Paper - The development of the pillar cells, tunnel space, and Nuel's spaces in the organ of Corti (1919): Difference between revisions

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==A CONTRIBUTION TO THE STUDY OF VASOMOTOR REFLEXES==
D. OGATA AND SWALE VINCENT Physiological Laboratory, University of Manitoba, Winnipeg, Canada
NINETEEN FIGURES
CONTENTS
1 . Introduction 355
2. The influence of respiratory movements upon blood-pressures 357
3. The effect of the strength of the stimulus upon vasomotor reflexes 331
4. The influence of the frequency of stimulation upon vasomotor reflexes.:. 364
5. The effects upon vasomotor reflexes of stimulating nerve trunks of dif ferent categories (sensory, motor, and mixed nerves) and of different sizes t 366
6. Vasomotor reflexes from nerve terminations 370
7. The influence of the ductless glands upon vasomotor reflexes 374
8. The question as to which vascular areas are constricted or dilated on
central stimulation of somatic nerves 375
9. Summary 376
I. INTRODUCTION
General blood-pressure is affected reflexly by central stimulation of various sensory nerves (reflex vasomotor action). This subject has been studied already by a number of authors. A complete list of the older investigations may be found in Tigerstedt's Lehrbuch der Physiologie des Kreislaufs,'*'^ papers by Asher^ and Bayliss^ in Ergebnisse der Physiologie, and in Nagel's Handbuch der Physiologie (Hofmann^O- The history up to November, 1914, is given by Vincent and Cameron. ^^ As to more recent important investigators of this problem, we may refer to Porter,^^-^-' Martin, "-26 Ranson,35-36 Gruber,^'^ and their respective co-workers, also to Domitrenko^ and Hunt.'^'^^
Even among these recent investigators there seems to be considerable difference of opinion as to what may be regarded as the usual or normal response to afferent impulses. Thus Porter
355
356 D. OGATA AND SWALE VINCENT
and Quinby^^ say: "It is sometimes urged that in shock the blood-pressure falls instead of rising on stimulation of afferent nerves. This abnormal reaction was observed in several of our experiments." This statement clearly involves the assumption that a rise is the normal effect, though it is recognized that the fall is a not very unusual occurrence. Vincent and Cameron'*^ seem to be of opinion that the usual effect of stimulating the central end of the cut sciatic nerve is a rise, and the fall due to a pure vasomotor reflex is rather rare. So also Hunt. On the other hand, Martin and Lacey^^ having observed regularly a definite drop in blood-pressure by weak stimulation and a rise by far more strong stimulation, became doubtful of the truth of the generally accepted doctrine that pressor responses are the normal results of sensory stimulation.
These differences of opinion must be due to some factor or factors other than the strength of stimulus. Besides the factors most usually considered, such as different modes of stimulation, different nerves, different conditions of the same nerve, different narcotics, drugs, etc., there are two important considerations recently brought fo*rward which unmistakably affect the vasomotor reflexes or complicate the problem of their elucidation.
In 1915 Vincent and Cameron"*^ called attention for the first time to a fall of blood-pressure caused by increased respiratory movements. They write: "While anaesthesia is fairly complete the effect of stimulating the central end of the cut sciatic nerve is a pure and distinct rise. As the effect of the anaesthetic begins to pass off, the effect of stimulation will be a rise of bloodpressure followed by a more or less pronounced fall. Respiratory movements will now be found to have been markedly increased, and the extent of the fall of pressure appears to be at any rate proportional to the violence of the respiratory activity."
Martin and Lacey^' investigated the influence of the interruption of the primary current at widely varying rates, but failed to notice any effect, as also did Hunt^^ in his earlier work. Only quite recently it was clearly pointed out by Gruber^ that with the same strength of stimulus, pressor and depressor results
VASOMOTOR REFLEXES 357
were obtainable by varying the rate of stimulation from 1 to 20 stimuli per second. This was later incidentally confirmed by Hunt.i«
Thus, for the investigation of the complicated problem of vasomotor reflexes, it became very necessary to investigate each possible factor separately. In this way only would one be able to answer correctly for the normal vasomotor response.
The present investigation was undertaken for the purpose of studying some of the factors separately, of confirming previous investigations, and of trying, if possible, to reconcile contradictory views as to the conditions which determine any particular vasomotor response.
We beg to acknowledge our indebtedness to Mr. John Carmichael for his valuable assistance in all our experiments.
2. THE INFLUENCE OF RESPIRATORY MOVEMENTS UPON BLOODPRESSURES
It is well known that the respiratory center can easily be affected by central stimulation of sensory nerves. Thus HowelP* writes in his Textbook of Physiology that stimulation of any of the sensor}^ nerves of the body may affect the rate or the amplitude of the respiratory movements. ' But no mention is made of the influence of these movements upon blood-pressure. The same applies to other text-books, except Starling's,^^ in which we find, The increased respiratory movements will also aid the venous' circulation and have a similar effect in increasing the systolic output," which would necessarily bring about a rise of blood-pressure. But, "A constant and immediate result of exaggerated respiratory movements is a fall of blood-pressure," and not a rise, as Vincent and Cameron pointed out. They found that "the extent of the fall of pressure appeared to be at any rate largely proportional to the violence of the respiratory activity," that the fall of blood-pressure was "brought about by performing rapid artificial respiration by compression of the thorax," that "deep voluntary breathing in the case of the human subject produced a regular and pronounced lowering of the blood-pressure," that "the more widely the thorax is opened
358 D. OGATA AND SWALE VINCENT
the more the fall of pressure tended to become replaced by a rise," and that the effect of artificial respiration was "a rise, and not a fall, when the animal was mider curare," i.e., when a stop was put to the spontaneous respiratory movements. Thus the fall of blood-pressure as a result of increased respiratory movements seems to have been sufficiently established.
By the majority of previous observers curare was thought to be an indispensable drug in the study of the problem of vasomotor reflexes, with or without any consideration of its action on the vasomotor center itself. But we know that narcotics and other drugs are not always free from influence upon these reflexes, as pointed out by various previous investigators,^* ^^-^ and, therefore, in experimental work they should be reduced to as few as possible or altogether eliminated (Vincent and Cameron). The change in character of the respiratory movements, especially their increase, becomes thus an almost unavoidable complication in the study of vasomotor reflexes when curare is not used and the narcosis is not deep enough. If this complication be left out of consideration, erroneous conclusions may be reached.
Since the appearance of Vincent and Cameron's paper several writers have referred to the influence of the increased respiratory movements. Unfortunately, they are not in complete harmony with one another. Ranson and Billingsley^^'^^ say, With stronger stimulation the greatly increased respiratory movements may no doubt play an important part in the drops in bloodpressure," but Gruber and Kretschmer^ write that their experiments do not support Vincent and Camerofi's theory that the fall in blood-pressure is brought about by movements of respiration which interfere with the heart's activity." This latter statement seems to deny definitely the respiratory role upon blood-pressure. Vincent and Cameron did not positively deny that there is a true vasomotor fall of blood-pressure under certain conditions as a result of central stimulation of afferent fibers. But they insisted, and rightly, too, that many apparent vasomotor falls are really due to increased respiratory movements. We shall see later that the fall of blood-pressure with weak stimuli is a commoner occurrence than Vincent and
VASOMOTOR REFLEXES 359
Cameron were inclined to believe. At any rate, the matter is so importan: that we have repeated the experiments to test the effects of respiration on blood-pressure.
We have stimulated electrically the central stump of several cut nerve trunks (saphenous, tibial, peroneal, sciatic, ulnar, and median) with various strengths of stimulus.
When the narcosis (with ether or chloroform) was not deep enough, the respiratory movements were always increased by strong stimulation. The most frequent response of the bloodpressure to stimulation, e.g., of the sciatic nerve, may be illustrated bj^ figure 1.
With weak stimulation there is practically no increase of respiratory movements either in amplitude or in frequency, and the blood-pressure is either a fall or a fall followed by a more or less marked rise. When stronger stimuli are applied, the respiratory movements increase either in amplitude or in frequency, or in both, and the blood-pressure rises, instead of falling, and is followed by a marked fall. The rise of bloodpressure increases usually in proportion to the development of the strength of stimulation.
In figure 2 the anesthesia was made much deeper with the same animal as in figure 1, and stimuli of several strengths were applied to the same nerve.
There is very little increase of respiratory movements on each stimulation, and the response of blood-pressure is also small in degree. The latter, as seen from the figure, is either a fall or a rise according to the strength of stimulation, and the marked fall after the rise which is observed in figure 1 simultaneously with the increased respiration cannot be seen. This suggests at once that the marked fall accompanied by remarkably increased respiratory movements might be ascribed, at any rate, mainly, to the influence of the latter movements caused by sensory stimulation. Moreover, under brain compression it is not very difficult to stop the respiratory movements entirely, and in this case only very strong stimulation will initiate spontaneous respiratory movements. Under these conditions a fall of bloodpressure of the same character as that observed with an in
360 D. OGATA AND SWALE VINCENT
creased respiration never occurs. Thus it would not be unreasonable to assume that figure 2 shows real vasomotor reflexes, even though weak, not complicated by the increased respiratorymovements, while figure 1 represents the vasomotor reflex masked by the effects of increased respiration.
It is often very difficult or almost impossible to obtain any rise of blood-pressure when the respiratory movements are very violent. In those cases a marked fall is the only result of central stimulation of afferent fibers.
How these increased respiratory movements affect the bloodpressure was very carefully investigated by Vincent and Cameron. After pointing out several possible causes, they came to the conclusion that this fall is due to direct mechanical interference with the heart's action and with the return of the blood to the heart.
In order to confirm this theory, we opened the thorax in the middle line as did Vincent and Cameron, and found that the falls disappeared. The contrast is clearly shown in figures 3 and 4.
In these two cases the same nerve of the same animal was stimulated with the same strength of stimulus, in figure 3 in the intact animal and in figure 4 with thorax open.
In addition to opening the thorax we cut both vagi, both phrenici, and as many intercostals as possible on both sides, without obtaining very different results from those obtained by merely opening the thorax.
In a very few cases a marked fall of a similar character to that due to the increased respiratory movements, was observed in animals with thorax wide open. It was, however, soon discovered that this fall was produced by compression of the inferior vena cava by the heart which became more freely movable than before through opening the thorax. The heart fell back upon the soft-walled vein, and thus diminished the flow of blood to the right heart.
But it is certainly true that by means of almost pure vasomotor reflex, i.e., without any or with very little increase of respiratory movements one can obtain a marked fall preceded by a rise, as shown in figure 5.
VASOMOTOR REFLEXES 361
Therefore we do not conclude that such a fall of blood-pressure is always produced by increased respiratory movements. But the important point for us at present is the undoubted fact that increased respiratory movements can and do cause a fall of blood-pressure, and that this fall can be easily eliminated by opening the thorax sufficiently wide.
These observations, along with various others quoted above from the paper by Vincent and Cameron both on animals and on human subjects, confirm fully their statement as to the occurrence of a fall of . blood-pressure brought about by increased respiratory movements, and probably explain the nature of this fall. We believe that the increased respiratory movements caused by sensory stimulation form a very important complication which has often led to misunderstanding of the true vasomotor reflexes.
Gruber and Kretschmer, as mentioned before, deny this respiratory effect upon blood-pressure. They used a slow rate of stimulation and the fall of blood-pressure was the usual effect. But the fall is generally thought to be a result of weaker stimulation, and they do not deny that the increased respiratory movements to a certain degree cause a fall of blood-pressure when the stimulus is strong enough as to produce them.
Our experiments were made on thirty- three dogs.
3. THE EFFECT OF THE STRENGTH OF THE STIMULUS UPON
VASOMOTOR REFLEXES
After repeated experiments by numerous investigators, the generally accepted view as to the effect upon the vasomotor reflexes of different strengths of stimulus seems to coincide with Knoll's^^ original statement, i.e., that a depressor effect is usually the result of a weak stimulation, while a pressor effect follows, as a rule, a stronger stimulation. Reid Hunt^* pointed out that weak stimulation was one of the methods of obtaining a reflex fall of blood-pressure, and Vincent and Cameron noticed the same fact.
Among more exhaustive investigations on this point we should refer to those by Porter,29-34 Martinj-^-^^.^o and their respective
362
D. OGATA AND SWALE VINCENT
co-vvorkers. The former writer seems to regard a rise of bloodpressure as the normal vasomotor response, while the latter holds a different view. Martin and Lacey's^^ experiments were conducted on cats either under brain pithing, decerebration or brain compression, or under ether or urethane. The nerves stimulated were the sciatic, radial, median, ulnar, and saphenous. The results of their experiments were very definite. "In every one of the experiments the stimulation was repeated many times over a range of stimuli from the threshold value to three or four times the threshold. Well-marked drops of pressure followed all such stimulations," save in one exceptional case. Thresholds for pressor reflexes were much higher than those for depressor reflexes. Thus the experiments of these workers support Knoll's statement.
Our own experiments consisted in stimulating various nerves (sciatic, tibial, peroneal, saphenous, median, ulnar, and vagus) with induction shocks on dogs under ether, chloroform, and brain compression. As to the method, we have to mention that the different effects of weak and strong currents, respectively, were satisfactorily attained by means of sliding the secondary coil up to or away from the primary, but that on many occasions more than one battery was used to obtain a stronger stimulus. The rate of stimulation was 38 to 54 in a second.
The fall of blood-pressure due to the increased respiratory movements being taken into consideration, the main results of our experiments may be summarized as in the following table :
ANAESTHETICS
FALL WITH
WEAK
STIMULATION.
RISE WITH
STRONG
STIMULATION
FALL WITH
WEAK
AND STRONG
STIMULATION
RISE WAS ONLY
RESULT OF
STIMULATION
Ether
20 14
12
2 4
2
4
Chloroform
4
Brain compression
Total . . .
46
8
8
The term ' fall' in the table comprises also a fall followed by a rise and 'rise' also a rise followed by a fall.
VASOMOTOR REFLEXES 363
«
In forty-six cases out of sixty-two in total, weak stimulation produced a fall or a fall followed by a rise, and strong stimulation caused a rise or a rise followed by a fall. A typical response is shown in figure 6.
The animal was under ether and the thorax was very wide open in the middle line in order to eliminate the disturbance from increased respiratory movements. Figure 7 shows a similar response under chloroform.
In the remaining sixteen cases the response was either a fall or a rise through all strengths of stimulation which we used, and the different effects with weak and strong stimulation were not observable.
Thus it does not seem to us unreasonable to conclude that weak stimulation of the central stump of the cut nerve produces usually a fall of blood-pressure and a strong stimulation produces usually a rise.
From these conclusions it may naturally be understood that from the threshold of stimulation up to a certain point the fall of blood-pressure increases with the development of the strength of stimulus, and then the fall gradually decreases until a neutral point is reached, where the vasoconstriction and dilatation just counterbalance each other, and finally the rise appears, which increases usually with the increase of the strength of stimulus, but cannot continue very long, since powerful stimuli would elicit vigorous reflex movements of the animal and obscure the true vasomotor reactions unless indeed the animals were deeply under curare. As we have been unable so far to find any attempt by previous investigators except Stiles and Martin'* ° to describe this rather peculiar course of vasomotor responses, we though it worth while to emphasize it in this place (fig. 8).
In our experiments we have employed also stimuli of other kinds than electric induction shocks, namely, mechanical, thermal, and chemical. In this series thirty-eight out of sixty-seven stimulations were effective, and of these thirty-five caused a fall of blood-pressure and only three produced a rise. As the calibration of these stimuli was not so practicable as with induction shocks, we cannot draw any very positive conclusions, but we
364 D. OGATA AND SWALE VINCENT
are inclined to believe that a greater number of pressor responses could be obtained if we could improve the method of stimulation so that the sensory fibers might be stimulated more strongly.
It thus appears from our experiments that the depressor effect of weak stimuli is much more common than Vincent and Cameron thought, though these observers were careful not to deny its occurrence. Reid Hunt/ in a recent paper, seems to have had the same difficulty that Vincent and Cameron encountered in obtaining the depressor effect of weak stimulation, which he ascribed to the different frequency of stimulation they employed.
The fact that a weak stimulation of a sensory nerve causes, as a rule, a reflex fall of blood-pressure and a strong stimulation a reflex rise, together with the statement of Bayliss^ that the orthodox effect due to the stimulation of the depressor nerve (nerve of Cyon^) can be converted into a rise by the action of strychnine, led us to inquire whether a pressor response could be obtained by strong stimulation of the depressor nerve. So far as our results inform us, neither such a strong current as would injure the nerve nor induction shocks up to eighty per second frequency could reverse the depressor response. The response to the stimulation after injection of strychnine was sometimes increased and sometimes decreased, but the reversal of the response did not appear in our experiments even with a dose which caused general convulsions on weak stimulation.
4. THE INFLUENCE OF THE FREQUENCY OF STIMULATION UPON
VASOMOTOR REFLEXES
That the frequency of stimulation has a certain effect upon vasomotor reactions seems to have been known to the older investigators. In 1883 Kronecker and Nicolaides'-" noticed that the vasomotor centers could more easily be affected by changing the frequency of stimulation than by changing its strength. They write: One can never attain such a strong vasoconstriction by increasing the intensity of the stimulating current as by increasing the frequency of the current of moderate .ntensity." We have not been able to consult the original paper of these writers.
VASOMOTOR REFLEXES 365
From a reference in Ergebnisse der Physiologie by Asher/ it is not clear whether the stimulation was directly upon the nerve centers or reflexly through afferent nerves.
But the credit of pointing out clearly that the frequency of stimulation has an effect upon vasomotor reflexes must be ascribed to Gruber.^ This writer remarks: That summation takes place with rapid rates of stimulation is undisputable, but it does not seem probable where the strength is more than 400 times threshold that the phenomenon of summation can explain the different effect obtained with these rates of 1 per two seconds and 20 per second interruptions." The similar effect of frequency of stimulation was afterward proved incidentally by Reid Hunt,^^ who considers it convenient to use the infrequent rate of stimulus to obtain a reflex fall of blood-pressure.
Our experiments on this subject have been carried out on dogs with rates of stimuli of 1, 2, 5, 10, 20, 40, and 80 per second upon various nerves, under chloroform and curare or under brain compression. Though our results were not so conclusive as those obtained by Gruber (in fifteen out of forty stimulations similar results to his were obtained), still we do not hesitate to ascribe an important role to the frequency of stimulation. According to Martin's^^ investigations, the intensity of stimulation in Z-units is directly proportional to that of the current in the primary circuit. We arranged the apparatus in such a way as to get a current of a certain strength and one ten times stronger as we desired. With the former current we obtained a fall by stimulating five times per second, and a distinct rise by stimulating ten times per second, while with the latter current we observed a fall with the rate of stimulus one per second and a rise with five per second stimulations. A selected record is shown in figure 9, where one and the same nerve was stimulated with the same intensity but with different frequency.
Much more remarkable were the rates of stimulation at which the maximum pressor response was reached.
366
D. OGATA AND SWALE VINCENT
RATE OF STIMULI PER SECOND
1
2
5
10
4
20
18
40
80
Number of experiments whose maximum pressor response was reached at the rate of stimuli mentioned above
12
4
As is seen from the table, in 78.9 per cent the maximum response is reached between twenty to forty per second stimulation, and in one-third at the rate of forty per second. Beyond these points the effect increased only in four cases. This phenomenon may be seen a^so in figure 9.
Kronecker and Nicolaides^o observed the fact that the effect of stimulation of the vasomotor centers increased with the frequency of stimuli up to twenty to thirty per second, but not beyond this point. Tur^^ also pointed out that the effect of stimulation of the lingual nerve increased until the stimuli reached forty per second, beyond which, however, the effect diminished. These observations coincide fairly well with our own.
5. EFFECTS UPON VASOMOTOR REFLEXES OF STIMULATING NERVE TRUNKS OF DIFFERENT CATEGORIES (SENSORY, MOTOR, AND MIXED NERVES) AND OF DIFFERENT SIZES
According to the investigations of some authors, different nerves, apart altogether from the depressor nerve, respond differently to central stimulation. Hofmann,!^ in Nagel's Handbuch, says: There are single nerves, which for the most part (glossopharyngeal) are depressor, and others which are exclusively (splanchnic) or preponderatingly (sciatic, facial, infraorbital, cervical nerves) pressor." Vincent and Cameron studied the effect of stimulating the main trunk of the sciatic, as well as its common peroneal, lateral cutaneous, and purely muscular branches, the saphenous, median of axilla, the hypoglossal, the glossopharyngeal, the superior laryngeal, and the vagus. But the different nerves all produced similar or comparable results on the blood-pressure. They were strongly tempted to the hypothesis that an equivalent stimulation of a roughly equal number of afferent fibers will yield similar reflexes.
VASOMOTOR REFLEXES
367
Our experiments also have led to the conclusion that there is no essential qualitative difference between the various nerves subjected to stimulation (sciatic, tibial, peroneal, median, ulnar). A possible exception may be made in the case of the saphenous. It may be that there is a greater tendency to a fall on stimulating this nerve than in the case of others. Whatever the nerve may be, whether a purely sensory nerve, as the saphenous; a mixed nerve, as the sciatic, peroneal, tibial, ulnar, or median, or a purely motor nerve, such as a muscular branch of the femoral, the course of response to weak and strong stimulation was in most cases a fall and a rise as already described in section 3.
A few examples are quoted in tabular form where a selected purely sensory nerve and a purely motor or a mixed nerve apparently of the same size were stimulated in turn under entirely similar conditions.
Sensory and motor nerves compared
ARRANGEMENTS OF STIMULATION
1 battery coil at 30 cm. 15 seconds. 1 battery coil at 25 cm. 15 seconds. 1 battery coil at 20 cm. 15 seconds. 1 battery coil at 15 cm. 15 seconds. 1 battery coil at 10 cm. 15 seconds. 1 battery coil at 5 cm. 15 seconds . 1 battery coil at cm. 15 seconds,
RIGHT SAPHENOUS
—4 mm. Hg.
— 10 mm. Hg.
-10, +2 mm. Hg.
+4, -12 mm. Hg.
+6, —14 mm. Hg. +16, -18 mm. Hg. +22, -14 mm. Hg.
BRANCH OP RIGHT FEMORALIS
0, mm. Hg.
-1, +2 mm. Hg.
-4, +2 mm. Hg,
+4, mm. Hg.
+8, -2 mm. Hg.
+ 14, -12 mm. Hg.
+26, -14 mm. Hg.
(From experiment 47. Under brain compression.)
The sign ' — ' means a pure fall of blood-pressure, '+' a pure rise, and ' — , +' or ' + , — ' a mixed response, namely, a fall followed by a rise or a rise followed by a fall, respectively.
Sensory and mixed nerves compared
ARRANGEMENTS OF STIMULATION
1 battery coil at 30 cm. 15 seconds. 1 battery coil at 25 cm. 15 seconds. 1 battery coil at 20 cm. 15 seconds. 1 battery coil at 15 cm. 15 seconds . 1 battery coil at 10 cm. 15 seconds. 1 battery coil at 5 cm. 15 seconds. 1 battery coil at cm. 15 seconds.
RIGHT SAPHENOUS
mm. Hg.
—6, mm. Hg.
+2, -10 mm. Hg.
+6, -12 mm. Hg.
+8, -12 mm. Hg. +22, -10 mm. Hg. +22, -12 mm. Hg.
RIGHT PERONEAL
mm. Hg.
— 1, mm. Hg.
—4, +4 mm. Hg.
+2, —6 mm. Hg. + 14, -10 mm. Hg. +20, -14 mm. Hg. +22, -20 mm. Hg.
(From experiment 47. Under brain compression.)
THE JOURNAL OF COMPARATIVE NEDROLOGY, VOL. 30, NO. 4
368 D. OGATA AND SWALE VINCENT
With the increase of the strength of stimulus the respiratory movements also increased, though not very markedly, and therefore some of the falls following the rise on stronger stimulation might have been more or less due to this complication. But in the main it seems that the purely sensory nerves have somewhat lower threshold than other kinds of nerves (fig. 10).
Whether this is due to a large number of afferent fibers con' tained in the sensory nerve than in those of the other kinds of the same size could only be decided by more numerous experiments and more elaborate methods than those we have employed, as, for example, the measurement of resistance of each nerve and more satisfactory methods of controlling the intensity of stimulation in each case.
In connection with the problem as to different kinds of nerves we have studied the influence of the size of the nerve upon vasomotor reflexes. The hypothesis of Vincent and Cameron is quoted at the beginning of this section. A similar problem was taken up also by Stiles and Martin,^° who compared the effect of stimulating two nerve paths at the same time with that of exciting each by itself. They found that stimulation of two afferent paths at the same time has often a more marked vasomotor effect than the stimulation of either path alone with an equivalent strength of current. The degree of summation was only moderate." This shows that the stimulation of a larger number of afferent fibers will produce often a more marked effect than that of few fibers.
We stimulated two nerves of the same category but of different sizes separately one after another under conditions as similar as possible, a different number of afferent fibers being assumed to be present in the nerves of different sizes.
The results may be represented as follows, page 369.
These few examples show that the results were not very conclusive. We can say only so far with some confidence that when the responses were in the same sense, i.e., when the fall or the rise was the result of corresponding equivalent stimulations, the reflex change of blood-pressure was on the whole more marked with the nerve of larger size than with those of smaller size (fig. 11).
VASOMOTOR REFLEXES
369
Mixed nerves compared
with each other
ARRANGEMENTS OP STIMULATION
EIGHT SCIATIC
RIGHT PERONEAL
1 battery coil at 30 cm. 15 seconds. .. 1 battery coil at 25 cm. 15 seconds. . . 1 battery coil at 20 cm. 15 seconds. .. 1 battery coil at 15 cm. 15 seconds. .. 1 battery coil at 10 cm. 15 seconds. ..
-6, -14,
12,
18,
mm. Hg.
4 mm. Hg.
mm. Hg. -22 mm. Hg. — 16 mm. Hg.
mm.
-10, mm.
—8, 4 mm.
8, —6 mm.
12, -10 mm.
RIGHT TIBIAL
Hg. Hg. Hg. Hg. Hg.
RIGHT SCIATIC
1 battery coil at 25 cm. 15 seconds. . . 1 battery coil at 30 cm. 15 seconds. . . 1 battery coil at 15 cm. 15 seconds. . . 1 battery coil at 10 cm. 15 seconds. . .
-2,
-6,
16,
22,
2 mm. Hg.
4 mm. Hg. -18 mm. Hg. -22 mm. Hg.
-2,
-2,
10,
20,
2 mm.
mm.
—8 mm.
— 16 mm.
Hg. Hg. Hg. Hg.
(From experiment 47. Under brain compression.)
Sensory nerves compared with each other
ARRANGEMENTS OF STIMULATION
RIGHT S.*.PHENOUS
A BRANCH OF THE RIGHT SAPHENOU.S
1 battery coil at 25 cm. 10 seconds. . . 1 battery coil at 20 cm. 10 seconds.. . 1 battery coil at 15 cm. 10 seconds.. . 1 battery coil at 10 cm. 10 seconds. . . 1 battery coil at 5 cm. 10 seconds. . . 1 battery coil at cm. 10 seconds.. .
mm. Hg.
-6, mm. Hg. -18, mm. Hg. -22, mm. Hg. -18, mm. Hg.
+4, mm. Hg.
mm. Hg.
mm. Hg. -2, mm. Hg. -8, mm. Hg. +2, mm. Hg. — 4, mm. Hg.
RIGHT SAPHENOUS
A BRANCH OF THE RIGHT SAPHENOUS
1 battery coil at 25 cm. 10 seconds. . . 1 battery coil at 20 cm. 10 seconds. . . 1 battery coil at 15 cm. 10 seconds. . . 1 battery coil at 10 cm. 10 seconds. . . 1 battery coil at 5 cm. 10 seconds. . . 1 battery coil at cm. 10 seconds. . .
mm. Hg. —4, mm. Hg. -8 ,0 mm. Hg. —4, mm. Hg. +4, mm. Hg. +2, mm. Hg.
mm. Hg.
mm. Hg.
mm. Hg. —4, mm. Hg. —4, mm. Hg. -6, mm. Hg.
(From experiment 48. Under brain compression.)
These conclusions coincide with the experience of Stiles and Martin and lend some support to the hypothesis of Vincent and Cameron.
It may not be amiss to add to these conclusions that in few cases when the stimuli were very strong the smaller nerve reacted more vigorous!}' than the larger one, which phenomenon may perhaps be explained partly by different resistances of different-sized nerves to currents of similar strength.
370 D. OGATA AND SWALE VINCENT
Thus, SO far as our experiments go, we are inclined to conclude provisionally that among nerves of different categories there are no essential qualitative differences of response, and the greater the number of afferent fibers stimulated, the more marked is the response of blood-pressure within a limited range of strength of stimulation.
6. VASOMOTOR REFLEXES FROM NERVE TERMINATIONS
Several investigators have stimulated the nerve terminals instead of the nerve trunk itself.
WTien we apply a stimulus to a surface such as the skin we should bear in mind that we may actually be stimulating either the end-organs alone or these structures as well as the nerve fibers, according to the mode of stimulation. Any physiologically appropriate stimulus, though mild, applied to the endorgans would give rise to more highly effective impulses than inappropriate ones. Thus the study of vasomotor reflexes in response to stimulation of the sense organ with its most appropriate stimulus is highly desirable. But even with other kinds of stimulation we may learn much that is valuable, because any stimulus which plays a part in our normal daily life comes usually through the end-organs on the outer or inner surface of the body, and not by way of exposed nerve trunks, as in the foregoing experiments.
The skin, the mucous membrane of the nose, muscles, the intestine, and other abdominal organs were employed frequently by previous investigators. We have selected the skin, muscles, and the intestine as representative of regions containing different modes of nerve endings. The stimuli used were mechanical (incision, scratching, pinching, kneading), thermal (hot or boiling water and cold water or lumps of ice), chemical (10 per cent solution of sulphuric acid), and electrical (induction shocks of various strengths). The animals (dogs) were under ether, chloroform, or brain compression.
The results of a first series are presented in the following tables :
VASOMOTOR REFLEXES
371
Mechanical stimulation of the skin
ANESTHESIA
STIMULATED PORTION
MODE OF STIMULATION
REFLEX RESPONSE OF BLOOD-PRESSURE
No
effect
Fall
Rise
Ether <
Chloroform •
Brain compres- f sion \
L. inn. thigh R. inn. thigh R. inn. thigh
L. inn. thigh R. inn. thigh
Abdomen Over saphenous, ulnar, and sciatic nerves
L. inn. thigh R. inn. thigh
Pinching
Scratching
Incision
Pinching Scratching
Incision
Scratching Incision
2
1
2
3
2 5 6
1 3
4
2 3
Total
8
26
Thermal stimulation of the skin
Ether.
Chloroform.
Brain compression
Total.
L. L.
L. L.
inn. thigh inn. thigh
inn. thigh inn. thigh
L. inn. thigh L. inn. thigh
65°C. — boiling water Ice— 15°C. water
Boiling water Ice
Boiling water Ice
5
3
2
3
1
1
2
7
10
1
Electrical stimulation of the skin
Ether
Chloroform
Brain compression..
L. inn. thigh L. inn. thigh L. inn. thigh
Strong induction shocks Strong induction shocks Strong induction shocks
8 4 4
5
2 2
Total
.... ....
16
9
372 D. OGATA AND SWALE VINCENT
As is clear from the tables, almost every stimulation in this series, produced a reflex fall of blood-pressure, and no significant qualitative difference is observable either with different modes of stimulation or with different methods of anaesthesia or with different portions of the skin.
That this statement is applicable almost without any modification to the results of stimulation of muscles and intestine will immediately be understood from the tables on page 373.
Thus it is fairly clear that the stimulation of nerve terminals in the skin, muscles, and the intestine produces usually a reflex fall of blood-pressure, as was reported by Vincent and Cameron.
But the threshold of stimulation for the nerve-terminals in the skin is very much higher than that for the exposed nervetrunks. Thus a stimulus which is to be reckoned a strong one for the exposed nerve-trunk is to be considered a weak one for the surface of this skin. This fact explains the previously described results. So far the effects have all been those of a weak stimulation, namely, a fall of the blood-pressure.
If, now, we take steps to secure a considerably greater amount of stimulation by simultane6us scratching of large areas in different regions, it is not difficult to satisfy oneself that the same general law applies for the nerve-terminals as for one exposed nerve-trunk. Thus, if we scratch a limited area with a moderate degree of vigor, we get a fall, while more violent application of the instruments to a large area, will give a rise (fig. 19).
In the last section we compared the effects of stimulating two nerves of the same category but of different sizes, and showed that the nerves of greater size usually surpass those of smaller size in their power of evoking vasomotor reflexes, and referred to Vincent and Cameron's hypothesis that the number of afferent nerve fibers is an important factor. In our stimulation of nerve endings, as a rule, we could only apply the stimulations to a small portion of the surface. Now the nerve fibers spread widely from the nerve trunk, and the stimulation of a nerve would be equivalent to the stimulation of the entire surface to which the nerve is distributed. In other words, the stimulation of a small portion, e.g., of the skin, corresponds to that of a
VASOMOTOR REFLEXES
373
Mechanical stimulation of muscles
ANESTHESIA
STIMTJLATED MDSCLE
MODE OF STIMULATIOX
REFLEX
CHANGE OP
BLOOD PRESSUKE
Ott:
3
4
7
1
1 2 3
15
DO
s
Ether <
Chloroform
R. add. mag. R. sartor. R. sartor. L. semitend.
R. add. mag. or 1 semitend.
Scratching Scratching Kneading Scratching
Scratching
Total
Stimvlation of the intestine
Ether.
Chloroform.
Brain compression
Small intestine
Interior surface of small
intestine Interior surface of small
intestine Interior surface of small
intestine
Small intestine
Small intestine
Small intestine
Interior surface of small
intestine Interior surface of small
intestine Interior surface of small
intestine
Interior surface of small intestine
Kneading Induction shocks
Pinching
Boiling water applied
Distension
Kneading
Kneading Induction shocks
Scratching
Boiling water applied
Ice piece applied
Total.
1
3
1
1
1
1
4
2
4
1
1
1
1
2
20
374 D. OGATA AND SWALE VINCENT
small number of sensory nerve fibers. If the stimulation of a few fibers be the equivalent of a weak current, the fall of bloodpressure caused by stimulating the nerve terminals may be ascribed to the fact that we are stimulating only a few fibers.
Under morphia and curare a rise of blood-pressure is more easily obtained than a fall on stimulation of nerve-endings. But under morphia at any rate it is not difficult to obtain a rise with a strong stimulus and a fall with a weak one (fig. 19). GaskelFs^ discovery that in mammals A large dose of curare will remove both the contraction of the muscle and the dilatation of its blood-vessels upon stimulation of the nerve," may possibly account for the greater tendency towards a rise when the animal is under this drug.
The paralysis of the vasodilator nerves by curare seems to necessitate the taking of certain precautions in interpretation of the results of experiments.
It seems probable that if it were found possible to increase very considerably the energy and extent of the stimulation in the cases of kneading of muscle and of the intestine, we should have to record a rise of pressure instead of the fall with which we are familiar.
7. THE INFLUENCE OF THE DUCTLESS GLANDS UPON VASOMOTOR
REFLEXES
The extracts of some of the ductless glands (adrenal body, thyroid, and pituitary) have been alleged to affect the vasomotor irritability on one way or another.^^-'^^-^^'^'^* Since the results of the previous investigations are not conclusive, we thought it might be worth while to investigate the matter again. It is to be feared that our experiments are not much more convincing than those of previous workers on this subject.
The change of blood-pressure (augmentation or diminution) due to the injection of the extracts of these glands is an undesirable complication. In cases where an augmented blood-pressure is the result, as with adrenin and pituitrin, the decreased pressor reaction to the stimulation of a nerve may most properly be ascribed to the diminished response of the already more or
VASOMOTOR REFLEXES 375
less contracted blood-vessels, or possibly to the additional contraction of the blood-vessels of the small areas other than those previously affected by the drug.
But the comparison of the vasomotor reflexes before and after the injection of adrenin ("adrenalin" Parke, Davis & Co.) seems to show that the pressor reflex is slightly decreased in the latter case. The results of Hoskins and Rowley were similar and more definite. The elimination of the function of the suprarenal glands by tying them off gave no clear results.*
The injection of thyroidin (Parke, Davis & Co.) and the extirpation of both thyroid glands do not appear to have any distinct influence upon vasomotor reflexes.
Pituitrin (Parke, Davis & Co., surgical) showed scarcely any significant results.
All these experiments were performed on dogs under brain compression for the purpose of excluding any influences from the increased respiratory movements and those of anaesthetics and other drugs.
8. THE QUESTION AS TO WHICH VASCULAR AREAS ARE CONSTRICTED OR DILATED ON CENTRAL STIMULATION OF SOMATIC NERVES
The fall of blood-pressure produced by stimulation of the depressor nerve is effected chiefly by dilatation of the splanchnic area,-i though, as Bayliss has shown, the vessels of the limbs, head, and neck also partake in the relaxation. The latter writer showed also that the rise of blood-pressure on stimulation of the central stump of the splanchnic (?) nerve was, for the most part, due to the constriction in the splanchnic area. The reflex rise of blood-pressure due to the stimulation of the
That is to say, when the nerves to the limb are intact. In the denervated limb there is a very important difference according to whether or no the adrenal bodies are eliminated. Mr. Pearlman and myself have recently found that when the central end of the sciatic is stimulated in such a way as to give a pressor response the intact limb follows passively the blood-pressure while the denervated limb constricts. After removal of the adrenal bodies the denervated limb also dilates. These results are explained more fully in a paper about to be published in 'Endocrinology.' — S. V.
376 D. OGATA AND SWALE VINCENT
sensory nerves of the skin, too, depends mainly on the constriction of the blood-vessels in the same area,^^ and Hofmann^^ writes: The rise of blood-pressure on stimulation of sensory nerves is produced by the constriction of the blood-vessels of abdominal organs as in asphyxia. At the same time the bloodvessels of the brain, skin, and muscles dilate, as a rule, and an increase of the volume of limbs takes place." Thus the splanchnic area plays a principal part in the reflex changes of bloodpressure on stimulation of the somatic as well as the splanchnic nerves.
We have made some experiments on this point, and can confirm the above statements. The dogs had both vagi cut and were under morphia and curare or brain compression, and the sciatic or saphenous nerve was stimulated with induction shocks. The volume changes of the limbs (hind and fore) and of the abdominal organs (small intestine, kidney, and spleen) were recorded.
The rise of blood-pressure, when sufficiently high, was always accompanied by a remarkable diminution of the volumes of abdominal organs and a pronounced dilatation of limbs (fig. 18).
The pronounced fall of blood-pressure with weak stimulation when the animal was under brain compression was seen to be accompanied by a distinct increase of the volume of the intestine. Thus it appears clear that a reflex rise and fall of bloodpressure on stimulation of a somatic nerve (sciatic, saphenous) is brought about chiefly by constriction and dilatation of the blood-vessels in the splanchnic area.
9. SUMMARY
1. In dogs under ether or chloroform, stimulation of sensory nerves (saphenous, tibial, peroneal, sciatic, ulnar, and median) causes usually increased respiratory movements when narcosis is not profound or curare is not employed. These increased movements produce a fall of blood-pressure, and when they are very violent, one cannot obtain any pressor reflex even with a strong stimulation. When much increased respiratory movements are prevented by very deep narcosis or brain compression, fall of
VASOMOTOR REFLEXES 377
blood-pressure due to this cause does not occur. Mechanical interference with the circulation as a result of the increased movements of the thoracic walls seems to be the main cause of this fall, since it can be eliminated by opening the thorax. When this complication is not taken into careful consideration the results of vasomotor experiments are liable to be misinterpreted.
2. In dogs under ether, chloroform, or brain compression, a weak stimulation of the central end of the cut nerves (sciatic, saphenous, tibial, peroneal, median, ulnar, and vagus) produces usually a fall and a strong stimulation, a rise of bloodpressure. With a gradual increase of the strength of stimulus up from the threshold, the reflex fall of blood-pressure first increases, then decreases, and gradually becomes converted into a rise, passing through a neutral point. We have failed to obtain a pressor effect by the strongest stimulation of the depressor nerve of Cyon.
3. The frequency of stimulation has an effect upon vasomotor reflexes. With a rapid rate of stimulation a rise is obtained and with a slow rate of stimulation in many cases a fall of bloodpressure. Of the different rates of stimulation w^e employed (one to eighty per second), the maximum pressor response is reached at twenty to forty per second.
4. No essential qualitative difference was found among various nerves (sciatic, tibial, peroneal, median, ulnar, branch of femoral nerve) subjected to stimulation. The saphenous nerve has a greater tendency to give a fall than those above mentioned. A purely sensory nerve seems to have a somewhat lower threshold than other kinds of nerves. Between nerves of the same category but of different sizes, the larger one produces usually a more marked response within a limited range of the strength of stimulation.
5. When the animal is under ether, chloroform, or brain compression, stimulation (mechanical, thermal, chemical, and electrical) of nerve terminations, such as those in the skin, muscles, and the intestine, causes a fall of blood-pressure in the great majority of cases, but violent or extensive stimulations of the
378 D. OGATA AND SWALE VINCENT
skin produce a rise. Under morphia and curare, on the contrary, a rise is a usual response, due clearly to a specific pharmacodynamical influence of these drugs. But under morphia a weak stimulus will produce a fall.
6. The influence of the ductless glands (adrenal, thyroid, and pituitary) upon vasomotor reflexes is not clear.* The injection of adrenin, thyroidin, and pituitrin and tying off or extirpation of the glands produced in our experiments no distinct effect.
7, The reflex change (fall or rise) of blood-pressure on stimulation of the somatic nerves (sciatic, saphenous) is produced chiefly by the dilatation or constriction of the blood-vessels in the splanchnic area, as in the cases of the stimulation of the splanchnic and the depressor nerve.
'See footnote, page 375.
VASOMOTOR REFLEXES 379
BIBLIOGRAPHY
1 AsHER, L. 1902 Ergebnisse der Physiologie, Jg. 1, Abt. II,
2 Bayliss, W, M. 1906 Ergebnisse der Physiologie Jg. 5, S. 319.
3 1908 Proc. Roy. Soc, B., vol. 80, p. 353.
4 Brunton and Tunicliffe 1894 Journ. Physiol., vol. 7, p. 373.
5 Cyon, E. de 1900 In Richet's Dictionnaire de Physiologie, p. 774.
6 DoMiTRENKo, L. F. 1912 Dissert., Odesse, p. 312. (Physiol. Abstracts,
1917, vol. 2, p. .30).
7 Gaskell, W. H. 1916 The involuntary nervous system, p. 90.
8 Gruber, C. M. 1907 Amer. Journ. Physiol., vol. 42, p. 214.
9 Gruber, C. M., and Kretschmer, O. S. 1918 Amer. Jour. Physiol., vol.
46, p. 222.
10 Grutzner and Heidenhain 1878 Arch. f. d. gesammt. Physiol., Bd. 16,
(quoted from Stiles and Martin) .^*
11 HoFMANN, F. B. 1909 In Nagel's Handbuch der Physiologie desMenschen,
Bd. 1, S. 318.
12 HosKiNS, R, G., AND Rowley, W. N. 1915 Amer. Journ. Physiol., vol. 37,
p. 471.
13 Howell, W. H. 1915 Text-book of physiology, p. 688.
14 Hunt, R. 1895 Journ. Phy.siol., vol. 18, p. 406.
15 1918 Amer. Journ. Physiol., vol. 45, p. 197.
16 1918 Amer. Journ. Physiol., vol. 45, p. 231.
17 Kepinow 1912 Arch.f.exper. Pathol., Bd. 67, S. 247 (Zentralbl.f. Physiol.,
1913, Bd. 27, S. 129).
18 Kleen, 1887 Skand. Arch. f. Physiol., Bd. 1, S. 247 (quoted from Reid
Hunt).
19 Knoll 1885 Sitz. Acad. Wiss., Wien, Math. Naturur. Kl., Bd. 92 (iii),
S. 448 (quoted from Reid Hunt).
20 Kronecker, H., and Nicolaides, R. 1883 Du Bois-Reymonds Arch.
(quoted from L. Asher).^
21 LuDwiG AND Cyon 1866 Bar. d. Siichs. ges. d. Wissenschaften (quoted
from Bayliss, Journ. Physiol., 1893, vol. 14, p. 303).
22 Martin, E. G. 1912 The measurement of induction shocks, p. 34.
23 Martin, E. G., and Lacey, W. H. 1914 Amer. Journ. Physiol., vol. 33,
p. 212.
24 Martin, E. G., and Mendenhall, W. L. 1915 Amer. Journ. Physiol.,
vol. 38, p. 98.
25 Martin, E. G., and Stiles, P. G. 1914 Amer. Journ. Physiol., vol. 34.
26 1916 Amer. Journ. Physiol., vol. 40, p. 194.
27 Oswald, A. 1916 Arch. f. d. gesammte Physiol., Bd. 164, S. 506-582.
" (Physiol. Abstracts).
28 1916 Arch. f. d. gesammte Physiol., Bd. 166, S. 169-200 (Physiol. Abstracts).
29 Porter, W. T. 1908 Boston Med. and Surg. Journ., vol. 158.
30 1910 Amer. Journ. Physiol., vol. 27, p. 276.
31 1915 Journ. Physiol., vol. 36 p. 418.
380 D. OGATA AND SWALE VINCENT
32 Porter, W. T., and Storey, T. A., 1907 Amer. Journ. Physiol., vol. 18,
p. 181.
33 Porter, W. T., and Turner, A. H. 1916 Amer. Journ. Physiol., vol. 39,
p. 236.
34 Porter, W. T., and Quinby, W. C. 1908 Amer. Journ. Physiol., vol. 20,
p. 503.
35 Ransox, S. W., and Billingsley, P. R. 1916 Amer. Journ. Physiol., vol.
42, p. 16.
36 1917 Amer. Journ. Physiol., vol. 42, p. 16.
37 SoLMANN, T., and Pilcher, J. D. 1910 Amer. Journ. Physiol, xxvi,
p. 233.
38 Starling, E. H. 1915 Principles of human physiol., p. 1006.
39 Stewart, G. N., and Laffer, W. B. 1913 Arch. Int. Med., vol. 11, p.
365.
40 Stiles, P. G., and Martin, E. G. 1915 Amer. Journ. Physiol., vol. 37,
p. 102.
41 Tigerstedt, R. 18 3 Lehrbuch der Physiologic des Kreislaufs (quoted
from Asher,2 p. 349).
42 TuR 1898 Hermann's Jahresb., S. 61 (quoted from Nagel's Handbuch).*
43 Vincent, S., and Cameron, A. T, 1915 Quart. Journ. Exper. Physiol.,
vol. 9, p. 48.
PLATES
381
PLATE 1
EXPLANATION OF FIGURES
Fig. 1 The effect of increased respiratory movements upon vasomotor reflexes. Bitch. 9 kilos. 10/5/1918. Ether. The left sciatic nerve was stimulated at intervals, the stimulus increasing from left to right. Upper curve, respiratory movements. Lower curve, blood-pressure. Base line is that of zero pressure, with periods of stimulation. The height of the blood-pressure in mm. Hg. is indicated by the cm. measured out and numbered. Time in seconds. For further explanation see text.
Fig. 2 Increased respiratory movements prevented by very deep narcosis. Same bitch as in figure 1. For explanation see text.
Fig. 3 Effect of increased respiratory movements upon blood-pressure. Bitch. 14 kilos. 30/5/1918. Ether. 'Thorax intact. The left sciatic nerve was stimulated. The result is a marked fall of blood-pressure.
Fig. 4 Effect of increased respiratory movements upon blood-pressure prevented by opening the thorax. Same bitch as in figure 3. Thorax wide open in the middle line. The same nerve was stimulated with the same strength of stimulus as in figure 3. The result is a marked rise of blood-pressure.
382
VASOMOTOR REFLEXES
D. OOATA AND SWALK VINCENT
PLATE 1
iliiiJliiiiliiiilniiliiiiliiiiliiMliiiihuiliii
iiiliiiilimliiulmilimij
383
JuiUiililiiiiliiiili
PLATE 2
EXPLANATION OF FIGURES
Fig. 5 A marked fall of blood-pressure which is apparently not due to increased respiratory movements. Dog. 12 kilos. 9/5/191S. Ether. The left sciatic nerve was stimulated.
Fig. 6 An example of vasomotor reflexes upon weak (left) and strong (right) stimulations. Same bitch as in figure 3. Thorax was very wide open in the middle line to eliminate the influence from increased respiratory movements. A weak stimulation caused a fall and a strong stimulation caused a rise of bloodpressure.
Fig. 7 Vasomotor reflexes under chloroform. Bitch. 7 kilos. 28/5/1918. Thorax wide open. A weak stimulation produced a fall (left) and a strong stimulation produced a rise (right) of blood-pressure.
Fig. 8 Effects of weak and strong stimuli, respectively, under brain compression. Dog. IS kilos. 15/8/1918. Brain compression and artificial respiration. Right ulnar nerve stimulated. The fall of blood-pressure increased at first with the development of the strength of stimulus and then passed over to a rise crossing a neutral point. •
Fig. 9 Effect of frequency of stimulation upon vasomotor reflexes. Bitch. 13 kilos. 18/7/1918. Chloroform and curare. Right saphenous nerve was stimulated. The frequency employed 1, 2, 5, 10, 20, 40, and 80 per second, respectively, frorn left to right. The one per second stimulation caused a fall, the two per second stimulation showed practically no effect, and the other stimulations produced a rise. The maximum pressor response was reached at forty per second stimulation in this case.
Fig. 10 Stimulation of a sensory (saphenous) and a motor (a branch of the femoral) nerve. Dog. 9 kilos. 10/10/1918. Brain compression and artificial respiration. Stimulation of a sensory nerve gave a more pronounced fall than that of a motor nerve.
Fig. 11 Stimulation of nerves of the same category but of different sizes. Same dog as in figure 10. The stimulation of a larger nerve (sciatic) produced a more marked response than that of a smaller nerve (peroneal).
386
VASOMOTOR REFLEXES
O. OGATA AND SWALE VINCENT
388
389
PLATE 3
EXPLANATION OF FIGURES
Fig. 12 Scratching of the skin. Dog. 12 kilos. 9/5/191S. Ether. A marked fall of blood-pressure. Respiratory movements practically unaffected.
Fig. 13 Application of heat (boiling water) on the skin. Dog. 10 kilos. 25/10/1918. Brain compression and artificial respiration. A fairly marked fall of blood-])ressure.
Fig. 14 Electrical (strong) stimulation of the skin. Dog. 11 kilos. 14/5/1918. Ether. A very marked fall of blood-pressure. Respiratory movements affected very slightly.
Fig. 15 Scratching of muscle (right sartorius). Bitch. 10 kilos. 22/10/1918. Ether. A fairly marked fall of blood-pressure. Respiratory movements show no increase.
Fig. 16 Kneading of muscles (lateral muscles of the right thigh). Dog. 10 kilos. 25/10/1918. Brain compression and artificial respiration. Gentle kneading produced a pure fall (left) and violent kneading a fall followed by a rise (right). Artificial respiratory* movements affected mechanically by the manipulation.
Fig. 17 Kneading of the small intestine. Same dog as in figure 16. A fairly marked fall of blood-pressure. Artificial respiratory movements slightly affected mechanically by the manipulation.
Fig. 18 Simultaneous tracings of the carotid blood-pressure and the volumes of kidney and hind limb. Dog. 10 kilos. 13/12/1918. Morphia and curare. Artificial respiration. Strong stimulation of the right sciatic nerve caused vascular constriction of the kidney, dilatation of the limb, and a rise of bloodpressure.
Fig. 19 Dog. 10 kilos. Ether. Effects of weak, moderate, and very strong stimulation of the skin. The weak stimulation gives a pure fall, the moderate stimulation a rise followed by a fall, while one very strong stimulation gives a pure rise.
392
VASOMOTOR REFLEXES
D. OGATA AND SWALE VINCENT
PLATE 3
■%%
%*#V^'
393
394
attthor's abstract of this paper issued by the bibliographic service, june 30
METABOLIC ACTIVITY OF THE NERVOUS SYSTEM
III. ON THE AMOUNT OF NON-PROTEIN NITROGEN IN THE BRAIN
OF ALBINO RATS DURING TWENTY-FOUR HOURS
AFTER FEEDING
SHIGEYUKI KOMINE
The Wistar Institute of Anatomy and Biology
THREE CHARTS
Hatai ('17) found in the nervous system of adult albino rats that the amount of non-protein nitrogen is surprisingly constant if the rats are examined under uniform nutritional conditions. It was thought interesting to determine whether or not there is a variation in the amount of non-protein nitrogen in the brain during the various stages of metabolism which occur in the course of the twenty-four hours following feeding. Should such variations be found, the study of them would throw some light on the very intricate problem of the metabolism of the nerve tissue.
As a first step I have undertaken to determine, therefore, the changes in the amount of non-protein nitrogen in the brain of the albino rat at intervals during the twenty-four hours which follow the last feeding, and the results so far obtained seem to me sufficiently interesting to publish.
MATERIAL
Albino rats were used for this study. The rats, which are usually fed at about 9 a.m., were removed after feeding into another cage, and kept without food, excepting water, until the following morning. The time during which the rats actually experienced lack of food is then approximately twenty-four hours. After twenty-four hours without food, the control rats were killed and examined, while those belonging to the group
397
398 SHIGEYUKI KOMINE
to be tested received food, and after the required number of hours, were in turn examined for the non-protein nitrogen in the brain. It was found without exception that at the end of twenty-four hours without food the digestive tract is practicallyempty, although the lower part of the tract often contains some chyme.
In order to furnish a uniform diet for the test rats, we have always given them Uneeda biscuit mixed with condensed milk. Since some rats eat immediately, while others do not, we have placed enough food in the cage and left it there for exactly one hour, after which time the surplus food was entirely removed.
Our calculation of the time after feeding was started from the time when this surplus food was removed. We found the stomach always completely filled after one hour.
I have selected rats of more than 100 days old because Hatai ('17) states that the amount of non-protein nitrogen shows very slight age variation after the rats have passed this age. The younger the animal, the greater is the normal content of nonprotein nitrogen in the brain in relation to its solids. The sexes were not distinguished in this* investigation, but the control and test rats were taken from the same litter.
TECHNIQUE
The rats were killed with ether and the blood was removed by severing the carotid artery, followed by complete evisceration. The brain was removed as quickly as possible and the left half was used for the determination of the non-protein nitrogen, and the right for the water estimation. From the dried residue the total nitrogen was determined by the usual Kjeldahl method.
For the determination of the non-protein nitrogen I have followed the method adopted by Hatai ('17). According to this method, the brain was finely ground with 2.5 cc. of an aqueous solution of trichloracetic acid and then transferred to an Erlenmyer flask (50 cc.) with a small amount of distilled water. The amount of trichloracetic solution taken was always twenty times the brain weight in grams, which was expressed in volume, while the amount of water used was five times the brain weight
METABOLIC ACTIVITY OF NERVOUS SYSTEM 399
similarly expressed in volume. The mixture of tissue and reagents in the flask was shaken repeatedly during the first hour and then left for twenty-four hours at room temperature. The clear filtrate obtained from this extraction was now analyzed by Folin and Farmer's micro-method ('12) as modified by Benedict and Bock ('15). In all cases the nitrogen was estimated by means of the Duboscq colorimeter.
The water content of the brain was determined by drying it at 98°C. for one week and the total nitrogen by the usual Kjeldahl method.
It should be stated that as soon as the filtrates were completed the designation on each flask was replaced by a conventional mark put on by some other member of the laboratory, and thus the observer made the non-protein nitrogen determination in entire ignorance as to whether the specimen belonged to the control or to the test series. Thus no personal prejudice entered into the determinations, and since the difference between the controls and tests is not large, such a precaution was highly important.
THE NON-PROTEIN NITROGEN
Using forty controls and thirty-five test rats of both sexes, I have studied the amount of non-protein nitrogen in the brain during the twenty-four hours after feeding. The results together with several other incidental observations are recorded in table 1.
The number of milligrams of non-protein nitrogen per 100 grams of moist brain is evidently more uniform in the controls than in the test rats. This is of course to be expected if the non-protein content in the brain varies under different physiological conditions. However, when entire averages are taken, the values of the non-protein nitrogen given by both control and test rats are found to be identical. The average value of 157 mg. thus obtained unexpectedly coincides with the value found by Hatai in his determinations which were made on seventy-five brains of adult albino rats. This exact coincidence of the values is unquestionably accidental, but it shows that there is a sur
400
SHIGEYUKI KOMINE
TABLE 1
Showing the data on the amount of non-protein nitrogen of the brain, together with several other observations during twenty-four hours after feeding
Controls A
BODY WEIGHT
AGE
NUMBER RATS USED
BRAIN WEIGHT
WATER IN BRAIN
TOTAL NITROGEN IN BRAIN
NON-PROTEIN NITROGEN IN
TOTAL NITROGEN
NON-PROTEIN NITROGEN PER 100 GRAMS BRAIN
DIFFERENCE
grams
days
grams
per cent
per cent
mgms.
66.8
123
2
1.519
78.6
2.11
2.57
156
24
66.8
120
3
1.519
78.6
2.11
2.37
157
23
70.0
118
5
1.508
78.6
2.12
2.52
165
10
74.4
131
6
1.514
78.3
2.09
2.40
162
7
92.2
135
4
1.585
78.7
2.08
2.34
150
-11
96.3
135
6
1.590
77.9
2.09
2.33
148
3
98.6
112
3
1.573
78.7
2.13
2.36
152
-19
97.3
123
2
1.608
79.6
2.06
2.53
149
-21
99.6
161
3
1.611
78.4
2.11
2.57
163
-10
106.7
202
2
1.606
77.7
2.21
2.66
166
-5
131.6
173
2
1.670
79.2
2.22
2.20
135
-6
129.5
147
2
1.679
78.9
2.24
3.04
182
Average 94.5
140
40
1.582
78.6
2.13
2.49
157
Tests B
NON-PROTEIN NITROGEN PER 100 GRAMS BR.^IN
NON-PROTEIN NITROGEN IN
TOTAL NITROGEN
TOTAL NITROGEN IN BRAIN
WATER !N BRAIN
BRAIN , WEIGHT
NUMBER RATS USED
AGE
BODY WEIGHT
NUMBER OF HOURS
AFTER FEEDING
mgms.
per cent
grams
days
grams
180
2.88
2.16
78.4
1.590
2
123
89.1
2
180
2.84
2.22
79.0
1.533
3
120
85.9
3
175
2.72
2.21
79.6
1.548
5
118
91.5
4
169
2.64
2.16
78.4
1.533
5
131
83.0
5
139
2.25
2.13
78.8
1.623
3
•a35
93.5
6
151
2.40
2.16
79.0
1.590
4
135
109.5
7
133
2.24
2.08
78.0
1.675
2
112
144.4
8
128
2.31
2.15
78.6
1.549
2
123
121.6
9
153
2.48
2.19
78.1
1.615
3
161
133.1
10
161
1.70
2.23
77.9
1.701
2
202
137.8
11.5
129
2.32
2.26
78.8
1.590
3
173
133.8
16
182
3.03
2.24
78.3
1.668
2
147
116.7
20
Average 157
2.57
2.18
78.6
1.601
35
140
111.2
8.5
METABOLIC ACTIVITY OF NERVOUS SYSTEM
401
prising uniformity of non-protein nitrogen in the entire brain of the albino rat. Although the average values for the non-protein nitrogen content thus agree in both the control and test rats, yet the successive differences during the twenty-four hours after feeding are not at all similar, but the test rats show an interesting deviation from the controls. This deviation, or the difference between control and test rats, is well brought out by a graphic representation (chart 1).
JU III!
-T-i — 1 — r— I— ' 1 1 I
3n-protein Nitroger
MGMb.
Difference in Amount of Nc
1
per 100 grams of Brain Weight
j! *""';
1 1 1 r 1 1 1 I ,
on t t
20 ^ r^ T
1 ^
/ r
^ I
n 1 in - / .
A ^
r ^,
1 C
z i 4
,
_ .. 1
n -.U ^
"^L
-■>
11
^— "'^ '
U t\
_.J — "
t_. ' ^-
. — ■ — "
U ^
• in
t r- -,^
' t 7
I t
X -.
J 7
90
^ /
^,
No. of Hours
1 ] 1 1 1 i 1
10
15
20
24
Chart 1 Showing the differences in the amount of non-protein nitrogen in the brain of test rats from that found in the controls.
From chart 1 we see clearly that the amount of non-protein nitrogen in the test animal increases very rapidly at first, and so far as the present data show, it reaches its maximum within two hours after feeding. This high content of non-protein nitrogen in the test brain soon begins to diminish, however, and within the next five or six hours the content reaches the original level found in the brain of the controls.
The diminution of the non-protein nitrogen in the test rats still*continues steadily, and at eight or nine hours after feeding reaches a minimum, showing just as great a difference — in the
402 SHIGEYUKI KOMINE
opposite direction — as was found between these two series at two hours after feeding. Soon after the content of non-protein nitrogen of the test brain has reached a minimum, at about eight or nine hours, the value begins to increase again, and finally at twenty-three hours after feeding the value for the nonprotein nitrogen reaches once more the original level for the control rats.
The sort of variation just described was enth-ely unexpected, although an increase immediately after feeding and a diminution later were anticipated. The form of the graph at once indicates the periodic nature of the alteration in the content of non-protein nitrogen during the interval chosen. This periodic variation in the non-protein nitrogen content must of course be interpreted. It is evident that for a satisfactory understanding of this interesting phenomenon we must further analyze the non-protein nitrogen into its components, such as amino acids, urea, ammonia, creatinine, etc., as such a determination would reveal what sort of nitrogen was actually responsible for the variations shown by the graph. Although I have not as yet made an analysis of this kind, yet from the data given by numerous investigators, I feel justified in suggesting the following general interpretation.
The metabolic products from the digestive tract, which have been absorbed and then carried by the circulation, are reabsorbed from the blood by the brain tissue until this is saturated to a maximum degree with these metabolites.
In support of this view, there are abundant experimental data which show a quick absorption by the organs and tissues of non-protein substances injected into the circulation. For instance, Folin and Denis ('12) always obtained an increase in the non-protein nitrogen of muscle of the cat following the injection of amino acids, amino-acid digestion mixture, or Witte's peptone, into a ligated loop of intestine. Van Slyke and Meyer ('13-'14), by the now standard nitrous method, found an increase of amino acids in the muscles and several visceral organs of dogs after the injection into the venous blood of amino a^ids and protein digestion mixtures. Van Slyke and Meyer came to
METABOLIC ACTIVITY OF NERVOUS SYSTEM 403
the conclusion that the tissues rapidly absorb amino acids from the blood when their concentration in the fluid is increased.
The products thus absorbed in excess from the blood are probably utilized in part for rebuilding broken-down tissue, while at the same time the surplus which is not utilized, as well as the products formed by the catabolism of the brain tissue, are carried away by the circulation. This double process goes on in the brain until all the wear and tear is restored. The second period is represented by a marked reduction in the amount of non-protein nitrogen during the three to eight or nine hours after feeding. At the same time the amount of material to be absorbed from' the intestine diminishes.
Here we encounter a difficulty arising from the fact that the amount of non-protein nitrogen in the test brains falls below that in the brain of the controls. It seems, however, probable that during the process of resynthetization of the polypeptides, some of the missing amino acids might be supplied by the amino acids that appear in the catabolic products constantly present in the brain tissue. The withdrawal of these amino acids from the normal content of metabolites in the brain tissue for utilization might be responsible in part at least for this smaller amount of non-protein nitrogen in the test brains at this period of active reconstruction.
This is, however, a pure hypothesis and therefore must await experimental examination.
When the minimum has been reached, and when no fresh supply of non-protein bodies is coming into the brain, catabolism becomes evident, and as a consequence, the amount of the metabolites again shows an increase. The slow rise from eight or nine hours up to twenty-four hours after feeding may represent this last period.
From the observations of Van Slyke and Meyer ('13-' 14), that fasting increases the content of amino acids in the tissues and organs owing probably to autolysis, we may expect that the rise in the content of non-protein nitrogen after it has reached a minimum was also due to autolysis or to the phenomena of fasting.
Taking all these facts together, we conclude that after feed
404 SHIGEYUKI KOMINE
ing, the non-protein nitrogen content in the brain shows a definite periodic change. Under the same conditions, similar periodic phenomena should occur in other organs, and it will be highly interesting to test this inference on some other occasion. In this connection the observations made by Pepper and Austin ('15) on the content of non-protein nitrogen in the blood of dogs are very valuable. Pepper and Austin found that by feeding a dog with a moderate amount of meat, the blood nitrogen reaches a maximum within about two hours after feeding, and returns to the original level in about ten to fourteen hours.
When, however, the dog was allowed to fast, the blood nitrogen first falls gradually below the original level until it reaches a minimum at thirty to forty-eight hours, and then begins to rise gradually during a few hours, after which it tends to persist.
From the functions of the blood we can at once appreciate the relations of the observations of Pepper and Austin to the present study, because the blood receives the digestive products and distributes these to the tissues and organs.
When autolysis begins in the tissues and organs as the result of fasting, these autolytic products are again poured into the circulation. Consequently, what Pepper and Austin observed in the blood reveals what is probably happening in the organs and tissues. In reality the content of non-protein nitrogen in the blood indicates the periodic changes following first feeding and then fasting, as w^as observed by me in the brain of rats. Owing to the differences in the body size, as well as to the different degrees of activity of these two animals, the exact time relations found by Pepper and Austin cannot be directly compared with what has been found in the rat brain.
Very recently Mitchell ('18) studied the partition of nonprotein nitrogen in the entire body, as well as in some organs of the albino rat at birth, before and after feeding. Mitchell noted in the younger rats a decided increase of non-protein nitrogen soon after feeding when compared with that in the rats fasting for twenty-four to forty-eight hours. In the case of adult rats, however, this increase was not as conspicuous as in the younger rats. The observed period following feeding was
METABOLIC ACTIVITY OF NERVOUS SYSTEM 405
•
not extensive enough (only up to seven hours after feeding), and thus a periodic variation in the non-protein nitrogen content, such as I have found in the brain, did not appear. Mitchell's observations, however, show clearly that the amount of amino acids and urea, as well as ammonia, are regularly higher in the fed than in the fasting animal, and furthermore, these fed rats show rapid increase in the non-protein nitrogen during the first five hours, and then slight decrease at seven hours, not only in the entire body, but in such organs as the kidneys, liver, and muscles. Despite the somewhat greater irregularities of the data given by Mitchell, the increase in the content of these simpler nitrogenous bodies during active protein digestion is well indicated.
RELATION OF THE NON-PROTEIN NITROGEN TO THE TOTAL
NITROGEN
From the amount of non-protein nitrogen in the four divisions of the central nervous system of the rats, Hatai ('17) came to the following conclusions. "Although the amount of nitrogen given by the non-proteins — the amino acids, the urea and ammonia — in relation to the solids is higher in the cerebellum and cerebrum than either the stem or the spinal cord, these nitrogen values become constant in all the four parts when they are computed in relation to the protein nitrogen. This is interpreted to mean that the nitrogenous organic extractives are intimatelj^ related with the active cell substance, and not at all with the lipoid substance."
Following the statement of Hatai, it was thought desirable to find the relation which exists between the amount of non-protein nitrogen in the brain and the amount of total nitrogen, because by this means we can find a much closer relation between the active cell substance which is expressed in the present instance in terms of total nitrogen and metabolic products, owing to the elimination of myelin substance mainly.
The data for this determination are given in table 1, and I shall now discuss this observation as it appears in the graph given in chart 2.
406
SHIGEYUKI KOMINE
It must be stated that in the total nitrogen those nitrogen values which belong to the non-protein substances, as well as those belonging to the lipoids, are also included. Any disturbance which may arise from such treatment should be very slight, owing to the greater amount of protein nitrogen contrasted with the nitrogen of the metabolic products. We find in chart 2 that in general the variation of non-protein nitrogen in relation to the total nitrogen is essentially similar to the relation found between entire brain substance and the non-protein nitrogen. This is to be expected, since the ages of rats examined are close to each other, and hence the water content, as well as the'amount
0.5 0.4
0.3
0.2
0.1
-0.1
-0.2
-0.3
-0.4
-0.5
11
■
1 1 1 — 1 — r
1 1 1
-1 — 1 — 1
1 — 1
1 — 1
—.1
1 1 1 1
/\
Difference in Percentage of Non-protein Nitrogen .
y
\
i_
/
\
A
^
/
\
/
\
_«=-"
L- ^ iJ
^
-__
/
\ ^^
.* '
■^^ ^
"^ 1j
A /
i^
7
1
■ '-I
iL
^
/
Cnntfnl Vnhioc 1 1
L ^
7
\y
^.
~
No. of Houis
- 1 1 i 1 1 1 1
10
15
20
24
Chart 2 Illustrating the relation between the total nitrogen in the brain and metabolic products found during the twenty-four hours following feeding.
of myelin substance present in each brain, should not differ so much as they would, for instance, if the cerebrum was compared with the spinal cord of the same animal. This result was pleasing, since the close similarity found here, and that found in connection with the relation of the entire brain to the metabolites, shows the trustworthiness of the present technique which is suited to such experiments where very slight differences between the control and test animals are present.
We conclude from these additional facts that the non-protein nitrogen in the brain shows a periodic alteration during the twenty-four hours after feeding.
METABOLIC ACTIVITY OF NERVOUS SYSTEM
407
TABLE 2 Percentage of water in the brain
OBSERVED
CALCULATED
NUMBER OF HOURS
AFTER FEEDING
Control
Test
Difference from control
Difference from control
Test
Control
2
78.6
78.4
-0.2
-0.1
78.1
78.2
3
78.6
79.0
-0.4
0.4
78.6
78.2
4
78.6
79.6
1.0
1.1
79.3
78.2
5
78.3
78.4
0.1
0.1
78.0
77.9
6
78.7
78.8
0.1
0.1
78.5
78.4
7
77.9
79.0
1.1
1.1
78.7
77.6
8
78.7
78.0
-0.7
-0.7
77.6
78.3
9
79.6
78.6
-1.0
-1.1
78.2
79.3
10
78.4
78.1
-0.3
-0.3
77.8
78.1
11
77.7
77.9
0.2
0.4
77.7
77.3
16
79.2
78.8
-0.4
-0.5
78.4
78.9
23
78.9
78.3
-0.6
-0.6
78.1
78.7
Average 8.5
78.6
78.6
-0.3
-0.1
78.3
78.3
PERCENTAGE OF WATER IN THE BRAIN
It is conceivable that following the greater or less accumulation of the metabolic products in the brain, the water content might show a change also. We have determined the amount of water in the brain of the control and test rats and found that while their average observed values are identical, both being 78.6 per cent (tables 1 and 2, 'Observed'), yet their successive differences during the twenty-three hours after starvation are not identical, but show interesting deviations. In dealing with the values on the percentage of water, we must consider, in the interest of precision, the influence of brain size on the water content.
Donaldson ('16) finds that although the percentage of water in the brain is a function of age, nevertheless, within the same age, the heavier brain gives relatively less water than the lighter brain, and vice versa.
Since, then, the brain weights given by both the control and test rats are not identical, we thought it advisable to transform the observed values of the percentage of water to the theoretical
408
SHIGEYUKI KOMINE
values in which the influence of the difference in brain weight is entirely eliminated. The exact method of correction is given by Donaldson ('16) and for it the reader is referred to that paper. The percentage of water in each instance, as thus calculated, is also given in table 2. We notice that the differences obtained when the calculated values are used are practically identical with those obtained by the use of the observed values, but since the observed brain weights were small for their age, the absolute observed values for the percentage of water in both control and test rats run above those which are calculated. We have, however, chosen the calculated values of the percentage of water for the construction of chart 3.
2 „ 1 1 1 1 1 i 1 I 1 1 1 1 1 1 1 1 1 1
" "> ' Difference in Percentage of Water
1 -jL J
^\ 1-v A
J \ \ Or,ntrr.l U-nllir": .
T~ \ t 2"^^^
o~"=--r r -y-- " v-
\ -/ ^^
7 ^•
., J- ^ ±
1 v2
■1 ^f
_j No. of Hours —I
•0
15
20
24
Chart 3 Showing the differences in the calculated water content of the brains of the test rats compared with those of the control rats.
It is evident at once that the relative water content in the brain of the test rats is definitely higher during the first seven hours, and then falls below that of the controls and reaches its minimum at about nine hours, and so far as the present data are concerned, the relative water content of the test brains again rises, tending at the end of the period to approach the original level. On account of the irregularities, it is not desirable to place too much stress on the exact form of this graph; nevertheless, it gives unquestionable evidence of an increase in the water content during the first seven hours, followed first by a sharp fall and then by a return toward the normal or control value.
METABOLIC ACTIVITY OF NERVOUS SYSTEM 409
It is also evident that this variation in the water content as the result of feeding is in general similar to the variation which is shown by the content of non-protein nitrogen in the brain, although the exact time relations of the rise or fall of the curves are not identical.
From these results I am inclined to think that the percentage of water in the brain rises with the increase in the non-protein nitrogen and falls with its decrease, but this is merely a tentative conclusion which must await more careful study.
CONCLUSIONS
1. During the twenty-four hours after feeding the increase of non-protein nitrogen in the rat's brain reaches its maximum at from two to three hours after the taking of food. This rapid rise is followed by a steady diminution which reaches a minimum at about eight or nine hours. The non-protein nitrogen then shows a steady but slow increase and reaches the original level at about twenty-three hours. In other words, non-protein nitrogen in the brain shows a periodic alteration and completes one period within about twenty-four hours after feeding.
2. This periodic change depends on the ingestion of food. Non-protein nitrogen appears in the brain as the result of the absorption of such material from the digestive tract and the formation of such bodies by the catabolic activity of the brain tissue itself. Its amount is diminished by the anabolic process in the brain tissue and by excretion. The variation of its amount in the brain is a resultant of these several processes.
3. A similar periodic relation is shown when the non-protein nitrogen is compared with the total nitrogen instead of the entire brain mass.
4. The course of the percentage of water in the brain follows that of the non-protein nitrogen.
410 SHIGEYUKI KOMINE
LITERATURE CITED
Bock, J. C, and Benedict, S. R. 1915 An estimation of the Folin-Farmer method for the colorimetric estimation of nitrogen. J. Biol. Chem., vol. 20.
Donaldson, H. H. 1911 An interpretation of some differences in the percentage of water found in the central nervous system of the albino rat and due to conditions other than age. Jour. Comp. Neur., vol. 21. 1916 A revision of the percentage of water in the brain and in the spinal cord of the albino rat. Jour. Comp. Neur., vol. 27.
Folin, O., and Denis, W. 1912 Protein metabolism from the standpoint of blood and tissue analysis. J. Biol. Chem., vol. 11.
Hatai, S. 1917 Metabolic activity of the nervous system. I. Amount of non-protein nitrogen in the central nervous system of the normal albino rat. Jour. Comp. Neur., vol. 28. t
Mitchell, H. H. 1918 The influence of protein feeding on the concentration of amino acids and their nitrogenous metabolites in the tissue. J. Biol. Chem., vol. 36.
Pepper, O. H. P., and Austin, J. H. 1915 Experimental studies of urinary and blood nitrogen curves after feeding. J. Biol. Chem., vol. 22.
Van Slyke, D. D., and Meyer, G. M. 1913-1914 The effect of feeding and fasting on the amino acid content of the tissue. J. Biol. Chem., vol. 16. 1913-1914 The fate of protein digestion products in the body. 2. The absorption of amino acids from the blood by the tissue. J. Biol. Chem., vol. 16.
THE JOURNAL OF COMPARATIVE NEUKOLOGY, VOL 30, NO 5
Resumen por el autor, James Stuart Plant. Instituto Wistar de Anatomia y Biologia.
Factores que infiuyen en el comportamiento del cerebro de la rata albina en el liquido de Miiller.
El cerebro de la rata albina sufre un cambio tipico cuando se fija" en el liquido de Miiller, compuesto de 2 por ciento de bicromato potasico y 10 por ciento de sulfate sodico. El cerebro aumenta rapidamente de peso, al que sigue una perdida de peso lenta y continua, hasta que al cabo de setenta y cinco dias, al pesar el cerebro se comprueba que pesa de 20 a 30 por ciento mas que el cerebro fresco. 1. La edad es la principal condici6n que influye en esta reaccion del liquido de Miiller. Los cerebros de ratas vie j as aumentan mas en peso y retienen este aumento durante los setenta y cinco dias. 2. El peso inicial del cerebro, o sea su tamafio, es la condicipn de mas importancia despues de la apuntada mas arriba. Del mismo modo que con los cerebros de la misma edad, los cerebros mas ligeros aumentan mas de peso durante la primera parte de su permanencia en el liquido de Miiller. Esta diferencia disminuye gradualmente y desaparece al cabo de los setenta y cinco dias. 3. La edad pr6ximamente semejante (entre ciertos limites) tiene casi el mismo valor determinante que la igualdad de edad. 4. El sexo es un factor sin importancia, del mismo modo que la composici6n hereditaria (relacion dentro de un tronco determinado) .
Translation by Jose F. Nonidez Carnegie Institution of Washington
AtJTHOR's ABSTRACT OF THIS PAPER ISSUED BY THE BIBI.IOORAPHTC 8EUVICE, JUNE 30
FACTORS INFLUENCING THE BEHAVIOR OF THE BRAIN OF THE ALBINO RAT IN MULLER'S
FLUID
JAMES STUART PLANT Neurological Laboratory of The Wistar Institute of Anatomy and Biology
The brain of the albino rat, placed for a period in Miiller's fluid, exhibits a typical change. In the course of time it not onl}^ hardens, but also markedly increases in weight. There is a rapid increase to a maximum in about one week's time, after which there is a slow, steady loss until the seventy-five day weighing, at which time the brain weighs 20 to 30 per cent more than when fresh. It was thought that changes in this typical curve might be induced in the brains of rats previously anesthetized for prolonged periods, and it was hoped that this criterion would be more delicate than the microscopic or analytic tests which had, so far, failed to demonstrate a change. The work was done at The Wistar Institute of Anatomy during the academic year 1913-1914.
PLAN
The main question of the effect of the anesthetic on the typical curve remains unanswered. From the start, however, it was recognized that various factors influenced the reaction of 'control' brains to Miiller's fluid — factors which are inherent in the material. It is these factors and their influence on the reaction with which the present paper deals.
PROCEDURE
The brains studied belonged to 'stock' albino rats. The animals were killed with ether and the brains quickly removed with every care not to damage them. They were immediately weighed and then suspended in 50 cc. of Miiller's fluid. The
411
412 JAMES STUART PLANT
whole was kept in a black cardboard case in a dark closet. Subsequent weighings were as follows. The brain was removed from the solution and placed for about ten seconds on a drypiece of filter-paper. The string by which it had been suspended was during this time removed. The brain was then placed on a watch-glass and immediately weighed. It was returned to the Miiller's fluid as quickly as possible. The watch-glass was then weighed. Reweighings were carried out at 24 hours, and at 7, 14, 30, and 75 days after killing the rat. On completion of the weighings the percentage of water in the brain at the final weighing was determined. This procedure involved placing the brain, immediately after its last weighing, in a small glass vial of known weight. This was kept in a drying oven (temperature, 97°) for one week. On removal, the vial was cooled in a desiccator at room temperature and weighed.
The Mtiller's fluid used was made up in 1000 cc. lots. To 25 grams of potassium bichromate c. p. and 10 grams of sodium sulphate c. p. was added 1000 cc. of distilled water. Time was given for dissolving the salts and, after thorough agitation, the solution was divided into two '500 cc. lots and kept for one month before being used. In every instance 'pairs' of brains were fixed in fluid from the same bottle. The Miiller's fluid was always kept in a dark closet.
No attempt was made to control the temperature during the reaction of fixation other than that all specimens were kept in the same dark closet at room temperature. Thus the results may be considered as comparable.
Necessarily our original results are in terms of absolute weights and absolute gains. In the presence of so diverse initial weights it seemed, however, best to state all gains in weight as percentages of the original weight. This makes the data comparable. Corresponding to this, all statements of the relation of one brain's gain to that of another are in terms of a percentage of the percentages of gain of the heavier brain. This leads to higher figures, in the relations of the gains, than would be the case were the actual differences between the absolute gains stated.
ALBINO RAT BRAIN IN MULLER S FLUID
413
OBSERVATIONS
If we consider the brains of pairs (rats of the same age, sex, and litter, i.e., as similar as possible), there appears a very distinct tendency for the brains of older rats to gain more in Miiller's fluid than do those of the younger rats. Table 1 presents fifteen pairs arranged on the basis of their age. In ten of the fourteen possible comparisons the brains of older rats gain more in twentyfour hours. Also in ten of the comparisons the older brains
TABLE 1
Percentages of gain of pairs of albino rat brains arranged according to age and weighed at intervals from twenty-four hours to seventy-five days
SEX
AGE
INITIAL
WEIGHT
AVERAGI
PERCENTAGES OF GAIN OF BOTH BRAINS IN MULLERS FLUID
1
2
24 hours
7 days
14 days
30 days
75 days
days
grams
grams
9
52
1.465
1.368
19.0
28.2
26.2
23.5
22.9
9
55
1.615
1.578
20.8
32.1
28.1
26.8
25.4
c?
57
1.757
1.671
20.6
31.2
26.0
25.9
25.7
d"
59
1.635
1.519
21.2
33.1
29.9
27.0
26.7
&
61
1.834
1.715
18.0
32.2
28.0
25.9
25.0
9
61
1.699
1.581
20.0
29.5
27.3
24.1
23.0
c?
62
1.490
1.477
18.6
31.8
28.3
25.3
24.4
d^
62
1.662
1.656
19.3
28.2
24.6
23.1
22.7
9
62
1.497
1.496
20.5
31.7
29.1
25.7
25.3
&
62
1.831
1.699
20.8
32.8
30.8
28.0
27.8
9
64
1,677
1.606
20.9
32.0
28.6
26.2
25.7
&
67
1.651
1.587
21.7
32.6
29.7
26.6
26.4
9
72
1.610
1.492
27.6
33.6
30.7
27.9
26.7
d^
160
1.791
1.752
25.1
37.2
35.8
32.8
32.2
cT
218
2.008
1.824
28.3
40.0
38.6
35.7
34.6
gain more in seventy-five days, though this does not in every case involve the same comparisons as were favorable to the older rats at the twenty-four hour weighing. Table 2 presents a summary of the data of table 1. The averaged figures for the youngest three pairs and for the oldest three pairs are given. The data show that age is a very important factor in the reaction of the brain of the albino rat to Miiller's fluid.
Brain weight increases as a function of age, and there exists between these two characters a very high coefficient of correla
414
JAAIES STUART PLANT
TABLE 2 Percentages of gain of averaged entries of table 1
PAIRS AVERAGED
First three entries. Last three entries.
days
55 150
INITIAL WEIGHT
1
grams
1.612 1.803
grams
1.539 1.689
.A.VERAGE PERCENTAGES OF GAIN IN MGLLERS FLUID
24
hours
20.1 27.0
7 days
30.5 37.0
14 days
26.8 35.0
30 days
25.4 32.1
75 days
24.7 31.2
tion. If we make a comparison of the individual brains of the respective pairs involved in table 1, however, we maj^ study the effect of initial brain weight in animals of like age. The data are given in table 3. In place of the percentage of gain of the lighter brain there is entered at the several columns marked 'Per cent deviation of 2' — under 'Time in Miiller's fluid' — only the relation of that percentage to the percentage of gain of the heavier brain. Of the fifteen pairs involved, it will be noted that in twelve the lighter brain gains more in the first twentyfour hours (represented by a + in the second column). Also in twelve of the fifteen pairs, the heavier brain later 'catches up' — that is, the relative gain of the heavier brain is greater at seventy-five days than at one day. This phenomxcnon is clearly demonstrated in the 'averages' at the bottom of the table. The results may be summarized as follows:
1 DAT
7 DATS
14 DATS
30 DAYS
75 DAYS
Average difference
+3.9 ±6.1
+ 1.5 ±3.9
+1.3 ±5.0
+1.2 ±5.2
+0 2
Standard deviation
±6 5
Thus it appears that the lighter brain gains more in the early part of the stay in Miiller's fluid, but that this difference practically disappears at the seventy-five-day weighing. It is to be noted that the standard deviations are lowest at the seven-day weighing. This seems to represent the period of least individual variation. As this represents the time of maximum increase, we may consider that as the more stable period in the
ALBINO RAT BRAIN IN MULLER .S FLUID
415
curves and think of the rise and fall in percentage of increase as periods more subject to individual variation.
While increasing age presupposes increase in brain weight, it is apparent that these two — age and brain weight — act as opposing factors in the determination of the reaction of the brain to Miiller's fluid. That is, the older brains (these are heavier)
TABLE 3
The effect of initial brain weight — albino rat — on the percentage of gain of paired brains. Pairs arranged according to age. Deviation of the lighter brain
AGE
INITIAL WEIGHT
TIME IN MULLERS FLUID. PERCENTAGES OF GAIN
SEX
1
grams
1.465 1.615 1.757 1.635 1.699 1.834 1.490 1.497 1,662 1.831 1.677 1.651 1.610 1.791 2,008
2
24 hours
7 days
14 days
30 days
75 days
1
Per
cent deviation of 2
1
27.9 31.7 30.5 33.0 28,7 31.5 32.4 31.4 27.4 31.5 32.6 33,3 33.3 38.0 40.1
Per
cent deviation of 2
1
Per
cent deviation of 2
1
Per
cent deviation of 2
1
23.5 25.1 24.8 27.2 21.6 24.7 25.0 25.1 22,2 26.3 26,2 28.0 27.4 32.5 34.7
Per
cent deviation of 2
9
9
& 9 &
9 & d" 9
9
days
52 55 57 59 61 61 62 62 62 62 64 67 72 160 218
grams
1.368 1.578 1.671 1.519 1.581 1.715 1.477 1.496 1.656 1.699 1.606 1.587 1.492 1.752 1.824
19.3 20.4 19.5 20.5 18.8 17.9 18,8 20,5 19.2 18.9 21.1 21,6 26.9 25.0 28.0
- 2.8 + 3.6 +11.1 + 6.5 +13.3 + 1.3
- 1.8 + 0.3 + 0.9 + 19.6
- 1.2 + 0.8 + 5.2 + 0.3 + 2.0
+2.1 +2.5 +4.1 +0,2 +6.0 +4.2 -3.4 +1.7 +6.0 +8.4 -3.5 -4.6 + 1,3 -4.0 +1.2
26.8 27.7 25.5 30.3 26.2 27.5 29.3 28.7 23,7 29.4 29.4 30.5 30.4 35.3 38.5
-4.2 +2.6 +4.1
-2.2 +8.8 +4.1 -7.2 +2.1 +7,2 +9.8 -4.9 -5.1 + 1.7 +2.4 +0.9
24.1 25.8 25.2 27.4 23.3 25.4 26.3 25.9 22.6 26.7 26.8 27.3 28.3 32.6 34.8
- 4.9
+ 8.2 + 5.1
- 2,4 + 7.0 + 3.8
- 7.3
- 0.7 + 4.4 +10.3
- 4.3
- 4.7
- 2.3 + 1.1 + 4.9
- 5.7 + 2.1 + 6.8
- 4.0 +12.9 + 1.8
- 4.8 + 2.1 + 4.5 +11.4
- 4.3 -11.2
- 5.2
- 2.2
- 0.8
Average
StanHnrd devia
tion
+ 3.9 ± 6.1
+1.5 ±3.9
+1.3 ±5.0
+ 1.2 ± 5.2
+ 0.2 ± 6.5
gain more than do the younger ones; yet the lighter brains gain more than do the heavier ones if we can eliminate age as a factor, as was done in table 3. The curve of increase in weight may be considered as capable of solution into at least two curves — expressing these two factors. Age appears to be by far the more potent factor.
416 JAMES STUART PLANT
A further study was made of fifty-nine brains arranged according to increasing brain weight but without regard to sex or litter. In table 4 these are arranged according to initial brain weight in three age groups. Within these groups two phenomena are apparent (shown in the averages under the vertical column, 'Percentage difference from the following value'). These are the early greater gains for the lighter brains (this does not hold clearly for the ten brains of the youngest group where there is practically no difference); and the fact that at the seventy-fiveday weighing the lighter brains show relatively a less percentage of increase than they do at the twenty-four-hour weighing. Since these facts are just those which determine the curve when brains of rats of the same age, sex, and litter are compared, we may conclude that in the reaction of the brain to Miiller's fluid:
1. Sex is negligible.
2. Inherited composition is negligible.
3. Approximate similarity of ages (the range being limited) may be considered as having the same effect as though the ages were identical.
The data on the percentage' of water — in the last column of table 4 — will be discussed later.
A group of four brains — all belonging to young rats — was subjected to an additional procedure. The brains, immediately upon removal, were separated into cerebrum, cerebellum, stem, and olfactory bulbs. Each part was then treated as were the whole brains of the other series. The data are given in table 5 in this way, that that percentage of the whole brain weight represented by the weight of each part at each weighing is recorded. The figures for the four brains show but slight variation, and table 5 therefore presents onl}^ the averages of the four. The relative weights of the various parts undergo considerable change in Miiller's fluid, but this change is mainly consummated in the first twenty-four hours. Thus we may assume from this study that, while the relations of the various parts are altered in the fixing solution, the length of time, after the first twenty-four hours, during which the parts are subjected to this treatment, is a matter of minor import.
ALBINO RAT BRAIN IN MULLER S FLUID
417
TABLE 4
The effect of inilial brain weight — albino rat — on the percentages of gain of brains arranged in age groups, regardless of sex or litter
initial weight (gr.^ms)
NUMBER OF CASES
AVERAGE AGE
24 HOURS
IP. O
" «
a b w
%^ Z ai
5 H "* 6.
AVERAGE PERCENTAGES OF GAIN
7 days
14 days
30 days
■■voS
75 days
c c g .
C3 S a)
Ik
50 to 60 days
1.35-1.40
1
52
18.7
- 2.8
28.5
25.7
22.9
22.2
- 5.6
79.3
1.40-1.50
1
52
19.3
-11.1
27.9
26.8
24.1
23.5
-11.2
79.3
1.50-1.60
3
53
21.7
-1- 5.8
33.2
29.2
27.5
26.5
+ 1.2
80.1
1.60-1.65
2
57
20.5
- 2.2
32.3
29.0
26.6
26.2
- 3.0
79.9
1.65-1.70
2
58
20.9
+ 3.2
32.8
28.8
27.7
27.0
+ 8.7
80.3
1.70-1.80
1
57
19.5
30.5
25.5
25.2
24.8
80.3
Average. 1.58
10
55
20.5
- 0.9
31.7
28.1
26.3
25.7
- 2.1
80.0
60 to 70 days
1.40-1.50
7
64
21.7
+ 1.9
32.9
29.8
26.6
25.7
+ 0.5
79.7
1.50-1.60
5
65
21.3
- 8.6
32.1
29.1
26.2
25.6
- 9.3
79.3
1.60-1.65
6
69
23.3
+12.3
34.1
31.5
28.8
28.2
+ 10.6
80.2
1.65-1.70
11
68
20.7
+ 2.9
31.5
28.5
26.0
25.5
+ 3.1
79.7
1.70-1.80
5
65
20.1
+ 8.2
31.6
28.2
26.4
24.7
- 1.8
80.1
1.80-1.90
3
63
66
18.6
31.5
27.9
25.7
25.2
80.7
Average . 1 . 65
37
21.1
+ 3.9
32.3
29.2
26.6
25.9
+ 1.8
79.8
180 to 240 days
1.65-1.70 1.70-1.80 1.80-1.90 1.90-2.00
1 4 5 2
189 191 239 206
213
28.7 26.5 25.7 23.7
+ 8.5 + 2.8 + 8.7
42.4 38.2 37.6 35.6
39.2 36.2 36.4 33.0
36.1 33.2 32.4 30.4
35.4 32.4 31.5 29.7
+ 9.3 + 3.2 + 6.1
81.6 80.5 80.5
78.4
Average . 1 . 83
12
25.9
+ 6.1
37.9
36.0
32.7
31.8
+ 5.6
80.2
The percentages were obtained originally by the use of values carried to three places. These have now been reduced to one-place numbers and there are therefore some apparent discrepancies in the percentage-difference columns. These differences, however, are not significant.
418
JAMES STUART PLANT
TABLE 5
Averaged percentage weight relations of the parts of four albino rat brains during the course of their reaction to Miiller's fluid
AGE
WEIGHT OF
WHOLE BRAIN
AVERAGE OF
FOUR
PERCENT.VGE
REPRESENTED BY CEREBRUM
PERCENTAGE
REPRESENTED BY STEM
PERCENTAGE
REPRESENTED BY CEREBELLUM
PERCENTAGE REPRESENTED BY OLFACTORY BULBS
TIME IN
mulleb's
FLUID
52
1.610
65.17
17.40
13.80
3.63
Initial
2.127
63.10
17.68
15.08
4.14
24 hours
2.171
64.82
17.21
14.33
3.63
7 days
2.094
63.77
17.76
14.72
3.75
30 days
Difference between per
centages at initial and
24-hour weighing
-2.07
+0.28
+1.28
+0.51
Difference between per
centages at 24-hour
and 30-daj' weighing. . . .
+0.67
+0.08
-0.36
-0.39
WATER RELATIONS
We have studied the water relations after seventy-five days in fifty-nine whole brains and after thirty days in the parts of three brains. There is evidently, in' the reaction of the brain to the Miiller's fluid, a deposition of salts in the brain tissue. This is shown in table 6. Part A deals with the fifty-nine whole brains; Part B with the parts of three brains (all belonging to young females). The deposition of salts at the end of seventy-five days in the whole brain is from 3.9 per cent to 4.3 per cent of the total water of the brain despite the fact that the salts in Miiller's fluid are present in a concentration of but 3.5 per cent. This shows a deposition of salts in the tissues. If, in addition, there is some diffusion of solids from the brain to Miiller's fluid — and, from inspection, this appears to be the case — the percentage of salts deposited must be even higher than that indicated by the figures given.
The final percentage of water in a given brain at the seventyfive-day weighing is only slightly greater than that of a fresh brain belonging to a rat of the same age, sex, and litter. In view of the 20 to 30 per cent net increase in weight in Miiller's
ALBINO RAT BRAIN IN MULLER S FLUID
419
TABLE 6
Part A. Water relations at the seventy-five day weighmg of fi,Jty-nine whole brains
{sec table 4)
Average fresh brain weight
Percentage of water (from Donaldson, '16)
Calculated amount of water represented in fresh
brain
Final brain weight
Final amount of water
Percentage of water — observed
Increase in weight due to water, gms
Increase in weight due to salts, gms
Percentage of salts in total increase in weight
Percentage of salts in the total water in brain
UNDER 60
60 TO 120
D.\TS
DAYS
1.582
1.651
79.1%
78.9%
1.252
1.303
1.988
2.078
1.590
1.659
80.0%
79.8%
0.338
0.356
0.068
0.071
16.6%
16.7%
4.2%
4.3%
OVER 120 DATS
1.826
78.1%
1.426 2.407 1.932
80.2% 0.506 0.075
13.0% 3.9%
Part B. Water relations at the thirty-day weighing of parts of three brains
OLFACTORY BULBS
0.058
82.3%
0.048 0.078 0.067 85.6% 0.019 0.001*
4.9%
1.5%
Average fresh brain weight
Percentage of water (from Donaldson, '16) Calculated amount of water represented in
fresh weight
Final weight
Final amount of water
Percentage of water — observed
Increase in weight due to water, gms
Increase in weight due to salts, gms
Percentage of salts in total increase in weight
(for total brain 14.8%)
Percentage of salts in total water in the part
(for total brain 4.3%)
CEREBRUM
STEM
CEREBELLUM
1.097
0.292
0.235
80.0%
76.1%
79.7%
0.878
0.222
0.188
1.403
0.386
0.329
1.139
0.301
0.266
81.2%
77.9%
80.8%
0.261
0.079
0.078
0.044
0.016
0.015
14.5%
16.4%
16.4%
3.9%
5.0%
5.8%
fluid, this seems a striking fact, though where the initial percentage of water is so high, it is evident that it takes a relatively large difference in the absolute water content to markedly affect the percentage value.
In the three brains divided into their parts the salts are deposited in the following percentages after thirty days:
420 JAMES STUART PLANT
Cerebellum 5.8 per cent
Stem 5.0 per cent
Cerebrum 3.9 per cent
Olfactory bulbs 1 .5 per cent
Average of whole brain 4.3 per cent
With the exception of the cerebellum, the percentage of salts deposited is in direct relation with the proportion of myelin in the part involved. As none of the parts were washed, it may well be that the interstices of the cerebellum were the site of large deposits of salts, a physical factor which may account for this anomalous result.
CONCLUSIONS
General reaction of the brain to Miiller's fluid, or type curves. The brain of the albino rat undergoes a typical change when 'fixed' in Miiller's fluid (2.5 per cent potassium bichromate and 1 per cent sodium sulphate). There is a rapid increase in weight followed by a slow, steady loss until at the seventy-fiveday weighing the brain weighs 20 to 30 per cent more than when fresh.
Factors affecting this reaction, or components of the type curve.
1. Age is the main condition controlling this reaction to Miiller's fluid. The brains of older rats gain more and retain this higher relative gain throughout the seventy-five days.
2. Initial brain weight, or size, is the condition of next importance. As between brains of like age the lighter brains gain more during the earlier part of their stay in Miiller's fluid. This difference is gradually lessened, and it disappears at the seventyfive-day weighing.
Thus, while age and initial brain weight are highh^ correlated, they constitute factors which, when taken alone, influence in opposite ways the reaction of the brain to Miiller's fluid. The early greater increase of the smaller brain of two rats of the same age may be due to one or both of the following factors:
a. If we consider the brain as a sphere and the fluids as penetrating at a fixed rate, then in the smaller brain a slightly greater
ALBINO RAT ERAIN IN MULLER's FLUID 421
proportion of the brain will be penetrated — that is, swollen by the fixing fluid — at any given instant in the early part of the reaction. This would give a more rapid enlargement in the smaller brain.
6. The smaller braili has a higher percentage of water which might make diffusion more rapid. If this is a controlling factor, the matter of a higher percentage of water must be of more immediate importance than is that of a lesser percentage of myelin.
3. Approximately similar age (the range being limited) has nearly the same determining value as equality of age.
4. Sex is a negligible factor, as is also inherited composition (relationship within a given strain).
FINAL WATER RELATIONS
The increase in weight is due mainly to the taking up of water, but the percentage of salts deposited in the fixed tissues is much greater than that in the fixing fluid. With the exception of the cerebellum, the deposition of salts is proportional to the myelin present in the part of the brain.
RELATION OF RESULTS
The curve of reaction of 'control' brains to Mliller's fluid is evidently of such constancy in character as to make it a satisfactory criterion for a judgment as to alterations produced in the brain by various experimental procedures. It seems that in problems involving changes not available to such microscopic or analytical tests as we have, this reaction might furnish a valuable means of study. It appears from the foregoing results that when tests are made for the experimental modification of the response of the brain to Mliller's fluid, it is necessary to have the test and control brains of the same age and, in those cases in which the brains differ in weight, to allow sufficient time for the compensation of this difference.
422 JAMES STUART PLANT
LITERATURE
While various observations bearing upon this problem have been made, none have employed exactly our experimental procedure. Various solutions of jDotassium bichromate have been used— Donaldson ('94), Fish ('93), and King ('10)— but a study in the changes in the reaction to Miiller's fluid has not been made. A similar study was carried out by Hrdlicka ('06) in which various formalin preparations were used as fixing reagents. The effect of age upon this reaction has been discussed in a general way — Donaldson ('94) and King ('10) — but it seems that the extent of its control over the reaction has not been previously stated.
BIBLIOGRAPHY
Donaldson, H. H. 1894 Preliminary observations on some changes caused in the nervous tissues by reagents commonly employed to harden them. Jour. Morph., vol. 9.
1916 A revision of the percentage of water in the brain and in the spinal cord of the albino rat. Jour. Comp. Neur., vol. 27.
Fish, P. A. 1893 Brain preservation with a resume of some old and new methods. Wilder Quarter-C^nturj^ Book, Ithaca.
Hrdlicka, A. 1906 Brains and brain preservatives. Proc. U. S. Nat. Museum, vol. 30.
King, H. D. 1910 The effects of various fixatives on the brain of the albino rat, with an account of a method of preparing this material for a study of the cells in the cortex. Anat. Rec, vol. 4.
Resumen por el autor, Olof Larsell. Universidad de Wisconsin.
Estudios sobre el nervio terminal : la tortuga.
El autor describe las relaciones perifericas y la distribuci6n del nervio terminal en el embrion de la tortuga. Un plexo del nervio, situado en el tabique nasal, esta intimamente mezclado con un plexo de la rama oftalmica del nervio trigemino. El autor compara las celulas ganglionares del nervio terminal, en lo referente al tamano y caracteres morfologicos generales, con las celulas de los ganglios sensorio y del simpatico de los mismos embriones. Las celulas del nervio terminal presentan una semejanza sorprendente, tanto en, el tamano como en la forma, con las celulas halladas en los ganglios del simpatico, especialmente las de los situados en la region cefalica. Las celulas de los racimos ganglionares del nervio terminal no pueden diferenciarse de celulas emigrantes que se presentan a lo largo de la rama oftalmica del trigemino. Todo esto indica que el nervio terminal esta relacionado con el sistema del simpatico.
Translation by Josd F. Nonidez Carnegie Institution of Washington
AUTHOR S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JUNE 30
STUDIES ON THE NERVUS TERMINALIS: TURTLE^
O. LARSELL
Department of Anatomy, University of Wisconsin
SIXTEEN FIGUKES
The present contribution was begun as part of a comparative study of the nervus terminahs in several groups of vertebrates. The work had not progressed far before it was found advisable to confine attention to one group at a time, so that the greater portion of the present report embraces the results of observations made since the studies on the nerve in mammals by the author ('18) was published.
The somewhat extensive literature of the nervus terminalis was reviewed in the previous article, and only two papers which have appeared on the subject during the past year will be mentioned briefly. These papers are by Van Wijhe ('18) and Ayers ('19).
Van Wijhe's paper reviews much of the literature of the nervus terminalis in the various groups of vertebrates briefly and homologizes the nerve with one he noted a number of years ago ('94) in Amphioxus, which he termed at that time the 'nervus apicis.' He states: "Before the homologue of the profound ophthalmicus there is in Amphioxus still another nerve which supplies the utmost point of the snout. On account of this and because it arises from the morphological fore-end of the cerebral ventricle I called it the nervus apicis."
Ayers ('19), in continuing his studies of Cephalogenesis, begun long ago, has found the nervus terminalis (Van Wijhe's 'nervus apicis') in Amphioxus, and calls attention to its large size as
^ Contribution from the Zoological Laboratory of Northwestern University, William A. Locy, Director, and from the Anatomical Laboratory, University of Wisconsin,
423
THE JOURNAL OF (XJMPARATIVE NEUROLOGY, VOL. 30, NO. 5
424 O. LARSELL
compared with the olfactory nerve in that form. He finds it also in Cyclostomes and states that in Bdellostoma the nerve presents an intermediate stage, as respects its size and relations, between Amphioxus and the selachians. He calls attention to the distinction between the vomeronasal nerve, which he terms the 'nervus septalis' and the olfactory nerve, and suggests a new classification of the cranial nerves in which the nervus terminalis would be number I, and, with the 'nervus septalis', would be added to the list of twelve cranial nerves usually recognized. The nervus terminalis is considered a sensory nerve which has to do with a group of chemical sense organs, and is related physiologically to the vomeronasal (his septal) nerve. The conclusions are reached from a study of Amphioxus and cyclostomes. Doctor Ayers believes that in higher forms the nerve has undergone considerable modification due to changes in head structure.
I am under great obligation to Doctor Ayers for opportunity to read his manuscript prior to publication and for permission to make use of his observations. He also afforded me opportunity to read Van Wijhe's paper, which I had not previously seen. It is a pleasure to express here my sense of indebtedness to him. My acknowledgments are also due Prof. William A. Locy, of Northwestern University, under whose direction the general problem was originally begun and who has since continued his interest.
MATERIAL AND METHODS
Embryos of the painted turtle (Chrysemys marginata) were used. Most of these had been fixed in a formol-bichromateacetic fluid, some in formalin of 10 per cent, others in Tellyesniczky's fluid, and a few living embryos were obtained and prepared by the Cajal and the Vom Rath methods.
Stages beyond 10- to 11-mm. carapace length had become chitinized to such an extent in the rostral region that intact serial sections could not be obtained. Chiefly for this reason, the present contribution is confined to a description of the nervus terminalis in embryos up to 11-mm. carapace length (about 17 mm. greatest length.
NERVUS TERMINALIS: TURTLE 425
Numerous dissections of embryos and of newly hatched turtles were made with the aid of the binocular microscope. The head was split slightly to one side of the midsagittal plane, and the soft parts were then sufficiently removed to expose the nerve and its adjacent structures.
The embryos sectioned were cut in the sagittal plane or transversely, and were stained by various methods. The most generally satisfactory stain for older stages was found to be iron-hematoxyUn, but some of the most instructive series were obtained by overstaining with Delafield 's hematoxylin, followed by a counterstain of saturated aqueous orange G to which two drops of glacial acetic acid were added for each 50 cc. of stain.
The serial sections studied were -as follows:
1 series 6-mm. embryo, sagittal, stained with iron-hematoxylin.
2 series 6.3-mm. embryo, transverse, stained with hematoxylin and Congo red.
3 series 7.5-mm. embryo, transverse, stained with hematoxylin and Congo red. 1 series 8-mm. embryo treated by the Cajal method, cut sagittally.
1 series 9-mm. embryo, sagittal, stained with hematoxylin and Congo red. 1 series 9-mm. embryo, sagittal, treated by Vom Rath method.
1 series 9.o-mm. -carapace-length embryo, stained with hematoxylin and Congo
red, sagittal plane.
2 series 10-mm. -carapace-length embryos, sagittal, stained with iron-hema toxylin. 1 series 10.5-mm. -carapace-length embryo, sagittal, stained with hematoxylin
and erythrosin. 1 series 11-mm. -carapace-length embryo, sagittal, stained with hematoxylin and
Van Gieson's stain. 1 series 11-mm. -carapace embryo, sagittal, stained with hematoxylin and
orange G.
DESCRIPTIVE
The nervus terminalis in the turtle has its origin by several small roots from the ventromesial surface of the forebrain, just caudad to the olfactory bulb. It can be demonstrated by dissection in suitably prepared material. Figure 1 represents a dissection of an embryo of 11-mm. carapace length, showing the left nervus terminalis and its relation to neighboring structures. In the specimen figured the rootlets were not evident until some of the overlying brain tissue had been removed by brushing. By this process three roots were demonstrated. In other dissections but two roots were brought to light, usually after some brushing.
426
O. LARSELL
mes
NERVUS TERMINALIS: TURTLE
427
In sections of corresponding stages, cut in the sagittal plane, two or three roots were observed to enter the brain substance, but their fibers could be followed within the brain for only a short distance (fig. 2).
On following these roots distally from the brain, they are seen to unite (figs. 1 and 2) and a ganglionic swelling was invariably found just beyond their point of union. In the dissection figured it will be noted that the two more dorsal roots unite to form a single short trunk before entering the ganghon, while the ventral root enters the ganghon directly. In sections a number of ganglion cells (figs. 2 and 3) may be observed in this mass, but in none of the embryos examined did this ganglion appear to have as many cells as others located more rostrally, especially the one marked gn. (fig. 3).
Rostrally from the ganglionic mass which lies at the junction of the rootlets, the nerve continues as a compact trunk as far as the most anterior part of the olfactory bulb, midway between the
Fig. 1 Dissection of head of turtle embryo of 11-mm. carapace length to show the nervus terminalis and its relation to neighboring structures. The left lateral half of the brain is viewed from the mesial aspect. X ca. 15.
Fig. 2 Central roots of the nervus terminalis at their point of junction with the brain. Turtle embryo of 11-mm. carapace length. Hematoxylin and orange G stain. X 410.
Fig. 3 Reconstruction of the right nervus terminalis of turtle embryo of 11-mm. carapace length, stained with hematoxylin and orange G. The nervus terminalis is represented as lying on the medial surface of the vomeronasal nerve for all of that part of its course which is parallel to the latter. Fart of its course, however, as shown in figure 1, is lateral to the vomeronasal nerve. X ca. 16.
ABBREVIATIONS
hl.v., blood-vessel
hu.olf., olfactory bulb
cer.hem., cerebral hemisphere
cr.cav., cranial cavity
gn., main ganglionic mass (ganglion
terminale) of nervus terminalis gn.', accessory ganglion terminale gn.c, ganglion cells gn.cl., ganglionic clusters mes., mesencephalon m.ob.do., dorsal oblique muscle
m.ob.ven., ventral oblique muscle
m.r.ant., anterior rectus muscle
n.olf., olfactory nerve bundles
n.oph., ophthalmic branch of V nerve
n.ter., nervus terminalis
n.vom., nervus vomeronasalis
na.ant., anterior naris
na.po., posterior naris
olf.epith:, area of olfactory epithelium
op.chi., optic chiasma
ret., retina
428 O. LARSELL
dorsal (vomeronasal) bundle and the main bundle of olfactory fibers proper, as shown in figure 1. At this point it turns to follow these bundles, passing between them in such a manner that it could not be traced further by the method of dissection. Sections, however, reveal the further course of the main bundle of the nerve and indicate the presence of numerous ganglionic cells scattered along its trunk as it passes over the mesial surface of the olfactory bulb. These cells form clusters of various sizes. One of the largest of these clusters is no doubt indicated by the swelUng (fig. 1, gn.') shown in the dissection. Another and larger ganghonic mass is shown (fig. 3, gn.) at the point where the terminaUs passes lateral to the vomeronasal bundle. This corresponds to the position usually occupied by the largest cluster of cells in the majority of the embryos which were sectioned.
From the position of this ganglion distally the nerve could not be followed further by the method of dissection, because its strands became too intimately mingled with those of the olfactory and vomeronasal nerves. Fortunately, however, in some of the series of the older stages studied, a differential stain was obtained by the method previously described, so that the terminahs bundles could be distinguished from those of the other two nerves and, further rostrad, from the fibers of the ophthalmic branch of the V nerve. This differentiation was aided by the fact that the olfactory and vomeronasal nerves appear as compact bundles of wavy fibers with few nuclei scattered among them. The strands of the nervus terminalis are smaller and much less compact and present relatively numerous sheath nuclei as well as larger ganglionic cells. Where the strands of the trigeminus were intermingled on the septum, they also had a characteristic appearance, apparently due to the process of myelination, as well as to differential staining. These characteristics, however, could not be noted with any degree of certainty in the smaller tracts, so that the reconstruction represented in figure 3 indicates only the larger bundles and their grosser ramifications.
In some of the preparations the nerve was seen to divide intracranially at the ganglion gn. into two strands, which, however, reunited to form a compact trunk before the nerve left the brain
NERVUS TERMINALIS: TURTLE 429
cavity. This condition is illustrated in figure 7. There were some indications of much finer strands also in this region, but they were not sharply enough differentiated from the olfactory strands to justify inclusion in the figure as part of an intracranial plexus of the terminalis.
After emerging from the cranial cavity, the nervus terminalis is composed of several well-marked strands which continue parallel with the vomeronasal bundles, mesial and in part dorsal, to the latter. A short distance from the point of emergence of the nerve, its strands begin to form a plexus over the nasal septum. Lack of silver preparations of the older stages made it impossible to follow this plexus for any considerable distance, especially in its more rostral part, where it becomes more complex due to entrance of fibers from the trigeminus. It was very evident that both the nervus terminalis and rami from the ophthalmic branch of the V nerve take part in the formation of a plexus on the nasal septum.
Clusters of ganglionic cells (fig. 3) were scattered throughout this plexus. Along the vomeronasal nerve there was a nearly continuous mass of cells from the point where the nerve turned ventrorostrally at the bulbus olfactorius, to the point where the more profuse spreading out of the septal plexus began. A somewhat similar arrangement of cells along the vomeronasal nerve was found by Johnston ('13) in Emys, but apparently the cells were not so numerous as in Chrysemys.
A fortunate Cajal preparation of an 8-mm. -total-length embryo gave a very clear demonstration that the trigeminus forms a more important portion of this septal plexus than might have been anticipated. As shown in figure 4, which represents a reconstruction from fourteen serial sections cut in the sagittal plane, the trigeminal portion of the plexus is formed by the ramifications of the ophthalmic nerve. The fibers were stained quite uniformly brown or black. They could be followed to the gasserian ganglion, with the beautifully stained cells of which they united.
The nervus terminalis in this preparation is represented by a few clusters of cells and some yellowish fibers. The cells could not with certainty be distinguished from the mesenchymal cells,
430
O. LARSELL
T) Q.ant.
NERVUS TERMINALIS: TURTLE 431
but sections of embryos of approximately the same stage which were stained by other methods, indicate that the clusters are composed of ganglionic cells, which from their position no doubt belong to the nervus terminalis.
Some details of this plexus of the ophthalmic nerve are illustrated in figures 5 and 6. As shown in the figures, relatively small strands of fibers meet at nodal points, from which the individual fibers are redistributed to bundles diverging at various angles. No ganglionic cells could be observed about these nodal points at this early stage. In older stages, however, cell clusters of various sizes are numerous at the points where the ophthalmic nerve ramifies on the septum, and are found as far forward (figs. 3 and 7) as the most rostral part of the septum. It seems likely that the more rostral of these cells correspond to clusters of sympathetic cells described and figured by Willard ('15) in Anolis. The observation of Rubaschin ( '03) of a 'ganglion olfactorii ner.vi trigemini' on one of the branches of the ophthalmic nerve in the chick appears also to be related.
At various points the branches of the trigeminus and of the terminalis become so intimately related that the two cannot be told apart, and the smaller strands of the plexus which continue from these points, appear to contain fibers from both nerves. As shown in figure 7, which represents a reconstruction from nine serial sections, from which the finer strands of the plexus are omitted, the nervus terminalis anastomoses with one of the larger branches of the ophthalmic nerve just dorsal to the vomeronasal nerve. At the point of anastomosis is a large ganglionic cluster. The two nerves had the characteristic different appearance, previously noted, proximal to their point of union, but their
Fig. 4 Reconstruction of the septal plexus of the ophthalmic branch of the trigeminal nerve of a turtle embryo of 8 mm. greatest length, prepared by the Cajal method. The figure was reconstructed from sections 79 to 92 of the series. The numerals indicate the sections from which the adjacent structures were projected. For the sake of simplicity, section 79 is indicated by the numerals 1, section 92 by 14, and intervening sections accordingly. X 80.
Fig. 5 Portion of the plexus from section 81, at point marked 3* in the previous figure, to show details of structure. X 385.
Fig. 6 Portion of the plexus from section 88 of the series, at point marked 5* in the reconstruction, to show detail. X 385.
432
O. LARSELL
mpQ
^Ai?
n.vom
■^ i!^ k/f /f^ ^< '■ 'S rM^'
NERVUS TERMINALIS: TURTLE 433
strands could not be told apart distal to this point. The relation of the two nerves and something of their appearance are represented more highly magnified in figure 8, but this does not show the slight although clearly apparent differentiation of staining which the preparations themselves reveal in the larger bundles. The trigeminal fibers appear coarser and more compactly collected into bundles than do those of the terminalis, but in the smaller strands these characteristics are not apparent, probably because of the small number of fibers composing them.
The olfactory and vomeronasal bundles, which are also represented in the figure, resemble each other in being composed of very delicate fibers with a characteristic wavy appearance which could not be well represented in the drawing.
Huber and Guild ('13) in the rabbit and Brookover ('17) in the human found indications of such an anastomosis of terminalis and trigeminal strands on the nasal septum, and the present writer ('18) partially demonstrated it in the cat. It is rather striking that three nerves, the terminalis, the olfactory (Read, '08), and the trigeminus, should each form a plexus on the nasal septum. Two of these plexuses overlap to a marked degree. The vomeronasal nerve is not plexiform, except as the large bundles composing it branch and reunite to a slight extent in their course to the vomeronasal organ. In this connection a statement from the previously cited paper of Ayers is of interest. He states: "The nasal chamber in man therefore contains the surface distribution of these three (terminalis, septalis, olfactorius) cranial nerves as well as the surface terminations of invading branches of a fourth and more recent cranial nerve, the trigeminus.
jj
Fig. 7 Reconstruction from ten sections (175 to 185) of the series from which figure 3 was reconstructed, to show anastomosis between rami of the nervus terminalis and of the ophthalmic branch of the trigeminal nerve. X 44.
Fig. 8 Reconstruction from four of the sections (sections 182 to 185) included in figure 7, to show at higher magnification the anastomosis of one of the rami of the ophthalmic plexus with a bundle of the nervus terminalis. This figure also gives some idea of the appearance of the fiber bundles. X 180.
434
O. LARSELL
GANGLION CELLS
An effort to reach some conclusion as to the character of the ganghon cells of the termmalis in the turtle embryos was made by comparing them with cells of other ganglia, cranial, spinal and sympathetic. The gasserian, sphenopalatine, and ciliary ganglia were studied in the head region. The spinal ganglia, beginning with the first thoracic, and the sympathetic chain ganglia were studied in the body region. Measurements were made of the nuclei with the aid of an ocular micrometer, the results of which are indicated in table 1. The cells measured were taken at random. The only selection exercised was in measuring nuclei
TABT.F, 1
AVER
NUM
SIZE OF
SIZE OF
AGE
BER
LARG
SMALL
SIZE OF
POSITION OF CELLS
MEAS
EST NU
EST NU
ENTIRE
REMARKS
URED
CLEUS
CLEUS
NUMBER
M
M
M
Periphery of spinal
48
12.3
8.2
9.8
Many unipolar.
ganglia
Central part of same
52
8.2
5.3
7.3
Bipolar, approaching unipolar
ganglia
condition
Above combined
100
12.3
5.3
8.5
Gasserian ganglion
52
11.4
7.0
9.0
Two sizes present, but small cells not many
Ciliary ganglion,
50
12.4
7.0
9.0
Nuclei large in proportion to
large cells
entire cell
Ciliary ganglion,
22
7.9
5.3
6.4
small cells
Above two combined
72
12.4
5.3
8.3
Sympathetic chain
50
8.1
5.3
6.2
Five nuclei larger than 6.7 m
ganglia
Sphenopalatine
50
8.1
5.3
6.74
ganglion
N. terminalis, periph
50
8.8
5.3
6.72
Some of these cells probably
eral clusters
belonged to clusters related to the trigeminus
N. terminalis, central
30
8.4
5.3
6.7
Of the 80 nuclei measured in
root clusters
«
both peripheral and central clusters three were larger than 8.1 m and three were smaller than 5.5 m
NERVUS TERMINALIS: TURTLE 435
which were spherical or nearly so. In the case of some cells, especially many in the terminalis ganglia, the nuclei were so elongated that it was necessary to measure the greater and the lesser diameters and take the mean of the two.
All of the measurements and the drawings of the ganglion cells represented were made from a single embryo of 10-mm. carapace length, stained with iron-hematoxylin. This was done for the sake of uniformity, although the statements hold in general for all the embryos examined which were sufficiently advanced to show any pronounced differentiation of the various types of nerve cells. In drawing the figures the outlines of the cells and nuclei were traced with the aid of the camera lucida, and the same combination of lenses was employed in each case, so that the figures represent directly the variations in form and size of the various types.
Comparison of embryos at different stages of development indicated that in embryos of 10-mm. total length, the spinal ganglion cells were on the average somewhat larger than those of the sympathetic chain ganglia. There was, however, but slight difference in the size of the individual cells within the spinal ganglia. The sympathetic chain ganglion cells had stiU much the appearance of indifferent cells. In the gasserian ganglion of embryos at this stage of development, many of the ceUs were larger than the spinal ganglion cells of the same embryo.
In embryos of 9.5 to 11-mm. carapace (15.5 to 17 mm. greatest length) there is a marked difference between the size of the largest sensory ganglion cells and those of the sympathetic chain ganglia. The latter (fig. 12) are of pretty uniform size, but the spinal ganglia and, in less marked degree, the cranial ganglia, showed two fairly distinct sizes of cells. The larger type in the spinal ganglia (fig. 9) was found near the periphery of the ganglion. Many had already reached the unipolar condition, but others showed various transitional stages from the primitive bipolar cells.
Of one hundred cells measured from three different ganglia, one in the thoracic region, one in the lumbar, and one in the sacral, the largest nucleus had a diameter of 12.3 m and the
436
O. LARSELL
10
B
16
14
NERVUS TERMINALIS: TURTLE 437
smallest was 5.3 m in diameter. The average size of the entire one hundred nuclei was 8.5 n. These cells were divided into two groups, those with a nuclear diameter greater than 8.2 yu and those whose nuclear diameter was less than this, down to 5.3 M which was the smallest found. While this division was somewhat arbitrary, there was sufficient ground for it in the character of the cells, aside from their size, to make it appear justifiable. The smallest cells (fig. 10) had considerably less cytoplasm surrounding the nucleus, both relatively and actually. Only various stages of the bipolar condition were observed in these smaller cells. They were found closely packed together in the central portion of the ganglion, while the larger cells were nearer the periphery.
As indicated in the table, the fifty-two cells which showed a nuclear diameter less than 8.2 fx had an average diameter of 7.3 /x. The forty-eight cells whose nuclear diameter was greater than 8.2 ^t were found to have an average nuclear diameter of
9.8 m.
The smaller cells (fig. 10) may correspond to the small ganglion cells found in the spinal gangha of mammals by Dogiel ('08), Ranson ('12), and other workers, but it seems likely that some of them at least represent cells of the larger type which have not
Fig. 9 Cells from the peripheral portion of the first thoracic spinal ganglion of a turtle embryo of 10-mm. carapace length. Iron-hematoxylin stain. X 500.
Fig. 10 Cells fropi the central portion of the same ganglion from which figure 9 was drawn. Turtle embryo 10-mm. carapace length, iron-hematoxylin stain. X 500.
Fig. 11 Cells from the gasserian ganglion of turtle embryo of 10-mm. carapace length. Iron-hematoxylin. X 500.
Fig. 12 Cells from the third thoracic sympathetic chain ganglion of turtle embryo of 10-mm. carapace length. Iron-hematoxylin. X 500.
Fig. 13 Cells from the ciliary ganglion of turtle embryo of 10-mm. carapace length. Cells of smaller type indicated at A, and the larger type at B. Ironhematoxylin. X 500.
Fig. 14 Cells from the sphenopalatine ganglion of turtle embryo of 10-mm. carapace length. Ii-on-hematoxylin. X 500.
Fig. 15 Cells from the peripheral cell clusters of the nervus terminalis of a turtle embryo of 10-mm. carapace length. Iron-hematoxylin. X 500.
Fig. 16 Cells from the central root clusters of the nervus terminalis of a turtle embryo of 10-mm. carapace length. Iron-hematoxylin. X 500.
438 O. LARSELL
yet reached the same degree of development. This view appears to be favored by the extremely crowded condition of these cells ^^■ithin the ganglia. This would appear to result in a reduction both of the amount of space and of nourishment which the individual cell may obtain, thus retarding its growth. The fact that the larger cells were found near the periphery of the ganglia, where there would appear to be space for greater expansion of the cells during growth as well as more abundant nourishment, may account for their larger size at this stage of development. There were also cells of intermediate size between the two groups, but these were not so numerous as the cells of either group.
In the gasserian ganglion (fig. 11) of the older stages the number of small cells was not so great as in the spinal ganglia, but here also some were present, as the figure indicates.
The sympathetic chain ganglia of the older stages, as in the younger, contained cells of rather uniform size and appearance (fig. 12). Two or three processes were observed on most of the cells which were examined on this point. The nuclei were smaller than those of the small spinal ganglion cells, showing an average size for fifty cells of 6.2 fx, as compared with 7.3 ^ for the latter type. The relative amount of cytoplasm surrounding the nucleus appeared to be about the same in the two types. No difference in the size of the peripherally located cells, as compared with those situated nearer the centers of the sympathetic ganglia, was observed.
The ciliary ganglia showed two groups of strongly contrasting cells. Without entering into a detailed description of this ganglion or attempting to review the large amount of literature w^hich has accumulated concerning it, the present purpose will be served by calling attention to Carpenter's ('06) excellent study of it in the chick and adult fowl. He finds two distinct sizes of cells. The smaller cells were arranged in a definite group on the dorsal side of the ganglion. This group composed about one-third of the entire mass.
A similar group of small cells (fig. 13, A) was present in many of the turtle embryos, but not in all, studied by the present
NERVUS TERMINALIS: TURTLE 439
writer. In all cases, however, whether or not arranged in a distinct group, small ganglionic cells were found in the ganglion. These differed not only in size, but also in form, from the larger more typical cells. Carpenter considers the large cells in the chick to be derived from the midbrain and to have migrated along the oculomotor nerve to the ciliary ganglion. The small cells he believes to be sympathetic cells which have migrated forward along the ophthalmic nerve.
Fifty of the large cells were measured. The largest had a nuclear diameter of 12.4 n, the smallest of 7 ix. The average nuclear diameter of the fifty cells was 9 /i. While the size of these nuclei approached that of the large spinal ganglion cell nuclei, and the largest found in the ciliary ganglion even exceeded the largest observed in the spinal ganglia, the amount of cytoplasm surrounding the nuclei of the ciliary ganglion cells was considerably less, as may be seen by comparing figures 9 and 13, B. The actual size, therefore, of the ciliary ganglion cells was somewhat less than that of the large spinal ganglion cells.
Twenty- two cells of the smaller type were measured. These were found in several adjacent sections, forming a distinct mass in the embryo on which the measurements were made. The largest of these cells had a nuclear diameter of 7.9 i^. The smallest was 5.3 m, and the average of the twenty-two was 6.4 /x. As figure 13 (A) indicates, there were also differences in the form of the cells and in the amount of cytoplasm siu"rounding the nucleus, as compared with the large cells.
There remain the cells of the sphenopalatine ganglion, which form a relatively smaU cluster. These cells are of quite uniform size and structure (fig. 14) with spheroidal, eccentrically located nuclei. Most of them were in the primitive bipolar stage of differentiation. Except that there was less individual variation among these cells than among those found in the clusters of the nervus terminaUs, to be described, the sphenopalatine cells may be said to resemble the terminalis cells more closely than any of the other ganghonic cells studied. Of fifty sphenopalatine cells which were measured, the largest had a nuclear diameter of 8.1 iJL. The smallest was 5.3 n, and the average for the fifty nuclei was 6.74 n.
THE JOUBNAL OF COMPARATIVE NBUBOLOQT, VOL. 30, NO. 5
440 O. LARSELL
With these measurements as criteria, a comparison may be attempted between the ganghonic cells of the nervus terminalis and these other cells of well-recognized function, with the purpose in mind of throwing more light on the relationship of the nervus terminalis.
Many of the terminahs cells (figs. 15 and 16) have a peculiar elongated form not met with elsewhere in the ganglia which were subjected to observation. This is well illustrated in some of the cells represented in figure 15. Only two processes were observed in any of these cells, and these were in most cases continuous with the longer axis of the cell. In view of the findings of McKibben ('14) in the dogfish and of the various authors who have made observations on the ganglion cells of the terminalis in the mammals, it seems likely that this bipolar condition is developmental in the turtle.
The nuclei were elongated in many cases and many were also considerably distorted otherwise, as the figures indicate. This was more frequently the case in the peripheral clusters than in those withm the cranial cavity. These conditions made the terminalis cells more difficult 1:0 measure, so that the results obtained represent the mean of several measurements on many of the nuclei. In order to determine the difference in size, if any, between the cells which were located in the ganglionic swelling near the central roots of the nerve and those along the olfactory and vomeronasal nerves outside the cranial cavity, the measurements were tabulated separately for the two groups. Thirty cells from the centrally located ganglion (fig. 16) indicated an average nuclear diameter of 6.72 m, the largest being 8.4 ix, and the smallest 5.3 m (table 1). The fifty cells of the peripheral clusters (fig. 15) which were measured indicated an average nuclear diameter for the entire number of 6.7 ju, practically the same as that of the centrally located cells. The largest nucleus of the peripheral clusters was 8.8 m in diameter, the smallest was 5.3 fx. A larger number of measurements was not made because the variation in size was not pronounced. Only three of the eight}^ nuclei measured were larger than 8.1 m? i-e., more than 1.4 yu larger than the average. This amount indicates the
NERVUS TERMINALIS: TURTLE 441
variation above the average size which the smallest cells, which had nuclear diameters of 5.3 ju, showed below the average size. Three cells of this smallest size were found to be included among the eighty measured.
Reference to the table will indicate that the cells of the gasserian, spinal, and cihary ganglia have an average nuclear diameter of 8.3 n or more, even when the smaller cells, which in the spinal and cihary ganglia may with some show of reason be considered as a separate group, are included with the larger in computing the average. If the two types are considered separately, the average size of the larger cells, which may be considered as the typical cells in the three ganglia under consideration, is considerably increased, being 9 m for the ciliary ganglion and 9.8 IJL for the spinal and gasserian ganglia. The sympathetic chain ganglia, the small cells of the ciliary, and the sphenopalatine ganglion cells, together with those of the nervus terminahs, have an average nuclear diameter of approximately 6.7 m or less.
WTien these facts are considered in connection with the difference in form of the cells of the terminahs clusters, and their manner of distribution in more or less irregular clusters instead of in the compact ganglia of the sensory nerves, it seems reasonable to conclude that the majority of cells at least, which are found in the ganghonic clusters of the nervus terminahs in the turtle, are related to the sympathetic nervous system.
The writer 's previous work on various mammals, together with Huber and Guild's ('13) comparison of terminahs cells with those of the gasserian ganglion in the rabbit, points in the same dhection for mammals, as does the work of McKibben ('14) on the terminahs ganglion of Mustelus, for the selachians, and certain of Brookover's observations in ganoids for that group of vertebrates.
At this point attention may also be called to the position of some of the ganglion cells at the point of entrance of the nerve roots into the brain, as illustrated in figure 2. The position of these cells is interesting. As shown in the figure, some of them appear to be migrating outward from the brain wall. Both cells and nuclei are elongated, and the cells lie just at the border of
442 O. LARSELL
the limiting membrane of the brain. The Hmiting membrane in this region is bulged forward to a considerable extent. Some of the cells appear to have passed beyond this membrane, at least they lie outside of it, but their processes extend into the brain substance. Similar cells were found along the entire course of the nerve between the entrance into the brain substance of the central roots and the rostral end of the olfactory bulb. The position of a few of these cells is indicated in figure 7.
These cells strongly suggest migratory cells, such as have been pointed out in the ventral roots of the spinal nerves of the turtle embryo by Kuntz ('11 b) and of the pig embryo by Carpenter and Main ('07). These writers conclude that the ceUs which migrate from the cord into the ventral nerve roots have a part in the formation of the sympathetic ganglia, together with migratory cells from the dorsal root ganglia. Gaskell ('16) takes a similar view.
A condition similar to that illustrated in figure 2 was observed in a kitten one day old, and was illustrated in the previous paper (fig. 23) on the nervus terminalis in mammals. No comment was offered at the time on th6 point now under consideration, since the observation had not been repeated in more than one preparation. Such a relationship of cells is present in all of the turtle embryos which are far enough advanced in development to show distinct differentiation of ganglionic cells.
CONCLUSION
The nervus terminalis of the turtle takes part in the formation of a plexus on the nasal septum, comparable to that found in mammals. In the turtle fibers from the trigeminal nerve form a more important share of this plexus than has yet been demonstrated to be the case in mammals.
There is a pronounced resemblance of the cells of the ganglionic clusters of the nervus terminalis to the cells of the various sympathetic ganglia. This resemblance is apparent not only in size, but also in their structure and manner of distribution and to some extent, so far as the developmental evidence goes, in their apparent origin as migratory cells from the central nervous system.
NERVUS TERMINALIS : TURTLE 443
The ganglionic cells in the regions which appear to be occupied solely by fibers of the terminalis, i.e., without intermingling of trigeminal fibers, resemble migratory cells which are found along the ophthalmic branch of the trigeminal nerve so closely that they cannot be told apart. It is probable that the ganglionic clusters of the septal plexus are composed of cells from both sources, as the plexus itself is composed of nerve fibers from the two sources.
LITERATURE CITED
Ayers, Howard 1919 Vertebrate Cephalogenesis IV. Jour. Comp. Neur.,
vol. 30, pp. 323-342. Brookover, Chas. 1917 The peripheral distribution of the nervus terminalis
in an infant. Jour. Comp. Neur., vol. 28, pp. 349-360. Carpenter, F. W. 1906 The development of the oculomotor nerve, the ciliary
ganglion, and the abducent nerve in the chick. Bull. Mus. Comp.
Zool. Harvard Coll., vol. 48, pp. 139-230. Carpenter, F. W., and Main, R. C. 1907 The migration of medullary cells
into the ventral nerve-roots of pig embryos. Anat. Anz., Bd. 31, S.
303-306. DoGiEL, A. S. 1908 Der Bau der Spinalganglien des Menschen und der Sauge thiere. Jena. Gaskell, W. H. 1916 The involuntary nervous system. London and New
York. Huber, G. C, and Guild, Stacy R. 1913 Observations on the peripheral distribution of the nervus terminalis in Mammalia. Anat. Rec, vol. 7,
pp. 253-272. Johnston, J. B. 1913 The nervus terminalis in reptiles and mammals. Jour.
Comp. Neur., vol. 23, pp. 97-120. Kuntz, Albert 1911 b The development of the sympathetic nervous system
in turtles.. Amer. Jour. Anat., vol. 11, pp. 279-312. Larsell, O. 1918 Studies on the nervus terminalis: mammals. Jour, Comp.
Neur., vol. 30, pp. 1-68. McKibben, p. S. 1914 Ganglion cells of the nervus terminalis in the dog-fish
(Mustelus canis). Jour. Comp. Neur., vol. 24, pp. 437-443. Ranson, S. W. 1912 The structure of the spinal ganglia and of the spinal
nerves. Jour. Comp. Neur., vol. 22, pp. 159-175. Read, Effie A. 1908 A contribution to the knowledge of the olfactory apparatus in the dog, cat and man. Am. Jour. Anat., vol. 7, pp. 17-47. Rubaschin, W. 1903 tjber die Beziehimgen des Nervus trigeminus zur Riech schleimhaut. Anat. Anz., Bd. 22, S. 407. Van Wijhe, J. W. 1894 Over de herzenzenewen der Cranioten bij Amphioxus.
K. Akad. van Wetenschappen te Amsterdam. Natuurkundige Afdeel ing, Deel III, pp. 108-115.
1918 On the nervus terminalis from man to Amphioxus. K. Akad.
van Wetenschappen te Amsterdam, vol. 21. Willard, William A. 1915 The cranial nerves of Anolis carolinensis. Bull.
Mus. Comp. Zool. Harvard Coll., vol. 59, no. 2.
Resumen por el autor, C. G. MacArthur.
Universidad de Illinois y Escuela Medica de Stanford.
Cambios qmmicos cuantitativos del cerebro humano durante
el crecimiento.
Durante el crecimiento, las protemas, fosfatidos, sulfatidos, cerebr6sidos, colesterol y s61idos totales aumentan en tanto por ciento. Solamente hay un ligero cambio en el tanto por ciento de los extractivos, mientras que el agua decrece regularmente hasta la edad adulta. La mayor parte de los compuestos cerebrales, con la excepcion de los cerebrosidos y sulfatidos, se descomponen con mayor rapidez en el recien nacido, y cuando se suman diariamente, su cantidad en miligramos es la siguiente: agua 3270, s61idos 494, lipinas 165, fosfatidos 85, colesterol 70, sulfatidos 7.7, cerebr6sidos 1.9, proteinas 186, extractivos organicos 100, extractivos inorganicos 44, azufre 2.3, fosforo 8.5. Probablemente durante el crecimiento la medula espinal contiene la mayor cantidad, en tanto por ciento, de s61idos totales, lipinas totales y de cada lipina, pero la menor cantidad de proteina, extractivos y agua. El cerebro difiere poco de la medula, mientras que el cerebelo difiere mucho. El analisis quimico esta de acuerdo con la existencia de tres estados en el crecimiento del cerebro: 1) Aumento del numero de celulas; 2) su crecimiento, incluso el de los cilindro ejes; 3) la medulaci6n. Aunque la cantidad absoluta de cada uno de los componentes sumados es mayor durante el periodo medio de crecimiento (en el recien nacido), tiene lugar un aumento mayor de crecimiento en el tejido mas j6ven. El crecimiento del cerebro no es necesariamente autocatalitico. El cerebro entero, del mismo modo que cada componente, aumenta con el desarrollo, tal como sucederia a una masa determinada de protoplasma que construye mas material semejante a el en un ambiente que cada vez es mas desfavorable. Parece una necesidad 16gica que ni aiin depende de la vida.
Translation_,by Jos6 F. Nonidez Carnegie Institution of Washington
AUTHOR S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JULY 21
QUANTITATIVE CHEMICAL CHANGES IN THE HUMAN BRAIN DURING GROWTH
C. G. MACARTHUE, and E. A. DOISY
The Biochemical Laboratory of the University of Illinois, and Stanford Medical
School
THREE CHARTS
It seemed desirable to develop a more complete growth series of quantitative determinations of the important constituents in the human brain than has heretofore been published (KochMann, '07- '08). During fetal life and infancy these changes are most interesting, but least studied. The chemical differentiation during growth in young pigs (Koch, '13) and young rats (W. and M. L. Koch, '13) has received attention, but early human life has been curiously neglected.
METHOD OF ANALYSIS
The method of analysis is essentially the same as that used by others in quantitative brain work.^ The outline on page 446 gives the main points.
LIMITATIONS AND ERRORS
It needs to be kept in mind that disease caused the death of the people whose brains were analyzed. Though the brain in no case showed appreciable lesions, yet it is always possible that chemical alterations might have taken place before death.
Only one brain (except in the case of the three-month fetal brains) was analyzed for each of the ages given. Of course there is no guarantee that each was an average brain. Moreover, we have, because of the few analyses, no means of finding out the
^ For detail of method and formulae for calculation of results see Koch, W., J. Am. Chem. Soc, 31, 1329, ff. 1909; Koch, M. L., and Voegtlin, C, Hyg. Lab. Bull. No. 103, p. 67. 1916.
445
446
C. G. MACARTHUR AND E. A. DOISY
Moist brain tissue
Add alcohol and extract alternately with alcohol and ether
EXTRACT^ (fractions 1 AND 2)
RESIDUE (fractions 3 AND 4)
Evaporate to dryness, emulsify with water, precipitate with CHCI3 in 0.5 per cent HCI solution
Dry, weigh, and extract with hot water.
Precipitate (fract.l): (Colloidal)
Filtrate (fract 2): (Crystalloidal)
Filtrate (fract 3) : (Crystalloidal)
Residue (fract. 4): (Colloidal)
Lipoids
Organic constitu
Inorganic constit
Proteins
Phosphatids
ents:
uents :
Nucleoprotein (a)
Cerebrosides
Hypoxanthin
Ammonium
Nucleoprotein (b)
Sulphatids
Tyrosin
Iron
Neurokeratin
(Cholesterol, etc.)
Leucin
Sodium
Urea
Potassium
Inosit
Calcium
Taurin
Magnesium
Peptones
Chlorides
Sarcolactic acid
Inorganic constit
Organic constitu
uents (see fract. 3)
ents (see fract. 2)
Lipoid sulphur
Neutral sulphur
Inorganic sulphur
Protein sulphur
(Sulfates)
Lipoid phosphorus
Organic extractives
Inorganic phos
Protein phosphorus
Phosphorus
phorus (Phosphates)
Lipoid nitrogen
Organic nitrogen
Inorganic nitrogen
Protein nitrogen
2 Substances in italics were determined in this investigation.
average deviation. Not until many such series have been developed shall we be able to state what normal brain growth really is. To a certain extent, a smooth curve averages the data, but this may introduce larger errors than it attempts to average. There is no way of knowing definitely that a curve should be regular as given or made up of a series of waves of different sizes. This also can be determined only by a larger number of investigations.
If the thirty-five-year-old brain is normal, there is a rather wide range of variability. It will be noticed that the total solids in this brain are 5 per cent higher than in the other adult
CHEMICAL CHANGES IN HUMAN BRAIN 447
brains. This cannot be an analytical error, because the same amount was obtained in two analyses made at different times in the series.
The brain marked '8-24 mo.' was labeled '2 years' when sent for analysis. There seems to be no good reason for questioning the accuracy of this information. However, the weight of the brain indicates an infant of a few months. Its water content suggests an infant of about eight months, while some of the phosphatids would favor a slightly greater age, as would the weight of cerebellum and stem. Very likely this brain was two years old, but subnormal. In spite of the uncertainty concerning the brain, it is included in this series because it was the only one of this age available, but it is considered with the greatest reservation in forming general conclusions.
No brains about five and twelve years of age could be obtained. This leaves the series incomplete.
Unfortunately, it was not known until the investigation was nearly finished that the method used for sulphur determination gave low results. This vitiates to a certain extent the reports on the various forms of sulphur, the sulphatids, and because of the methods of calculating the data, the cerebrosides, and the undetermined cholesterol. It was planned to make direct cholesterol estimations, but the series took so much longer than expected that these estimations were omitted.
Analyses 7 and 10 were made together. A combination of circumstances tendered their phosphatid determinations somewhat unreliable. If the solutions to be precipitated by chloroform and hydrochloric acid become too warm and are allowed to stand too long, the phosphatids are incompletely precipitated. This gives not only an error in phosphatids, but, by difference, in 'cholesterol, etc' and in extractives.
Many ways were found of improving the method after beginning this series, but they could not be adopted because of the effect on comparisons of results. One always sees many ways of improving an investigation after it is finished, and that is unusually true of this investigation.
448 C. G. MACARTHUR AND E. A. DOISY
DESCRIPTION OF MATERIAL
Dr. H, Gideon Wells, of the Pathological Department of the University of Chicago, very kindly arranged to help us in this investigation. Without his cooperation, this series would have been very incomplete.
Upon receipt of a brain the meninges and blood were removed from the brain and it was divided into forebrain, cerebellum, and brain stem, and each division weighed. Samples were then taken and placed in enough 95 per cent alcohol to make the concentration 85 per cent alcohol. The specimens were as follows:
Three-month fetus. Male. Two three-month fetuses, referred to as normal, were united in order to furnish material enough for one good analysis. These brains were not divided into forebrain, cerebellum and brain stem.
Seven-month jetus. Female. The mother of this stillborn fetus entered the hospital five days before the dehvery with signs and symptoms of placenta praevia. A brain of this age also give^ too small amounts if separated into divisions, so such separation was not made.
One-month child. Male. This child died of bronchopneumonia. The forebrain was separated from the rest of the brain, the cerebellum and brain stem were analyzed together because there was not enough in either to make a satisfactory separate analysis.
Three-month child. Male. Died of bronchopneumonia and marasmus.
Eight to twenty-jour month child. Male. Though there was no record of this child having been abnormal, the brain was found to be decidedly underweight for the two-year age reported. The child may not have been two years old, but younger. More Hkely it was subnormal.
Twenty-one year adult. Male. Died of pneumonia. The autopsy did not take place for three days after death, but the weather was cool, so the brain was in good state of preservation when received.
Thirty-three year adult, (negro). Male. Died of acute pneumonia. No other disease was present.
Thirty-five year adidt (Hungarian). Male. Cause of death not reported. Brain was very high in solids, but not pathological in any evident way.
Sixty-seven year adult. Male. Died of tuberculosis.
The weights of the different divisions obtained from these brains were as follows :
CHEMICAL CHANGES IN HUMAN BRAIN
449
Whole brain: Weights of divisions in grams
Whole brain. Forebrain. . .
Left
Right
Cerebellum. . Brain stem. .
FETUS
3
MONTHS
FETUS
7
MONTHS
CHILD
1 MONTH
CHILD
3
MONTHS
CHILD'
8
MONTHS
ADULT
21 YEARS
ADULT
33 YEARS
ADULT
35 TEARS
17.08
119.0
457.4
585.2
492.5
1122.4
1221.3
1158.3
395.0
514.0
409.0
950.0
1026.0
986.0
200.0
263.0
206.0
485.0
516.0
510.0
195.0
251.0
203.0
465.0
510.0
476.0
(37.4)4
42.6
48.2
111.4
130.6
110.8
(25.0)4
28.5
35.3
61.0
64.7
61.5
ADULT
65 TEARS
1297.9
1075.0
535.0
540.0
145.4
77.5
^ See section on Limitations for explanation.
Analyzed together because of small amounts of each. DISCUSSION OF RESULTS Water and total solids
The water determinations show that though the absolute amount increases (table 12, fig. 1), the percentage of this constituent decreases continually (table 10) until growth is completed. The relative amount of water present is an indication of the rate of activity. Water, like the inorganic salts and the simpler organic substances (table 10), decreases relatively rather regularly with the approach to adult condition and its decrease in rate of metabohsm.
The percentage of total solids of course varies inversely with that of water. The increase is largely due to the formation of the colloidal substances. They increase in absolute amounts (table 12, fig. 1) and in percentage (table 10), while the simpler molecules, as a rule, decrease in percentage, but increase in absolute quantity. It will be noticed from table 13 and figure 2 that the solids are formed most rapidly soon after birth; at least 0.5 grams a day are then being added. This coincides with the period of most rapid myelination.
It does not necessarily follow that this is the time of greatest protoplasmic activity, because under other conditions the products of the reaction may be removed, while during the period of myelination a part may form the sheath.
It needs to be remembered that the substances produced in myelination are not to be thought of as active protoplasmic
450
C. G. MACARTHUR AND E. A. DOISY
Wtinqms.
80
72
64
56
48
+0
32
i+
16
1 1 1 1 1
Kjrdpn I
^^
r totems ^*^ -^
^XT_ ^'^
^ /
<^ ."^
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. ^ Lipins^ ^
/ 7
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.^ ^"^
^^ ^^
-y^lt ^^
y y x
/ ^
/ ^ x^
. t' Hk , ,r ,/,',/, - — V ^n^
y ^ rnosphatias y^
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-f f- ^ -~] LXcf^ct/ves~
tt ^-*' ^—
t ^^"2"
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Jt^ ^^ ^ it
jfe ^
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24- 32
1/ me In months Fig. 1
40
48
56
compounds in the same way as the extractives, certain lipins, and nucleoproteins. This is true whether one considers the sheath as nutritional or insulating in function. In comparing nerve activity with other tissues, it would probably be more exact, therefore, to use data on the axis cylinders and not the whole nerve (Donaldson, '16).
CHEMICAL CHANGES IN HUMAN BRAIN
451
Milligrams added perday
lay
1 M 1 M 1 1 11 1 1 1 M 1
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40
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12 16 20
T/me in months Fig. 2
24
28
Phosphatids
Lecithin (MacArthur and Darrah, '16), cephalin (MacArthur, '16), sphingomyelin and myeUn are the principal phosphatids found preformed in the brain. Some or all of these compounds are present from very early fetal life (table 1). They gradually increase (table 12) until myelination becomes rapid, then they are formed at the maximum rate of 0.1 gram per day (table 13). They continue to be formed rapidly until two years of age; after
452 C. G. MACARTHUR AND E. A. DOISY
this the rate decreases to adult age. During adult life they probably increase very slowly and are one of the colloidal factors to be considered in retarding metabolism in old age.
We have some reasons for beUeving that lecithin is the phosphatid largely found in the nerve cells (Cowdry, '14). It looks as though it were rather closely associated with the nucleoproteins in carrying on vital activities. Cephalin is probably present in both the cells and axis cylinders, though more largely in the latter. Very little is known about sphingomyelin, but it it is probably largely to be found in the sheaths. It would be very interesting to study the increase in each of these phosphatids during growth.
Lecithin, and especially cephalin, because of their auto-oxidation characteristics, are believed to be closely related to nervetissue oxidation (Signorelli, '10; MacArthur and Jones, '17).
Cerebrosides
Phrenosin (Levine and Jacobs, '12) and kerasin (Rosenheim, '13), the two brain cerebrosides, may be parts of an unstable complex made up of sulphatid, phosphatid, and cerebroside. If this is true, the data in this paper would indicate that with growth this complex increases in complexity (fig. 1), because the cerebrosides as analyzed do not appear until birth (tables 2, 5, and 8), when myelination becomes the dominant brain activity. This may mean that they are in some way dependent on the presence of other constituents for their production. They are peculiar in the rapidity with which they assume such a prominent place in developing nerve tissue.
The cerebrosides are probably more directly related to sheath formation than any other constituent (Smith and Mair, '12- '13). Their maximum rate of formation does not occur as early as in the case of the other brain constituents (at four months instead of at birth) (table 11). Then about 0.025 gram are added each day.
CHEMICAL CHANGES IN HUMAN BRAIN 453
Sulphatids
Because of uncertainty concerning the sulphatids found in the brain, the report for this constituent is open to question. It is assumed, however, that it is a cerebroside and phosphatid fastened together by a sulphate radicle (Koch, '10). The sulphatids are very closely related to the cerebrosides in physiological function and anatomical distribution. The sulphatids seem to be more fundamentally necessary because they are found earlier (table 10). They may be related to conductivity in axis cylinders. Small amounts are present in very early fetal life (tables 1, 4, and 6). Soon after birth the amounts formed are greatest (table 12), but there is no time during life when this compound is not being produced. Probably it is concerned in the rivalry between structure and function, helping the former to victory in stability of activity, and finally in death. This is one of those substances so necessary for highly specialized brain work, but so detrimental to continued growth.
Proteins
The most important protein of the brain, because of its greater lability, is probably nucleoprotein a (McGregor, '17). One would expect it to be associated with the vital functions. It probably is a combination of the globulin a, globulin b, and the nucleoprotein of an earlier worker (Halliburton, '94). Nucleoprotein b is mUch more stable and may be the protein of the chromatin and Nissl bodies, thus related to the hereditary quality of the nerve cell. Neurokeratin is stable and probably is connected with the structure of the nerve sheaths. It is highly important to know how these different proteins increase with growth, but we have only indirect evidence of what these changes are. From the data on total protein, protein phosphorus and protein sulphur (table 11), we can get an idea, of what is happening, however, Thus indirectly we can suggest that neurokeratin approaches a maximum percentage at two years of age, but is present in very small amounts in even early fetal life. Nucleoprotein b is probably present in largest percentage amounts in
454 C. G. MACARTHUR AND E. A. DOISY
early fetal life, but continues to increase in absolute amounts (table 12) until maturity, when there is about twice as much as of nucleoprotein a and half as much as of neurokeratin (McGregor, '17). Nucleoprotein a possibly is always present, but probably is largest in percentage amounts when nerve growth and activity are greatest. It would not do even to guess how these last two proteins are distributed in the brain.
The total protein curve (fig. 1) indicates that some particular protein (possibly nucleoprotein b) is an especially important factor in the subsequent growth of the brain. It seems to lead in the increases that take place.
Extractives
The separation of extractives into organic and inorganic, as given in the data, is of but little value because of the fact that such a separation, based on the solubility of organic constituents in alcohol and the insolubility of the inorganic ones in alcohol (or on the residue after ignition), is very unreliable. The data given are merely suggestive. Howev-er, the determination of total extractives is rather accurate. Inosit, urea, leucin, tyrosin, taurin, hjTDOxanthin, and peptones are a few of the organic substances present in this fraction. In general it may be stated that the larger the percentage of these simpler crystalloidal molecules, the more rapid the metabolism and the younger the tissue. Various inorganic salts of sodium, potassium, ammonia, calcium, magnesia, and iron have about the same significance. While the rate of growth is high, these constituents are present in larger percentage amounts (table 11), but with a decrease in rate of development they rapidly decrease in rate of formation, until after two years of age they are but very slowly increased in absolute amount (table 12).
They are present in larger amounts in cells than in axis cylinders. Potassium salts and chlorides are supposed to be related to nerve conductivity (Alcock and Lynch, '11).
In drawing conclusions concerning the rate of activity of nerve tissue from the percentage amounts of extractives, one needs to
CHEMICAL CHANGES IN HUMAN BRAIN 455
remember that it may be more accurate to leave the sheath substances out of the reckoning. The calculations would then be based on the assumption that but three-fifths of total solids are directly concerned.
Sulphur compounds
By estimating sulphur in the various fractions we obtain information about the relative amounts of several important brain compounds. The lipin sulphur is a measure of the amount of sulphatid, and is therefore largely concerned in sheath development. In consequence it obtains a maximum rate of formation at about three months of age. Protein sulphur represents the amount of cystin in protein combination. Cystin is present in much smaller amounts in the nucleoproteins of the brain than in neurokeratin. So protein sulphur gives us a rough estimate of the amounts of neurokeratin being formed. It will be noticed that this form of sulphur follows very closely myelin formation (table 10). Neutral sulphur may represent an intermediate oxidation product of cystin or possibly taurin; an increase in this form might indicate a decreased oxidative ability in the cells (W. and M. L. Koch, '13). Of the total sulphur, neutral sulphur forms a greater portion in the younger tissue, while the portion of protein sulphur increases with age. The inorganic sulphates are the final sulphur oxidation products. They remain rather constant in percentage amounts (table 11). This may be due to the fact that, they are readily eliminated from the cell. Total sulphur increases in percentage of fresh tissue until adult age, then remains rather constant. The maximum addition, of about 3 mg. per day, takes place at about three months of age (table 13).
Phosphorus compounds
The amount of phosphorus in the lipins is used to determine the amount of the phospho-lipins. It is added most rapidly at birth (table 13, fig. 2) (at least 3.5 mg. per day), but like most of the other constituents, its rate of addition per unit of reaction substances is greatest in the youngest tissue (tables 14 and 15, fig. 3).
THE JOURNAL OF COMPARATIVE NECROLOGT, VOL. 30, NO. 5
456
C. G. MACARTHUR AND E. A. DOISY
Percentage Increese Per Day
24
?2
20
1.3
1^
I.+
U
t.O
"\ ' "1 ■" 1 1 T r
\ KjrapnJ
V \t ilnh^i'irtn
\
k r
vV .- \. - pL L^f.:J^
vv \ lospnduQS
\\\ p- ,
Y^ Y Lxtractives ""
1 \V \ 1 1 1 1
"t t| "T rroteins
\ w'
I V\\
\ \\\
\ \i\
i\i\
WW
Ml L
— &L\
niT LXudcwes ic)
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w\\\ \
im '
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111 1
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ill N
jtC t — • 1
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'Si **'^5-. Jp"-* — ^
_ ^^5*=-|,.^^^^::^^^-=== L
VL 16 20 24
Time inrnonths Fig. 3
28
32
36
Protein phosphorus represents the amount of nucleoprotein. This probably closely approximates the changes in the activity of the protoplasm, both nucleus and cytoplasm. This form increases in amount with age, but the percentage changes less than any other kind.
CHEMICAL CHANGES IN HUMAN BRAIN 457
Under the heading of organic phosphorus is included a number of comparatively simple compounds of phosphorus with organic radicles. There is no definite separation of this group from the following one (Emmett and Grindley, '06). It is representative of the amount of protein metabolism and bears a definite relation to the two colloidal forms of phosphorus mentioned above.
Inorganic phosphates are also a measure of rate of activity. In fact, the sum of these last two forms the best criterion of rate of phosphorus metabolism. It is worth noticing that in terms of percentage of total phosphorus the amounts of colloidal phosphorus compounds are increasing with growth, while those of the crystalloidal forms are decreasing (table 11). In fresh tissue the percentage of extractive phosphorus increases slightly, then decreases to a certain extent; but these small variations from a constant may not be significant. The figures, however, indicate rather definitely that these simpler forms of phosphorus do not closely follow the change in percentage of water, as one would expect if the quantity of fluid determined the amount of extractives. From table 11 it is evident that extractive phosphorus, like other extractives, markedly decreases in the percentage of total solids.
Comparison of forebrain, cerebellum, and brain stem
In the adult brain the cerebellum contains the largest percentage of water, the forebrain slightly less, the brain stem least. A high percentage of solids indicates slower but more highly dfferentiated metabolism; therefore the cerebellum acts fastest, while the brain stem is most stereotyped. During growth the rate of increase in the percentage of solids is in the following order, brain stem (table 4), forebrain (table 1), and cerebellum (table 7) ; this is, of course, to be expected.
In the stem the phosphatids are in larger amounts (table 5) and are probably laid down earlier than in the other two divisions of the brain. The cerebellum contains the smallest amount of this group of constituents (table 8) , indicating that it is probably less highly specialized than other parts.
458 C. G. MACARTHUR AND E. A. DOISY
The cerebrosides and sulphatids are present in slightly larger amounts in the stem (table 5) than in the forebrain (table 2), but in very much larger quantities than in the cerebellum (table 8). This would indicate that one of the main differences in chemical constitution between the cerebellum and the rest of the brain is in the amount of meduUation.
Cholesterol, etc., is found most largely in the stem and least in the cerebellum.
The total lipins are not only largest in amount in the stem (table 5) and least in the cerebellum (table 8), but possibly are formed slightly earlier and at a more rapid rate in the same order.
In all divisions the proteins show in general variations exactly opposite to that of the lipins. Thus with age there is a decrease in percentage of solids (tables 2, 5, and 8). The total proteins exist in but slightly different percentages in the different parts of the fresh tissue (tables 1, 4, and 7), and they seem to be formed at approximately the same rate in all.
In an attempt further to analyze the meaning of these variations in protein content, the data frpm protein sulphur and protein phosphorus are of value. It is probable that the greater amount of protein -sulphur is in neurokeratin, a constituent of supporting tissue, while the protein phosphorus is very largely present as nucleoprotein b (0.6 per cent P), only a relatively small amount being present as nucleoprotein a (0.11 percent P). (The percentage amount of the former, 10 per cent, is about twice that of the latter, 5 per cent in adult tissue.) From tables 1, 4, skid 7, then, it will be noticed that there is more than twice as much nucleoprotein in the cerebellum as in other parts of the brain. This difference prevails throughout growth. The stem and forebrain differ but little in this respect. On the other hand, protein sulphur (neurokeratin) is a little greater in the brain stem than in the forebrain or cerebellum. To judge from these data, one might state that the number of nuclei, or at least the amount of nuclear material, is considerably larger in the cerebellum, while the amount of supporting tissue is not very different in the
CHEMICAL CHANGES IN HUMAN BRAIN 459
various parts of the brain. Concerning the extra nuclear proteins (nucleoprotein a) it is difficult to make more than a guess — that they would vary with the other functioning protein (nucleoprotein h).
Neutral sulphur is a rough measure of the amount of protein metabolism taking place. It is rather striking that the greatest rate of protein metabohsm is in the forebrain (table 2) and cerebellum (table 8) and least in the brain stem (table 5). The rate in all remains rather constant in spite of the fact that the amount of protein increases with age.
Throughout growth the total extractives are present in much larger amounts in the cerebellum (table 8) than in the rest of the brain. The stem has a slightly larger percentage than the forebrain. In all divisions the maximum addition per day is reached at about three months of age (tables 2, 5, and 8, fig. 2). After this there is a slow decrease in amount of daily additions till old age, indicating that aging is a regular decrease in rate of metabolism. The larger part of the extractives in the cerebellum is composed of inorganic constituents. This is probably to be expected from the larger protein content in this division of the brain.
The total sulphur increases more rapidly and attains a somewhat greater percentage in the brain stem than in the forebrain (table 1) and a considerably greater percentage than in the cerebellum (table 7). This seems to be largely due to the relatively larger amounts of lipins in each division in the order named. The inorganic sulphates gradually increase in each division at about the same rate.
The amount of total phosphorus does not differ much in different parts of the brain, but the lipin phosphorus is greater in the stem (table 5) and forebrain (table 2) than in the cerebellum (table 8). The inorganic phosphates seem to be closely related to nucleoprotein h, because they are represented to a much greater extent in the cerebellum than in the other divisions of the brain.
460 C. G. MACARTHUR AND E. A. DOISY
GENERAL DISCUSSION 1 . Dominance of the nervous system
There seem to be no facts presented in this paper that are inconsistent with the theory that the chromosomes are very important in the early differentiation of cells. In fact, the data suggest that nucleoproteins (tables 10 and 11, fig. 1) then phosphatids and simple extractive molecules dominate in the youngest tissue. It is probable that there is a metabolic gradient in the fertilized cell that is important in determining which will be the head end of the organism (Child, '12). Very early in growth nervous tissue differentiates in this head region. Because of this early formation, much of the later development in other parts of the body is rather dependent on the nervous system. It needs to be emphasized that this dominance of one substance over another (or one organ over another) is but relative. Most of them are developing together, but their influence on each other is very different. Very probably growth consists in the formation of continually larger quantities of the respective cell constituents in a more or less definite order, commencing with the nucleoproteins (table 11, fig. 2) of the chromosomes. In the brain there is rapid formation of certain substances, then the presence and formation of the substances influence the rate of formation of another substance, this another, and so on till all have come into adjustment with the new conditions. While these changes are occurring in the brain, and partly because of them, other areas of differentiation are split off, which are to become other organs. Then in these areas a similar cycle of changes will occur. Probably each of the organs has periods of maximum growth. When such unusually large amounts of material are being formed rapidly, in comparison with the rate of formation at other periods of growth, we get an irregularity in the main curve of growth that produces a so-called growth cycle.
During growth substances are regularly coming to the various organs through the blood. We do not now believe that the amount of either these food substances or the oxygen determines the rate of growth, though they do influence it. The
CHEMICAL CHANGES IN HUMAN BRAIN 461
cells in any organ seem to grow somewhat in unison, but they are influenced by cells of other organs, both through the blood and through the nerves. There is no doubt that each organ directly influences the growth of every other in both of these ways. Certain glands secrete substances and discharge them into the circulation that have a disproportionately large effect in influencing growth in most tissues. But it is not correct to assume that growth is determined by these; it is only altered by them. Growth seems to be a general cell process, and these substances simply change the rate of this general cell development.
The growth of the brain is probably less under the influence of internal secretions than other organs. Indeed, there is strong evidence that the secretions are largely under the influence of the nervous system. There is no indication of internal secretions in simple organisms, yet these organisms have a similar form of growth.
2. Relation of these data to four physiological facts
In any interpretation of brain growth it is necessary to keep in mind several important facts:
1. A larger amount of early differentiation occurs in nervous tissue than in other tissues. Though this fact is associated with some definite differences that exist in the fertilized cell, the subsequent chemical supremacy is important in evolving the marked specialization. The early formation of colloidal substances such as the nucleoproteins, phosphatids, and sulphatids (table 10) give a peculiarity to young nerve tissue that permits it to influence rather markedly the development of other tissues. It is more than theoretical to assume that the early start of nervous tissue allows it to differentiate more than other parts of the body.
2. The nerve cells, unlike other cells, do not regenerate. It is very probable that a nerve cell if tested for regenerative power early enough in its growth would regenerate just as other undifferentiated tissues do. It very soon reaches a stage, however, w^hen it is so highly specialized by the elaboration of colloidal complexes (table 11) that regeneration is impossible.
462 C. G. MACARTHUR AND E. A. DOISY
3. The number of nerve cells remains constant. Rather early in growth, probably as early as the seventh fetal month (table 10) the number of nerve cells is largely determined. No amount of functioning produces an increase. This would indicate that the chemical changes in brain growth are fixed within rather close limits. It also suggests that development in the brain is essentially different than elsewhere. Probably the main processes are determined at the time of the formation of the cells. The organization is such, however, that smaller but no less important (speaking physiologically) change occurs during later activity.
4. Nervous tissues remain constant in composition under conditions that markedly alter many other tissues. The chemical composition of the nervous system must be related to this supremacy. The large amounts of several of the lipins (table 10) seem to be of importance. Though the large amount of colloidal material in the form of lipins and proteins is often supposed to be indicative of slower metabolism and a lack of dominance (Child, '11), the chemical condition in the brain would suggest that colloidal structure is equally important wdth the rate of metabolism in maintaining dominance. It te conceivable that in the case of the brain its early importance, due to the high rate of metabolism, should be maintained through specialized activity, even when this rate is no longer greater than the rate in other tissues. The highly specialized nerve fiber and cell are made of many compounds that are but slightly available to other tissues, because such substances are in almost irreversible equilibrium with metabolizing substances elsewhere.
Another factor that is undoubtedly involved is the selective nature of the membranes surrounding the cells of the nervous system. Very probably such membranes or surfaces are much more common than is supposed, thus providing means of keeping the various tissues in equilibrium with each other. If the membranes in the nervous system are more nearly irreversible than in other parts, the condition exists that is favorable for maintenance under circumstances that use up other tissues.
No other tissue has a chance to supplant the nervous system with its highly specialized pathways to all parts of the body.
CHEMICAL CHANGES IN HUMAN BRAIN 463
It does not need to depend on its rate of activity for supremacy; the conditions it has developed for its maintenance assure this dominance though the means used for obtaining it no longer exist.
3. Concerning three periods of growth
There are three distinct processes to be distinguished in brain development. The one that takes place first is cell division. This is probably almost completed at the time of the sevenmonth-old fetus. It is worth noting that there is no evidence of sheath development up to this point. There are no cerebrosides; the amounts of sulphatids are increasing slowly. The phosphatids do not show any dominance. The relatively large amounts of protein, and especially the nucleoproteins, suggest that chromosome formation is very prominent. The large quantity of extractives emphasizes the fact that metabolism is very rapid during this period.
From the seven-month fetus to about the time of birth, cell growth is the important process. At this time the phosphatids come into prominence, while the proteins and extractives retain their earlier dominance. Cholesterol, though present, is not important. The same is true of sulphatids, while the cerebrosides are lacking entirely or are present in but small amounts. These changes arfe what one would expect in growing cells and enlarging axis cylinders. How important the axis cylinders are in accounting for brain growth is indicated by the fact that about two-fifths of the brain consists of them.
The third period is that of meduUation. It becomes prominent soon after birth, reaches its maximum a few months after birth, and slowly decreases in importance. The sheaths comprise about two-fifths of the brain substance, so it is not surprising that cerebrosides, sulphatids, and some of the phosphatids become so prominent. The proteins and extractives are skill of importance, but thoroughly masked by the new process. It is probable that when the nerve cells reach the stage at which conditions are proper for sheath formation, there is a release of energy or an alteration in metabolism through the extension of
464 C. G. MACARTHUR AND E. A. DOISY
the field of local dominance that is large enough to amount to a slowing down temporarily of the rate of loss of growth power.
By comparing these results with those obtained in a growth series on the brain of the albino rat (W. and M, L. Koch, '13) a great similarity is evident. The nature of the process, the division into periods, the relative amounts of the various constituents, and their order of development are much alike. The main differences are found in the larger percentage amounts of lipins, with a corresponding decrease in proteins and a great lengthening of the periods of growth. Thus the changes occurring in the rat brain are much more rapid than in human brain, but the rat brain does not attain quite the same degree of differentiation. By comparing the data in the two series for extractives as a whole, and the various extractives, no marked differences are evident, indicating that the changes in rate of metabolism with growth are similar in both, though the time necessary to change from one corresponding physiological age to another in the rat is probably but about one-thirtieth of that in the human.
4. Nature of the' growth process
One of the most interesting points which these data raise is that of the nature of the growth process in nervous tissue. The curves show that the brain as a whole, as well as each of the individual substances or groups of substances (tables 12 and 13, figs. 1 and 2) estimated, increases slowly in absolute amounts per unit of time during the first part of development. Later the increase is larger, and is then followed by a period when the amounts are continually smaller. If, however, one observes the curve for the amount added per unit mass of substance during a given period of time (tables 14 and 15, fig. 3) it is seen that the rate of addition is greatest in the youngest tissue. This rate of addition diminishes most at first, then more slowly, and is followed by a somewhat greater comparative rate of loss of growth power. The first curves (absolute amounts) are smilar to those reported for growth of the whole organism (Robertson, '08). Such curves are by some authors supposed to indicate that the process they
CHEMICAL CHANGES IN HUMAN BKAIN 465
represent is an autocatalytic one (a chemical reaction that increases in speed at first because of the catalj^zing effect of a product of the reaction, and then slows down, because of the retarding effect of larger amounts of a product of the reaction and decrease in the original substance). Can the second curve, however, be reconciled with this theory? From this curve it would seem that the rate of reaction is fastest at first and slows down continuously during growth (Meyer, '14).
There is no inconsistency between these two facts (1st, that the absolute amount of substance added is greatest during the middle period of development; 2d, that the amount of substance added per unit mass is greatest at an early period of development) if we make certain assumptions. It is necessary to assume that all or nearly all of the substance (or group of substances, or total brain, or total organism) is a product of this reaction or determined by some other reaction. The substances .weighed are entirely (within limits of error in data) the product of something not weighed or too small to make a significant difference in the weighing. This means that the cytoplasm, and probably nucleus (Loeb, '06), is a product of something else either present and very small, or absent, or not weighable. The easiest interpretation of this difficulty is to invoke the aid of vitalism. This would furnish our unweighable element that determines the growth of even the protoplasm. However, if something a little more substantial is required, one can assume the presence in the brain (or in some other part of the body connected physiologically with the brain) of a very small amount of a substance that in some way determines the formation of all other substances in the tissue (or organism) considered. This substance probably would decrease during growth. One or more of its products would catalyze its effect on formation of other substances. It might exert its control over other reactions by operating over a longer period of time or by having an unusual nature. It is conceivable that a hormone or enzyme-like compound might have such unlimited power. This would assume that at fertilization, or soon after, this substance was made, and that subsequent development is essentially a product of it. Aging would mean the using
466 C. G. MACARTHUR AND E. A. DOISY
up of this substance or an interference with its rate of reaction. Any variations in growth would be due to alterations in the general growth produced by other substances or conditions.
Though the hormones and the active principles in the internal secretions are very popular these days, it seems rather too much to expect that one and only one of them possesses such vitalistic properties. It seems more rational to suppose that they are active in bringing about alterations in growth, but that the main process is independent of them. There is practically no evidence that such substances are determiners of growth in unicellular organisms. If one accepts the autocatalytic theory, it seems necessary to give up the protoplasmic theory, for protoplasm, too, should be simply a product and does not possess growing power. As a result of this and other work, it can be stated with considerable certainty that neither nucleus nor cytoplasm causes growth to take place autocatalyticaUy, If one believes that the evidence for the living, growing nature of the protoplasm as a whole is well founded, chemical autocatalysis should be discarded. The data agree so well with the theory, however, that there must be some reason why a substance in a living organism, as well as the whole tissue or organism should add largest absolute amounts of substance (table 13, fig. 2) during the middle of the growth period. It is worthy of mention, though it is probably not a fundamental explanation to say that protoplasm has an inherent power, when unimpeded by the lack of food or too much of the products of its activity, to increase in a geometrical ratio. As is well known, bacteria and unicellular organisms increase in number and in absolute weight (when retarding factors are small) in this 1, 2, 4, 8, 16 ratio. If such numbers are plotted against tune, the first part of the S-shaped curve is obtained. The latter half of the curve is produced through decreasing the geometrical ratio by the retarding effect of lack of food or production of toxic products. By analogy, such a curve should be produced in a multicellular organism, through the division and development of the cells producing it. It is thus seen that the essential characteristics of autocatalysis are the necessary result of cell division in an imperfect environment. Of course, one of the reasons why cells do
CHEMICAL CHANGES IN HUMAN BRAIN 467
not divide so often when there are more of them in a more unfavorable medium is that the individual cells do not grow to the dividing stage so quickly. However, one can apply the geometrical ratio idea to the development of the individual cell if that is found to increase in absolute weight fastest during the middle period of growth. In fact, it is rather to be expected that such would be the form of its growth, without its being in any way related to autocatalysis. For if the protoplasm formed on cell division is thought of as a unit of protoplasm, it would form two units in a certain period of time; then these two would form four, and so on through the geometrical series, if no retarding factors were present. But there are undoubtedly such factors, so we get essentially the autocatalytic phenomenon. The size of the cell, the relation of size of the nucleus to that of the cytoplasm, the amount of cell differentiation, complexity of colloidal substratum of cell are large factors in determining this form of growth. There seems to be a physiological state that is rather definite for any kind of cell, which, unaltered, tends to make the cells increase. One sees the necessity, granting the power of protoplasm to produce more material like itself, in an increasingly unfavorable environment, for the S-shaped curve of growth. This is independent of the question why growth takes place; it is true, irrespective of the nature of the growth impulse. It is probably not wise, however, to speak of such growth as autocatalytic, because it probably does not have a chemical autocatalytic basis. Though enzymes seem to play a part in it, it is not necessarily enzymic at all, much less autocatalytic and monomolecular. Probably anything that can increase geometrically, put under progressively less favorable conditions, whether living or not (say, the growth of a crystal in a slightly supersaturated solution), would give an autocatalytic form of increase.
SUMMARY
1. During growth the proteins, phosphatids, sulphatids, cerebrosides, cholesterol, and total solids increase in percentage amounts. There is but slight change in the percentage of ex
468 C. G. MACARTHUR AND E. A. DOISY
tractives, either organic or inorganic. Water decreases regularly to maturity.
2. In percentage of solids each of the lipins increases rapidly until a few months after birth, then more slowly until maturity. (Cerebrosides are not present in free condition till about the time of birth.) The proteins slowly decrease in percentage of solids with growth, but the extractives, both organic and inorganic, very rapidly decrease.
3. At birth most of the brain compounds are being laid down most rapidly. Cerebrosides and sulphatids, however, have the greatest daily additions about three months after birth.
The following amounts, in milligrams, are added per day in a new-born child: water 3270, solids 494, lipins 165, phosphatide 85, cholesterol + 70, sulphatids 7.7, cerebrosides 1.9, proteins 186, organic extractives 100, inorganic extractives 44, sulphur 2.3, phosphorus 8.5.
4. The brain stem contains the largest percentage amounts of total solids, total lipins, and of each lipin, but the least protein, organic extractives, inorganic extractives, and water. The forebrain is not much different frQm the stem. The cerebellum, however, varies largely. In development, the brain stem differentiates chemically first and fastest. The forebrain follows closely. The cerebellum never attains to such a high degree of specialization.
The data may indicate that the cerebellum is not only the slowest and least meduUated, but that it remains the youngest division of the brain with the highest rate of metabolism.
5. It is suggested that, because of the early marked chemical differentiation of the brain in the head end of the organism, further development is greatly influenced by the central nervous system.
6. An attempt is made to correlate the data obtained with the early differentiation of specialized nerve tissue and its constancy in number of cells and composition.
. 7. The chemical analyses agree that brain growth consists of 1) increase in the number of cells; 2) their growth, including that of the axis cylinders, and 3) medullation.
CHEMICAL CHANGES IN HUMAN BRAIN 469
8. The data show that, though the absolute amount of each of the constituents added is greatest during a middle period of growth (birth), the greatest rate of growth is in the youngest tissue. It is not believed that brain growth is necessarily autocatalytic. The whole brain, as well as each constituent, increases with development, as is to be expected if it is assumed that a given mass of protoplasm makes more material like itself in an increasingly less favorable environment. It seems to be a logical necessity, not even dependent upon life.
LITERATURE CITED
Alcock, N. H., and Lynch, G. Roche. 1911 On the relation between the physical, chemical, and electrical properties of the nerves. III. Total ash, sulfates, phosphates. J. Physiol., vol. 39, p. 402.
Child, C. M. 1911 A study of senescence and rejuvenescence based on experiments with Planaria dorotocephala. Arch. Entw. Mech. Org., vol. 31, p. 571.
1912 Studies on the dynamics of morphogenesis and inheritance in experimental reproduction. IV. Certain dynamic factors in the regulatory morphogenesis of Planaria dorotocephala in relation to the axial gradient. Jour. Exp. Zool., vol. 13, p. 103.
CowDRY, E. V. 1914 The comparative distribution of mitochondria in spinal ganglia cells of vertebrates. Am. Jour. Anat., vol. 17, p. 1.
Donaldson, H. H. 1916 A preliminary determination of the part played by myelin in reducing the water content of the mammalian nervous system (albino rat). Jour. Comp. Neur., vol. 26, p. 443.
Emmett, M. D., and Grindley, H. S. 1906 The chemistry of flesh (third paper). A study of the phosphorus content of flesh. J. Am. Chem. Soc, vol. 28, p. 25.
Halliburton, W. D. 1894 The proteids of nervous tissues. J. Physiol., vol. 15, p. 90.
Koch, M. L. 1913 Contributions to the chemical differentiation of the central nervous system. I. A comparison of the brain of the albino rat at birth with that of the fetal pig. J. Biol. Chem., vol. 14, p. 267.
Koch, W. 1910 Zur Kenntnis der Schwefelverbindungen des Nerven Systems. II. Uber ein Sulfatid aus nerven Substance. Z. Physiol. Chem., vol. 70, p. 94.
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. Biol. Chem., vol. 15, p. 423.
Koch, W., and Mann, S. A. 1907-08 A comparison of the chemical composition of three human brains at different ages. Am. J. Physiol., vol. 36, p. xxxvi.
470 C. G. MACARTHUR AND E. A. DOISY
Levene, p. a., and Jacobs, W. A. 1912 On sphingosine. J. Biol. Chem., vol.
11, p. 548. LoEB, J. 1906 Weitere Beobachtungen iiber den Einfluss der Befruchtung und
der Zahl der Zelkerne auf die Saurebildung im Ei. Biochem. Z., vol.
2, p. 34. MAcARTHtfR, C. G- 1914 Brain cephalin: I. Distribution of the nitrogenous
.hydrolysis products of cephalin. J. Am. Chem. Soc, vol. 36, p. 2397. MacArthur, C. G., and Darrah, J. E. 1916 Nitrogenous constituents of
brain lecithin. J. Am. Chem. Soc, vol. 38, p. 922. MacArthur, C. G., and Jones, O. C. 1917 Some factors influencing the
respiration of ground nervous tissue. J. Biol. Chem., vol. 32, p. 259. McGregor, H. H. 1917 Proteins of the central nervous system. J. Biol.
Chem., vol. 28, p. 403. Meyer, A. W. 1914 Curves of prenatal growth and autocatalysis. Arch.
Entw. Mech. Org., vol. 40, p. 497. Robertson, T. B. 1908 On the normal rate of growth of an individual and
its biochemical significance. Arch. Entw. Mech. Org., vol. 25, 581. Rosenheim, O. 1913 The galactosides of the brain. I. Biochem. J., vol. 7,
p. 604. SiGNORELLi, E. 1910 Tiber die Oxydation-processe der Lipoide des Riicken marks. Biochem. Z., vol. 29, p. 25. Smith, J. Lorrain, and Mair, W. 1912-13 The development of lipoids in the
brain of the puppy. J. Path. Bact., vol. 17, p. 123. The lipoids of
the white and gray matter of the human brain at different ages. J.
Path. Bact., vol. 17, p. 418.
TABLE 1
Forebrain: Constituents in -percentage of fresh tissue
FETUS
3
MONTHS
(13)1
FETUS
7
MONTHS
(12) =
CHILD
1
MONTH
(11)
CHILD
3
MONTHS
(V)
CHILD
8
MONTHS
(4)3
ADULT
21 YEARS
(23)
ADULT
(33)
YEARS
(28)
ADULT
35 YEARS
(3)
ADULT
67 YEARS
(22)
Water
91.91 8.09
1.04
0.16 0.58
90.56 9.44
1.24
0.27 0.97
88.09 11.91
1.94
0.25 1.53
87.03 12.97
(1.74)4 .30 .50 1.70
85.81 14.19
3.17 0.49 0.50 0.91
77.32
22.68
5.68 1.29 1.84 3.63
77.06 22.94
6.00
1.28 0.66
4.81
72.85 27.15
6.86 2.58 1.72 4.08
78.47
Solids
21.53
Phosphatids
Cerebrosides
Sulphatids
Cholesterol
6.54 1.72 1.35 2.55
Total lipins
1.78
2.48
3.71
4.24
5.06
12.44
12.75
15.23
12.15
Total proteins
Organic extractives
Inorganic extractives
3.77
1.54 1.00
3.98
1.77 1.21
4.57
2.44 1.19
5.29
2.38 1.06
6.09
2.01 1.03
8.03
1.19 1.02
8.11
1.11 0.96
8.99
2.03 0.91
7.53
0.88 0.96
Total extractives. .
2.54
2.98
3.63
3.44
3.04
2.21
2.07
2.94
1.84
Lipin suiphur... . Protein sulphur. Neutral sulphur. Inorganic sulphur
0.003 0.026 0.015
0.001
0.005 0.028 0.018
0.002
0.005 0.033 0.022
0.004
0,010 0.038 0.026
0.005
0.010 0.069 0.018
0.012
0.036 0.053 0.013
0.002
0.013 0.052 0.007
0.003
0.034 0.039 0.022
0.009
0.027 0.061 0.015
0.003
Total sulphur
0.045
0.053
0.064
0.079
0.109
0.104
0.075
0.104
0.106
Lipin phosphorus
Protein phosphorus ,
Organic phosphorus
Inorganic phosphorus
0.044 0.025 0.026 0.056
0.054 0.009 0.028 0.062
0.080 0.005 0.036 0.082
0.078 0.006 0.055 0.074
0.127 0.008 0.032 0.055
0.256 0.013 0.012 0.058
0.284 0.011 0.027 0.091
0.300 ,0.014 0.049 0.048
0.254 0.012 0.008 0.053
Total phosphorus..
0.151
0.153
0.203
0.213
0.222
0.339
0.363
0.411
0.327
1 The two brains of this age that were used for this analysis were not separated into cerebellum, forebrain, and stem, because the brains were too small to make good samples of these divisions. For purposes of comparison the whole brain was arbitrarily divided into cerebellum 10 per cent, brain stem 10 per cent, and forebrain 80 per cent.
2 The same is true of this brain.
3 See section on limitations.
4 See section on limitations for explanation of this low figure.
471
THE JOURN.VL OF COMPARATIVE NEUROLOGY, VOL. .30, NO. 5
472
C. G. MACARTHUR AND E. A, DOISY
TABLE 2 Forebrain: Constituents in percentage of solids
FETDS
FETUS
CHILD
CHILD
CHILD
ADULT
ADULT
ADULT 35 YEARS
ADULT
3
7
1
3
8
21
33
67
MONTHS
MONTHS
MONTH
MONTHS
MONTHS
TEARS
YEARS
YE.\.RS
(13)1
(12)1
(11)
(V)
(4)1
(23)
(28)
(3)
(19)
(22)
Phosphatids. .
12.90
13.12
16.27
(13.40)1
22.33
25.06
24.67
25.26
25.34
27.19
Cerebrosides..
2.32
3.43
5.67
5.59
9.50
7.13
8.00
Sulphatids ....
1.95
2.85
2.06
3.87
3.53
8.12
2.89
6.32
6.90
6.26
Cholesterol. . .
7.24
10.28
12.86
13.09
6.39
16.02
22.45
15.01
18.47
15.03
Total lipins
22.09
26.25
31.19
32.68
35.68
54.87
55.60
56.09
57.84
56.48
Total proteins . .
46.56
42.15
38.31
40.78
42.91
35.40
35.36
33.10
32.27
34.97
Organic ex
tractives
19.04
18.75
20.52
18.35
14.18
5.23
4.85
7.46
6.12
4.08
Inorganic ex
tractives —
12.31
12.85
9.98
8.19
7.23
4.50
4.19
3.35
3.77
4.47
Total extrac
•
tives
31.35
31.60
30.50
26.54
21.41
9.73
9.04
10.81
9.89
8.55
Lipin sulphur.
0.039
0.057
0.041
0.077
0.071
0.163
0.058
0.127
0.138
0.125
Protein sul
phur
0.314
0.294
0.277
0.^7
0.483
0.235
0.225
0.146
0.279
0.279
Neutral sul
phur
0.187
0.183
0.190
0.195
0.128
0.057
0.029
0.081
0.022
0.070
Inorganic
sulphur
0.013
0.022
0.038
0.037
0.085
0.011
0.015
0.032
0.029
0.015
Total sulphur...
0.553
0.556
0.546
0.596
0.767
0.466
0.327
0.386
0.468
0.489
Lipin phos
phorus
0.538
0.563
0.676
0.597
0.886
1.136
1.017
1.110
1.120
1.179
Protein phos
phorus
0.306
0.100
0.041
0.045
0.056
0.055
0.048
0.052
0.063
0.057
Organic phos
phorus
0.322
0.300
0.306
0.422
0.225
0.053
0.117
0.180
0.226
0.035
Inorganic
phosphorus .
0.678
0.648
0.693
0.565
0.384
0.258
0.394
0.172
0.189
0.244
Total phos
phorus
1.844
1.611
1.716
1.629
1.551
1.502
1.576
1.515
1.598
1.515
iSee table 1.
TABLE 3
Forebrain: Weights of constituents in grams
FETUS
FETUS
CHILD
CHILD
CHILD
ADULT
ADULT
ADULT
ADULT
3
7
1
3
8
21
33
35
67
MONTHS
MONTHS
MONTH
MONTHS
MONTHS
TEARS
YEARS
YEARS
YEARS
(13)1
(12)1
(11)
(7)
(4)1
(23)
(28)
(3)
(22)
Brain
17.08
119.0
457.4
585.2
492.5
1122.4
1221.3
1158.3
1297.9
Forebrain..
13.664
95.2
395.0
514.0
409.0
950.0
1026.0
986.0
1075.0
Water
12.56
86.16
347.9
447.3
351.0
734.5
790.7
718.3
843.6
Solids
1.104
9.04
47.1
66.68
58.0
215.5
235.3
267.7
231.5
Phospha
tids —
0.1424
1 . 1808
7.660
8.940
12.96
53.95
61.56
67.64
70.33
Cerebro
sides...
0.0000
0.0000
0.000
1.542
2.00
12.26
13.13
25.44
18.49
Sulfa
tids....
0.0216
0.2568
0.980
2.570
2.05
17.48
6.77
16.96
14.52
Choles
terol.. .
0.0792
0.9232
6.043
8.738
3.72
34.48
49.34
40.24
27.42
Total
lipins
0.2432
2.3608
14.660
21.80
20.69
118.1
130.80
150.28
130.60
Total pro
teins
0.5152
3.7888
18.05
27.19
24.91
76.28
83.20
88.65
80.97
Organic
extrac
tives . .
0.2104
1.6848
9.638
12.24
8.22
11.31
11.39
20.02
9.46
Inor
ganic
extrac
tives...
0.1368
1.152
4.700
5.45
4.21
9.69
9.85
8.97
10.32
Total ex
tractives
0.3472
2.8368
14.338
17.69
12.43
21.00
21.24
28.99
19.78
Lipin
sul
phur. . .
0.0004
0.0048
0.020
0.051
0.041
0.342
0.133
0.335
0.290
Protein
sul
phur.. .
0.0035
0.0266
0.130
0.195
0.282
0.504
0.533
0.385
0.656
Neutral
sul
phur.. .
0.0021
0.0171
0.187
0.134
0.074
0.123
0.072
0.217
0.161
Inor
ganic
sul
phur...
0.0002
0.0019
0.016
0.026
0.048
0.019
0.031
0.088
0.032
Total sul
phur
0.0062
0.0505
0.253
0.406
0.446
0.988
0.770
1.025
1.140
473
TABLE 3— Concluded
FETUS
3
MONTHS
(13)'
FETUS
7
MONTHS
(12)1
CHILD
1 MONTH
(11)
CHILD
3
MONTHS
(7)
CHILD
8
MONTHS
(4)'
ADULT
21 YEARS
(23)
ADULT
33
YEARS
(28)
ADULT
35 TEARS
(3)
ADULT
(67)
YEARS
(22)
Lipin phosphorus
0.0060
0.0514
0.316
0.401
0.519
2.432
2.401
2.958
2.731
Protein
.
phosphorus
0.0034
0.0086
0.020
0.031
0.033
0.124
0.113
0.138
0.129
Organic phosphorus
0.0025
0.0266
0.142
0.283
0.131
0.114
0.277
0.483
0.086
Inor
ganic phosphorus
0.0077
0.0590
0.324
0.380
0.225
0.551
0.934
0.473
0.570
1
Total phosphorus
0.0206
0.1457
0.802
1.095
0.908
3.221
3.724
4.052
3.516
^ See table 1.
TABLE
Brain stem: Constituents in percentages of solids
FETUS
3
MONTHS (13)1
FETUS
7
MONTHS
(12)1
CHILD
1 MONTH
(14)2
CHILD
3
MONTHS
(20)
CHILD
8
MONTHS
(21)1
ADULT
21 YEARS
(27)
ADULT
35 TEARS
(18)
ADULT
67 YEARS
(26)
Phosphatids
Cerebrosides
Sulfatids
12.90 0.00 1.95 7.24
13.12 0.00
2.85 10.28
17.29 1.95 0.44
16.32
(25.86) 1.08 4.48
13.38
16.67 2.33 5.40
19.75
22.85
(0.81) 7.45 28.48
15.73
4.58
8.40
31.86
30.86 9.83 7.52
Cholesterol
11.87
Total lipins
22.09
26.25
36.00
44.80
44.15
59.59
60.57
60.08
Total proteins
46.56
19.04 12.31
42.15
18.75 12.85
39.21
17.31 7.66
40.55
8.61 6.04
40.52
9.50 5.83
31.41
5.45 3.55
29.96
5.78 3.69
32.01
Organic extractives. . . Inorganic extractives .
4.01 3.90
Total extractives
31.35
31.60
24.97
14.65
15.33
9.00
9.47
7.91
Lipin sulphur
Protein sulphur
Neutral sulphur
Inorganic sulphur
0.039 0.314 0.187 0.013
0.057 0.294 0.183 0.294
0.009
0.158 0.036
0.090 0.238 0.069 0.013
0.108 0.272 0.093 0.008
0.148 0.264 0.009 0.015
0.168 0.211 0.023 0.018
0.150 0.289 0.037 0.009
Total sulphur
0.553
0.556
0.410
0.481
0.436
0.420
0.485
Lipin phosphorus
Protein phosphorus. . . Organic phosphorus. . . Inorganic phosphorus.
0.538 0.306 0.322 0.678
0.563 0.100 0.300 0.648
0.674 0.138 0.252 0.595
1.100 0.068 0.119 0.435
0.753 0.064 0.168 0.425
1.035 0.041 0.064 0.314
0.778 0.052 0.184 0.199
1.343 0.044 0.147 0.238
Total phosphorus
1.844
1.611
1.659
1.722
1.410
1.454
1.213
1.772
1 See table 1. ^ gee table 4.
474
CHEMICAL CHANGES IN HUMAN BRAIN
475
TABLE 4 Brain stem: Constituents in percentage of fresh tissue
Water
Solids
Phosphatids
Cerebrosides
Sulfatids
Cholesterol
Total lipins
Total proteins
Organic extractives. . . Inorganic extractives .
Total extractives
Lipin sulphur
Protein sulphur
Neutral sulphur
Inorganic sulphur
Total sulphur
Lipin phosphorus
Protein phosphorus. . . Organic phosphorus. . . Inorganic phosphorus.
Total phosphorus
FETUS
3
MONTHS
(13)1
91.91 8.09
1.04 0.00 0.16 0.58
FETUS
7
MONTHS
(12)1
1.78
3.77
1.54 1.00
2.54
0.003 0.026 0.015 0.001
0.045
0.044 0.025 0.026 0.056
0.151
90.56 9.44
1.24 0.00 0.27 0.97
CHILD
1
MONTH
(14)2
2.48
3.98
1.77 1,21
2.98
0.005 0.028 0.018 0.002
0.053
0.054 0.009 0.028 0.062
0.153
86.15 13.85
2.40 0.27 0.06 2.26
4.99
5.43
2.37 1.06
3.43
0.001
0.022 0.005
0.094 0.019 0.035 0.083
0.231
CHILD
3
MONTHS
(20)
84.24 15.76
(4.07) 0.17 0.71 2.11
MONTHS
(21)'
7.06
6.40
1.35 0.95
2.30
0.014 0.037 0.011 0.002
0.064
0.172 0.011 0.019 0.068
0.270
82.71 17.29
2.89 0.40 0.94 3.41
7.65
7.01
1.64 1.00
2.64
0.019 0.047 0.016 0.002
0.084
0.131 0.011 0.029 0.074
0.245
ADULT
21 YEARS
(27)
73.60 70.34 26.40 29.66
ADULT
35
YEARS
(18)
ADUI/r
67
YEARS
(26)
6.03 (0.21) 1.97 7.52
15.73
8.29
1.44 0.94
2.38
0.039 0.069 0.002 0.004
0.114
0.273 0.011 0.017 0.083
0.384
4.69 1.36 2.49 9.43
17.97
8.90
1.71 1.10
2.81
0.050 0.062 0.007 0.005
0.124
0.231 0.015 0.055 0.059
0.360
76.26 23.74
7.33 2.33 1.78 2.83
14.27
7.60
0.95 0.93
1.88
0.036 0.069 0.009 0.002
0.116
0.317 0.011 0.035 0.058
0.421
1 See table 1.
2 Brain stem and cerebellum analyzed together, because of small sample.
476
C. G. MACARTHUR AND E. A. DOISY
TABLE 6 Brain stem: Weight of constituents in grams
FETtTS
FETUS
CHILD
CHILD
CHILD
ADULT
ADULT
ADULT
3 MONTHS
7 MONTHS
1 MONTH
3 MONTHS
8 MONTHS
21 YEARS
35 YEARS
67 YEARS
(13)>
(12)1
(14)2
(20)
(20)1
(21)
(18)
(26)
Brain
17.08
119.0
457.4
585.2
492.5
1122.4
1158.3
1297.9
Brain stem .
1.708
11.9
25.0
28.5
35.3
61.0
61.5
77.5
Water
1.57
10.77
21.54
24.00
29.20
44.90
43.26
59.10
Solids
0.138
1.125
3.46
4.50
6.10
16.10
18.24
18.40
Phospha
tids ....
0.0178
0.1476
0.600
1.160
1.024
3.678
2.884
5.681
Cerebro
sides . . .
0.0000
0.0000
0.068
0.048
0.141
0.128
0.836
1.806
Sulphatids
0.0027
0.0321
0.015
0.202
0.332
1.202
1.531
1.380
Choles
terol . . .
0.0099
0.1154
0.565
0.601
1.204
4.587
5.799
2.193
Total lipins.
0.0304
0.2951
1.248
2.012
2.700
9.595
11.052
11.059
Total pro
teins
0.0644
0.4736
1.358
1.814
2.475
5.057
5.474
5.890
Organic
extrac
tives... .
0.0263
0.2106
0.592
0.385
0.579
0.878
1.052
0.736
Inorganic
extrac
tives... .
0.0171
0.144
0.265
0.271
0.353
0.573
0.677
0.721
Total ex
tractives..
0.0434
0.3546
0.857
0.656
0.932
1.451
1.729
1.457
Lipin sul
phur
0.0001
0.0006
0.0003
0.0040
0.0138
0.0238
0.0308
0.0279
Protein
sulphur.
0.0004
0.0033
0.0105
0.0244
0.0421
0.0381
0.0535
Neutral
sulphur.
0.0003
0.0021
0.0055
0.0031
0.0007
0.0012
0.0043
0.0070
Inorganic
sulphur.
0.0000
0.0002
0.0013
0.0006
0.0014
0.0024
0.0031
0.0016
Total sul
phur
0.0008
0.0063
0.0182
0.0402
0.0695
0.0763
0.0900
CHEMICAL CHANGES IN HUMAN BRAIN
477
TABLE 6— Continued
FETUS 3 MONTHS
(13)'
FETUS
7 MONTHS
(12)1
CHILD 1 MONTH
(14)2
CHILD 3 MONTHS
(20)
CHILD
8 MONTHS
(21) J
ADULT 21 YEARS
(27)
ADULT 35 YEARS
(18)
ADULT 67 TEARS
(24)
Lipin
phosphorus..
0.0008
0.0064
0.0235
0.0490
0.0462
0.1665
0.1421
0.2457
Protein
phosphorus..
0.0004
0.0011
0.0048
0.0031
0.0039
0.0067
0.0092
0.0085
Organic
phosphorus..
0.0004
0.0033
0.0088
0.0054
0.0102
0.0104
0.0338
0.0271
Inorganic phosphorus..
0.0010
0.0074
0.0207
0.0194
0.0261
0.0506
0.0363
0.0450
Total phosphorus. . .
0.0026
0.0182
0.0578
0.0769
0.0865
0.2342
0.2214
0.3263
1 See table 1.
2 See table 4.
TABLE 7 Cerebellwn. Constituents in percentage of fresh tissue
FETUa 3 MONTHS
(12)1
FETUS 7 MONTHS
(12)1
CHILD 1 MOXTH
(14)2
CHILD 3 MONTHS
(17)
CHILD
S MONTHS
(1)'
ADULT 1 YEAR
(25)
ADULT 1 35 YEARS
(10)
ADULT 67 YEARS
(29)
Water
91.91 8.09
1.04 0.00 0,16 0.58
90.56 9.44
1.24 0.00 0.27 0.97
86.15 13.85
2.40
(0.27P (0.06)3 2.26
85.05 14.95
2.70 0.00 0.86 1.33
84.56 15.44
2.58 0.26 0.75 1.54
78.83 21.17
6.66 0.98 0.94 0.89
77.99 22.01
(2.84) 0.84 1.02 4.12
80.64
Solids
19.36
Phosphatids . . . Cerebrosides. . .
Sulphatids
Cholesterol ....
4.07 0.54 0.96 3.10
Total lipins
1.78
2.48
4.99
4.89
5.13
9.46
8.82
8.67
Total proteins . . .
Organic extractives
Inorganic extractives
3.77
1.54 1.00
3.98
1.77 1.21
5.43
2.37 1.06
6.97
1.90 1.19
6.94
2.10 1.28
8.95
1.55 1.23
8.60
(2.97) 1.61
7.66
1.68 1.36
Total extractives
2.54
2.98
3.43
3.09
3.38
2.78
(4.58)
3.04
Lipin sulphur. . Protein sulphur
0.003 0.026 0.015 0.001
0.005 0.028 0.018 0.002
0.001
0.022f 0.005
0.018 0.041 0.020 0.001
0.015 0.051 0.014 0.002
0.019 0.053 0.014 0.002
0.020 0.067 0.037 0.009
0.019 0.058
Neutral sulphur
0.006
Inorganic sulphur
0.002
Total sulphur
0.045
0.053
0.080
0.082
0.088
0.133
0.085
Lipin phosphorus
Protein phosphorus
Organic phosphorus
Inorganic phosphorus . .
0.044 0.025 0.026 0.056
0.054 0.009 0.028 0.062
0.094 0.019 0.035 0.083
0.122 0.045 0.020 0.075
0.115 0.046 0.039 0.086
0.278 0.042 0.022 0.110
0.140 0.036 0.080 0.114
0.178 0.028 (0.009) 0.106
Total phosphorus
0.151
0.153
0.231
0.262
0.286
0.452
0.370
0.321
1 See table 1.
2 See table 4.
' Earlier in the paper it was stated, that sulphur determinations were occasionally low. This is the case here. Because of the sugar content of sulphatids, if the latter is too low, these may be reported for cerebrosides when there are none free.
478
CHEMICAL CHANGES IN HUMAN BRAIN
479
TABLE 8
Cerebellum: Constituents in percentage of solids
FETUS
3
MONTHS
(13)'
FETUS
7
MONTHS
(12)1
CHILD
1 MONTH
(14) •i
CHILD
3
MONTHS
(17)
CHILD
8
MONTHS
(16)1
ADULT
21 YEARS
(25)
ADULT
35 YEARS
(10)
ADULT
67 YEARS
(29)
Phosphatids
Cerebrosides
Sulphatids
12.90 0.00 1.95 7.24
13.12 0.00
2.85 10.28
17.29
(1.95)3 (0.44)3 16.32
18.03 0.00 5.76
8.92
16.71 1.65 4.85 9.99
31.42 4.64 4.37 4.18
12.90 3.81 4.64
18.72
21.00
2.77 4 98
Cholesterol
16.05
Total lipins
22.09
26.25
36.00
32.71
33.20
44.61
40.07
44 70
Total proteins
Organic extractives . . . Inorganic extractives .
46.56
19.04 12.31
42.15
18.75 12.85
39.21
17.13 7.66
46.63
12.71 7.95
44.92
13.59 8.29
42.29
7.26
5.84
39.12
13.51 7.30
39.51
8.68 7.01
Total extractives
31.35
31.60
24.79
20.66
21.88
13.10
20.81
15.69
Lipin sulphur
0.039 0.314 0.187 0.013
0.051 0.294 0.183 0.022
0.009
0.158 0.036
0.115 0.273 0.129 0.009
0.097 0.323 0.092 0.015
0.088 0.252 0.064 0.007
0.093 0.307 0.168 0.044
100
Protein sulphur
Neutral Sulphur
Inorganic sulphur
0.296 0.032 0.009
Total sulphur
0.553
0.556
0.526
0.527
0.411
0.612
437
Lipin phosphorus
Protein phosphorus. . . Organic phosphorus. . . Inorganic phosphorus.
0.538 0.306 0.322 0.678
0.563 0.100 0.300 0.648
0.674 0.138 0.252 0.595
0.812 0.302 0.130 0.502
0.741 0.297 0.249 0.554
1.310 0.198 0.104 0.519
0.642 0.163 0.364 0.522
0.912 0.146 0.046 0.546
Total phosphorus
1.&44
1.611
1.659
1.746
1.841
2.131
1.691
1.650
1 See table 1.
2 See table 4. » See table 7.
480
C. G. MACARTHUR AND E. A. DOISY
TABLE 9 Cerebellimi: Weights of constituents in grams
FETUS
FETUS
CHILD
CHILD
CHILD
ADULT
ADULT
ADULT
3 MONTHS
7 MONTHS
1 MONTH
3 MONTHS
S MONTHS
21 TEARS
35 TEARS
67 TEARS
(13)'
(12)'
(14)2
(17)
(16)1
(25)
(10)
(29)
Brain
17.08
119.0
457.4
585.2
492.5
1122.4
1158.3
1297.9
Cerebellum .
1.708
11.9
37.4
42.6
48.2
111.4
110.8
145.4
Water
1.57
10.77
32.22
36.23
40.76
87.80
86.41
117.25
Solids
0.138
1.125
5.18
6.40
7.44
23.58
24.39
28.15
Phospha
tids
0.0178
0.1476
0.898
1.150
1.244
7.418
3.147
5.918
Cerebro
sides . . .
0.0000
0.000
0.101
0.000
0.125
1.091
0.931
0.785
Sulphatids
0.0027
0.0321
0.022
0.366
0.362
1.036
1.130
1.396
Choles
terol . . .
0.0099
0.1154
0.845
0.567
0.742
0.991
4.565
4.507
Total lipins.
0.0304
0.2951
1.866
2.084
2.473
10.536
9.772
12.606
Total pro
teins
0.0644
0.4736
2.031
2.969
3.345
9.968
9.529
11.138
Organic
extrac
tives . . .
0.0263
0.2106
0.886
0.810
1.012
1.704
3.291
2.443
Inorganic
extrac
tives... .
0.0171
0.144
0.397
0.507
0.617
1.370
1.784
1.977
Total ex
tractives..
0.0434
0.3546
1.283
1.317
1.629
3.074
5.075
4.420
Lipin sul
phur . . .
0.0001
0.0006
0.0004
0.0077
0.0072
0.0212
0.0222
0.0276
Protein
sulphur.
0.0004
0,0033
0.0175
0.0246
0.0590
0.0742
0.0843
Neutral
sulphur.
0.0003
0.0021
0.0082
0.0085
0.0067
0.0156
0.0410
0.0087
Inorganic
sulphur.
0.0000
0.0002
0.0019
0.0004
0.0010
0.0022
0.0100
0.0029
Total sul
phur
0.0008
0.0063
0.0341
0.0395
0.0980
0.1474
0.1236
1 See table 1.
2 See table 4.
TABLE 9— Continued
PETDS 3 MONTHS
(13)'
FETUS 9 MONTHS
(12)1
CHILD 1 MONTH
(14)2
CHILD 3 MONTHS
(17)
CHILD 8 MONTHS
(16)1
ADULT 21 YEARS
(25)
ADULT 35 YEARS
(10)
ADULT 67 YEARS
(29)
Lipin phosphorus .
0.0008
0.0064
0.0352
0.0520
0.0554
0.3097
0.1551
0.2588
Protein
phosphorus .
0.0004
0.0011
0,0071
0.0192
0.0222
0.0468
0.0399
0.0407
Organic phosphorus .
g.ooo4
0.0033
0.0131
0.0085
0.0188
0.0245
0.0886
0.0131
Inorganic phosphorus .
0.0010 0.0026
0.0074
0.0311
0.0320
0.0415
0.1225
0.1263
0.1541
Total phosphorus . . .
0.0182
0.08&4
0.1116
0.1378
0.5035
0.4100
0.4667
TABLE 10 Whole brain: Constituents in percentage of fresh tissue
FETUS
3
MONTHS
FETUS
7
MONTHS
CHILD
1 MONTH
CHILD
3
MONTHS
CHILD
8
MONTHS
ADULT
21 YEARS
ADULT
35 YEARS
ADULT
67 YEARS
Water
Solids
91.91 8.09
90.56 9.44
87.81
12.19
86.75 13.25
85.47 14.53
77.25 22.75
73.20 26.80
78.58 21.42
Phosphatids
Cerebrosides
Sulphatids
1.04
0.00
.16
.58
1.24
0.00
.27
.97
2.00 .04 .22
1.63
1.92 .27 .53
1.68
3.09 .46 .56
1.15
5.80 1.20 1.75 3.57
6.35 2.35 1.69 4.36
6.30 1.62 1.33
Cholesterol
2.62
Totallipins
1.78
2.48
3.89
4.42
5.26
12.32
14.75
11.87
Total proteins
3.77
1.54 1.00
3.98
1.77 1.21
4.69
2.43 1.18
5.47
2.29 1.08
6.24
1.99 1.05
8.14
1.24 1.04
8.95
2.10 0.99
7.54
Organic extractives
Inorganic extractives . . .
0.98 1.01
Total extractives
2.54
2.98
3.61
3.37
3.04
2.28
3.09
1.99
Lipin sulphur
0.003 0.026 0.015 0.001
0.005 0.028 0.018 0.002
0.004 0.029 0.022 0.004
0.011 0.038 0.026 0.005
0.012 0.066 0.016 0.010
0.035 0.054 0.013 0.002
0.034 0.043 0.023 0.009
0.026
Protein sulphur
0.061
Neutral sulphur
Inorganic sulphur
0.014 0.003
Total sulphur
0.045
0.053
0.059
0.080
0.104
0.104
0.109
0.104
Lipin phosphorus
Protein phosphorus
Organic phosphorus
Inorganic phosphorus. . .
0.044 0.025 0.026 0.056
0.054 0.009 0.028 0.062
0.081 0.007 0.035 0.082
0.086 0.009 0.051 0.074
0.124 0.012 0.032 0.060
0.259 0.016 0.013 0.064
0.280 0.016 0.052 0.055
0.248 0.014 0.010 0.059
Total phosphorus
0.151
0.153
0.205
0.220
0.228
0.352
0.403
0.331
481
482
C. G. MACARTHUR AND E. A. DOISY
TABLE 11 Whole brain: Constituents in percentage of solids
FETUS
3
MONTHS
Phosphatids
Cerebrosides
Sulphatids
Cholesterol
Total lipins
Total proteins
Organic extractives
Inorganic extractives . . .
Total extractives
Lipin sulphur
Protein sulphur
Neutral sulphur
Inorganic sulphur
Total sulphur
Lipin phosphorus
Protein phosphorus
Organic phosphorus
Inorganic phosphorus. . .
Total phosphorus
22.09
12.90 0.00 1.95 7.24
26.25
46.56
19.04 12.31
42.15
18.75 12.85
31.35
0.039 0,314 0.187 0.013
0.553
0.538 0.306 0.322 0.678
1.844
FETUS
7
MONTHS
13.12
0.00
2.85
10.28
31.90
38.46
19.93 9.68
31.60
0.057 0.294 0.183 0.022
0.556
0.563 0.100 0.300 0.648
CHILD 1
MONTH
16.40 0.33 1.80
13.37
33.30
41.30
17.29 8.15
29.61
0.033 0.238 0.180 0.033
0.184
0.664 0.057 0.287 0.672
1.611
1.680
CHILD 3
MONTHS
14.50 2.04 4.00
12.76
21.26 3.16 3.85 7.91
36.19
42.93
13.69
7.22
25.44
0.083 0.287 0.196 0.038
0.604
0.649 0.068 0.385 0.559
1.661
CHILD
8
MONTHS
25.52 5.28 7.70
15.70
54.20
35.82
5.46 4.58
20.91
0.083 0.454 0.110 0.068
0.715
0.853 0.083 0.220 0.413
1.569
ADULT
21 YEARS
23.69 8.77 6.30
16.26
55.01
33.38
7.83 3.69
10.04
0.154 0.238 0.057 0.009
0.458
1.140 0.070 0.057 0.282
1.549
ADULT
35
YEARS
29.42 7.56 6.21
12.24
55.43
11.52
0.127 0.160 0.086 0.034
0.407
1.044 0.060 0.194 0.205
1.503
ADULT
67 TEARS
35.21
4.58 4.72
9.30
0.121 0.285 0.065 0.014
0.485
1.158 0.065 0.047 0.276
1.546
CHEMICAL CHANGES IN HUMAN BRAIN
483
TABLE 12 Whole brain: Weights of constituents in grams
FETUS
3
MONTHS
FETUS
CHILD
CHILD
CHILD
ADULT
ADULT
ADULT
7 MONTHS
1 MONTH
3 MONTHS
8 MONTHS
21 YEARS
35 YEARS
67 YEARS
Whole brain
17.08
119.0
457.4
585.2
492.5
1122.4
1158.3
1297.9
Water
15.70
107.7
401.7
507.5
421.0
867.2
848.0
1020.0
Solids
1.38
11.25
55.74
77.58
71.54
255.2
310.3
278.0
Phospha
tids ....
0.178
1.476
9.158
11.250
15.228
65.05
73.67
81.93
Cerebro
sides . . .
0.000
0.000
0.169
1.590
2.266
13.479
27.207
21.081
Sulpha
tids ....
0.027
0.321
1.017
3.138
2.744
19.718
19.621
17.296
Choles
terol . . .
0.099
1.154
7.453
9.906
5.666
40.058
50.604
34.120
Total lipins.
0.304
2.951
17.774
25.896
25.863
138.23
171.02
154.27
Total pro
teins
0.644
4.736
21.439
31.973
30.730
91.305
103.65
98.00
Organic
extrac
tives . . .
0.263
2.106
11.116
13.435
9.811
13.892
24.363
12.639
Inorganic
extrac
tives . . .
0.171
1.440
5.362
6.228
5.180
11.633
11.431
13.018
Total ex
tractives..
0.434
3.546
16.478
19.663
14.991
25.525
35.794
25.657
Lipin
sulphur.
0.0005
0.0060
0.0207
0.0627
0.0620
0.3870
0.3880
0.3453
Protein
sulphur.
0.0044
0.0333
0.1300
0.2230
0.3310
0.6051
0.4973
0.7938
Neutral
sulphur.
0.0026
0.0214
0.1007
0.1456
0.0814
0.1398
0.2623
0.1767
Inorganic
sulphur.
0.0002
0.0024
0.0192
0.0270
0.0514
0.0236
0.1011
0.0365
Total sul
phur.. .. . .
0.0077
0.0631
0.2706
0.4583
0.5248
1 . 1555
1.2487
1.3525
484
C. G. MACAETHUR AND E. A. DOISY
TABLE \2— Continued
FETUS
3
MONTHS
FETUS
7
MONTHS
CHILD 1
MONTH
CHILD 3
MONTHS
CHILD
8
MONTHS
ADULT
21
TEARS
ADULT
35 TEARS
ADULT
67 TEARS
Lipin phosphorus .
0.0075
0.0643
0.3747
0.5020
0.6206
2.0980
3.2550
3.2360
Protein
phosphorus .
0.0043
0.0102
0.0319
0.0533
0.0591
0.1775
0.1871
0.1782
Organic phosphorus .
0.0044
0.0333
0.1639
0.2969
0.1600
0.1489
0.6054
0.1262
Inorganic phosphorus .
0.0096
0.0738
0.3758
0.5415
0.2926
0.7241
0.6356
0.7691
Total phosphorus . . .
0.0258
0.1821
0.9463
1.2836
1.1323
3.9585
4.6831
4.3095
Figure 1 was plotted from the data in this table relating to the earlier period of growth.
CHEMICAL CHANGES IN HUMAN BRAIN
485
TABLE 13 Whole brain: Milligrams added per day
UP TO 3-MONTH
FETUS
3-MONTH
TO
7-MONTH
FETUS
7-MONTH
FETUS TO
1-MONTH
CHILD
1-MONTH
TO
3-MONTH
CHILD
3-MONTH
TO
8-MONTH
CHILD
8-MONTH
TO 21-YEAR
Whole brain
Water
190.0
174.0 15.3
1.98 0.0 0.30 1.10
848.0
766.0
82.3
10.80 0.0 2.45
8.79
3764.0
3270.0 494.0
85.3 1.88 7.73 70.0
2127.0
1763.0 364.0
34.9 23.7 35.4 40.9
501.6
417.0 84.6
26.3 4.07 2.99 0.74
131.2 113
Solids
18 2
Phosphatids
5.4
Cerebrosides
1.4
Sulphatids
2 2
Cholesterol
4 4
Total lipins
3.38
22.04
164.9
135.1
33.9
13 4
Total proteins
7.16
2.81 1.90
34.1
15.4 10.6
185.6
100.1 43.6
175.6
38.7 14.4
38.5
7.2 5.2
5 1
Organic extractives
Inorganic extractives ....
-0.6 -0.3
Total extractives
4.81
26.0
143.7
53.1
11.4
-0.3
Lipin sulphur
0.006 0.049 0.029 0.002
0.05 0.24 0.16 0.02
0.16 1.07 0.88 0.19
0.70 1.55 0.75 0.13
0.08 0.61 0.01 0.11
0.04
Neutral sulphur
Inorganic sulphur
0.0 -0.01
Total sulphur
0.086
0.47
2.30
3.13
0.81
0.03
Lipin phosphorus
Protein phosphorus
Organic phosphorus
Inorganic phosphorus. . . .
0.083 0.048 0.049 0.107
0.47 0.081 0.24 0.54
3.45 0.24 1.45 3.36
2.12 0.36 2.22 0.93
1.01 0.09 0.0 0.17
0.27
0.01
-0.02
0.03
Total phosphorus
0.287
1.30
8.50
5.62
1.27
0.29
A part of these data are plotted in graph 2.
TABLE 14 Whole brain: Average percentage increase per day
Whole brain
Water
Solids
Phosphatids
Cerebrosides
Sulphatids
Cholesterol
Total lipins
Proteins
Organic extractives. . Inorganic extractives Total extractives
3-MONTH
FETUS
7-MONTH
FETUS
1-MONTH
3-MONTH
2.3
1.7
0.88
0.26
2.1
1.7
0.88
0.23
2.4
1.7
1.0
0.36
2.3
1.8
0.79
0.36
0.0
0.0
4.7
1.6
2.7
1.9
1.3
1.1
2.5
1.8
1.1
0.1
2.2 •
1.8
1.3
0.32
2.3
1.6
1.1
0.5
2.3
1.7
0.42
0.13
2.2
1.6
0.56
0.26
2.3
1.7
0.6
0.15
8-MONTH
0.028
0.04
0.046
0.03
0.057
0.072
0.02
0.03
0.02
0.00
0.00
0.00
These data were estimated from curves similar to, but larger than curves (1). These were plotted from data in table 10.
In the calculation from the curves a period of one-half month before and onehalf month after each age was used. It is believed that the enormous figures for rate of growth sometimes presented for early fetal life are due to the method of calculating from the weight at the beginning of the period. When the growth rate is changing rapidly, the error in such calculations is very large.
The curves in graph 3, excepting extractives (c), are plotted from this table,
TABLE 15 Whole brain: Average percentage increase per day
8 MONTH21 YEARS
Whole brain
Water
Solids
Phosphatids
Cerebrosides
Sulphatids
Cholesterol
Total lipins
Proteins
Organic extractives. . . Inorganic extractives. Total extractives
3-7
MONTH FETUS
7 MONTH
FETUS 1 MONTH
1 MONTH3 MONTH
3 MONTH8 MONTH
1.25
1.31
0.41
0.067
1.24
1.28
0.39
0.065
1.30
1.47
0.55
0.081
1.31
1.59
0.34
0.13
0.00
(2.23)
2.69
0.14
1.41
1.16
1.70
0.075
1.46
1.63
o;47
0.073
1.36
1.59
0.62
0.093
1.27
1.42
0.66
0.087
1.28
1.52
0.32
0.046
1.32
1.28
0.25
0.067
1.30
1.44
0.29
0.048
0.013
0.014
0.01
0.012
0.016
0.018
0.018
0.014
0.007
0.0
0.0
0.0
Calculated from data in table 11. The average number of milligrams added per day was divided by the average weight for the given period, instead of the weight at the beginning of the period, as is usually done. When there are rapid changes in weight, this method is not as accurate as that used in table 12. It is believed that the temporary rise in growth at about the seventh month of fetal life is due to the method of calculation.


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Larsell O. Studies on the nervus terminalis: Mammals. (1918) J Comp. Neurol. 30(1): 1-

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Mark Hill.jpg This historic 1919 paper by Van der Stricht describes development of the cellular components of the organ of Corti of the inner ear.



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The Development of the Pillar Cells, Tunnel Space, and Nuel's Spaces in the Organ of Corti

O. Van Der Stricht

Department of Anatomy, Johns Hopkins Medical School, Baltimore, Maryland

Eighteen Figures

Introduction

In spite of numerous thorough and exliaustive investigations concerning the earhest stages of development of the organ of Corti, our knowledge of the origin of the tunnel space is still very limited and vague. This is true also of the formation of the heads and cephalic appendages of the pillar cells, and almost nothing is known concerning the origin of the so-called spaces of Nuel. This observer (78) described in the organ of Corti in adult mammals a system of intercellular channels, and his findings have been confirmed by Retzius ('84) and other more recent authors. These spaces or channels are situated between the outer rods of Corti and the neighboring outer hair cells, and between the three rows of outer acoustic elements. The}^ contain fluid and are traversed by the phalanx processes of the cells of Deiters, intercommunicating through clefts between the sensory elements, and communicating with the tunnel space through interstices between the outer pillars. The view taken by Nuel, that they communicate with the lumen of the cochlea "par I'entremise de lacunes en fonne de rames de la mambrane reticulaire," must be regarded as erroneous.


The development of the tunnel space between the two spiral rows of rods of Corti appears to be verj^ difficult of observation. Indeed, most authors, referring to its appearance in embryonic material, state that it originates in the form of a narrow cleft between the inner and outer pillars, but give no details concerning the significance of this primitive interstice. Is it intercellular or intracellular? From what source is derived the fluid contained within the cleft? Those observers who clearly specify that the space appears between two neighboring pillars, as do Gottstein (72), Retzius ('84), Vernieuwe ('05), N. Van der Stricht ('08), and Hardesty (.'15), give no explanation as to the origin of its fluid. Alluding to the development of the space in rabbits two daj^s after birth, Retzius states (p. 303) :


Von besonderer Bedeutung ist nun die enge Spalte, welclie zwischen den beiden Pfeillerzellen reichen, ungefahr in der Mitte der Zellenhohe, nach oben vom spiralen Nervenbiindel entstanden ist und den Anfang des Tunnelramiis dai*stellt; diese Spalte ist in der Basalwindung — wo indessen noeh eine geringe Neigiing der Pfeillerzellen nach aiissen hin vorhanden ist — noch viel weiter entwickelt, und man sieht hier deutlieh, dass die durch Einziehung (Verdtinnung) der beiderseitigen Pfeillerzellen entstanden ist. Gleichzeitig ist aber auch die Anlage der Pfeiler in der Zellen als helleglanzende Streifen nunmehr wahrnehmbar. Nach aussen von der ausseren Pfeilerzellenreihe sieht man deutlieh auch die Anlage der Nuelschen Raums.

According to Vernieuwe, the timnel space is produced by the separation of the bases of the two pillar cells, due to elongation of the pillars, increase in size of the nuclei, and chiefly by the extension of the subjacent basilar membrane. Referring to the trend of the spiral organ of Corti towards the axis of the cochlea, Hardesty ('15, p. 52) states: The normal spaces between the elements of the spiral organ, including the large Nuel's space, no doubt result in part from this movement of the organ axisward." Other authors, Rosenburg ('68), Boettcher ('69), and Pritchard ('76) describe two neighboring inner and outer pillars as derived from a single original cell, the nucleus of which divides in two, and by a process of liquefaction of the undivided cytoplasm, the tunnel space is produced within it. This space is originally intracellular and its fluid is a protoplasmic product. Rickenbacher ('01, p. 402) seemingly ascribes a similar origin to the fluid of the space of Nuel in the adult guinea-pig : ' ' Bei der Schnecke des ausgewachsenen Tieres hat der Prozess der Verfltissigung zur Bildung des Nuelschen Intercellularraume und des Leiterepithels gefiihrt." According to Kishi ('02), the tunnel space is due to the spiral course of the nerve fibers after they have passed through interstices between the inner pillar cells. The formation of tunnel and intercellular clefts is considered by Held ('09) to be the result of 'ungleichen Wachstumbewegungen' of different epithelial cells. His so-called 'outer tunnel,' the spaces between the outer hair cells, and the space of Nuel outside the outer pillars are sheer intercellular channels, 'reine Intercellularspalten;' but the tunnel between the pillars is originally intracellular.


Eine reine intrazellular Spalt, da die ersten Nervenfasern, die hier spiralig abbiegen und weiter ziehen, nicht in der Zwischengrenze zwischen Aussenr und Innenpfeiler liegen, sondern im Protoplasma der Innenpfeilerzellen randstandig eingebettet sind, was audi fiir die unten den inneren sowie ausseren Haarzellen resp. zwischen den Deiterschenzellen imd in ihren Intel zellularbriicken gelegenen Formation eines intraepithelialen Nervusplexus gilt.


The development of the tunnel and the pillar cells is closely connected wdth the formation of the pillar heads, the appearance of the 'head-plates' of the inner pillars, the phalanx processes of the outer pillars, and the extension of the membrana reticularis. The superficial structures of the rods of Corti in adult mammals have been exhaustively investigated by many observers: Max Schultze ('58), Koelliker ('59), Boettcher ('59, '72), Deiters ('60), Hensen ('63, 71), Gottstein (70, 72), Nuel (78), Tafani ('84), Retzius ('84), and by most of the more recent authors; but the appearance and extension of these structures and the mechanical factors taking part in their fonnation require more careful study. N. Van der Stricht has shown that the head-plate of the inner pillar is originally represented by a very small square field, the apex of the cell, which becomes fibrillated and extends over the enlarging head of the outer pillar, the foniier undergoing great pressure from the latter The outer pillar cells originally belong to the first spiral row of outer sensory elements. As development advances they are pressed out from this row towards the inner rods of Corti and form a new row of outer rods, the apices of which always remain fixed between those of the outer acoustic elements of the first row. Hence there persists an apical segment of the outer pillar, which runs obliquely from the apex or phalanx of the cell, downward and inward toward the future head of the pillar. This oblique process contains a bundle of fibrils which, issuing from the head, passes between two outer acoustic elements and spreads out upon the phalanx — the head-plate of the outer piljar. By enlargement of the headj the fibrillar bundle 'gradually acquires a more horizontal position. Held ('09, p. 109) seemingly ascribes the head-plafce of the inner pillar not only to the apex, but also to the superficial portion of the cell, der obere Zellteil welche die Faserrohre enthalt," and which is pressed flat from the developing head of the outer pillar. Although he did not recognize the original position of the outer pillar cells within the first row of outer acoustic elements, he nevertheless observes the squeezing of their 'Kopfplatte,' which becomes thinner from compression between two hair cells, and also of the bundle of fibrils, which at first run obliquely, then at right angles to the intermediate piece of the outer pillar, due to pressure from the elongating pillar cell.

In the present paper the appearance of the tunnel space, the development of the heads and cephalic appendages of the pillar cells, and the formation of the Nuel spaces will be dealt with in order.


Appearance of the tunnel space

Sections tangential to the surface of the organ of Corti, and always somewhat oblique, affect transversely series of neighboring inner and outer pillars at various and successive levels of their length, from the superficial membrana reticularis toward the basilar membrane (fig. 1). As illustrated in figures 1 and 2, one may distinguish in the prismatic lamv-^Uar pillars three portions, although they are not sharply marked off: a basal or nucleated part, the largest, which is lamellar in shape or flattened out in a radial direction from mutual compression; an intermediate part; and a superficial part, the narrowest, which is compressed between the inner and outer hair cells, and hence more or less flattened out in a spiral direction {ip and op). The basal and intermediate portions are each made up of two cytoplasmic zones, the larger being clear and vacuolated, and occupying the area of the cell body close to the future tunnel; the smaller compact and fibrillated, and occupying the axial side of the inner pillar and the lateral side of the outer pillar. The superficial segment of the two rods of Corti contains no vacuolated protoplasm; it consists of a more homogenous, compact cytoplasm, which in the inner pillar is traversed by a bundle (fig. 2, ip) or a tubule (fig. 3, ip) of fibrils, and in the outer, encloses a bundle of fibrils which pass between neighboring outer hair cells and give rise to a small band, the phalanx process of the outer pillar (figs. 1, 2, and 3, oph), connected with the superficial apex of the cell, the phalanx. In the adult organ a part of this fibrillar bundle is a characteristic constituent of the superficial portion of the head, and thus its early presence in a definite portion of the outer pillar is very important in enabling one to determine, from the earliest stages of development, a very narrow but long superficial portion of the cell (figs. 1, 2, and 3, op), which later enlarges and becomes transformed into a part of the bulky head. It is also obvious that the adjoining portion of the inner pillar, which in figures 1, 2, and 3 is in close contact with this future head of the outer pillar, must be considered as the segment which will become converted into the so-called head of the inner pillar.


The outlines of all the pillars are very sharp, not only between the cells of the same row, but also between the neighboring elements of the two rows. While at the level of the superficial segments this outline is represented by an intercellular material (figs. 1 and 3, tb), which in its staining capacity and chemical constitution agrees with that of the superficial terminal bars, between the two lower segments of the pillars it is composed of a paler, more fluid, or true intercellular cement, in addition to which a very thin superficial cytoplasmic film can be brought into view. This outline and film are lacking along the axial surfaces of the inner pillars and the lateral surfaces of the outer. The spiral nerve bundle (A^") occupies an intercellular position between the nucleated parts of the outer and inner pillars, sometimes encroaching somewhat upon the lower interstice which separates their intermediate portions. The nuclei of the pillar cells are surrounded by vacuolated cytoplasm. The nuclei of the inner pillars are much smaller than those of the outer and much more flattened out radially.


When the tunnel space is about to appear, there occurs a characteristic alteration in the cytoplasm adjacent to this future cleft, the vacuoles running together and thus increasing in size (figs. 1 and 2, t). A common vacuolated mass soon appears (figs. 3 and 4, t) ; at certain places it remains fused with the cell body from which it is derived, at others it is independent, so that one cannot determine to which of the neighboring pillars it belongs. From this moment there exists a narrow intercellular cleft, filled with a small amount of extracellular, vacuolated material, a common mass which doubtless represents the first trace of the intratunnelar fluid, and w^hich gradually increases in quantity by the coalescence of adjoining portions, partly incorporated in the original pillar and partly free or extracellular. Although from the earliest stages of the appearance of the space small extracellular, vacuolated masses can be found between the intermediate segments of the pillars (fig. 4, t), the larger part of the tunnel is generally seen around and close to the spiral nerve bundle, that is to say, between the nucleated portions of the inner and outer rods of Corti (fig. 5, t, T), never ungefahr in der Mitte des Zellenhohe," as Retzius asserts and manyinvestigators illustrate. It is a perinervous space. Later it extends between the intemiediate portions of the pillar cells.

According to this description, the tunnel space must be held to be a true intercellular cleft, the fluid contents of which are developed by a process of secretion from the neighboring parts of the pillars and a simultaneous partial cytolysis of the latter. The space enlarges at the expense of the cell bodies. In the earhest stages of development of the organ of Corti there appears in the pillars not only a fibrillated sustentacular apparatus related to their function of support, but also a large, clear, vacuolated cytoplasm, the bulk, of their cell bodies. This portion of the protoplasm is glandular in nature, and from the blood plasma of the subjacent vas spirale (fig. 1, vs), it derives its nutritive material, which is elaborated and converted into clear vacuoles. The products of secretion are discharged along with a partial liquefaction of the surrounding cytoplasm.


During the extension of the tunnel space the superficial seg-ments of the outer pillars undergo considerable enlargement, and their radial diameters soon correspond to those of the intemiediate portions (fig. 4, ohd). At that time the process of cytolysis obviously extends along these segments (figs. 4 and 5, t), from below upward, involving a rapid reduction of their radial diameters. The intermediate segment of the outer and inner pillars, previously broad and formed of a small fibrillated part and a large vacuolated portion next to the future cleft, becomes gradually converted into a slender band, the so-called 'body' of the pillar. In figure 7 (opb) these bodies are shown cut at successive levels through fifteen outer pillars. In their lower portion, as seen in nine sections, they are reduced to thin cylindrical fibrillar strands, part of their apparatus of support, from around which the clear cytoplasm has disappeared. In their upper part, as seen in the next four sections, the pillar bodies are still composed of the original two zones, the vacuolated portion having been somewhat reduced. Close to the future heads are seen two sections (connected with sections of inner pillars), the structures of which have undergone no change. The process of cytolysis is completed at the level of the first nine elements; it is progressing in the following four and has not yet begun in the last two. On comparing these structures with more advanced stages, and especially with those in the adult cochlea, it is plain that the body of the outer pillars acquires its final form and structure by a process of secretion and cytolysis along with the elongation of the intermediate segment. In young cats, bats, common and white rats it becomes a slender fibrillated strand, destitute of clear cytoplasm (figs. 10, 13^, 14, 15, 17 and 18, oph). Betw^een the pillar bodies, as aheady noted by Nuel, are large clefts through which the fluid of the tumiel space and the neighboring space of Nuel intercommunicate.


The intermediate portions of the inner pillars undergo similar, but never such marked changes. The greater part of their clear cytoplasm disappears, only a very narrow zone of it persisting, so that in young and adult animals the body of the pillar becomes lamellar in shape (fig. 18, ipb) and flattened out in a spiral 'direction. It is composed of a, fibrillar lamella and a thin layer of clear protoplasm (fig. 17, iph). Besides the pores traversed by the nerve fibers, no true intercellular clefts sever the inner pillars.


Along with these alterations and elongation of the pillar bodies, the tunnel space enlarges gradually but considerably, and very soon its radial diameter surpasses that of the two original clear zones belonging to two contiguous pillars. In other words, the fluid accumulated within the cleft exceeds the am.ount of disintegrated protoplasm. Indeed, the cytolysis occurs in a merocrine glandular cell, which, although undergoing partial liquefaction, is able to elaborate new clear secretion products at the expense of material derived from the vas spirale. Hence the tunnel fluid is the result not only of a sheer cytolysis, but also of a true elaboration and subsequent discharge. In the earliest stages of development the process of cytolysis seems to be more prevalent, since the contents of the cleft are seen in the form of a coagulated, vacuolated mass; afterwards the larger space is usually filled with a clear unifomi fluid, which seems to arise from a more active, true secretion. This secretion may continue even in the adult organ, for, according to all the investigators, the nucleus of each pillar cell is surrounded by a clear cytoplasm which extends over the floor of the tunnel. This protoplasm is vacuolated and represents the rest of the original bulky, glandular portion of both sustentacular and secreting cells.

Development of the heads and the cephalic appendages of the pillar cells

According to the results published in a previous paper (now in press) and those obtained by N. Van der Stricht, at the earliest stage of the development of the organ of Corti the outer pillar cells are located within the first spiral row of outer hair cells, and their superficial segments occupy interstices between two neighboring acoustic elements. By the rapid enlargement of the latter, these superficial elements are pressed out of the row and pushed towards the inner pillars, although their apices remain fixed between those of the hair cells. From this tune the inner and outer rods of Corti constitute a scaffolding, which is made up of two spiral rows of sustentacular elements and is triangular in shape on vertical section. The rapidly enlarging base of the triangle abuts against the basilar membrane, and the apex is interpolated within the superficial membrana reticularis, separating the apices of the supporting and sensory elements of the inner spiral row from the apices of those of the first outer spiral row. In a section tangential to the organ of Corti the summit of the scaffolding is represented by a spiral row of very narrow fields, the apices of the inner rods of Corti, separated from one another and from the neighboring fields of the reticular membrane by deeply staining terminal bars, which extend into the d^pth between the superficial portions of the inner and outer pillar cells. Each of these narrow fields contains a diplosome, and will gradually enlarge by a process of compression from the underlying expanding heads of the outer pillars.


Outer pillars. In the superficial part of the entirely developed outer pillar, as seen in the adult organ of Corti, three different portions are distinguishable: 1) The apex or 'phalanx,' fonning a part of the membrana reticularis. This consists of a lateral, expanded segment (fig. 8, oplv), which constitutes a portion of the roof of a subjacent intercellular interstice, through which course the phalanx processes of the cells of Deiters of the first row (paper in press); and a medial, constricted segment (op/i") lying just between two apices of the outer hair cells of the first row {oh>). 2) A fibrillated band or the phalanx process (fig. 13'", oph\ oph", oph), which runs nearly horizontal and unites the apex to the head. 3) The head proper, or the enlarged superficial part of the pillar, in contact with the inner pillar (figs. 13^, 17, and 18, ohd). This is a cubical segment; in sections tangential to the surface it is square (fig. 13'", ohd) or somewhat lengthened out radially (fig. 18, ohd). Its upper portion is traversed by the fibers of the phalanx process (fig. 17, ohd), and its larger, lower part by a fibrillar bundle belonging to the body of the pillar (fig. 13'^, ohd). Thus two different fibrillated fasciculi spread out, and fade off into the head; there is no direct continuity between the fibrils of the two bundles (figs. 14 and 15, ohd).


In the first stage of development, which may last until the. tunnel space is about to appear and before there is any marked increase in the site of the future head (figs. 1, 2, and 3, op), the three parts of an adult pillar just referred to are recognizable. The apex acquires the appearance illustrated in figure 4, oph. The phalanx process is short and a nearly vertical, dejeply stainmg bundle of fibrils (figs. 1 and 3, oph) which is traceable between the cell bodies of the outer hair cells {oh'), and a little deeper between these sensory elements and the future head. The future head is a thin tapering part of the pillar, composed of a more or less homogeneous cytoplasrh which encloses in its upper two-thirds the rootlets of the phalanx fibrils, and in its lower one-thu'd the summit of the bunch of fibrils of the pillar body (figs. 2 and 3, op). Indeed, in figures 1, 2, and 3, two or three fields, cross-sections of the future head, contain parts of the two fibrillated hands. This rather deep portion of the pillar, situated at the level of the lower poles of the outer hair cells (oh^), doubtless belongs to the developing head. From this it is evident that the superficial, thin, tapering segment of the outer pillar cells, which gives rise to both the phalanx process and the head, attains more than one-third (figs. 1 and 3, op) or about one-half of the entire length of the cell, or about the length of the outer acoustic element {oh^), although no distinct demarcation can be observed between the future head and the pillar body.


Two other features lend support to this view: the existence of an abundant, vacuolated cytoplasm along the intermediate portion of the cell, the future pillar body, which only slightly encroaches upon the lower part of the future head, and the presence of terminal bars or rather true intercellular, obturating partitions. These have been observed and termed 'bandelettes obturantes' by N. Van der Stricht ('08) and 'Kittsubstanz' or 'Kittlinie' by Held ('09). This material stains intensely with iron hematoxylin like the superficial terminal bars with which it is in continuity, and corresponds to them in nature and chemical constitution. It gives rise not to 'lines' or 'bars,' but to true septa, uniting parts of the cells and obturating the subjacent intercellular spaces. These partitions exist not only between contiguous developing and definitive heads of inner and outer pillars, but also between the apical surface (that turned toward the apex of the cochlea) and basal surface (that turned toward the base of the cochlea) of the heads of each spiral row. On the other hand, they are altogether lacking along the medial surfaces of the heads of the inner pillars and the lateral surfaces of those of the outer (figs. 1 and 3, tb).


The second stage of development is characterized by a rapid enlargement of the future head of the outer pillar (fig. 4, ohd), so that it reaches the site of the intermediate portion or even surpasses it, when the process of cytolysis progresses along the tunnel space (fig. 7, op). At first the head remains smaller next to the surface, but soon this portion expands and becomes somewhat larger than the deeper part (fig. 4, ohd) and acquires a cubical or prismatic shape, the larger base of which touches the surface of the organ of Corti, its tapering apex blending with the much smaller pillar body. In cross-sections the prism is square or quadrangular in shape.


During this process of enlargement of the head, many remarkable changes occur. 1) A considerable shortening of the head segment (fig. 7) as if the compact substance of the lower parts had been pushed upward. Moreover, there can be no doubt that, at the same time, the vacuolated cytoplasmic zone of the intermediate portion of the pillar extends upward along the primitive head, so that the pillar body becomes longer at the expense of the latter. 2) A peculiar transformation of the protoplasm of the heads of the outer and inner pillars, close to and through the agency of the obturator septa. Primitively compact, homogeneous, or granular, entirely different from the vacuolated or fibrillated cytoplasm above referred to, the protoplasm of the head becomes converted into a denser material, staining intensely with iron hematoxylin. These changes occur in succession, first within the heads of the outer (figs. 4 and 7, ohd), then within those of the inner pillars (fig. 8, ihd), in proximity of the obturator septa separating their apical from their basal surfaces ; later, along the medial surfaces of the heads of the outer pillars, and finally ^long the lateral surfaces of the heads of the inner pillars, close to the obturator partitions which separate these two elements (fig. 9, ohd and ihd). In sections tangential to the surface of the organ of Corti these altered cytoplasmic portions are seen in the form of deeply staining uniform, planoconvex masses, the planar surface of the clump of one head adjoining that of another mass belonging to a contiguous head (fig. 11). In reality, each planoconvex clump is the section of a vertical band or semicolumn. Thus in each head there appear three semicolumns, which at first are separated from one another, but in more advanced stages Coalesce to fonn a single band or imperfect collar open toward the side of the head where the obturator material is lacking (figs. 7, 8, and 9). What mechanical factors cause these structures to appear is uncertain. It can only be stated that this dense and horny like exoplasmic head-collar develops and extends in close contact with the intercellular septa, as if the material elaborated at the periphery of the cytoplasm to increase the amount of extracellular cement were prevented from leaving the cell and retained within this collar, the staining capacity of which gradually increases, while the more central protoplasm, the endoplasm, becomes clearer and paler- This head-collar has been described in the embryonic pillar cells by N. Van der Stricht as 'plaque cuticulaire;' in the adult organ by Schwalbe ('87) as 'ellipsoider Einschlusskorper,' by Joseph ('00) and v. Spee ('01) as 'Kopfeinschluss,' and by Held ('02) as 'Kopfkorper.' 3) A change in the direction of the phalanx process and the intracephalic rootlets of its fibrils. Previously (fig. 1) inchned almost vertically, this fibrillar bundle gradually takes a more obhque course (fig. 3, oph), becoming in time nearly horizontal (figs. 4 and 6, oph) not only outside the head, but also within it, the fibrils occupying its superficial part. This alteration is caused doubtless by the shortening and considerable enlargement of the head and constitutes a striking evidence that this enlargement is the result not only of a sheer expansion, but also of a process of stretching of its lower parts in a more horizontal and radial direction, as if pushed upward by the strain of the elongating pillar body. At the same tune, this pressure involves a conspicuous shortening of the previous cephahc segment. The peculiar change in the direction of the phalanx process has been observed by N. Van der Stricht and by Held ('09).

Inner pillars

In the adult organ of Corti the superficial portion of the inner pillar can be divided into three parts:

The apex, or 'Kopfplatte' of Held, the ' Innenpfeilerzellenschnabel' of v. Spee and Kolmer ('09), the 'plaque cephahque ou membrane fibrillaire' of N. Van der Stricht. This is a very thin, quadrilateral membrane (fig. 13'), elongated radially and stretched between the apices of the sustentacular cells (originally the outer pillars) and the sensory cells {oh') of the first outer row and the apices of the supporting {is') and acoustic {ih) elements of the inner row of hair cells. It constitutes a part of the membrana reticularis and is fibrillar in structure, the fibrils running parallel to the axis of the plate and in continuity with those of the head.

The so-called 'head' is formed of at least two segments, the smaller superficial one being in close contact with the head of the outer pillar. In the bat, the upper part appears to be reduced, from compression between neighboring elements, to a simple fibrillated lamella (fig. 13"'"'^, ihd), while in the lower part there is a thin cytoplasmic layer lateral to the fibrils (fig. 13^, ihd). In the white rat (fig. 17, ihd), the common rat (fig. 18, ihd), and particularly in the cat (fig. 9, ihd) twelve days after birth, this superficial lamella is obviously thicker, its lateral cytoplasmic layer being larger. In the bat (fig. 13"^, ihd) and other mammals this layer increases in breadth at the level of the lower part of the head, whence, without any demarcation, it blends with a larger, deeper segment. This is not connected with the outer pillar, but is situated below the head of the latter. It is a little shorter than the superficial segment and gradually tapers and continues with the body (ipb) of the pillar.

During the first stage of its development the future head of the inner pillar is a four-sided, somewhat flattened prism (figs. 1, 2, and 3, ip), nearly uniform in diameter, although tapering to its apex. It is composed of a granular or homogeneous cytoplasm and a bundle of fibrils, which occupy the medial side of the lower part of the prism and the central area of its superficial portion where, in the earliest stages of development, the fibrils are arranged in the fonii of a hollow tubule (fig. 3, ip) which later gives rise to a solid bundle. During the second stage of development the future head undergoes no very marked changes. By compression from the outer pillar head its superficial segment becomes somewhat thinner — lamellar in shape (figs. 4 and 6, ip) — while its lower segment maintains its previous size or enlarges slightly in the neighborhood of the pillar body. At the same time the transformations above mentioned are occurring in its cytoplasm in the proximity of the obturator septa. In order to clearly recognize the lamellar shape of the superficial segment of the head, cross-sections are needed. A longitudinal fibrillation as illustrated in figures 7 and 8 {ihd) indicates an oblique or more or less longitudinal section of the pillar, and such preparations are liable to misinterpretation.


The most remarkable changes occur at the level of the free apices of the inner pillars, the summit of the pillar scaffolding. The gradual development of the head of the outer pillar, situated just beneath this summit, produces a radial extension of the latter, and the transformation of a very small square field (figs. 1, 2, and 3, aip) into a long narrow fibrillated membrane or headplate. This gradual extension is clearly shown in figures 4 (aip), 7 (ipl) and 8 (ipl\ ipl'^), whereas no enlargement in a spiral direction is noticeable. On measuring the radial diameters of the fibrillated head-plates in figures 3, 4, 7, and 8, and comparing them with the radial diameters of those portions of the membrana reticularis included between the plates and the outer border of the apices of the third row of acoustic elements, it is found that the former are respectively represented by about 1/11, 1/2.75, 1/2, and 1/1.64 of the latter. This statement gives a rather accurate picture of the rapid enlargement of the head of the outer pillar and the subsequent extension of the superficial inner pillar plate; that is, of the portion of the membrana reticularis formed by the latter during the development of the tunnel space.


From this description it is also evident, according to N. Van der Stricht (p. 610), that the extension of the apex of the inner pillar is due solely to a mechanical factor, a compression by the underlying enlarging head of the outer pillar. This view has been corroborated by Held ('09). He does not mention the description given by N. Van der Stricht but states (p. 212): Je mehr der Kopf des Aussenpfeilers sich bildet und in seiner Masse wachst, um so diinner wird iiber ihm die Kopfplatte des Innenpfeilers." In its extension the head-plate undergoes important structural changes. Originally foniied of a clear cytoplasmic field (figs. 1 and 3, aip) containing a diplosome or two central corpuscles, the elongating plate becomes subdivided into two zones, a lateral, small, clear zone, enclosing the diplosome (fig. 7), and a medial, more extensive, fibrillated one. This continues to lengthen and is composed of several parallel horizontal fibrils, which, close to the apex of the inner hair cells, are continuous with the more vertical fibrils of the subjacent head lamella. Such structures depend upon the extension of the head-plate in a definite direction, i.e., from a fixed point corresponding to the seat of the central corpuscle close to the outer hair cells, towards the inner acoustic elements.


Some sections tangential to the surface of the organ of Corti give pictures which prove that the head-plate is formed of two superposed planes, one deeper and fibrillated (figs. 8 and 9, ipP'-), the other more superficial, destitute of fibrils, and composed only of a clear homogeneous cytoplasm (ipl^), imperfectly enclosed by a part of the above-mentioned firm head-collar (fig. 8, ipl).

Structure of the heads of the inner and outer pillars in adult animals

The ultimate structural changes undergone by the heads of the pillars consist mainly in a broadening and extension of their collars. This band not only becomes thicker, but also extends over the head, to form the roof of the outer and inner pillar head (fig. 10, ohd, ihd). This roof appears to correspond to the 'plaque cultioulaire' of N. Van der Stricht. Due to such transformation, the collar becomes converted into a head cap, a firm exoplasmic zone which circumscribes a clear granular endoplasmic zone, except at the medial side of the inner, and at the lateral side of the outer pillar. In other words, the remainder of the previous cytoplasm having now become much clearer, occupies a cephalic notch (fig. 18,- ohd, ihd) which extends from the head roof towards the pillar body; the bottom and the lips of the groove are represented by the broadened head collar. The clear endoplasm filling up the notch is traversed by the fibrillar bundles of the heads, which in selected preparations stain deeply with iron hematoxylin.


The head of the inner pillar in the white rat (fig. 17, ihd) and in the conmion rat (fig. 18, ihd) is thus fonned of a superficial thinner, and a deeper, enlarged segment, both composed of a medial groove containing a lamella of fibrils and a lateral layer of firm, dark, homogeneous cytoplasm — the walls of the notch.


This lateral layer enlarged rapidly toward the lower pole of the head and tapers downward to blend with a small unifonn protoplasmic zone of the pillar body (fig. 17, ipb). In the cochlea of the adult bat, similar structures are seen (fig. 13'^, ihd), but near the surface of the head (fig. 13'""*^, ihd) only a very thin fibrillated lamella is recognizable. However, in vertical spiral sections showing the longitudinal fibrils throughout the length of the pillars, a part of the head cap is visible (fig. 16, ihd).


The notch of the outer pillar head enlarges from the roof towards the pillar body and presents a true asyimnetrical position (figs. 17 and 18. ohd), and so is the structure of the head cap itself. On cross-sections the lips of the groove differ in thickness, the apical (i.e., that turned toward the apex of the cochlea) being obviously thinner than the basal (i.e., that turned toward the base of the cochlea). The clear cytoplasm of the notch is traversed by the nearly horizontal fibrils of the phalanx process (fig. 17, ohd), the rootlets of which merge obliquely into the apical lip. The other bundle of fibrils, running vertically from the pillar body toward the surface of the head, also shows an asymmetrical position. On passing into the head this bundle proves to be bipartite, being formed of a smaller and a larger fasciculus (fig. 13'^-^, ohd). Within the lower and wider portion of the notch (fig. 13'^, ohd) the subdivision into two unequal fasciculi is more evident, and the two bands are more closely connected respectively with the apical and basaJ lip of the groove. At the level of the head roof the horizontal fibrillated bundle courses through the cleft between the two vertical fasciculi, each of which merges into its neighboring lip (figs. 13 and 17, ohd). Most of these asymmetrical structures may be recognized during the development of the head (figs. 4 and 7, op). In vertical spiral sections of the adult organ, the asynometry is very conspicuous. In figure 14 the apical surface (the surface turned toward the apex of the cochlea) of the head is clearly indicated by the course of the apical filament of the cells of Deiters {ap, d^) in the direction of the apex of the cochlea. In such sections (figs. 14 and 15) can be seen the clear, eccentric oval notch, which contains a cross-section of the horizontal bundle (op/i) and is outlined by a thinner apical border or wall, and a larger basal border, the bulk of the head. On penetrating into the head the fibrils of the pillar body (opb) become divided into two fasciculi, a thinner apical, and a broader basal one. The fonner seems to be shorter and its fibrils spread out obliquely through the corresponding lip; the latter is longer and its fibrils spread out fanlike (fig. 15, ohd) through the basal portion of the head, and seem to encroach upon the more homogeneous head roof. When the two systems of fibrils are not stained, the head roof can be more clearly seen to continue into the two borders of the notch. In the cochlea of young animals (fig. 10, ohd) the groove is much larger and its lips may be mistaken for sections through two different separate bodies, the 'ellipsoider Einschlusskorper' of Schwalbe and Joseph. These bodies do exist in earlier stages of development, but later, with the roof, they form one structure — the head cap.

The elongation of the phalanx process of the outer pillar will be dealt with in the next chapter.

The development of the spaces of Nuel

With the exception of very short references, such as those alluded to above, no investigations have been carried out to determine the formation of the spaces of Nuel. Fence the problem appears to be a very knotty one and abiiost insolvable.


In the cochlea of adult animals the largest of these spaces is represented by a spiral cleft between the outer pillars and the, cell bodies of the hair and supporting cells of the first outer row. This space may be termed the first space or the first spiral interstice of Nuel. Another cleft, which may attain considerable size, is the fourth space or spiral interstice of Nuel. This contains the phalanx processes of the cells of Deiters of the third row and is included between the hair cells of the third row and the so-called cells of Hensen. It is the 'external tunnel' of Held ('02). A second and a third space or spiral interstice of Nuel contain the phalanx processes of the cells of Deiters of the first and second rows, respectively, the former situated between the outer acoustic elements of the first and second rows, the latter between those of the second and third rows. The second and third spaces do not extend between the long subjacent cell bodies of the supporting elements.


Appearance of the first spiral space of Nuel. This doubtless develops before the others and before any trace of the tunnel of Corti. The first trace of its appearance may be seen rarely (fig. 1) before the enlargement of the future heads of the outer pillars, in the form of clear, vacuolated, prominent vesicles on the lateral surfaces of the outer rods of Corti. How these vesicles are produced is uncertain; they seem to be only transitory and appear rather abruptly, as though due to pressure within the clear fluid contained in the vacuolated medial zone of the outer pillars, and as though part of this fluid had been driven across the outer fibrillated zone of the cell to give rise to large prominent vacuoles. These are seen along the lateral surfaces of the intermediate, the basal, and occasionally even parts of the superficial portions of the outer pillars. In more advanced stages their outlines and connections with the secreting cells become indistinct, and the vesicles are replaced by a common fluid mass, pervaded by a few delicate trabeculae in process of disintegration or liquefaction (fig. 3, SN). This process is not unHke that of cytolysis by which the fluid of the tunnel is produced. It is noteworthy that a distinct outhne or a superficial membrane is never seen, either on the lateral surface of the outer pillars or on the medial surface of the inner pillars; so that under special conditions of intracellular pressure, fluid may exude and pass into intercellular channels. The cleft, filled up with this fluid, is the first space of Nuel. It enlarges gradually and extends toward the membrana basilaris, from which, even in the adult cochlea, it is separated by the lateral expansions of the feet of the outer pillars.


From this description it would appear that the initial dominant factor in the development of the cleft corresponds to a difference in pressure in two parts of the outer pillars: the large vacuolated medial zone, where clear fluid is being accumulated, and the surface of the fibrillated zone, where a pecuUar structure, the absence of a membrane, and a lower pressure, promote an exudation of fluid. In this respect a second important factor deserves due consideration, i.e., the shifting of the outer pillars. These structures originally are incorporated within the first row of outer hair cells, and although their extremities remain always fixed, their bodies are pushed inward and inside of the acoustic elements, so that at least a virtual, if not a true space appears below the first row of outer hair cells, between the nucleated portions of the cells of Deiters of the first row (fig. 1, d^) and the outer pillars (op) . This virtual cleft contains a spiral bundle of nerve fibers and represents the future space of Nuel.


The enlargement of the space of Nuel is doubtless promoted by a third peculiarity — a change in the shape of the outer pillar. When the shifting of the is latter completed, and before any appearance of a cleft, the lateral surface of the rod of Corti is a plane, represented in a vertical or oblique section by a straight line. Along with the lateral extension of the foot upon the basilar membrane (fig. 3, op) and the appearance of the head (figs. 4 and 7, ohd), and by a considerable elongation of the intermediate portion (the future body, fig. 7, opb), which is only possible by virtue of a curvation, the straight line becomes markedly curved, its concavity being turned towards and embracing the cleft. This may be a more important factor than appears at first sight. Indeed, in previous investigations (in press) it has been noted that the shifting of the cells of Deiters may be " completed (that is to say, the sustentacular elements of the first outer row may be situated beneath their corresponding hair cells) before smy appearance of a tunnel space (fig. 9 of the previous paper), or even of the true space of Nuel. In such figures, the original straight line persists, although the heads of the outer pillars are large, but the lateral extension of the feet is delayed.


Structure and transformation undergone by the phalanx processes of the outer pillars. Should any doubt be entertained as to the process of cytolysis along the lateral lower parts of the outer pillars, the structures and the transformation undergone by their phalanx processes afford striking evidence of such a Uquefaction. In the above description of these apical bands which unite the phalanges to the outer pillar heads, the most distinct constituent, the fibrillated bundle, alone has been mentioned. In the early stages of the development the band is composed of fibrils collected into a fasciculus, which is surrounded by a clear granular cytoplasm. Before the space of Nuel reaches the membrana reticularis this phalanx process proper is very short, being limited to the portion running between two neighboring hair cells (figs. 4, oph; 8 and 11, oph), and the portion lying under the phalanx itself (figs. 8 and 11, opU^). In other words, the enlarged head contains the longest part of the fibrillar bundle (figs. 8, oph'^; fig. 11, oph, oph'^) and covers completely the head plate of the inner pillar. The roof of the developing space of Nuel is made up of two strata, the lateral, thinnest part of the outer pillar head (fig. 11, oph; compare with figs. 6 and 12), and the lateral part of the superficial striated membrane (fig. 8, ipl). When the first interstice of Nuel has attained its entire extent in the adult organ its roof is composed of the lateral part of the head plates of the inner pillars (figs. 13', 17, and 18, ipl) strengthened by equidistant, parallel, fibrillated bundles, portions of the ultimate phalanx processes (figs. 13, 17, and 18, oph), which run in an oblique direction toward the spiral rows of pillars (fig. 13).


Figures 13', 13, and 13' illustrate the structures of this roof at these successive levels in the adult organ of Corti. Between the apices of the inner hair cells (ih) and the outer sensory elements {oh') they show respectively a superficial plane — the striated head-plates of the inner pillar cells (fig. 13', ipl") — an intermediate plane composed of parts of the preceding plates (fig. 13, ipl) and parts of oblique subjacent fibrillar bundles, and a deeper plane (fig. 13) showing from the axial to the lateral side, the row of fibrillated lamellae, heads of the inner pillars (ikd), the row of outer heads," a gap nearly as large as the preceding row and bridged across by equidistant fibrillar bundles {oph), entirely devoid of clear cytoplasm. The gap is the upper floor of the space of Nuel {SN'), which is covered by the equidistant bundles and the lateral part of the uninterrupted striated membrane, formed of the head plates of the inner pillars. This description is corroborated by vertical spiral sections. In figures 14 and 15, above the heads of the outer pillars (ohd), is seen a dotted, very delicate membrane {ipl"), subdivided into short segments by coarser spots. This is the fibrillated membrane formed of the head-plates of the inner pillars ; the fibrils have been cut transversely and the spots represent the sections of terminal bars which separate the plates. This dotted membrane extends over the neighboring space of Nuel {SN') and covers equidistant coarse granules (op/i'^'), the cross-sections of the phalanx processes of the outer pillars. In figure 15 is seen, above the dotted line, a very fine, pale, uniform covering, which doubtless represents the homogeneous superficial zone {ipl') already mentioned.


The phalanx process of the outer pillar, represented in early stages by two portions, is formed in the adult cochlea of three segments, the original two — a subphalanx (fig. 13, oph') and an intercellular segment (oph) which courses between two hair cells— and a subsequently developed one, the submembranous stalk {oph) which is derived from a part of the original intracephahc bundle (fig. 11, oph). Indeed, transitional stages can be observed. In figure 11 an uninterrupted extracephahc clear protoplasmic layer, a kind of a pale veil, developed from the embryonic heads of the outer pillars (fig. 8, ohd) by a process of differentiation, unites three upper fasciculi (fig. 11, oph) and assumes a festooned appearance around three lower bundles. This festooned appearance has been observed by N. Van der Stricht in the cochlea of a guinea-pig one day after birth. This investigator described (p. 641) each festoon as une sorte de voile triangulaire a sommet dirige vers la rangee des cellules acoustiques externes et a base en continuity avec la tete du piUer externe," and beheves that the veil corresponds to the "Schwelling des Aussenpfeilerschnabels" of v. Spee. This clear protoplasmic sheath of the extracephahc bundle is seen also in vertical spiral sections (fig. 10, oph). In the cochlea of a dog about four or five months of age it seems to persist, but unquestionably disappears by a process of cytolysis in the adult bat (fig. 13 ^ 14 and 15, oph') and the white rat (fig. 17). Evidences of such disintegration are shown in figures 9 and 10 (cy). The result is that a part of the clear cytoplasm which belongs to the originally enlarged heads of the outer pillars (fig. 8, ohd) undergoes a process of liquefaction. A portion of the intracephalic horizontal fibrillar band becomes free or at least partially destitute of protoplasm, so that the fluid of the large space of Nuel, in direct communication with that of the tunnel space through the wide interpillar clefts, comes in close contact with the very fine, superficial, fibrillated membrane of the head of the inner pillars. This membrane separates the fluid in question from that of the cochlea duct.


The physiological importance of these structures is evident, for the vibratory waves may be readily transmitted from one to another fluid through the intermedium of the striated membrane. As regards the transmission of vibrations from thefibrillated basement membrane of the membrana basilaris to the contents of the first interstice of Nuel, it is noteworthy that the fonner, at the level of the floors of the Nuel and tunnel's spaces, is separated from the latter by only a very thin cytoplasmic covering which belongs to the laterally expanded feet of the outer pillars and to the feet of the outer and inner pillars. Hence the transmission can be readily carried out.


Development of the second, third, and fourth spaces of Nuel. As shown in previous investigations, the phalanx processes of the cells of Deiters of the first and second rows are represented, in the earliest stage of development, by long apical segments of the cell bodies, which segments are included, respectively, in the second and third row of outer hair cells, within which they run between two neighboring acoustic elements. In length these apical segments agree with the hair cells. Due to the rapid enlargement of the latter, the apical segments are pressed out from their original row and shifted into interspaces; those of the first row of Deiters cells reaching the second future interstice of Nuel and those of the second row of Deiters cells reaching the third interval. All around the phalanx processes of the cells of Deiters of the third row, which remain in situ between the third row of sensory elements and the cells of Hensen, will appear the large fourth interstice.


Before the appearance of any space the phalanx process is composed of a clear cytoplasmic sheath, enclosing a darker, axial, mitochondrial strand, which by juxtaposition and fusion of the chondrioconts becomes gradually transformed into an axial, fibrillated filament. The process is larger at its base, which issues from the nucleated cell body, and tapers to the superficial membrana reticularis.


In a kitten nine days old, at the level of the apical spiral turn of the cochlea, the second, third, and fourth sustentacular interstices are still filled up with the unmodified phalanx processes, so that intercellular spaces are absent. In the- second turn, narrow channels appear and are somewhat larger near the surface of the epithelium than towards the base of the processes. Inversely the processes have become reduced in diameter at the expense of their clear protoplasm. At the level of the basal or third turn the enlargement of the spaces of Nuel and the reduction in size of the cytoplasmic sheath of the phalanx processes are much more marked. It must be noted that the thinning out of the latter is not the result of a sheer concomitant elongation, for these alterations are accompanied by a considerable elongation of the nucleated cell bodies of the sustentacular elements, involving a subsequent shortening of the supported hair cells, hence of the neighboring phalanx processes. These become more slender on account of a process of elaboration and secretion and a subsequent extrusion of clear fluid from the protoplasmic sheath. Whether, as many preparations seem to prove, this discharge is accompanied by a process of true cytolysis is uncertain, for these structures are very delicate and the shrinkage caused by the reagents might give rise to artefacts liable to misinterpretation.


The fourth space of Nuel de^'elops in the same manner as the second and third, and when it appears, is but little larger than the others. It is occupied by the apical processes of the ceUs of Deiters of the third row. Originally as long as the neighboring sensory elements, these processes are more numerous (previous, paper) and larger than those of the first and second supporting rows. Situated outside the hair cells of the third row, they are not squeezed and impeded in their lateral expansion like the others. It is not to be wondered at that the products of secretion or cytoplasmic disintegration around the apical fibrillated bundles are more abundant and result in an expansion of the fourth interstice. Nevertheless, the process of development is identical with that of the two preceding spaces and therefore the application of a special term, external tunnel, to designate this formation is unnecessary. However, there can be no doubt that phalanx processes of the cells of Deiters of the third row retain their cytoplasmic sheath much longer than the others, and may show parts of it in the adult cochlea, as pointed out by Held ('02) for the 'apical type' of these cells in guinea pig, cat, dog, and even the mouse. In such cases these processes are in closer connection with the outer wall of the space than with the medial.


In the basal spiral turn of the cochlea of a kitten twelve days after birth (fig. 12), the floor of the second, third, and fourth spaces of Nuel is fonned by parts of segments of the cel^s of Deiters {d\ d^\ d) supporting their corresponding sensory elements {oh\ o}v\ oh). The medial and lateral boundaries of the second interstice are represented, respectively, by the lateral surfaces of the hair cells of the first row and the medial surfaces of those of the second. The medial and lateral boundaries of the third interstice are represented, respectively, by the lateral surfaces of the hair cells of the second row and the medial surfaces of those of the third. The adjoining surfaces of the acoustic elements of each sensory row are separated by narrow clefts, through which all of the spaces of Nuel intercommunicate. These channels, originally occupied by the phalanx processes of the sustentacular cells (those of the first sensory row being the superficial segments of the outer pillars), are liberated after the shifting of the phalanx processes. At first very narrow and virtually obliterated by the process of enlargement of the hair cells, these intercellular clefts become wider by the reduction in size of the acoustic elements. The medial and lateral boundaries of the fourth interstice of Nuel are represented, respectively, by the lateral surfaces of the sensory elements of the third row and the medial surfaces of the so-called cells of Hensen, which, according to previous investigations, should be held as atrophied hair cells {aoh).


The roofs of the spaces of Nuel are made up of parts of contiguous apices of the supporting elements (fig. 9). The roof of the second space is formed of alternating lateral and medial segments of phalanges of the outer pillar {aop), and the Deiters cells of the first row {d^). The roof of the third space is composed of alternating lateral and medial segments of the phalanges of Deiters cells of the first (d') and second row (rf'Ol the roof of the fourth space is represented by the lateral segments of the phalanges of Deiters cells of the second row and the apices of those of the third (c?'"). The roofs of the intercellular clefts between the hair cells of the first, second and third sensory rows are formed, respectively, of the medial or constricted part of the phalanges of the outer pillars (fig. 9, aop), the middle part of those of Deiters cells of the first row (d'), and the middle pari of those of Deiters cells of the second row (d^'). The fluid contents of the first, second, third, and fourth spaces of Nuel intercommunicate through the intercellular clefts. Like the fluid of the first interstice, that of the others is separated from the contents of the cochlear duct only by very thin membranes, partially fibrillated, since the fibrillar bundles of the phalanx processes of the sustentacular elements spread over the under surface of their corresponding phalanx, according to the investigations of Held ('02) and of N. Van der Stricht. Such structures doubtless are able to promote the propagation of vibratory waves from the membrana basilaris to the fluid and the membrana tectoria within the cochlear canal.

Summary

  1. The tunnel space is developed around the spiral nerve bundle, which runs between the nucleated portions of the inner and outer pillar cells. It is originally an intercellular cleft, the fluid contents of which are elaborated in the vacuolated cytoplasm of the pillar cells and discharged into the adjoining space. Parts of this secreting protoplasm undergo a process of cytolysis or liquefaction, so that the cleft enlarges and the fluid contents increase in quantity at their expense.
  2. The tunnel extends upward along the vacuolated zones of the intermediate portions of the pillars. This clear cytoplasm also disappears by a similar process of secretion and cytolysis. Ultimately the intermediate segments of the pillars become transfonned into pillar bodies, reduced almost to their fibrillar apparatus of support, the outer being represented by thin cyhndrical strands which are separated by large intercellular clefts, and the inner, lamellar in shape, being composed of a medial, thin, fibrillar lamella and a lateral narrow zone of clear cytoplasm.
  3. In the earhest stage of development the heads of the outer rods of Corti are represented by thin tapering segments, nearly as long as the neighboring outer hair cells. They are characterized by the presence of two fibrillated bundles, the intracephalic rootlets of the fibrils of the phalanx process and the apical intracephalic extremities of those of the future pillar body. Moreover, pecuhar structures, a more homogeneous cytoplasm and a system of obturator septa between the two rows of the future outer and inner heads and between the contiguous surfaces of the heads of each row, constitute important features, which enable one to recognize the *nature of these apical segments.
  4. In a more advanced stage the long superficial segment of the outer pillar enlarges rapidly into a shorter prismatic mass, the head proper, within which develop, in contact with the obturator septa, a firmer exoplasmic collar, and ulthnately a head cap, enclosing imperfectly a clearer endoplasmic mass, which is traversed by the nearly horizontal and the vertical fibrillated bundles, the rootlets of the fibrils of the phalanx process and the apical ends of those of the pillar body, respectively.
  5. The original head of the inner pillar is represented by a small, four-sided, somewhat flattened prism. Later by compression from the outer pillar's head, the superficial part of the prism assumes a more distinctly lamellar shape; the lower part enlarges and acquires its largest size at the level of the lower pole of the outer pillar's head, whence it tapers upward and downward. The so-called 'head' of the inner pillar extends down beyond that of the outer and furnishes a pad of support to the latter. The prismatic head is composed of a lateral uniform cytoplasm, which enlarges at the level of the lower, broader portion, and a medial fibrillated lamella. In contact with the obturator septa and within the homogeneous protoplasm the head collar develops.
  6. By compression from the underlying and enlarging head of the outer pillar, the free apex of the inner pillar undergoes a gradual and extensive elongation, becoming converted into a fibrillated, long head-plate. The constant position of the central corpuscle within this niembrane, close to the apices of the outer hair cells of the first row, proves that this elongation occurs in a definite direction, from the seat of the diplosome toward the apices of the inner hair cells.
  7. The heads of the outer pillars are asymmetrical in structure. The cephalic notch, imperfectly surrounded by the head cap, is traversed by two fibrillated bundles and has a parapical position; it is situated nearer to the apical surface of the head (i.e., the surface turned toward the apex of the cochlea) than to the basal, since the apical lip of the groove is thinner than the basal. The horizontal rootlets of the fibrillated phalanx bundle are also parapical and merge obliquely into the apical border of the notch. The vertical fibrillar cephalic bundle, arising from the pillar body, divides into two unequal fasciculi, a smaller and a larger, circumscribing a cleft through which run the horizontal fibrils. The smaller fasciculus merges into the apical lip of the notch and the larger one into the basal lip, the bulk of the head cap.
  8. The first space of Nuel, which, in the adult organ of Corti, is situated between the outer pillars and the outer hair cells and the cell bodies of the sustentacular elements of the first row, appears as an intercellular cleft, within which is accumulated a fluid discharge from the outer pillars. At the lateral surfaces of the latter project clear secretion vesicles, which undergo a process of cytolysis and liquefaction. The exudation of this fluid seems to be due to a difference of pressure, on the one hand within the medial vacuolated cytoplasmic zone of the outer rods of Corti, and on the other, at their lateral surface. It is promoted by the shifting of the embyronic pillars from the first spiral row of outer hair cells towards the inner rods of Corti, by the development of laterally enlarged segTiients at the tw^o extremities of the outer pillars, the foot and the head, and an elongation of the outer pillar bodies, only possible by virtue of an incurvation, the concavity being turned towards the cleft of Nuel.
  9. This process of cytolysis is manifest at the level of the heads of the outer pillars. Indeed the enlarged embryonic head is more bulky than the adult head, and the phalanx process, shorter in the earlier stages of development, later becomes longer. Originally the phalanx process is represented by two segments, a subphalangeal and an intercellular segment, the latter running between two hair cells. Later on, a submembranous segment appears. This lies beneath the lateral portion of the head plates of the inner pillars, and is developed from a lateral part of the enlarged outer head by a process of disintegration of clear cytoplasm, which encloses the horizontal intracephalic fibrillated bundle.
  10. The roof of the first space of Nuel in the earliest stages of development of this interstice is composed of two uninterrupted coverings, one superficial and very thin, the lateral portions of the head-plates of the inner pillars, the other deeper and much thicker, the lateral parts of the outer heads. In the adult cochlea this roof is made up of the same parts of the superficial head-plates and a largely interrupted covering, the equidistant submembranous segments of the phalanx processes.
  11. The second, third, and fourth spaces of Nuel are located, respectively, between the first and second, the second and third, and the third row of hair cells and the cells of Hensen (atrophied hair cells of a fourth row). These spaces do not extend do^^^l between the sustentacular elements, but communicate with each other and with the first space through clefts between the hair cells. These intercellular channels, originally occupied by the phalanx processes of the sustentacular elements, become free after the shifting of the latter into the neighboring medial spaces, the phalanx processes of the cells of Deiters of the third row remaining in situ.
  12. Each of these phalanx processes is composed of an axial, fibrillar filament and a peripheral, clear, cytoplasmic sheath. In the course of development this sheath becomes thinner and may disappear by a process of secretion, which gives rise to the fluid contents of the primitive second, third and fourth spaces of Nuel.
  13. The roofs of the second, third, and fourth spaces of Nuel and of the intercellular clefts between two neighboring hair cells of each sensory row are made up of delicate membranes, partially fibrillated, which betong to various parts of the phalanges of the sustentacular elements.
  14. The fluid contents of the tunnel and the first space of Nuel are separated from the fibrillated basement membrane of the membrana basilaris by a thin protoplasmic covering, beonging to the feet of the inner and outer pillar cells. They ntercommunicate through clefts between the outer pillars and communicate with those of the second, third, and fourth spaces of Nuel. The fluid of all the spaces of Nuel is separated from the endolymph of the cochlea duct by the roofs of these inter .stices, very thin membranes, entirely or partially fibrillated. Such structures doubtless promote the propagation of vibratory waves from the basilar membrane to the membrana tectoria, contained in the cochlear canal.


All the material and reagents necessary for the present investigations were suppUed by Dr. T. Wingate Todd, Director of the Anatomical Laboratory of the Medical School, Western Reserve University, Cleveland, Ohio. It affords the author great pleasure to express his deep gratitude to Dr. Todd.

Bibliography

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1869 tJber Entwickelung und Bau des Gehorlabyrinths nach Unter suchungen an Saugethieren. Dresden.

1872 Kritische Bemerkungen und neue Beitrage zur Literatur des Gehorlabyrinths. Dorpat. Deiters, O. 1860 Untersuchung uber die Lamina spiralis membranacea.

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1872 tJber den feineren Bau und die Entwickelung der Gehorschnecke der Saugetiere und der Menschen. Arch. f. mikr. Anat., Bd. 8.

Hardesty, I. 1908 The nature of the tectorial membrane and its probable role in the anatomy of hearing. Am. Jour. Anat., vol. 8, pp. 109-179.

1915 On the proportions, development and attachment of the tectorial membrane. Am. Jour. Anat., vol. 18, pp. 1-73.

Held, H. 1902 Untersuchungen fiber den feineren Bau des Ohrlabyrinths der Wirbeltiere. I. Zur Kenntnis des Corti'schen Organs u. der iibrigen Sinnesapparate des Labyrinths bei Saugetieren. Abhandl. d. k. Sachs. Ges. d. Wiss., Math.-phys. Kl., Bd. 28, S. 1-74.

1909 Untersuchungen liber den feineren Bau des Ohrlabyrinths der Wirbeltiere. II. Zur Entwicklungsgeschichte des Corti'schen Organs und der Maculaa acustica bei Saugetieren und Vogeln. Abhandl. d. k. Sachs. Ges. d. Wiss., Math-phys. Kl., Bd. 31, no. 5, S. 195-294.

Hensen, V. 1863 Zur Morphologie der Schnecke des Menschen und der Sauge thiere. Zeitschr. f. wiss. Zool., Bd. 13, S. 481-512.

1871 tJber Boettcher's Entwicklung und Bau des Gehorlabyrinths nach eigenen Untersuchungen. Arch. f. Ohrenh., Bd. 6, S. 1-34. Joseph, H. 1900 Zur Kenntnis der feineren Bau der Gehorschnecke. Anat. Hefte, Bd. 14, S. 447-486.

KiSHi, J. 1902 tiber den Verlauf und die periphere Endigung des Nervus cochleae. Arch. f. mikr. Anat., Bd. 59, S. 144-179.

Koelliker, a. 1859 Handbuch der Gewebelehre des Menschen. 3 Aufl., Leipzig. Kolmer, W. 1909 Histologische Studien am Labyrinth mit besonderer Be riiscksichtigung des Menschen, der Affen und der Halbaffen. Arch. f. mikr. Anat., Bd. 74, S. 259-310.

NuEL, J. P. 1878 Recherches miscroscopiques sur 1 'anatomie du lima^on des mammiferes. Mem. Couronnes et mem. des savants etrangers publics par I'Acad. roy. de Belgique, T. 42, n. 1.

Prentiss, C. W. 1913 On the development of the membrana tectoria with reference to its structure and attachments. Am. Jour. Anat., vol. 14, pp. 425-458.

Pritchard, U. 1876 The development of the organ of Corti. Journ. Anat. and Physiol., vol. 13, pp. 99-103, and Quart. Journ. Micr. Sc, n. s., vol. 14, no. 64, pp. 398-404.

Retzius, G. 1884 Das Gehororgan der Wirbelthiere. II. Das Gehororgan der Reptilien, der Vogel und der Saugetiere. Stockholm.

RiCKENBACHER, O. 1901 Untersuchungen liber die embryonale Membrana tectoria der Meerschweinchens. Anat. Hefte, I. Abth., Bd. 16 (Inaug. Dissert. Basel.)

Rosenberg, E. 1868 Untersuchungen liber die Entwickelung des Canalis cochlearis der Saugethiere. Diss. Dorpat.

ScHULTZE, M. 1858 tjber die Endigungsweise des Hornerven im Labyrinth. J. Mliller's Arch. f. Anat., Phys. u. wiss. Med., S. 363-381.

ScHWALBE, G. 1887 Lehrbuch der Anatomie des Sinnesorgane. Hoffmanns Lehrbuch der Anatomie des Menschen. 2. Bd., III. Abt., Erlangen.

V. Spee, F. 1901 Mitteilungen zur Histologie des Corti'schen Organs in der Gehorschnecke des erwachsenen Menschen. Verh. d. anat. Gesellsch., Bonn., S. 13-23.

Tafani, a. 1884 L'organe de Corti chez les Singes. Arch. ital. de Biol., vol. 6, pp. 207-247.

Van der Stricht, N. 1907 Lehistogenese des parties constituantes du neu roepithelium acoustique. Verh. d. anat. Gesellsch. Wurzburg, S. 158-170.

1908 L'histogenese des parties constituantes du neuroepithelium acoustique, des taches et des crates acoustiques et de l'organe de Corti. Arch, de Biol., T. 23, pp. 541-693.

Van der Stricht, O. 1918 The genesis and structure of the membrana tectoria and the crista spiralis of the cochlea. Contributions to Embryology (Carnegie Inst, of Washington), no. 227, pp. 55-86.

(In press.) The arrangement and structure of the sustentacular and hair cells in the developing organ of Corti. Contributions to Embryology (Carnegie Inst, of Washington), no. 272.

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Description Of Plates

All figures were outlined with a Zeiss camera lucida, at the level of the stage of the microscope, with the aid of a Zeiss ocular no. 3, and 2-mm., homog. immersion. Apert. 1.30, except figures 1, 2, 3, 4, 5, and 7, which were outlined with Zeiss ocular no. 1.

General Abbreviation

aip, apices of embryonic inner pillar cells

aoh^^, apices or bodies of atrophied hair cells of an outer fourth spiral row

aop, apices or phalanges of the outer pillar cells

cy, cytoplasm in process of cytolysis

c?\ d", d"', apices or bodies of cells of Deiters, respectively, of the first, second, and third rows

H, apex of the cell of Hensen

ih, apices or cell bodies of the inner hair cells

ihd, heads of the inner pillar cells

ij), inner pillars

iph, bodies of the inner pillars

ipl, head-plates of the inner pillars

ipl^, ipV\ respectively, the superficial homogeneous zone and the deeper fibrillated zone of the head plates of the inner pillars

t's', is", apices or cell bodies of supporting cells, respectively, of the first and second inner rows

A, Nerve bundle passing through the foramen nervinum

A'", spiral nerve bundle running between the inner and outer pillar cells or within the tunnel space

iV"', spiral nerve bundle running between the outer pillars and the cells of Deiters of the first row

nd, apices of non-differentiated cells of the greater epithelial ridge

m, nuclei of non-differentiated cells of the greater epithelial ridge

nih, nuclei of inner hair cells

niji, nuclei of inner pillar cells

nis^, nis", nuclei of inner supporting cells, respectively, of the first and second rows

nop, nucleus of an outer pillar cell, seated near the head (abnormality)

oh^, oh", oh}", apices or cell bodies of outer hair cells, respectively, of the first, second, and third rows

ohd, heads of outer pillars

op, outer pillar cells

oph, bodies of outer pillars

oph, phalanx processes of outer pillars

oph^, opd", opd"^, op<^^, respectively, the subphalanx, intercellular, submembraneous segments and the intracephalic roots of the phalanx process of the outer pillar

pd^, pd", pd"^, phalanx processes or apical filaments of cells of Deiters, respectively, of the first, second, and third rows

SN, SN\ the first space of Nuel

t, developing tunnel space

T, tunnel space

tb, terminal bars or obturator septa

VS, vas spirale

Plate 1

EXPLANATION OF FIGURES

1 Section tangential (and somewhat oblique) to the surface of the organ of Corti, through the second (middle) turn of the cochlea. New-born kitten. Fixation: osmic acid, 1 per cent aqueous solution for about one hour, followed by immersion in Zenker's fluid. Stain: Iron hematoxjdin, Congo red, light green.

2 Section tangential to the surface of the organ of Corti, through the second turn of the cochlea. Kitten 3 days, 12 hours after birth. Exposure of the cochlea, the bony wall of which had previously been provided, with two small openings, to vapors from a 2 per cent aqueous solution of osmic acid for approximately one hour, and subsequent treatment of the piece by trichloracetic acid 5 per cent in water. Iron hematoxylin, Congo red.

3 Section tangential to the surface of the organ of Corti, through the basal portion of the second turn of the cochlea. Dog 3 days, 18 hours after birth. Zenker's fluid. Iron hematoxylin, Congo red.

4 and 5 Sections tangential to the surface of the organ of Corti, through the basal portion of the second turn of the cochlea. Kitten 3 days after birth. Solution of trichloracetic acid 5 per cent in water. Iron hematoxylin, Congo red, light green.

6 Radial vertical section of the organ of Corti through the third (basal) turn of the cochlea. Kitten 3 days, 12 hours after birth. Exposure of the cochlea to vapors from a 2 per cent aqueous solution of osmic acid and subsequent treatment of the piece by trichloracetic acid 5 per cent in water. Iron hematoxylin, Congo red.


Plate 2

EXPLANATION OF FIGURES

7 Section tangential to the surface of the organ of Corti, through the third turn of the cochlea. Dog 3 days, 18 hours after birth. Solution of trichloracetic acid 5 per cent in water. Iron hematoxylin, Congo red.

8 Section tangential to the surface of the organ of Corti, through the third turn of the cochlea. Kitten 5 daj'^s, 12 hours after birth. Solution of trichloracetic acid 5 per cent in water. Iron hematoxylin, Congo red, light green.

9 Section tangential to the surface of the organ of Corti, through the basal portion of the first (apical) turn of the cochlea. Kitten 12 days after birth. Osn.ic acid 1 per cent aqueous solution for about one hour, followed by immersion in a 5 per cent aqueous solution of trichloracetic acid. Iron hem.atoxylin, Congo red.

10 Vertical spiral (parallel with the spiral rows) section of the organ of Corti, through the second turn of the cochlea. Kitten 11 days after birth. Osmie acid 1 per cent aqueous solution for a^bout one hour, followed by immersion in a 5 per cent aqueous solution of trichloracetic acid. Iron hematoxylin, Congo red.

11 Section tangential to the surface of the organ of Corti, through the basal portion of the second turn of the cochlea. Kitten 12 days after birth. Osmic acid 1 per cent aqueous solution for about half an hour, followed by immersion in a 5 per cent aqueous solution of trichloracetic acid. Iron hematoxylin, Congo red, light green.

12 Section tangential to the surface of the organ of Corti, through the third turn of the cochlea. Kitten 12 days after birth. Osmic acid 1 per cent aqucd»us solution for about half an hour, followed by immersion in a 5 per cent aqueous solution of trichloracetic acid. Iron hematoxylin, Congo red.


Plate 3

EXPLANATION OF FIGURES


13 Sections tangential to the surface of the organ of Corti through the third turn of the cochlea. Adult bat (Vespertilio fuscus). Zenker's fluid. Iron hematoxylin, Congo red. The figures illustrate structures at five successive levels of the organ of Corti.

14 and 15 Vertical spiral sections of the organ of Corti, through the second turn of the cochlea. Adult bat (Pipistrellus subflavus). Bouin's fluid. Iron hematoxylin, Congo red, light green.

16 Vertical spiral section of the organ of Corti through the second turn of the cochlea. Adult bat (Pipistrellus 'subflavus). Trichloracetic acid. Iron hematoxylin, Congo red, light green.

17 Section tangential to the surface of the organ of Corti. through the second turn of the cochlea. Adult white rat. Trichloracetic acid. Iron hematoxylin, Congo red, light green.

18 Section tangential to the surface of the organ of Corti, through the third turn of the cochlea. Adult rat (Mus decumanus). Bouin's fluid. Iron hematoxylin, Congo red, light green.



Cite this page: Hill, M.A. (2024, April 23) Embryology Paper - The development of the pillar cells, tunnel space, and Nuel's spaces in the organ of Corti (1919). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_development_of_the_pillar_cells,_tunnel_space,_and_Nuel%27s_spaces_in_the_organ_of_Corti_(1919)

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© Dr Mark Hill 2024, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
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