva than figure 14 of A. erectus. X 85.
Fig. IG Outline of a dorsal view of a larva of A. erectus about the same stage as figure 14. The brain is shown. X S5.
Fig. 17 Fourth stage larva of A. erectus from below. X 35.
Fig. IS Central ganglia of a larva of A. erectus with three pairs of walking legs. The drawing is from below. The upper area without nerves in the figure is the supraesophageal ganglion. X 35.
470
WILLIAM A. HILTON
sucli a. iKM'iod dial new ^;ms2;li()iii(* iiuiterial seoiiH to be tlevelopcd. Figures 9, 11 and 14 are drawn from such early stages. At a later moult more ganglia are evident, as in figures 10, 15, and U). Tlie ventral ganglia at first are mere groups of cells, as is shown in the frontal section from which figure 10 was taken. As maj^ be seen from the figures 10 and 15, ganglia are developed in each segment, a pair for each appendage and several for the cephalic region and a common mass of cells for the ab
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P^ig. 19 A longitudinal section through the central ganglia of Lecythorhynchus
marginatus, Cole. Two small abdominal ganglia show at the end of the last
thoracic ganglion. X 35.
Fig. 20 A longitudinal section through the supraesophageal ganglion of L. marginatus. The dorsal side is up, the cephalic side to the right. X 210.
dominal. In a stage just before this there are two pairs of ganglia on the dorsal side of the larva; these are shown in figure 16. They represent the brain.
At about the third moult, as shown in figures 12 and 17, the ganglia have developed central fibers, but still show their paired nature. There seems to be some indication of more ganglia than there are appendages, some of the caudal elements may not be evident in later stages, and the first ventral ganglion seems composed of two small pairs of elements. In the proboscis
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Fig. 21. Drawing of ilw ncrvi's and ganglia of the proboscis of L. marginatus. Slightly diagrammatic. No structures shown in the proboscis but nerves and ganglia. The drawing was made by Miss M. L. Moles from the first sketch taken from the dissection. Much enlarged.
471
472 WILTJAM A. HILTON
of (his st;ifi;c (luM-e seem to be two sin;ill i:)Mirs of ganglia. The dorsal ganglia are not shown in figure 17.
When the larvae moult again and leave the cavity of the hydroids they have all but one pair of legs. Figure 18 shows the whole central nervous system from below at such a stage. The brain above the esophagus is at the upper end of the figure, then follow the ventral ganglia, seven paired masses and a small unpaired caudal ganghon. There is a gradual fusion of these ganglia until the adult condition shown in figure 7 is attained.
The structure of the adult nervous system of pycnogonids is quite simple. There is the same general arrangement of cells that we find in other arthropods. The ventral ganglia have few cells on the dorsal side, but many on the lateral and ventral sides. The supraesophageal ganglion is sheathed in cells on the lateral and dorsal sides. Nerve fibers connect the ganglia and certain regions but in no place is there a concentration of the fibers. The fibrous mass is not particularly dense at any point. There do not seem to be many long tracts and the supraesophageal ganglion is not more complicated than other parts so far as could be determined. There are no marked decussations of nerve fibers and the nerve cells present a uniform appearance. Among the nerve cells are many nuclei of neuroglia networks which form the framework of the ganglia especially in the area of the cells.
Although there are indications of special groups of cells and fibers, there was no indications of mushroom bodies.
The animals do not seem to have a special brain. The supraesophageal ganglion is not a very special center. The movements of the animals agree with this; they move sideways, forwards or backwards when stimulated. No part of the body seems to lead in the locomotion.
NERVOUS SYSTEM OF PYCNOGONIDS 473
BIBLIOGRAPHY
Aldkrz, G. 1888 Bidrag till Pantopodernas Morphologi och Utvecklings
historia. Bihaiig till k. Svenska Vetenskap Akad. Hand., Bd. 13,
afd 4, no 11, Stockholm. Dahl, F. 1913 Vergloich. Phys. und Morph. der Spinncnticrc, Erster Teil,
Jena. DoHRN, A. 1870 Untcrsuch. liber Bau und Entw. der Arthropoden. 2. Pjxno goniden. Jen. Zeit. f. Nat., Bd. 5.
1881 Die Pantopodcn des Golfes von Xeapel. Fauna und Flora des
Golfcs von Neapcl. Monog. 3, Leipzig. Hallkk, B. 1912 t)ber das Zrntralncrvcnsysteni des Scorpions und der Spinnen.
Arch. f. IMicr. Anat.. Bd. 79. Abt.'l. Hallez, p. 1905 Observations sue le parasitisme des larves de Phoxichilidium
chez Bouganvilla. Arch. zool. e.xp. et gen., 4me scrie, t. 3. Hilton, W. A. 191(5 The life-history of Anoplodactylus ercctus Cole. Jour.
ent. and zool., vol. 8, no 1, March. Hokk, p. p. C. 1881 Report on the pycnogonida. \oyage of H. M. S. Challenger, Zoology, vol. 3.
1881 Nouvelles Etudes sur les pycnogonids. Arch. Zool. exp., t. 9,
Paris. HoDfiK, G. 1862 Observations on a species of pycnogon (Phoxichilidium coc cineum) with an attempt to explain the order of its development.
Ann. mag. nat. hist. (3), vol. 9. Khoykh, H. 1842 Notes sur les metamorphoses des pycnogonides. Ann.
so. nat. 2e scr., t. 17. Lendknfeld, R. von 1883 Die Larvtiicntwickiung von Phoxichilidium plumu larie. Zeit. Wiss. Zool., Bd. 3.s. Melsenheimeh, J. 1902 Beitrage zur Kntwick. der Pantoijodcii. Zeit. f.
Wiss. Zool.. Bd. 72. Mekten."^, pi. 190() Eine auf Tethj-s leporina, parasitisch lebende Pantopodcn
larva (Nymphon parasitica n. sp.). Mitt, aus der Zool. Stat. Neapel,
Bd. 18. Meineut, Fr. 1898 Pycnogonida den Daniske Ingolf-exi)e(lition. M()H(;an, T. H. 1891 Contribution to the embryology and phylogeny of pycnogonids. Stud. biol. lab. Johns Hopkins Univ., vol. o.
EVIDENCE OF A MOTOR PALLIUM IN THE FOREBRAIN OF REPTILES!
J. B. JOHNSTON
University of Minnesota
OXE FIGURE
At the title indicates, the purpose of this note is not to describe the structure of the reptiUan motor cortex nor to discuss the locaHzation of function within this area. The purpose is onh' to give such evidence as is now at hand that a speciaHzed area comparable to the mammalian motor cortex probably exists in the reptilian brain and to indicate the general ]-)osition and extent of this area.
The writer has indicated in a jirevious i)a])er ('15 b) that the rostral portion of the dorsal pallium in the turtle differs structurally from the rest and has suggested the possibility that this area may correspond to the motor cortical area in the mammalian brain.
This hy])()thesis is being tested by degeneration methods and by cortical stimulation. Sufficiently definite results have been obtained by the latter method to indicate that motor and siMisory fields can be (hstinguished in the reptilian ])allium.
Three species of turtle (Chelydra serpentina, Cistudo Carolina, and Chrysemys marginata) and one lizard (Gerrhonotus) have been studied. Two methods of stimulation were used : induction shocks with two-])oint electrodes, the two i)oints closely approximated; induction shocks with one point electrode, the second electrode being formed by a copper plate covered with moist cloth on which the body of the animal rested.
An important factor in the experiments is the degi'ee of anaesthesia employed. The clearest results have been obtained with very deep anaesthesia. Some of the turtles were given a dose of morphine by hypodermic injection, and all were anaesthet ' Neurological Studies, University of Minnesota, no. 22. June 29, 1916.
47.")
476 J. B. JOHNSTON
izod l)y inoiius of cliloroforin phiccd on cotton in the mouth or injected directly into the trachea or both. Although the resistance of these animals was well known, in several cases the anaesthesia was not carried far enough and the reactions to cortical stimulation were prolonged contractions of neck muscles or struggling movements of the limbs indicative of pain. In these cases occasional responses, as of the eye muscles or temporal muscle, appeared to be due to local excitation of the cortex but the more extensive and prolonged movements were elicited from any part of the cortex and were not distinguishable from the responses to stimulation of the dura mater, the lower brain regions or even the tissues exposed in the head. Under conditions of deep anaesthesia the responses consisted of contraction of a small set of muscles, and of short duration, and these were obtained from a certain region of the pallium only. The lizards are not so difficult to anaesthetize, but long continued application of chloroform is necessary (one and a half to two hours in a closed dish containing a wad of cotton wet in chloroform). Another factor presenting difficulty is the small size of the brain in these animals. This makes it necessary to use a single point electrode or to have the two points very close together. It is of the greatest importance that the meninges be carefully removed from the whole hemisphere and that the brain and surrounding tissues be kept as dry as possible without injury to the brain tissue. If the electrode comes too close to the dura mater or if fluid facilitates the spread of the current to the dura, the muscles, thalamus or midbrain, the results are vitiated because of responses coming from two or more sources of stimulation. The responses which follow stimulation of the dura or brain stem are very different from those produced by cortical excitation, being usually more vigorous and prolonged as well as more extensive. In the lizards and in several turtles, after the dura was laid back and the olfactory and optic connections were cut, the entire forebrain was raised out of the skull so as to be free from contact with other tissues. Under these conditions the entire surface of the hemisphere was explored with the electrodes.
MOTOR PALLIUM TX REPTILES 477
With the induced current minimal stimuli were applied at first and increased later in the experiment when it became necessary in order to obtain responses. In many cases the brain fatigued quickly and in some it recovered with equal promptness. Individuals differed greatly as to the recovery. Often when the strength of stimulus was increased struggling movements resulted and the experiment had to be discontinued. In some cases also, the animals gave definitely localized responses to the first stimulation and later, probably because of recovery from the anaesthesia, gave irregular and prolonged reactions.
COIMICAL REACTIONS IN TIHTLKS
If we take into account only the short contraction of restricted groujis of muscles, the following jiarts of the hemisi)here have been found to give nmscular responses: dorsal surface of the olfactory bulb, retraction of the neck, extension of the legs, movements of eyeball and eyelid: dorsal surface of jiallium near olfactory ])eduncle and lateral liorder of pallium in the anterior one-half or two-thirds of hemisphere, movements of eyes, jaw, neck, legs and tail; striatal area, movements of all parts. Xo responses were ol^tained from any other part of the dorsal surface or llic medial w;ill or from the tuberculum olfactorium, the amygdaloid eminence or elsewhere, except that contractions typical of thalamic stimulation were sometimes obtained when the caudal pole or amygdaloid eminence was being ex]>lored. These were probably due to spread of current to the thalanuis.
The responses from the striatal area were presumably due to the direct stimulation of descending fibers in the crus. The responses from the olfactory bulb may be due to the close proximity of the 'motor area' which is indeed overlapped by the caudal border of the olfactory formation. Thus it appears that a somewhat comma-shaped area involving the rostral and lateral boi'ders of the general pallium (fig. 1) may be regarded as a motor area in the turtle's pallium. This area corresponds roughly to the 'pallial thickening' described in a previous paper ('15 b).
Not enough experiments have been conducted under uniform conditions to furnish a basis for the discussion of localization
478
.1. n. JOHNSTON
witliiii t.liis ai'oa. As the whole area is only a few millhneters in extent, it is obvious that ^I'cat care must be exercised in accepting as conclusive any api^arent indications of special function of particular parts of this area. For this reason I mention only tentatively and with reserve that leg movements have been obtained most often from the anterior part of this area and eye, jaw and neck movements from the lateral portion. Further experiments will be made to determine whether a locahzation within the niotoi' area clearly exists. In certain experiments
Fig. 1 Sketches of the dorsal surface of the right hemisphere of the turtle (left) and lizard fright) on which the motor area is shaded.
the eye and jaw^ movements were homolateral only or more frequently, while the neck and leg movements were mostl}^ heterolateral.
Excitation of the dorsal ventricular ridge in the turtle was carried out by cutting away the whole dorsal pallium and applying the electrodes to the ventricular surface of the ridge. Muscular responses were often obtained, such as retraction of the eyeball, contraction of temporal muscle and twitching of fore leg, but there is the double danger of spread of stimulus to the closely adjacent pallial thickening and of stimulation of the underlying crus.
MOT(3R PALLIUM IX REPTILES 479
MOTOR RESPONSES IN I'HE LIZARD
Only four animals have been examined, two of which gave much clearer and more definite results than those obtained from most of the turtles. From the area shown in the accompanying sketch one animal gave movements of the fore leg, jaw and eye mijscles, neck and throat muscles, and anterior body musculature. Stimulation of the olfactory tubercle also caused contraction of small nmscles in the throat, which was felt as the head was held in the fingers. Stinmlation of the crus in the striatal area produced mo\'ements of legs and body muscles. Movements of the fore leg were regularly heterolateral, although homolateral movements occurred occasionally. Just the opposite was true of the eye and jaw muscles. The other animal responded to stinuilation of the anterior part of this area by strong contraction of ])elvic muscles and movements of the hind legs and by weak movements of the fore legs. Stimulation of the lateral border of the right pallium in this animal produced a definite torsioji of the fore part of the body.
(lerrhonotus is n rather large lizard and its brain is fairly satisfactory to woik witli. The results in these two cases were so definite that I regard them as strong evidence that a motor area exists in the lizard pallium essentially similar in j^osition and extent to that in the turtle.
Early in my studies I examincMl \\\c brain of a single specimen of Alligatoi' about a foot and a half in length, and obtained motor res})onses from the anterior \vdvt of the dorsal pallium, but I ha\'e no written record of this experiment.
Fiom these experiments, together with the study of the structure of the hemis])here already reported, I am strongl}' inclined to belie^•e not only .'hat reptiles possess a general or somatic pallium which has been in dispute until recently, but also that in that pallium are to be distinguished definite sensory and motor areas in the sense in which those terms are commonly used of the mammalian pallium. Localization within these areas and the significance of these areas in the evolution of the mammalian paUium are subjects for further study.
THE JOUHNAI. OF COMPARATIVE NF.UKOLOGY, VOL. 26, NO. 5
THE DEVELOPMENT OF THE DORSAL VENTRICULAR RIDGE IN TURTLES'
J. B. JOIIXSTOX Universili/ of Minnesota
TWE.VTY-SK\ KV FKilKKS
In tlic adult tuitlc (Jolinstdii. "l.")!)) the lar^c ridge projecting into the ventiiclc from \\\o iaicial wall of the hemisphere has strong fiber conncclion-- with tlie cerebral ])edunele and has obvious close relations with the cortical layers of the pallium m the caudal ])ole. Indeed, in its caudal piirt this ridge a])pears to be a fold of tlic whole thickness of the brain wall and is marked externally by tiie "amygdaloid fissure.' Here the cell layers of the general pallium turn in to become continuous with the cell masses of the ridge. It is evident from these facts that the relations of the doi'sal vcntiiculai- ridge aic cliiefly with the general ])allium. Its |)osition lateral and dorsal to both the caudate and lentiform miclei shows that it does not belong to the corpus striatum. Its independence from other cell masses in its rostral part gi\'es one the impression (from the study of adult material alone), that the ridge has been formed by infolding from the caudal region ('lob, p. 417).
The origin of this ridge in the embryo shoukl throw light not only on its relations in the turtle but also on its signihcance in the evolution of the manunalian brain. I am able to give the main outlines of the development on the basis of a series of early embryos of Chelydra serpentina, for which I am indebted to Dr. C. E. Johnson of this University; and of sections of 17, 20, and 28 mm. embryos of Chrysemys, belonging to the Anatomical Laboratory of Washington L^niversity for the loan of wliicli 1 am indebted to Dr. Edwin A. Baumgartner.
I Neurological Studies, University of Minnesota. No. 23, July 1, 1916.
481
482 J. B. JOHNSTON
Tho luatoriiil in botli cases was boMutifully sectioned and stained. The brains of freshwater turtles are so similar that the younger stages of Chelydra can be readily compared with the later stages of Chrysemys for the rather general purposes of this study.
ARIOAS OF PROLTFP]RATION IN THP^ EARLY EMBRYO IN RELATION TO CELL MASSES OF THE ADULT BRAIN
Broadly speaking, three periods are to be recognized in the development of cell masses in the telencephalon: a period of evagination of the hemispheres and formation of indifferent cells by mitosis of the germinal cells; a period of form changes and further proliferation of cells in which the successive formation of functionally ind pendent cell masses is noticeable; and a period during which the growth of fiber bundles and other factors bring about the definitive form of the adult cell masses.
In the first period the wall of the telencephalon consists of a deep layer of spongioblasts and dividing germinal cells, a thick layer of indifferent cells and a superficial clear layer or marginal veil of very varying thickness. The thickest part of the marginal veil forms the floor of the groove along the lower border of the hemisphere, at its junction with the brain stem. This is the course of the future lateral forebrain bundle or crus cerebri. The indifferent cells form a continuous layer with no indication of individual masses.
The second period begins with the appearance of independent cell masses separated by cell-free zones which show in sections as clear lines or areas. Such cell-free zones in adult brains have been commonly regarded as boundary lines between individual cell masses which are presumed to have different functions; i.e., centers or nuclei. Similar cell-free zones in the brains of lower vertebrates have been used in recent years as landmarks between important morphological regions, as the pallial and basal areas. The writer has pointed out the need of caution in interpreting these zones in this connection ('13 b, p. 382). It is now to be noted that the formation of individual masses of cells is dependent upon the proliferation of cells from different regions of the germinal cell layer and upon the proliferation
DORSAL VENTRICULAR RIDGE 483
of cells at successi^'e periods of development. Thus the proliferation of cells in the dorsal wall gives rise to pallium, that in the basal wall gives rise to various olfactory centers, coi-pus striatum, etc. Also, certain centers owe their origin to the prohferation of cells in an early stage, other centers to proliferation in later stages. The former come to he superficial to the latter and the two are separated by cell-free zones. A further factor is the shifting or spreading of cell masses, whether due to cell-migration or to mechanical forces. These several factors must be taken into account in any attemj^t to use the embryological method in tlie study of tlio significance of cell-grouping in the brain.
(;ni;i.vi)i;A si;i{Im:.\ tixa
In the oldest Chclydra embryo studied, it is possible to recognize several of the important cell masses and fiber bundles of the telencejohalon and in part the boundary lines between jiallial and basal areas. Examination of the model (figs. 1, 2) and of sections (figs. 6 to 14) shows that the telencejihalon and diencephalon are narrow from ^itle to side and higii dorso-ventrally. The hemisphere is a simple sac which ])rojects much farther rostrad than caudad. The intei-ventricular foramen is still very large. Behind it, the tliin wall which rei)resents the choroid plexus extends nearly half way to the caudal ])ole. This stage illustrates very clearly, both in its general form and its internal structure, how much the rostral \)o\e of the hemis}ihere precedes the caudal pole in de\'elopment. The outer surface of the brain shows few of the landmarks wliich are seen in later embrj^os and adults. The stem-hemisphere sulcus is of course the most prominent. The olfactoiy peduncle is not yet formed, but the broad depression on the lateral surface may represent the beginning of constriction. The fissura prima and diagonal band are recognizable at the medio-basal angle in front of the preoptic recess. Thei-e is a shallow furrow over the rostral part of the foramen which may be the beginning of the sulcus fimbrio-dentatus.
484 .1. n. JOHNSTON
Tlio \'cntricle shows a xcvy sharp ventral groove leading forward from the foramen and much deepened in the region of the future tuberculum olfactorium. The middle ventricular groove is clearly marked in the region rostral to the foramen, while the dorsal groove has not yet appeared.
In spite of the simple form of this brain, the internal structure shows considerable advance in differentiation.
The sections illustrated in figures 6 to 14 can be understood best by beginning with that which passes through the rostral border of the foramen interventriculare (fig. 9). In the medial wall above the foramen are recognized the primordium hippocampi and the fimbria, one of the early fiber tracts to be formed in the embryo. The dorsal wall is occupied by pallium, the medial portion by hippocampal, the lateral portion by general pallium. In the lateral wall the pallium seems to be limited by a ventricular groove and by a cell-free space. This boundary line strongly reminds one of the lateral zona limitans of the selachian and frcg brains (Johnston, '11 a). The later embryos show this groove to be the middle ventricular groove of the adult brain and the clear area becomes the cell-free zone separating the dorsal ventricular ridge and the nucleus lentiformis in the adult. In the lower half of the lateral wall the following structures are seen in the figure: an active proliferation which gives rise to the nucleus lentiformis, the nucleus caudatus, the lateral and medial forebrain bundles, the anterior commissure bundle, the fiber layer of the diagonal band, and the lateral olfactory area. Although I have not sufficient stages to enable me to follow the history of these structures, there is evidence that the superficial cell layer which represents the lateral olfactory nucleus has been derived from the layers of indifferent cells in the early embryo and that the mass of the caudate nucleus represents a later proliferation. The embryo shows clearly that the lentiform nucleus is beginning to form when the caudate is already completely formed or nearly so. As the other sections are studied it will become evident that the len:iform nucleus is formed by a proliferation distinctly dorsal to the caudate and that the changes in later development bring
DORSAL VENTRICULAR RIDGE 485
it into a position lateral to the caudate. There is some indication of the superficial layer of the olfactory area spreading upward beyond the middle ventricular groo\-e (at the point marked l.pyr).
Passing caudad from this level, in the next section drawn (fig. 8) the same structures are to be seen in the lateral wall. The middle ventricular groove has almost disappeared, however, and the proliferating area for the lentiform nucleus is much smaller. The next section dra^^^l (fig. 7) is the last one in which the lentiform proliferation can be recognized and with it the locus of the i^allial iDorder. In the lower part of the hemispheie the lateral olfactory area is represented by a large collection of cells closely related to the caudate nucleus and the diagonal band. This is the nucleus of the lateral olfactory tract and the next section shows the close relation of l)()th this and the diagonal band to the stria medullaiis. Caudal to this the nucleus of the latei'al olfactory tract soon disai)pears and the hemispjiere wall ])resents the aj^j^earance of an imdifferentiated pallium, except for the fimbria and tlie thin choioidal area in the meilial wall.
The next section rostrad from the one first described i)asses through the anterior commissure (fig. 10). The lentiform i)roliferation ajij^ears larger, the diagonal band fibers approach those of the olfacto-hypothalamic system. In figiu'es 9, 10. and those of sections farther rostrad, one of the most noteworthy features is the mass of cells proliferating from the deep layers of the pallium. The medial border of the pallium does not show active jiroliferation but in the lateral three-foin*ths many new cells are forming ami these a]:>])ear to be streaming laterad into the thick wall just dorsal to the middle ventricular groove. Indeed the proliferation and st reaming of these cells is the active cause for the thickening of this part of the wall and the formation of the middle ventricular groove. Further thickening of this part of the wall deepens this groove and produces the dorsal ventricular groove at the point indicated in figure 9.
In figures 9 to 12 appear important relations of the dorsal border of the olfactory area to the pallium. Here it is quite
486 .). H. JOHNSTON
clear iliai the siipci licial layor of (*ells of the olfactory iinvd extoiuls II]) oil tlic ouUm" surfMce of the pallium and that it is accompanied b,y a special bundle of fibers. The crowding of cells in this stage is such that it is impossible to determine whether the pyriform lobe cells have spread or migrated up from the lateral surface in the basal region or have been formed here by the proliferation which at this stage is giving rise to the pallium. Farther caudad and in later stages the relations are such as to make the former appear more probable. The ceils of the pyriform lobe probably all come from the layer of indifferent cells seen in earlier stages and formed by proliferation in the lateral and basal region before the pallial proliferation seen in this stage began. Pallium and pyriform lobe are still visible in a section (fig. 14) through the peduncle in which the olfactory formation appears in the dorsal wall. The olfactory formation extends far back on the dorsal surface, as is already well known.
CHRYSEMYS
Models were made of the 17 mm. and 28 mm. stages. Most of the chief landmarks of the adult brain are already visible in the 17 mm. embryo (figs. 3, 4, 15, 16, 17). The caudal pole still lags behind the rostral portion in development. The caudal pole projects much farther back than in the oldest Chelydra embryo and in the 28 mm. stage the olfactory area (lobus pyriformis) extends relatively farther back than in the 17 mm. stage. The tuberculum olfactorium is well developed and the ventral groove of the ventricle dips deep into it. The middle ventricular groove (fig. 4 and sections) is very sharp and deep in the middle part of the brain and stops abruptly just caudal to the level of the foramen interventricukre. The dorsal ventricular groove appears in the 17 mm. embryo (figs. 4, 17) as a slight groove in the lateral wall some distance from the medio-dorsal angle of the ventricle.
Three sections caudal to the foramen are drawn from the 17 mm. embryo and nine sections from various levels of the 28 mm. embryo. The internal differentiation has already progressed so far in the 17 mm. embryo that it will be unnecessary
DORSAL VENTRICULAR RIDGE 487
to speak of it separately. In both sts.ges many features of the sections can readily be compared \\'ith the structures seen in corresponding sections of the adult brain. For this purpose the reader should consult the description of the cell masses in the brain of Cistudo in this Joui-nal for October, 1915. The necessary description of indi\'idual sections is given in connection with the figures. Here I shall take up the relationships of the lateral margin of the pallium and the dorsal ventricular ridge.
Corpus striatum und lateral olfactorj/ area. In the younger embryos the loosely arranged cells of the lateral olfactory confers cover the outer surface of the caudate and lentiform nuclei and form a continuous layer over the lateral surface of the hemisphere (except dorso-caudally). This is shown in figures 7 to 12, and this interpretation is given to the models. In the 28 mm. embryo there is a small area ojiposite the foramen in which the striatum and the crus bundle contained in it come to the surfiice (fig. 21) and the olfactoiy centers form the pyriform lobe above and the diag(jnal band below. This small area is painted blue in the model and appears white in figure 5. Sections show that it is the elbow of the crus, with certain large cells contained in it ('15 b, ]). 405), which comes to the surface here. This is j^resinnably due to the rapid growth of both ascending and descending fibers in the crus. The crowding thus ]:)roduced leads to the shifting of the cells of the olfactory center upward and downward. This process must go much further in later development until the large striatal area is left exposed in the adult. The embryonic history thus seems to supi)ort very clearly the hypothesis ('15 b, p. 428, 429) that the exj)osed striatal area in the turtle had been covered at an earlier stage of evolution by the lateral olfactory area.
Relations of pyriform lobe and pallial jnargin. In the caudal half of the hemisphere the pja-iforin lobe forms a layer external to the corpus striatum and extends up to a thin edge superficial to the dorsal ventricular ridge and the margin of the pallium. This overlapping of the pallial margin by the pyrifoi'in lobe persists in the atlult. At about the level of the interventricular
488 J. H. JOHNSTON
foniiueii the i)yrif()rni lobe begins to extend higher on the hiteral surface. At the same time thickening of the hiteral border of the pallium moves dorsally, becomes crowded and folded on itself and sinks in, forming a projection into the ventricle (figs. 22 to 25). These changes take place rapidly from behind forward so that in thirty or forty sections of 10 microns the place occupied by the thick border of the pallium comes to be taken by the pyriform lobe. From this point forward the pyriform lobe forms a characteristic ridge filled with a dense \ayev of cells, very much as in the adult.
Origin of dorsal ventricular ridge. In the Chelydra embryo above described the dorsal part of the lateral wall is thickened by cells coming from a proliferating area in the dorso-lateral wall. The groove which bounds this thickening was identified with the middle ventricular groove of the adult. In the older embryos of Chysemys the groove is readily identified but has been very greatly deepened by further thickening of the mass just above it. Likewise the thickening of this mass has produced a groove above it, the dorsal ventricular groove. The thick mass, then, is the dorsal ventricular ridge and in earlier stages it is indistinguishable from the dorsal pallium. In the caudal part of the hemisphere of the older embryos the relations of the dorsal ventricular ridge are not essentially changed from those seen in the Chelydra embryo. Caudal to the level of the stria mednllaris (figs. 18, 19) this ridge bulges into the ventricle and is covered outside and below by the thick mass of the nucleus of the lateral olfactory tract. In the caudal pole the ridge merges gradually with the general palhum caudal to the level at which the pyriform lobe ends In the rostral part of the hemisphere (figs. 22, 23) the dorsal ridge has become practically independent of the pallial thickening, very much as it is in the adult. From these embryos it is quite clear that the dorsal ventricular ridge arises as a thickening of the lateral border of the pallium and that its continued growth causes ic to project into the ventricle and produces the middle and dorsal ventricular grooves. In the 28 mm. embryo the mass of cells which in the adult was called the core-nucleus of the ridge
DORSAL VENTRICULAR RIDGE 489
is clearly recognizable in the rostral part, back to the level of the caudal border of the foramen interventriculare.
Between the pallial thickening and the cells of the dorsal ventricular ridge appears in the sections a clear space filled by fibers. In the caudal ]3art of the hemisphere, where the dorsal ventricular groove is already formed, this bundle of fibers is seen to hold the same position as an important bundle in the adult. In the adult many fibers from the crus enter this bundle or, more probably, pass through it on their way to the pallium. Here in the embryo the crus is not yet sufficiently develo])ed to enable one to trace it up through the stiiatum to this le\'el. The strong development at this stage of the bundle here mentioned indicates that it is comjiosed chiefly of fibers connecting parts of the hemisphere; an association bundle, in other words.
Relations of dorsal ventricular ridcje to basal structures. In the rostral part of the hemis]ihere the ridge is from the first clearly marked ofi" from the stiiatum below by the middle groove and by a prominent cell-frcn^ zone. In later embryos this cellfree zone ])econies comjiressed into a nanower line as seen in section and scattering cells are found in it. There is. however, always a clear boundary between the ridge and the developing lentiform nucleus. In section the cell-free zone inclines dorsad toward the lateral surface and ]iasses al)ove the lateral olfactory area. In the rostral i^art, where the i)yriform lobe crowds far dorsal this limiting zone is bent into a semicircular or VshajKHl line (figs. 23 to 27). When the writer first studied the adult turtle brain he found it im])ossible to establish a direct comparison between the lateral zona limitans of the selachian, frog and other simpler vertebrates, and either of the cell-free zones seen in the lateral wall of the turtle brain. It is now evident that the clear zone which in the adult brain passes from a point near the middle ventricular gi'oove toward the lateral surface and then bends dorsad to reach the surface in the sulcus rhinalis above the lobus pyriformis, is the zona limitans of the embrj'o and corresponds to the zona limitans lateralis in the selachian and frog. (See Johnston, '15 b, figs. 19 to 22 and compare with '11a, fig. 75). Since the relations of the p^'ri
490 J. B. JOHNSTON
form lobe, corjius striatum and pallium in the turtle are readily comparable with those in the manunal, the identification of the lateral zona limitans in the turtle completes the history of this important landmark throughout the series of vertebrates. The boundary lines between the pallial and basal portions of the hemisphere are now clear in both medial and lateral walls in fishes, amphibians, reptiles and mammals (Johnston, '11a, '13 b, '15 b).
The relation of the dorsal ventricular ridge to the amygdaloid complex requires further comparative study. In the adult, it will be remembered, the caudal part of this ridge is separated from the rest by a broad shallow groove which continues in the general direction of the deep middle ventricular groove ('15 b, figs. 4 and 10). This caudal portion of the ridge is made up of the medial amygdaloid nucleus and of an apparent infolding of the general pallium. Even in the oldest of these embryos the dorsal ventricular ridge extends into the tip of the caudal pole as a simple thickening of the general pallium. It seems probable, although the evidence is not sufficient for a definite conclusion, that the caudal portion of the ridge including the large-celled medial amygdaloid nucleus is formed from this thickening of general pallium.
For the photographs of the models I am indebted to Dr. W. F. Allen of the University of Oregon.
LITERATURE CITED
Johnston, J. B. 1911 a The telencephalon of selachians. Jour. Comp. Neur., vol. 21.
1913 b The morphology of the septum, hippocampus, and pallial commissures in reptiles and mammals. Jour. Comp. Neur., vol. 23. 1915 b The cell masses in the forebrain of the turtle, Cistudo Carolina. Jour. Comp. Neur., vol. 25.
DORSAL VENTRICULAR RIDCE
491
I IGIHES
AI«HRKVI.\TIO^•^
a. p., :irc:i parolfactoria
b.o., hulhus olfantorius
c.a., cominissura anterior
c.h., commissura hippocampi
ch. op., chiasnia oi)ticuni
c.p.a., commissura pallii anterior
cr«.s, cms ccrchri or lateral forebraiii
hundlc est., corjjus striatum d.b., diagonal hand of Hroca d.v.r., dorsal ventricular ridge fi., fimbria
f.o., formatio olfactoria /.p., fissura i)rima for.i., foramen interveiitriculare f.rh., fissura rliiiialis g.p., general i)allivim g.s., gyrus suhcallosus h., hippocampus hy., hypothalamus I. pyr., lobus pyriformis 1. 1., lamina tcrminalis m.fh.hdl., medial forel)rain bundle
H.c, nucleus caudatus
n.d.b., nucleus of the diagonal band
n.L, nucleus Icntiformis
/(.'>., nervus olfactorius
n.rot., nucleus rotundus
n.lr.dlf.ldl., nucleus of the lateral olfactory tract
/«;.. ])allium
pn.lh., pallia! lliickening
p.li., primordium hii)i)ocampi
P.O., peilunculus olfactorius
r.p., recessus praeoi)ticus
r..s., recessus superior
s.f-d., sulcus fimbrio-dentatus
•s.m., stria medullaris
s.v.d., dorsal ventricular sulcus
a.v.tn., middle ventricular sulcus
S.V.V., ventral ventricular sulcus
thai., thalamus
I.e., tuberculum olfactorium
Ir.olf-hy.. tractus olfacto-hypothalamicus
Ir.op., tractus opticus
492
J. B. JOHNSTON
Fig. 1 Chelydra serpentina. The size of this embryo is indicated by the
owner by the words '8.5 mm. carapace.' Lateral view of a model of the right
hemisphere. Description in the text.
This and the following models have been merely rubbed with a blunt instrument to smooth the surface. The external surface has been painted so that the olfactory centers and hippocampus appear dark and the general pallium light. The diencephalon has been left with the gray color of the modelling paper and the ventricular surface of the hemisphere has had no treatment whatever. The lamina terminalis and line of attachment of the choroid plexus have been painted white. All the models were made at a magnification of 50 diameters and are reduced one-third in reproduction
DORSAL vp:xtri(T'lar ridge
493
ifi. J Medial view of I luMiind.-l •<1i..\vm in limirc 1. Description iti the text.
494
J. IJ. JOHNSTON
DORSAL VENTRICULAR RIDGE
495
Fill, '.i CMirysiMiiys I'lnl.ryo of 17 iiiiii. Medial view of a iiiodol of tlu" rinlit
hemisphere. The narrowing of the interventricular foramen seems to have
taken place from above downward on account of the expansion of the hippocampus and general jiallium. The white area labelled r.h., is the cut surface of
thcprimordium hippocam])! in the median plane. The thin portion of the medial
hemis))here wall which will form the choroicF fissure and plexus is l)ounded hy a
white line.
rig. 4 Same model as figure 3, with the thalanuis. hippocampus antl primordium hijipocampi removed. The lateral wall of the ventricle shows the dorsal ridge hounded ijelow hy the deep middle ventricular groove as described in the text. The dorsal groove is shallow. There is no evident boundary between the dorsal ridge and the pallial thickening at the rostral end.
Fig. 5 Chrysemys, 28 mm. Lateral view of a model of the right hemisphere. Note the great elongation of the pj-riform lobe in the caudal pole as compared with the condition in figure 1. With regard to the exposure of the corpus striatum on the lateral surface, compare figures 1 and 5 with the figures in '15 b.
THE JOURNAL OF COMPARATIVE NECROLOGY, VOL. 26, NO. 5
49() J. B. JOHNSTON
Figs. 6 to 14 Transverse sections through the right hemisphere of the Chelydra embryo illustrated in figures 1 and 2.
Fig. 6 Section just caudal to the foramen interventriculare. This is the most caudal section in which the nucleus caudatus can be recognized and here it practically fuses with the nucleus of the lateral olfactory tract. Both are undoubtedly parts of the primitive basal olfactory centers which have been carried out into the caudal pole of the hemisphere evagination. There is in this section no apparent boundary between these nuclei which enter into the amygdaloid complex and the general pallium.
Fig. 7 Section through the caudal part of the foramen, 120 microns rostral to figure 6. The nucleus caudatus is much more distinct and the proliferation of cells for the nucleus lentiformis begins to appear. There is now a slight indication of the latera* boundary of the pallium. The letters s.v.m. in this and figure 8 indicate the position in which the middle ventricular sulcus is to be formed, although it is not actually present in these sections.
Fig. 8 Section through rostral part of the foramen, 100 microns rostral to figure 7. The lateral olfactory area is represented chiefly by cell masses adjacent to the diagonal band. The nucleus caudatus begins to be separated from this area by the crus entering the hemisphere. The lentiform proliferation is larger and the clear space just above it, which corresponds to the zona limitans lateralis in lower vertebrates, is present from this level forward.
Fig. 9 Section through the rostral wall of the foramen. The position of the foramen is represented by the light space at the medio-ventral angle of the ventricle. The crus has now separated the nucleus caudatus from the nucleus of the diagonal band and both are somewhat removed from the larger mass of the lateral olfactory area which is here labeled l.pyr. The middle ventricular sulcus which is present in this section extends farther caudally in order embryos and adult.
/^
498 J. B. JOHNSTON
Fig. 10 Section through the anterior commissure. Although the hiteral olfactory area is continuous in these sections and even the nucleus caudatus is nowhere wholly separated from it, one can clearly see the beginnings of the segregation of the pyriform lobe, diagonal band and caudate nucleus by reason of the enlargement of the lateral forebrain bundle (cms). This process will be completed in later stages by the development of the lentiform nucleus and the further enlargement of the crus.
Fig. 11 Section just rostral to the preoptic recess where the diagonal band turns up in the medial wall. The dark mass on the medial surface of the area parolfactoria represents the beginning of the nucleus of the diagonal band, which corresponds to the gyrus subcallosus of the mammalian brain. The nucleus caudatus is connected beneath the ventricle with the area parolfactoria. From this level rostrad the olfactory area or lobe is quite continuous from its lateral border at l.pyr. to its medial border near fi.
Fig. 12 Section 60 microns rostral to figure 11, where the diagonal band fibers rise up in the medial wall to join the fimbria. Although the cells of the pyriform lobe and pallium lie immediately in contact, there appears in many sections a very sharp line as if it were caused by a delicate septum running dorsad from the small fiber bundle beneath l.pyr.
Fig. 13 Section through the tuberculum olfactorium. Note that the ventral ventricular sulcus here descends nearer to the surface than elsewhere so that the somewhat bulging tuberculum contains a large pouch of the ventricle.
Fig. 14 Section through the olfactory peduncle. The ventral part of the section cuts the rostral wall of the tuberculum while its dorsal part cuts the olfactory formation.
DORSAL VEXTRICULAR RTDGE
499
500 J. H. JOHNSTON
Figs. 15, 16, 17 Three transverse sections of the right hemisphere of the 17 mm. Chrysemys. Figure 17 falls in the caudal part of the foramen and figure 15 represents next to the last section in which the connection of the hemisphere with the thalamus is seen. Figure 16 is between these two.
In this embryo the mass formed by the pallial proliferation of the earlier stage has become divided into a general pallium and the dorsal ventricular ridge. The ridge projects into the ventricle forming a deep middle ventricular sulcus and a shallow dorsal sulcus. Note that the dorsal sulcus is quite independent of the dorso-medidl angle of the ventricle. The dorsal ventricular ridge therefore is clearly a thickening of the lateral wall above the lateral zona limitans. This zona limitans is the light streak in the drawings running diagonally between d.v.r. above and n.l. and l.pyr. below. The nucleus of the diagonal band is very large and definite in figure 17, while in figure 15 and 16 its substance is fused with the nucleus of the lateral olfactory tract. The lentiform jiroliferation is still in progress, especially from the dorsal lip of the middle sulcus. The peculiar arrangement of ependyma cells seen at this point in the adult already begins to be apparent here.
Figs. 18 to 27 Transverse sections of the right hemisphere of the 28 mm. embryo of Chrysemys.
Fig. 18 Section at about the point where the fimbria curves down behind the choroid area. Caudal to this the nucleus of the lateral olfactory tract soon disappears and the dorsal ridge continues on into the caudal pole as a simple thickening of the lateral walls. At g.p.? is a partly segregated mass of cells which probably represents general pallium extending from the caudal pole forward to this point in the basal wall as it does in the adult.
DORSAL VENTRICULAR RIDGE
501
'ntrolflat
502 J. n. JOHNSTON
Fig. 19 Section 120 inicirons rostral to figure IS. In botli these sections the connection of the dorsal ridge with the pallium is clear and they illustrate the more embryonic conditions which prevail in the caudal pole than in the rostral part of the hemisphere.
Pig. 20 Section at the point of connection of the hemisphere with the thalamus where the stria medullaris passes into the thalamus. In this and the next two figures the lateral zona limitans appears as a diagonal light line running dorsolaterally from s.v.m. to the outer surface.
Eig. 21 Section through the foramen interventriculare. The lentiform and caudate nuclei, neither of which was distinguishable in figure 20, are here quite distinct. The crus in this figure separates the caudate from the nucleus of the diagonal band. From this point rostrad for thirty to forty sections the crus lies practically exposed on ths lateral surfac3. This condition is represented by a small striatal area in the model of this brain (fig. 5).
Fig. 22 Section through the anterior commissure. Here the general pallium begins to be decidedly thickened. The motor area of the adult includes this region and extends farther caudad. Forward from this level the olfactory area is again continuous on the lateral, basal and medial surfaces.
DORSAL VENTRICULAR RIDGE
m
504 J. H. JOHNSTON
Fig. 23 Section through the rostral wall of the preoptic recess and the nucleus of the diagonal band (gyrus subcallosus). Between the level of figure 22 and this the pyriform lobe has rapidly pushed up over the outer surface of the pallial thickening as described in the text. The dorsal ridge is decreasing in size and the arrangement of its cells in this and in figure 22 suggests the core-nucleus of the adult. The dorsal ridge and the pallial thickening form a common large ridge, while in the adult they are separated by a groove (see '15 b, fig. 10).
Fig. 24 Section through the rostral end of the dorsal ridge and through the fissura prima, in which the fibers of the diagonal band are turning up into the medial wall. Both pallial thickening and pyriform lobe are massive.
Figs. 25, 26 and 27 Sections through the anterior part or motor area of the general pallium. The whole of what is called general pallium here becomes more massive in the adult and is included in the pallial thickening. An important feature of these sections is that the general pallium especially at the dorso-medial angle is composed of conspicuously large cells. If the results of experiments reported elsewhere in this journal are to be credited, this is certainly a part of the motor area and probably is concerned with the control of the limbs. The presence of the large cells is readily understood if this is true.
DORSAL VEXTRICULAR RIDGE
505
THE MORPHOLOGY AND MORPHOGENESIS OF THE
CHOROID PLEXUSES WITH ESPECIAL REFERENCE TO THE DEVELOPMENT OF THE
LATERAL TELENCEPHALIC PLEXUS
IN CHRYSEMYS MARGINATA
l'i;i{(I\AL MAILi:V From llic Aualoinirnl Lnlxunlni!/ of Die Xorthirrslrnt r>iii'crsilif Medical School^
TWKNTV-SKVKN FKiURES
(•(».\ii;Ni-s
Introduction 507
History 5f;S
Material and Methods . 519
Doscri|)tioii 520
Discussion 525
Conclusions . 528
IN ll{< (DICTION
111 ;i rectMit coiiiinuiiicataoii. llic aullun- ( I^ailcy, 'L')) presented an inteipietation of the latertil telenceplialie elioroid plexus in the human embryo based on ontoj:;enetie and pliylop;enetic evidenee. The phylog;enetie evidence ])resented consisted of the most accurate descriptions a\'aihible of the de\'elopment of this structure in the lower vertebrates, viz., Warren's ('05) account of Necturus maculatus and Tandler and Kant or 's ('07) account of Platydactylus mauritanicus. With a \'iew to confirming, and strengthening if possible, the phylogenetic evidence it was determined to in\'estigate more definitely the de^'elopment of the prosencephalic choroid plexuses in others of the lower vertebrates. The following scheme taken from Wilder was accepted
1 Contrihutioii No. JJ. Suhinittcd for publication July 20, 1916.
507
5()S PEHCIVAL HAILKV
to represent as accui-ately as possible the phylogenetic stages of greatest value:
Ainphioxus
Cyclostoines
Selachians
Ganoids
Urodeles (for Stegocephali)
Chelonia (for Theromorphs)
Monotremes (for Pantatheria)
Marsupials (for primitive Insectivora)
Insectivora
Lemurs (modern Mesodonta)
Cercopithecoidae (tailed moniveys of Old World)
Tailless Apes (Gorilla, etc.)
Pithecanthropus (extinct)
Homo primigenius (extinct)
Homo sapiens
Below the Urodeles, the lateral telencephalic choroid plexus is very rudimentary if it appears at all. Its condition in the Urodeles has been thoroughly elucidated by Warren's ('05) account of Necturus maculatus. For the Chelonia, Chrysemys marginata was selected because it was readily accessible and also because a great part of the work had already been admirably done by Warren ('11) in the course of his investigation of the Paraphysis and Pineal Region in Reptilia." It stands a little closer to the line of ascent of the mammals than the hzard, Platydactylus mauritanicus, described by Tandler and Kantor. Of the forms above the Chelonia, only Didelphys virgin iana representing the Marsupials, was available. The present communication is concerned with Chrysemys marginata and a review of the literature, leaving Didelphys for a later paper.
HISTORY
There are no choroid plexuses in Amphioxus (Burckhardt, '94).
Ahlborn ('83) describes well developed plexuses in the roof of the hindbrain of Petromyzon planeri, and also in the roof of the midbrain. The plexus of the diencephalic roof is not so well developed and no reference is made to any telencephalic plexuses. Burckhardt ('94) writes of Petromyzon fluviatihs:
MORPHOGENESIS OF THE CHOROID PLEXUSES 509
Aiich hier wie bei Teleostiern bleiben die Plexus inferioris (median telencephalic choroid plexus) nur in Gestalt einer Querfalte nachweisbar, die Paraphyse ist eine blosse Kuppel, der caudalen Rand wir als rudinientares \'elum auffassen.
There are no lateral telencephalic choroid plexuses in Cyclostomes.
Among the Selachians, ho^ve^"er, there is a well formed plexus inferioris in Xotidanus according to Burckhardt ('94), but no lateral telencephalic plexuses appear. On the other hand, I infer from Kappers and Carpenter ('ID tliat in Chiniaera monstrosa the lateral plexuses are present.
Der ependyniale Theil der Sciiluszplatlc \V()ll»t sicli in ihiciii tVoiitalsten Al)schnitt ctwas iiber deni Niveau des (ieliinies hiuaus cine Art paraj)hyse darslt'ilciul uiu sicli daini plotzlieh wieder eiuzufalteii und in den unparen X'entrikel eindrinjicnd den plexus chorioideus vent rieuli imparis zu bildcn. wovon audi jicrinRe Auslaufci- in den schnialen Seit(MiventriUe!n ilvt^ (lehirns eindrinjien.
And Minot ('01) in dosciihing Acantliias lemarks:
verA
The velum has now distinctly the character of a choroid |ilcxus, beiu}!; _ry irregular in the form of its surface, rich in blood vessels, covered by a thin ependyma ami projecting far into the ca\'ity of the brain. Laterally the projections from its surface are much more developed and as the organ has grown forward alongside the median para])hysal arch, it has produced what we can now easily identify as the jilexus of the lateral ventricle. These i)lexuses are therefore to be interpicted morphologically as scH'ondary modifications or appendages of the primary velum transversum Attention should be jiaid to the
two lateral projections. L.ch., of the ependyma on the anterior sui-face of the velum, because these j^rojections not only fix the lateral boundaries of the paraphysal arch but also are the anlages of the choroid plexuses of the lateral ventricles. These anlages from this stage (28.0 mm.) on rapidly increase both in size and in comjilication of form.
D'Erchia ('9(3) shows in Torpedo the velum transformed into a plexus and the tela chorioidea diencephali practically nonexistent. In fact, this seems to be the tendency of these two structures in the entire Selachian group. There is a i)lexus in the roof of the fourth ventricle in all Selachians.
No plexus develops in the tela chorioidea telencephali medii in Ganoids, but in Acipenser, von Kupffer ('93) figures a plexus formation arising from the anterior wall of the velum trans
510 PERCIVAL BAILEY
versum, and Terry ('10) describes such a formation in Amia, as well as folds which appear on the caudal wall of the velum. The tela chorioidea diencephali itself forms a large thin-walled dome with no plexus formation. There is a plexus in the roof of the fourth ventricle. Lateral telencephalic choroid plexuses according to Burckhardt ('94) are absent, but Hill ('94) writing of Amia says: "It (the paraphysis) may be thought of as an isolated portion of the roof of the fore-brain which owes its existence to the formation of the folds marked Pl.chr. in figure 20, and which are themselves the representatives of the choroid plexuses of the lateral ventricles."
Of the Urodeles, Burckhardt ('91) has described extensive plexuses developing both from the tela chorioidea telencephali medii and from the tela chorioidea diencephali in Ichthyophis. Warren ('05) shows the enormous diencephalic plexus of Necturus absorbing also the entire caudal wall of the velum transversum. There is a plexus in the roof of the fourth ventricle. The lateral telencephalic plexuses are present and arise from the base of the median telencephalic plexus (plexus inferioris) as has been attested by Mrs. Gage ('93), Studnicka ('93), Warren ('05) and Burckhardt ('91). Burckhardt ('91) says of Ichthyophis: "Die Plexus der Hirnhemispharen aber spalten sich je in zwei Stamme von denen der eine sich gegen das Zwischenhirn ausstreckt, und in der Folge zuerst sich in Zweige spaltet, indess der andere in den Hemispharenventrikel eindringt und sich sodann in zwei Zweige spaltet, einen nach rtickwarts umbiegenden, welcher den Ventrikeltheil des Temporallappens und einen, welcher das iibrige Vorderhirn versorgt." And Warren ('05) writes concerning Necturus: "The telencephalic plexus develops from the paraphysal arch " "The plexuses of the hemispheres arise on either side from the origin of the telencephalic plexus and pass into the lateral ventricles."
Concerning the Chelonia, Humphrey ('94) shows a plexus arising from the tela chorioidea telencephali medii in Chelydra serpentina, as does also C. L. Herrick ('91) in Cistudo. Warren ('11) states that the plexus is not present in Chrysemys marginata and with this statement my observations agree. The "two
'MORPHOGENESIS OF THE CHOROID PLEXUSES 511
paired masses growing backward from the origin of the lateral plexus into the diencephalon" of which Warren writes do not seem to me to be at all homologous with the median telencephalic plexus. They arise too far posterior on the plexus; are at no time connected with the roof plate; and are merely prolongations of the lateral plexus. The diencephalic plexus is not so well developed as in Urodeles but is present in all forms, as is also the plexus of the fourth ventricle. Humphrey ('94) shows in Chelj'dra serpentina the lateral telencephalic plexus arising from the base of the median telencephalic plexus and passing into the lateral ventricle. According to Warren ('11), in Chrysemys marginata, "The plexus chorioideus laterahs springs from the paraphysal arch immediately in front and lateral to the mouth of the paraphysis, figure 25, and invaginates the dorso-mesial wall of the hemisphere."
In considering the Mammalia, considerable space will be given to interpreting accurately the velum and paraphysis. This is rendered necessary if one is profitably to homologize the lateral telencephalic plexus of Mammalia with the same structm-e in the lower vertebrates. Since, as we shall see later, the jiarai^h}'sis never appears in Mammalia excei)t as an arch of the roof plate of the telencephalon in early stages of development, it will be referred to as the paraphysal arch. ^Nlinot ('01) used the term 'to apply to the entire roof plate of the telencephalon between the velum transversum and the lamina terminalis, a sense in which it is no longer used.
The only observations of value dealing with the Monotrenies are those of Th. Ziehen ('05) on Echidna hystrix and G. Elliot Smith ('97) on Ornithorhynchus, and these leave much to be desired. Smith describes the origin of the lateral telencephalic plexuses and a structure which he says "constitutes the paraphysis of Selenka." I do not know what ex'idence he had for stating that the structure he describes is the paraphysis of Selenka for so far as I ha^'e been able to determine, Selenka has nowhere a description of the paraphysis but merely the bald statement that it is present in Marsupials. Smith's statement probably implied no more than that he interpreted this structure as the
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5
512 PERCIVAL BAILEY
paraphysis. Extracts from this paper follow and will be discussed later.
The lamina suprancuroporica takes a sudden bend backwards (fig. 3) to form a horizontal band, which gives origin in many lowly vertebrates to the plexus inferioris, and in higher animals to the plexus lateralis as well, or exclusively. In the specimen under consideration, however, although the plexus laterales do not actually spring from this lamina, they are formed from the caudal prolongation of its lateral
parts In describing the structures met with in a medial
sagittal section it was mentioned that the dorsal part of the (actual) anterior wall of the median cavity of the forebrain was bulged out to form a large sac. The corresponding structure is well seen in the early
embryo of Parameles In the Platypus embryo, however
(fig. 2), a well developed choroidal fold extends from the superior commissure to the lamina from which the lateral plexus arises, completely invaginating the paraphysis (figs. 7, 9 and 15) in the middle line. In Platypus the transition from optic thalamus to paraphysis is a very gradual one, so that in examining a series of coronal sections the lateral walls of the diverticulum would seem to be merely the forward continuation of the ependymal layer of the Fliigelplatten (fig. 15).
As a matter of fact, the appearances in this series of coronal sections are not deceptive at all, the sac being undoubtedly just what it appears to be, an anterior pouch of the choroid plexus of the diencephalon, the lateral walls of the pouch being actually the forward continuation of the Fliigelp'atten. A glance at figures 1, 2 and 3 will make this quite apparent. The similarity would be much more obvious if figure 3 were from a coronal instead of a transverse section. In front of this pouch lies the velum transversum and then the roof plate of the telencephalon (lamina supraneuroporica as he calls it), from the caudal prolongations of the lateral parts of which arise the lateral telencephalic plexuses. In a later article, Smith ('03) reaffirms his belief that the anterior extremity of the lateral telencephalic plexus arises from the roof plate. The embryo is doubtless too far advanced to show the true paraphysal arch.
Ziehen's work on Echidna is eminently unsatisfactory from the standpoint of this discussion, because the sections he shows invariably skip the anterior end of the lateral telencephahc plexus. He has but one suggestive statement referring to this region:
MORPHOGENESIS OF THE CHOROID PLEXUSES
513
Im Bercich des Sulcus hemisphaericus ist die mediale Hemispharenwand verdiinnt und taschwartig in das Heniispharenlumen eingestiilpt. Diese Tasche entspricht dem Plexus chorioideus ventriculi lateralis. Sic offnet sich also in den Sulcus hemisphaericus (und zwar in seine laterale Wand) in der Decke des Foramen Monroi und conmiunieirt sowohl mit der Sichelspalte wie mit dem hinten obsteigenden zwischen Zwischenhirn und Heniisphareuhirn gelegenen Absclmitt des Sulcus hemisphaericus.
P'ig. 1 Coronal section from the forebrain of an oinhryo of Paramelea nasuta.
Copied from Smith. Labels mine.
Fig. 2 Coronal section from the forebrain of a foetal Ornithorhynchus. Copied from Smith. Labels mine.
Fig. 3 Transverse section through the forel>rain of a 10 mm. human embryo. H 173, Chicago Eml)ryological Collection. Slide L'l, Section 11. X 13 J.
REFERENCE LETTERS
a.a.c.l.L, anterior area chorioidea tel encephali lateralis CO., chiasma opticum c.p., commissura posterior C.S., corpus striatum d-t.gr., di-telencephalic groove ep., epiphysis f.c, fissura chorioidea f.M.. foramen interventriculare hem., hemisphere wall hy., hypothalamus Li., lamina terminalis mes., mesencephalon n.a.L, nucleus anterior thalami n.o., nervus opticus p.a.c.t.l., posterior area chorioidea tel encephali lateralis
p.a., p.t.L, pars anterior i)le.\us telen cephali lateralis par., paraphysis p. p., p.t.L, pars posterior ple.xus telen cephali lateralis r.inf., recessus infundibuli r.m., recessus mamillaris r.posl., recessus postopticus r.pre., recessus preopticus Led., tela chorioidea diencephali t.c.t.in., tela chorioidea telencephali
medii i
l.f., taenia fornicis th., thalamus
t.r-p., telencephalic roof-plate t.L, taenia thalami v.L, velum transversum
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PERCIVAL BAILEY
The 'Sichelspalte' I understand to be the cleft between the hemispheres in front of the diencephahc roof. The lateral telencephalic plexus, then, must extend anteriorly beyond the velum transversum into the roof plate of the telencephalon.
Ziehen also is inclined to interpret the anterior extremity of the diencephalic roof in Echidna as the paraphysis. He remarks:
Das ganze Bild erinnert an die Paraphyse mancher Reptilien. Ich stehe auch nicht an, diese leichte fordere Zuspitzung des hinteren Kuppelgebietes der Paraphyse homolog zu sitzen, wie im vergleichenden Abschnitt specieller erortert werden soil. Eine tiefere Einsenkung hinter der Zuspitzung — etwa im Sinne eines Velum transversum — f ehlt. Vor der Zuspitzung beginnt sofort die Einsenkung der Fossa interhemisphaerica.
ted
Fig. 4 Section from the forebrain of a 6| mm. embrj^o of Echidna hystrix. X 30. Copied from Ziehen. Labels mine.
Fig. 5 Transverse section from the forebrain of a 14 mm. pig. no. 18, Chicago Embryological Collection. Slide 5, section 19. X 25.
In an older embryo of which he says, "Sehr bemerkenswerth is wiederum das Verhalten des Kuppelgebietes des Zwischenhirns gegen die Fossa praediencephalica hin," he describes nothing resembling a velum transversum and a comparison of figures 4 and 5 will show the resemblance of his 'Kuppelgebietes' to the anterior pouch of the tela chorioidea diencephali of a pig embryo. If the plane of section in figure 5 were homologous with that of figure 4 the resemblance would be much more striking. If it be really the paraphysis, it forms a good transition stage to the condition found in higher Mammalia.
MORPHOGENESIS OF THE CHOROID PLEXUSES
515
Observations on the Marsupial brain are remarkably meagre. I have been able to find but two statements bearing on the subject. Broom ('97) says of a 14.8 mm. embryo of Trichosurus vulpecula: "The choroid folds into the lateral ventricles, is partlj^ formed, and the paraphysis well marked." He has no figures of sections. Selenka ('91) has a simple statement that the paraphysis is present in Marsupials, presumably in the opossmn. Bis jetzt habe ich die Paraphyse bei embrj'onen von Haifischen, Reptihen, und Beuteltieren beobachtet, zweifle jedoch nicht, dass sie alien Wirbeltieren gemeinsam ist." That is all, but I hope soon to fill this gap.
Fig. 6 Sagittal section of the forcbraiii of a 15 mm. pig. Copied from Johnston. X 10. Labels mine.
Fig. 7 Sagittal section of the forebrain of a 11 mm. embryo of Erinaceus europaeus. Copied from Gronbcrg. X 10. LabeLs mine.
Thanks mainly to Gronberg, the brain of the Insectivora is better known. His work was done with Erinaceus euro])aeus. Ziehen ('06) says of Tupaja, "Die Verhilltnisse gleichen den fiir den Igel beschriebenen in hohem Masse."
Gronberg's figures 25, 26, and 27 show clearly the velum transversum and paraphysal arch although he is loath to call them so. A comparison of figures 6 and 7 will make this plain. He describes also plexuses in the diencephalon and fourth ventricle. Of the lateral telencephahc plexus he writes:
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PERCIVAL BAILEY
Die Adergeflechtsfurche entstcht am friihesten. Sie ist schon bei meinom Stadium C (11 mm.) vorhandcn und sowohl ausgebildet, dass ihre crsto Entstehung sicher aiif cinem Ijedeutend jungern Stadium zu suchen ist. Doch fiiidct sich auf Stadium B (8 mm.) noch keine Spur einer Faltenbildung. Die Form und das Aussehen der Falte ergeben sich aus den Figg. 52 und 53. Man sieht, dass sie in ihrem vordern Theil weiter in die Hemispharenhohle hineinreicht, als es mehr caudalwiirts der Fall ist. Es zeigt sich auch, wenn man eine Schnittserie durchmustert, dass die Falte nach hinten allmahhch kleiner wird und schlicsslich nur eine leichte Einbuchtung darstellt, welche nach hinten ganz allmahlich verstreicht. Der vordere Theil dagegen verhallt sich ganz anders. Die Falte ist hier tief und erstreckt sich mit ihrem freien Rand weiter nach vorn als die mit der iibrigen Hirnwand in Verbindung
stehende basis Auf den folgenden Stadien vergrossert sich
die Adergeflechtsfalte bedeutend, besonders in ihr vorderer, freier Theil, ....
Fig. 8 Cross section through the forebrain of a 15 mm. embryo of Erinaceus
europaeus. Copied from Gronberg. X 10. Labels mine.
Fig. 9 Cross section through the forebrain of a 32 mm. human embryo. H41, Chicago Embryological Collection. X 25. Slide 29, Section 6.
One would conclude from this description that the anterior end of the lateral plexus had arisen the earlier, and that it arises from the roof plate of the telencephalon is clearly shown in his figure 54, which is here reproduced with a section from a human embryo for comparison (figs. 8 and 9). Again if the section of the human embryo were a coronal instead of a transverse section, the similarity would be more obvious.
MORPHOGEXESIS OF THE CHOROID PLEXUSES 517
Of the forms between Insectivora and Man nothing of value is known. Ziehen ('06) shows sections of the lateral telencephalic plexus of Tarsius spectrum, a prosimian, but not in the proper plane to be of value, and again describes the anterior pouch of the diencephalic roof as the paraphs-sis.
Sehr beachtenswert ist auch, dass unmittelbar hinter der Fossa praediencephalica die epitheliale Dccke des Z\\-ischenhirns sich zu einer stulen Falte, welche an die Parapliyse erinnert, erliebt und dass erst einige Schnitte weiter occipitahviirts diese steile Falte durch den nicdiane Plexus des 3. Ventrikels eingestiilpt wird.
In an earlier paper, the author (Bailey, '15) called attention to the true homologue of the paraphysis in the human embryo and insisted upon its position in the telencephahc roof plate just anterior to the velum transversum. Previously, Streeter ('12), Francotte ('94) and D'Erchia ('90) had written of the paraphysis in the human embryo. Streeter homologizes the anterior pouch of the diencephalic roof with the paraj^hysis, and from a comparison of Francotte 's figures witli sections of embryos of approximately the same stage, I am convinced that he mistook the same structure for the paraphysis. D'Erchia shows a section from a 30 mm. human embryo in which he labels a structure 'paraphysis' which seems to me to be merely an oblique section of the lateral telencephalic i)lexus.
In his model of a LS.O nmi. human embryo. His shows clearly the lateral telencephalic plexus coming off lateral to the paraphysal arch, but his statement ('04) of the origin takes no account of this fact. "Sein deni Thalanms angehefteter Randstreifen bleibt epend\^nal und in ihm bildet sich die Fissura chorioidea, von der aus die Epithelfaltungcn des Corpus chorioideum in den Seitenventrikel sich einstiilpen."
Hochstetter ('13) in his account of the development of the lateral telencephalic plexus in the human embryo shows one figure (fig. 6) of a section through the plexus which passes also through the telencephahc roof plate, but his description contains nothing concerning the origin of this part of the plexus.
D'Erchia ('96) considers the lateral telencephalic plexus to be derived from the \'elum transversum: Per questa parte volga
518 PERCIVAL BAILEY
tutto quello che si e detto per la cavia. Dal velum per diverticoli lateral! di formano i plessi emisferici inferiori destro e sinistro, dei qiiali uno e disegnato nella fig. 28."
Finally the author (Bailey, '15) has presented an account of the development of the lateral telencephalic plexus which it is the purpose of the present piper to supplemant.
Concerning these structures in other groups of vertebrates this discussion is not so much concerned, but a few words at least about the lateral plexus in other vertebrates will not be out of place. This plexus is not present in Teleosts (Burckhardt, '94) nor Anura (Herrick, '10). In Lacertilia its development has been described by Tandler and Kantor ('07) and Warren ('11) and agrees in all essential respects with that hereinafter detailed for Chrj^semys marginata. In examining some young alligators in the collection of Dr. Elizabeth Crosby, the posterior part of the lateral telencephalic plexus was found to be present, but very small and poorly developed. The brain of an adult pigeon was also examined and the lateral telencephalic plexus was seen arising from a plexus formation in the roof of the telencephalon, but of the posterior part of the plexus there was no sign except a plexus of blood vessels along a thin but uninvaginated medial hemisphere wall. Dr. C. Judson Herrick suggested that this may represent a stage in the phylogenetic development of the plexus. Mrs. Gage ('95) figures the brain of the embryo of the English sparrow. Both the figures and the context show that the' lateral telencephalic plexuses arise from the median telencephalic plexus and just lateral to the paraphysis, apparently not invaginating the medial hemisphere wall. She writes : In the young sparrow ( g. 2) the paraphysis occurs in the midst of a mass which gives off the paraplexuses, and it opens directly dorsad of the portas, i.e., into the aula. . . . It is noticeable in the older embryo that 1;he union of the auliplexus with the paraplexuses lies dorsad of the porta (fig. 14)." The works of Neumayer ('99) and Hochstetter ('98) contain nothing of value for the present discussion. The two invaginations which they describe are temporaTy and unimportant. A similar condition is shown in my figure 19 (Bailey, '15). D'Erchia ('96)
MORPHOGENESIS OF THE CHOROID PLEXUSES 519
considers the lateral plexus in the guinea pig to be derived from the velum transversum. "E dal velum che si origina il plesso del III ventricolo e i plessi coroidei emisferici destro e sinistro." It is interesting also to note that although Tilney ('15) correctly labels the paraphysal arch in models of the brains of young cat embryos, in later stages he invariably transfers the label to the anterior pouch of the diencephalic roof. Johnston ('09) has correctly interpreted the paraphysis in pig embryos, but his statement that the lateral telencephalic plexus appears as a folding of the anterior wall of the velum transversum is misleading for ho immediately follows with the statement that it is separated from the velum by tlic i^araphysal arch.
In the angle between the vesicle and the dicncepluilon appears the chorioid plexus pushing into the lateral ventricle. It appears as a folding of the anterior wall or limb^jf the velutn transversum and its lateral prolongation. In this way ai)pe:ir.s the chorioid fissure whose further history need not be traced. Near the median line the plexus apjiears as a fold projecting into tiic interventricular foramen and separated from the velum transversum by the paraphysal arch.
MATKI{IAL AND .MHTHODS
The material on which this study is based consists of a series of twelve embryos of Chrysemys marginata from mj'- own collection, ranging in size from 5.1 mm. greatest length to one having a carapace 10.0 mm. long, and an 8.8 mm. embryo from the Harvard Embryological Collection, very kindly loaned to me by the Harvard Laboratory. All of my material was fixed in Zenker; stained in bulk with borax carmine; embedded by the celloidin-paraffin method; cut at 10m in transverse series; and counterstained on the slide with orange G. The excellent preservation of the form relations of the delicate membranous portions of the brain may be attributed to the method of embedding. The embrj'os were all passed from 95 per cent alcohol to etheralcohol; then through 0.5, 1 and 2 per cent celloidin; hardened in chloroform alcohol, and cleared in benzol, before embedding in paraffin. The Harvard embryo is cut in 10 m sagittal sections and stained with borax carmine and eosin.
620 PERCIVAL BAILEY
The forebrains of four embryos — 1 a, greatest length 5.1 mm.; H. E. C. no. 1433, greatest length 8.8 mm. ; 5 b, carapace 8.6 mm.; and 4 b, carapace 10.6 mm. — were reconstructed by the Born method at a magnification of 100 diameters. Millimeter plates were used and every section drawn. It was necessary to dissect the models rather extensively to expose- the fissura chorioidea. The models were stacked from a side view of the head drawn from a photograph, with the exception of the Harvard embryo. This latter embryo was cut sagittally and the stacking was guided by the epiphysis and paraphysis. Since the embryo was not cut in an exactly sagittal plane, after the paraphysis and epiphysis had passed out of the plane of section, most of the lateral telencephalic plexus had been stacked and the remaining sections were added with no other guide except comparison with other models.
DESCRIPTION
This description will be confined to the lateral telencephalic plexus, since concerning the other plexuses I find no reason to differ from Warren's account, with the exception noted in the history.
The main landmarks of the region in which the lateral telencephalic plexus develops are already laid down in an embryo of 5.1 mm. greatest length. Figure 26 shows the roof plate of the forebrain in such an embryo and figure 10 shows the same region schematically represented. The roof plate of the telencephalon, back of the lamina terminalis, appears as a triangular area (only half of it shown in figure 26) its base formed by the velum transversum and its apex lying at the posterior end of the lamina terminalis while from its center arises the paraphysis. The lateral sides of the triangle are formed by the taeniae fornices. At the lateral angles of the triangle, taenia fornicis, velum transversum, taenia thalami and di-telencephalic groove meet. It is not possible accurately to determine this point in the embryo under consideration because the roof-plate has been but imperfectly differentiated histologically, but in later stages these angles of the triangle may be easily located.
MORPHOGENESIS OF THE CHOROID PLEXUSES
521
The earliest unquestionable appearance of the lateral telencephalic plexus is in the Harvard enibrj'o, 8.8 mm. in length. It appears here as a crescent-shaped ridge projecting into the lateral ventricle, with two more strongly developed points showing as small elevations (fig. 22). The anterior extremity of the plexus lies clearly in the roof of the telencephalon lateral to the paraphysis and medial to the taenia fornicia. The taenia fornicis appears in figure 22 as a ridge apparently in the medial hemisphere wall. It bears here a very close superficial resemblance to the hippocampal ridge in a young human embryo, but its later development and internal structure show plainly that
Fig. 10 Diagniiii of the region around the paraphysis in tlie roof of the forebrain of a 5.1 mm. emiiryo of Chrysemys marginata.
Fig. 11 Diagram of the same region in an embryo of 8.8 mm.
Fig. 12 Diagram of the same region in an embryo having a carapace of S.6 mm.
it is the taenia fornicis. Its apparent position in the medial hemisphere wall is an illusion produced by the invagination of the plexus between it and the paraphysis. The triangular area of the telencephalic roof plate is well marked and the lateral angles can be determined. A sagittal section through the triangle is practically a straight line, except for the evagination of the paraphysis (figs. 13 and 21) as are also parasagittal sections (fig. 14). Nevertheless the lateral angles are depressed and the sides of the triangle are concave outward. Transverse sections through this region are therefore curved and convex upward (fig. 21). The velum trans versum sharply dehmits the telencephalic roof plate at the base of the triangle (fig. 14).
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Figure 15 shows ;i parasagittal section still farther laterally through the triangle. The velum transversum is obvious, the plexus invaginating the roof plate medial to the taenia fornicis and tilting the portion of the roof between itself and the taenia. Above and lateral to the taenia fornicis the brain wall is histologically differentiated; medial it is ependymal. This difference is much more apparent in later stages (fig. 17). Posteriorly the plexus is less well developed and as we approach the lateral angles of the triangle, plexus, velum, and taenia fornicis tend to merge into one groove which becomes continuous with
Fig. 13 Sagittal section no. 135 from an 8.8 mm. embryo of Chrysemys marginata. Harvard Embryological Collection, no. 1433. X 33j. Fig. 14 Parasagittal section no. 130 from the same embryo. Fig. 15 Parasagittal section no. 124 from the same embryo.
the di-telencephalic groove. Lateral and anterior to the point where the taenia thalami meets the di-telencephalic groove and velum transversum the hemisphere wall is massive and uninvaginated. It would seem, then, that in this embryo, the lateral telencephahc plexus lies entirely in the roof plate of the telencephalon. The condition is diagrammatically represented in figure 11.
Figures 23 and 24 depict the region around the foramen of Monro in an embryo with a carapace 8.6 mm. long, about 14 mm. greatest length. Figure 23 looks at the foramen of the right half of the brain from the medial side. The cephalic end of the
MORPHOGENESIS OF THE CHOROID PLEXUSES
523
model is, consequently to the left. The paraphysis which had been removed is represented by a line of white dashes. Figure 24 looks at the same foramen from the lateral side. The model is therefore reversed and the cephalic end points to the right.
The condition of the roof-plate is diagrammatically represented in figure 12. The differentiation between hemisphere wall and
Fip;. IG Transverse section from the forebrain of an embryo of Chrysemys
marginata having a carapace 8.6 mm. in length. Embryo 5 b, slide 13, sect. 11.
X 33 J.
Fig. 17 Transverse section of the same embryo; section IG, slide 13. X 33J.
roof plate is clear and the taenia fornicis obvious (figs. 10 and 17). The anterior extremit}' of the lateral telencephalic plexus arises plainly from the telencephalic roof plate lateral to the paraphysis and medial to the taenia fornicis (fig. IG). Figure
17 shows a section posterior to figure 16 through the main body of the plexus, the plexus still b'ing in the roof plate. Figure
18 is of a section still farther posteriorly. The plexus is here shown crossing the taenia fornicis into the medial hemisphere wall. The taenia fornicis is now medial to the plexus and drop
524
PERCIVAL BAILEY
ping down to meet the anterior nucleus of the thalamus, which it does in a few sections and becomes continuous with the taenia thalami. In figure 19, much farther posteriorly, the taenia thalami is present, the plexus being entirely in the medial hemisphere wall. The portion of the plexus arising from the medial hemisphere wall is very poorly developed (fig. 19) but its area of invagination is extensive (fig. 24).
In later stages this posterior part of the plexus develops more rapidly and overshadows the other. Figure 25 shows a lateral
Fig. 18 Transverse section of the same embryo as figure 16. Slide 13, section 22. X 33 J.
Fig. 19 Transverse section of the same embryo; section 5, slide 14. X 33|.
view of the region around the foramen of Monro in an embryo with a carapace of 10.6 mm. A pen sketch of the entire model is appended (fig. 27) showing the region represen ed in figure 25. (Figure 24 is of a homologous region in a younger embryo.), The fissura chorioidea appears merely as a big hole in the medial hemisphere wall; all the landmarks are lost. In still later stages, this hole becomes reduced to a long narrow slit.
The development of the plexus in size and shape is so well discussed and figured by Warren ('11) that it will not be considered here.
MORPHOGENESIS OF THE CHOROID PLEXUSES 525
DISCUSSION
From the foregoing history and description, and from an analysis of the remaining hterature, for in the history are included only the most important papers and especially those deaUng with the lateral telencephahc plexuses, it will be seen that there are certain definite regions of the brain wall wherein choroid plexuses develop. These regions and the plexuses which develop from them may be tabulated as follows:
Tela chorioidea telencephali medii— Plexus telencephali medius Anterior area chorioidea lateralis telencephali — Plexus telencephali lateralis (below Chelonia)
{Anterior area chorioidea lateralis telencephali — Plexus tclencoi)hali lateralis (Chelonia and above) Posterior area chorioidea lateralis telencephali (heraispherici) — Plexus telencephali lateralis (Chelonia and above) Velum transversum — Plexus velares Tela chorioidea diencephali — Plexus dicncephali Tela chorioidea mcsencephali — Plexus mesenccphali Tela chorioidea myelenccphali — Plexus myclencephali
Tilney ('15) has suggested that the saccus vasculosus should be reckoned with the choroid plexuses in the forms where it is present and in this opinion I concur. It is best developed in those forms in which the diencephalic plexus is rudimentary or absent, i.e., in Cyclostomes, Selacliians and Ganoids. The plexus formation is ver}' poorly de\'cloi)ed in Urodeles and never again present. There should be added to the above therefore:
Rccessus infuiuiibularis (posterior wall) — Saccus vasculosus.
The myelencephalic ]ilexus arises in the roof of the fourth ventricle, tela chorioidea nwelencephali, in every known vertebrate above Amphioxus.
The mesencephalic plexus is found only in Petromyzon, where it arises from the mesencephalic roof, tela chorioidea mesencephah.
The diencephalic plexus arises from the tela chorioidea diencephali. Appearing first in Cyclostomes, but very poorly developed, it disappears almost entirely in Selachians, where the tela chorioidea diencephali is almost completely absorbed
526 PERCIVAL BAILEY
by. the overgrowth of the vekim transversum. In Ganoids, the tela chorioidea diencephaU begins to emerge, forming a thin walled sac, and in Urodeles is again invaginated by an enormous diencephalic plexus. It may be that the diencephalic plexus in Selachians and Ganoids is represented by the choroidal folds on the posterior limb of the velum, developing from the anterior portion of the tela chorioidea diencephali which has been drawn down into the velum transversum by the overgrowth of the latter. The diencephahc plexus is present in all forms above Urodeles but is never again so well developed.
The velar plexuses are present only in Selachians, Ganoids, and Urodeles. They may involve either the diencephalic limb or telencephalic limb, or the entire velum transversum. The choroidal folds on the diencephalic limb have been homologized, in Selachians and Ganoids, with the diencephalic plexus of other forms, and with some reason. The choroidal folds on the anterior limb are not homologous with either the median or lateral telencephalic plexuses.
The median telencephalic plexus arises from the tela chorioidia telencephali medii, just in front of the paraphysis (paraphysal arch of Mammalia), from the Selachians to the Chelonia, inclusive, with the apparent exception of the Ganoids. It is not constantly present in Chelonia and is never found in Mammalia. Its development seems to be in inverse ratio to the degree of development of the lateral telencephalic plexus and the latter in direct ratio to the size of the hemispheres.
The lateral telencephalic plexus is found in all groups of vertebrates from the lowest to the highest in the line of ascent of Mammals with the exception of the very lowest, Amphioxus and the Cyclostomes. Apparently sporadically and imperfectly developed in Selachians and Ganoids, it is present constantly thereafter. We have stated previously that it is absent in Teleosts and Anura, with which we are not concerned. In all forms below Chelonia, it develops in what I have called elsewhere (Bailey, '15) the anterior lateral telencephalic chorioidal area, in the roof plate of the telencephalon between the paraphysis and the taenia fornicis of the medial hemisphere wall. Where the median
MORPHOGENESIS OF THE CHOROID PLEXUSES ;V2<
telencephalic plexus is strongly developed the lateral plexus appears to be an appendage of it (Necturus, Warren); where the velum is involved in an extensi\'e plexus formation, the lateral plexus appears to be an appendage of it (Acanthias. Minot); but always arises from the region of the telencephalic roof plate described above, and medial to the taenia fornicis.
With the Chelonia comes a change. The lateral plexus arises in the anterior area chorioidea lateralis telencephali as has been previously described but in its later de\elopment crosses the taenia fornicis and invaginates also the i)osterior area chorioidea lateralis telencephali in the medial hemis]ihere wall. Such a condition is found also in the (Jecko brain (Tandler and Kantori. This imohement of the medial hemisphere wall comes more and more to i:)redominate in the development of the lateral plexus as the hemis])heres come more and more to dominate the devol(»))inent of the telencephalon, but even in the highest Mannnaha the anterior extremity of the lateral choroid ])!exus dev(^loi)s from the loof plate of the telence]ihnlon and th(^ evidence ni:iy briefly be summarized hci'e:
(a) Elliot Smith ('S)7) in describing the br.-'iii of a foelal Oi uiIhorhyncus described the lateral choroidal plexus as arising from the continuation backwards of the horizontal jxirt of the lamina sui)raneui'oporica, which term he used to include all the i-oof plate of the telencej^halon between the velum transversum and the lamina terminalis. Again in 190.'?. he reiteiated his belief that the anterior extiemity of the latei:il chomid;!! j)Iexu< :iii>c^ from the roof plate.
(6) Th. Ziehen's figures of l^chidii;', do not show l\w anterior extremity of the lateral plexus, but he states that it opens into the Sichelspalte. that is, into the cleft between the hemisi)heies anterior to the diencephalon, as well as the sulcus hemisphaerieus (di-telencephalic groove) .
(c) Observations on the ]\laisu]iials up to i)resent ie\eal nothing.
(d) Crronberg states of Erinaceus euro])aeus that the anterior extremity of the lateral plexus is better developed in his earliest stage than the posterior extremity and appai-ently arises fii-st.
THE JOURXAI. Ol- COMPARATIVE XEUROI.OGV, VOL. 26, NO. 5
528 PERCIVAL BAILEY
'J'his Iiokls true of all iiiMininals and is what one would expect from the phylogenetic history of the plexus. His figure 42 of a section anterior to the velum shows clearly the anterior extremity arising from tlie roof plate of the telencephalon.
(e) The earliest appearance of the plexus in the human embryo occurs in His' embryo of 13.6 mm. and his model shows clearly the plexus arising just lateral to the paraphysal arch.
(/) Hochstetter ('13) figures a section of a human embryo showing the anterior extremity of the plexus, and the author recently (Bailey, '15) has described three human embryos showing the plexus extending into the roof plate alongside the paraphysal arch anterior to the velum transversum.
(g) Collateral evidence is derived from the development of the lateral plexus in other mammals, for example, Johnston ('09) states that in the pig the plexus arises from the anterior limb of the di-telencephalic groove but is separated medially from the velum transversum by the paraphysal arch.
CONCLUSIONS
1. The lateral telencephalic plexus of Chrysemys marginata arises from the anterior area chorioidea lateralis telencephali in the roof plate of the telencephalon between the paraphysis and the taenia fornicis of the medial hemisphere wall and in its later development oversteps the taenia fornicis just anterior to the point where taenia fornicis, taenia thalami, velum transversum and di-telencephalic groove meet, and invaginates also the posterior area chorioidea lateralis telencephali in the medial hemisphere Wall, just anterior to the di-telencephalic groove. The pars anterior plexus telencephali lateralis which develops from the anterior area chorioidea lateralis telencephali in earlj^ stages is much larger and better developed than the pars posterior plexus telencephali lateralis, but in later stages develops much less rapidly than the pars posterior.
2. All the data at present available support the conclusion that the lateral telencephalic plexus arises from the anterior area chorioidea lateralis telencephali in the roof plate of the telencephalon between the paraphysis (or paraphj'-sal arch) and the
MORPHOGENESIS OF THE CHOROID PLEXUSES 529
taenia fornicis in all vertebrates where it is present, but that in Chelonia and the forms above, it oversteps the taenia fornicis and invaginates the posterior area chorioidea laterahs telencephaU in the medial hemisphere wall and that this latter portion comes more and more to dominate its development as we ascend the vertebrate scale.
I wish here to express my gratitude to Dr. S. Walter Ranson for his unvarying kindness and interest throughout the progress of this work.
530 piokcival ijailkv
i.itki{ati;kk citkd
Ahlbohn, F. 1883 Untersuchunpicii iihcr das (Idiirti dci- I'cdoinyzDiitcii.
Zcits. wiss. Zool., Bd. 39. Haii.kv, p. 1915 The inorpholofj;y of the roof i)Iat(' of the forchraiii and the
lateral choroid i)lexuse.s in tho human ondjiyo. Jour. ('onip. X(!ur.,
vol. 2(;. Bl.\kk, J. A. 1000 Tlic roof aiui lateral recesses of I he fourth ventricle, considered int)i'i)liolojiically and enihryologically. Jour. Coinp. Neur.,
vol. 10. Bkoom, J. 1897 The embryology of Trichosurus vulpecula. Proc. Roy. Soc.
of N. S. Wales, vol. 27. BuRCKHARDT, R. 1891 Untcrsuchuiigen am Gehirn und (leruchsorgan von
Triton und Ichthyophis. Zeitschr. f. wiss. Zool., lid. 52.
1894 Der Bauplan des Wirbeltiergehirns. Morjjh. Arheiten von G. Schwalbe, 4.
D'Erchia, Fl. 1896 Contributo alio studio della volta del cervello intermedio e della regione parafisaria in embrioni di pe.sci e di Mammiferi. Monitore Zoologico, vol. 7, pp. 118 and 201.
Francotte, p. 1894 Note sur I'oeil parietal, I'cpiphyse, la ])araphyse et les plexus choroides du troisieme ventricule. Bull, de I'Acad. roy. de Belg., no. 1.
Gage, Mrs. S. P. 189 ; The brain of Diemyctylus viridescens. Wilder (QuarterCent. Book. ■ 1898.
1895 Comparative morphology of the brain of the soft-shelled turtle (Amyda mutica) and the English sparrow (Passer domesticus). Prf)c. Am. Micr. Soc, vol. 17.
Gronberg, Gosta. 1901 Die Ontogenese eines niedern Siuigergehirns nach
Untersuchungen an Erinaceus europaeus. Zoolog. Jahrb., Abth. Anat.,
Bd. 15, p. 261. Herrick, C. J. 1910 The morphology of the forebrain in Amphibia and Reptilia.
Jour. Comp. Neur., vol. 20. Herrick, C. L. 1891 and 1893 Topography and histology of the brain of certain
reptiles. Jour. Comp. Neur., vols. 1 and 3. Hill, C. 1894 The epiphysis in teleosts and Amia. Jour. Morph., vol. 9, p. 237. His, Wilhelm. 1904 Die Entwicklung des menschlichen Gehirns. Leipzig. HocHSTETTER, F. 1898 Beitrage zur Entwickelungsgeschichte des Gehirns.
Bibliotheca Medica., Abthg. A., Heft. 2.
1913 liber die Entwickelung der Plexus Chorioidei der Seitenkammern
des menschlichen Gehirns. Anat. Anz., Bd. 45, pp. 225-238. Humphrey, O. D. 1894 On the brain of the snapj>ing turtle. Jour. Comp.
Neur., vol. 4. Johnston, J. B. 1909 On the morphology of the forebrain vesicle in vertebrates.
Jour. Comp. Neur., vol. 19. Kappers and Carpenter 1911 Das Gehirn von Chimaera monstrosa. Folia
Neuro-Biologica, Bd. 5. Kingsbury, B. F. 1897 The encephalic evaginations in Ganoids. Jour. Comp.
Neur., vol. 7.
MORPHOGEXESIS OF THE CHOROID PLEXUSES 531
KuPFFKU, C. VOX 1S93 Die Entwickliing des Kopfes von Acipenser sturio.
Studien zur vergloichendcn Entwicklungsgoschichte des Kopfes der
Kranioten. Heft. I., Miinchen.
1903 Die Morphogenio des Xervensystems. Oskar Hortwig's Hand buch. MiNOT, C. S. 1901 On the morphology of the pineal region based upon its
development in Acanthias. Am. Jour. Anat., vol. 1. Xeumayer, L. 1899 Studien zur Entwicklung-sgeschichte des Gehirns der
Ssugetiere. Fcstschr. z. 70 Geburt.stag von C. von Kupffer. Jena.
Fischer. Selenka, E. 1891 Das Stirnorgan der Wirb.'iticre. Biol. Centralbl.. Bd. 10. Smith, G. Elliot 1897 The brain of a foetal oniithorhynchus. Quart. Jour.
Micros. Sci., n. s., vol. 39.
1903 On the morphology of the cerebral commi.ssures. Trans, of
the Linnaean Soc, London., vol. S. part 12. Stekzi, CJ. 1907 II sistema nervoso centrale doi vertebrate. Vol. 1, Ciclostomi. Stueetek, G. L. 1912 The development of the central nervous system. Keibel
and Mall's Human Embryology. Studnicka, F. K. 1S93 Zur Lisung einigcr Fragen aiis der .Morphologic des
\'orderhirns der C'ranioten. .\nat. .\nz.. Bd. 7. Tandlkh, J. A\u KanToh, H. 1907 Beitnige zur Entwickelung des Vertebrat engehirns. I. Die Enfwickelungsgeschichtc des Geckohirnos. .Vnat.
Heflc. Bd. 33. Heft 101. Teiuiy, H. .1. I'.tlO 'i'lic inorpliology of the pineal region in teleosts. Jour.
.M..rpl... vol. L'l. TiL.N'EV, FuEDEUKK 191.^ The morphology of the diencejihalic floor. Jour.
Conip. Xeur.. vol. 2o, no. 3, pp. 213-2S2. Wahhe.n, John- 190") The development of the paraphysis and pineal region in
Xecturus maculatus. .\m. Jour. .Vnat., vol. o, |)p. 1-29.
1911 The development of the paraphysis and |)ineal region in reptilia.
Am. Jour. Anat.. vol. 11, pp. 313-392. Ziehen, Tii. 1904 \'erhand. Kon. .\k. van Wetensch. te .Vmsterdam. 26 Nov.
(pp. 331-340).
1905 Zur Entwickclungsgeschichte des Centralnervensystems von
Echidna hystrix. Jenaischc Denkschriften, Bd. VI, 2. Theil.
190G Morphogcnie des Centralnervensystems der Sjugetiere. Oskar
Hertwig's Tlaiidbuch.
PLATE 1
EXPLANATION OF FIGUKES
The reference letters for all figures are found on page 513. It is impossible to portray all the topography of the region around the foramen interventriculare by means of drawings. For aid in orientation, the position of the principal landmarks is indicated on the models as follows: the taenia fornicis by a line of dashes; the velum transversum by a line of crosses; the taenia thalami by dots; and the di-telencephalic groove by dots and dashes alternating. The cross sections will also aid. On figures 22 and 23 the positions of the sections are indicated by arrows. Since the models are tilted, the arrows indicate accurately only the points where the sections cross the lateral telencephalic plexus.
20 Median view of the fore brain of a 5.1 mm. embryo of Chrysemys marginata. Embryo 1 a. X 50.
532
MORPHOGENESIS OF THE CHOROID PLEXUSES
PEKCiVAL B.ULEY
e p.
,rri es.
533
PLATE 2
EXPLANATION OF FIGlIRKh
21 Median view of the forebrain of an S.8 mm. embryo of Chrysemys marginata. Embryo 1433. H.E.C. X 40.
22 Lateral view of the forebrain of an 8.8 mm. embryo of Chrysemys marginata. Embryo 1433. H.E.C. X 40. Lateral hemispheio wall I'omoved.
534
MORPHOGENESIS OF THE CHOROID PLEXUSES
PERCIVAI, BATTF.V
t.c.t.nn.
he>ri
r.c.cL
.p.t(
M»>i;niiM;i:.\i:sis nv iiri':
^■f.
t.clr^x.
It'-
t.c.cL.
n.a.t.
EXPLANATION OF FIGURES
23 Median view of the region around the foramen interventriculare in an embryo of Chrysemys marginata having a carapace 8.6 mm. in length. Embryo 5 b. X 100. In order to show the fissura chorioidea it was necessary to remove the paraphysis and represent the fissure slightly higher than it really is.
536
MORPHOGENESIS OF THE CHOROID PLEXUSES
PERCIVAL BAILEY
hem.
fc.
Ff'F
t.i
EXPLANATION OF FIGURES
24 Lateral view of the same region as in figure 23. Lateral wall of the hemisphere and plexus telencephali lateralis removed, exposing the fissura chorioidea. Embryo 5 b. X 100.
537
PLATE 5
EXPLANATION OF FIGURES
25 Lateral view of the region around the foramen interventriculare in an embryo of Chrysemys marginata having a carapace 10.6 mm. in length. Embryo 4 b. X 66|. Lateral hemisphere wall and lateral telencephalic plexus removed, exposing the fissura chorioidea.
26 Dorsal view of the forebrain of a 5.1 mm. embryo of Chrysemys marginata. Embryo 1 a. X 80.
27 Pen sketch of model of entire forebrain of embryo 4 b, showing the area from which figure 25 was taken. X 25.
538
MORPHOGENESIS OF THE CHOROID PLEXUSE>
PERCIVAL BAILEY
C.c.t.vn
539
THE STRUCTURE OF THE THIRD, FOURTH, FIFTH,
SIXTH, NINTH, ELEVENTH AND TWELFTH
CRANIAL NERVES
SUMNKIl L. KOCH
From the Analomical Laboratory of the Xorthivestern University Medical School
FIVE FIGURES
FoUowmg the denioiistration of uiiinyeliiiatccl fibers in the spinal Rentes and in the vagus nerve by means of the pyridinesilver technique (Ranson, '11 and '12; Chase and Ranson '14), Professor Ranson suggested the appUcation of the same method to the study of certain of the cranial nerves, with especial reference to the presence or absence of unmyelinated fibers.
The nerves studied were the oculomotor, trochlear, trigem'nal, and abducens of the dog and of man; and the glossophaiyngeal.. accessory, and hypoglossal nerves of the dog, the cat and the rabbit. The nerves were obtained by Ufting off the skull cap and following them distally from their cerel)ral origin by chipping away the base of the skull about their foramina of exit. The di^sccted specimens were laid on glass slides and prepared by different methods. Some were stained by the p>Tidine-silver method; others were placed in 50 per cent pyridine solution for seven days, washed, and then treated with silver nitrate, water, and pyrogallic acid as in the j^yricUne-silver method; others were stained by the Pa'-Weigert method and the osmic acid method. All were cut and mounted serially.
The oculomotor, trochlear, and aixlucent nerves form a natural group formerly described as pureh' motor and consisting of large and small myelinated axons, but now recognized as containing somatic afferent as well as efferent fibers. The nerves are described as communicating with the ophthalmic division of the trigeminal nerve and with the cavernous plexus of the sympathetic system.
' Contribution no. -40.
541
542 SUMNIOIl L. KOCH
(l;isk(>ll ('S<)) stii(li(Hl the ci'anijil nerves with especial reference to their relation to a ty])ical spinal nerve. The oculomotor, trochlear and abducent nei'A'c^s he described as pure efferent nerves. The oculomotor was composed of large and small myelinated fibers, in the dog 14.4 to 18 micra and 3 to 5 micra in diameter. The smaller fibers passed to the ciliary ganglion, the larger to the extrinsic muscles of the eye. The trochlear nerve consisted of large myehnated fibers, 14.4 to 18 micra in diameter, supplying the external oblique muscle, and small myelinated fibers 3.6 to 5.4 micra in diameter, of unknown function. The abducens consisted of large myelinated fibers 14.4 to 18 micra in diameter, and a few smaller ones, but contained no distinct group of small fibers as did the Ilird and IVth nerves. In the roots of these nerves he saw fibrillar structures which he interpreted as the remains of ganglion cells present at an earlier stage of their development, and representing the afferent portion of the nerves.
Barratt ('98, '99, and '01) described the oculomotor nerve as composed of large and small myelinated fibers, 11 to 15 micra and 3 to 5 micra in diameter, in about the ratio of three to one. He found unmyelinated fibers also, both in the fibrous sheath and in the main trunk; in the latter situation usually at the periphery." He did not find any communication with the cavernous plexus nor with the ophthalmic division of the trigeminal nerve. The trochlear nerve was composed of large and small myelinated fibers 12 to 19 micra and 4 micra in diameter, in the ratio of three to one. There were no unmyelinated fibers present. The abducent nerve he described as composed of large and small myelinated fibers, 11 to 17 and 3 to 6 micra in diameter. A few small twigs composed of small myehnated and unmyehnated fibers joined it 25 mm. from its superficial origin, and left it again 10 mm. distalward.
Carpenter ('06) studied the development of the oculomotor and abducent nerves in the chick. He found that the oculomotor nerve was composed of myelinated axons 3 to 15 micra in diameter. The trunk was composed of comparatively large axons, with a few scattered small axons, and a zone of small
STRUCTURE OF THE CRANIAL NERVES 543
axons at the periphery which passed into the ciUary ganglion. He found a ccmmunicating branch extending from the ophthalmic division of the trigem'nal nerve to the ciliary nerve, but none to the undivided oculomotor nerve. He did not speak of a direct communication between the nerves studied and the s>in pathetic system, but referred to Jegorow's suggestion that a distribution of abducent fibers to the eyeball in birds might be accounted for by sympathetic fibers joining the abducens as it passed through the cavernous sinus.
Boughton ('06) found an almost regular increase in the number and size of myehnated fibers in the oculomotor nerve of the white rat and the cat at different ages. In rats of 730 days weighing 414 grams the large fibers averaged 8 1 micni in diameter, the small 4.3 micra. In cats of 2893 grams the large fibers averaged 13.5 micra in diameter, the small 7.2 micra.
Kopsch ('07) described the oculomotor nerve in m;>n as composed of about 15,000 mostly large myeliiuited fibers grou])e(l in a number of sccondaiy bundles. In the roots between the fibers were isolated, branched, s])herical nerve cells. The trochlear nerve he described as ccmposed of about 1200 myelinated fibers, the abducens of about 2600 myeUnated fibers. All three nerves received ccn.niunicating branches from the carotid ])lexus of the sympathetic system, and from the ophthahnic nerve.
THE OCULOMOTOR, THOUIILIvVlJ AND AHOUCIIX T XI:RVIvS
Specimens of the oculomotor nerve of the dog and of three human adults were studied in serial sections. Upon dissection at least two fine branches from the sym])athetic system could be seen joining the nerve. ^Microscopically sections showed in each case clear pictures of a tj^oical motor nerve (fig. 1) composed of large and small mj'eUna ed axons. In the human the ratio of large to small axons was about three to one; in the dog it varied between two to one and three to one. In osmic acid preparations the large myelinated fibers of the dog averaged 12 to 16 micra, the small 3 to 6 micra. Close to its peripheral origin the fibei's of the nerve were grouped in one large bundle, with incomplete septa extending toward the center of the nerve. As
THE JOUKXAL OK COMPAKATIVE NEUROLOGY, VOL. 26. NO. 5
r)44
lUMN'KK I,. KOCH
I he ii(M\(' ;(|)|)i'()uc.lu>{l the cavernous sinus tlio septa became ('()iu|)Iet(>, and divided it into from five to eiglit fasicles of varying .size. Nowhere along the course of the undivided nerve were unmyeUnated fibers seen within its substance. In sections of the intra-ca^'ernous portion of the nerve a few clusters of unin>elinated fibers were seen approaching it, but could not be followed into its substance. The characteristic grouping of
Ocu.
small fibers at the periphery of the nerve, noted by Gaskell and Carpenter, could be seen most clearly in osmic acid preparations; but not all of the small fibers were grouped at the periphery, nor did they all leave the nerve to pass to the ciliary ganglion. The cellular structures in the roots of the nerves, mentioned by Gaskell and other observers, were not observed in our preparations, as even in the most proximal sections the roots had already united to form a connnon trunk.
STRUCTURE OF THE CRANIAL NERVES 545
Microscopic sections of the trochlear nerves of the dog closely resembled those of the oculomotor nerve; large and small myelinated fibers, 11 to 15 and 2 to 6 micra- in diameter, in about the ratio of three to one, with a few fibers of intermediate size. No communicating branches from the cavernous plexus or the ophthalmic nerve were seen.
The abducent nerve of the dog near the brain stem consisted of a single group of large and small myelinated fibers. 11 to 15 and 3 to 6 micra in diameter, in about the ratio of three to one. Within the cavernous sinus the ner\-e was joined by a large bundle of sympathetic fibers, the majority of which formed an intimate union with three of the six or seven fasicles which made up the nerve at this ])()int (fig. 2). More distally the main grou]) of sympathetic fibci-s left the ner\(' to ]mrsue an inde])(Mident course.
The striking cliaracteristic of these llnce ii('i\-es is their similarity in structure, botli as to the size of the fibers and the ])ro])ortion of large and small fibers. (Jaskell and Carjienter noted the presence of small fibci-s in tlie oculomotor nerve, and considered the ciliary ganglion ;is their destination, though they did not say that all the small lilxM's entered the ganglion, (laskell further noted tlie similarity in structure between the Ilird and IVth ncrNcs, but said the destination of the small fibers of the latter was as yet unkjiown. Tli(> Xltli nerve he described as ('om])()sed of large myelinated fibers '"with a few smaller ones; with no .sign of any distinct group of small fibers as in the Ilird and I\'th nerves." In our preparations all three nerves showed strikingly similai- cliaracteristics as to \\\v siz(> of the fibers and the ratio of tlie small and large fibers. The \Tth nerve could not be distinguished from the other two by a diminished number of small myelinated fibers, but was characterized by the presence of unmyelinatetl fibers from the sym])athetic system which came into inthnate contact with tlie myelinated fibers and travelled distalward with them as far as the nerve was followed. The only clue to an explanation of this fact is Jegorow's suggestion that a distribution of fibers of the Vlth nerve to the eyeball in the birds might be accounted for by the presence of syinpa
546
SUMNER L. KOCH
thetic fibers which joined the nerve as it passed through the cavernous sinus. No such communications from the sympathetic system were seen in connection with the Ilird and IVth nerves. Branches of the cavernous plexus, which on gross dissection could be seen to join the Ilird nerve, on microscopic
M
Fig. 2 Section of the abducent nerve of the dog, showing bundles of sympathetic fibers, a, Pyridine-silver. Ocu. 0, Obj. 6.
examination were seen to accompany the nerve for a short distance and again separate from it farther distalward, without entering into intimate contact with its fibers. No communication whatever between the IVth nerve and the sympathetic system was seen.
STRUCTURE OF THE CRANIAL NERVES 547
The unmyelinated fibers seen in connection with the Ilird and Vlth nerves nowhere showed the characteristic arrangement of the unmyehnated fibers seen in the spinal nerves and the vagus. In the Ilird nerve the unmyelinated fibers remained grouped in the nerve sheath and did not enter its substance. More distally they separated from the nerve completely. The unmyelinated fibers which joined the Vlth nerve and entered into intimate association with the myehnated fibers were not uniformly distributed throughout the nerv^e substance, but remained at the periphery, definitely grouped in two or three of the fasicles of the nerve. In no sections did these unmyelinated fibers from the sympathetic system appear evenly and uniformly distributed throughout the nerve substance as do tlie unmyelinated fibers in the spinal nerves and the vagus.
THE THICKMIXAL XKRVE
The intracranial portion of the trigeminal nerve of the dog and the cat, and of one human specimen was studied. Pyridinesilver sections showed large and small myelinated fibers, and in addition small munbers of unmyelinated fibers, appearing in hygest numbers in two fasicles of the sensory portion of the nerve. No sj-mpathetic fibers were seen joining the nerve within the cranium. In osmic acid preparations of the Vth nerve of the dog the large myelinated fibers measured 12 to 16 micra in diameter, the small 3 to 6 micra. In the cat the small fibers measured 4 to 7 micra, the large 12 to 16 micra, with occasional fibers 18 micra in diameter.
Till': GLOSSOPIIAUYXGEAL, ACCESSORY AND HYPOGLOSSAL NERVES
The glossopharyngeal nerve is usually described as a mixed nerve, and is recognized, according to Herrick's classification, as containing both general and special v'sceral efferent fibers, and general and special visceral sensory fibers, as well as somatic fibers with sensory function. The accessory and hj^Doglossal nerves are pure motor nerves, the former containing general and special visceral fibers, the latter special somatic fibers.
548
SriVINIOk L. KOCH
(Jaskcll (lescrilxMl thv small myeliiuitod fibers in the <;l()ss()pluiryngeiil nerve as 1.8 to 3.G micra in diameter, the large as not exceeding 10.8 micra. He found that large myelinated fibers were present in all the roots of the accessory nerve, but that the small fibers were confined to the bulbar and upper cervical roots. I^arratt described the IXth nerve as composed chiefly of small myelinated fibers 4 micra in diameter. Kopsch described the IXth nerve as consisting of a motor and sensory portion, and
Fig. 3 J'rom a section of the accessory nerve <jf tlie clog, showing a bundle
of sympathetic fibers joining the nerve. Pyridiiie-silvor. Ocu. 0, Obj. S.
receiving sympathetic fibers from the superior cervical ganglion. He described the accessory nerve as arising in two parts, the accessorius vagi, which -joined the vagus as the ramus internus; and the accessorius spinalis, which as the ramus externus received fibers from the jugular ganglion of the vagus. Chase and Ranson found numerous unmyelinated fibers in the roots of the vagus but thought the bulbar rootlets of the accessory nerve contained few if any unmyelinated fibers. Kopsch described the hypoglossal nerve as arising hi 10 to 15 root bundles, and
STRUCT!' RE OF THE CRANIAL NERVES
549
forming anastomoses with the vagus, the upper three cervical nerves, and the superior cer\'ical ganghon of the sympathetic system.
Pyridine-silvei' sections of the roots of the glossopharyngeal nerve showed large and small myelinated fibers, and a few
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Fin. ^ Si-ction of tlu" accessory ncivc of the dofi. showing bundles of darkly stained uiiiiivelinated fil.ers. Pyridine-silver. Ocu. 0. Ohj. 8.
unmyelinated fibers. Sympathetic fibers could be followed into the trunk of the IXth nerve between its two gangha. In the trunk of the nerve the small myeUnarted fibers outnumbered the large fibers nine to one. In osmic acid preparations the large fibers measured 9 to 12 micra in diameter, the small 3 to
550
SUMNER L. KOCH
5 micra. Sections of a pyridiiie-silvcr preparation of the IXth nerve of the cat, just proximal to the superior ganghon, showed few large myelinated fibers 12 to 15 micra in diameter", many small myelinated fibers, 3 to 6 micra, and a few unmyelinated fibers. Sections of the IXth nerve of the rabbit showed a similar picture. No connections with the sympathetic system distal to the petrous ganglion were seen in any preparations.
The bulbar rootlets of the accessory nerve are composed chiefly of small myelinated fibers 2 to 5 micra in diameter. The spinal
Fig. 5 From a section of the hypoglossal nerve of the dog, showing bundles
of darkly stained unmyelinated fibers joining the nerve. Pyridine-silver. Ocu.
0, Obj. 8.
portion was composed of large and small myelinated fibers in about the ratio of five to one. The majority of the large fibers were 9 to 12 micra in diameter, with a few larger fibers of 14 to 16 micra; the small fibers 2 to 5 micra. In several preparations at the point of its separation from the vagus, a few unmyelinated fibers could be seen in the ramus externus, arranged in three or four groups. Bundles of sympathetic fibers could be traced for some distance in the sheath of the Xlth nerve, finally entering it (fig. 3) and forming well marked clusters of fibers within its
STRUCTURE OF THE CRANIAL NERVES 551
substance (fig. 4). In Pal-Weigert preparations the bluish-black myelin rings were separated in places by small unstained areas, corresponding closely with the location of the unmj'elinated fibers seen in pyridine-silver preparations. No essential differences were seen in the Xlth nerve of the cat and rabbit. The large myelinated fibers of the spinal root of the accessory of the cat were the largest of all the fibers measured, some of them having a diameter of 18 micra.
The hypoglossal nerve of the dog showed a pictm-e closely resembling that of the oculomotor nerve, large and small myelinated fibers in about the ratio of three to one, measuring 11 to 15 micra and 3 to 6 micra in diameter. No unmyelinated fibers were seen within the roots of the nerve, but slender bundles of sympathetic fibers could be followed as they approached the nerve, entered its sheath, and finally joined the nerve substance (fig. 5). The hypoglossal of the cat and rabbit showed similar pictures.
Nowhere did the umnyelinated fibers seen in sections of the Xlth and Xllth aj^jiear evenly distiibuted among the myelinatetl fibers, as in the sj)inal nerves and the vagus; but always grouped in clusters, as hi the \'Ith nerve, and in largest numbers near the periphery.
SUMMARY
1. Unmyelinated fibers are present in the Vth, Vltli. IXth, Xlth and Xllth cranial nerves. Those in che Vlth, Xlth and Xllth are probably all derived from the sympathetic system. In these nerves they have an arrangement characteristic of sym]iathetic fibers; they are grouped in clusters, and are most numerous near the periphery of the nerve.
2. The oculomotor and trochlear nerves are strikingly similar in composition; they are composed of large and small myelinated fibers, without any accessions or unmyelinated fibers from the sympathetic system.
3. The abducens is similar to the Ilird and R'th nerves as regards its myelinated fibers, but in addition receives a large number of unmyelinated fibers from the sympathetic system,
.);)2 SUMNKK L. KOCH
some of whicli outer the nerve sheath and travel distalward with the myelinated fibers.
4. The accessory and hypoglossal nerves are composed of large and small myelinated fibers, and are similar in appearance and structure to the Ilird and IVth nerves. Like the Vlth nerve, they receive considerable numbers of unmyelinated fibers from the sympathetic system which can be followed to the termination of the nerves in the muscles which they supply.
LITERATURE CITED
Bakhatt, J. O. W. 1899 Observations on the normal anatomy of the 9th, 10th, nth, and 12th cranial nerves. Arch, of Neur. from the Path. Lab. of the London County Asylums, vol. 1, p. 537.
1899 On the anatomical structure of the 9th, 10th, 11th, and 12th cranial nerves. Brit. Med. Jour. 1899, part II, p. 837. 1901 Observations on the structure of the third, fourth, and sixth cranial nerves. Jour. Anat. and Phys., vol. 35, n. s. 15, p. 214.
BouGHTON, T. H. 1906 The increase in the number and size of the medullated fibers in the oculomotor nerve of the white rat and of the cat at different ages. Jour. Comp. Neur., vol. 16, p. 153.
CARPENTER, F. W. 1906 The development of the oculomotor nerve, the ciliary ganglion and the abducent nerve of the chick. Bull. Mus. Comp. Zool. Harv. Coll., vol. 48, no. 2.
Chase, M. R. and Ranson, S. W. 1914 The structure of the roots, trunk, and branches of the vagus nerve. Jour. Comp. Neur., vol. 24, p. 31.
Gaskell, W. H. 1889 On the relation between the structure, function, distribution and origin of the cranial nerves; together with a theory of the origin of the nervous system of vertebrates. Jour. Phys., London, vol. 10, p. 153.
KopscH, Fr. Rauber's Lehrbuch der Anatomie des Menschens, Abt 5, Leipzig.
Ranson, S. W. 1911 Non-medullated fibers in the spinal nerves. Am. Jour. Anat., vol. 12, p. 67.
1912 The structure of the spinal ganglia and of the spinal nerves. Jour. Comp. Neur., vol. 22, p. 159.
THE BKAIX AM) THE FRONTAL GLAND OF THE
CASTES OF THE 'WHITE AXT,' LEUCO TERMES FLAVIPES, KOLLAR
CAROLINE HLRLING THOMPSON De-pnrtmenl of Zoology, Wellesley College
TWKN TV-SIX FKMKES
CO NIK NTS
Introdiicticjn 553
Material and methods 554
The itU'inl)(M-.s of the colony of L. Havipes
1. The adult castes and the younn oiin
2. Distinguishing characters of the five adult castes and of three types
of nymphs 55S
li. Means of distinguishing the sexes 560
General anatomy of the termite hrain ... 561
The finer structure of the hrain . . 564
1. The hrain sheath 564
2. The protocereliral lobes 564
li. The mushroom bodies 566
4. The cent ral body 570
5. Ocelli and ocellar nerves 570
6. The optic lobes 572
7. The anteiuial lobes 574
S. The tritocerebral lobes and the tritoct-rebral conmiissure 574
9. The frontal ganglion 574
10. The ventral coiuiectives and the subesopluigeal ganglion 576
11. The frontal or fontanel glan<l and the fontanel nerve 576
Summary of results 589
Bibliograi)hy 592
INTHODIC'TION
This paper was iintlcitaken with the idea of studying the finer structure of the brains of the tUfiferent castes of the common termite or 'white ant,' and to compare them with the brains of the castes of true ants, the chief point of interest being that both termites and ants are social insects, Uving in communities
553
554 CAROLINE B. THOMPSON
c()ini)()se(l of many types of indivitluals, hut difforins greatly in their degree of specialization and in intelligence. As the work progressed it was found necessary to study the frontal gland in detail, and it is now my intention to follow this paper with a second, dealing with the development of the frontal gland and the differentiation of the different types of brains in the recently hatched nymj^hs.
The only form to be discussed in the present paper is the common termite, Leucotermes flavipes, Kol. As is well known, this insect is not an ant, but belongs to the order Isoptera, family Mesotermitidae, Holmgren ('11). The species, flavipes, was described by Kollar ('37) ; the genus Leucotermes, formerly included in the Linnaean genus Termes, was established by Silvestri ('01).
In the northeastern United States there is only this one genus and species of termites; in the southern states there is a second species, L. virginicus, with three different genera in the Gulf states, Snyder, and several other genera occur in California and the southwest, Heath ('03).
MATERIAL AND METHODS
My material was collected in Wellesley, Mass., in May, 1915, and June, 1916, beneath and within some old planks of wood. The fixatives used were Bouin's Fluid, and Gilson's Fluid. The former, however, is decidedly better for termite nervous tissue, and three hours in the fluid gives a good fixation. Whole mounts of all the heads were made by staining a long time, twenty-four to thirty-six hours, in Conklin's picro-haematoxylin, and then destaining for a day or longer in acid alcohol, clearing in cedar oil and mounting with the frontal side up. The heads of workers and of both types of nymphs, were embedded in hard paraffin, three to four hours, and were sectioned at a thickness of G/JL. The frontal or horizontal plane proved the most satisfactory for sections of the head. In the case of the soldier, where the chit in of the head is very thick, the brain was first dissected out, under a dissecting lens, stained, embedded, and then sectioned. This was also done for the worker, in addition to sections of the entire head, the great hardness of the chitin making
BRAIN OF THE 'WHITE AXT' 555
the sections of this caste frequently imperfect. \Miole mounts of the true adult and other brains were also made. It should be stated here that on account of the internal chitin of the tentorium, in addition to the chitin in the cuticula of the skin, the termite head is a difficult object to section. Cnly the prolonged time in paraffin has given good results. The sections were stained on the slide with (1) Ehrlich's acid haematoxylin and eosin, or (2) iron haematoxylin and orange G.
For the adult sexual forms, I am indebted to Mr. A. D. Hopkins and Mr. T. E. Snyder of the division of forest entomology of the U. S. Department of Agriculture, who have kindly furnished me with young and old adults of both the 'ti-ue' and the neoteinic type with short wing pads, and with much other termite material that will be utilized later. I wish here to exjiress my thanks to both ]\Ir. Hoi)kins and Mr. Snyder for thcii- kindness in sending me this material.
'iiii'] Mi;.MUi;i{s oi' iiii: colons oi- i.. flampks /. The ndult easier and the young
The colony of Leucotermes flavipes is composed of many difTerent kinds of individuals, some of which are adult or final stages, the castes, while others are merely the young, or the developmental stages of the castes, their presence, in some degree, depending upon the season of the year Each caste contains both males and females, caste not corresponding with sex, as in the bees and ants.
The term adult is here used in the general and biological sense to denote an insect which has undergone its last molt, and which has, in general, acquired its final definitive form and structure. The phrase "in general" is inserted to co\'er the cr.se of those older females whose abdomens have become greatly enlarged in the course of egg-laying. I am aware that some biologists would exclude from the category of adult stages the neoteinic reproductive forms and the sterile workers and soldiers, on the ground that the former do not possess the outer bodily structure characteristic of the species, as seen in the 'true' adult,
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BRAIX OF THE 'WHITE A XT 557
and that the latter have undeveloped sex organs. Que disadvantage of excluding these forms is that we are then left with no general term to denote the full grown stage, which after all is the original sense of the word adult.
The adult stages may be divided into two grou])s: (1) forms with functional and mature sex organs, the 'kings' and 'queens,' of which there are three different types, (a) kings and queens with long functional wings, the 'true' royal pair, (b) kings and queens with short wing pads, the neoteinic substitute pair with short wing pads; (c) kings and (jueens with no wing pads, the neoteinic substitute wingless pair, sometinuv^ known as ergatoid or workerlike; (2) forms with imdeveloped sex organs, (d) the 'workers,' (e) the 'soldiei^.' making in all five adult castes. To these five types of adults, tliicc inoic. lepresenting phases of a later development, may be added, namely: the enormously distended fertilized egg-laying (|ueens of the three (lueen types (table 1).
The young, or the (ieveloi)mental stages of the five adult castes, occur in many stages, the number of these differing with the caste. From tlic egg hatch forms which are said to be all alike and undiffeicntiated, (Irassi. Snydei', and from these undiffei'entiated nyin))li-. laiAae'of Snyder. 'develop nym])hs with large heads and nymphs with small heads. The large-headed nymphs develop "after a seiies of molts and (|niescent stages of comparatively short duration,'" the wiiole j^rocess requiring less than one year, Snyder ('!()), into the sterile workers and soldiers. The small-h(vided nym])hs undergo a more com])lex development, which "api)arently recjuires two seasons," and develop into: (1) nymphs of the 'first foiin.' witli long wing pads, which become the true royal pair, the sexual adults with long wings, (2) nj'mphs of the 'second form,' with short wing pads, which become the sul)stitute sexual forms with short wing ])ads, (3) nymphs without wing pads, whose devek)pment is not fully understood, and whicli become the substitute wingless sexual adults.
' llu' term nymph i.s used in tlii-s pajjor to donote any cl('vclo|)nicntaI stage of an insect with incomplete nietamorpliosis. whether the form po.ssesses wing inula or not. Snyder, in accordance with the older authors, applies the term 'larva' to the younger nymphs which possess rudiments of wing pads that can only be distinguished with magnification. He uses the term nymph to designate forms with wing pads that can l)e distinguished with the naked ey<\
558 CAROLINE B. THOMPSON
2. Dif<(inguishing characters of the five adult castes and of three types of nyinphs
Before beginning the discussion and comparison of the brains of the different forms of L. flavipes some descriptions of the types themselves and the means of distinguishing them may be of interest.
Forms with sex organs developed in the adult. 1. Nymphs. Nymphs of the first form, with long wing pads. These nymphs are most numerous in the early spring before the transformation into the adult has taken place. According to Snyder they attain a length of 7.5 mm. before the last molt, but they may be distinguished while much shorter. The color is creamy white, with a pale pink or rose pigmentation of the compound eyes. The hind wing pads extend back as far as the fourth abdominal segment.
Nymphs of the second form, with short wing pads. These nymphs are found at the same time and in about equal numbers with the long- winged nymphs. They likewise attain a length of 7.5 mm. before the last molt. They may be readily distinguished from the 'first form' by the short rudimentary wing pads which hardly reach the second abdominal segment. The color is creamy white and the compound eyes show no trace of pigment.
Nymphs with no wing pads. I have never found these nymphs and therefore quote from Snyder, Another type of substitute or neoteinic reproductive form, which greatly resembles the worker ('ergatoid') is developed from young larvae of the sexed forms."
2. Adults. The true royal pair, with long wings. The true winged adults are found in the nest or in the air in late spring and early summer. The head and body are dark brown, of about the same size as the nymphs of the first form, the long filmy wings are nearly twice the length of the body. The compound eyes aie black surrounded by a lighter rim of unpigmented skin. The two lateral ocelli are visible as small light spots in front of the compound eyes and behind the antennae. The opening of the frontal or fontanel gland lies in the median line of the frontal surface of the head.
BRAIN OF THE WHITE ANT 559
The egg-laying true queen. The old egg-laying true queens are always found within the nest, often in large 'queen cells' or chambers, and they may attain a length of 14.5 mm., Snyder ('16). The head and thorax are of normal size and brown in color, the abdomen is enormously distended, and is straw-colored with intersegmental bars of brown. The broken off stubs of the long wings remain attached to the thorax.
The neoteinic substitute pair with short wing pads. This pair is found within the nest and is similar in length to the nymphs of the second form. The body color is light bro\viiish and the 'compound eyes are browTi. The wing pads are now merely short, transparent chitinous plates.
The egg-laying substitute queen with short wing pads. This old substitute queen with short wing pads attains a length of 9 mm., Snyder ('16). The abdomen is distended and is creamy white with darker chitinous bars, the head and thorax are of noimal size. The compound eyes are brown. ^
The neoteinic substitute wingless pair. As I have never seen this form, I will again quote from Snyder ('16, p. 7), "Of a pale yellowish or grayish color, and having no wing pads (fig. 3; fig. 4c), individuals of this larval supplementary reproductive form are apparently blind and never leave the parent colony, except by underground tunnels."
The egg-laying substitute wingless queen. The old egg-laying substitute wingless queen attains a length of 9 mm. and has a distended abdomen, Snyder ('16).
Forms with sex organs not developed in the adidt. 1. The workers. Workers are found in termite colonies tlu'oughout the year. The smallest of all the castes, the body measures only 5 mm. in length. The head is considerably broader from side to side than that of the nymphs. The abdomen is shorter, softer, and covered with a more transparent skin. The color of the abdomen is usually grayish, owing to particles of wood in the alimentary canal seen through the skin. No eyes are visible in
^ The above descriptions of the enlarged egg-laying queens are based upon the alcoholic specimens sent to me by Mr. A. D. Hopkins and Mr. T. E. Si.yder of the U. S. Dept. of Agriculture.
THE JOURNAL OF COMP.VRATIVE NEUROLOGY, VOL. 26, NO. 5
560
CAROLINE B. THOMPSON
the worker until after staining, when small rudimentary compound eyes may be distinguished. No ocelli are present.
2. The soldiers. Soldiers are found in the colony throughout the year. The entire body measures 6 to 7 mm. in length, but the head is much longer and the abdomen shorter than that of the worker. No eyes can be seen until after the head is stained, when rudimentary compound eyes even smaller than those of the worker become visible. The opening of the frontal gland is in the median line of the frontal surface of the head.
Fig. 1 The abdomens of three individuals seen from the ventral surface.
A, adult male; B, adult female; C, female nymph with long wing pads; 2 to 10,
2nd to 10th abdominal segments. Obj. 32, oc. 6, reduced one-half.
III. Means of distinguishing the sexes
The only differentiation between the two sexes in any caste of L. fiavipes is in the size of the abdomen, and in the relative size and arrangement of the sternites, i.e. the ventral parts, of the posterior abdominal segments.
The sex of any termite may be distinguished by the size and the shape of the seventh and eighth abdominal sternites.^
In the male viewed from the ventral surface (fig. 1, ^) the seventh and eighth abdominal segments measure about the same in length, i.e., in an antero-posterior direction, and the seventh
' The ventral surface of the first abdominal segment is entirely covered by the metathorax, so that the actual seventh segment is apparently the si.xth.
BRAIN OF THE 'WHITE ANT' 561
segment is shorter than in the female (B). The posterior margin of the ninth segment is concave and two slender cerci are present on the lateral sm-faces of the tenth segment.
In the adult female (fig. 1, B) the seventh segment is very long, with a rounded posterior edge, and completely covers the eighth and part of the ninth segment. Jointed cerci are borne on the sides of the tenth segment.
In all female nymphs (fig. 1, C) and also in the female workers and soldiers, the seventh segment, although long, does not completely cover the small indented eighth segment. Cerci similar to those of the male are borne on the median surface of the ninth segment and also on the lateral surfaces of the tenth segment.
fJENERAL ANATOMY OF THE TERMITE BRAIN
The present paper will discuss the brains of the nymphs of the first and second forms, the true sexual adults, and the workers and soldiers.
It is generally thought today that the insect brain includes six regions, corresponding to the embryonic segments of the head, three of which are supracsophagcal, namely: the protocerebrum, the deutocerebrum, and the tritocerebrum, and three subesophageal, the mandibular, the maxillary, and the labial ganglia. Holmgren ('09) finds these six regions in the termite brain and discusses at some length the origin of the labrofrontal nerves, which he decides is in the tritocerebrum. It need only be remarked here that the same six regions may be distinguished in Leucotermes, and that in agreement with Holmgren I find the labrofrontal nerves arising from the tritocerebrum. Table 2 names the regions and the parts that are present in the brain of L. flavipes.
Figure 2 is a diagram of the brain of L. flavipes, as seen from the frontal surface. The supraesophageal ganglion is drawn from an entire mount of a brain dissected out from the head of a nymph with long wing pads; the ventral connectives, v.c, and
562 CAROLINE B. THOMPSON
The parts of the brain
I. The supraesophageal ganglion.
A. Protocerebrum
The protocerebral lobes
_,,,,, . J anterior, dorsal
The protocerebral commissures < .11 [ posterior, dorsal
[anterior roots The mushroom bodies -! central body roots
[porterior roots The central body The optic lobes
B. Deutocerebrum
The antennal lobes
C. Tritocerebrum
The tritocerebral lobes
The tritocerebral commissure (subesophageal)
II. The subesophageal ganglion The ventral connectives The mandibular
The maxillary f gangli The labial
The nerves of the head
A. The optic nerves The ocellar nerves The fontanel nerve
B. The antennal nerves
C. The labrofrontal nerves to [the nerve to the protocerebrum
the frontal ganglion i the labral nerves
[the recurrent nerve
The mandibular nerves The maxillary nerves The labial nerves
the subesophageal ganglion, sb.g., are drawn from sections of the same form.*
- The heads of all termites are held in a somewhat slanting position, making
a large obtuse angle with the long axis of the body, so that the morphologically dorsal surface of the head has become frontal or anterior. The slant of the head is much less in termites than in ants, and in the termite soldier the head is almost horizontal, the so-called frontal surface being practically dorsal, but for the sake of clearness the same terms of direction will be applied to all the castes of termites. In describing the entire heads the terms anterior and posterior imply toward and away from the frontal surface, in the same sense that Hesse (1901 b) uses the terms rostral and caudal ; in like manner dorsal and ventral imply toward or away from the vertex of the head.
BRAIN OF THE 'WHITE ANT' 563
The insect brain froms a ring of nerve tissue which encircles the esophagus and which is composed of the supraesophageal ganglion,, the ventral connectives, and the subesophageal ganglion.
The three parts of the supraesophageal ganglion, the protocerebrum, the deutocerebrum, and the tritocerebrum, are merged into a single mass which constitutes the principal part of the brain. To the protocerebrum belong the protocerebral lobes, p.L, the optic lobes, o.L, connected by the fibers of the optic
/(J .^=^ /
tn b.
P.l. """ -^ -^
ol
t r.cm.\,...jz:;Xl >^. '/fQ'^
md.n
sb.
^■c. 'ifn.
mx.n
Ib.n.
Fig. 2 Diagram of the brain of a nymph with long wing pads, as if seen in frontal optical section. A.I., antennary lobe;/.f7., frontal gland; /.«.. fontanel ncrvo; fr.gn., frontal ganglion: If.n., abrofrontal nerve; Ib.n., labial nerve; m.b., mushroom body; ni'Z.n., mandibular nerve; mx.n., maxillary nerve; or. n., oc liar nerve; o.L, optic lobe; p.'., protocerebral lobe; sb.g., subesophageal ganglion; tr.cm., tritocerebral commissure; v.c, ventral connective.
nerves with the compound eyes, the two mushroom bodies, in.b., the ocellar nerves, oc.n., connected with the two lateral ocelli, and the fontanel nerve, f.n., connected with the frontal gland, f.g. To the deutocerebrum belong the antennal lobes, a.L, and the antennal nerves of the antennae. The tritocerebrum consists of the tritocerebral lobes, and the tritocerebral commissure, tr.cm., beneath the esophagus. The labrofrontal nerves, l.f.n., or the tritocerebral nerves, arise on the inner, median surfaces of the tritocerebral lobes, and running upward and fonvard unite in the frontal ganglion, fr.gn., which lies anterior to the main
564 CAROLINE B. THOMPSON
part of the brain and directly above the mouth opening. The connection of the frontal ganglion and the labrofrontal nerves is not shown in the diagram nor is the recurrent nerve which runs backward from the frontal ganglion.
The delicate unpaired nerve from the dorsal surface of the frontal ganghon to the protocerebral lobes, and the labral nerves from the ventral surface of the frontal ganglion, are figured but not labeled. Posterior to the tritocerebral commissure the two slender ventral connectives, v.c, run first backward (which cannot be shown in this diagram), then downward, and unite to form the large subesophageal ganghon, a single mass, consisting of the fused mandibular, maxillary, and labial gangUa, and from which arise the mandibular, maxillary, and labial nerves. Posterior to this ganghon the thoracic connectives, not shown in the figure, pass upward and then backward into the thorax.
THE FINER STRUCTURE OF THE BRAIN
/. The brain sheath
The entire brain is surrounded by a membranous sheath composed of a single layer of cells resembling mesenchym cells, their fibrous processes making a continuous double walled membrane between which the cell bodies and the nuclei are situated.
//. The protocerebral lobes
The protocerebral lobes (fig. 2, p.l.) form the central part of the supraesophageal ganghon; they are continuous on their dorsal surface with the mushroom bodies, on their lateral surfaces with the optic lobes, and on their ventral surface with the antennal lobes, and are also connected by a slender nerve with the frontal ganglion, fr.gn., which lies anterior and somewhat ventral to the protocerebrum. The ocellar nerves, from the lateral ocelli, and the fontanel nerve, from the frontal gland, also enter the protocerebral lobes, and will be considered in more detail under those headings. Like other parts of the brain the protocerebral lobes consist of a central fibrous core and an outer investing layer of nerve cells. Most of these nerve cells are small
BRAIN OF THE 'WHITE ANT' 565
with round nuclei and a round cell body which appears as a mere rim of cytoplasm (fig. 3, A,B), but in the .median dorsal region, the intercerebral region of Haller, and here and there on the ventral surface, the cells are larger with pear-shaped cell bodies (fig. 3, C, D). The smallest nerve cells of the protocerebral lobes are, however, almost twice as large as the adjacent nerve cells belonging to the mushroom bodies (fig. 3, E) and to the optic lobes.
In a series of frontal sections, beginning with the frontal or anterior surface (figs. 13 to 20) it will be seen that the proto
c O
^M^^
- ^^mr
Fig. 3 Nerve cells from the protocerebral lohes and ihe mushroom body. A, small cells, from the lateral region of the protocerebral lobes; B, medium sized cells, from the same region; C, large cells, from the median ventral region of the protocerebral lobes; D, large cells, from the intercerebral region; E, cells from the mushroom bod}*. Homog. immers. 1.8 mm., oc. 6.
cerebral lobes are at first entire (fig. 13), then, farther back in the series (fig. 14) the two great anterior roots of the mushroom bodies penetrate deep into the fibrous core of the protocerebral lobes, dividing them into a median and two lateral portions, and this division is further continued by the stalks of the mushroom bodies (figs. 15, 16).
a. The protocerebral comiiiissures. About the middle of the protocerebral lobes, in the plane of the central body (fig. 15) the lateral and median parts of the lobes are again connected by a stout fiber tract passing from side to side beneath the central body and connected also with the antenna! lobes. This tract or commissure is homologous with the so called 'ventral com
566 CAROLINE B. THOMPSON
missure' of the hymenoptera and other insects. The lateral portions of the protocerebral lobes are also connected by two dorsal protocerebral comrhissures, the anterior, a delicate strand of fibers, lying above the central body (figs. 15, 16, a.cm.) the posterior, on the same level as the first, lying immediately posterior to the central body, fig. 18, 'p.cm. These two commissures are homologous with the two dorsal commissures found in ants, and it is interesting to note that the posterior one, the 'Briicke' of the older authors, has the same characteristic wide inverted n shape.
In figures 15 to 18, the connection of the optic lobes with the protocerebral lobes may be seen.
Figures 19 to 20 show the relation between the protocerebral lobes, the deutocerebrum or antennary lobes, and the tritocerebrum.
///. The mushroom bodies
The two mushroom bodies, or the globuli, as Holmgren terms them, are now generally considered the chief centers of intelligence in insects. They appear, in frontal views of the head (figs. 8 to 12, m.b.), as two slightly grooved projections from the dorsal surface of the brain, the frontal gland occupying the space between them. Each mushroom body consists typically of two lobes, which in the termites are not distinctly separated but form one continuous mass of tissue, the lobes being indicated on the exterior of the brain merely by a slight groove or depression of the surface. In sections (figs. 15 to 18) it may be seen that the mushroom bodies are composed of an outer nerve cell layer and an inner fibrous portion which forms the cups or calyces, the stalks, and the three pairs of roots.
a. The nerve cells of the mushroom bodies. The nerve cells of the mushroom bodies are all of the same size and are among the smallest cells of the brain (fig. 3, E). They have a round or oval nucleus and so small an enveloping layer of cytoplasm that it cannot be distinguished, even with the immersion lens, except at the distal end where the axon is given off. The chromatin masses of the nucleus are all about the same size, forming a
BRAIN OF THE 'WHITE ANT' 567
peculiar characteristic pattern by which these cells may be recognized.
Although the nerve cells are of similar size throughout the mushroom bodies, they are differentiated into groups, or zones, according to their position. In the center of each cup or calyx, as seen in section (fig. 4), lies an oval mass of cells, /, which is homologous in position with the central oval mass of large cells found in the bees, Jonescu ('09), and in the ants, Pietschker ('11), Thompson ('12), and which I have termed Group I. On each side of Group I, are broad wedge-shaped masses of cells (fig. 4,
III II
I
II, III .
Fig. 4 Diairram of a mushroom body, showing the nerve cell layer flivided
into groups, the calyces, and tho bos^inning of the stalk. /, cell group I; //, cell
group II; ///, cell group III; gl., glia cells.
//), which occupy most of the dorsal surface of each lobe and whose inner margins overlap and enclose the central group I. These masses, which appear separate in sections, form a continuous zone if seen in surface view, and are homologous with Group II of ants. Again, in each lobe, on each side of Group II lie smaller masses of cells which form the lateral surfaces of the lobes (fig. 4, ///). There are only three, instead of four, of these cell masses, because the inner lateral surfaces of the two lobes are in contact and their cells are continuous. These groups are equivalent in position to Groups III and IV of the ants.
b. The fibrous core of the mushroom bodies. The cups or calyces of the inner and outer lobes of the mushroom bodies are composed (1) of converging bundles of fibers, the axons of the three
568 CAROLINE B, THOMPSON
zones of nerve cells just described, and (2) of masses of glia cells (fig. 4, gl.) resembling the 'glomeruli' of the antennal lobes, which envelop the fiber bundles -on all sides and unite the calyces of the two lobes into one continuous whole.
The stalks of the mushroom bodies are the continuations downward and inward of the fiber bundles of each lobe. These lie side by side, the fibers from the inner lobe on the median side of those from the outer lobe, and for a short distance they remain distinct. The two stalks, surrounded by a dehcate sheath, penetrate deep down into the fibrous core of the protocerebral lobes and then run forward. There is no 'decussation' of fibers such as is seen in the hymenoptera. In the same frontal plane with the central body (fig. 15) the distal ends of the stalks lie beneath the central body, and at this point each stalk gives rise to three roots, the anterior, the central body, and the posterior roots. The central body roots, which are the shortest, pass upward and directly into' the central body (fig. 16, c.h.r.). The anterior roots which are the longest, bend sharply upward and forward, making an elbow with the stalks from which they have arisen (fig. 14, a.r.m.b., m.b.s.). In sections that are farther forward in the head, the anterior roots may be seen as two great bundles of fibers curving latero-dorsally and dividing the protocerebral lobes in the manner already described. On reaching the dorso-lateral margins of the protocerebral core, the anterior roots curve in a posterior direction and are seen in sections as two detached masses on the outer sides of the mushroom bodies (figs. 15, 16, a.r.m.b.), then (fig. 14) returning again forward, the narrow distal ends turn downward and terminate among the nerve cells of the anterior part of the protocerebral lobes.
The posterior roots branch off from the stalks together with the central body roots, and, after the latter have passed into the ventral surface of the central body, pass dorsalward, posterior to the central body and to the posterior dorsal commissure, there expanding into two large and very prominent lobes which nearly fill the intercerebral region (figs. 19, 20, 23, 26, p.r.m.b.). The ventral part of these lobes or roots is connected with the protocerebral fibrous core, the dorsal part, however, projects back
BRAIN OF THE 'WHITE ANT 569
ward for some distance, evidently giving fibers to and receiving fibers from the posterior cells of the mushroom bodies, and then ending in about the same plane with, the beginning of the tritocerebral lobes.
c. Comparison of the mushroom bodies of the different castes of L. flavipes. The mushroom bodies differ Uttle in the different castes except in size, and even in this respect there is not a very great dissimilarity (figs. 8 to 12).
The worker has the largest mushroom bodies, largest by actual measurement and in the estimated number of nerve cells; the nymphs of the fir>t and second forms have mushroom bodies about similar in size, but slightly smaller than those of the worker; the soldier has the smallest mushi'oom bodies of any caste. The cells of the mushroom bodies of the worker- and soldier penetrate into the intercerebral region (figs. 22, 25), but merely border upon it in those of the njTuphs of the first and second forms.
My only true adult material, as already stated, was alcoholic and much of it seemed to have shrunk. It will be noted that in the true adult brain outlined in figure 9 the mushroom bodies are farther apart than in the n>aiiph of the first form from which this adult has developed (fig. 8) and the frontal gland has increased in size. Some other better preserved true adult brains are considerablj' larger than the brain of a npuph of the first form, and the muslu-oom bodies are likewise larger.
Holmgren ('09) emphasizes the great deterioration of the brain tissue which he observes in the older enlarged and egg-laying queens. IVIy material has been preserved in alcohol and is not the best for finer study, but the examination of several old and enlarged queens leads me to believe that in L. flavipes this deterioration of the brain has not taken place.
d. Comparison of the 7?iushroom bodies of termites and hymenoptera. A comparison of the Leucotermes mushroom body with those of ants and bees shows, as one would naturally expect, that the termite muslu-oom body is much more simple and primitive. This primitive condition is apparent in the small and uniform size of all the nerve cells, especially in the cell
570 CAROLINE B. THOMPSON
group I; in the presence of three zones of cells instead of the four found in ants; in the incomplete differentiation of the two lobes, whose cells are not separated by a deep furrow, as in ants, and whose two cups or calyces are completely fused by intervening masses of gha cells; in the shallowness of the cups; finally, in the smaller size of the entire mushroom bodies and their sUght differentiation in the different castes.
IV. The central body
The central body is situated in the central frontal plane of the protocerebral lobes, embedded in their fibrous core, directly beneath the intercerebral region. In form as well as in' position the central body of L. flavipes resembles that of bees and ants. It has two parts (fig. 15, c.b.), a curved dorsal portion, composed of fiber bundles that are radially arranged with intervening spaces, and a flatter ventral part, also fibrous. No nerve cells are found within the central body, as in ants, where small nerve cells occupy the spaces between the radial bundles of fibers, but a few scattered nerve cells are occasionally found along the outer surface.
The structure of the central body is the same in all the castes, but the size vanes with the size of the different brains (figs. 15, 22, 25).
Lying beneath the central body are two small round bodies which were formerly known as the 'tubercles of the central body" and also as 'ocellar glomeruli.' I have shown, Thompson ('12, '14), that in ants and in the bumble bee these previously misinterpreted bodies are the posterior roots of the mushroom bodies. In L. flavipes these rounded bodies are in connection with the central body roots of the mushroom bodies (figs. 15, 16, c.b.r.), which, it will be remembered, arise from the distal ends of the stalks in close proximity to the posterior roots.
V. Ocelli and ocellar nerves
Two simple eyes or ocelli are present in the adults and nymphs of the sexual forms, and are situated on the lateral surfaces of the head, in front of the compound eyes and behind the antennae.
BRAIN OF THE 'WHITE ANT' 571
In the head of the true adult (fig. 9, ox.) the ocelU may be readily seen as small colorless spots which stand out sharply from the surrounding brown skin, but in the heads of nymphs (figs. 8, 10), whose skin is not pigmented, the ocelU are not visible except in sections.
The ocelli are of a very simple and primitive tj^pe, Hesse ('01 b), without a lens and with httle or no pigment. The outer surface of the hypodermis above the ocelU is slightly convex, the inner surface is very concave, the latter caused by the sudden thinning of the inner cuticula, and in this concavity the bulbhke ocelli are situated. In general contoui* the ocelli resemble the tactile buds found in the skin of Amblj'stoma punctatum. The visual cells are long slender and curving, with spaces between their bases. As to the finer intracellular structure, I am not prepared to make a statement at this time. I shall also leave for further study the question whether the distal ends of the visual cells lie between the hypodermal cells, a primitive position, according to Hesse, or whoUj', and secondarily, beneath them; although the first view would seem to be upheld l^y my present material.
In a frontal section of the anterior part of the brain of a nymph with long wing pads, the oceUi (fig. 13, oc.) may be seen on each side of the section, just beneath the hypodermis, and covered by a thinner layer of cuticula, the inner cuticula, i.cu., being absent at this point. From the ocelli the slender ocellar nerves, lying just outside the brain sheath, run in toward the median line and enter the nerve cell layer of the protocerebral lobes just above the anterior roots of the mushroom bodies. Passing down into the intercerebral region, the ocellar nerves (figs. 15 to 18, oc.n.) run backward and finally enter the dorsal surface of the protocerebral lobes just behind the posterior dorsal commissure and in the same frontal section as the fontanel nerve from the frontal gland (fig. 19, oc.n., f.n.). At no point along their entire length do the ocellar nerves expand into ocellar lobes such as are present in the bees and ants. After my first cursory examination of the brain sections of L. flavipes, I was inclined to assign the role of ocellar lobes to the large lobes.
572 CAROLINE B. THOMPSON
p.r.m.h. seen in figures 19, 23, 26, on the ground that these lobes occupy a position similar to that of the ocellar lobes of ants and are in the neighborhood of the entering ocellar nerves. A very careful examination of a large number of sections, however, proved that there is no connection between the ocellar nerves and these adjacent lobes, and that there is direct connection between the mushroom body stalks, the protocerebral lobes, and the lobes in question, which are, as shown in a preceding section, the posterior roots of the mushroom bodies.
VI. The optic lobes
The optic lobes are situated on the lateral surfaces of the protocerebrum and are present in all the castes of L, flavipes, although they are well developed only in the sexual forms (figs. 8 to 12, O.I.). They are continuous with the protocerebral lobes and consist of an outer layer of very small nerve cells, similar in size to those of the mushroom bodies, and an inner fibrous portion that is subdivided into the outer, middle, and inner fiber masses, and the outer and inner crossings of fibers found in the typical insect brain, Berlese ('09).
The optic lobes of the nymph with long wing pads are the largest and most highly developed of any caste studied. The outer fiber masses (fig. 17, o.f.) are slender and elongated in a dorso-ventral direction. Nerve fibers pass in to them from the compound eyes, and continue inward, as the outer crossing, to the middle fiber mass, mf., the largest of the three masses. Fibers again cross between the middle and the inner fiber masses, i.f., making the inner crossing. The small inner fiber masses are directly continuous with the fibrous core of the protocerebral lobes.
In the nymph with short wing pads (fig. 10) the optic lobes and the compound eyes are slightly smaller than in the nymph just described, but the relative size and arrangement of the parts is the same.
In the worker, although the optic lobes are very much reduced in size, they are readily seen in surface views (fig. 11) as small
BRAIN OF THE 'WHITE ANT' 573
projections from the lateral surfaces of the brain, and a slender optic nerve passes from each greatly reduced compound eye to the optic lobe. In a frontal section of the worker brain (fig. 25) the small inner, middle, and outer fiber masses may be distinguished in the optic lobes, and also the fibers of the distal part of the optic nerve. By carefully following the optic nerve in subsequent sections it may be traced to the vestigeal compound eye.
The condition of the compound eyes evidently varies considerably in different termites. Holmgren ('09), describing the optic lobes in the worker of Eutermes, ^\Tites as follows:
Die Reduktion der Facettaugen hat eine entsprechende Rediiktion der Sehganslien mitgefdhrt, Die Sehnerven sind vielleicht vorhanden aber enthalteri nur wenige Nervenfiidchen iind von den bei den Geschlechtstieren, bcsonders den jungen, so wohlentwickelten Seliganglien kann man gar nichts cntdccken. Die Seiten des Protocerebralganglions sind somit kreisformig abgcrnndct.
The optic lubes of the soldier, like those of tlie worker, a])])ear as small rounded projections on tlie lateral surfaces of the jwotocerebrum (fig. 12, o.L), with optic nerves extending to the still smaller vestigeal compound eyes, c.e. On account of the very hard chitin of the soldier's head no attempt was made to section the entire head. The brain was dissected out, under a dissecting microscope, stained, embedded, and then cut in frontal sections. A section through the optic lobes (fig. 22, o.L) shows that the inner, middle, and outer fiber masses, although small, are clearly distinguishable, and that fibers enter the outer fiber mass from the optic nerve.
Holmgren ('09) states of the soldiers of Eutermes:
Die Soldaten sind ja blind und deshalb sind die Sehganglien voUstiindig vcrschwunden. Man kann von denselben kein Spur entdecken, obwohl ein sehr dinner n. opticus meistens vorhanden ist. Dieser fungiert aber als Hautnerv. Zufolge des Fehlens der Sehganglien sind die Seiten des Protocerebrums beinahe ki-eisrund.
The optic lobes of the true adult have not been studied in sections.
574 CAROLINE B. THOMPSON
VII. The antennal lobes
The antennal lobes compose the deutocerebrum, or the second brain segment, and are continuous with the anterior and ventral part of the protocerebrum (fig. 2, a.L). In form they are elongated masses that continue into the antennae as the antennal or olfactory nerves. On their inner lateral surfaces the antennal lobes are continuous with the third brain segment, the tritocerebrum.
In sections it may be seen that the antenna! lobes have an outer nerve cell layer containing both large and small cells, and an inner fibrous core that contains scattered masses of glia cells, the so-called 'gjomeruh.'
The relative size of the antennal lobes differs very slightly in the different castes of L. flavipes (figs. 8 to 12). The antennal lobes are largest in the nymphs with long wing pads and in the worker, but are smaller in the nymph with short wing pads and in the soldier. Holmgren states that these lobes are much larger in the worker of Eutermes than in the other castes: "die Deutocerebralganghen der Arbeiter sind verhaltnissmassig grosser und enthalten eine auch absolut bedeutend grossere Zahl GangUenzellen als bei den Geschlechtsindividuen."
VIII. The tritocerehral lobes and the tritocerebral co7nmissure
The tritocerebral lobes are small lobes extending ventrally along the sides of the esophagus and continuous with the inner, lateral, surfaces of the antennal lobes (fig. 20). From the inner median surfaces of the tritocerebral lobes arise a pair of nerves that ran forward alongside of the esophagus and unite in the frontal ganglion; these are the tritocerebral or labrofrontal nerves (fig. 2, l.f.n.). Beneath the esophagus the tritocerebral lobes unite in the slender tritocerebral commissure (fig. 2, tr.cm.), which is almost devoid of any investing nerve cells.
IX. The frontal ganglion
The frontal ganglion is a small mass of nerve tissue situated just beneath the clypeus, the sclerite of the head to which the labrum is attached, and above the mouth opening, somewhat antero-ventral to the brain (figs. 2, S,fr.gn.).
BRAIN OF THE ' WHITE ANT' 575
In sections (figs. 13, 21, 24) the frontal ganglion is rather triangular in appearance, the apex composed of nerve cells and the base of nerve fibers. The nerves connected with the frontal ganglion are (1) the paired tritocerebral or labrofrontal nerves, l.f.n., (2) the paired labral nerves, la.n., (3) an unpaired nerve to the protocerebrum, (4) the unpaired recurrent nerve, r.n.
The tritocerebral or labrofrontal nerves, l.f.n., arise from the trjtocerebrum as described under that heading, and their anterior ends are connected with the latero-ventral surfaces of the front.al ganglion.
From the median ventral surface of the frontal ganghon a bundle of nerve fibers emerges that runs first ventralward for a short distance and then divides into delicate right and left branches that pass to right and left through some muscle fibers in the clj^peus, continuing down and forward into the labrum as two delicate but distinct nerves. Those branches are distributed throughout the labrum. I have termed these nerves the labral nerves. In this I am apparently not in agreement witli Holmgren ('09) who does not describe the labral nen'es as arising from the frontal ganglion but speaks as tliough the labmm were innervated directly from branches of the labrofrontal nerves, which is not the case in L. flavipes. In all the sections which I have examined the nerves of the labrum have no direct connection with the labrofrontal nerves but arise indirectly, from the base of the frontal ganglion as described above. To quote Holmgren :
Ganglion frontale liogt iinmittelbar vor dem obcren Schlundganglion und ist mit diescin durch einen iinpaaren Nervenfaser, dcr iin Protocerebrum eintritt, verbunden, Vor dcm Ganglion frontale gehen nach vorn Nerven aus, welche einige clypealen Muskcln innervicren. .
An den Seiten des Ganglion frontale konimen die tritocercbralen Konnective ein, und hintcn lauft der ncrvus re^urrens aus, um oberhalb des Darmes sich nach hinten zu begeben.
Aus TritocGrebruni werden folgende Teile inn6rviert:
1) Die ganze oben beschriebene Labralmuskulatur von n. labrofrontalis
2) Die Labraldriisen von n. labrofrontalis.
In agreement with Holmgren I find a verj^ delicate unpaired nerve running between the dorsal apex of the frontal ganglion
THE JOURNAL OF COMPARATIVE NErROLOGY, VOL. 26, NO. 5
r)7() CAROLINE B. THOMPSON
and the ventral surface of the protocerebral lobes, also the unpaired recurrent nerve which arises from the posterior surface of the frontal ganghon. This latter nerve (figs. 14 to 26, r.n.) mns backward above the esophagus into the thorax and at a short distance posterior to the supraesophageal ganglion expands into the socalled 'esophageal ganglion.'
X. The ventral connectives and the subesophageal ganglion
The two slender ventral connectives (fig. 2, v.c.) arise from the posterior surface of the tritocer. bral commissure. They continue backward side by side, their inner surfaces touching, beneath the esophagus and above the bread tentorial band of chitin that is present in the interior of the head. They become still smaller, and pass down through the tentorial aperture, finally merging into the dorsal surface of the subesophageal ganglion.
The subesophageal ganglion (fig. 2, sb.g.) is large and consists of a fibrous core and a thick investing layer of nerve cells on the ventral surface. From the anterior end arise the mandibular nerves, md.n., from the posterior end, the maxillary, mx.n., and the labial, Ib.n., nerves. The branches of these nerves, described by Holmgren, have not been traced. Immediately behind the point of exit of the labial nerves the subesophageal ganglion ends, or rather is continued into the thoracic connectives, which pass upward and out of the head through the great tentorial aperture.
XL The frontal or fontanel gland and the fontanel nerve
The frontal or fontanel gland of the termites has long been an object of much interest and speculation. It is situated in the median line of the frontal surface of the head beneath the so-called 'fontanel,' which is defined by Brues ('15) as "sl small, depressed pale spot on the front of the head between the eyes (isoptera)."
In surface views of the heads of the castes of L. flavipes, (figs. 8 to 12) the frontal gland appears as a rather triangular or rounded mass lying in the space between the two mushroom bodies. In the adult sexual forms and in the soldier a tiny round
BRAIN OF THE 'WHITE ANT' 577
opening may be observed in the cuticula above the anterior end of the gland, but I have not been able to distinguish any such opening in the worker or in the two nymphs. The opening is situated in a shallow depression of the surface, the fontanel, which is paler than the surrounding skin.
The frontal gland differs greatly in size in the various castes. It is smallest of all in the worker (fig. 11) and greatly elongated in the soldier (fig. 12). In neither of these castes does the frontal gland completely fill the space between the mushroom bodies. In the sexual forms the true adult (fig. 9) has the largest frontal gland; the nymph with long wing pads (fig. 8) has a gland of similar proportions but smaller; the njmphs with short wing pads (fig. 10) has an even smaller gland. In all the sexual forms the frontal gland completely fills the space between the mushroom bodies.
Looking down into the frontal gland as it is seen in surface views is like looking into a shallow crater. The edges rise into folds which approach each other and nearly meet at the anteroventral end and enclose the central lumen. Sections show that the marginal cells of the gland are directly continuous with the hypodernuil cells and that the cuticula above the center of the gland is slightly depressed and thin, consisting only of the primary or outer cuticula, the secondary, inner, cuticula ending at the gland margin. Tliis depression and thinning of the cuticula is the structural explanation of the pale 'fontanel' spot seen on the outer surface of the head.
In the series of sections seen in figures 13 to 20, the first indication of the frontal gland is seen in a group of elongated hjiDOdermal cells (fig. 13,f.g.) lying in the median line directly beneath the cuticula, wliich is here of normal thickness. This group of cells forms the anterior margin of the frontal gland and it should be noted that it occurs in the same frontal section as the simple eyes, or ocelli, oc, a point wliich will be further discussed later. Figure 14, which is six sections farther back in the series, includes a section of the anterior part of the frontal gland, f.g. The high columnar cells of the gland are continuous with the low cuboid hjTDodermal cells; the cuticula is slightly depressed and
578
CAROLINE B. THOMPSON
the secondary, inner, layer is lacking above the lumen of the gland. In figures 15, 16, 17, the deeper, central, part of the gland is shown; figures 18, 19, contain the posterior outpocketing of the gland, not connected with the margin in the figures, and since these sections are considerably farther back in the series, in the longest part of the head, the cuticula and hypodermal
Fig. 5 Frontal section of the frontal or fontanel gland of a nymph with long
wing pads. B.ni., basement membrane; hyp., hypodermis; i.cu., inner cuticula;
o.cu., outer cuticula; /, long slender cells, probably not yet secretory in function;
2, swollen cells, probably glandular. Homog. immers. 1.8 mm., oc. 6, reduced
one fifth.
cells are now some distance from the brain, and, for considerations of space, are no longer included in the figures. Figure 20 shows the posterior wall of the frontal gland which ends in the next section.
a. The finer structure of the frontal gland. A closer study with the immersion lens shows that the frontal gland is composed of
' BRAIN OF THE 'WHITE ANT' 579
the following parts, which are present, with slight modifications, in all the castes: (1) the epithelial cells, (2) the basement membrane, (3) muscle fibers attached to the basement membrane, (4) the fontanel nerve, an unpaired nerve running from the frontal gland to the protocerebral lobes, and here described for the first time.
1. Nymph with long wing pads. The epitheUal cells (fig. 5, 1, 2) are of two kinds, of which the first far outnumber the second: 1, long slender columnar cells, with rounded distal ends, the distal cytoplasm taking a slightly deeper stain than the proximal portion, and the large oval nucleus situated near the base, 2, larger swollen cells whose cytoplasm contains vacuoles and irregular masses of a deeply staining substance. The nuclei of these cells are evidently basal but they are so crowded by the adjacent cells that I have been unable to distinguish them.
It will be noted that the lateral cells are more closely crowded together than those on the floor of the gland, in the median line, where the outlines of several cells maj' be clearly traced. It will also be noted that the marginal cells of the gland are lower and are directly continuous with the hj-podermal cells. Mitotic figures are present here and there among the cells of the frontal gland in both nymphs, but no cutiqula on the distal surfaces of the cells and no extracellular secretion have been observed. The only cells which appear capable of producing a secretion in the frontal gland of the nymph with long wing pads are the few scattered swollen cells marked 2, and these seem to have not yet discharged their secretion. This and the fact that there is no external opening may indicate that the frontal gland is not 3'ct functional in this nymph.
The basement membrane (fig. 5, h.m.) upon which the epithelial cells of the frontal gland rest, is continuous with that of the hypodermal cells and is considerably thickened along the lower, ventral, surface. This membrane is composed of mesenchjTn cells and fibers and is beset with many irregular openings. In some specimens it is very delicate but thick and firm in others, and takes a deep pink stain with eosin, yellow with orange G. The ventral surface of the basement membrane near the posterior
580 CAROLINE B. THOMPSON
end of the frontal gland is prolonged into a slender median process that passes downward posterior to the protocerebrum and connects the frontal gland with the great tentorial membrane that divides the interior of the head into the two parts in which lie the supra- and the subesophageal ganglia, and through which the ventral connectives pass. The basement membrane is similar in structure in all the castes.
Muscle fibers may be seen attached to the posterior or caudal surface of the basement membrane in the true adult, in surface \'iews, but I have not observed them in either of the nymphs; muscle fibers are also attached to the basement membrane in the soldier and in the worker.
The fontanel nerve (figs. 2, 19, 23, 26, f.n.) is the slender unpaired nerve which makes its exit through apertures of the median ventral process of the basement membrane of the frontal gland, and which passes vertically downward and enters the median dorsal surface of the protocerebral lobes at a definite point, namely: between the large posterior roots of the mushroom bodies and in the same frontal plane in which the ocellar nerves enter the protocerebral fibrous core. This is the first tim.e that this nerve has been described or figured in any termite brain. It is present in a similar position in all the castes and phases of L. flavipes that have been examined, and it is undoubtedly a strand of nerve fibers, although a very delicate one. I have provisionally termed this nerve the fontanel nerve, and we shall return to it again after a discussion of the frontal gland in the other castes.
2. The true adult. I have not yet succeeded in making good sections of the true adult's frontal gland, but in a surface view of an entire brain and frontal gland dissected out and mounted it is most evident that the inner surface of the frontal gland is lined with a wavy chitinous cuticula, the channels of which converge toward the anterior opening on the surface of the head.
Although I have unfortunately not studied the true adult frontal gland in sections and can not therefore describe the epithelial cells, the facts that it is larger than in the nymph with long wing pads, and that it has acquired a chitinous cuticula.
BRAIN OF THE WHITE ANT
581
and an opening to the exterior, seem to indicate that this gland probably produces secretion and is therefore functional. The true adult frontal gland in this respect would represent a later phase in development than that of the n>Tnph wdth long wing pads.
3. The njTiiph with short wing pads. The frontal gland of this nymph is similar in all respects except size to that of the nymph with long wing pads. Although this gland as a whole is smaller (fig. 6, A), its component epitheUal cells are similar in height and width to those of the gland just ascribed. The two types of cells, the numerous long slender ones, 1, and the few swollen gland (?) cells, 2, are also present. No cuticula is dis
Fig. 6 Sections of the frontal gland. ^1, the median part of the ghind of a nymph with short wing pads; li, the lateral part of the gland of a soldier, Homog. immers. 1.8 mm., oc. 6, reduced one fifth.
tinguishable on these cells and there is no external opening. This gland is evidently not yet functional.
4. The soldier. The cells of the elongated frontal gland of the soldier form a continuous sj'ncytium, a part of which is shown in section in figure 6, B. The syncytium rests upon a thin basement membrane, the nuclei are rather widely separated, and the distal, inner, surface is bordered by a wavy glassy and porous cuticula. The entire inner surface is thrown into folds but the average height of the component cells is lower in the soldier than in the nymplis. The cells are all of one kind, and are evidently glandular in function, the abundant cytoplasm contain
582
CAROLINE B. THOMPSON
ing a network of intracellular canals that open into the lumen of the gland by pores in the cuticula. In surface views of the soldier head strands of muscle fibers seem to be connected with the lateral and posterior surfaces of the frontal gland, and, as was stated above, an opening in the cuticula of the head above the gland may be clearly seen. Although no secretion was
Fig. 7 Frontal section of the frontal gland of a worker. B.m., basement
membrane; ms., mesenchyme; /, epithelial cells. Homog. immers. 1.8mm., oc. 6,
reduced one-fifth.
actually observed in the lumen of the gland, the intracellular canals and the pores in the glandular cuticula seem to indicate that the frontal gland in the soldier is in active secretion.
5. The worker. The frontal gland of the worker as seen in surface view (fig. 11) is very small and is surrounded by an empty space. Sections (figs. 7, 25) show that this gland, although bearing a morphological resemblance to that of the
BRAIN OF THE 'WHITE ANt' 583
nymphs of the sexual forms, is in a less highly developed condition, a condition that seems to be secondarily, indeed regressively, modified, rather than primitive. In the area between the mushroom bodies we find (fig. 7) a cup formed by basement membrane, b.m., which, although smaller, is somewhat similar in outline to that of the two nymphs just described. The space within the basement membrane is not, however, entirely filled by the cells of the frontal gland. The cells, 1, occupy only the central part of the cup, the remainder consists of a membranous network of mesenchjmi, 771s., similar to and indeed continuous with the tentorial membrane which lies posterior to the frontal gland and to the supraesophageal gangHon in all castes. The cells of the frontal gland as shown in figure 7, 1, are cut in a plane parallel to their distal upper surfaces, the cell bodies polygonal in outline and closely pressed together, and only a few nuclei are present. In the following sections from deeper portions of the gland, many more nuclei occur. The cells are all of the slender elongated tjT^e, but mucli smaller in every dimension than the slender elongated cells of the two nymphs above described. None of the swollen secretory cells are present. From the general appearance it may be inferred that this gland in the worker is not only nonfunctional but degenerate in structure.
b. General discussion of the frontal gland. To summarize the different structural and functional conditions of the epitheUal cells of the frontal gland in the various forms of L. flavipes, we find that in the soldier the frontal gland is evidently functional and in active secretion. An external openmg is present and the epithelial cells are all glandular, united in a syncytium containing a network of intracellular canals and covered b}' a porous cuticula.
In the true adult the frontal gland is probably functional. An external opening is present above the gland; the gland has enlarged since the last nj-mphal phase, and a cuticula resembling that of the soldier is present upon the inner surface of the epithelial cells. No statement as to the cells themselves can be made now, since no sections of this gland have been studied.
584 CAROLINE B. THOMPSON
In the two n>Tiiphs with long and short wing pads the frontal gland seems not yet functional. There is no external opening above the gland; the cells which appear capable of producing secretion are few and scattered, while most of the cells are narrow and slender with no evidence of any contained secretion, but resembling rather sensory cells in their form and contents. The last nymphal molt will remove the outer cuticula, and the absence of the inner cuticula, described above, will produce the external opening seen in the true adult. At the same time the epithelial cells which were not glandular in the nymphal phases may form a cuticula on their inner surface, acquire the power of secretion, and become fully glandular in function in the adult condition. It would therefore seem that the frontal gland of nymphs with long and short wing pads represents an earher phase of development than that of the true adult or soldier. On the other hand, it is apparent that the frontal gland of the worker is in a secondarily modified and regressive condition. It is evidently nonfunctional, the cells showing no signs of secretion; it is also degenerate in structure, for the cells, although of the same form as in the nymphs, are much smaller, are numerically fewer, and are partly replaced by a network of mesenchym.
The study of the frontal gland might end here with the frequently asked and still unanswered question : What is the function of the frontal gland? The question, however, that has presented itself most forcibly throughout my study of this organ is. Why should most of the epithelial cells of the frontal gland in the nymphal phases bear such a striking, resemblance to sensory cells? Young gland cells may usually be recognized as such even in their early phases, and they are rarely so slender and elongated. If, as the biogenetic law teaches us, ancestral structure is frequently to be observed in young, that is, in developing organs, is this then an instance? It is possible that the frontal gland of the termite, whose function is unknown and whose structure needs further investigation, may represent an ancestral organ, highly developed and secondarily modified in some individuals and vestigeal in others, but whose primitive
BRAIN OF THE 'WHITE ANT' 585
structure and perhaps even whose origin may be revealed in the developing nymphal stages.
This supposition leads naturally to the query, What then was the frontal gland if it were originall}^ not a gland? In answer I wish to offer the suggestion that the frontal gland of the termites may be a modification of the ancestral frontal ocellus or simple eye that is found in many insects, and of which Berlese ('09) says that if the third eye is not present it should be considered as having disappeared. "Ordinamente sono in numero di tre e giacciono sul vertice o sulla fronte — . ... In taluni casi se ne osservano solo due (Grillidae ecc.) ma il terzo devesi considerare come abortito."
Several naturaUsts of the nineteenth ccntur>% notably Jol}' ('49) and Lesp^s ('56), in their study of termites observed the pale depressed spot now known as the fontanel and described it as the third, median, ocellus.
Hagen ('55-'C0) in his description of Tennes flavipes, Kollar, states that there is "Mitten auf dem Scheitel ein flachcn Eindruck mit einem erhabenen helleren Fontanellepunkt." Referring to the earlier view that this 'fontanel point' was a thiid ocellus, he WTites:
Ein drittes Nebenauge fchlt l:)estiinmt; dass das so oft dafiir angegcbenc Organ (dem iibrigcns stets cine gew ll)te Hornliaut fchlt) kcin Ncbenauge sein kann, liatte man schon aiis seiner Lago abnohmen konnen. Es liegt niimlich immer bctrachtlich h iher auf clem Scheitel als die Nebenaugen, ein Verhiiltniss, dass bei den Insektcn ohnc Belspiel ist; das dritte Ncbenauge liegt bei alien bckannten Insektcn n-iher dem Miinde, als die seithchen.
Nassonoff ('93) described the structure of the frontal gland in a termite soldier.
Czerwinsky ('97) studied a number of termites, including some tropical forms. He described the structure of the frontal gland as follows:
Die Stirndriise liegt hinter dem Oberschlundganglion ein wenig iiber demselben and geli irt immer zu den mehrzelligen Driisen, in der Bildung aber stellt sie mannigfiiltige Grade der Entwicklung dar. In dem einfachen Fiille besteht sie aus einer Schieht in die Liinge verzogener Hypodermiszellen. Solchen einfachen Bau findet man bei vielen
586 CAROLINE B. THOMPSON
Arbeitarn (Eutcrmes sp., Eutermcs Ripperti, Termes Miillori). Einen komi)lizicrtcrcii Ban finclcn wic bei einer anderen Gruppe der Arbcitcr (Termes lucifiigus, Eutcrmes capricornis, Termes dirus) und boi den gefliifjelten Insekten. Die Driisenzellen sind hier stark in der Lange
- uissezof;en und bilden zusammen ein Sackchen. Das Sekret der
Driisenzellen hauft sich zwischen denselben und dem Chi tin und gelangt wahrscheinlichcrst durchdie Korperoberflilche nach aussen. Das Chitin ist hier sehr dAnn und bildet ausserhalb einen weissen Fleck (Fontanelpunkt der Beschreibungen). Dieser Fontanelpunkt wurde sogar von einigen Verfassern als ein drittes Nebenauge angenommen.
Hagen leugnet die Existenz des dritten Nebenauges, aber erklart nicht die Bedeutung des Fontanellpunktes. Der Fontanellpunkt niit der anliegenden Oberflache l:>ildet eine wenig deutliche Verticfung. Durch tiefere Einsenkung der Driisenzellen ins Innere mit der dariiberliegenden Cuticula kann sich ein Reservoir bilden aus dem das Secret durch eine Oeffnung heraustritt. Eine solche Bildung stellt die Stirndriise bei den Soldaten dar. Den kompliziertesten Bau der Stirndriise finden wir bei den soldaten Nasuti und Arbeitern Nasuti, bei denen ausser dem schon beschriebenen Bau ein Ausfiihrsgang in der Nase vorkommt.
Holmgren, '09, finds the fontanel gland similar in structure in the imago and worker of Eutermes chaquimayensis. The glands consist of a single layer of elongated cells. Two muscles, the mm. retractores fontanellae, are attached near the tip of the gland. No actual secretion was observed. In the soldier, the frontal gland consists of two clearly defined parts, (1) the glandular part, of high cylindrical cells, with a row of short rods (Stabchen) on the distal ends, (2) the duct of low and culticulated cells.
Feytaud ('12), in a study of 'Le termite lucifuge,' writes:
La glande fontanellaire ou glande frontale est formee par la differentiation d'une groupe de cellules hypodermiques au niveau de la partie superieure du front, au-dessous de la zone plus claire designee sous le nom de fontanelle. Dans beaucoup de cas, la glande frontale n'est representee que par une differentiation des cellules hypodermiques, sans depression. Enfin, dans certaines formes, elle est completement absente.
Feytaud cites the nymph of the second form as an instance of a slightly differentiated frontal gland, stating that hke that of the worker the frontal gland in this nymph is merely a zone of elongated hypodermal cells. In the conclusion of this paper Feytaud asks What is the role of the frontal gland?"
BRAIN OF THE 'WHITE ANT' 587
Wasmann ('02), in describing the new genus Speculitemies states that it is " Ausgezeichnet durch die grosse, unpaare Stirnocelle, welche nicht nur bei der Imago, sondern auch beim Arbeiter vorhanden ist."
Now, although I am firmly convinced that the fontanel spot with its opening is not an ocellus or simple eye, I feel that there is considerable evidence that the frontal gland of the termites may have developed phylogenetically from an ancestral median eye and its ocellar nerve. In other words, the frontal gland may have first arisen as a modification of the hj^podermis at the point where the ancestral median eye was formerly situated, and then may have extended inward into the head along the course of the former median ocellar nerve, the proximal part of which maj^ still persist in the nerve which I have termed provisionally the fontanel nerve.
The evidence in support of this view, gained from the study of the different forms of L. flavipes, ^^^ll now be presented.
1. The anterior surface of the frontiil gland of the n}inphs and adults of the sexual forms hes in the same frontal \)hu\e as the lateral ocelli. The posterior end of the frontal gland lies in the region in which the lateral ocellar nerves enter the protocerebral fibrous core. The frontal gland has therefore the same linear extent as the lateral ocelh and the ocellar nerves.
2. The nerves from the lateral ocelli run from the ocelli in toward the median line on the outside of the brain sheath, directh' beneath the h>T3odermis, then pierchig the brain sheath, they run downward and backward within the nerve cell layer of the brain, finally entering the fibrous core of the protocerebral lobes at a definite point, namely: immediately behind the posterior dorsal commissure. The fontanel nerve from the frontal gland enters the protocerebral fibrous core in the median line, immediately behind the posterior dorsal commissure, and, according to the present theory, may represent the median ocellar nerve (fig. 19).
3. The lateral ocelli are derived from the hypodermis, and the tips of the \'isual cells still lie in contact with and perhaps between the h^-podermal cells. The frontal gland is directly con
588 CAROLINE B. THOMPSON
tiniious ^vith the hypodermis, and its cells are modified hypodermal cells.
4. The inner cuticula is thinner at the margins and lacking above the center of the lateral ocelH (fig. 13). The same is true of the inner cuticula above the frontal gland (fig. 16).
5. The cells of the frontal gland in the adult stages are secretory in function and resemble other gland cells in structure, but in the developing nymphal stages the cells produce no secretion and bear a striking resemblance to the slender elongated visual cells found in insect ocelli, Hesse ('01 b). According to our theory this ontogenetic phase corresponds to a primitive, phylogenetic, condition. In this connection it is significant that in very young n5rmphs the frontal gland is barely distinguishable in surface views, while the brain and eyes are quite well developed. The farther study of these youngest nymphs by means of sections may give additional evidence.
6. The phylogeny of the termites upon the basis of the morphology of the frontal gland suggested by Holmgren ('09) may be referred to as additional evidence. Holmgren has divided all adult sexual termites into three groups: The first group, including all the higher termites, consists of those forms with a sack-like frontal gland (sackformige Druse) and a fontanel opening; the second group, including part of the lower termites, consists of those forms with a plate-like nonglandular 'gland' (Fontanellplatte) and no fontanel opening; the third group, including the remainder of the lower termites, consists of forms in which both the plate-Uke nonglandular 'gland' (Fontanellplatte) and the fontanel opening are lacking. Holmgren beheves that the sack-like fontanel gland and the nonglandular plate are morphologically equivalent, and that both are derived from a common ancestor without either gland or plate. He further remarks that no fontanel is found in the Blattidae, which in this respect are more nearly related to the lower termites. To quote his exact words:
Versuchen wir nun eine Phylogenie der Termiten auf dem Verhalten der Fontanelle zu griinden, so m ichte erstens bermerkt werden, dass diejenigen Formea, welche Fontanelldriisen (Fontanellplatte und sack
BRAIN OF THE 'WHITE ANT' 589
formige Druse) besitzen, aus Formen ohne driisige Fontanellplatte stammen miissen. Da es wenig wahrscheinlich ist, dass eine driisige Fontanellgewebe unabhanig bei verschieden Termitengruppen entstanden ist, so miissen wir die jenigen mit Fontanelplatte und die jenigen mit schlauchformiger Fontanelldrlise von einer gemeiasamen Ausgangspunkt ableiten.
7. It is always questionable whether comparisons should be drawn between invertebrates and vertebrates, but it does not seem wholly improbable that the frontal gland of the termites may have had a phylogenetic history similar to that of the vertebrate pineal gland. The disappearance of the simple median vertebrate eye may be understood in conjunction with the increasing complexity of the compound ej'es, and a brief survey of termite habits may throw light upon the causes of the disappearance of this median invertebrate eye. The true sexual adults are the most conservative and primitive in structure and in habits of all the castes. They are deeply pigmented, possess long functional wings, and for a short time lead an aerial existence like other tj^^ical winged insects, and unlike all the other members of the conununity. After the short period in the air, which termite observers tell us is not a nuptial flight, the true adults abandon their normal ancestral mode of life and begin a secondarily acquired existence within the total darkness of their burrows. Here the long wings are shed, mating takes place, and from henceforth they live ^^^thin the dark and narrow chambers of the nest. It is possible that this sudden and marked change of environment may account in part for the disappearance of the median eye in the ancestral termite and that the frontal gland may have arisen in response to some new need.
SUMMARY OF RESULTS
The purpose of this study is to compare the brains of the different castes of L. flavipes, and, further, to compare them with those of the castes of ants, since both termites and ants are social insects with a highly organized conununity life, but with a very different degree of specialization and intelligence.
The forms whose brains are here discussed are: the nymph of the first form, with long wing pads, the njnnph of the second
590 CAROLINE B. THOMPSON
form, with short wing pads, the soldier, the worker, and, to some extent, the true adult.
1. There is no differentiation between the brains of the males and females of any caste or stage of L. flavipes.
2. There is very little differentiation between the brains of the different castes and stages of L. flavipes here discussed.
a. The most marked differentiation is in the optic apparatus, a correlation existing between the degree of development of the compound eyes and the size of the optic lobes. The latter are large in the nymphs and adults of the sexual forms, but are greatly reduced in the worker and soldier.
h. The mushroom bodies differ very little in size, by actual measurement and in the estimated number of cells. They are largest in the worker, smallest in the soldier, and are intermediate in size in the sexual forms, although the mushroom bodies of the true adult are nearly as large as those of the worker.
c. The antennary lobes are very similar in size, but are largest in the worker and in the sexual forms and slightly smaller in the soldier.
3. The mushroom body stalks do not end beneath the central body, as was formerly thought, but divide into three roots, the anterior, central body, and posterior roots. The latter are expanded into large and prominent lobes.
4 The labral nerves arise from the ventral surface of the frontal ganglion, and not directly from the labrofrontal nerves.
5. The termite brain as a whole is very similar in structure to the brain of ants, with the notable exception of the mushroom bodies which are of a much more simple and primitive type. This is apparent in : (a) the nerve ceUs, which are all very small and of equal size, (6) the separation of these cells into three groups, instead of the four of ants, (c) the incomplete separation of the two lobes.
Only the two lateral simple eyes or ocelli are present in the nymphs and adults of the sexual forms, and none are found in the worker or soldier. These ocelli are very simple and primitive in structure, without lens or pigment, and the ocellar nerves do not expand into ocellar lobes as in ants.
BRAIN OF THE VhITE ANT' 591
6. The frontal gland is a gland found in all the castes of L. flavipes, situated on the postero-dorsal surface of the brain in the space between the mushroom bodies. The gland is composed of epithelial cells which are continuous with the hypodermal cells, A nerve, termed provisionally the fontanel nerve, runs from the frontal gland into the brain.
In the ti-ue adult and soldier the frontal gland is evidently functional; in the worker it is nonfunctional and degenerate in structure; in the nymphs with long and short wing pads it is evidently not j'et functional, a few cells are glandular in appearance with contained secretion, the large majority of the cells are slender and elongated and resemble sensory, visual, cells.
7. The suggestion is made that the frontal gland may have arisen phylogenetically from the ancestral median ocellus wliich is now lacking in the termites, and that the 'fontanel' nerve may be a vestige of the former median ocellar nervT.
The arguments for this view are based upon the position and the structural resemblances of the frontal gland and the lateral ocelli, upon the presence of the 'fontanel' nerve in the same frontal section in which the lateral ocellar nerves enter the brain, upon the resemblance of the frontal gland cells of the developing njmiphal phases to visual cells, and upon the facts collected by Holmgren regarding a phylogeny of the termites based upon the morphology of the frontal gland.
WELLESLEY, MASS. AUGUST 18, 1916
THE JOURNAL OF COMPAUATIVE NEUnOLOCV, VOL 26, NO 5
'>92 CAROLINE B. THOMPSON
BIBLIOGRAPHY*
Banks, W. 1907 A now species of Termes. Ent. News, vol. IS, no. 9.
Berlese, a. 1909 Gli Insetti, vol. 1.
Bbues, C. T. and Melander, A. L. 1915 Key to the families of North American insects.
Bugnion, E. 1913 La differentiation des castes chez los termites (Nerv.) Bull. Soc. Ent. de France, No. 8.
GzERWiNSKY, K. 1897 Beitriige zur Kenntniss der Termiten. Zool. Anz., Bd. 20.
Feytaud, J. 1912 Contribution h I'etude du termite lucifuge. Archives d'anat. microsc, T. 13.
Grassi, B., and Sandias, A. 1893 The constitution and development of the society of termites; observations on their habits; with appendices on the parasitic protozoa of Termitidae and on the Embiidae, translated bj' T. H. Blandford. Quart. Jour. Micros. Sci., vol. 39, n. s., April, 1897.
Hagen. H. a. 1855-60 Monographic der Termiten. Linn. Ent., Bd. 10, 12, 14.
Heath, H. 1903 The habits of California termites. Biol. Bull., vol. 4.
Hesse, R, 1901a Ueber die sogen. einfachen Augen der Insekten. Zool. Anz., Bd. 24.
1901 b Untersuchungen iiber die Organe der Lichtempfindung bei niederen Thieren. 7. von den Arthropoden-Augen. Zeit. f. wiss. Zool., Bd. 70.
Holmgren, N. W. 1909 Termiten Studien. 1. Anatomische Untersuchungen. K. Svenska Vetensk. Akad. Hand., Bd. 44.
1911 Termiten Studien 2. Systematik der Termiten. K. Sv. V. Ak. Hd., Bd. 46.
1912 Termiten Studien 3. Systematik der Termiten. Die Familie Metatermitidae. K. Sv. V. Ak. Hd., Bd. 48.
1913 Termiten Studien 4. K. Sv. V. Ak. Hd., Bd. 50.
JoLY 1849 Recherches pour servir a I'histoire naturelle et a I'anatomie des
Termites. Mem. Acad. Sc. Toulouse, t. V. .Jonescu, C. 1909 Vergleichende Untersuchungen iiber das Gehirn der Honig biene. Jenaische Zeit., Bd. 45. Kollar 1837 Naturgeschichte der schiidlichen Insekten. Lespes, C. 1856 Recherches sur I'organisation et les moeurs du Termite
lucifuge. Ann. Sci. Nat. Zool., 4 serie, t. 5. Marlatt, C. L. 1908 The white ant (Termes flavipes, KoU.) U. S. Dept. Agr.
Bur. Ent. Circ. no. 50. Nassonoff, 1893. Ueber Eigenthumliche auf den Nestenbau beziigl. Organi sationsverhaltnisse bei den Termiten. Entom. Untersuchungen, Warsaw. PiETSCHKER, H. 1911 Das Gehirn der Ameise. Jenaische Zeit., vol. 47. Silvestri, F. 1901 Boll. Mus. Torino, vol. 16, no. 389.
1903 Redia, vol. 1, p. 37.
' More extensive bibliographies of the termites may be found in the works of Holmgren ('11), Feytaud ('12), and Snyder ('15).
BRAIN OF THE 'WHITE ANT' 593
Strickland, E. H. 1911 A quiescent stage in the development of Termes
flavipes, Kollar. Journal of N. Y. Ent. Soc. Snyder, T. E. 1915 Biology of the termites of the Eastern United States,
with preventive and remedial measures. U. S. Dept. Agr., Bur. Ent.,
Bull. no. 94, pt. II.
1916 Termites, or 'White Ants,' in the United States: their damage,
and methods of prevention. U. S. Dept. Agr. Bur. Ent., Bull. no. 333. Thompson, C. B. 1913 A comparative study of the brains of three genera of
ants, with special reference to the mushroom bodies. Jour. Comp.
Neur., vol. 23.
1914 The mushroom bodies of the worker of Bora bus sp. Jour. Comp.
Neur., vol. 24. Wasmann, E. 1902 Termiten, Termitophilen und Myrmekophilen. Zool.
Jahrbuch., Abt. f. Syst., vol. 17.
1908 Zur Kastenbildung und Systematik der Termiten. Biol. Cen tralblatt., vol. 4§. Wheeler, W. M. 1904 The phylogeny of termites. Biol. Bull., vol. S.
PLATE 1
EXPLANATION OF FIGURES
8 and 9 are drawn from whole mounts of the heads. Figure 10 is a combination drawing of the outline of the head and the brain, the latter added from a dissected and mounted brain. The stippling represents the nerve cell layer, the fibrous matter is blank. Obj. 16, oc. 6, stage level, reduced one-third.
8 The head and brain of a nymph with long wing pads.
9 The head and brain of a true adult.
10 The head and brain of a njonph with short wing pads.
EXPLANATION OP PLATES »
All figures are drawn with the Spencer camera lucida and with Spencer lenses
ABBREVIATIONS
a.cm.. anterior dorsal commissure
a.l., antennary lobe
a.r.m.h., anterior root of mushroom
body h.m., basement membrane C.6., central body c.h.r., central body root c.e., compound eye cu., cuticula f.g., frontal gland f.n., fontanel nerve fr.gn., frontal ganglion gl., glia cells hyp., hypodermis i.cu., inner cuticula i.f., inner fiber mass la.n., labral nerve Ih.n., labial nerve l.J.n., labrofrontal nerve m.h., mushroom body
m.b.s.. mushroom body stalk
fiid.n., mandibular nerve
m.f., middle fiber mass
ms., mesenchym
mx.n., maxillary nerve
oc, ocellus
oc.n., ocellar nerve
o.cw., outer cuticula
oe., oesophagus
o.f., outer fiber mass
O.I., optic lobe
p.l., protocerebral lobe
p.r.m.b., posterior root of mushroom
body '
r.n., recurrent nerve sb.g., subesophageal ganglion tr.cm., tritocerebral commissure tr.L, tritocerebral lobe V.C., ventral connective
594
BRAIN OF THE 'WHITE ANT'
CAROLINE B. THOMPSON
J r.gn
10
595
PLATE 2
EXPLANATION OF FIGURES
11 and 12 are drawn from whole mounts of the head. The stippling represents the nerve cell layer, the fibrous matter is blank. Obj. 16, oc. 6, stage level, reduced one third.,
11 The head and brain of the worker.
12 The head and brain of the soldier.
596
BRAIN OF THE 'WHITE ANT'
CAROLINE B. THOMP80>J
PLATE 3
EXPLANATION OP FIGURES
13 to 20 are taken from a series of frontal sections of the head of a male nymph with long wing pads, beginning at the frontal surface. The fibrous core is mottled, the nerve cell layer is blank. Obj. 16, oc. 6, table level.
13 Section through the anterior part of the protocerebral lobes, p.?., showing the lateral ocelli, cc, and the anterior margin of the frontal gland, f.g.
14 Section showing the connection of the anterior roots, a.r.m.b., and the stalks, m.b.s., of the mushroom bodies.
15 Section through the central body, c.b., and the central part of the mushroom bodies, m.b.
16 Section showing the entrance of the central body roots, c.b.r., into the central body.
BRAIN OF THE 'WHITE ANT
CABOLINB B. THOMPSO;^ Pl.
hyp
-- a.rm_i,
PLATE 4
EXPLANATION OF FIGURES
17 Section through the posterior part of the central body, showing the origin of the posterior roots of the mushroom bodies, ■p.r.m.h.
18 Section showing the posterior dorsal commissure, p.cm. The exit of the labrofrontal nerve, l.f.n., from the nerve cell layer is also shown.
19 Section showing the entrance of the ocellar nerves, oc.n., and of the fontanel nerve, /.n., into the protocerebral fibrous core. The large lobes of the posterior roots of the mushroom bodies, p.r.m.h., are very prominent, and are also connected with the protocerebral fibrous core.
20 Section showing the connection between the protocerebrum, deutocerebrum, and tritocerebrum.
BRAIN OF THE 'WHITE ANT'
CAROLINE B. THOMPSON
p.r. Vl.h
PLATE 5
EXPLANATION OF FIGURES
21 to 26, obj. 16, oc. 6, table level.
21 Frontal section through the anterior part of the brain of a soldier.
22 Frontal section through the central part of the brain of a soldier.
23 Frontal section through the posterior part of the brain of a soldier.
24 Frontal section through the anterior part of the brain of a worker.
25 Frontal section through the central part of the brain of a worker.
26 Frontal section through the posterior part of the brain of a worker.
BRAIN OF THE 'WHITE ANT'
CAROLINE B. THOMPSON
/r,gn._2^
. ar»nj»
21
24
l.p
Z2
25
f-9s
26
23
SUBJECT AND AUTHOR INDEX
A cms. The taste of 453
Action of light. Changes in the rocI-vis\ial
cells of the fros flue to the 429
.Activity on the hi.stolonical structure of nerve cells. The efTc--t of 341
Afferent system of the head of Amblvstoma. Correlated anatomical and physioloaical studies of the irrowth of the nervous system of Amphibia. If. The 247
^\lhino rat). A preliminary determination of the part played by myelin in reducing the water content of the mammalian nervous system 443
.\lle.v, William F. Studies on the spinal cord and medulla of cyclostomes with special reference to the formation and expansion of the roof plate and the flattening of the spinal cord 9
.\ml)lystoma. I. The forebrain. Regeneration in the br.iin of 203
.\mphibia. II. The afferent system of the head of Amblvstoma. Correlated anatomical and pliy.sli)logioal studies of the urowth of the nervous system of. 247
.\nt, I.eucotcrmes flavipes, Kollar. The brain and the frontal gland of the castes of the "white" 553
AuEY, Leslie B. Changes in the rod-visual cells of the frog due to the action of light.. 429
.\key, Leslie R. The function of the efferent fibers of the optic nerve of ti.shes 213
.VnEY, Leslie H. The influence of light and temperature upon the migration of the retinal pigment of I'lanorbis trivolvis 359
Akey, Leslie H. The movements in the visual cells and retinal pigment of the lower vertebrates 121
B.\ILKY. Percival. Morphology of the roof plate of the forebrain and the lateral choroid plexuses in the human embryo. 79
Bailey, Percival. The morphologv and morphogenesis of the choroid plexuses with especial reference to the development of the lateral telencejihalic ple.xus in Chrysemys marginata 507
Brain. A study of a Plains Indian 403
Brain and the frontal gland of the castes of the 'white ant,' Leucotermes flavipes, Kollar. The 553
Brain of Amblystoma. I. The forebrain.
Regeneration in the 203
Brain of reptiles. Evidence of a motorpallium in the fore- 475
Bl-rr, H. Sv.>:to.v. Regeneration in the brain of Amblystoma. L The forebrain 203
CASTES of the 'white ant,' Leucotermes flavipes, Kollar. The brain and the
frontal gland of the 553
Cells and retinal pigment of the lower vertebrates. The movements in the visual... . 121 Cells of the frog due to the action of light.
Changes in the rod-visual 429
Celb. The effect of activity on the histological structure of nerve 341
Central nervous system of the woodchuck (Marmota monax) during hibernation. Ab.sence of chromatolytic change in the.. 391
Change in the central nervous system of the woodchuck (.Marmota monax) during hibernation. Absence of chromatolytic 391
Chase, Martin R. .\n experimental study of the vagus nerve 421
'Chemical sense' in vertebrates. Regarding the existence of the 'common' 1
Choroid plexu,se,s in the human embryo. Morphology of the roof plate of the forebrain and the lateral 79
Choroid plexuses with especial reference to the development of the lateral telencephalic plexus in Chrysemys marginata. The morphology and morphogenesis of the 507
Chromatolytic change in the central nervous system of the woodchuck (Marmota monax) during hibernation. Absence of 391
Chrysemys marginata. The morpliology and morphogenejiis of the choroid plexuses with especial reference to the development of the lateral telencephalic plexu.s in 507
Common chemical sense' in vertobnites.
Regarding the existence of the 1
CoGUiLL, fi. E. Correlated anatomical and physiological studies of the growth of the nervous system of Amphibia. II. The afferent system of the head of .Vmblvstoma 247
Content of the mammalian nervous system (albino rat). A preliminary determination of the part played by myelin in reducing the water 443
Cord and medulla of cvclostomes with special reference to the formation and expansion of the roof plate and the flattening of the spinal cord. Studies on tic spin.il. . 9
Cranial nerviv. The structure of the third, fourth, fifth, sixth, ninth, eleventh and twelfth 541
Crozier, W. J. Regarding the existence of the 'common chemical sense' in vertebrates 1
CROiiER, W. J. The taste of acids 453
Cyclostomes with special reference to the formation and expansion of the roof plate and the flattening of the spinal cord. Studies on the spinal cord and medulla of 9
DEVELOPMENT of the dorsal ventricular ridge in turtles. The 481
Development of the lateral telencephalic plexus in Chrysemys marginata. The morphology and morohozenesis of the charoid plexuses witt especial reference to the 507
DoN\LDsov, H. H. A prelinninary determination of the part plaved by myelin in reducing the water content of the n.ammalian nervous system al ino rati 443
Dorsil ventricular ridge in turtles. The development of the 481
605
600
INDEX
EFFECT of activity on the histoloi;ical striicturo of nerve cells. The 341
Efferent fibers of the optic nerve of fishes.
The function of the 213
Rnibryo. Mornholocv of the roof plate of the foro'irain and the lateral choroid ple-\usesin the human 79
FIBERS of the optic nerve of fishes. The function of the efferent 213
Fishes. The function of the efferent fibers of the optic nerve of 213
Flattenins; of the spinal cord. Studies on the spinal cord and medulla of cycloston'es with special reference to the formation and expansion of the roof plate and the . 9
Flavice?, KoUar. The brain a,nd the frontal Rland of the castes of the 'white ant,' Leucotermes 553
Forebrain and the lateral choroid plexuses in the human ernbryo. Morphology of the roof plate of the 79
Forebrain of reptiles. Evidence of a motor pallium in the 475
Forebrain. Reseneration in the brain of Amblystoma. I. The. 203
Frog due to the action of light. Changes in the rod-visual cells of the 429
Frontal gland of the castes of the 'white ant,' Leucotermes fiavipes, Kollar. The brain and the 553
GLAND If the castes of the 'white ant,' Leucotermes flavipes, Kollar. The brain and the frontal 553
HEAD of Amblystoma. Correlated anatomical and physiological studies of the nervous system of Amphibia. II.
The afferent system of the 247
Hibernation. Absence of chromatolytic change in the central nervous system of the woodchuok (Marmota monax) during 291 Hilton, William A. The nervous system of pycnogonids 453
TNDIAN brain. A study of a Plains 403
JOHNSTON, J. B. Evidence of a motor pallium in the forebrain of reptiles 475
Johnston, J. B. The development of the dorsal ventricular ridgs in turtles 481
KEEGAN, J. .1. A study of a Plains Indian brain 403
KocH,S[j\iNEH L. The structure of the third, fourth, fifth, sixth, ninth, eleventh and
twelfth cranial nerves 541
KocHER, R. A. The effect of activity on the histological structure of nerve cells 341
LIGHT and temperature upon the migration of the retinal pigment of Planorbis
trivolis. The influence of 359
Light. Changes in the rod-visual cells of the
frog due to the action of , 429
Leucotermes flavipes, Kollar. The brain and the frontal gland of the castes of the 'white ant' 553
MAMMALIAN nervous system (albino rat). A preliminary determination of the part played by myelin in reducing
the water content of the 443
Marginata. The morohology and morphoeenesis of the choroid plexuses with especial reference to the development of the lateral telencephalic plexuses in Chrysemys 507
fMarinota monax) during hibernation. Absconce of chromatolytic chance in the central nervous system of the woodchuck... . 391
Medulla of cyclostome.^ with special reference to the formation and expansion of the roof plate and the flattening of the spinal cord. Studies on the spinal cord and 9
Migration of the retinal pigment of Planorbis trivolvis. The influence of light and temperature upon the 359
Monax) during hibernation. Absence of chromatolytic change in the central nervous system of the woodchuck (Marmota. . 391
Morphogenesis of the choroid plexuses with especial reference to the development of the lateral telenceohalic plexus in Chrysemys marginata. The morphology and 507
Morphology anrl morphogenesis of the choroid plexuses with especial reference to the development of the lateral telencephalic plexus in Chrysemys marginata. The.... 507
Motor pallium in the forebrain of reptiles. Evidence of a 475
Movements in the visual cells and retinal pigment of the lower vertebrates. The 121
Myelin in reducing the water content of the mammalinn nervous system (albino rat). A preliminary determination of the part played by 443
My*;rs, J. A., Rasmussen, A. T. and. Absence of chromatolytic change in the central nervous system of the woodchuck (Marmota monax) during hibernation 391
NERVE. An experimental study of the vagus 421
Nerve cells. The effect of activity on the histological structure of 341
Nerve of fishes. The function of the efferent fibers of the optic 213
Nerves. The structure of the third, fourth, fifth, sixth, ninth, eleventh and twelfth cranial 541
Nervous system (albino rat). A preliminary determination of the part played by myelin in reducing the water content of the mammalian 443
Nervous syst'em of Amphibia. II. The afferent system of the head of Amblystoma. Correlated anatomical and physiological studies of the growth of the 247
Nervous system of pycnogonids The 453
Nervous system of the woodchuck (Marmota monax) during hibernation. Absence of chromatolytic change in the central 391
OPTIC nerve of fishes. The function of the efferent fibers of the 213
P.4LLIIJM in the forebrain of reptiles. Evidence of a motor 476
Pigmentof Planorhistrivolvis. The influence of light and temperature upon the migration of the retinal 359
Pigment of the lower vertebrates. The movements in the visual cells and retinal 121
Plains Indian brain. A study of a 403
Planorbis trivohis. The influence of light and temperature upon the migration of the retinal pigment of 359
Plate and the flattening of the spinal cord. Studies on the spinal cord and medulla of cyclostomes with special reference to the formation and e\pan.«ion of the roof. . 9
Plate of the forebrain and the lateral choroid plevuses in the human embryo. Morphology of the roof 79
INDEX
607
Plexus in Chrysemys marginata. The morphology and morphogenesis of the choroid plexuses with especial reference to the development of the lateral telencephalic. . . 507
Plexuses in the human embi./o. Morphology of the roof plate of the forebrain and the lateral choroid 79
I'lexuses with espeiial reference to the development of the lateral telencephalic plexus in Chrysemys marginata. The morphology and morphogenesis of the choroid ... 507
Fycnogonids. The nervous system of 453
RASMUSSEN, A. T. and Myers, J A. Absence of chromatolytic change in the central nervous system of the woodchuck (Marmota monax) during hibernation 391
Rat). A preliminary determination of the part played by myelin in reducing the water content of the mammalian nervous system falbino 443
Regeneration in the brain of Amblystoma. I. The fore'jrain 203
Reptiles. lOvidcnce of a motor pallium in the forebrain of 475
Retinal pigment of Planorbis trivolvis. The influence of light and temperature upon the migration of the 359
Retinal pigment of the lower vertebrates. The movements in the visual cells and . 121
Ridge in turtles. The development of the djrsal vonlricular 4*<1
Rod-visual ct'll.s of the frog due to the action of light, f "liangos in the 429
Roof plate and the flattening of the spinal cord. Studies on the spinal nord and medulla of cyclostomcs with special reference to the formation and expansion of the 9
Roof plate of the forebrain and the lateral choroid plexu.ses in the human embryo. Morphology of the 79
SENSE' in vertebrates. Regarding the existence of the 'common chemical I
Spinal cord and moduU.i of cyclostomes with special reference to the formation and expansion of the ro :)f plate and the flattening of the spinal cord. Studies on the 9
System of Amphibia. II. The afferent system of the head of .\mblystoma. Correlated anatomical and physiological studies
of the growth of the nervous 247
Systen of pycnogonids. The nervous 453
System of the woodchuck {.Marmota monax) during hibernation. .-Vbsence of chromatolytic change in the central nervous 391
nPASTE of acids. The 453
Telencephalic plexus in Chrysemys marginata. The morphology and morphogenesis of the choroid plexuses with espe<"i il reference to the development of tiie lateral 507
Temperature upon the migration of the retinal pigment of Planorbis trivoh-is. The influence of light and 359
Tho.mi>so\, Caroline Btrlino. The brain and tlie frontal gland of the castes of the 'white ant,' I.eucotermes fla\-ipes, Kollar 553
Trivolvis. The influence of light and temperature upon the migration of the retinal pigment of Planorbis 359
Turtles. The development of the dorsal ventricular ridge in 481
VAGI'S nerve. An e.tpcrimental study of the 421
Ventricular ridge in turtles. The development of the dorsal 4S1
Vcrtel>ratos. Regarding the existence of the 'common chemical sense' in 1
Vertebrates. The niovements in the visual cell.s and retinal pigment of the lower. .. 121
Visual cells and retinal pigment of the lower vertebrates. The movements in the 12 I
W.VTER content of the mammalian nervous system (albino rat). A preliminary determination of the part played
by myelin in re.lu -ing the 443
Woo Ichuck (.Marmota monax) during hil)ernation. Absence of chromatolytic change in the central nervous system of the 331
