Talk:Book - Contributions to Embryology Carnegie Institution No.11

The Structure of Cchromophile Cells of the Nervous System

CONTRIBUTIONS TO EMBRYOLOGY, No. 11.

By E. Y. Cowdry

Introduction

It has long been known that certain peculiar nerve-colls, well characterized by their structural appearance, occur in the normal human brain, and indeed in the brains of all the vertebrates which have been examined. In fixed preparations they are slightly shrunken, they stain deeply with both acid and basic dyes, and their nuclei are obscure and hard to define. Flesch (1887, p. 196) called them "chromophile" cells. Nissl (1896, p. 1154) thought at first that they were artefacts of some sort, but Cajal (1909, p. 210) and others brought forward strong evidence against this view. Cajal (1909, p. 211) concluded thatthey were resting cells. On the other hand, in the light of Dolley's (1910, p. 333) work, they would seem to be in the initial stages of fatigue, as evidenced by the increase in the amount of Nissl substance in them and by their obscure, deeply-staining nuclei. Our knowledge of their structure is incomplete so far as the mitochondria and the canalicular apparatus are concerned. Busacca Archimede (1913, p. 332), alone, has observed that the mitochondria in certain cells in the brain of Testudo grwca stain particularly intensely with iron hematoxylin, and in some cases seem to lose their definite outlines and to form homogeneous masses. Rina Monti (1915, p. 39) has made a comprehensive study of the canalicular apparatus ("apparati di Golgi") in nerve-cells, but she does not mention cells in the chromophilic condition. I shall consequently venture to present in this paper my observations on these two structures in the chromophile cells in the brain of the white mouse.

Materials and Methods

White mice were employed because they are the smallest mammals which can be conveniently used in the laboratory for experimental purposes. The small size of their nervous system permits the study of the distribution and the arrangement of chromophile cells in serial sections. All the mice were of known age and care was taken that they were perfectly normal.

A modification of the methods of Altmann (1890, p. 27), Galeotti (1895, p. 466), Regaud (1910, p. 296), Bensley (1911, p. 309), and Shirokogoroflf (1913, p. 523) was devised for the study of mitochondria. The method has many advantages. In the first place, the use of a mixture of formalin and ])otassium bichromate as a fixative (Regaud) gives a much more uniform preservation of mitochondria than the osmic acid containing fixatives in general use. The application of the fixative by

'The work was aided by the Department of Embrj-ology of the Carnegie Institution of Washington, and part of it was done at the Marine Biological Laboratory. Woods Hole. Massachusetts, where, through the kindness of the Director, Dr. Lillie. a room was placed at my disposal.

iiijrctiuii tlinuitili the hlood-vi'ssi-ls (r^hirokogorotTi cliniiiiiitcs tiiany vciy ohjectionahk' artrfacts dui' (u faulty poiu'tration. The use of pcnuaiiganatc and oxalic acid (Benslfv) facilitates the staining of the mitochondria with the anilin fuchsin (Altmann), and the countcrstaining with methyl green (Galeotti) ix'rniits of the demonstration of the Xissl substance in the same cell with the mitochondria. The fact thatthe method gives good results in the case of other tissues where the mixtures of Altniann. Flomming. and others are useless on account of their ])o(»r i)()W('rs of penetration, justifies the following detailed statement:

Fixation:

Chloroform tlic aiiiiiial. Inject wanned U.8o ix-r cent Na( '1 sDlutiim into the aorta tliroufjli the ventricle. If tlie lirain alone is to be stuciieil clamp the descending aorta. If the entire nervous system is to be fixed, clamp the cceliac, the renals, the superior antl inferioi- mesenteries, the iliacs, and the brachials. Continue the injection until the salt solution is returned uncolored through the jugulars. During this time lay t)are the arch of the aorta and the carotids from connective tissue, so that they may expand easily ami carry more fluid to the brain. (Jravity pressure of not more than 6 feet may be employed. Cut the inferior vena cava and the jugulars so that the salt .solution may run through e:i.-;ily.

Follow the .salt solution with the formalin and bichromate mixture: 3 per cent i)otassium bichromate. 4 parts; neutral formalin. 1 i)art. The pota.ssium l)ichromate acts best when freshly preparetl. Neutral formalin is made from the commercial variety l)y the addition of magnesium carljonate, a deposit of which should always remain at the i)Ottom of the formalin ijottle. It is important that tlie pressure should be at the maximum when the mixture is first injected, so that the blood-ve.s.sels may be fixed in a state of dilation. If the pressure is low when the fixative comes in contact with the ve.ssel-walls they will l)e fixed in a condition of collap.se. It will then be difficult, or even impo.ssil)le, to obtain a complete injection. The injection should be continued for about an liour.

The brain is then dissected out antl iniinersed in the fluid. In the ca.sc of the mouse's brain it is sufficient to divide it longitudinally into halves. The fixative must be changed every day for 4 or ') days, otherwi.se it undergoes a ciiange eviilenced by a darkening in color. This change is accelerated by light and by heat, so that the tissue should be kept in the dark and in a cool place. Fixation may also be effected by simple immersion of the tissue in the fixative, instead of by injection, but this procedure is not recommended.

After this prolonged fixation the tissue is mordanted in a fresh 3 per cent solution of potassium bichromate, in which it remains for 8 or 9 days, changing every .second day.

Wasli in running water for 24 hours. The object of this careful washing is to remove most of the formalin ami bichromate, for otherwise the tissue will be extremely lirittle and hard to cut.

hrhijdratiitu find einbeddimj:

.')0 per cent alcohol 12 hours; 70 jx-r cent and 9") jier cent alcohol 24 hours each; absolute alcohol G to 12 hours: half al>solute an<l xylol (> hours; xylol .'^ hours: paraffin ()()° ( '. 3 hours: cut in 4/i serial sections.

Staining:

(1) PaKS the .sections, niounleil on slides, duuii 1 hrowgli toluol, absolute, it."), 7(1. .iimI .">() per cent alcohol to distilled water.

(2) 1 jK'r cent u(|ueous .solution of pola.'^siuiii pcrnianganalc 30 seconds; but ihi^ tinie must lie determined experiment, -dly.

(3) .') jMT cent a(|ueous .solution of oxalic acid also about 30 .secomU.

(4) Hin.se in .s<'veral changes of distilled water about a minute. Imnnipletc washing prevents the staining with fuchsin.

(o) Slain in .Mtmatm's anilin fuchsin, which is to lie niaile up as follows: Make a saturatecl Milution of anilin oil in distilled water by shaking the two together (anilin oil goes into solution in water in about 1 per cent). Filti'r aiul add 20 grams of acid fuchsin to 100 c.c. of the filtrate. The stain should 1)C ready to use in about 24 hours. It goes bad in about a month. To stain, dry the slide with a towel, except the small area to which the sections are attached. Cover the sections on the slide wit h a small amount of the slain .-ind heat over a spirit lamp until fumes, smelling strongly of anilin oil.

conic off. .Vlluw to cool. Let the stain icmain on the section.-* for about minutes. Return the stain to the bottle.

(()) Dry ofT most of the stain with a towel and rinse in distilled water, so that the only stain remaining is in the sections. If a large amount of the free stain remains it will form a troublesome precipitate with the methyl green; on the other hand, if too much stain Ls removed the coloration of the mitochondria will Ix; impaired.

(7) Again dry the slide with a towel, except for the area covered by sections. Allow a little 1 per cent methyl green, added with a pipette, to flow over the sections, holding the slide over a piece of white pajicr so that the colors may be seen. Apply the methyl green for about o seconds at first and then modify the time to suit the needs of the tissue.

(8) Drain off excess of stain antl plunge the .slide into 9.5 per cent alcohol for a second or two. then rinse in absolute for the same time, clear in toluol, and mount in balsam.

Several difficulties may be met with: (1) The methyl green may remove all the fuchsin. even when it is only aii]>lied for a short time. This is due to incomplete mordanting of the mitochondria by the chrome salts in the fixative. It may often be avoided, either by omitting the treatment with permanganate and oxalic acid, or by treating the sections with a 2 per cent solution of pota.ssium bichromate for a few minutes immediately before staining (as advised by Bensley). The action of the permanganate and oxalic is to remove the excess of bichromate. (2) Or the fuchsin may stain so intensely that the methyl green removes it verj' slowly or not at all. This, on the other hand, is due to too much mordanting. It may be corrected by prolonging the action of the permanganate and oxalic. (3) Sometimes, after obtaining a good differentiation, the methyl green is wa.shed out before the slide is placeil in toluol. This may be avoided by omitting the 95 per cent alcohol, by passing from the methyl green to the ab.solute direct. (4) Unfortunately the stain Ls not very permanent. Under favorable conditions it will last for 3 or 4 j-ears. The fading in color is hastened by light and by heat, and it proceeds verj' rapidly in a damp atmosphere.

Cajal's (1912, p. 211) uranium-nitrate method was emploj'ed for the canahcuhir apparatus in its original form, except for the substitution of nu^thyl sreen in the place of carmakmi as a counterstain.

Control preparations were fixed in a variety of Huids and were stained in many ways, as will appear later.

The figures have been made from specimens prepared by the above-mentioned fuchsin-methyl green method, by which the mitochondria are stained red, the Xissl substance green, while the canalicular apparatus remains uncolored; and also from specimens made by the uranium-nitrate method, which blackens the canalicular apjxiratus and colors the Ni.ssl substance green.

OBSERVATIONS. Chromophile cells, as the name implies, possess an unusual affinity for stains, which may be either acid or basic. Their structure is variable. A glance at the figures is sufficient to show this. The variations may represent stages in a process, which, when pushed to an extreme, results in a cell in an advanced stage of chromophilia, but of this we have no conclusive i)roof. Neither can we as.scrt that the process proceeds in this direction, for the changes observed may ecjually well be interpreted as taking place in the reverse order. We do not yet know whether the series is homogeneous; that to say, whether we are not arbitrarily grouping several processes of different nature under the same heading. For instance, a mitochondrial increase (figures 1 and 2) may not precede a diffuse staining of the whole cell with mitochondrial d^es (figure 6), which may be brought about in an entirely different waj'. Nevertheless, the cells are all chromophile in the sense already defined, that they stain deeply.

Some chromophilo cells differ only from other cells hy a slifrlit increase in the amount and in the intensity of the staining of the mitochondria ( figure 1 ). There is apparently no correspontling change in the Xissl substance and the morphology of the mitochondria is unaltered.

Other cells show a remarkable increase in the number of mitochondiia. l"or examjile, a cell (figure 2) frequently contains three or four times as many mitochondria as its neighbor: this increase in mitochondria is associated with a slight but perceptible increase in the amount of the diffuse Nissl substance in the cytoplasm and with a darker staining of the acidophilic and basophilic nucleoli and the grovnul-.^ubstance of the micleus. Cells in this condition show no evidence of shrinkage. They may be recognized in Cajal preparations (figure 7) by the changes in the nucleus and the Nissl substance. The Cajal preparations show that the canalicular apparatus is unaltered.

There may be a great increase in the Nissl substance, which is present as a diffuse dei)Osit (figure 3). At the same time some of the mitochondria often lose their discrete outlines and .seem to merge into the surrounding cytoplasm. Mitochondria may not be very numerous in cells of this kind. The nucleus stains intensely and a few clear canals may be seen in its vicinity. The cell has ai^parently shrinkage spaces on either side of it. Preparations, made by fixing in alcohol and staining with tohiidiii l)lue, contain cells in which the Nissl substance is in this condition and Cajal ])reparations show that the canals are unaltered.

Figure 4 illustrates a cell in a rather more advanced stage of chromopliilia. In this cell there is an unusually large amount of Nissl substance and there are further evidences of the disappearance of formed mitochondria, esi^ecially in the cell process. The outlines of the nucleus can barely be made out. The canalicular apparatus shows no modifications either by this method or bj' the Cajal technique.

A very interesting condition is shown in figure 5. Here, with this degree of differentiation, only a few tyjiical mitochondria i)ersist near the origin of the cell ])rocess. The Nissl substance is overshadowed by a cloud of material staining the .same way as the formed mitochondria do in adjacent cells. Figure S illustrates a similar cell in a Cajal prei)arati(m. The Nissl substance in it is increased and there is no modification in the blackened canalicular apparatus. Cells in this condition are often shrunken. Il is difficult to determine whether the shrinkage is the expression of an actual diminution in the size of the cells during life, or whether it is simply the result of a difference in the reaction of chromo])hile cc^lls to the fixation and subse(|uent treatment. The presence of what apjx'ar to be shrinkage spaces around the cells seems to indicate that it is in reality due to the techniiiue em])loyed, because if. on the other hand, it was due to a decrease in the size of the cell during life, one would expect the space to hv filled up by a shifting of neighboring structures. It may be emi)ha.sized that the fact that other cells, in actual contact with chromophile cells, .show no signs whatever of shrinkage must be regarded as one of the distinctive |)roperties of cells in the chromophilic condition. There is. of course, still another interpn'tation. namely, thai the sjjaces in (luestion are unusually large perineuronal si)aces, the enlargement being in some way connected wild the <lilTereiice in the phvsiological c(»ndition of chromophile ci'lls as contrasted with other cells.

THE STUUCTURE OF CIIKOMOPHILE CKLL.S OF THF. NFRVOFS SYSTEM.. 33

The mitochondria maj- apparently- disappear more or less completely in certain cells, and their place be taken by a mass of amorphous material with the same staining properties (figure 6). The nucleus may or may not be visible. Cajal preparations of cells in the same condition (figure 9) show that the canals are unaltered. The nucleus is obscured by the cloud of Xissl substance. The appearance of these cells, in advanced stages of chromo])hilia, would jjerhaps lead one to suppose that they arc degenerating and that their nuclei have disapjjeared. That this is not the case may be seen if one of the mitochondrial preparations is stained with hematoxylin and eosin. The hematoxylin and eosin does not color either the amorphous deposit or the Nissl substance, which, in the mitochondrial and in the Cajal preparations, hides the nuclei. The nuclei have in reality distinct and definite outlines and apjiear to be quite unaltered, except that they contain rather more than the usual amount of chromatin. In fact, the change in the mitochondria and the increase in the amount of the Xissl substance would never have been suspected if hematoxylin and eosm had alone been used.

The distribution of chromophile cells is important. They often occur singly. They may be surrounded on all sides by cells which show no tendency toward an assumption of the clironiophilic condition. They may, on the other hand, occur in clumps. The clumps vary greatly in size. They contain cells in all stages of chromophilia in addition to a variable number of unaltered cells, which are always present, scattered among them.

The neuropil in which the chromophile cells are embedded differs m no way from the neuropil elsewhere. It seems, by all the mitochondrial methods, to be studded with mitochondria. But it must not be thought that the mitochondria occur in anything like equal num])ers in the neuropil of different regions, because there is a remarkable variation in this respect. The mitochondria appear to be intercellular, but unhapi)ily a source of error is introduced by the fact that the unmedullated, and to a les.ser extent the medullated, processes stain in much the same way as the mitochondria, so that in .some cases it is impossible to distinguish between them. Undoubtedly a large number of the mitochondria in the neuroi^il are contained in nerve-cell processes cut in section, but there is no a priori reason wh}' they should not occur free from the cells as an intercellular deposit. This important question can only be .solved by a detailed study of staming reactions, possibly by the elaboration of new methods, or b}' taking advantage of the differential solubilities of mitochondria. It has a direct bearing upon the role of intercellular material in the metabolism of the central nervous sj'stem.

Cells in the chromophilic condition are comparatively rare in the olfactory bulb as compared with the cerebral cortex. In fact, they are more abundant in the cerebral cortex than in any other part of the brain. Clumi)s of them are more common here than in other regions. The clumps vary in size, in shape, and in position in the brains of animals from the same litter, apparently treated in exactly the same way. Chromophile cells are also numerous in the hippocampus. They are, on the contrary, comparatively rare in the corpus striatum and in the thalamus, in both of which they are more frequentlj'^ met with singly than in groups. In the midbrain they are found in about the same number. It is interesting to note that they are

34 THE STRUCTURE OF CIIUOMOPHILE CELLS OF THE NERVOUS SYSTEM

(juite numerous in the eerebellar cortex. The Purknije cells are ])articularly lial)le to show this condition. They are infrequent in the medulla and they scarceh' ever occur in the spinal cord, in the spinal ganglia, or in the sensory ganglia of the cranial nerves, as, for example, the Gasserian ganglion. In other words, this remarkable condition of the nerve-cell is more prevalent in the higher centers than in the lower ones. This is particularly true of chromophile cells in advanced stages of chromophilia.

The question at once arises as to whether these changes in the appearance of the cells are indicative of real alterations in the cells themselves or whether they are merely the result of the treatment to which they have been subjected.

Unfortunatclj' it w^as found impossible to confirm these observations by the stud}' of unstained, living cells by reason of the difficulties met with in attempts to isolate the cells without injuring them. Attempts to stain the mitochondria in living cells by injecting a solution of janus green into the brain through the bloodvessels did not yield satisfactory results because the janus green was almost immediately reduced, first to the leucobase, and then to the red diethylsafranin, by the reducing action of the brain-substance and the absence of an adequate supply of atmospheric oxj'gen, so that observations could not be made. Pure ox3^gen was bubbled through the janus-green solution while it was being injected, in the hope that the reduction of the janus green might thus be retarded, but without success. Attempts to tease out indi\idual cells in the nervous system and to stain them bj' simple immersion in the janu.><-green solution resulted, of course, in a coloration of the mitochondria, but it was on the whole unsatisfactory on account of the unavoidable injury to the cells. Consequently I have had to rely solely upon the study of fixed material.

The results obtained with the fuchsin-methyl gr(>en method and with the Cajal techni(|ue have been confirmed by the detailed examination of material stained liy the Benda method, the Altmann method, and with iron hematoxylin. Chromophile cells are, I think, not artefacts due to alcohol fixation, as Barker (1899, p. 124) suppo.ses, becau.se I have observed them in tissues fixed in a great variety of fluids not containing alcohol. Moreover, Flesch (1887, p. 197) found years ago that they could be identified in the fresh, unstained condition as well as in tissues stained vitally with methylene blue.

The fact that the chromophile cells are very abundant in the superficial layers of the cortex would at first .s(>em to indicate, as some investigators believe, that they arc artefacts due to mechanical maniinilation. The clusters of chromojjhile cells are .sometimes cone-shaped, with the base on the surface of the cortex and the apex of the cone extending inwards, which looks as if they might have been produced by pressure from without which radiated inwards. But isolated clumjis of chromojihile cells occur in deeper i)arts of the brain, which can not be explained in this way. Moreover, a number of other facts seem to be incompatible with this view. In the first place, since all the brains were fixed, l)efore removal, by the injection of the fixative through the blood-vessels, it follows that there could be no mechanical injury until after fixation. The invariable occurnnice of unaltered cells, side by side with the chromophile cells, is hard to explain on tiie basis of mechanical injury, because wh:itever pressure had been brought to bear upon the t i.ssue must necessarily

THE STRUCTURE OF CHROMOPHILE CELLS OF THE NERVOUS SYSTEM. 35

have acted upon both; but one shows the condition and the other does not (as is shown in all the figures). Furthermore, if mechanical injury is the cause of the condition, it is difficult to understand why chromophile cells are so rare in the spinal cord and in the ganglia of the cranial nerves, which are l)f)und down by membranes and which in removal are consequenth^ subjected to greater mechanical injury than the cortex of the brain.

In order to settle the question the results of intentional mechanical injury brought about by bruising the cerebrum and the spinal ganglia with a blunt instrument were studied. It was found that the lesion produced was characterized by the flattening or comin-e.ssion of many cells in the same direction, at right angles to the direction in which the pressure had been exerted. All the cells in the area were uniformly affected. Normal cells were not ."scattered among them. The injured cells stained intensely, but they did not simulate the chromophile cells. The neuropil between them showed marked changes and could readily be distinguished from the neuropil elsewhere in the same section.

Chromophile cells are not the result of differences in the time or in the degree of fixation. The whole brain is uniformly fixed by the methods of technique employed. The distribution of chromophile cells is not related to the arrangement of the bloodvessels, which are the avenues of approach of the fixative. Neither do the mitochondria vary in number with the vascularitj' of the region.

The condition is not due to irregular mordanting with the potassium bichromate, because complete extraction of the bichromate by prolonged treatment with permanganate and oxalic acid does not essentially modifj- the appearance of the chromophile cells when the sections are stained.

Another possibility is that the intense staining of the chromophile cells results from incomjjlete differentiation. Even if this were the case the differences in the rate of decolorization must be the visible expression of real differences in the cells themselves. I have found, however, that the same differences obtain in undifferentiated specimens stained lightlj' with fuchsin, crystal \'iolet. and iron hematoxylin. I have made a number of experiments to determine whether more complete differentiation would bring to light formed mitochondria in cells in which they appear to have been replaced by the amorphous deposit which stains in the same way.

Specimens were stained in the usual fashion with fuchsin and methyl green and were mounted in balsam. Drawings were then made of chromophile cells which had been stained intenselj^ with the fuchsin and in which no formed mitochondria could be seen. The cover-glass was then dissolved off and the slide was passed down through toluol and graded alcohols to water. It was then restained with fuchsin. differentiated more strongly with the methyl green, mounted in balsam, and examined. The same condition was apparent, except that the homogeneous deposit had a distinctly greenish color. The same process was repeated as many as five times with the same cell, increasing each time the extent of differentiation, until the cell stained intenseh' with methyl green and very httle trace of the fuchsin was ■left; still no formed mitochondria were observed; this was repeated with other cells with the result that in some of them formed mitochondria were brought to hght, while in othei-s thev were not.

Similar experiments were performed with individual cells stained a homogeneous black color with iron hematoxylin. The results obtained are easier to interpret because the differentiator, iron alum, does not itself color the tissue like the methyl green. This advantage is counterlialanced by the fact that both the mitochondria and the Xissl substance stain in the same way and it is often difficult to distinguish between them. In many cases, jjarticularly in slightly undifferentiated specimens, the extraction of the stain from chromophile cells by further differentiation brought to light a variable number of formed mitochondria. Moreover, it is worthy of note that the chromophile cells in the cerebral cortex are the last to become decolorized and that the differentiation occurs with unequal rapidity in different parts of the cell, thus indicating that the homogeneous deposit is not present in the same concentration in all parts of the cell.

The end-result of this experimentation is that chromophile cells, particularly those in advanced stages of the condition, contain a diffuse deposit, which stains in a tyi^ical way with all mitochondrial dyes, and which is probably formed by the solution of some of the mitochondria in the cell.

The condition is not due to technique and it is not associated with a visil)le pathological change on the part of the animal.

All the mice employed were api)areiitly normal. They ate well and showed no signs of sickness. They were killed with chloroform, and it may at once be said that the changes are not due to acute chloroform poisoning, because animals killed in other ways, by decapitation, for example, showed the same condition. The mice were not excited, or disturbed or exercised in any unusual way before they were killed. A careful autop.sy of each mou.se was made to make sure that it was quite normal. Some were found to contain a parasite, present in the cysticercus stage in the liver; these were invariably discarded. The chromophile cells were found in mice of both sexes in almost all seasons of the year. They were found in mice Aarying in age from 25 days to adults, so that they can not be regarded as an expres,sion of .senility. It was thought that they might occur in consequence of abnormal conditions due to captivity. In order to settle this point a wild field-mouse was captured alive and in good condition and its brain was prepared in the usual way. It, also, showed chrom()i)hile cells.

An apparentlj'^ analogous partial solution of mitochondria was observed in liver-cells poisoned with i^hosphorus by INIayer, Rathery, and Schaeflfer (1914, |). G09). Accordingly, W. J. M. Scott tried the effect of experimental phosphorus ])oisoning on the nervous system of white mice. The chromophile cells were apparently entirely unaffected and a solution of mitochondria was not brought about. Dr. Bensley made the interesting suggestion to me that this partial solution of mitochondria in chromoi)hile cells might be due to a swing of the reaction in them toward the acid side, with the liberation of free fatty acids. I therefore made some preliminarj^ experiments on acidosis in mice produced by the subcutaneous injection of dilute hydrochloric acid, all of which yielded negative results as far as the chromf)phile cells were concerned. I have, further, found that slight exercise does not alter the appearance of the chromophile cells in the brains of white mice to any noticeable extent.

It seems highly probable, therefore, that chromophile cells occur normally in the bram of the white mouse and that we have to reckon with a partial solution of mitochondria just as we have for many years recognized a chromatolj'sis, or solution of the Nissl substance.

Discussion

This work on chromophile cells has, I believe, an important bearing upon (1) the question of differential nerve-cell activity; (2) the phenomena of chondriolysis and hyperchromatism; (3) the functional independence of the mitochondria and the canalicular apparatus; and (4) our conception of the structure of hving nerve-cells.

(1) The distribution of chromophile cells in the different parts of the brain is interesting. The fact that they occur most abundantly in the cerebral cortex and in the cerebellum, and that they are rarely found m the lower centers hke the spinal cord, would seem to indicate that the central neurones differ in some way from the more peripheral ones. The difference may be one of lability, for DoUey (1914, p. 56) has found that more highly speciahzed cells are more prone than less specialized ones to respond with structural changes to physiological experimentation. Moreover, the occurrence of these cells in groups, which \'ary m size and in position in different brains, is in accordance with our conception of the alternation of rest and activity in the higher centers and may well have some bearing upon the vexed problem of cortical locahzation, for as yet neither the mitochondria nor the canahcular apparatus have been considered in this connection.

(2) We must recognize a "chondriolysis, " or a partial solution of mitochondria, in nerve-cells as well as a "chromatoh'sis." The word "chondriolysis" was first employed by Romeis (1912, p. 139) to describe the disintegration of certain mitochondria which escaped from the cells into the uterine fluid of Ascarifi. It is, to my mind, more appropriate than the term "chromatolysis," which is frequently apphed to the so-called solution of Nissl bodies, for the simple reason that I am of the opinion (1914, p. 20) that the Nissl substance is usually in solution in the hving nerve-cell, whereas the mitochondria are assuredly present as definite formed bodies (except of course in the chromophilic condition).

Chemical changes are undoubtedly involved in the phenomena of conduction (Tashiro and Adams, 1914, p. 329) and, in view of the distinct differences in the chemical constitution of the mitochondria and of the Xissl substance, the one being of a lipoid albumin nature (Faurc-Fremiet, Mayer and Schaeffer 1910, p. 95) and the other bemg apparently a complex nucleoprotein containing iron (Scott, 1905, p. 507), it seems probable that the studj^ of mitochondria and the changes which they undergo may bring to light variations in the activity' of the nerve-cell which could never be detected by the stud}' of the Xissl substance alone. Quite apart from the role of the nucleus in the elaboration of the Xi.s.sl substance and the purely cytoplasmic nature of mitochondria, there is further evidence of a functional diversity between the two structures. I have found that m the nerve-cells of the mouse the mitochondria s cry directly ^dth the volume of the cytoplasm and that the Xissl substance varies inversely wdth the nucleus cytoplasmic ratio; also that the mitochondria are of more general occurrence in nerve-cells than the Xissl substance.

They are present in the granule-cells of the cerebellum, as is also evident from the earlier work of Altmann (1890. i)late xiii, figure 1) and Nageotte (1909, i). 826), and in the granule-cells of the olfactory bulb of mice and rats, which are well known to he devoid of Xissl substance. ^Moreover, in certain cell-groups, under normal conditions, there is often a variation in the mitochondria, as between different cells, without any corresponding change in the Xissl substance, ^litochondria occur abundantly throughout the length of the axone, where no Nissl substance has ever been seen. They also occur in certain dendritic processes which do not contain any Xissl substance. Evidence of this sort may be multiphed.

Just how the mitochondria are concerned with the activity of the nervous system is unknown. I have presented evidence elsewhere (1914, p. 18) that they jilay a part in the basic i)rocesses of metabolism which are common to all cells, but this is unfortunately a very broad statement and we natvu'aliy desire to learn something rather more specific about them. Coghill's (1915, p. 350) belief that the mitochondria are concerned in the constructive (anabohc) side of metabolism is of interest in this connection, particularlj- since it falls so well in line with the wellknown "eclectosome" theory of Regaud (1911, p. 699), which, in turn, is an exteni^ion of the "side chain" theory of Ehrlich. M. R. and W. H. Lewis (1915, p. 393) make the interesting suggestion that the mitochondria take part in cellular respiration, wliich is also a fundamental process common to all cells.

We may confidently expect that this new avenue of approach to the study of the activity of the nervous system will yield results of importance, not only because our histological methods of technique are now sufficientl}' accurate to permit of the actual enumeration of the mitochondria, a thing which can not be accompli.shed in the case of the Xissl substance, but also because AValdemar and JNIathikle Koch (1913, p. 427) have recently succeeded in devising chemical methods for the (jualitative and quantitative estimation of substances, ^■ery closely related, perhaps identical with mitochondria, in the nervous system. These substances are phospholipins. Hoppe Peyler long ago pointed out that lecithin (a typical phospholipin) and cholesterol are to be found almost everywhere that life jihenomena exist. In fact, a great wave of revi\-ed interest is manifested in recent chemical and pathological literature in these com])lex comjiounds of fatty acid, phosjihorus, and nitrogen. Mathews (1915, p. 88) very aptly remarks that tlic i)li(is])h<)lii)ins are the most important substances in living matter :

" For they arc found in all cells, and it is undoubtedly their fiinctimi to ])n)(iuee, with eliole.sterol, the peculiar .semifluid, seniisohd state of protopla.sni. Tlie hitler iiolds niucli water in it, but it does not dissolve. Indeed it may be said tiiat the pliospholipins witii cholesterol make the essential substratum of living matter. This physical substratum of ])hosi)holii)in dilTers in ditTerent cells and prohal)iy in tlie same type of cell in different animals, but everywhere, from the lowest plants to the hinlily differentiated brain cells of mammals and of man himself, it jiosstisses certain fundamental chemical and physical properties. In all cases tiie ])hospholipin sutistratum is soluble in alcohol containini; some water," etc.

In view of these considerations it is interesting to iiuiuire whc>ther the distribution of mitoclu»nrlria in cells corresponds with that of the i)hospholi])ins. It is certainlv true that mitochondria are more widclv distributed than anv other kind of cell grauulution now known to us. They occur in almost all cells. Yet certain cells, like the full}- differentiated non-nucleated red blood-cell, unquestionably contain a large amount of phospholii)in, though no formed mitochondria can be seen. The mitochondrial substance is probal)ly ])resent in solution, just as it appears to be in chromophile cells, for it would olniously l)c absurd to state that it must always occur in that state of condensation which makes it visible with the aid of certain powers of the microscope. The recent investigations of Levene (1915, p. 41) on cephalin are of interest. A new field of investigation is evidently opened up. It may thus be possible to pursue this line of work with chemical as well as with histological and physiological methods, a combination which has been but rarely effected.

A\'ork along these lines seems the more desirable since, as will be seen, it may throw new light upon certain problems in the pathological anatomy of the nervous system as well. Wells (1907, p. 460), in his discussion of mental fatigue, writes:

"Since the lecithin forms so important a part of the nervous system, it is tempting to imagine that in fatigue excessive quantities of its toxic decomposition product, cholin, and the still more toxic derivative of cholin, neurin, are formed in considerable amounts and cause part, at least, of the

intoxication."

Now we have seen that, in the opinion of certain investigators, mitochondria are largelj^ composed of lecithin. It is possible, therefore, if Wells's reasoning is correct, that the symptoms of mental fatigue are the result of their decomposition. Moreover, Halliburton (1907, p. 74) and others are convinced that organic diseases of the nervous system may be distinguished from functional neuroses on account of the formation of cholin in the one and not in the other. This opens up the possibility of a differentiation between these two great groups of diseases on the basis of cell structure, as to whether or not there is a change in the mitochondria.

(3) The persistence of the canalicular apparatus in chromophile cells is of interest in general cj'tology. In chromophile cells, in which there are marked structural changes, the canahcular apparatus remams without SLuy great modification. This is rather surprising, since investigators have gradually come to regard the canahcular apparatus as the most labile cell organ; but it is m conformity with Key's as yet unpublished observations on degenerative changes in spinal ganglion cells. Key finds that the canalicular apparatus persists without much modification for from 12 to 24 hours after death in spinal-ganglion cells left in the animal.

I have shown (1912, p. 494) that a canahcular apparatus, in the form of a system of clear, uncolored canals, occurs in the same cell with tj^iical mitochondria and that consequentlj'^ the canalicular apparatus and the mitochondria are structurally distinct. This conclusion is strongly supported by my observation that they may likewise be seen together in chromophile cells, the difference being that while the mitochondria are greatly changed, the canalicular apparatus remains with httle or no modification, so that thej' arc functionally as well as structurally different. ]My positive impregnations of the canahcular apparatus hy the uranium-nitrate method of Cajal confirm this observation.

Now, Cajal (1908, p. 123) is so certain of the identity of the clear canals (described originally by Holmgren) and the "Apparato reticolare interno" of Golgi

40 Tin: .><Tui( Ti IU-: or ciiuomoi'Hilk cells of thk nkhvous syste.nl

that he refci^i to them us "coiukiits de Golgi-Huhugieii." But Kina Monti (,1915, p. 40) has made the statement that the large internal reticular ai)paratus corresponds to the chondriome (/. c, to mitochondria) in the nerve-cells of mammals; to (juote her own words: "II grande apparato reticolare interno dal Golgi ncUe cellule nervose di mammiferi corrisijontle adunque al condrioma, comme il grande api)arato descritto dal Pensa nelle cellule cartilaginea." If Cajal is correct in his identification, it would ai^jiear that the canalicular apparatus and the mitochondria are identical. I have already discussed (1912, p. 490) the older statements of Popoff (1906, p. 258), Smirnow (1906, p. 153), Van Durme (1907, p. 84), Meves (1908, p. 846), and Hoven (1910, p. 479), who are incUned to believe this to be the case.

It is hard to see how these two views can be reconciled. I am incUned to think that the well-known lack of specificity of the methods of silver imj^regnation which Pensa (1913, j). 5(30) and Rina ]\lonti (1915, p. 45) have employed are the cause of the confusion. I do not beUeve that the Golgi method can be trusted invariably to demonstrate a certain structure within the cell, like the canalicular apparatus; and, for this reason, I can not accept unreservedly Cajal's identification of the canalicular apparatus with the Golgi apparatus. I agree with Duesberg that a more l)recise definition of the "Apparato reticolare interno" is highly desirable, but I do not agree with him in his attempt (1914, p. 37) to define it in terms of its relation to the centrosome, because our knowledge of the centrosome itself is so deplorabh' inadequate, ^^'e retiuire. above all else, more accurate methods before the matter can be cleared up.

(4) This discussion of the structure of chromophile cells may be profitablj' concluded by a statement of our present knowledge of the cytoplasmic structure of living nerve-cells of vertebrates not in the chromophilic condition, ^litochondria unquestionably occur and may be seen as such in living nerve-cells even without any vital stain. The Nissl substance is usually {)resent in solution, not in the form of discrete masses as seen in fixed prei)arations. I believe that there is also an amorl)hous argentophilic material which (when treated by appropriate but very capricious methods) as.sumes the form of fibrils. The canalicular apparatus, like the neurofiV)rils,is an unknown ([uantity in living nerve-cells, although it maybe demonstrated in fixed tissues with considerable regularity. These structures, or more correctly speaking substances, are distinct and should not be confu.sed with one another. Although the mitochondria alone have a definite morphology and can u.sually be seen in living nerve-cells, under ordinary conditions, with the i^rescnt means at our disposal, it would be arbitrary in the extreme to say that the others can never be seen. Pigment, fat, li])oid, etc., may of course be seen in variable amount in living nerve-cells. It is the more fundamental constituents with which we are concerned.

The recent work in l)io-cli(niistry, sunnnarized by F. (iowland Hopkins (1913, p. 663) in his presidential address before the Physiological Section of llu> British As.sociation, has, I believe, an imi)ortant bearing here. The cell is regardeil as a dynamic system of co-existing phases in more or less stable equililirium, the condit ion of which is altered, from moment to moment, by jirocesses of oxidation, reduction, desaturation, conden.sation, etc., which naturally result in physical changes in the cell, with the building-up and breaking-down of molecular aggregates which may or may not bo visible with tlie ]nicrosco]Jc or the uhni-iiiicroscopc. The Xissl substance, argentophilous niaterial, etc., doubtless undergo changes of this sort from liquid to fluid and semi-solid phases. It seems right and proper, therefore, to steer an intermediate course, as I have done, between those, on the one hand, who assert thattheNissl substance and the neuro-fil)rils occur in li\irig cells in approximately the same form as they appear in fixed and stained i)reparations, and those, on the other hand, who claim that they are artefacts pure and sim])le and that they can never be seen in the living cell. Our problem is more one of material than it is of form. In this connection the solution of mitochondria in chromophile cells is a phenomenon of considerable significance. In addition to variations in the chemical constitution of mitochondria, there is also evidence to the effect that there may be variations in the condensation or density of the mitochondrial sub.stance (vide Duesbcrg, 1915, p. 40). This is a factor which has been too often ignored. We are inclined to look for mitochondria in all cells which arc functionally active and in which metabolic changes are taking place. The fluidity of the mitochondrial substance varies and I am prepared to beUeve that further investigatioii wUl bring to light cells which are active functionally, but in which no trace of formed mitochondria may be seen.

Conclusions

(1) Chromophile cells occur under normal conditions in the brains of white mice.

(2) They are distributed luiccjually in the different parts of the nervous system. They are most abundant in the cerebral cortex. The}' are progressively less abundant in the cerebellum, corpus striatum, thalamus, midbrain, and medulla. They are of very rare occurrence in the spinal cord, spinal ganglia, and sensory ganglia of the cranial nerves.

(3) This restriction of the chromophile cells to the higher centers is in full accord with the well-known lability of the central, more highly specialized cells as contrasted with the more primitive, peripheral neurones.

(4) Chromophile cells, as seen in fixed and stained preparations, vary greatly in structure. There is usually more or less shrmkage of the cell-body. The nucleus vasiy also be shrunken. The acidophUic and basophilic nucleoli are particularly prominent and the ground-substance of the nucleus stains intensely with both acid and basic dyes. There is an increase in the amount of Xissl substance. The Nissl bodies become confluent and form a homogeneous mass. The cell is hyperchromatic. The canalicular apparatus is unaltered. The mitochondria either increase in number and stain more intensely', or else some of them lose their discrete outlines and form a diffuse deposit which stains intensely by the mitochondrial methods of technique. This change in the mitochondria occurs in the cell processes in the neighborhood of the cell, as well as in the cell-body. Although the nucleus may be completely obscured by this cloud of mitochondrial substance, it still remains and stains in the usual way with hematoxylm and eosm.

(5) The labilit}' of the mitochondria and the constancy of the canaUcular apparatus in chromophile cells confirms my earher contention by showing that the two structures are physiologically as well as anatomically distinct.

Explanation of Figures

All the figures have been drawn with Zeiss aiKxhromatic objective 1.5 mm. compensating ocular C and camera lucida giving a magnification of l.-'iOO diameters. The figures have not been reduced in reproduction. In all of them unaltered cells are represented side by side w-ith chromophile cells just as they occur in the preparations.

Figures 1 to represent cells in the cerebral cortex of a male white mouse. 26 days old and weighing o grams. The brain was cut into serial sections 4 /u in thickness and stained with fuchsin and methyl green. .\11 the figures were drawn from cells in the same section to insure uniformity in the action of the stain and of the differentiator. The mitochondria are stained red, the Xissl substance green, and the canalicular apparatus persi.«ts, in some of the cells, in the form of clear, uncolored spaces.

Figures 7 to 9 represent cells from the cerebral cortex of a male white mou.se, 29 days old and weighing 10 grams. Portions of the brain were prepared by the uranium-nitrate method of Cajal and were cut into serial sections 4 /i thick. These figures were also drawn from a single section to insure uniformity in the .action of the counterstain, methyl green. The canalicular apparatus is in the form of a blackened network and the Nissl substance is colored green.

Fig. 1 . Two cells, having a distinct increase in amount and intensity of the staining of the mitochondria. This change may mark the first stages in the assumption of the chiomophilic condition.

2. X much greater increase in amoimt of mitochondria and a slight increase in intensity of the staining of the Kissl substance and the nucleus.

.3. The Xissl substance is more abundant. It is diffuse and stains more brightly. The outlines of the mitochondria are indistinct. The nucleus stains darkly. -A few clear canals are visible near it. There is what appears to be a shrinkage space on either side of the cell.

4. Still greater changes. The mitochondria appear to be going into solution; outlines of nucleus barely distinguishable.

o. The mitochondria have almost all gone into solution. The Nissl substance is almost entirely obscured by the cloud of mitochondrial material which stains with the most energetic of the two dyes, acid fuclisin. The nucleus is invisible.

(). .V complete " chondriolysis" or solution of the mitochondria. The canalicular apparatus is present in the vicinity of the nucleus.

7. The increase in amount of the Nissl substance indicates a slight degree of chromophilia. The canalicular appa ratus is blackened and shows no changes.

8. Greater increase in tlie Ni.-vsl substance. It is diffuse, with marked hyperchromatisin. The nucleus stains

diffusely willi methyl green. Its outlines are obscure. The canalicular apparatus, in black, is unaltered and the cell as a whole is shrunken.

9. Cell so intensely stained with the methyl green that the nucleus can not be seen. Canalicular apparatus slightly coiidciisid, othcnvise unchanged. There is a considerable shrinkage of the cell.

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