‘2 a F rH * i! ‘ate ee * aa ‘ S . a] eee ‘ ¢ ‘ait ‘ - “> o ‘ 4 2-6 2-4 26 8 ~ CG > a2 “s » i * ui 7% . Pe oe ee & i) * 22 @¢ 2s enetet AG =é. * ee . > - 2 x + fc a J * * aK > yt ee . eee > * wn oe a ¢ vane .? 2 Se e o*# e + ee rs < ast Te * <4 2 -« ta 4 6 a a we ¢ “sr aes . —- Ce ee “2 ¢ * ’ + 4 Ca e-s-@ 6 . -". « . . Sides « Pu % 2a te Pd + + s*-e * * aa (a MARA sae Sete oo 2 * a * te 4 oes i ae? - ae ee ane 4 ** A 4-4 2 ie ke of 4-¢ * ee * «4 i %% t~ ¢ * + -* *- s 6 ae e « ¢ * or ne a 2 4 ofa *? 2-22 2+ 2? ree ie * «of @ er @ * aie 86 ** * * as ee ee ee 2 4-82-44 ¢ 24-2 © erGoetaerr ah? 2 os « * err eres eee “o 4 i 72 8 6-o 22-6 2&8 a Pa * eed t 2-2. ¢-¢ o-* ae 2 a« ona « e - Ct . teat ?-2-<¢ 2e- 2 ¢ +26 a4 ‘ eee #2 © & a a ea ee ee ew ee en * > Ses 24a 6 98 een ++ . © * OS RRS * >? im gener a ate te as 4<@e or 2 , 4a-nneeesat ~ - “« 2 2 a 2 2 i *-ee Ne & hed = ers e © 2 * * o-* - e x <: -” ~@ 2-0 @ ee oon “ea et 24 * * +2 +e ¢ ¢ A PRP PDAS *. ee *. ware ary hey ' sy i “ b i A * 4 a gtk THE JOURNAL OF COMPARATIVE NEUROLOGY EDITORIAL BOARD Henry H. DoNALDSON ApoLF MEYER The Wistar Institute Johns Hopkins University J. B. JoHNsTON Outver 8S. STRONG University of Minnesota Columbia University C. Jupson HERRICK, University of Chicago Managing Editor VOLUME 26 1916 PHILADELPHIA, PA. THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY COMPOSED AND PRINTED AT THE WAVERLY PRESS By THE Witiiams & WILKINS Company BatrmoreE, Mp., U.S. A. CONTENTS 1916 No.1 FEBRUARY W. J. Crozimr. Regarding the existence of the ‘common chemical sense’ in vertebrates 1 Witu1am F. Auten. Studies on the spinal cord and medula of cyclostomes with special reference to the formation and expansion of the roof plate and the flattening of the spinal cord. Eighty-seven figures... St iidw tidee wie seek See side he ghk Ete Od 9 PerctvaL Baitry. Morphology of the are plate of the fore-brain and the lateral choroid plexuses in the human embryo. Thirty-one figures... . ; ay eee 79 No. 2 APRIL Leste B. Arry. The movements in the visual cells and retinal pigment of the lower vertebrates. Thirty-seven figures.... 4 Se BE H. Saxton Burr. Regeneration in the brain of Ambly stoma. I. The fore-brain. Four RR bs Warn are Sot cao alan a'ornhd do» Rx, «os >:s +> - geile di o 16s werha tls dec 208 No.3 JUNE Lesuiz B. Arey. The function of the efferent fibers of the optic nerve of fishes. Twelve SAREE TU OMENS RRL ALG oo 5 oa 5 he a Savdsn « « « » «GUMS atau Clad sowie Mpls es SiS 213° G. E. Coeguinty. Correlated anatomical and physiological studies of the growth of the nervous system of Amphibia. II. The afferent system of the head of Amblystoma. Seventy-nine figures. . 8 On Ee oe eer ere ee. ee 247 R. A. Kocurr. The Bieet ca activieea on phe histalamteal serhis ture of nerve cells......4 241 No. 4 AUGUST Lesuiz B. Arey. The influence of light and temperature upon the migration of the ret- inal pigment of Planorbis trivolvis. Nine figures.................sccccsec eee r cree 359 A. T. Rasmussen and J. A. Myers. Absence of chromatolytic change in the central nerv- ous system of the woodchuck (Marmota monax) during hibernation. Six figures BS PMAE ES hg tecerd eae eta 9 Siew Sign =/<) SRS other ti A Sw es pee ved > 2 ide RY 391 J. J. Kenean. A study of a Plains Indian brain. Eight figures.................-....- 403 Martin R. Coase. An experimental study of the vagus nerve. Four figures. ......... 421 Lestiz B. Anny. Changes in the rod-visual cells of the frog due to the action of light. Two figures. AE ee aa Se Ee ee ak 429 iv CONTENTS H. H. Donatpson. A preliminary determination of the part played by myelin in reduc- ing the water content of the mammalian nervous system (albino fat). One chart.. 448 W. J. Grozipr. The taste of acids. Two figuresitiis. hes t cn. 1 > > «ener ne peo 453 No.5 OCTOBER Wiutiam A. Hitron. The nervous system of pyenogonids. Twenty-one figures....... 463 J. B. Jonnston. Evidence of a motor pallium in the fore-brain of reptiles. One figure. 475 J. B. Jounston. The development of the dorsal ventricular ridge in turtles. Twenty- OVO TOTITOR 2 ooh. si 8 oe ccc acackle-« 5 pine cre eerie Sete cae Ne Sento te ta a 481 PercetvAL Barney. The morphology and morphogenesis of the choroid plexuses with especial reference to the development of the lateral telencephalic plexus in Chry- semys marginata. ‘Twenty-seven figures... 000.5 oa cee i ae 507 Sumner L. Kocu. The structure of the third, fourth, fifth, sixth, ninth, eleventh and twelfth cranial nerves. Five figures: 22.053. 625 hls - oe ee ee 541 CAROLINE BurtING THompson. The brain and the frontal gland of the castes of the ‘white ant,’ Leucotermes flavipes, Kollar. Twenty-six figures...................... 553 CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. No. 268. REGARDING THE EXISTENCE OF THE ‘COMMON . CHEMICAL SENSE’ IN VERTEBRATES! W. J. CROZIER I. In a recent paper by Coghill (14) exception is taken to the view held by Herrick (’08), Sheldon (’09), Cole (’10), and Parker (’12), that there is in vertebrates a set of receptors (free nerve terminations) which are responsible for reactions to rather high concentrations of chemicals when applied to moist periph- eral surfaces. The theoretical significance attached to this ‘common chemical sense’ (Parker, 712) makes it appropriate to bring forward certain facts which may to some extent serve to clear up the situation. The evidence adduced by Coghill is this: that in Amblystoma larvae, before the establishment of the definitive nervous system, tactile and chemical irritability appear concomitantly and, so far as studied, remain completely parallel in development; and that if larvae be placed in irritating solutions of hydrochloric acid (as dilute as jo 900), the epithelial cells become visibly disrupted, the reaction of the larvae being correlated with the disintegration of the skin and a gradual disappearance of reaction to tactile stimuli. His conclusion, that there is not present in the skin any normal irritability to acid, is probably correct for the larvae experi- mented upon; but it by no means follows that the same con- ditions obtain in the adult. Coghill argues, however, that the permanently embryonic cells of the germinative layer in the skin of amphibians and fishes when exposed to the high con- centrations of acid (,)) used by Parker and Sheldon act as do the superficial ciliated cells of the Amblystoma larval skin. Because they are bound down by a ‘‘thick, less sensitive, and 1 Contributions from the Bermuda Biological Station for Research. No. 40. 1 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 1 FEBRUARY, 1916 2 W..J. CROZIER more impervious layer of cells,’’ the disintegration of the sensi- tive cells would cause exceedingly violent disturbances, pre- sumably affecting tactile and pain terminals; and further, that it has not been satisfactorily demonstrated that it is possible (as claimed by Sheldon and by Cole) to effect a separation of tactile and ‘common chemical’ irritability in fishes and amphibians. . This general argument is somewhat strengthened by the fact (to which Coghill does not refer) that aquatic vertebrates pos- sessing a soft slimy skin—cyclostomes, eels, catfish, Necturus— are known to react to local irritation by chloroform and other substances by the expulsion of mucus and even of entire gland cells. The reaction time of this response is not known accu- rately, though it is judged to be much greater than that, for example, of the catfish as a whole when stimulated by hydro- chlorie acid applied to its trunk region; still, it is not incon- ceivable that mechanical. deformations brought about in this way might provide stimuli for tactile nerve endings, provided the mucus response were sufficiently sharp and prompt. II. The exact manner in which solutions are to produce the hypothecated effects upon the cells of the germinative layer is left entirely untouched by Coghill. The rapidity of the re- sponses given by the catfish and Necturus immediately negatives the idea that osmotic transfer of water is the agency of stimu- lation. In the case of the spinal frog, as studied by Braeuning (04), Loeb (’05), and Cole (10), though the reaction times are rather long, there is abundant evidence that other than osmotic factors are at work. The most important point which arises for consideration is the extent to which chemical stimulants actually penetrate the skin, 1.e., the degree to which the cells of the germinative layer are exposed to the action of the stimu- lant. In addition to the rapidity of the reactions under discus- sion, it is to be remembered that they are excited by acids, salts, alcohols, and a variety of other-substances. That the skin of aquatic animals is not to any appreciable degree damaged by the agents responsible for ‘common-chemical- sense’ reactions, is clearly indicated by such facts as the following: COMMON CHEMICAL SENSE 3 1. High concentrations of acids (#4), salts (2N), alkalies ( 3) and alkaloids (;5)), which evoke prompt and vigorous reactions from the earthworm, Eisenia foetida, cease to cause any dis- turbance the moment the stimulant is washed off by immersing the animal in water. If any serious disintegration were produced by these solutions, it would be reasonable to expect the continu- ance of activity after the external supply of the stimulant had been removed.’ 2. Holothurians, such as H. surinamensis (Crozier, ‘15 ¢) and Stichopus moebii (Crozier, 15 b), possess skin pigments whose loss is a fairly good indicator of changes in permeability. Yet in no case on the application, by the pipette method, of the stimuli used by Parker (’12) was there any indication of pigment loss accompanying reaction.* This is true also of the nudi- branch, Chromodoris zebra. III. The value of the intracellular indicator of Chromodoris in the study of cell penetration by acids has been pointed out elsewhere (Crozier, °15b). When small volumes of acids, even in +; solution, are used to stimulate Chromodoris, there is no visible evidence that they penetrate the skin. In fact the time required for the penetration of the acids is quite high (see also Harvey, ’14), the most rapid rate observed being with 3» 1so-valeric acid, where penetration requires 1.5 minutes. ? Experiments with earthworms also disclose certain important deficiencies in’ the method whereby the animals to be tested are entirely immersed in the stimulating solution. If the reactions are to be studied quantitatively, there are features undoubtedly obscured by this procedure. For example, the time elapsing between the complete immersion of an earthworm (Hisenia foetida in a >) lithium chloride solution and the instant the first writhing movement appeared, was found to be at least ten times greater than the reaction time of the same worm when part of it (the anterior end) was stimulated. The speed of reaction is in part conditioned by the number of receptors affected, but this does not analyze the situation completely. It is quite probable that the re- sponses observed by Coghill are not at all due directly to sensory stimulation by acid. 3T have observed that under certain circumstances Ptychodera sp.—a Ber- mudan enteropneust—reacts to chemical irritation by extruding a yellowish pigment. There is ground, however, for believing that this is an instance com- parable to that of Lumbriconereis (Kschischkowski, ’12), in which Ix salts have apparently a more or less specific action in producing the response. | W. J. CROZIER For sy HNO; the penetration time found (Chromodoris tissue) was 3.5 minutes; for 4, 4.3 minutes; whereas Necturus, under water, was found to react to 7 HNO; (0.5 ee. of the solution being applied with a pipette) in 1.5 seconds when stimulated on the dorsal surface of the head, 5 seconds on the lateral mid-body surface, and 2 seconds on the tail; with HNO; the reaction times for corresponding locations were 5, 10, and 6 seconds respectively.!. Even here, though the pipette tip was held within approximately a centimeter of the animal’s surface, the concen- tration of the solution in the pipette does not represent the con- centration which actually reaches the stimulated surface, as already noted by Parker (12, p. 222). The conclusion must therefore be, that acids do not penetrate the skin with sufficient rapidity to affect the cells of the germinative layer. This con- clusion must also be extended to alkalies, since Harvey (10) and others have shown the high impermeability of cells to strong hydroxides. IV. In the light of the evidence just discussed, it seems im- probable that the high concentrations of irritants employed by Parker and others in studying reactions attributed to the com- mon chemical sense produce any violently disruptive effects when applied to the skin of aquatic animals. Indeed, so far as concerns the skin as a whole, they do not penetrate at all,° and the cells of the germinative layer cannot be held to be ex- posed to their action. This conclusion was verified, in the case of the spinal frog, by experiments of the following type: 1. The reaction time for the withdrawal of the frog’s foot from 7 CuSQ,, is about 7 seconds (at 24°9). After being withdrawn from the solution, the feet continue for some time to be spasmodically contracted. These subsequent contractions are entirely inhibited the instant a foot, just retracted from CuSO,, * 4 The reaction times were measured at 20°, while the penetration of the acid was studied at 27°; the speed of penetration decreases markedly with falling temperature. > The mode of action of the stimulating agent on the individual receptor is entirely another question. ‘ COMMON CHEMICAL SENSE 5 is dipped into a weak solution of K,Fe(CN)., which precipitates the copper held by the mucus of the foot. A similar result was obtained with copper acetate. Washing the foot with distilled water does not lead to a cessation of the contractions, because, after exposure of the foot to certain solutions (Loeb, 05), water stimulates. 2. After two to four stimulations of the frog’s foot by relatively strong solutions of either copper acetate or ferric sulphate, the stimulated area was sectioned .and tested microchemically for the penetration of the copper or iron. The metals were found in the mucus of the surface of the foot, and in several instances doubtful traces were discovered between cells of the extreme outer portion of the epidermis. No evidence was found of any disruption of the germinative layer. 3. As a stimulating agent whose penetration would readily be visible, the action of picric acid was studied. The outcome of experiments with this substance at several concentrations may be illustrated by the records here copied: Experiments Hand L. Picrie acid, %. R. T.=Reaction time in seconds. Successive tests at 5 min. intervals. N. R.=No reaction. R.T NO, os NOTES H I iL sate oa Bee 7.8 ay No staining. BSAA DN «6 seer d the 12.8 6.6 ’| Slight staining. 153 SRe, ee: 16.8 7.8 20.9 Oe SRR 30.2 11.8 Staining progressively re 40.0" 24.6 Drtenee ee ee aes 45.0 | 43 .2* ties ce ER Seed devas bees N.R. | N.R. No reaction to ¥ eshte acid. * Not reactive to pinching beyond this point. From these and similar tests, it was concluded that the pene- tration of the stimulant rapidily renders the frog’s foot less re- 6 W. J. CROZIER active by killing the superficial cells. In the case of picric acid it is probably the H ion which is mainly concerned in stimulation, since 3; ammonium picrate is entirely ineffective, neither does it easily stain the frog’s foot. This does not, however, signify that other acids behave as does picric, since as many as 20 suc- cessive reactions may be obtained from * HCl. Moreover, the staining is not directly correlated with the stimulating effect, since the skin of the frog’s foot was stained by immersion for 3 minutes in 39) picric acid without producing any reaction. The skin of Necturus and the catfish may be stimulated with 3) picric acid without producing any visible stain. V. I have repeated on the frog’s foot the experiment of Shel- don (’09) and Cole (10) regarding the separation of tactile irritability and responsiveness to irritating solution, by treat- ment with cocaine. A 0.5 per cent solution of cocaine hydro- chloride was used, and the tests were made by comparing at intervals the reactions of the cocainized foot with those of the untreated one. After about 20 minutes’ immersion, the reaction time of the cocainized leg to + formic acid (chosen as a powerful stimulant) was usually twice that of the normal foot; after about an hour, varying in some tests to an hour and a half, the cocainized foot no longer reacted to pinching, but gave good responses to the acid with reaction times of 10-15 seconds, still about twice the reaction time of the non-narcotized leg. It is therefore possible, I believe, to effect experimentally a separation of sensitivity to mechanical and to chemical stimu- lation in the frog’s foot. There can be no question of the distinctness of the human sensation attributed to the common chemical sense (Parker, 712; Parker and Stabler, ’13) as compared with any tactile sensation; and from tests made upon cocainized areas of my own mouth,® I am certain that the two sets of receptors are not only qualita- tively distinct as regards the sensations with which they are connected, but also may be separated by the use of cocaine. * These tests concerned mostly the inner surface of the cheek. COMMON CHEMICAL SENSE 7 SUMMARY 1. There is positive evidence, in the case of alkalies, acids, and certain salts, that solutions supplying stimuli for the com- mon chemical sense do not penetrate the skin of aquatic animals, nor when applied from a pipette do they damage the skin to any extent. 2. There is consequently no ground for Coghill’s assumption that the cells of the germinative layer of the epithelium of fishes and amphibians are exposed to the action of the stimulating agent and thereby disrupted; and there is no histological evi- dence of disruption. 3. The reactions attributed to the common chemical sense depend upon a group of sense organs distinct from those sensi- tive to mechanical stimulation. REFERENCES Bragunina, H. 1904 Zur Kenntnis der Wirkung chemischen Reize. Arch. ges. Physiol., Bd. 102, p. 163-184. Coauitt, G. E. 1914 Correlated anatomical and physiological studies of the growth of the nervous system of Amphibia. I. The afferent system of the trunk of Amblystoma. Jour. Comp. Neur., vol. 24, p. 161-233. Coz, L. W. 1910 Reactions of frogs to chlorides of ammonium, potassium, sodium, and lithium. Jour. Comp. Neur., vol. 19, p. 273-311. Crozier, W. J. 1915 a The behavior of an enteropneust. Science, N. S., vol. 41, p. 471-472. (Note—Lines 5 and 6 [p. 472] have been accidentally interchanged by the printer. ) 1915 b The rhythmic pulsation of the cloaca of holothurians. Ibid., p. 474. 1915¢c The sensory reactions of Holothuria surinamensis. Zool. Jahrb., Abt. Physiol. [Not yet published]. 1915 d On cell penetration by acids. Science, N.8., vol. 42, p. 735- 736. Harvey, E. N. 1910 Studies on the permeability of cells. Jour. Exp. Zodl., vol. 10, p. 507-556. 1914 Cell permeability for acids. Science, N. 8., vol. 39, p. 947-949. Herrick, C. J. 1908 On the phylogenetic differentiation of the organs of taste and smell. Jour. Comp. Neur., vol. 28, p. 157-166. 8 W. J. CROZIER Kscuiscuowskl, KX. 1912 Neue Beitrige zur Pigmentabsonderung bei Anne- liden. Zentralbl. f. Physiol., Bd. 26, p. 528-582. Lorn, J. 1905 Production of hypersensitivity of the skin by electrolytes. In: Studies in General Physiology, pt. II, Chicago. pp. 748-765. Parker, G. H. 1912 The relation of smell, taste, and the common chemical sense in vertebrates. Jour. Acad. Nat. Sci. Phila., Ser. 2, vol. 15, p. 221-234. Parker, G.H., ano Stasier, EH. M. 1918 On certain distinctions between taste and smell. Amer. Jour. Physiol., vol. 32, p. 230-240. Suetpon, R. E. 1909 The reactions of the dogfish to chemical stimuli. Jour. Comp. Neur., vol. 19, p. 273-311. STUDIES ON THE SPINAL CORD AND MEDULLA OF CYCLOSTOMES WITH SPECIAL REFERENCE TO THE FORMATION AND EXPANSION OF THE ROOF PLATE AND THE FLATTENING OF THE SPINAL CORD! WILLIAM F. ALLEN Department of Anatomy, University of Minnesota EIGHTY-SEVEN FIGURES CONTENTS SP AUNOUUCHLON: aed eerie s cies ta ways ena oso e a «
70.
12 From a section 645 microns behind figure 11, and not far from the caudal
end of the first model (figs. 1 and 2). At this point the first roof plate expansion
and its enclosed cavity attain their greatest width, which cavity is broadly con-
nected with the central canal. X 70.
13 Transverse section S80 microns behind figure 12 and at the very beginning of
model 2 (figs.4and5). The dorsal wall of the first roof expansion is very wide and
extremely vascular, and the convexity of its walls and the arch of the cavity bear
evidence of moderate internal pressure from cerebro-spinal fluid. The wall,
while still quite thick, contains a lesser number of ependymal cells, but more
connective tissue. Note especially the absence of any direct connection between
the cavity of the roof expansion and the central canal. A chain of ependymal
cells still connects the two, and may be indicative of a former embryonic con-
nection that has been lost. From this region, caudad, there is no communica-
tion between the cavity of the roof expansion and the central canal. That a
former connection occurred may be indicated by the fact that at various inter-
vals ependymal cells are scattered between the two. A sensory root can be seen
sending its fibers inward toward what is believed to be the substantia gelatinosa.
xX 70.
13 A, represents a small portion of the first roof plate expansion and its in-
closed cavity from a section taken 410 microns behind figure 13. Observe es-
pecially the rich blood supply for the dorsal wall and the ease by which diffusion
could take place between a blood vessel and the cavity. A large roof plate cell
is drawn separately, highly magnified, directly to the left of 13 A. Note the
fine granules in cytoplasm, which gives evidence of being secretory. 125.
ABBREVIATIONS
B.V., blood vessel M.R., motor or ventral spinal nerve
C.C., central canal root
C.C.Ex., central canal extension into N.A., membranous neural arch
roof plate expansion N.C., notochord
Ep.N., layer of ependymal nuclei R.P.Ex., roof plate expansion
M.C., motor or effective cells S.C.F., cerebro-spinal fluid
M.D.C., median dorsal cartilaginous S.G., substantia gelatinosa
bar S.R., sensory or dorsal spinal nerve
M.F., Miillerian or giant fiber root
50
SPINAL CORD AND MEDULLA OF CYCLOSTOMES PLATE 4
WILLIAM F. ALLEN
At
Wey
we
”
LATE
PLATE
EXPLANATION OF FIGURES
14 From a transverse section taken 19 microns behind figure 138A. It repre-
sents a condition found throughout the entire posterior end of the first roof
expansion, where a series of cavities are connected with one another.
root is seen issuing from the cord. X 70.
15 to 17 Three transverse sections, anterior, middle, and posterior through
what has been designated as the second outcropping of the roof plate, which
in model 2 (figs. 4 and 5) appears immediately behind the first roof expansion
already figured. In figure 15, which is through the anterior end of this roof
plate expansion, the outcropping is one of cells only. In figure 16 a distinet cay-
ity containing coagulum is visible, connected below with the central canal. Ob-
serve the vascularity of its walls, of the ependyma surrounding the central canal
and the diverticula of the same, which doubtless serve as a modified chorioid
plexus for producing cerebro-spinal fluid. In figure 17, which is through the
posterior end of ths roof expansion, a cavity is still present. Its walls, which
are mainly connective tissue, are rich in blood vessels. 70.
21 to 23 represent three transverse sections taken through the extreme pos-
terior, non-nervous, end of the spinal cord, which is composed solely of support-
ing tissue and undifferentiated embryonic cells. Figure 21 is the most cephalic,
and passes through the spinal cord a short distance behind the abnormal sinus
terminalis (figs. 9 and 20, S.7'.). The spinal cord is still flattened here, but not
indented ventrad, and contains a normal central canal. In figure 22 there has
occurred a marked reduction in the size of the notochord. Observe that the
spinal cord has not become flattened as it has more anteriorly, where the noto-
chord is massive. With figures 21 and 22 compare figures 59 and 60, which are
taken from a similar region of the spinal cord from a 70 mm. Polistotrema embryo.
Figure 23 is the last of this series of drawings. It passes through the extreme
posterior end of the spinal cord, some 45 microns caudad of the last trace of the
notochord. Note the presence of a central canal, and that the cord has separated
into several processes, which further on become lost in the surrounding con-
nective tissue. X 70.
PLATE 7
EXPLANATION OF FIGURES
24 to 31 From transverse frontal sections through the medulla and spinal
cord of several human embryos, drawn with the aid of an Edinger-Leitz draw-
ing apparatus, and reduced 3 diameters in reproduction.
24 and 25 From transverse sections through the rhombic brain (frontal
through the embryo) of a 23 mm. human embryo; figure 25 passes through the V
root and posterior end of the cerebellar rudiments (lateral lobes), while figure 24
is from a more anterior section. The roof expansion (chorioid plexus) is shown
as a conspicuous black line in these figures. Everywhere within the boundaries
of the roof expansion, the cavity is filled not only with coagulated cerebro-spinal
fluid, but with embryonic red corpuscles. Whether these entered through veno-
lymphatic openings (C) or are the result of extravasations was not determined.
Wherever mesenchyme borders the roof expansion it is very vascular. It is
apparent that the roof expansion is under moderate internal pressure. At first
glance the roof expansion will show resemblance to the so-called first roof plate
expansion of the spinal cord of the 20 cm. Polistotrema, already figured, but its
later mode of development was shown to be very different. 10.
(Continued on page 56)
54
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
PLATE 6
WILLIAM F. ALLEN
ABBREVIATIONS
B.V., blood vessel
Myo., myotomes
C.C., central canal
Ne., notochord
S.C.F., cerebro-spinal fluid.
Sp.M., spinal cord
S.R., sensory or dorsal spinal nerve
root
C.N.A., cartilaginous neural arch
C.T., white fibrous connective tissue
Ep., ependyma
M.D.C., median dorsal cartilaginous bar
M.V.C., median ventral cartilaginous bar S.T., sinus terminalis
55
(Continued from page 54)
26 and 27 Represent two transverse sections through the medulla, passing
through the posterior end of the fourth ventricle of the same series from which
figures 24 and 25 were drawn. Note especially the expansion of the roof plate
and compare with the so-called second roof plate expansion of the 20 em. Polis-
totrema spinal cord (fig. 16). It is questionable whether the openings (C) are
artifacts or not. As was noted previously, the mesenchyme outside the roof
plate is very vascular and the roof plate has the appearnce of being under a moder-
ate degree of internal pressure. > 10.
28) A rather oblique frontal section through the medulla of a 15 mm. human
embryo (Inst. of Anat., trans. series, H 23). In this region the contour of the
rhombic brain is such that the posterior part of the roof expansion of the fourth
ventricle is cut transversely; while the more anterior portion, seen below, appears
more or less in frontal section. More anterior sections would show the roof
plate to be continuous. The posterior end of the fourth ventricle will admit of
direet comparison with the second roof expansion of the 20 cm. Polistotrema
spinal cord (fig. 16). Had the fourth ventricle been empty, as was the case of
the rubber tubing in His’ experiments, there would be absolutely no grounds
for believing that the anterior portion of the roof plate would be expanded as
it is by the appearance of a pontine flexure. It might on the contrary have been
folded up within the ventricle. > 16.6.
29 A transverse section through the extreme posterior end of the fourth
ventricle of the same seriesas figure 28. There ishereaslight roof plate expansion
containing no cavity. Compare with figure 15. X 16.6.
30 Transverse section through the thoracic spinal cord, taken from the same
series as figure 28. Note that the roof plate consists of ependyma only, while
the floor is reinforced by white matter, and even at this late stage if any marked
increase In pressure occurred from the cerebro-spinal fluid of this region, an
expansion of the roof plate would have been entirely possible. X 16.6.
31 Similar to figure 28, but from an 8 mm. human embryo (Inst. of Anat.,
series H4). In this plane the posterior end of the fourth ventricle is cut nearly
transversely, and is directly comparable with the second roof expansion of the
20 cm. Polistotrema spinal cord (fig. 16). The cavity was full of coagulum, its
walls have the appearance of being under moderate internal pressure, and the
adjacent mesenchyme is very vascular. X 46.6.
ABBREVIATIONS
B.V., blood vessel Ol., inferior olive
C., an apparent communication be- F.Hx., roof plate expansion
tween the veno-lymphatics and the &.L., rhombic lip
fourth ventricle R.P., roof plate of the central nervous
C.C., central canal system
Ch n., chondrocranium S.C., semicircular canals
C.P., choroid plexus of the fourth S.R., sensory or dorsal spinal nerve
ventricle root
Crb.L., lateral lobes of the cerebellum T.S., tractus solitarius
C.T., white fibrous connective tissue VJ///.G., auditory ganglion
Ep., ependyma V.L.S., veno-lymphatie sinus
Ex.Ar., external arcuate fibers V.R., trigeminal root
I.L., inner or ependymal layer of nuclei W.R.F., white reticular formation
Mar.L., marginal layer XI.R., accessory nerve root
M.L., mantle layer
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WIL' IAM F. ALLEN
5)
7
PLATE 7
PLATE 8
EXPLANATION OF FIGURES
32 to 538 A series of transverse sections through the region of the V, VIII,
and X ganglia in embryos of Petromyzon of ages varying from 10 to 26 days. It
will be seen from these sections that Petromyzon develops an extensive roof
expansion without the aid of a pontine flexure, and the cranial and spinal ganglia
are well-formed while the central nervous system is a solid cord. All of the
figures were drawn with the aid of an Edinger-Leitz drawing apparatus. With
figure 54 a magnification of 76.6 diameters was used, while 250 diameters was used
for the others. In reproduction they were all reduced one-half.
32 Transverse section through the medulla in the region of the auditory
vesicle from a 10 day Petromyzon. This is my oldest embryo in which the cen-
tral nervous system has remained a solid cord. Ordinarily it becomes tubular
during the seventh day. This section shows the medulla to consist of a syney-
tium of protoplasm, consisting of a mass of round nuclei, much yolk, and a few
fibers in the marginal layer. The nuclei have migrated a short distance to either
side of the median dorso-ventral line. A seam (C.C.S.) has appeared here, which
marks the position and beginning of the embryonic central canal. The proto-
plasm bordering the central canal seam is finely granular and may be assum-
ing a secretory function. The acustic ganglia and fibers are shown to be well-
differentiated on both sides. 125.
33 to 35 Three transverse sections passing through the medulla region of
another 10 day Petromyzon embryo, in which the central canal has been somewhat
retarded in development. These sections pass through the V, VIII, and X gan-
glia respectively, and with the exception that the central canal furrow or seam
(C.C.S.) has expanded into small dorsal and ventral cavities (no dorsal cavity
has appeared in fig. 35) the general structure of the medulla is about the same
as in figure 32. Later these cavities will become the dorsal and ventral ex-
pansions of the embryonic central canal of the medulla. The protoplasm in the
region of this seam is granular and may be secreting an embryonic cerebro-
spinal fluid. From these figures it will be seen that the beginning of the central
canal occurs at the same time throughout the entire rhombic brain. The anterior
portion of the spinal cord, while not figured, contains a central canal furrow in
the same stage. X 125.
(Continued on page 60)
ABBREVIATIONS
Aud.V., auditory vesicle or otocyst Mes., mesencephalon
Br.A., branchial arch M.L., mantle layer
B.V., blood vessel Myo., myotomes
C.C., central canal Ne., notochord
C.C.C., central canal closure, caused P.B., pineal body
by fusion of lateral plates R.P., roof plate of the central ner-
C.C.S., central canal seam or furrow, vous system
in Petromyzon Sp.M., spinal cord
Ep.N., layer of ependymal nuclei Sy.P., syneytium of protoplasm
F.L.P., fused lateral plates of the Tel., telencephalon
spinal cord V.G., Gasserian or semilunar ganglion
F.P., floor plate of the central nervous VJJI.VII., acustico-fascialis ganglion
system; W.M., white matter
G.C., germinal cell X.G., vagus ganglion (nodosum).
Mar.L., marginal layer ’Y., yolk granules
58
PLATE 8
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILLIAM F. ALLEN
ecreky
(Continued from page 58)
36 to 38 From a Ll day Petromyzon embryo through the same regions as those
shown in figures 33 to 35, and to facilitate comparison were placed directly un-
der them. Considerable progress has occurred everywhere. Note 1) that
the central canal seam with its small dorsal and ventral cavities in the 10 mm.
series has given place to a typical embryonic central canal, which is much wider
at the top and bottom than at the center. The constricted portion of course
represents the last place for the protoplasm to give way or to be disintegrated.
2) The floor plate is slightly thicker and less expanded than the roof plate, being
reinforced on the outside by white matter and by a rapidly growing notochord
below. 3) A few more nerve fibers and nuclei have appeared in the lateral
plates. 4) The number of dividing germinal cells has increased while the num-
ber of yolk granules remains about the same. 5) The first blood vessels have put
in appearance directly outside the meningeal membrane (fig. 37, B.V. and
nearer the roof plate on the opposite side). > 125.
39 Transverse section through the spinal cord of the same 11 day Petromyzon
series as figures 36 to 38, showing the so-called typical embryonic spinal
(c@yeels — << Mey
40 and 41 Somewhat oblique transverse sections from a 12day Petromyzon,
passing in figure 40, through the V ganglionon one side and the VIII ganglion on
the opposite side, and in figure 41 through the X ganglion on one side and a
region behind the X ganglion on the opposite side. They can readily be compared
with the 11 day series above (figs. 36-38). The lateral plates have apparently
increased notably in the number of nerve fibers and nuclei, some of which, however,
will have to be attributed to the fact that the sections are cut quite obliquely.
Also the numbers of nerve fibers have increased in the floor plate. Throughout,
the central canal has increased in width. Of especial interest is a small central
mass of protoplasm (Sy.P.) in figure 40, which for a space of 50 microns per-
sists as the last remnant of a once solid mass of protoplasm in the center of the
medulla. It is obvious at this stage that some factor must have produced suf-
ficient internal pressure to prevent the closing up of the ventricle on account
of the rapid increase of cells and fibers in the lateral plates. It is fair to assume
that this factor is internal pressure from cerebro-spinal fluid. X 125.
54 of this plate will be described in its proper place, opposite the next plate.
PLATE 9
EXPLANATION OF FIGURES
42 to 44. Three transverse sections through the medulla of an 18 day Petromy-
zon embryo, passing through the V, VIII, and X ganglia, and for the sake of com-
parison preserving the same order or arrangement as was used for the earlier
embryos. A slight increase in the white matter is to be noted for the lateral and
ventral plates over the 12 day series; but little, if any change has taken place in
the central canal, unless possibly the central portion has increased slightly in
width. Absolutely no further expansion of the roof plate has occurred. Since
sections through the medulla of a 14 and 16 day series presented about the same
appearance as figures 42 to 44 none were figured. X 125.
45 Transverse section through the cephalic portion of the spinal cord from
the same series as figures 42 to 44. Observe especially the beginning of the dorsal
closure of the central canal (C.C.C.), showing the central canal to consist of dor-
sal and ventral cavities and a central seam, strongly resembling the stage when
it first appeared in the spinal cord. This dorsal closure of the embryonic central
canal begins in the anterior portion of the spinal cord much earlier than the cor-
responding ventral closure of the embryonic central canal in the medulla, occur-
ing in my series of 14 and 15 days. It is obviously caused by the ingrowth of
the lateral plates due to the great increase in the number of nerve fibers. & 125.
46 to 48 Three transverse sections through the same region of the medulla of
a 20 day Petromyzon as is shown above in figures 42 to 44 for the 18 day embryo.
It will be seen that many noticeable changes have taken place, due primarily
to a marked increase of nerve fibers in the lateral and ventral plates, and to a
slight increase in the number of cells in the lateral plates. The shape of the
medulla has become more compressed (flattened out in a dorso-ventral plane).
Unquestionably the increase of fibers in the lateral plates has occurred largely
in the median and ventral portions. Note the result on the embryonic central
canal, which has been completely closed, except for a small dorsal triangular
‘avity (C.C.) the early fourth ventricle. Its roof plate, however, has expanded
somewhat. Observe that true ependymal cells are beginning ¢ > take on form
about the ventricle, and those in the roof plate may soonassume a secretory func-
tion, if they are not already active. Also the blood vessels have become more
abundant outside of the medulla, especially in the region of the roof plate, which
would make infiltration and diffusion through the roof plate into the ventricle
sasy. X 125. ;
49 From a transverse section through the anterior portion of the spinal cord.
Observe the great increase in fibers in the lateral and ventral plates, and note
that the cleft-like embryonic central canal has become entirely closed, but for
a small portion, which will remain as the adult central canal. It is obvious that
the two portions of the embryonic central canal which persist in the medulla
and the spinal cord are the opposite, being the dorsal in the medulla and the ven-
tral in the cord. X 125.
50 to 53. ~Four transverse sections through the medulla of a 26 day Petromy-
zon embryo, taken through the V, VIII, and X ganglia, and through a region
behind the fourth ventricle where the central canal is passing ventral to assume
its characteristic position in the spinal cord. A comparison with the 20 day
series above (figs. 46 to 48) will demonstrate a marked increase in the size of the
medulla and the fourth ventricle, and a greater expansion and convexity of the
(Continued on page 62)
61
(Continued Jrom page 61)
roof plate, in consequence of which the dorsal tips of the lateral plates are widely
separated. There is a marked increase in the number of nerve fibers in the lateral
plates, especially in the median and ventral portions. Of prime importance is
the great expansion of the fourth ventricle and the roof plate, which apparently
in Petromyzon can be explained only from internal factors, the most obvious
of which is the mechanical expansion due to an increase in the cerebro-spinal
fluid. It will be seen that these forces were sufficiently strong to more than
offset the thickening of the lateral plates which would tend to obliterate the
dorsal portion of the embryonic central canal as it has the ventral portion. It
is apparent that this internal pressure has pushed the lateral wall apart in the
dorsal region, where the lateral plates are thinnest and weakest. As was pointed
out in the 20 day series the ependymal cells are becoming differentiated and
probably have assumed a secretory function. Likewise the increase in the num-
ber of blood vessels above the roof plate favors filtration and diffusion into the
fourth ventricle. X 125.
54 (See preceding plate.) Median longitudinal section through the head
region of a 26 day Petromyzon embryo introduced for a comparison with the
transverse sections in figures 50 to 53. Note especially that the marked convexity
of the roof plate of the fourth ventricle is suggestive of expansion from an in-
crease of cerebro-spinal fluid. Absolutely no pontine fiexure is to be seen, the
little convexity that occurs in the floor plate can easily be attributed to an in-
crease in the number of nerve fibers. Observe that the fourth ventricle (C.C.)
is the remains of the dorsal portion of the original embryonic central canal, while
the central canal of the spinal cord is the remains of the ventral portion. The
ventral portion of the embryonic central canal of the medulla has been obliter-
ated through the fusion of the ventral portions of the lateral plates. 38.3.
ABBREVIATIONS
Aud.V., auditory vesicle or otocyst Myo., myotomes
B.V., blood vessel N.C., nerve cell
C.C., central canal R.Ex., roof plate expansion
C.C.C., central canal closure, caused R.P., roof plate of the central nervous
by fusion of lateral plates system
Ep., ependyma V.G., Gasserian or semilunar ganglion
Ep.N., layer of ependymal nuclei VIIT.VII., acustico-fascialis ganglion
G.C., germinal cell W.M., white matter
G.M., white matter X.G., vagus ganglion (nodosum).
M.L., mantle layer
PLATE 10
EXPLANATION OF FIGURES
55to62 Taken from various transverse sections through embryonic and larval
Polistotrema and Entosphenus (Pacific coast lamprey). Introduced to show the
effect of the developing notochord on the spinal cord in Cyclostomes. They
were drawn with the aid of an Edinger-Leitz drawing apparatus and reduced
one-half in reproduction.
55 Transverse section through the caudal region of a 20 mm. Polistotrema
embryo. It will be seen at this stage that the notochord has produced very
little visible effect on the spinal cord. Cyclostome embryos of this stage (com-
pare fig. 39 for Petromyzon) presenta nearly cylindrical spinal cord; while that
of all other vertebrates is more or less elliptical in cross section, the greater
(Continued on page 64)
62
PLATE 9
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILLIAM F. ALLEN
(Continued from page 62)
limmeter being dorso-ventral. It should be noted that the spinal cord is en-
veloped tightly by a meningeal membrane, more or less fused with connective
tissue outside that will form the neural arch, which is firmly attached to the
notochord below. Immediately above, the mesenchyme is proliferating rapidly
and migrating to the center where it will form the median dorsal cartilaginous
bar. Little progress has occurred in the formation of the myotomes at the
side, and elsewhere there is only loose mesenchyme. > 70.
56 Similar transverse section to figure 55, but from a 27 mm. Polistotrema
embryo. ‘This slightly later stage shows considerable growth of the notochord
and a median indentation on the ventral surface of the spinal cord as the result.
Note that the conditions surrounding the development of the notochord previous-
ly enumerated under the description of figure 55 are instrumental in assisting the
notochord in producing the gradual flattening (depression) of the spinal cord
seen in the next figure. 70.
57 Transverse section of the spinal cord of a 60 mm. Polistotrema embryo
from the same region as figure 56. It will be seen that the spinal cord is en-
closed in a membranous canal of dense connective tissue, attached below to the
notochord and above to the median dorsal cartilaginous bar. Above this there
are developing cartilaginous rays surrounded by dense connective tissue. The
developing myotomes rest against the neural arches both laterally and dorsally.
The notochord has increased greatly in size and, pushing up against the soft
spinal cord, produces the depression and ventral indentation of the spinal cord
exhibited in this figure. It should be noted that the roof plate is still ependyma
and an expansion of the roof plate could take place even in this late stage if the
mechanical factors enumerated for the medulla of Petromyzon were operative
here. The thickening of the lateral plates has about obliterated the central
portion of the embryonic central canal, leaving only the dorsal and ventral
portions, in which there is a fibrillar feltwork, probably representing both cere-
bro-spinal fluid and ependyma cilia. Reissner’s fiber is visible in the ventral
or permanent central canal. X 70.
58 ‘Transverse section through the tail region of a 20 mm. Entosphenus larva.
It will be observed that the spinal cord is further developed than in the 27 mm.
Polistotrema embryo (fig. 56). It 1s apparent that the same factors are involved
in flattening the spinal cord as were enumerated for Polistotrema. The noto-
chord has made fully as much growth and the structures surrounding the spinal
cord are the same as in Polistotrema, with the exception that instead of a median
dorsal cartilage for the attachment of the membranous neural arch there is a
membranous neural spine. To some extent this may reduce the dorsal resistance,
but on the other hand it may be compensated for by a greater development of
the myotomes above the neural arch. X 125.
ABBREVIATIONS
C.A., caudal artery M.V.C., median ventral cartilaginous
C.C., central canal bar
C.H., caudal heart Myo., myotomes
C.V., caudal vein N.A., membranous neural arch
D.R., dorsal cartilaginous rays Ne., notochord
Ep.N., layer of ependymal nuclei P.M., pia mater or meningeal mem-
L.S., lateral veno-lymphatie sinus or brane of the younger stages
anlage of the same R.P., root plate of the central nervous
Mar.L., marginal layer system
M.D.C., median dorsal cartilaginous Sp.G@., spinal ganglion
bar V.7T., ventral veno-lymphatic trunk
W.AL., white matter
64
PLATE 10
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILLIAM F. ALLEN
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, No. 1
PLATE il
EXPLANATION OF FIGURES
59 and 60 ‘Two transverse sections only 290 microns apart through the ex-
treme posterior end of the spinal cord of a 70 mm. Polistotrema embryo. In
the more anterior section (fig. 59) the spinal cord is depressed, contains develop-
ing nervous elements, and the notochord is large proportionately. In the pos-
terior section (fig. 60) the diameter of the notochord is much reduced and only
supporting elements appear in the spinal cord. As a result no flattening of the
spinal cord has taken place in this region. Compare with figures 21 and 22, which
are similar sections through an adult Polistotrema. X 70.
61 and 62 Taken from two transverse sections 480 microns apart, through
the medulla oblongata of an adult Polistotrema. In both sections no nervous
structures have appeared that were not present in the spinal cord. Note as you
pass caudad (figs. 62 to 61) that the medulla becomes flattened ventrally and dor-
sally in direct proportion to the increase in size of the notochord. This rela-
tionship can be shown fully as marked in more anterior sections, and in sections
taken from a similar region of larval Petromyzon. X 25.
ABBREVIATIONS
Aud.V., auditory vesicle or otocyst
B.V., blood vessel
C.C., central canal
E.N., undifferentiated embryonic nu-
clei
Ep., ependyma
Ep.N., layer of ependymal nuclei
L.S., lateral veno-lymphatic sinus or
anlage of the same
M.D.C., median dorsal cartilaginous
bar
M.F., Miillerian or giant fiber
M.V.C., median ventral cartilaginous
bar
Myo., myotomes
N.A., membranous neural arch
N.C., nerve cell
P.M., pia mater or meningeal mem-
brane of the younger stages
P.P., parachordal plate
Sp.G., spinal ganglion
W.M., white matter
X.G. in figure 62 should be Sp.G.
X.N., vagus nerve
M.L., mantle layer
PLATE 12
EXPLANATION OF FIGURES
63, represents a diagrammatic reconstruction of the fourth ventricle from an
adult Polistotrema series, and the planes from which the transverse sections were
drawn for figures 64 to 66 are indicated by lines bearing those figures. Observe
how the large fourth ventricle of the embryo has been reduced to a small central
canal, having a posterior dilation (PyV), and how the anterior end breaks up
into two or more small longitudinal canals that soon terminate in the sinus
mesoccelicus.
64 and 65, represent transverse sections, taken at different levels of the fourth
ventricle of Polistotrema. These sections were drawn with the aid of an Edin-
ger-Leitz drawing apparatus and were reduced one half.
64 From a transverse section through the caudal portion of the mesencephalic
lobes (cerebellum of Miss Worthington). Exact plane indicated by line 64 in
figure 63. The section passes through the posterior mesoccele or cerebellar ven-
tricle (7’’.) and the sinus mesoccelicus (anterior dilation of the fourth ventricle
of Miss Worthington), a short distance behind the branching off of the posterior
mesoccele. The cavity contains a fibrillar feltwork, which is in part coagulated
cerebro-spinal fluid and in part ependymal cilia. The ependyma surrounding
the fourth ventricle is rich in blood vessels, which derives its arterial supply from
(Continued on page 68)
66
PLATE 11
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILLIAM F. ALLEN
(Continued from page 66)
two medulla arteries (/.A.). Here as elsewhere, the ependyma surrounding
the fourth ventricle doubtless functions as a modified chorioid plexus, discharg-
ing cerebro-spinal fluid into the fourth ventricle. It will be seen that the cavity
of the fourth ventricle is smaller than the peculiarly modified central canal and
roof expansion cavity of the Polistotrema spinal cord portrayed in figures 10 to
13. X 25.
65 A more caudal section through the extreme tip of the posterior lobes of
the mesencephalon (cerebellum of Miss Worthington), its exact plane being
indicated by line 65 in figure 63. It will be seen that the fourth ventricle of the
embryo has in this region of the adult become reduced to three small longitudinal
canals (A,V.), which are imbedded in a rather large, dense, and vascular epen-
dymal mass. The most dorsal of these canals contains Reissner’s fiber. Here
again the ependymal walls are probably functional as a modified chorioiod plexus.
Very shortly these canals reunite and continue some little distance caudad as
a small central canal, no larger than the central canal of the spinal cord. X 25.
66 Transverse section through the posterior end of the medulla of the same
series as figure 64. The exact plane of the section is indicated by line 66 in figure
63. It passes through what has been designated as the posterior dilation of the
fourth ventricle (P;V), which is nothing more than a fair-sized centrally lo-
cated cavity, the remains of a much larger embryonic fourth ventricle, surrounded
by a great mass of vascular ependyma. The center of this cavity contains a
fibrillar feltwork (S.C.F.) composed largely of coagulated cerebro-spinal fluid
and some ependymal cilia. Here as more anteriorly we probably have a modi-
fied chorioid plexus, the ependymal walls and their blood vessels secreting and
filtering cerebro-spinal fluid into the fourth ventricle. > 25.
ABBREVIATIONS
A4V., anterior fourth ventricle M.A., medulla artery
B.V., blood vessel Mes’., posterior lobes of the mesen-
Ep., ependyma cephalon, cerebellum of Miss Worth-
Hab. B., habenular body ington
Inf., infundibulum P,V., posterior fourth ventricle
M., mesoccele or mesencephalic ventri- S.C.F., cerebro-spinal fluid
cle S.M., sinus mesocelicus
M’., anterior portion of the mesocele V.M.R., motor V root
or sub-commissural canal of Nicholls 4V., fourth ventricle
M"’., dorsal portion of the mesoccele VII/I.G., auditory ganglion
or optocoel and posterior portion of | X.R., vagus root
the optocoel of Nicholls
PLATE 13
EXPLANATION OF FIGURES
67 to 69 Three transverse sections through the brain region of a 37 mm.
Amphioxus. No blood vessels were seen in any of these sections, but the mem-
branous neural canal is surrounded on three sides by enormous veno-lymphatic
sinuses, and the structure of the central nervous system is to a considerable extent
made up of rather coarse supporting tissue, making infiltration an easy method
for nourishing the brain. Drawn with an Edinger-Leitz drawing apparatus and
reduced one-half in reproduction.
(Continued on page 70)
68
PLATE 12
SPINAL CORD AND MEDULLA OF CYCLOSTOMES
WILLIAM F. ALLEN
(Continued from page 68)
The most anterior section, figure 67, passes through the anterior ventricle at
its highest point, which is a short distance behind the neuropore. This ventri-
cle has no dorsal dilation suggestive of the fourth ventricle. What dilation occurs,
is median and ventral. Cilia-like processes from the border of the cells enter
the cavity. If the ependymal cells are not secretory it is possible that the cere-
bro-spinal fluid of Amphioxus does not differ from the serum of the adjacent
veno-lymphatie sinuses. If nerve cells occur in this region they are small, and
in ordinary preparations indistinguishable from ependymal cells. > 125.
68 60 microns behind figure 67. The large central canal of the embryonic
brain has evidently become reduced in this region to a ventral central canal
(C.C.) and a small dorsal isolated cavity (V 2.). This isolated dorsal cavity can
not be compared with the fourth ventricle of higher vertebrates. It is rather
to be looked upon as a vestigeal structure, which may aid in the infiltration of
lymph from the outer veno-lymphatic sinuses. 125.
69 Taken from a section 530 microns behind figure 68. It passes through
that part of the brain in which there are accumulated a great number of giant
cells (J/’.C’.) in the region of the roof plate. As in figure 68, there is an isolated
cavity near the dorsal surface, which was probably a portion of the large embry-
onic central canal, but which in the adult is separated from the central canal and
from the more anterior isolated dorsal cavity by ependyma. It seems best to
the writer to regard this and the preceding dorsal cavity as vestigeal structures.
x 125.
70 (See next plate.) Transverse section through the anterior spinal cord
from the same series as the three previous figures. Observe that the Amphioxus
spinal cord is not depressed as is the Cyclostome spinal cord, but is indented ven-
trally by the notochord. The central canal, which in some places exists as a
dorso-ventral cleft, is almost obliterated here by the ingrowth of ependymal
tissue. X 125.
71 Cephalic transverse section through a portion of the spinal cord, menin-
geal membranes, neural arch, notochord, spinal ganglion,and sensory root of
an adult Polistotrema. Observe the depression of the spinal cord, its ventral
indentation, the ventral or permanent central canal (C.C.), which contains Reiss-
ner’s fiber, and immediately above, the dorsal portion of the embryonic central
canal, which is here more or less filled with ependymal cells and their processes.
It will be seen that the gray matter is as much flattened out as is the cord it-
self, and the ventral horn and motor cells are crowded laterad, while the dorsal
horn, substantia gelatinosa (S.G.), is apparently median and dorsal. Within
the neural arch there is abundant room for a spherical spinal cord. The cord is
held in place by the usual meningeal membranes. X 70.
ABBREVIATIONS
Ar., Arachnoidea Ne., notochord
C.C., central canal P.M., pia mater or meningeal mem-
C.T., white fibrous connective tissue brane of the younger stages
D.M., dura mater S.G., substantia gelatinosa
D.S., dorsal veno-lymphatic sinus Sp.G., spinal ganglion
Ep., ependyma S.R., sensory or dorsal spinal nerve
L.S., lateral veno-lymphatie sinus or root
anlage of the same Suba.S., subarachnoid cavities
M’.C’., Miillerian or giant cells Subd.S., subdural spaces
M.F., Miillerian or giant fiber W.M., white matter
Myo., myotomes V.1., anterior ventricle Amphioxus
N.A., membranous neural arch V.2., vestiges of the embryonic central
N.C., nerve cell canal in Amphioxus
70
PLATE 13
SPINAL CORD AND MEDULLA OF CYCL ISTOMES
WILLIAM F. ALLEN
PLATE 14
EXPLANATION OF FIGURES
72 to 80 represent a number of transverse sections through the medulla of shark,
amphibian, and pig embryos for the purpose of demonstrating various stages of
roof plate expansion.
72 Rather oblique transverse section through the medulla of a 19 mm. Squa-
lus embryo in the region of the VIII ganglion (from series No. 2 of Professor
Scammon’s collection). Note the well-formed fourth ventricle, and the broadly
expanded and very much stretched roof plate. Its collapsed appearance in this
section is doubtless due to fixation or preparation. On account of a great pro-
liferation of cells and nerve fibers the lateral plates have fused ventrally, as
in Petromyzon, obliterating that part of the embryonic central canal. Attention
should be called to the fact that the medulla roof plate in sharks begins to expand
much earlier than it does in Petromyzon. This figure shows a well-expanded
roof plate, while the cells in the mantle layer are no more differentiated and there
are no more nerve fibers in the marginal layer than appear in a 12 day Petromy-
zon-embryo (fig. 40), where there is no fourth ventricle and no expansion of the
roof plate. X 70.
73 Transverse section through the medulla of a 15 mm. Necturus taken
through the VIII ganglion. Observe the wide fourth ventricle and the broadly
expanded and greatly stretched roof plate, and the coagulated appearance of
the cerebro-spinal fluid in the ventricle. Like Squalus, the fourth ventricle
begins relatively much earlier than in Petromyzon. It should be noted that no
blood vessels have reached the level of the roof plate or entered the medulla;
hence the coagulum in the ventricle must be largely a product of secretion. X 39.
74 From a transverse section of a very young Amblystoma embryo in the
region of the auditory vesicle (Professor Johnston’s series No. 50). Here there
has occurred a dorsal and a smaller ventral excavation of the cleft-like central
canal. It will be seen that the larger dorsal cavity, the beginning of the fourth
ventricle, possesses no thinner roof plate than does the spinal cord (fig. 83).
Also in this section (fig. 74) the roof and floor plates are about equally thick.
What has taken place dorsally and ventrally throughout the embryonic central
canal has been a migration of the cells outward. The fact that in this section
of the medulla the roof plate is no thinner than in the section of the spinal cord
(fig. 83), taking note that the spinal and cranial ganglia are well-formed, is evi-
dence against the hypothesis, that the greater migration of the neural crest
cells of the medulla was the prime cause of the thinning out of the roof plate of
the rhombic brain. X 70.
(Continued on page 74)
ABBREVIATIONS
Aud.V., auditory vesicle or otocyst M.L., mantle layer
B.V., blood vessel Myo., myotomes
C.C., central canal or cast of the same N.A., membranous neural arch
C.C.C., central canal closure, caused JN.C., nerve cell
by fusion of lateral plates Ne., notochord
Ep., ependyma P.C., pigmented or eye cells of Amphi-
Ep.N., layer of ependymal nuclei oxus
F.P., floor plate of the central nervous R.F2z., roof plate expansion
system R.P., roof plate of the central nervous
G.C., germinal cell system
L.S., lateral veno-lymphatic sinus or 8S.C.F., cerebro-spinal fluid
anlage of the same V.G., Gasserian or semilunar ganglion
Mar.L., marginal layer VITI.G., Auditory ganglion .
M’.C’., Miillerian or giant cells W.M., white matter
M.F., Miillerian or giant fiber 4 V., fourth ventricle
72
SPINAL CORD AND MEDULLA OF CYCLOSTOMES PLATE 14
WILLIAM F. ALLEN
(Continued from page 72)
75 to SO represent five transverse sections through the developing fourth
ventricle and roof plate expansion in pig embryos from 5 mm. up to 14 mm.
With the exception of figure 75, which is from my collection, the remaining figures
are from frontal series belonging to the Institute of Anatomy. That there is
a direct relationship between the amount of visible coagulum in the form of a
fibrillar feltwork and the expansion of the roof plate is evidenced by the fact
that this coagulum does not appear in the early embryos before the roof plate
has assumed the appearance of an organ capable of the production of cerebro-
spinal fluid (as indicated by vascular supply and granular appearance of the
cells). It may be inferred that the earliest non-coagulable cerebro-spinal found
in the earliest stages is an embryonic fluid which differs in no way from the ordi-
nary intercellular juices, but that the appearance of coagulum at the time when
the roof plate has attained the appearance of a functional chorioid plexus is
indicative of a chemical change in the fluid, which if a product of secretion is
capable of producing a marked increase of internal pressure in the cerebro-spinal
fluid and consequent expansion of the roof plate.
75 From a transverse section through the medulla of a 5 mm. (or less) pig
embryo, in the region of the auditory vesicle. It will be seen that the peripheral
branches of the intersegmental blood vessels have about reached the roof plate,
but no blood vessels have entered the medulla. The protoplasm of the inner
margin of the ependymal cells is sufficiently granular to suggest a secretory
function. The small amount of coagulum in ventricle is probably the result
of secretion, but the cerebro-spinal fluid has probably not exerted much inter-
nal pressure. X 70.
76 Transverse section of a 6 mm. pig medulla through the widest portion
of the fourth ventricle, namely, at the level of the V ganglion. This is the only
portion of the roof plate to have undergone any stretching of its cells, and this
is confined solely to the most centrally located cells. This section shows a con-
siderable increase in the size of the fourth ventricle and expansion of the roof
plate, together with some increase in the amount of coagulable cerebro-spinal
fluid (S.C.F.) and an increase in the number of blood vessels above the roof plate;
but no blood vessels have entered the substance of the medulla. At this stage
the pontine flexure could not have been a factor in producing the roof expansion.
The collapsed appearance of the roof plate at its center is not natural, but rather
a result of the preparation of the material. 39.
PLATE 15
EXPLANATION OF FIGURES
77 ‘Taken from a transverse section through the V ganglion of a 7 mm. pig
embryo. Note the increase in the number of blood vessels above the roof plate,
which together with the increase in the coagulable cerebro-spinal fluid suggests
a functional chorioid plexus. The pontine flexure in this stage is too slight to
have any effect on the expansion of the roof plate. It should also be recorded
that a few blood vessels have entered the outer surface of the medulla. Asin
the previous series the sections have suffered a collapse of the roof plate from
fixation or later preparation of the material. X 39.
78 From a transverse section of a 10 mm. pig embryo through the region of
the V ganglion. The increased vascularity of the mesenchyme above the roof
plate together with the enormous amount of coagulated cerebro-spinal fluid
GS.C.F.) in the ventricle are evidences of the factors which have produced the
increased expansion noticed in the roof of the ventricle. Also at this stage the
pontine flexure has increased to such an extent that its action on a fourth ventri-
cle full of cerebro-spinal fluid, itself under a moderate pressure, would produce
a further expansion of the roof plate. As in the preceding sections the roof plate
has suffered a collapse in the preparation of the material. X 39.
74
SPINAL CORD AND MEDULLA OF CYCLOSTOMES PLATE 15
WILLIAM F. ALLEN
3 — Fr = eens REx.
RX BY.
~ V.
Ms pty.
us Ri iA
ie yr thee,
79 and 80 Two transverse sections through the anterior and posterior ends
of the fourth ventricle from a 14 mm. frontal series of a pig. A more advanced
stage in the development of the chorioid plexus together with « more pronounced
pontine flexure has produced a much larger fourth ventricle and expanded roof
plate than is shown in the previous series (fig. 78). The thickening of the lateral
walls of the medulla is taking place as it did in Petromyzon and Squalus, but the
greater expansion of the ventricle in the pig (fig. 79) has prevented the walls
from fusing ventrally. Nevertheless, the thickening of the ventral portion of
the lateral plates would increase the pressure of the cerebro-spinal fluid. 25
and 39.
ABBREVIATIONS
B.V., blood vessel R.Ex., roof plate expansion
C.S.F., cerebro-spinal fluid S.C.F., cerebro-spinal fluid
Ep.N., layer of ependymal nuclei V.G., Gasserian or semilunar ganglion
Mar.L., marginal layer 4 V., fourth ventricle
M.L., mantle layer X.R., vagus root
R.C., embryonic red corpuscle
75
PLATE 16
EXPLANATION OF FIGURES
S81 to 87 Represent true transverse sections through what has been termed
in the text, the typical embryonic spinal cord, from a number of different verte-
brates, all of which have developed a tubular nervous system after the neural
fold method. They were drawn with the aid of an Edinger-Leitz drawing appara-
tus and reduced one half in reproduction.
81 From a transverse section through the anterior portion of the spinal cord
of a 10 mm. Squalus embryo (Professor Scammon’s series No. 16). This so-
called typical embryonic spinal cord is decidedly compressed. An earlier stage
possessed an elliptical cord with its greatest diameter from side to side. The
floor plate is slightly thicker than the roof plate. Each contains a single layer
of nuclei. The ventral portion of the cleft-like central canal is expanded into
a cavity, which persists as the permanent central canal. 125.
82 Transverse section of the spinal cord of a 19 mm. Squalus embryo (from
Professor Scammon’s series No. 2). Note that the dorsal closure of the lateral
plates, due to fiber and cell proliferation, is the same as was figured for Petromy-
zon. They meet in a seam, leaving dorsal and ventral cavities, of which only
the ventral one persists. As in Cyclostomes this method of closure would tend
to throw a large part of the embryonic cerebro-spinal fluid into the brain cavities.
x 70.
83 and 84 ‘Transverse section through the anterior portion of the spinal
cord of an Amblystoma and a turtle embryo. The former (taken from Professor
Johnston’s series No. 50) is a rather early representative of the so-called typi-
cal embryonic stage; while the latter is a rather late representative of this stage.
Both cords may be said to be compressed (elliptical, having its greatest diameter
dorso-ventral), but only slightly so, when compared with birds and mammals
(figs. 85 and 86). As a result, granting an equal proliferation of fibers and cells
in the lateral plates, it would be expected that the adult cord in Amblystoma and
the turtle would be more depressed, which is found to be the case. X 70 and125.
85 and 86 From anterior transverse sections of the spinal cord of a 93 hour
chick anda 5mm. pig. Both are good illustrations of the so-called typical em-
bryonic stage, the pig being in a slightly more embryonic state. In these we have
the most compressed of all embryonic cords examined, while the adults cord are
nearly cylindrical. XX 125.
87 Transverse section through the caudal end of the same spinal cord shown
in figure 86. Observe spherical appearance which is indicative of an earlier phase
in its development. X 125.
76
SPINAL CORD AND MEDULLA’ OF CYCLOSTOMES
WILLIAM F. ALLEN
ABBREVIATIONS
C.C., central canal
C.C.C., central canal closure, caused
by fusion of lateral plates
Ep.N., layer of ependymal nuclei
F.P., floor plate of the central nervous
system
G.C., germinal cell
Mar.L., marginal layer
M.L., mantle layer
M.R., motor or ventral spinal nerve
root
Ne., notochord
R.P., roof plate of the central nervous
system
Sp.G., spinal ganglion
Sy.P., syneytium of protoplasm
MORPHOLOGY OF THE ROOF PLATE OF THE FORE-
BRAIN AND THE LATERAL CHOROID PLEXUSES
IN THE HUMAN EMBRYO
PERCIVAL BAILEY
From the Anatomical Laboratory of the University of Chicago
THIRTY-ONE FIGURES
CONTENTS
IMAGCOCWUCELOMS «ss bien iea's wee TG ee ieee 15, MASE ey
PAM E eectai aiken cine s Sid'y hd ues ao White ois wa tie WG bien ai Ee Oe ee Aaa 80
Material and methods..... Ar tre OPE Neer etl toe th ea Seto 84
PPDRGUIDWONS ¢ «<0 hk ss00is ies oh ao ie or er Park Soo a ew oe
1. The 19 mm. embryo...... fry ree areeise rte ears te. Reta z 86
Pe DEO INN OM DLT Osa < scum eho sxe was ale lee conte Meee eae anal 9]
3. The 32 mm. embryo...... .y e eS ; ee Ror Mie Come Ce 96
MIB EUB SION so w.¢ Medasos abe aves SPA? SCR Be err es Be 99
1. Telencephalon...... a t Wyswaie ek, ac nlig atk are pat erp RU Ses Pere aks 99
2. Diencephalon........... de ele tcsie le iS: ae. < Wis 9 ok OR RRO LS AG ton in et cal ea 108
YANO LINO; te isin re ne eeia ee tre, I
- eau Ti Pee | ROD INNE
NUMBER OF TEMPERATURE ras . TO EXTERNAL es . Yee hts
near, on TO EXTERNAL ciMrria CONE MYOID MEMBER
: LIMITING SSS (MAXIMUM)
MEMBRANE Sah i
ees [Sas cae, oe ‘a =
1 5 54 78 8-14 50
2 25 88 100 8-14 75
(8) Fundulus. The retina of this fish is interesting because
of the presence of prominent ‘double cones.’ Such elements
are found in representatives of all the vertebrate classes, with
the exception of mammals (Greeff, 00). They consist of two
cones with fused inner members, although close examination is
necessary to demonstrate this union. One component is usually
larger and is known as the ‘chief cone’ (fig. 29; ell. con.), where-
as the smaller is the ‘accessory cone’ (fig. 29; con. acc.) The
chief cone alters its position independently of its accessory
cone, which remains close to the external limiting membrane and
is not moved to any great extent by the action of light or other
stimulating agents.
The rods, although rather small, are quite in evidence and
differ from those of Abramis in maintaining fairly uniform de-
grees of elongation.
In the tables (7 and 8) which show the characteristic results
of experimentation in the dark, the mean myoid length of the
chief cones at 5°C. (fig. 28) is only about one-third that at the
higher temperature (fig. 29). The differences in the accessory
cones are not striking, although the lower values are somewhat
increased at 25°C. Since the movements of the accessory cones
are very limited, this probably represents a significant elongation.
The contrast between the extension of the rod at 5° and 25°C.
is striking and, added to the evidence gained from other fishes,
158 LESLIE B. AREY
TABLE 7
Measurements from the retinas of five Fundulus which had been kept at &°C. in the
dark; the values are in micra and represent measurements taken along axes coin-
ciding with radii of the eyeball
NERVD FIBER
: CHOROID
NUMBER OF we role TO EXTERNAL | CHIEF CONE ACCESSORY ROD INNER
ANIMAL LIMITING . LIMITING MYOID CONE MYOID MEMBER
MEMBRANE MOM RAND
1 100 69 5-11 1-5 16-19
2 106 68 5 1-5
3 88 69 5 1-5
4 105 68 3 1-4 11-19
5 5: 52 5-9 1-5 16-23
IMGamen cates 95 65 5-7 1-5 14-20
TABLE 8
Measurements from the retinas of five Fundulus which had been kept at 25°C. in the
dark; the values are in micra and represent measurements taken along axes coin-
ciding with radii of the eyeball
NERVE FIBRE
CHOROID
NUMBER OF LATE TO EXTERNAL | CHIEF CONE ACCESSORY ROD INNER
ANIMAL pig ear at a LIMITING MYOID CONE MYoID | MEMBER
MEMBRANE ER ANe
1 120 87 14-22 2-5 24
2 120 87 15-22 2-6 31
3 130 100 17-26 6 7A
4 135 94 17-27 6 19
5 135 110 17-26 1-3 38
Meanie sss: 2. 128 96 16-25 3-5 27
indicates that in these animals a lengthening of the inner mem-
bers is favored by a high temperature.
A series of measurements of chief cones in the light failed to
show any differences at the extreme temperatures.
(4) Carassius. The retina of Carassius, as well as that of
Fundulus, has prominent double cones.
In the few eyes measured, the chief cone elongated with in
creased temperature (figs. 23, 24) but the accessory cone did not
change its position, at least to any extent.
Table 9 summarizes the results from typical retinas.
MOVEMENTS IN THE VISUAL CELLS 159
TABLE 9
Measurements from the retinas of four Carassius, two of which had been kept at 5°C.,
and two at 25°C. in the dark; the values are in micra and represent measurements
taken along axes coinciding with radii of the eyeball
NERVE FIBER |
LAYER CHOROID
NUMBER OF TEMPERATURE TO EXTERNAL | CHIEF CONE ACCESSORY
ANIMAL 5 oH se ates LIMITING MYOID CONE MYOID
es MEMBRANE
MEMBRANE 7
1 25 75 88 21 3-5
2 25 69 69 21 2-3
3 5 56 63 1-3 2
4 5 69 94 3-4 2-3
2. Frog. (1) Rana pipiens (adult). Gradenigro (’85), Ange-
lueci (90), Herzog (’05) and Fujita (’11) have all stated that at
a temperature of 30°C. or more, the cones of the frog shorten
until they assume the position characteristic of light. Thus,
Herzog (p. 419) says: “‘Aufenthalt im Briitschrank 1/2 Stunde
lang, Temperatur von 21° bis 30° C. ansteigend: . . . die-
selbe zeigt auch, dass die Zapfen maximal contrahiert sind. Die
Linge der Zapfen betrigt mit ganz vereinzelten Ausnahmen
0.0091 mm. (v. d. Lim. extern.—QOelkugel exel.).”
The action of low temperature was first tried by Herzog (05),
who makes the following statement (p. 424), concerning the re-
sult of two hours’ cooling to 0°C. in the dark: ‘“‘Dagegen sind
die Zapfen bereits nahezu héchstgradig verkiirzt. Ihre Linge
betragt fast durchweg 0.0078 bis 0.0091 mm.”’
Fujita experimented upon six frogs, which were kept at
a low temperature in an ‘Eisgefass’ for periods of 30 minutes
to 6 hours, after which the decapitated heads were fixed in
ice-cold fluid. His conclusion (p. 170) is diametrically opposed
to that of Herzog. ‘Das Resultat war in allen Fallen das gleiche:
ich konnte keine Hellstellung konstatieren. Die Zapfen waren
micht kkontrahiert. . . . .”
Measurements showing the elongation of the cone myoid at
intermediate temperatures (14° to 19°C.) are not given by Herzog,
who, however, states (p. 418) concerning retinas that had been
raised in the course of 15 minutes from 18° to 24°C.: ‘‘Ein
wesentlicher Einfluss is nicht zu erkennen. . . . . Die
160 LESLIE B. AREY
Zapfenlinge (von der Limitans externa bis zur Oelkugel im Ellip-
soid exclusive) betrigt im Durchschnitt maximal 0.034 mm., im
Minimum 0.0169 mm.”’
With apparatus and methods similar to those described in
connection with the experimentation upon frog’s retinal pigment,
an attempt was made to discover the exact responses of the cone
myoid to high, medium, and especially to low temperatures.
In table 10, which gives the measurements of visual cells
from a few typical preparations, it will be observed that the cones
are greatly shortened at 33°C. (fig. 36), but that at the other
temperatures they retain the elongated condition typical of
darkness (figs. 34, 35).1% Measurements of the cones gave two
modal lengths, which are approximately expressed by the figures
in the table representing the extreme values. The maintenance
of elongation at a low temperature is in agreement with Fujita’s
(711) experiments, but is opposed to Herzog’s conclusion, which
was apparently based upon exhaustive investigation. It is true
that the temperature of 0°C. which the latter worker used was
a few degrees less than the lower limit (3° to 5°C.) employed
TABLE 10
Measurements from the retinas of eight Rana pipiens which had been kept at 3°, 14°,
19°, and 33°C., respectively, in the dark; the values are in micra and represent
measurements taken along axes coinciding with radii of the eyeball
NERVE FIBER anencimn
NUMBER OF TEMPERATURE | ,, ae Ae TO EXTERNAL | CONE INNER ROD INNER
ANIMAL 2C: LRG: LIMITING MEMBER MEMBER
MEMBRANE MEMBRANE
1 3 140 66 12-20 10+
Z 3 160 75 13-22 Q=+
3 14 103 56 11-20 1+
4 14 88 50 11-16 10+
5 19 103 61 13-19 13+
6 19 94 63 14-23 10+
7 33 163 63 8-12 10+
8 33 126 63 7-10 11+
12 The actual values at medium and high temperatures vary somewhat from
those given by Herzog for R. temporaria (and R. esculenta?).
13 This experimentation upon the retinal elements of the frog was practically
completed before Fujita’s paper was known to me.
MOVEMENTS IN THE VISUAL CELLS 161
by me, yet it seems improbable that such a small difference would
cause the cones to shorten maximally. Any error that could
be introduced during these determinations would tend to shorten
the cones, hence Herzog’s results are at a disadvantage in this
respect. I have continued experiments for 6 hours, yet the
results were always the same; in no case was there found a gen-
eral shortening of the cones that in any way resembled the ex-
treme condition at 33°C.
From these results, which, in a general way, are the reverse
of those found in fishes, it is evident that the responses of the
cones are not comparable to those of the retinal pigment. The
pigment may indeed be under nervous control, so that stimulat-
ing agents such as heat, cold and light produce a migration ac-
cording to the principle of specific energies, yet if the cone cells
are influenced by the nervous system, these experiments can not
be said to furnish proof of such a relation.
In order that there should be no doubt concerning the effect
of low temperature upon the cone myoid, a further determina-
tion was made long after the results which are tabulated above
were obtained. In this later experiment rigorous precautions
were observed to eliminate possible errors. After frogs (R.
pipiens of various sizes), kept at a temperature of 18°C., had been
subjected to a preliminary treatment of darkness for 72 hours,
they were introduced into a vessel cooled to 1°C., where they
remained for a period of 4 hours. The eyes were then quickly
excised in dim, red light, the operation not requiring more than
15 seconds, after which they were returned into darkness where
fixation at approximately the freezing temperature ensued.
The average measurements of the cone myoids in these prepara-
tions were as follows: 12 to 14 »; 10 to 15.4 uw; 10 to 14 u; 10 to
11 4313 to 15.4 4;9 4. These values, although somewhat smaller
than those given in table 10, can not be said to prove that low
temperature shortens the myoids as does high temperature.
Gradenigro (’85) found that elevated temperature induced
a shortening of the rod in the dark. After measuring the myoid
length in a considerable number of preparations from retinas
subjected to various temperatures both in light and in darkness,
162 LESLIE B. AREY
I was unable to discover constant differences in length that
could be correlated with definite temperatures. The agreement
of mean values obtained from various retinas under identical
temperature conditions was not good, and since the rod myoid
measures only 6 » to 12 uw in length, even small variations fur-
nish serious obstacles in determinations of this kind. In any
one preparation, moreover, variability in the length of adjacent
myoids tended somewhat to mask a possible temperature effect.
If anything, my measurements showed the reverse of what
Gradenigro maintained, the elongation at 33°C. in the dark
being greater than at 5°C., but, as stated before, these results
are by no means trustworthy.
A shortening of the rod through the action of high tempera-
ture, as claimed by Gradenigro, is of interest because, accord-
ing to most investigators, ight produces the same result. With
this can be compared an analogous correlation in fishes, where
light causes an elongation of the rod myoid and, as I have shown,
elevated temperature does likewise. It is certain, on the con-
trary, that although the cones of both frogs and fishes shorten
in the light, heating produces unlike responses in the dark.
(2) Rana catesbiana (larvae). Although the cones in both
the 4.5 cm. and the 7.0 cm. larva of this frog are of large size,
clearly defined temperature responses were not observed; in-
deed, the difference between the positions assumed even in light
and darkness is not striking, the cone myoids in the light re-
maining well elongated in comparison to those of the adult R.
pipiens. The variability in length in different preparations is
considerable, yet if anything, the cones appeared more shortened
at 3°C. than at higher temperatures. There is no marked shorten-
ing at 33°C., for the cones under these conditions were as long
as at lower temperatures, and in some cases longer. It is
probable that the responses of the cones in adult R. catesbiana
will be found to agree with those in other species which have been
studied, although no experimentation was performed to deter-
mine this point.
3. Necturus. The rods, and the single and double cones of Nec-
turus are very large, yet positional changes with varying tempera-
MOVEMENTS IN THE VISUAL CELLS 163
ture (3° to 28°C.) were not observed. Individual cells vary more
or less in the height at which they are situated above the exter-
nal limiting membrane, yet no constant differences of significant
amount could be correlated with definite temperatures. With
these results should be compared Garten’s (’07) denial of a
change in the position of the cones of the ‘salamander’ through
the influence of light, such as Angelucci (’90) had previously
claimed. Stort (’87), however, described movements in both the
rods and the cones of Triton.
C. EXPERIMENTATION UPON EXCISED EYES
a. Effect of light and darkness
The results of a number of investigators since the first work
of Englemann (’85) have indicated that the retinal elements of
some vertebrates, and especially the frog, are subject to a nervous
control, the action of which is not well understood.
The réle which the nervous system plays either in producing
or in assisting the movements of the various retinal elements
is hard to demonstrate. Experimentation involving the direct
action of light on excised eyes can not be expected to solve the
problem decisively, for if no movements result, autoanaestheti-
zation, or some similar disturbance due to the interrupted blood
supply, may be the real cause. If, on the other hand, responses
are called forth by direct stimulation, it by no means follows
that a similar phenomenon necessarily occurs in the living animal,
any more than a demonstration of the direct stimulation of
muscle fibers proves that this rather than a nervous impulse is
the normal method of muscle stimulation. The limitations which
restrict a wide interpretation of results, however, do not lessen
the interest involved in determining the extent to which the
retinal elements can be directly stimulated.
Hamburger (’89) maintained that the cones and retinal pig-
ment in excised eyes of the frog assumed the positions characteris-
tic of light or darkness according to the conditions of the experi-
ment. Dittler (’07) working on isolated frog’s retinas obtained
164 LESLIE B. AREY
a shortening of the cones in localized areas through the action
of light but found darkness to be ineffectual. The spread of
the response to portions of the retina unstimulated by light, led
Dittler to investigate further the cause of cone retraction. He
was able to furnish experimental proof that weak acids, resulting
from catabolic processes in the retina, caused the cone myoid
to shorten; hence he concluded that the cone myoid was not of
itself ‘lichtempfindlich,’ as Englemann (’85) had believed, but
was stimulated to movement through chemical agents result-
ing from the action of light on the retina. Fujita (11), as a
result of very limited experimentation, stated that the pigment
of the excised eye of a frog expanded in the light but did not con-
tract in the dark.
Ringer’s solution, normal saline solution, and tap water were
used by me for the immersion of excised eyes. When the first
two media were employed the movements of the rods and cones
of Ameiurus, through the action of light, were never clearly
demonstrated; possibly such results are to be interpreted as
evidence of a chemical control somewhat comparable to that de-
seribed by Spaeth (’13) for the melanophores of Fundulus. Tap
water did not inhibit the movements of any of the retinal ele-
ments of Ameliurus and consequently it was used in all subse-
quent experimentation.
The pigment of Ameiurus, Abramis and Fundulus did not
contract when excised eyes from light-adapted fishes were sub-
jected to darkness for periods of 4 hours or less. At most there
was only evidence of a retraction of the distal accumulation of
pigment, which is characteristic of light-adapted eyes, to form
a more homogeneously pigmented zone (figs. 2, 10, 6). When
the reverse experiment (subjection to light) was performed,
the pigment of Ameiurus became maximally expanded in 2 hours
(figs. 1, 3). Only the slightest tendency toward expansion,
however, could be found after similar experimentation on the
two other fishes. It thus appears that light acts directly on
the pigment of Ameiurus only, while darkness is totally ineffec-
tive on all three animals.
MOVEMENTS IN THE VISUAL CELLS 165
When the rods and cones of Ameiurus and the cones only of
Abramis and Fundulus were tested, the following results were
obtained. The rods of Ameiurus moved both in light and in
darkness, whereas the cones were stimulated only by light, no
elongation occurring in the dark even when the experiment
continued for 4 hours. The cones of Abramis and Fundulus
did not change their positions to any extent either in light or
in darkness. In the light the cones of Abramis, which were more
carefully investigated than those of Fundulus, at most showed
only slight retraction and never closely approached the exter-
nal limiting membrane. If light did exert a direct influence
on the cones or retinal pigment of this fish, the changes would
be extremely easy to distinguish due to the wide difference
between the light- and dark-adapted phases.
Since neither the cone cells nor retinal pigment of Abramis
underwent movements under these conditions, it is possible
that the accumulation of catabolic products, occasioned by the
interruption of the blood supply, was responsible. Experimenta-
tion of the following kind shows the importance of maintaining
the vascular circulation. If the optic nerve only of Abramis is
cut, the retinal elements undergo their normal movements in
darkness and in light. If, however, all the blood vessels and
muscles are cut and the eye ball is attached to the body by the
optic nerve only, no movements result. The objection may be
made that some nervous mechanism is deranged by cutting
these muscles and blood vessels, but this is hardly probable,
as further experimentation, to be presented in a subsequent
paper, on this and other fishes has shown.
In eases like the movements of the pigment or cones in excised
eyes of Ameiurus through the action of light only, it is probable
that an inhibitory tendency is also present, but the response to
the stimulus furnished by light is sufficiently vigorous to over-
come it. Either a less vigorous stimulus or response may eX-
plain why no movements of the cones and pigment occur in
darkness.
An inhibition due to the presence of unremoved catabolic
products, as postulated here, would be merely a form of auto-
166 LESLIE B. AREY
anaesthesia. As will be shown, carbon dioxide and other anaes-
theties do, in fact, arrest the movements of all the retinal elements
of fishes.
Dittler (07) accounted differently for the absences of elonga-
tion in the cones of isolated frog’s retinas which were introduced
from light into darkness. In order to appreciate his way of
viewing this situation, it is necessary to understand the general
theory advanced by him to explain the movements of the cones.
In darkness an equilibrium was supposed to exist in the metab-
olism of the retina, the elongated cone myoid representing an
unstimulated condition. Through the action of light, however,
catabolic processes preponderate, and the accumulated acid
wastes chemically stimulate the myoid to shorten. These con-
clusions were based upon experimental evidence by which it
was shown that weak, free acids could be detected if isolated
retinas were subjected to light in limited amounts of Ringer’s
solution, and further that such an acid solution was capable of
causing other dark-adapted cones to shorten while still in the
dark. To return to the case under consideration, Dittler believed
that the accumulation of the catabolic products formed in the
light merely continued its contractile influence after the isolated
retina was removed into the dark, and since these products were
not removed, the metabolic equilibrium could never be restored
and consequently elongation failed to occur.
This theory of chemical stimulation is not supported by the
condition in Fundulus and especially in Abramis, where the
cones of excised eyes do not shorten even when exposed to light,
for under these favorable conditions the tendency toward the
production of a catabolic excess should be maximum. In still
another way Dittler’s theory does not explain a typical response
of the cones of fishes. The cone myoid in isolated retinas of
the frog shortens when the temperature is raised to 30°C. or
more in the dark (fig. 36), and this fact Dittler used to sup-
port his view in the following logical manner. It is well known
that most chemical reactions are accelerated by raising the
temperature; hence in this case the autonomic equilibrium
normally existing in the dark would become disturbed, the re-
MOVEMENTS IN THE VISUAL CELLS 167
sulting increase of catabolic products causing the cone myoid
to shorten.4 Although Dittler’s statement is not altogether
clear, it seems evident that low temperature was supposed not
only to reduce the metabolism of the retina to a low level,
but also to render the cone myoid less ‘empfindlich’ to chemical
stimulation.
The conditions in the dark-adapted cones of fishes, however,
are entirely different, for here not only do the cone myoids
elongate when the temperature is increased, but also elongated
cones can be made to shorten by the use of low temperatures.
If the shortening of the cones of fishes in the light were due to
a chemical stimulation, how can the elongation of these elements
in the dark through the action of heat be explained, since mani-
festly in this case the metabolic equilibrium tends to become
destroyed, the result being the formation of an excess of catabolic
wastes, which by analogy with the conditions in the frog, should
cause the cone myoids to shorten? Moreover, the efficiency
of low temperature in retracting elongated cones, and the cor-
relation between the uniform degree of myoid elongation and
the temperature gradient (p. 155) finds no explanation through
Dittler’s hypothesis.
It should be said, however, that Dittler strictly limited his
conclusions, the experimental evidence for which appears to
be well established, to the material upon which he worked, and
was even reserved in suggesting the occurrence of a similar
method of stimulation in living animals.
The reason why the rods of Ameiurus move in darkness, while
the cones do not, may be as follows. The rod myoid normally
shortens in the dark, whereas the cone myoid elongates. It is
probable that the contractile function of the myoid is more
vigorous than the reverse process of elongation. This is not
only substantiated by the fact that dark adaption of the rod
takes less time than light adaption, but also by experiments
14 Dittler did not formulate this conception in extenso as I have expressed it,
yet several statements (pp. 317-318) show that this was his belief. . A concluding
quotation reads: . . . . ‘der Einfluss der Temperatur iiberhaupt ganz nach
physikalischen Modus zu Wirken scheint, und berechtigt uns, seine Wirkung rein
in diesem Sinne zu fassen.”’
168 LESLIE B. AREY
in which the optic nerve was cut. In such cases the rod never
elongated in the light although it often showed a tendency to
shorten when introduced into the dark, whereas the behavior
of the cone cells in light and darkness was the exact opposite
to that of the rod, since they tended to shorten in the light but
remained unchanged in the dark. On these grounds, therefore,
an explanation is offered to show why it is that through the strong
stimulation produced by the direct action of light, both types
of cells show characteristic responses, while in the dark only
the more vigorous contractility of the rod myoid becomes effec-
tive. It must be remembered, however, that although the direc-
tion of the movements of the rod and cone cells are opposed,
the real response of the protoplasmic myoid may be similar in
both cases. If this were true, the apparent inconsistency in
the movements of these elements would be due to a difference
in the axis of contractility in the two kinds of myoids, and the
explanation just advanced would not stand. Reference will
be made to these possibilities in another place.
b. Effect of temperature
Previous attempts to determine the direct influence of tem-
perature upon the retinal elements have been confined to the frog.
Gradenigro (’85) found that if excised eyes of dark-adapted
animals were subjected to a temperature of 30° to 386°C. the
rods and cones shortened and the pigment expanded, both
end-results béing characteristic of light-adaption. Dittler (07)
was able to confirm Gradenigro’s discovery concerning the cone
cells. When the isolated retina was heated to 35° to 37°C. in
the dark for 50 to 60 minutes, the cone myoids shortened. After
retinas had been subjected to a temperature of 1° to 2°C. for
many hours, on the contrary, no shortening of the cone myoid
was observed.
The apparatus and methods used by me were similar to those
described in connection with the experiments upon living fishes.
The excised eyes were contained in test tubes which were sus-
pended in jars of water kept at appropriate temperatures. Eyes
MOVEMENTS IN THE VISUAL CELLS 169
from the same animal were used simultaneously, one at each
temperature extreme.
1. Effect of temperature upon retinal pigment. After many
trials it was found that sharp differentiation of the retinal pig-
ment of fishes was hard to secure at the extreme temperatures
when the initial temperature had been intermediate. Accord-
ingly, the expedient was employed of subjecting the living ani-
mals to a preliminary treatment either at 3°C. or at 25°C., and
as a result uniformly satisfactory differentiation was obtained.
The retinal pigment of Ameiurus behaved precisely as in
living animals. At a low temperature, both in darkness and
in light (figs. 3, 1), the degree of distal migration was greater
than that at a high temperature (figs. 4, 2). Particularly in
experiments conducted in the light was this strikingly appar-
ent, since in many preparations at 3°C. the pigment migrated
so far distally that the more proximal portions of the cells were
free of granules, while a sharp line of demarcation existed be-
tween the pigmented and non-pigmented zones.
A few experiments were made upon the dark-adapted eyes
of Abramis. In this case also, a greater pigment expansion
was found at 3°C. than at 25°C. (figs. 11, 12).
Reference has been made to the contention of many investi-
gators that there is an apparent nervous control over the move-
ments of the frog’s retinal elements. It has been shown that at
a medium temperature in the dark the pigment is maximally
contracted (fig. 18), whereas at higher and lower temperatures
(figs. 17, 19) a considerable degree of expansion is effected. Not
only is the amount of migration occasioned by temperature
much more extensive than that in fishes, but also the similarity
between the effects of the two temperature extremes as con-
trasted with an intermediate temperature, has no parallel among
other pigment cells or even in melanophores.
Since it is at least agreed that the pigment of excised eyes of
the frog expands in the light, it ought to be possible to observe
the effect of temperature, if this agent acts directly upon the
pigment cells. Excised eyes from animals that previously had
been at an intermediate temperature in the dark, were subjected
THE JOURNAL OF COMPARATIVE NBUROLOGY, VOL. 26, NO. 2
170 LESLIE B. AREY
to temperatures of 3°, 16°, and 33°C., but no changes were ob-
served in the position of the retinal pigment. These results,
which do not agree with Gradenigro’s statement, by no means
furnish conclusive proof that in the living frog temperature
operates through the nervous system, yet when supported by a
comparative study of the retinal pigment and melanophores of
other animals, the conclusion reached by Herzog (’05), that the
expansion of the frog’s retinal pigment under these circumstances
is of nervous origin, involving the principle of specific energies,
becomes highly probable.
According to Fujita (11), when excised eyes from dark-adapted
frogs are retained in the dark for 20 minutes, the pigment assumes
a partial light position. My observations do not confirm this
result, for no migration of any consequence occurred.
2. Effect of temperature upon visual cells. When a study of
the visual cells of Ameiurus was made, the effect of temperature
was found to be essentially similar to that upon normal animals.
When eyes from dark-adapted individuals of Abramis that
had previously been kept at a temperature of 25°C. were like-
wise excised and subjected to 5°C. and 25°C. in the dark,
those at 25°C. (fig. 27) retained the elongated position while
those at 5°C. (fig. 25) shortened to a considerable extent
although somewhat less than in living animals. Mention has
already been made of the significance of these results in con-
nection with the applicability of Dittler’s theory of chemical
stimulation to the cones of fishes. Although the rods of Abramis
at both temperatures showed a distribution extending over wide
limits, yet the shortest measured 12 u at 5°C. as compared with
20 wat 25°C., and the modal elongation at the same temperatures,
as judged by the eye, was 18 » and 25 u respectively. It is
interesting to note that in the excised eyes of dark-adapted
Abramis, temperature is able to produce changes in both the
retinal pigment and visual cells, notwithstanding the fact that
light and darkness are wholly ineffectual in this respect.
Table 11 summarizes these results from typical retinas.
A few experiments were performed upon the cone cells of
the frog. The results obtained were identical with those stated
MOVEMENTS IN THE VISUAL CELLS 171
TABLE 11
Measurements of the visual cells from the retinas of two Ameiurus and two Abramis,
of which one of each had been kept at 5°C. and the other at 25°C. in the dark; the
values are in micra and represent measurements taken along axes coinciding with
radii of the eyeball
FISH TEMPERATURE PG: CONE MYOID ROD INNER MEMBER
Ameiurus No. 1....... 5 4-16 5-6
Ameiurus No. 2:...... 25 19-32 8-12
IApramis No. b:.;.:3:..| 5 10-30 18
Abramis No. 2 7 25 35-50 25
by Dittler (07), who used isolated retinas. In dark-adapted eyes
which were placed in water at a temperature of 33°C. the cone
myoids shortened (fig. 36), while at 3°C. or 16°C. (fgs. 34, 35)
the myoids remained for the most part unchanged. These re-
sults are identical with those found by Fujita ('11) and myself
on the cones of living animals, and indicate that, unlike the pig-
ment cells, the movements of the cones are not dependent upon
nervous control. If an influence of the nervous system over
these elements exists in the normal animal, it is at least not mani-
fested as is the control over the retinal pigment, in which changes
at both high and low temperatures can be interpreted according
to the principle of specific energies.
D. EFFECT OF ANAESTHETICS
Various instances have been noted throughout this paper in
which the behavior of the retinal pigment and the visual cells,
when deprived of their blood supply, cast suspicion upon auto-
anaesthetization as being the factor causing suspension of move-
ment. Certain conditions discovered in the responses of melano-
phores in the web of the frog’s foot had previously suggested
such a possibility; indeed, it was this difficulty which led to
the abandonment of the frog’s melanophore as material for an
investigation somewhat similar to the present one. In this way
my interest was aroused to determine the effect of anaesthetics
on the movements of the retinal elements, both in normal animals
and through the more direct action upon excised eyes.
172 LESLIE B. AREY
The effect of certain drugs, as quinine and strychnine, upon
the retinal pigment (‘protoplasmagifte’) is in dispute. It is
clear, however, from the work of Ovio (95) and of Lodato (’95)
that cocaine can arrest pigment migration.
As a precaution against a possible source of error, animals
were never introduced from one condition of light or darkness
to the other without having been previously subjected to a
brief preliminary treatment of the anaesthetic which was to be
tested.
a. Retinal pigment
1. Carbon dioxide. The carbon dioxide used in these experi-
ments was a commercial soda-water product sold under the trade
name of ‘Pureoxia.’ Quantitative determinations of the con-
centrations used were made by titration with #4) sodium
carbonate, using phenolphthalein as an indicator.
In the first experiments made on Ameiurus the movement of
the pigment was arrested by a strong solution of carbon dioxide,
but since none of the animals survived such treatment the-
obvious objection exists that the pigment cells also may have
been killed.
A slight refinement in method consisted of revivifying the
fishes at intervals, by temporary removal to running water,
until opercular movements were restored. By this method
fishes were kept alive for 2 hours, during which time four or
five revivifying treatments were necessary. The migration of
retinal pigment was shown to be checked both in light and in
darkness, yet controls proved that the cells were not permanently
injured.
A method which gave more satisfactory results was devised
after repeated trials had given a mixture of tap water and car-
bonated water of sufficient strength to anaesthetize an Ameiurus
but not to prohibit opercular movements of greatly reduced
amplitude. The record of an experiment will well illustrate
both the method and the results.
Experiment 4.1.6. A dark-adapted Ameiurus was placed in a
mixture of 1 part of ‘Pureoxia’ to 4 parts of tap water, and after re-
MOVEMENTS IN THE VISUAL CELLS 173
maining 10 minutes in the dark the jar was removed into strong dif-
fuse daylight for 1{ hours. During this time, the fish was practically
motionless except for a very weak but rhythmical pulsation of the oper-
cular rims. At the end of the experiment one eye was removed and
fixed. The Ameiurus was allowed to recover until the next day when
the other eye was removed. The pigment in the eye which had been
subjected to carbon dioxide was in the typical dark position (cf. fig. 4)
while the pigment of the control eye was maximally expanded (ef.
fig. 2). Titration of the anaesthetizing solution showed that the
concentration of carbon dioxide had been in the ratio of 60.14 ec. per
litre of water.
In the converse experiment from light to darkness an Ameiurus
lived 3 hours in a similar solution (53.07 ee. of carbon dioxide
per litre) during which time the pigment retained its light dis-
tribution, whereas the control eye removed on the next day,
showed maximal contraction.
These results prove conclusively that in the presence of certain
concentrations of carbon dioxide the pigment cells are not injured
but are in a condition of anaesthetization whereby there is a
failure to respond to the normal stimulus causing contraction
and expansion. Such experimentation, however, does not show
whether this failure is due to a direct effect upon the pigment
cells or to an inhibition through the central nervous system.
To demonstrate which alternative is true, the effect of carbon
dioxide was tested on excised eyes of Ameiurus. If, under these
conditions, a migration occurs a direct influence of the anaesthetic
on the cell itself will be disproven, while on the other hand,
‘ if no migration ensues one can only infer that a similar direct
action on the pigment cell is responsible for the whole course of
events in the living fish, whereas an inhibition through the cen-
tral nervous system may be involved as well.
For such an experiment the excised eye of Ameiurus is well
adapted, since its pigment has been shown to migrate from the
dark to the light position, although the reverse process does
not occur. Excised eyes of dark-adapted fish were exposed to
light in a solution of carbon dioxide having a strength of about
60 ce. per litre. The pigment in each case was arrested in the
contracted position.
174 LESLIE B. AREY
The work described for living Ameiurus has been repeated
on both Abramis and Fundulus with identical results. In every
case the pigment maintained the position it occupied previous
to the application of the anaesthetic.
The results obtained in this study, as a whole, are very differ-
ent from those of Fick (’90), who concluded that the retinal pig-
ment of the frog expanded when the animals were subjected
to an atmosphere of carbon dioxide gas. Fick attributed this
result to asphyxiation and it is certain that the experimental
conditions in his work differed greatly from those in my tests.
In order to make the experiments more comparable, frogs should
be treated with a mixture of oxygen and carbon dioxide gases
in which they could live.
2. Ether. The anaesthetic effect of ether on the retinal pig-
ment was demonstrated by a series of tests that duplicate those
described with carbon dioxide. Care must be observed against
using an excess of ether since otherwise a partial or complete
disintegration of the pigment cells results.
Both dark and light trials were made on Ameiurus, Abramis,
and Fundulus. In each animal the pigment was found to be
completely arrested in whatever position it occupied at the be-
ginning of the experiment. Controls proved that ether, if
used in small amounts, does not permanently injure the pigment
cells.
Ether also checked the migration of pigment in excised eyes
of dark-adapted Ameiurus when such eyes were subjected to light.
3. Chloretone and urethane. ‘These substances are such satis-
factory narcotizing agents that their effect was tested upon the
retinal pigment of Ameiurus. Individuals lived in 0.1 per cent
chloretone or in 1.0 per cent urethane, but the pigment was not
arrested in its movement from the dark to the light phase. In
concentrations of 0.5 per cent chloretone and 2.5 per cent ure-
thane, the pigment migrated when fish were brought from dark-
ness to light although the animals died in both cases.
The results from all the foregoing experimentation are of
interest in showing the difference in the effect upon pigment
cells of four powerful anaesthetics, of which only two were
MOVEMENTS IN THE VISUAL CELLS 175
efficient. The experiments with chloretone and urethane also
prove that even though the animal as an organism dies, the
pigment, nevertheless, can expand independently.
b. Visual cells
Experiments similar to those just described were repeated
in order to determine the action of anaesthetics on both rod and
cone cells. Since the cone myoid is maximally elongated at
about 25°C. in the dark (figs. 25, 27), this condition was taken
advantage of in producing sharp contrasts between dark and
light phases. The cones of Abramis and Fundulus, on account
of their great contractility, were particularly favorable for ob-
servation, as were the rods of Ameiurus because of their large size.
The results of these experiments are shown in table 12.
TABLE 12
A tabulation of the effects of carbon dioxide and ether upon the movements of the
visual cells of Ameiurus, Abramis, and Fundulus; X indicates that the movements
of the elements were completely arrested; conditions corresponding to the blank
spaces were nol investigated
CONE ROD
FISH eS ee eee od ms “
Dark to Light | Light to Dark Dark to Light Light to Dark
Ameiurus....... 4 xX
Abramis......<.. 4 x xX . 4
MUNOUMISes <2. X x
The conclusion is, therefore, that both ether and carbon diox-
ide anaesthetize the visual cells of normal fishes to such an extent
that neither light nor temperature is effective in causing positional
changes.
A few experiments upon the excised eyes of Ameiurus showed
that both carbon dioxide and ether have the same anaesthetic
effect on the rods and cones as that described for normal animals.
Whether or not autoanaesthetization prevented movements
of the retinal elements, as was suspected in previously described
experiments when the normal blood supply was interrupted, it
is at least demonstrable that certain anaesthetics do act in a
176 LESLIE B. AREY
similar way. The effect of carbon dioxide is especially interesting
for, as it is the commonest catabolic product, it may have been
the agent that prevented movements in those cases. This con-
ception is opposed to Dittler’s (07) view, which assumes the
existence of a balance in the metabolism of the unstimulated
cone cells which is disturbed by the increased catabolism through
the action of light. The movement of the cone cells in the
isolated retina of the frog was stated by Dittler to be due to
the action of a weak free acid, the product of increased catabolism.
This conclusion, which was supported by experimental evidence,
is opposed to that postulated by me; nevertheless, it must be
pointed out that the responses of the retinal elements differ
considerably in fishes and in the frog, and while evidence for
autoanaesthetization is indirect yet the results obtained from
experimentation upon fishes can be consistently interpreted
in this way, whereas Dittler’s hypothesis does not meet all the
known facts. ~__
EIS Wb | Sp Be So
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,
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rin;
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195
PLATE 3
EXPLANATION OF FIGURES
Figures 17 to 22, which are photographs showing the influence of temperature
on the distribution of the retinal pigment of the frog (adults and larvae), are all
at a magnification of 170 diameters. The larval Rana catesbiana, from which
figures 20 to 22 were made, had a total body length of 7.0 em. A Leitz ;': homo-
geneous immersion objective was used in making figures 23 and 24, which are
magnified 715 diameters.
17 R. pipiens (adult), 3°C. in the dark.
18 R. pipiens (adult), 18°C. in the dark.
19 R. pipiens (adult), 33°C. in the dark.
20 R. catesbiana (larva), 3°C. in the dark.
21 R. catesbiana (larva), 16°C. in the dark.
22 R. catesbiana (larva), 32°C. in the dark.
23 Carassius; 27°C. in the dark.
24 Carassius, 3°C. in the dark.
196
MOVEMENTS IN THE VISUAL CELLS PLATE 3
LESLIE B. AREY
- a >
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——_
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19 292
. zi
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23 24
197
PLATE 4
EXPLANATION OF FIGURES
These photographs, taken with a Leitz y's homogeneous immersion objective,
are at a magnification of 715 diameters and show the responses of the myoids of
the cone cells of fishes when under the influence of temperature.
25 Abramis, 3°C. in the dark.
26 Abramis, 15°C. in the dark.
27 Abramis, 27°C. in the dark.
28 Fundulus, 3°C. in the dark.
299 Fundulus, 27°C. in the dark.
198
MOVEMENTS IN THE VISUAL CELLS PLATE 4
LESLIE B. AREY
---sl, ply.
_-ell. bac.
mb. lim.exz>
ell. con
st. nl. ex,
con, ace,
~~mb. lim. ex
~~-sl. nl, ex
{ : i
ee
mb. lum. ex
rem MN gare a
j , M jé ig. y,
i . iy’ ;
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ell. con.-
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CON. ACCz -- --=-===-=---
mb. lim. ez.
---st. nl, ex. bc Page od or
st. nl. ex~-- =
af 29
199
PLATE 5
EXPLANATION OF FIGURES
All drawings in this plate are at a magnification of 930 diameters, a Leitz
js homogeneous immersion objective being used.
30 Showing the positions of the rods and cones in a typical light-adapted
retina of Ameiurus.
31 Showing the positions of the rods and cones in a typical dark-adapted
retina of Ameiurus.
32 Showing the effect of low temperature (8°C.) on the position of the dark-
adapted rod- and cone-cells of Ameiurus.
33. Showing the effect of high temperature (27°C.) on the position of the dark-
adapted rod- and cone-cells of Ameiurus.
34 From the retina of a Rana pipiens that had been previously kept at a
temperature of 3°C. in the dark. The cone myoids remain elongated as at an
intermediate temperature.
35 From the retina of a Rana pipiens that had previously been kept at 18°C.
in the dark. The cone myoids are elongated.
36 From the retina of a Rana pipiens that had been kept at 33°C. in the
dark, showing the resulting shortening of the cone myoids.
200
MOVEMENTS IN THE VISUAL CELLS PLATE 5
LESLIE B. AREY
_-prs.dst.bac.
we prs.dst.bac.
prs.dst.con:------
prs.dst.con
--.ell.bac ell.con------
a gtt.ol
Penn prs.dst.bac..
cee ell. bac.
my .con------
oo my .bac <3
----prs.dst.con, = es fee aaery
inl ~~mb.lim.ex.
st.nl.ex.-----
y---H----ell.con. 34
ell.bac:----
---my.bac. prs.dst.bac.. my.bac:-----
prs.dst.con..
---MY.con,
ell.bac
my.bac. _ Px
= “nb.lim.ex.
~-st.nlex,
gtt.ol---- a
ellcon.—¥,
---.my.con,
prs.dst.bac. +---ell.bac.
my .con:---
SS _ell.bac. . 7
Sa mb.lim.ex:
---my.bac.
ell. bac. --4 :
SES my bac. __-
\ -----my.bac.
~--mb.lim.ex, ;
~~~st.nl.ex. 3 5 mb.lim.exs
sstnlets
201
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, No. 2
REGENERATION IN THE BRAIN OF AMBLYSTOMA
I. THE REGENERATION OF THE FOREBRAIN
H. SAXTON BURR
The Anatomical Laboratory of The School of Medicine, Yale University
FOUR FIGURES
Up to the present time the data dealing with regeneration in
the central nervous system have been exceedingly conflicting.
The early workers reported the regeneration of various definitive
parts after they were extirpated. As far back as 1890 Danielew-
sky found that the removal of the cerebral hemispheres of the
frog resulted in the formation of a ‘cerebral mass’ which he
believed contained embryonic nerve cells, though this mass
was in no way a new hemisphere. More recently Bell, in 1907,
removed the ‘lateral half’ of the brain of the frog and found
that the brain nearly always is reformed though never does it
reach the normal size.
On the other hand, Schaper (’98) found that the removal of
portions or of the entire brain of Rana esculenta was never
followed by regeneration. Rubin in 1903 working with Rana
fusca larvae found that no regeneration occurred after the re-
moval of the brain or of parts of it.
The results of experiments published elsewhere (Burr ‘16)
show conclusively that the nasal placode of Amblystoma and
also of the frog does not regenerate when it is completely re-
moved. In addition, the large amount of work by Lewis and
others has shown that the eye will not regenerate when all of
the anlage is removed. It was deemed probable, therefore,
that complete extirpation of the cerebral hemisphere would not
be followed by even a partial regeneration of the part removed.
The discrepancy in the results previously reported might be
due to incomplete operations. The regeneration of parts of
203
204 H. SAXTON BURR
the brain would then be due to the fact that the entire substance
of the hemisphere was not removed, enough being left to carry
the regeneration through to a considerable degree of complete-
ness. A condition similar to this has been shown by Harrison
to exist in the extirpation of limb buds in Amblystoma (Harri-
son 715).
So far as it has been possible to ascertain, no attempt has
previously been made to control the stimulus afforded by the
functional activity of the end organ normally connected with
the part of the central nervous system removed. Extirpation
of a portion of the brain has invariably carried with it the re-
moval of the end organ. It is a little difficult then to see why
a part should regenerate when its activity has ceased owing to
the removal of the end organ.
In order to obtain some answer to the above question the
following experiments were performed on Amblystoma larvae.
Two series of operations were undertaken on embryos possess-
ing neither peripheral nerves nor a circulatory system. In
the first of these the right cerebral hemisphere and the right
nasal plaéode were extirpated, the cut that severed the hemi-
sphere from its connections passing directly in front of the optic
stalk. In the second series the right telencephalon was removed
but the right nasal placode was left in position. This was ac-
complished by turning back the flap of skin containing the pla-
code and removing the underlying forebrain. The flap was
then returned to its former position and held in place until the
wound had healed. Great care was taken to remove all of the
cells of the cerebral hemisphere in all the operations.
The above experiments subject the brain tissue left by the
extirpation of the hemisphere to two conditions. In the first
series of operations the nervous tissue is left to regenerate with-
out the possibility of any stimulus from the end organ that is
normally connected with it. In the second series the end organ,
the nasal placode, is left in its normal position and may there-
fore act as a stimulus to the nervous tissue (Burr 716).
In the first series of experiments in which the right hemisphere
and the right nasal placode were removed the wound usually
BRAIN REGENERATION IN AMBLYSTOMA 205
healed within the first twenty-four hours. Five days after the
operation a new wall had formed connecting the wall of the
right diencephalon with the wall of the left telencephalon. At
first this wall consists of a narrow band of new cells bridging
the interventricular foramen. As growth proceeds the narrow
Fig. 1 Transverse section of embryo five days after operation, by which
the cerebral hemisphere was removed, leaving the nasal placode in place, show-
ing curtain of cells across the interventricular foramen. X 50.
band of cells is drawn out into a thin plate, never more than two
or three cells thick, stretching across the foramen (figs. 1 and 3).
A eareful inspection of a series of operated larvae makes it
quite clear that the new tissue thus formed is derived from the
primary ependymal cells that line the neural tube. Figure 2
drawn from a section of an operated larva of the second series
shows the origin of this new tissue from the margin of the dien-
206 H. SAXTON BURR
cephalon. It is composed entirely of the typical columnar
cells that make up the ependyma. As growth proceeds the
cells of the new membrane lose their columnar shape and _ be-
come metamorphosed into flattened quadrilateral cells. In the
three months old larva, a section of whose brain is shown in
figure 3, it is evident that this metamorphosis is accompanied
by a thinning of the tissue so that at this time the membrane is
Fig. 2. Portion of the regenerated curtain of an embryo seven days after
the same operation as in figure 1, showing regeneration from the margin of the
diencephalon. 50.
composed of only a single layer of cells whose somewhat elon-
gated nuclei are parallel to the surface.
It is evident from the above that the regenerated portion is
not made up of nerve cells but rather of the primitive germinal
epithelium which lines the neural tube, the only regeneration
that takes place being that necessary to close the wound made
by the removal of the hemisphere. We have then, forming as
a result of the operation, a curtain of primary ependyma across
the interventricular foramen. This is not nervous and hence
constitutes no true regeneration of the telencephalon.
BRAIN REGENERATION IN AMBLYSTOMA 207
Hardesty (04) has shown that the dividing elements of the
primary ependyma, the germinal cells, give rise to a nuclear
layer just outside of the ependyma from which later develop
the neuroblasts and the spongioblasts. It is conceivable that
the differentiation of the nuclear layer into nerve cells results
from the stimulus derived from the ingrowth of nerve fibers
eX ry ay
O09". e |
LR
are)
Fig. 3. Transverse section of embryo without the right hemisphere or nasal
placode three months after the operation, showing the thin curtain across the
interventricular foramen. X 50.
from other definitive parts of the nervous system. Evidence
of this is seen in the fact that the fiber tracts of the telencephalon
do not develop until the olfactory nerve fibers have grown into
the peripheral part of the hemisphere from the nasal placode.
The possibility of such a stimulus has been used by Kappers
(14) in his neuro-biotaxis theory of the phylogenetic migration
of nuclear centers in the central nervous system.
208 H. SAXTON BURR
On the other hand Harrison (10) has shown that embryonic
nerve cells taken from the medullary cord of frog larvae will
produce protoplasmic nerve processes when cultivated in vitro,
but it is manifestly impossible to determine by such methods
the extent to which development will proceed without the inter-
vention of functional activity. As has been shown elsewhere
(Burr 716), development and differentiation of the central ner-
vous system will progress to a certain point without the presence
of a functioning end organ. Beyond this poimt functional
activity is essential for continued growth. In the experiments
just referred to, the telencephalon was quite completely organ-
ized without the ingrowth of the olfactory fibers, but this nerve
is not the only one whose fibers penetrate the hemisphere, for
Herrick (10) has shown that in the posterior third of the telen-
cephalon of Amblystoma there are at least two centers that are
directly connected with the hypothalamus and the pars dorsalis
thalami by ascending projection fibers. Hence it seems probable
that the apparent self-differentiation of the telencephalon may
be due in some degree to the presence of the forward growing
nerve fibers which establish this connection, the functional stim-
ulus imparted by them being sufficient to carry differentiation
some distance. Experimental evidence that is in favor of this
view is seen in the fact that in the posterior margin of the cur-
tain which develops across the foramen of Monro on the re-
moval of the telencephalon and nasal placode, there appears in
the older larvae a rounded mass of nerve cells and fibers. This
mass is in direct communication with the nucleus habenulae
and the hypothalamus, a tract of fibers reaching it from each
of these regions. Evidently the neuroblasts at the posterior
margin of the interventricular foramen have been stimulated
to further development by the ingrowth of axones from lower
centers. This mass is, then, in all probability the rudiment
of the primitive pallium.
The second series of experiments involved the removal of the
right hemisphere without the removal of the nasal placode.
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PRELIMINARY
A number of observations have been recorded, chiefly upon
the frog, which indicate that the retinal pigment and visual
cells are at least partially under the control of the central nervous
system, although the manner and extent of this influence are
not well understood. An interrelation between the retinal ele-
ments of the two eyes has also been maintained by which stimu-
lating agents, such as light or salt crystals, applied to one retina
induce changes! in the other. It has not only been declared by
1 Extensive experimentation has demonstrated the existence of photomechani-
cal responses in the retinal pigment of most of the lower vertebrates. In all
eases in which movements of the retinal pigment are demonstrable, light causes
an expansion (i.e. a migration toward the external limiting membrane), and dark-
ness a contraction of the pigment (figs. 1 and 4). A portion of the cone’s inner
member, to which the appropriate term ‘myoid’ has been applied, is also capable
. of actively shortening when the retina is stimulated by light, a compensatory
213
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 3,
JUNE, 1916
214 LESLIE B. AREY
some workers that this interrelation is independent of the brain,
but also that it continues after the optic nerve is cut distal to
the chiasma. The latter assertion, however, has been the sub-
ject of much controversy.
Observations on the frog relative to these conditions have
- been presented by the following workers: Englemann (’85), Grijns
(91); Nahmmacher (’93); Angelucci (90; ’05); Lodato e Pirrone
(01); Chiarini (04) and Herzog (05). Similar statements were
made by Pergens (’96) for fishes, and by Birch-Hirschfeld (’06)
for the pigeon. The diversity of stimuli which have been reported
as being effective in producing positional changes in the cones
and pigment has caused Fick (’89; ’90; 791) to doubt the reflex
nature of the process, although some of his own experiments by
no means disprove many of the contentions of the other investi-
gators. More recently Fujita (11) has shown wherein the older
experimentation on the interdependence in the responses of the
elements of the two eyes through the action of light, is not
trustworthy.
Englemann (’85), who was the pioneer in asserting the pres-
ence of ‘retino-motor’ nerve fibers, further supported his view
with results obtained by illuminating the skin only of dark-
adapted frogs, whence changes in the cone cells and retinal pig-
ment were said to occur. This observation, however, is not in
agreement with those of Fujita (11) and myself (Arey, 716).
A number of other statements are on record which ascribe a
control over the movements of these elements to the central
nervous system. An enumeration of stimulating agents supposed
to act in this way would include such as the following: noises,
unilateral pressure on the eyeball, mechanical irritation (Ange-
lucci, ’90), and trussing up a frog for 24 hours (Herzog, ’05).
The first direct experimentation in which the relation of the
optic nerve to movements of the retinal elements was tested
elongation taking place when the retina is again subjected to darkness (figs. 10
and 11). In fishes and birds, at least, the rod myoid is likewise contractile (figs.
10 and 11), although the direction of movement in light and in darkness is the
exact reverse of that executed by the cones. For a review of the literature on
this question, reference may be made to an earlier paper by the writer (Arey,
715).
EFFERENT FIBERS OF THE OPTIC NERVE 215
was performed by Hamburger (’89), whose results were corrobo-
rated by Fick (91). After the optic chiasma of the frog was
severed, light- and dark-adaption occurred as in normal animals,
hence the pigment and cone cells were viewed as independent
structures, responding to direct stimulation, the movements of
which are not dependent upon the integrity of the optic nerve.
Arcoleo (’90) likewise found that the retinal elements of the
pithed toad exhibited photomechanical changes. On the other
hand, Nahmmacher (’93) showed that stimulation of the frog’s
optic chiasma with salt crystals induced changes in the cones,
only if the optic nerve was intact.
Hence it seems probable that, in the frog, the retinal elements
are capable of more or less independent movement, but over
this is superimposed a nervous (efferent) control, the nature of
which is not altogether evident.
Apart from the work of Pergens (’96), who believed that the
illumination of one eye of a fish induced cone contraction in the
other, practically no attempt has been made to determine the
conditions under which movements of the retinal elements of
fishes are accomplished. The negative results of Gertz (11)
concerning the effect of electrical stimulation of the eyes of
Abramis brama differ only slightly from those of Fujita (11),
who, however, maintained that induction shocks caused an insig-
nificant pigment contraction in the light-adapted eye of the
‘Weissfisch.’
STATEMENT OF THE PROBLEM
It has been shown that the retinal pigment of most fishes
expands to a marked degree when the normal animal is brought
from a situation of darkness to one of light, and that the pig-
ment again contracts when the procedure is reversed (figs. 1 and
4).
The writer (Arey, ’16) has also found that the pigment in the
excised eyes of Ameiurus, at least, is capable of expanding when
subjected to light, which, in this case, presumably acts as a direct
stimulus on the pigment cells; a contraction in darkness, how-
216 LESLIE B. AREY
ever, does not occur. In several other fishes (Abramis; Fundulus;
Carassius) a general pigment migration could not be demon-
strated under these conditions even in the light, and such a lack
of response was accounted for by postulating the occurrence of
an autoanaesthetization of the pigment cells, presumably through
the accumulation of catabolic wastes. Furthermore, the rods of
the excised eyes of Ameiurus undergo movements both in the
light and in the dark, whereas the cones move in the light only;
yet neither darkness nor light induces positional changes of the
cones in the excised eyes of certain other fishes (Abramis and
Fundulus). As will be seen farther on, the total absence of
response in the excised eyes of these fishes throws important
light upon the question of a probable autoanaesthetization of the
retinal elements.
If the optic nerve transmits afferent impulses exclusively, as
hitherto has been believed, no disturbance in the movement of
the retinal pigment should be introduced when the optic nerve
only is severed, and the retina is thereby freed from this source
of cranial innervation. The present paper will be devoted to
the description of experiments devised to test the validity of
this hypothesis.
In order that the reader may better interpret the various
experiments about to be described, as well as the significance of
their progressive sequence, it is advisable to state in advance
the thesis which this paper aims to establish. Experimental
evidence will be presented which offers physiological proof for
the existence in Ameiurus of two distinct components of a
mechanism, through the balanced action of which are move-
ments of the visual cells and retinal pigment alone possible. One
component involves the efferent nerve fibers of the optic nerve,
whereas the second component (possibly the ciliary, autonomic,
nerves) is closely associated with the eye muscles. This latter
set of nerve fibers exerts a constant. inhibition upon the move-
ments of the retinal elements, while the impulses in the efferent
optic nerve fibers, on the other hand, serve only as a block to
this tonic inhibition, thus allowing photomechanical responses
to occur.
EFFERENT FIBERS OF THE OPTIC NERVE 217
The following paper presents one phase of an investigation
upon the visual cells and retinal pigment, pursued at Harvard
University under the supervision of Prof. G. H. Parker. To Pro-
fessor Parker I am greatly indebted for the continued interest
and kindly criticism that characterizes his instruction.
MATERIAL AND TECHNICAL METHODS
The fishes used in this investigation were as follows: the com-
mon horned pout, Ameiurus nebulosus Lesueur; the shiner,
Abramis erysoleucas Mitchill; and the common killifish, Fundu-
lus heteroclitus Linn. A greater part of the experimentation
was done upon Ameiurus, since in this animal the activities of
the retinal pigment and visual cells proved to be especially
favorable under the experimental conditions imposed upon them.
Furthermore, the occurrence of responses in the excised eyes of
Ameiurus was a happy concomitant circumstance, since the
results were thereby controlled at several important points.
~The technical methods used in preparing retinas for micro-
scopical examination were simple. The eyes of Ameiurus were
excised directly, for the skin of this animal is soft and the eyes
are prominent. In the two other fishes, especially when rapidity
of operation was desirable, the following procedure was observed.
With heavy scissors the cranium was bisected in the sagittal
plane; following this, a transverse cut just posterior to the orbit
freed the two halves of the cranium, with the contained eyes,
from the rest of the body. In either case the operation was per-
formed in a few seconds, and the eye, without being handled, was
allowed to drop into the fixing fluid.
Perenyi’s fluid gave good fixation and was used exclusively.
Fixatives containing nitric acid have long been recognized as
favorable in the obtainment of faithful preservation of the retina.
The preparatory steps prior to embedding in paraffine demand
generous allowances of time, yet the processes of dehydration
and clearing should progress as rapidly as possible, since other- -
wise the sclera becomes extremely hard.
Two methods were used in removing the lens, one of which,
although longer, gave much more satisfactory results. The first,
218 LESLIE B. AREY
somewhat tedious, procedure consisted in paring away the front
face of the eyeball with a razor after the eye had previously been
imbedded in paraffine. After removing the face of the eyeball
slightly beyond the ora serrata, the lens was pried from its
paraffine matrix with a dissecting needle; following such manipu-
lation retmbedding was of course necessary. The second and
simpler method was to remove the face of the eyeball with fine
curved scissors after the eye had been hardened in absolute
alcohol; unless, however, the eye was sufficiently hardened and
the greatest care exercised, the retina proper easily separated
from the pigmented epithelium. On the whole, the first method
was preferred to the second because of the wrinkling of the retina
that usually accompanied the use of the latter.
Sections were cut 7p to 10 thick, and only those passing
through the region of the optic nerve were retained for examina-
tion. Preparations were stained with Ehrlich-Biondi’s triple
stain or were double stained in Heidenhain’s iron haematoxylin
and a plasma counterstain. Ehrlich-Biondi in some instances
gave excellent differentiation of all elements, while at other
times it would show the capriciousness for which it is notorious;
iron haematoxylin gave uniformly good preparations.
When it became necessary to bleach the pigment, in order to
study the visual cells, which would otherwise be masked by the
partially or completely extended processes, the method employed
was essentially that of Mayer, in which nascent oxygen? is the
effective agent.
EXPERIMENTAL PART
a. Experimentation upon Ameiurus
Ameiurus is well adapted for operations involving the optic
nerve. The soft skin and the relatively small eye allow easy
access to the orbit without causing the serious disturbance and
shock that is almost unavoidable when operating on fishes which
have eyes set in prominent bony sockets.
2 When potassium chlorate and hydrochloric acid interact, it is commonly
said that nascent chlorine is the agent causing bleaching. As a matter of fact
the reaction liberates free oxygen.
EFFERENT FIBERS OF THE OPTIC NERVE 219
The method of operation was as follows. With curved scissors
an incision was made in the skin ventral to the eye and the cut
carried upward around both sides of the ball until a semicircular
(or greater) incision resulted. The eyeball could now be rolled
back, and when thus displaced, no excessive strain was exerted
on the conjoined tissues. By dissecting through the aperture
thus exposed, the optic nerve was separated from the overlying
muscles and severed. If a delicate razor-edge scalpel, such as
ophthalmologists use in cataract operations, is employed, it is
comparatively easy to cut the nerve without injury to the sur-
rounding parts. Ameiurus has no large central artery and vein
in the optic nerve, as is the case in mammals. A blood-vessel,
however, runs beside the optic nerve, but if care is taken it need
not be injured by the operation.
1. Retinal pigment. My first effort was to discover the effects
upon pigment migration of the severance of the optic nerve only,
A typical experiment will illustrate the response of the pigment
when a previously dark-adapted fish was operated on and exposed
to light.
Experiment 8.1.12. The optic nerve of a dark-adapted fish was
severed and the animal allowed to recover from any shock effect until
the next day, when it was exposed to diffuse daylight for 2} hours.
At the expiration of this time both the operated and the normal eye
were excised. Subsequent examination showed that the retinal pig-
ment in the operated eye had retained the position typical of darkness
(fig. 4), while the pigment in the control eye had migrated normally
(fig. 1).
Thus it is seen that the integrity of the optic nerve must be
maintained in order that the retinal pigment of an otherwise
intact animal may undergo a positional change when stimulated
by light.
In successful experiments of this type the length of exposure
varied between 45 minutes and 3 hours, yet uniform results
were obtained.? A possible influence of operative shock, on ani-
mals that were subjected to experimentation directly after the
3’ Under normal conditions the pigment is fully extended by light in about 45
minutes (Arey, 716).
220 LESLIE B. AREY
optic nerve was severed, was shown to be non-existent. It was
also shown that roughly performed operations, in which various
mutilations of the eye muscles and the adjacent blood vessels
were intentionally made, did not interfere with the obtainment
of essentially typical results.
This type of experiment has been repeated very many times.
Among animals used during both the spring and the fall of 1913,
no deviation was found from the conditions recorded above.
When work was begun again on a new supply of Ameiurus
in the spring of 1914, the results secured from operated animals
brought from darkness into the light, were not always as decisive
as those of the preceding year. Some experiments showed a
complete retention of the pigment, while others resulted in a
partial migration, which, in some cases, was quite extensive
although never as complete as in normal light adaption. Further
experimentation on other animals procured in the fall of 1914
also gave inconsistent results, varying from complete to very
incomplete control.
I am unable to state the cause of these discrepancies. That
the results of the work done in the year 1913, which embraced
the larger part of this experimentation, are beyond question, I
feel confident; the remarkable consistency of a long series of
experiments about to be described substantiates this conviction.
On the other hand, the persistent occurrence of more or less
incomplete, mingled with complete control in the work of the
year 1914, was also undeniable.
It is possible that individual fish vary considerably in their,
ability to inhibit the migration of pigment after sectioning of
the optic nerve, or what amounts to the same thing, the activity
of the pigment in producing expansion may be variable. I shall
attempt to present evidence, based on experimentation to be
described later, that demonstrates an inhibiting mechanism which
prevents the pigment from migrating when the optic nerve is
severed. Since it has been proven (Arey, 716) that the action
of light on completely excised eyes can directly stimulate the
retinal pigment to movement, it is evident that there must be
a competition between light stimulation and this inhibiting mech-
EFFERENT FIBERS OF THE OPTIC NERVE 22a
anism for the control of pigment migration. If the inhibiting
mechanism is weak or becomes exhausted, or if the response of
the pigment cells to the direct action of light is especially strong,
the common result will be the production of a more or less exten-
sive movement of the pigment.‘
This, nevertheless, does not explain how different lots of ani-
mals, obtained in the spring and fall of one year, differed as a
whole from other lots, procured in corresponding seasons of the
following year. All the fish used were supplied by one collector,
who secured them from several small ponds, according to their
varying abundance from year to year. It is possible that the
stocks of different ponds may show more or less consistency, due
to common inheritance, in the behavior of their retinal pigment
under the previously mentioned experimental conditions. The
writer, however, wishes to present this suggestion merely as a
possibility that may or may not be true.
A second type of experiment, in which light-adapted animals
were subjected to darkness, is illustrated by the following record.
Experiment 8.1.49. One optic nerve only of a light-adapted Ameiurus
was severed. The animal was subjected to darkness for 18 hours,
after which both eyes were excised. No pigment contraction was
found to have occurred in the operated eye (fig. 2), whereas the pig-
ment of the control eye showed the extreme contraction of dark-
adapted retinas (fig. 4).
The greatest amount of contraction ever observed was a with-
drawal of the distal accumulation of pigment, which even at
room temperature is often characteristic of light adaption, to
form a uniform zone (fig. 2). Sometimes a slight thinning out
of pigment was seen, whereby the expanded mass appeared less
dense than normally. In no case, however, was there observed
a withdrawal of pigment, as a whole, from the zone usually
4 A few determinations of the rapidity of pigment adaption seemed to indicate
that the migration occurred more slowly in the fishes used during the first season,
which gave constant results, than in the animals used during the following year.
This fact not only tends to support the hypothesis that a variability in the
activity of pigment migration was responsible for the lack of consistency in the
later results, but also indicates that the résponse of the pigment in the animals
of the first year, taken as a whole, may have been less vigorous.
222 LESLIE B. AREY
occupied during light adaption. In this type of experiment,
therefore, it is also evident that severance of the optic nerve
restrains the normal pigment migration. The fact that the pig-
ment of excised eyes does not contract in darkness, and that
pigment contraction in general seems to involve a less vigorous
response than does expansion, explains why consistent results
were obtained in all these experiments, for the competition with
the inhibiting mechanism presumably was less keen.
To remove any doubt that the integrity of the optic nerve is
necessary for the migration of pigment in light and in darkness,
the nerve was cut inside the cranium near the chiasma. In order
to do this, a small aperture was made in the cranium over the
region of the optic nerve and the severance of the nerve was
accomplished by introducing a cataract scalpel through the open-
ing thus made. The removal of bone from the thick cranivm
had to be done with eare or the fish failed to recover from the
resulting shock effect. In successful experiments the results
obtained were similar to those previously recorded. Since the
eye and the surrounding parts were left intact during the whole
procedure, it is safe to conclude that in Ameiurus the optic nerve
is intimately related to the phenomenon of pigment migration.
A microscopical study of preparations demonstrating the rela-
tion of the optic nerve to the nerve-fiber layer of the retina shows
that the arrangement of nerve fibers is approximately of a radial
nature, and that the fibers from any sector of the retina are
extended into the adjacent side of the optic nerve. These rela-
tions probably become disturbed a short distance from the
emergence of the optic nerve from the eyeball. The optic nerve of
Ameiurus joins the retina by several roots (fig. 7, rdz. n. opt.),
hence this condition is not shown as diagrammatically as in
preparations of the eye of Abramis which cut the optic nerve
in aradial plane. Here the nerve fibers form a distinct V-shaped
‘parting’ as they pass to the two sides of the retina. On the
periphery of the nerve this is especially evident although at the
center there is undoubtedly more decussation, as was shown by
Kohl (’92) for several of the lower vertebrates.
EFFERENT FIBERS OF THE OPTIC NERVE 223
This relation should stand the physiological test imposed by
partial section of the optic nerve. In view of the results already
established, one would expect to find the pigment situated in
a sector adjacent to the cut to be unaffected by varying condi-
tions of light and darkness, while the pigment adjacent to the
intact portion of the nerve should expand or contract in a normal
manner. The description of an actual experiment will make this
clear.
Experiment 8.1.38. The optic nerve of a light-adapted Ameiurus
was two-thirds severed close to the eyeball, and the fish was trans-
ferred to darkness. After 2 hours the eye was excised, a pointed flap
of skin being left attached to the eyeball to insure proper orientation.
In a preparation, sectioned to include portions of the retina adjacent to
both the cut and the uncut fibers, a striking contrast was evident
(fig. 7). On the uncut side the pigment was contracted maximally,
while on the cut side it still remained in the expanded position charac-
teristic of light adaption.
I can think of no experiment that could be devised to support
better the view concerning the rdle which the optic nerve plays
in the movement of retinal pigment, than the one just described.
The similar treatment of dark-adapted fish which were sub-
jected to light did not give as decisive results as those of the
reciprocal set. The pigment on the ‘cut’ side of the eye was
not retained in a contracted state, but migrated to a considerable
extent (fig.6). In no case, however, was the expansion maximal,
that is, involving an accumulation near the external limiting
membrane, but at most only a broad evenly pigmented zone was
formed, which was markedly in contrast with the maximal expan-
sion of pigment in that half of the retina adjacent to the intact
portion of the nerve.
By the evidence of previous experimentation, a reason for this
difference in behavior is suggested. It has already been shown
that light is an efficient’ stimulus in producing expansion of the
pigment in excised eyes, whereas darkness does not contract
expanded pigment. Furthermore, after complete section of the
optic nerve, the pigment in certain cases still tended to migrate
when the animal was exposed to light. In the experiment last
described, it is probable that there was a more or less extensive
224 LESLIE B. AREY
decussation of fibers distal to the cut, and that these fibers,
although few in number, since they come from the intact portion
of the nerve and distribute themselves through the retina of the
opposite side are capable of allowing the pigment to migrate when
the eye is subjected to.the action of light. In producing this re-
sult, these fibers are doubtless servient to the direct action of light
on the pigment cells themselves. In darkness, without the aid of
some independent and efficient factor, such as the direct stimulus
of light, it may well be that these stray fibers are not sufficiently
potent to overcome the inertia of the pigment.
If the cause of the apparently inconsistent behavior described
in these two types of experiment is something of the nature of
that just outlined, then the evidence for the control of pigment
migration through the optic nerve thereby receives additional
support.
It was thought that if a small cut were made through the
eyeball and retina, near the entrance of the optic nerve, the por-
tion of the retina peripheral to the cut would be freed from all
connections with the optic nerve, and consequently some decision
could be reached concerning the réle of the decussating fibers
just considered. This method, if successful, would also corrobo-
rate the general conclusions which were drawn as to the control
of pigment migration by the optic nerve fibers.
On the whole, the results showed quite conclusively that in
the earlier experiments the presence of decussating fibers had
caused the pigment expansion on the side of the retina adjacent
to the cut optic nerve, when the eye was stimulated with light.
Cuts about 1.0 mm..in length were made with a cataract scalpel.
Although the blade of the scalpel was very thin, tapering to a
needle-like point and the edge was of great keenness, nevertheless,
in many cases the retina was torn away from the contracted
pigment layer. The ease with which a separation of the retina
and the pigment layer is effected, especially when the pigment
is not expanded to strengthen the union, is well known.
In successful operations (fig. 5), the pigment lying peripheral
to the cut showed but little expansion, although it was often the
case that toward the extreme periphery of the retina, pigment
EFFERENT FIBERS OF THE OPTIC NERVE 225
expansion was again found. Since it is probable that the fibers
of the nerve-fiber layer do not have a precise radial distribution,
the latter condition is readily explained by assuming the presence
in this peripheral expanded region of intact nerve fibers, which,
near the fundus, bordered on the incision.
The decrease in intraocular pressure necessitated by cutting
the eyeball has no effect on the activity of the pigment, for nor-
mal animals whose corneas have been punctured show typical
responses. ;
One is driven to the conclusion, by all the experiments hereto-
fore described, that there must be some mechanism, either in the
eye muscles or in the blood vessels of the eye, that exerts an inhi-
bition on the movements of the retinal pigment, for proper con-
trol experiments have eliminated the skin as a possible factor.
If this supposition is true, the retinal pigment should undergo
expansion when all the eye muscles and blood vessels of a dark-
adapted fish are cut, and the eye, connected to the body by the
optic nerve only, is subjected to light. This condition was indeed
realized, the pigment distribution being essentially like that in
totally excised eyes. The same experiment under reversed light
conditions did not result in a contraction of the pigment. As in
the similar failure of excised eyes to show contracted pigment
when subjected to darkness, I believe there is a strong proba-
bility of an anaesthetic action on the pigment cells due to the
accumulation of catabolic waste. If the vascular circulation
could be preserved, it is probable that the pigment cells would
contract in an experiment of this kind. Possibly with artificial
circulation the pigment of an excised eye would also contract in
the dark. '
The next step was to discover whether the presence of certain
eye muscles could be correlated with the inhibition of the pigment
response. When dark-adapted Ameiurus, having the optic nerves
cut, were brought into the light and the dorsal oblique and pos-
terior rectus muscles (those innervated by trochlear and abducens
nerves respectively) were severed, no pigment migration occurred.
It is evident, therefore, that the inhibiting mechanism does not
involve these muscles alone. Reciprocally, the dorsal, ventral
226 LESLIE B. AREY
and anterior rectus and the ventral oblique muscles (those inner-
vated by the oculomotor nerve) were cut, leaving the eyeball
attached to the body by the two remaining muscles. Under
these conditions, the pigment migrated much as in a normal
animal.
Hence it appears that the inhibitory mechanism has been
located as existing in association with the muscles (or possibly
the blood vessels of the muscles) which are innervated by the
oculomotor nerve. The objection may be raised, however, that
there is a possibility of the dorsal oblique and posterior rectus
muscles possessing an inhibitory function also, but that the inhibi-
tion produced when only two muscles are left in connection with
the eye is not sufficient to prevent the pigment response. The
evident check experiment which answers this criticism consists
in severing all the muscles except two which are innervated by
the oculomotor nerve. When this was done (the dorsal rectus~
and the inferior oblique being left intact) no movement of the
pigment was observed. This result indicates that the inhibi-
tory mechanism is found associated with those muscles inner-
vated by the oculomotor nerve and is not demonstrably asso-
ciated with the other eye muscles.
Since the dorsal oblique and posterior rectus muscles are not
potent in restraining pigment migration, it was possible to make
the following experiment. All the muscles excepting these two
were cut and light-adapted fish with the optic nerves either intact
or severed were placed in a dark situation. If the retinal pig-
ment contracted under these conditions, it could be reasonably
assumed that the presence of a partial vascular circulation had
been responsible for the change. However, no movement of the
pigment was detected, yet as the blood vessels in these muscles
are small in number and size, the results neither support nor
detract from the general view that absence of blood supply pre-
vents pigment contraction in the dark.
Although the relation of the autonomic fibers to the oculo-
motor nerve of teleosts has not been worked out as completely
as in some other forms, it is at least evident that from the ciliary
ganglion the so-called ciliary nerves pass to the eyeball, probably
EFFERENT FIBERS OF THE OPTIC NERVE 227
followivg the same general courses as do the branches of the
oculomotor nerve. In man, where the ciliary nerves have been
traced rather carefully, autonomic fibers supply the sclerotic and
choroid coats, ciliary muscle, iris, and cornea of the eyeball
(Carpenter, ’06). The ciliary ganglion of fishes according to
Onodi (’01) also receives fibers from the trigemival nerve, the
relation being more intimate than Schwalbe (’79) believed.
The action of atropine upon sympathetic nerves is to paralyze
the endings of the postganglionic fibers. If the mechanism that
inhibits pigment migration involves sympathetic nerves, it was
thought that it might be possible to eliminate its action by the
use of this drug.
By supplying fishes with water through a tube, they could be
retained indefinitely in the air. Dark-adapted fish with severed
optic nerves were brought into the light, and oecasionaly during
the course of an hour small amounts of a 0.5 per cent solution of
atropine sulphate were introduced into the orbit. Subsequent
examination of the retina showed that a migration of pigment to
the light phase had occurred.
The possibility of a direct stimulation of the pigment cells
presented a difficulty, however, that had to be tested, for Spaeth
(13) found that a 1.0 per cent solution of atropine caused a rapid
expansion of the melanophores of Fundulus. When dark-adapted
eyes were placed in a 0.5 or 1.0 per cent solution of the drug and
were returned to darkness for an hour or more, the pigment did
expand to an extent which nearly equalled that caused by in-
complete ight adaption.
It is certain, therefore, that any evidence gained from experi-
mentation of the kind described is valueless, and it is doubtful
if much more dependence can be placed on the significance of a
pigment migration when atropine was painted on to the eye
muscles in such limited amounts that a direct stimulation of the
pigment cells seemed improbable, or when small quantities of
atropine were injected into the cranial cavity.®
°> The use of nicotine, which paralyzes the synapse of the preganglionic fiber
with the sympathetic nerve cell, might furnish more interesting results, provided
it did not have a toxie effect on the pigment cells. Unfortunately, no experi-
ments of this kind were performed.
228 LESLIE B. AREY
An attempt was made to cut the oculomotor nerve and thus to
separate the sympathetic fibers which arise with it from the brain.
To make a large opening through the cranium leads to operative
shock from which Ameiurus does not recover. Accordingly, a
relatively small aperture was made, through which a cataract
scalpel was introduced, all the nerves presumably being cut on
one side of the brain between the optic and the trigeminal. A
dark-adapted Ameiurus with severed optic nerve, when exposed —
to the light after this treatment, showed pigment expansion.
This experiment, although properly controlled, was not of a
‘ refined type. It is possible that disturbances other than the
mere section of the oculomotor verve may have led to the ob-
served results.
It is not intended that any of the experimentation in which
an attempt was made to locate the fibers of the inhibiting mecha-
nism, should be received as conclusive. The results obtained
from cutting muscles and from the severance of the oculomotor
nerve suggest that autonomic fibers are involved, and that these
enter with the oculomotor nerve. , a. f hs
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CORRELATED ANATOMICAL AND PHYSIOLOGICAL
STUDIES OF THE GROWTH OF THE NERV-
OUS SYSTEM OF AMPHIBIA
Il. THE AFFERENT SYSTEM OF THE HEAD OF AMBLYSTOMA
G. E. COGHILL
Department of Anatomy, University of Kansas
SEVENTY-NINE FIGURES
CONTENTS
fn iS ABADOUMICAN WARG es da oy ssc sss 6 id ape o's ao ve eodls «SEDO MMMR aS RSs bi 248
PVE PRD DRLVO Los... sx 5 ancien wae oooh ee eRe SR Oy eelin oO
a; Die MONAMOUWLS BLAGE. . oso: 2 «sis olen sp s aie es ed setae sine 2 sk 5 ha 248
bu The early flexure stage...... 2... 6. ccs. ee ne continues mass se awe 251
c, The: coiled-reaction stage. ... 55... .-1 sana wists Cailege ahs oe anes sie 253
d. The early swimming stage............ 0-5. 6+ edesessecesesee. 200
2. The facial and auditory nerveS..............cceeeee eee daneeeeeeee 257
Br LNG MOMINOUIIC SLAB. .cbeccs ea uve sun cee oe PORE oh 258
b. The early flexure stage............0..s0seneeresenwstectccees 259
Go ENG COMEU-TEACLION SCARS... os cco eens oa:er oa bie Cinperee emia S aielsin (> 261
d. The early swimming stage................eeeseeer enews eeeees 264
3. The glossopharyngeal and vagus nerves.............000eee ee eee 266
A. Dhe mon-motile Stages ....0.. 65. cco ene ens cueess Smee ens ces 266
b. The early flexure stage........ 050. ..0 sendin an diwe espera es es 267
c.. The coiled-reaction stage... ..0 2.026. c rene enener meses cress 268
d. The early swimming stage..............0css cess eee e en eneees 270
4. The eye and optic nerve............. cece cece nsec cence neceeeeeeee 273
5. The olfactory organ and nerve.............eec cece cece ne eeeeeeeee 275
RA SMITE Ee oh Geddy cis Fach sia Wg « niet «sis aie Sp i he pu Slo eR NDS oe be 276
Pig tne Dhyslolopial PATE... .) vaccines So0 week Cea sxe eta smeNe Eye's > ch 4 280
1. Response to tactile stimulation...............00. see e cece ener ees 280
2. Respense to chemical stimulation. ............6---+e eee e erences 285
Si. Response, fo Tights... a5 <5 nies pa sic tes on ie aie nen gM RSE Bn yalo Sie ye oe 285
4. Response to olfactory stimulation.............--.0e0e ee eee e eens 288
5. The auditory and lateral line organS............,..02-sseeeeeeees 288
es SSUNINAEY sted. « 500. Ganglion cells
of the Gasserian ganglion, with axones reaching towards the brain. Other indif-
ferent cells appear around the ganglion cells. The yolk is indicated in the cyto-
plasm as in the last figure.
Figs. 20 and 21 Non-motile stage transverse section (No. 543, sections 3-3-16
and 13, respectively). > 500.
Fig. 20 is 15 caudad of figure 21, and shows root fibers of the trigeminal
nerve within the brain (7'r.Des.V). Both figures show cells of the root mass
(R.M.) Fig. 21 a large cell (D.C.) which has the anatomical features of Rohon-
Beard cells of the spinal cord sends a process out of the brain into the trigeminal
root. Asimilar cell (D.C.) appears in figure 20, in which figure the motor nucleus
of the trigeminus (Nue. vis.m.) is seen to be in a much more ventral position.
THE, NERVOUS SYSTEM OF AMPHIBIA ald
Nuc.vis.m.
F.D
- Be
316 G. E. COGHILL
R.L.L.VII
Asc.
AX RM.
= y 4 Be
Fig. 22 Early flexure stage (No. 550, section 1-2-14). X 200 The plane of
section of this figure is illustrated in figure 16. It illustrates a longitudinal sec-
tion of the medulla oblongata, in which the entrance of the trigeminal root ap-
pears (R.V). There is a short ascending trigeminal tract (Tr.asc.V) and a
descending trigeminal tract (T'r.des.V) which reaches to the level of the middle
of the auditory vesicle (Aud.V) The ventricular pits opposite the entrance of
the roots are conspicuous features of such a section.
Figs. 23 to 28 Early flexure state (No. 473, sections 1-5-14 to 19). X 500.
These figures are from six successive sections of the series from which figure 2
was made, and show the relations of the root bundles of the seventh and eighth
THE NERVOUS SYSTEM OF AMPHIBIA 317
nerves as they enter the brain. Figures 25 and 26 show the root fibers of the
lateral line component (?.L.L.V/I, a and b) and figures 24 and 23 show the ascend-
ing divisions of these root fibers (L.L.VII,Asc.). Figure 25 shows also the root
fibers of the visceral sensory component (R.VI/.vis.) of the facial nerve approach-
ing the brain amid cells of the root mass, and figure 27 shows this component
entering the brain. Immediately ventrally of this is the root of the eighth nerve.
In the next section caudad, figure 28, descending divisions of the lateral line
root fibers (L.L.VII,Des.) appear, but nothing can be seen of the other com-
ponents of this complex. The entire cephalocaudal range through which the
lateral line component can be recognized in this series within the brain is only
60 mu. ;
318 a. E. COGHILL
Figs. 29 to 31 Early flexure stage (No. 473, sections 1-6-16 to 18). X 500.
three successive transverse sections through the entrance of the root of the
glossopharyngeal nerve into the brain. The lateral line component (R.L.L.1X,X)
enters in figures 29 and 30, and the visceral sensory component (R.Vis./X) enters
in figures 30 and 31 at a slightly more ventral and caudal level than the entrance
of the lateral line component. No ascending or descending fibers of this complex
can be made out beyond the limits of these three sections, a range of 30 u.
Fig. 32 Early flexure stage (No. 473, section 2-1-12). .x 500. This figure
shows the only perceptible connection of the vagus nerve other than the lateral
line root with the brain at this time, a single nerve fiber (R.X.), which is of doubt-
ful nature. It may be motor.
THE
NERVOUS SYSTEM OF
AMPHIBIA
319
320 G. E. COGHILL
Fig. 33 Coiled-reaction stage (No. 561, section 1-3-14). X 500. The plane’
of section is shown in figure 17, in which the region of this figure is included
between the lines and indicated by a. The caudal end of the figure is to the left,
and is a great deal farther dorsad than is the rostral or right end of the figure.
The root of the trigeminal nerve (R.V) here approaches the brain in two divi-
sions, the root from the Gasserian ganglion (R.G.G.) and that of the ophthalmic
ganglion (R.oph.). The latter is entering the brain and bifurcating into a short
ascending tract (7’r.Asc.V) and a massive descending tract (T'’r.Des.V). In the
angle of this bifurcation is a large neurone (D.C.), the very large nucleus of which
is in sharp contrast with the surrounding nuclei. This cell lies in the dorso-
ventral level of the sensory root and is far removed from the motor nucleus,
several cells of which can be recognized in a more ventral position (Nuc.vis.m.).
Fig. 34 Coiled-reaction stage (No. 562, section 1-4-2). X 500. This is from
a transverse section located 56 u rostrad of the entrance of the trigeminal root.
It shows the presence at this level of ascending root fibers of this nerve (T'r.Asc.V).
In a more dorsal position there is a cell of the Rohon-Beard type (D.C.) which
has a long process extending into the immediate vicinity of or actually into the
ascending trigeminal tract.
Figs. 35 to 37 Coiled-reaction stage (No. 562, sections 1-4-9, 10 and 11).
< 500. Three successive transverse sections through the brain at the entrance
of the trigeminal root (R.V).
Figure 35 is the most rostral in position, and shows two or three cells of the
type described in the last two figures (D.C.). Another cell of this type appears
in figures 36 and 37 (D.C.), two sections through the same cell. In both figures
the peripheral process of the cell is shown entering the trigeminal root. In figure
37 there is a stump of a dorsally directed process, and in figure 36, evidence of a
ventrally directed process. Cells of the root mass (#.M.) appear in all these
floures.
321
THE NERVOUS SYSTEM OF AMPHIBIA
Nuc.vis.m.
NY
R. oph.
he
bas
: eben
322 G. E. COGHILL
Figs. 38 to 41 Coiled-reaction stage (No. 447, sections 1-5-10 to 13). > 500.
Four successive, serial, transverse sections through the entrance of the seventh
and eighth nerves into the brain. These figures are from the same specimen as
are figures 3 and 5, with which they should be studied. Figure 38 is the most
rostral of the series. In this figure the root of the lateral line ganglion on the
hyomandibular division of the nerve (R.L.L.VIT,b) is entering, and the ascending
fibers of the other division (L.L.VII,Asc.a) appear in a slightly more dorsal
position. These fibers are seen entering the brain in the three following figures
(R.L.L.VII,a). The visceral sensory component of the facial nerve is entering
the brain in figure 39, and its fibers appear as the fasciculus solitarius (Fas.Sol.)
in the two following figures. The auditory root (R.V/I/) enters in figures 40 and
41. All of these root systems lie dorsally of the descending trigeminal tract
(Tr.Des.V), which consists here of scattered fibers through a rather extensive
zone.
THE NERVOUS
=
f
mr
]
a
§
a,
SYSTEM OF
AMPHIBIA
324 G. E.. COGHILL
Fas.Sol.pe /
Des, ‘ '
Figs. 42 to 46 Coiled-reaction stage (No. 447, sections 2-1-6 to 10) X 500.
Five successive serial, transverse sections through the entrance of the glosso-
pharyngeal nerve into the brain. Figure 42 is the most rostral. In it can be
readily recognized the ascending fibers of the lateral line component of the ninth
and tenth nerves (L.L.IX,X,Asc.a and b), and ventrally of these the visceral
sensory component of the facial nerve as the fasciculus solitarius (Fas.Sol.).
The lateral line components are entering the brain in the three successive figures,
while the visceral sensory (R.Vis.X) enters in figure 44. In the two following
figures this component appears with the facial fibers as the fasciculus solitarius,
and in figure 46 the descending divisions of the lateral line roots occur (L.L.
IX,X,Des.a,b). The descending trigeminal tract (Tr.Des.V) appears as scattered
fibers immediately beneath the external limiting membrance throughout this
region. Neurones of the second order (New. I/) stretch across the whole mesial
face of this system of root fibers.
325
THE NERVOUS SYSTEM OF AMPHIBIA
»')
Ace 3°,
So fe
ys
nS
7 "
———
&
Fas.Sol
Nuc.vis.m.
ele ho eae
26, No. 3
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL.
326 G. E. COGHILL
Figs. 47, 48,49 Coiled-reaction stage (No 449, sections 1-6-17, 18,19). X 500
Three successive serial transverse sections through the brain at the level of the
entrance of the root of the vagus nerve. Figure 47 is the most rostral of this
set: In it the visceral sensory root of the vagus (R.Vis.X) is approaching the
brain surrounded by cells of the root mass (R.M.), and the fasciculus solitarius
appears in two clusters of fibers (Fas.Sol.), a more dorsal representing the facial
and glossopharyngeal portion and a ventral consisting of ascending fibers of the
vagus root. ‘This figure, as well as the following, extends ventrad to show the
relation of the sensory centers to the motor column (V 7’), and the visceral motor
nucleus (Nuc.vis.m.). The root fibers of the vagus enter in figure 48, and in the
following figure a few descending root fibers can be recognized (Fas.Sol.).
Fig. 50 Early swimming stage (No. 567, section 2-1-19). > 500. From a
transverse section through the medulla oblongata 35. caudad of the entrance
of the trigeminal nerve. Around the dorsal margin of the descending trigeminal
tract (7’r.Des.V) is a cluster of three or more large neurones (DG) of the giant
ganglion cell type. Their processes run ventrad towards the motor nucleus
of the trigeminus (Nuc.vis.m.).
THE NERVOUS SYSTEM OF AMPHIBIA 327
Tr.Des.V
328 G. E. COGHILL
Figs. 51 to 54 Early swimming stage (No. 557, sections 2-1-1,5,7,11). X 200.
Four sections selected from a transverse series through the brain in the vicinity
of the entrance of the root of the trigeminal nerve. The massing of the
ganglia against the brain is shown here, a relation which should be noted in the
study of figures 1-4. Figure 51 is the most rostral in this set, and figure 54 the
most caudal. In figure 51 the ophthalmic ganglion (G.oph.) appears with a
crescentic piece of the Gasserian ganglion (G.G.) on its. ventral surface. The
fibers from this ganglion enter in figure 52 (R.oph.), while the root of the Gasserian
ganglion enters in figure 53 (R.G.G.) more dorsally. This section is 28 4 caudad
of the last. In figure 54 the motor root of the nerve appears.
THE NERVOUS SYSTEM OF AMPHIBIA 329
Tr.Asc,. V— )
—
330 G. BE, COGHILL
Figs. 55 to 60 Early swimming stage (No. 496, sections 1-5-4 to 9). X 500.
Six successive serial transverse sections through the entrance of the roots of the
seventh and eighth nerves into the brain. Figure 55 is the most rostral of the
set. In this figure the ascending divisions of the lateral line component (L.L.
VII,Asc.a,b) and the roots of the visceral sensory component (R.VIT, Vis.) and
the auditory root R.VIII) are beginning to enter. The lateral line roots (R.L.
L.VII,a,b) enter in figures 56 to 59. In figure 58, 59 and 60 the fibers of the vis-
ceral sensory component have become the fasciculus solitarius (Fas.Sol.), and
the descending auditory root fibers (VIJI,Des.) appear along the dorsal margin
of the descending trigeminal tract (7r.Des.V). Neurones of the second order
(Neu. IT) here lie across the entire sensory zone. These figures should be com-
pared with figure 6.
331
THE NERVOUS SYSTEM OF AMPHIBIA
L.L.VIlAse.
Nuc.vis.m.
332 G, E. COGHILL
Fig. 61 Early swimming stage (No. 496, section 1-5-15). X 500. From the
same series as figures 55 to 60, at the level of the middle of the auditory vesicle
and endolymphatic appendage (Hnd). This should be compared with figure 6.
It shows the relations of the descending facial and ascending postauditory lateral
line components in this early stage; also the fasciculus solitarius (Fas.Sol.) and
the descending auditory fibers (VIII Des.). Mesenchymal cells are now pressing
in between the brain and the otocyst.
THE NERVOUS SYSTEM OF AMPHIBIA 333
Fas.Sol.__
~S
RVI ses
a) e
a
ViliDes.
334 G. E. COGHILL
Figs. 62 to 65. Early swimming stage (No. 496, sections 1-5-24 to 1-6-2).
< 500. Four successive transverse serial sections through the entrance of the
glossopharyngeal nerve into the brain. Figure 62 is the most rostral. The
lateral line roots (R.L.L.JX,X) enter in figures 63 to 65, and on their ventral sur-
face the visceral sensory root (R.Vis./X ) enters in figures 63 and 64. These figures
should be compared with figure 6.
THE NERVOUS SYSTEM OF AMPHIBIA 335
= ~~
Nuc.vis.m. a! Bs Ao ec
a Sy ma RAN
Z Se a
RiVisIX &, é
Tr.Des.V \
Nuc.vis.m,
336 G. EB, ‘COGHILE
Figs. 66 to 70 Early swimming stage (No. 496, sections 1-6-19 to 23). > 500.
Five successive serial transverse sections through the entrance of the root of
the vagus nerve into the brain. Figure 66 is the most rostral in position. In
this figure the fasciculus solitarius (Fas. Sol.) can berecognized between the
descending trigeminal tract (T'r.Des.V) ventrally and the ascending fibers of the
spinal Rohon-Beard cells dorsally (DT). The visceral sensory root of the nerve
(R.Vis.X) enters in figures 67 and 68 and the somatic sensory root (R.G.Jug.) en-
ters in figure 69; the visceral sensory root entering more dorsally. Figure 70 shows
fasciculus solitarius fibers caudally of the entrance in a relation like that in
figure 66, which is on the rostral side of the root. The somatic motor column is
shown in 67, 69 and 70 (V7). The visceral motor nucleus appears in all the
figures of this set, and lying between it and the external limiting membrane is a
tract (Tr.S) which can not be described in detail in this paper. It is probably
a bulbo-spinal tract. The figures of this set should be compared with figure 6.
THE NERVOUS SYSTEM OF AMPHIBIA BBYA
338 G. E. COGHILL
Nuc.vis.m,
Retinal Ganglion Cell
TI
Fig. 71 Early swimming stage of Amblystoma microstomum Cope (No. 635,
section 1-3-7). X 500. A transverse section of the embryo through the eye,
to show the nature of the ganglion cells of the retina and their fibers, which at
this time decussate in the most rostral portion of the postoptic commissure.
Silver impregnation after fixation in neutral formalin.
THE NERVOUS SYSTEM OF AMPHIBIA 339
gh
SAG. VIII
Se
IS
Figs. 72 to 79 From transverse sections through the eye and ear of the four
stages.
Figs. 72, 73 non-motile (No. 467).
Figs. 74, 75 early flexure (No. 473).
340 G. E. COGHILL
ows NS «
Ma reinks
eer
oO
Figs. 76, 77 coiled-reaction (No. 449); early swimming, figures 78, 79 No.
444). The sections through the eye were selected at levels which show the most
extensive area of the lens, and those of the ear were.selected at levels which
show the clearest differentiation of the endolymphatic appendage.
THE EFFECT OF ACTIVITY ON THE HISTOLOGICAL
STRUCTURE OF NERVE CELLS
R. A. KOCHER
From the Henry Phipps Psychiatric Clinic, Baltimore,’ and the George Williams
Hooper Foundation for Medical Research, University of
California, San Francisco
INTRODUCTION
The study of the effect of functional activity on the morphology
of nerve cells has been the subjectof numerous investigations
in the past. These researches have covered a considerable range
as to kinds of cells studied, degrees of fatigue, and as to methods
of treatment of the material. There is almost complete agree-
ment on one point, namely, fatigue results in appreciable changes
in cell structure. There is, however, the utmost divergence of
opinion as to the nature of the changes, and one who tries to
correlate the findings of the different workers in this field is
utterly confused.. The chief findings relate to*(a) size of cell
body and nucleus and (b) amount and distribution of chromatic
material. While one group of workers concludes that fatigue
results in decrease in the size of cell and nucleus (Hodge (1),
Van Durme (2), Luxemburg (3), Legendre and Piéron (4), Lugaro
(5), and Nissl (6) ), another group finds an increase in the size
or a change in the nucleus-plasma relation (Crile-and Dolley
(7), Mann (8), Vas (9) ). Lambert (10) noted no change in
the size of cell or nucleus. Increase in chromatic substance
-(hyperchromatism) as the chief result of fatigue has been noted
by Nissl. Most workers (Van Durme (2), Vas (9), Lugaro (5),
Mann (8) ) have deseribed a decrease in chromatic material as
1 These experiments were carried out at the suggestion and under the direc-
tion of Prof. Adolf Meyer, and completed in 1912. The computations were in
part made after leaving Baltimore. It g ves me great pleasure to express here
my thanks and appreciation to Doctor Meyer for his keen interest in the experi-
ments and for many helpful suzgestions.
341
THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 25, NO. 3
342 R. A. KOCHER
the result of exercise. Luxemburg (3) described a breaking up
of the chromatic bodies into granules, and Dolley tried to recon-
cile these contradictions by regarding the hyperchromatism as a
result of moderate activity, and the hypochromatism the result
of excessive activity or exhaustion. They find that both stages
may be present in the same animal simultaneously. Other
changes, such as wandering of the nucleus toward the periphery
of the cell (Magini (11), Lambert (10), Vas (9) ), rupture of the
nuclear and cell membranes (karyolysis and karyorhexis) have
been described. Dolley noted thirteen stages of cell change
from hyperchromatism to disintegration or death of the cell,
corresponding to the degrees of moderate activity up to com-
plete exhaustion. Eve (12), in a study of sympathetic nerve
cells, concludes that “there are usually some small differences
before and after stimulation, but these are nearly all inconstant
and generally reversible. Such divergence in the results is not
so surprising when we stop to consider the complicating factors
necessarily attending these experiments, such as, (1) difficulty
of separating the effects of normal activity from unavoidable
shock or injury to the nervous system in killing the animal (the
nervous system does not ‘die’ as soon as the heart staps beating) ;
(2) postmortem changes ensuing between the time of death and
complete penetration of the tissue by the fixing agent due to the
action of autolytic enzymes present in all tissue; (8) varying
- chemical action of fixing agents; for example, formaldehyde coag-
ulates protein by combination with the amino groups, alcohol
by dehydration, sublimate by formation of salts, ete.; (4) the
solvent action of materials used in fixation and in imbedding,
for example, alcohol, xylol, paraffine; (5) varying effects of chemi-_
cal reaction between basic or acid dyes used in staining and the
differént cell structures; (6) effect of subjecting tissue to tempera-
tures of 50° to 54° in the paraffine oven for a period of several
hours.
In the present series of experiments I attempted as much as
possible to minimize the formation of artefacts, having in mind
the above mentioned considerations. It was hoped that by
using special care in the handling of material, the use of im-
EFFECT OF ACTIVITY ON NERVE CELLS 343
proved technic in fixation and staining, and by varying the kind
of degree of activity in a long series of experiments, using differ-
ent animals and studying various kinds of nerve cells, that some
degree of uniformity of results might be obtained.
METHODS
A resting control animal was used in each experiment. The
animals were killed in most cases by bleeding after ether anaes-
thesia, the nerve material removed as quickly as possible, cut
into small pieces, and the control and fatigue specimens placed
in the same fixing solution, imbedded side by side in the same
block of paraffine, cut with the same stroke of the knife, mounted
and stained together on the same slide. The detailed data of
experiments showing animals used, kind of stimulation, length
of time, microscopic technic, ete., are shown in table 1. In all,
fifteen experiments were performed. Examination of the table
reveals the fact that almost every form of activity was used;
normal activity, forced activity, activity resulting from electrical
stimulation, both faradie and galvanic, chemical stimulation, and
shock being applied in the experiments. The kinds of animals
used were dogs, cats, rats, sparrows, pigeons, and frogs. The
microscopic technic used was varied considerably, not only as to
fixative but also as to staining fluid. The stain most frequently
used was Held’s modification of Nissl’s method.
The examination of the material for comparison was facili-
tated by having both control and fatigue specimens mounted on
the same slide. Changes such as have been previously described
as resulting from fatigue were carefully examined for; namely,
comparative amounts and distribution of chromatic substance,
size of granules, nucleus-plasma relation, relative size of cells
and nuclei, ete. In order to determine whether any change in
the size of the cell had resulted from activity, a large series of
camera lucida drawings were made. These drawings were made
of cells without selection. A field was taken, and every cell
showing a nucleolus was included. This precaution was neces-
sary in order to be assured that the cell was cut through a com-
KOCHER
R. A.
344
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346 R. A. KOCHER
parable plane. Cells were first drawn using a low power for
orientation, each cell numbered on the paper, then the one-sixth
objective or the one-twelfth objective was used to project the
cell, care being taken to draw always at the same distance from
the microscope. ‘The area of the cells was then computed with
the use of the polar planimeter. The data are tabulated in
table 2, and will be referred to later in connection with the indi-
vidual experiments.
Normal Activity
Experiment I. Dogs. The animals which served for this experiment
were two fox terrier puppies from the same litter, three months old.
A female was used for activity while the male served for the control.
The latter remained quiet in a cage, where he had been kept for several
weeks previously. The activity animal was led by a chain on a fast
walk into the country; the distance covered was fifteen miles in three
and a half hours. This was a considerable feat for a puppy of this
size, as the pace meant running all the way for her. At the end of
three and a half hours, she was so fatigued that she refused to go any
further, and had to be carried home. She was then killed less than
one hour after exercise had ceased, the brain and cord at once removed,
and sections taken from the lumbar and cervical enlargements, from
the cerebellum, and from the cruciate gyrus, and the sections placed
in 10 per cent formalin and in Held’s fluid. The control dog was killed
at the same time, in the same way, and corresponding sections taken
from the brain and cord, and placed in the same fixing fluid with those
from the ‘fatigue’ animal.
MICROSCOPIC STUDY OF THE NERVE CELLS
Cervical enlargement of the cord
The cells are uniformly stained, the Nissl bodies standing out
clear and distinct. In both control and fatigue specimens, there
is an occasional cell showing slightly clear areas about the nucleus,
but this is no more marked in either section, and these cells are
as numerous in the control as in the other. Drawings were made
with the Leitz camera lucida, using the one-twelfth objective
and no. 2 ocular. The camera lucida outlines of the cells and
nuclei were traced with a planimeter with the following result:
Control cells, 43 measured, average area 0.441 square inches.
347
EFFECT OF ACTIVITY ON NERVE CELLS
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348 R. A. KOCHER
Fatigue cells, 36 measured, average area 0.420 square inches.
These results are tabulated along with the measurements of
cells from other regions and experiments in table 2.
LUMBAR ENLARGEMENT OF THE CORD
Examination of sections stained according to Nissl’s original
method, e.g. “‘seifen methylen blue,” after fixation with 95 per
cent aleohol, and according to Held’s modification of this stain.
As regards amount and distribution of chromatic material, there
is no difference between the control and fatigue sections. Several
sections stained by Held’s method showed grouping of cells near
the dorsal part of the anterior horns where the chromatolysis
was slightly more marked in the fatigue specimen than in the
control. This paucity of chromatic substance was not only
around the nucleus but also extended to the dendritic trunks.
This was not constant in all sections nor throughout the same
section. Cells in the extreme anterior horn show no difference
from the control. It seems probable that the particular section
cut through a nucleus of the cord where the character of the cells
was slightly different, since the similar variation in favor of the
control cells was observed in some of the subsequent experi-
ments. The nuclei of the control and fatigue animals show no
difference whatever in the amount and nature of the staining of
the chromatic material.
CRUCIATE GYRUS
These cells are uniformly stained, the nuclei and nuclear mem-
branes and chromatic bodies being distinct. No difference in
the morphology of these cells can be made out with the highest
power of the microscope.
CEREBELLUM
The cells of the cerebellar cortex stained with Held’s method
are well defined, and show a distinct architecture. Numerous
cells were examined, but without any discoverable difference in
the staining reaction between control and fatigue specimens.
EFFECT OF ACTIVITY ON NERVE CELLS 349
Sparrows
Experiment 2. A male sparrow was shot and instantly killed with a
rifle ball at 6 a.m. The brain, cord, and brachial and dorsal ganglia
were removed one-half hour later. These were placed in Held’s fluid
and in 10 per cent formalin.
Fatigue bird. A male sparrow that had been flying about all day
was shot, and the brain, cord, brachial and dorsal ganglia removed
one-half hour later, and placed in Held’s fluid and 10 per cent formalin.
Microscopic study. Brachial ganglia—one-twelfth objective, no. 2
ocular, Leitz. The cell architecture is distinct in both the fatigue and
control specimens. Sections stained with Held’s method show the
cell bodies stained diffusely pink with distinct Nissl granules stained
dark blue. There is no difference as regards depth of stain of either
cell bodies or the amount and size or depth in staining of the Nissl
bodies. No cells in either section showed any crenation of the nuclear
membrane such as described by Hodge. Some cells show excentric
nuclei and nucleoli, but by actual count this occurrence is just as com-
mon in the control specimen. Examination of the anterior horn cells
of the spinal cord in the region of the cervical enlargement as well as
the examination of the Purkinje cells of the cerebellum show no varia-
tion from the control morning specimens with respect to size of cells
and nucleu§ or in the morphological markings.
Pigeons
Experiment 3. For this experiment pigeons were selected which were
in the daily habit of making long flights, sometimes remaining on the
wing for over an hour at a time. One of these pigeons was killed just
at dusk, and as a control, one approximately of the same age was
killed from the same flock at six o’clock the following morning. For
study the entire brain was removed as well as the brachial ganglia and
sections from the spinal cord in the region of the brachial and lumbar
enlargements.
Microscopic study. Brachial ganglia. The evening (fatigue cells)
show no crenation, no central chromatolysis, and there is no apparent
difference in the size or distribution of the granules from the controls.
Measurement of a large number of cells show that the differences in
size of the cell bodies or nuclei in the fatigue and control specimens fall
within the limit of “‘variation.”’
Anterior horn cells. Brachial cord. No difference in any respect
could be detected between control and fatigue specimens.
Cerebellum. A large number of the cells were studied from both the
evening and morning pigeons, but no constant variation in morpho-
logical markings could be detected.
350 R. A. KOCHER
Rats
Experiment 4. This and the following experiments differ from the
preceding in that here the activity was forced to the point of exhaus-
tion. In the previous experiments exercise was voluntary. Two half
grown white rats from the same litter served for this experiment. They
had previously been kept in a cage and well fed. The fatigue rat was
kept running in a revolving wheel for one-half hour, having become
tired, he refused to run, and clung to the wall of the wheel. The
exercise was then changed from running to swimming. The rat was
placed in a tank of lukewarm water, where he kept up constant swim-
ming in an attempt to escape. At the end of one hour he was quite
exhausted, was taken out, and allowed to rest for an hour. He was
then made to swim a half hour again, followed by a half hour of rest.
This was continued until the total time of swimming was three hours.
He was then killed at the same time as the control. The total brain
and portions from the cervical and lumbar cords were removed. The
brains were cut sagittally, and placed in 10 per cent formalin; the other
portions were fixed in Held’s fluid.
Microscopic examination. In this experiment a large number of sec-
tions were cut in series, and a thorough search made for constant
differences in staining reaction, amount of chromatic material, size of
cells and nuclei, etc. No such constant differences appeared as
would go beyond the limits of simple variation. In some slides one
might be quite sure of a preponderance of cells of a certain type; for
example, showing central chromatolysis; but on actual counting and
comparison, the number of such cells will be balanced by an equal
number of the same type in the control.
Forced activity
Experiment 5. Dogs. Four young fox terrier dogs of approximately
the same size and age were used for this experiment; one served for a
control, the other three were subjected to continuous running for
periods of one, two and a half, and five hours respectively. They were
killed immediately after the exercise, and the nerve tissue from the four
animals given identical treatment as to fixation, imbedding, staining,
and cutting, the four pieces being mounted side by side in the same
block of paraffine, and cut with the same stroke of the microtome
knife. In this way the effect of exercise of various grades of intensity
could be studied in the cells of the anterior horn of the cord, of the
posterior ganglia, and of the cerebellum.
Microscopic examination. Thorough study of all the sections num-
bering over a hundred, most of which were made in series, was made in
this experiment. The various types of cells described by Dolley were
particularly kept in mind, and an attempt made to correlate them with
various grades of fatigue. Dolley (7) describes thirteen different stages
of fatigue corresponding to different grades of work and over-work.
EFFECT OF ACTIVITY ON NERVE CELLS 351
Representatives of practically all these types of cells were
found in my specimens, from the resting control animal, as well
as from those animals exercised for one, two and a half, and
five hours. In order to determine as accurately as possible the
relative proportion of these types of cells in the different speci-
mens, a table was made out listing each of these cell types, and
then beginning with a section under the microscope, every cell
showing a complete nuclear membrane was taken in order, and
checked in the proper column. In this way over three thousand
cells were counted with the result listed in table 3.
As will be seen in the table, the number of a particular type
of cell varies considerably, but this variation is the same for the
different animals. There are neither progressive changes in the
morphology of the cells from rest to exhaustion nor are there
any qualitative or quantitative differences in type of cells from
resting and fatigued or even exhausted animals. The animals
- used in Dolley’s experiment exercised at most up to three to four
hours altogether. Dolley also describes these types of cells as
persisting in the effort to recuperate for from two weeks to several
months after exercise no more severe than that of two or three
hours running in a treadmill. All these types of cells are ad-
mittedly present at the same time (Dolley, American Journal of
Physiology, vol. 28, p. 151). Dolley describes thirteen stages
of fatigue in one animal where the animal exercised one hour in
a tread mill. Also, the cells selected by him for illustration of
these stages are taken from a single preparation of three sec-
tions in the same experiment. Obviously the observations were
not over a large enough range of sections nor sufficiently con-
trolled by actual counts of the various types of cells. In the
Journal of Medical Research, vol. 21, p. 104, Dolley says,
‘“Measurements were made of five cells of each type in five
anaemia experiments, one a fatal resuscitation, the other a re-
peated hemorrhage;’’ a little farther on, ‘‘Measurements were
made of ten cells in each of three groups.”’ A great many cells
were skipped (those not entire) in Dolley’s method of counting,
giving a large leeway for a personal factor in the selection of
types. Ibid, volume 20, page 291, ‘‘ Measurements were made
KOCHER
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EFFECT OF ACTIVITY ON NERVE CELLS Goo
of camera lucida outlines of cells and nuclei. This was done in
fourexperiments. . . . . Five cells of each type and after
each fixative seemed to give a fair average.’ Dolley’s material
in which consecutive stages were studied did not receive the same
treatment as to fixation, staining, mounting, and cutting. The
control and fatigue material was handled entirely separately
(Amer. Jour. of Physiol., vol. 25, p. 155). Slight unavoidable
variations in the exposure of the tissue to the various agents
and different thickness of the cut sections would make such
material worthless for comparative study. As pointed out above,
this objection cannot be applied to my own experiments, as the
handling of material was identical in all cases. Dolley’s method
of measuring size of cells and nuclei is open to serious objection.
In the Journal of Medical Research, volume 21, he states, ‘‘ While
not adapted for exact measurement on account of their shape,
the largest diameters (cells and nuclei) were always taken.”
In my own experiments the actual relative areas of the nuclei
and cells was computed. with the use of the polar planimeter,
which traces the entire outline of the cell and gives very accurate
results. As seen in the table, I found no difference in the size of
the cells of the control and exercise animals.
Electrical stimulation (galvanic)
Experiment 6. Pigeons. In this experiment a pigeon was fastened
by the feet in a standing position, while a wire from four 50 amperes
galvanic batteries was wrapped around the legs. A current was al-
lowed to pass through the wire once a second by a clock make and break
arrangement. With the entrance of the stimulus there was a strong
contraction of the leg and thigh muscles. After four hours the con-
tractions became feeble, owing to fatigue, and the pigeon was killed.
Rigor mortis of the leg and thigh muscles set in immediately. A con-
trol pigeon was killed at the same time.
Microscopic study revealed absolutely no constant morphological
differences in the anterior horn cells and in the dorsal ganglion cells
from the two birds.
Faradic stimulation
Experiment 7. Faradic stimulation of the sciatic nerve in a cat was
applied in this experiment. A cat weighing 2 kilos was anaesthetized
with ether, and decapitated according to the method of Sherrington by
354 R. A. KOCHER
tying off the carotids, ete. After a rest of about three-quarters of an
hour to give the animal a chance to recover somewhat from the shock,
a canula was inserted into the carotid artery, and connected with a
manometer for record. The left sciatic nerve was then exposed and
stimulated with a faradic current from two dry cells. The secondary
coil was placed at eight, later at six. Stimulation was applied at inter-
vals, fifteen seconds stimulation was followed by forty-five seconds
rest. Stimulation began at twelve o’clock and continued until five
pM. The heart was still beating strongly at the end of the experiment,
the blood pressure remained fair, and reflexes were obtained through-
out by stimulation of the sciatic, both in the right and left leg. The
contractions on the right were spasmodic, those on the left—the stimu-
lated side, were tetanic. The latter were feeble, and gave all signs of
fatigue. The three pairs of dorsal ganglia of the sciatic nerve as well
as the lumbar and sacral cord, were removed and placed in the fixing
solution. Microscopic examination of the cells and measurements of
cells and nuclei from the fatigued and unstimulated side of the cord
of the same animal failed to disclose any difference in morphology.
Experiment 8. Faradic stimulation of the sciatic nerve of the frog
was used in this experiment, the unstimulated dorsal ganglia cells and
anterior horn cells of the unstimulated side of the same animal as well
as corresponding material from a second resting frog killed at the same
time served as control. The two frogs were pithed and placed in a
moist chamber. One-half minute stimulation of the sciatic nerve was
followed by one minute rest. The electrodes were applied just above
the knee at the back. This interrupted stimulation was continued six
hours. The muscles showed response by tetanic contractions. At the
end of the period the muscles were still irritable. Three pairs of dorsal
ganglia corresponding to the sciatic nerve as well as the spina’ cord of
the same region were taken for study.
The stimulation produced no changes in the cell morphology that
could be detected by a measurement of the size of the cells and nuclei
or by study with various powers of the Leitz compound microscope.
Drug stimulation (strychnine)
Experiments 9 to 14... Verworn (13) attributed fatigue in nerve cells
in part at least to a local asphyxia of the cells due to an accumulation
of fatigue substances and an insufficient supply of oxygen. By per-
fusing fatigued frogs with oxygenated salt solution, inserting the canula
into the aorta, he was able to restore irritability of the nerve cells and
corresponding response of the muscles in contraction after they had
ceased to respond from fatigue. He succeeded in keeping the muscles
and nerve responsive to stimulation for many hours longer than would
ordinarily be the case. In experiments 9 to 14 this method was applied
in order to continue stimulation and corresponding nerve exhaustion
to a degree not possible by the usual methods. Strychnine in doses of
4 to + of a grain was given in each case by injecting into the subcu-
=
EFFECT OF ACTIVITY ON NERVE CELLS 355
taneous area of the lower back just after beginning perfusion with
normal salt solution and by adding to the perfusion solution. The
heart continued beating throughout the experiment. The strychnine
caused violent tetanic contractions at first, which later gave place to
continued fibrillary contraction. This served as an indication of the
condition of irritability of the spinal cord cells. To prevent direct
action of the strychnine on the nerve endings in the gastrocnemius
muscle, the femoral artery was tied off. This muscle continued in a
state of irritability for several hours. The experiments carried out by
this method were continued for a length of time varying from two to
ten hours. Controls were used, treated in the same manner, except
for the strychnine injections. The subsequent treatment of the nerve
material (sections of the spinal cord and from the thoracic and lumbar
regions) was identical. There is a striking change in both the control
and treated cells, which had been perfused for a long period. Many
cells as well as nuclei are swollen beyond the normal size, owing, doubt-
less, to a simple turgescence caused by a flooding of the circulatory
system with the salt solution, and the staining reaction is diffuse.
There are, however, no differences in morphological characters between
the cells of the strychnine stimulated and the control cells of the spinal
cord.
Shock (anaemia)
Experiment 15. A four kilogram dog was given morphine and bled
from the jugular vein; 200 ce. were removed, following which the dog
remained semi-comatose for five hours. At the end of this time he
was killed along with a control dog of four and a half kilograms weight.
Material was taken from the cruciate gyrus and cerebellar cortex, fixed
in 10 per cent formalin, and stained with polychrome methylene blue.
Examination with the microscope revealed no difference in the char-
acter of the nerve cells in the shock animal as compared with the
control.
DISCUSSION OF RESULTS
It has long been known that prolonged activity of mucous
gland cells results in characteristic histological changes in these
cells, owing to the disappearance from the cell of certain granules
(zymogen granules). This granular material is evidently used
to make organic material of the secretion. It was doubtless on
the basis of such observations that certain physiologists were
led to seek for similar alterations in the highly specialized nerve
cell following functional activity. Many of the investigations of
these physiologists were confined to one or two experiments, the
material was often not sufficiently controlled by normal tissue
356 R. A. KOCHER
for comparison, and frequently the histological technic was faulty.
In explanation of the diverse changes described by these workers
as resulting from fatigue, it has been assumed that certain mate-
rials present in the nerve cells have been katabolized to form
energy for the nerve impulse, and that the using up of this
material during activity can be detected histologically by the
depletion of the granules (chromatic substance), changes in size
of cell and nucleus. In a long series of carefully controlled
expetiments, I could find no evidence of any analogy between
the effect of activity in certain glands and activity in nerve cells.
In no experiment did the histological structure of the nerve cell
following activity show any constant deviation from that of the
corresponding resting cells of the controls. Some very sweeping
generalizations have been drawn from the conclusions of pre-
vious workers; namely, that fatigue, fear, shock and exhaustion
may lead to permanent damage and even disintegration of nerve
cells. Crile’s present theory of surgical shock and of certain
aspects of Graves’ disease, based essentially on these assu np-
tions, may be cited to show to what extremes these deduct ons
based on insufficiently controlled experiments of this kind have
led.
SUMMARY
The effect of various grades of activity on nerve cells was
studied in a series of fifteen separate experiments. The animals
used were dogs, cats, pigeons, sparrows, frogs, and rats. «very
experiment was carefully controlled by a resting animal of the
same species, of the same approximate age and size, and the
material from both given identical treatment, except for the
activity. The nerve cells studied were from the cruciate gyrus,
from the cerebellum, from the anterior horn of the spinal cord,
and from the dorsal ganglia. In one of the experiments over
thirty-five hundred nerve cells classified into thirteen types ac-
cording to histological characters were counted to determine the
relative frequency of characteristics which might be correlated
with grades of activity. There was no deviation from the nor-
mal in even the most advanced fatigue. Over a thousand cells
EFFECT OF ACTIVITY ON NERVE CELLS SOT
and nuclei were measured by computing the areas of the pro-
jected outlines with the planimeter. There was found to be no
constant difference in size of cells ornuclei resulting from activity.
Furthermore, no qualitative differences in histological charac-
ters could be found between fatigue and resting nerve cells.
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THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 3
CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT
HARVARD COLLEGE, No. 278, AND FROM THE ANATOMICAL LABORATORY OF THE NORTHWESTERN
UNIVERSITY MEDICAL SCHOOL, No. 36.
THE INFLUENCE OF LIGHT AND TEMPERATURE
UPON THE MIGRATION OF THE RETINAL
PIGMENT OF PLANORBIS TRIVOLVIS
LESLIE B. AREY
NINE FIGURES
CONTENTS
SULPGUUCTION BN BIGLOLICAL SUTVOY.«....cceces onus awancswvepENauneseawecccs 359
Serer ett YAEL NEDRHIONNRS p 2's Wiis viva a vedic 3 WKS aes so o's ope ERM ET ate «iain 362
Wesersption of the eye of Planorbis. .... 2. 6...0. 500s cewensie see ghia ae sles so 365
RNP MIATN PORES 5 cane yc ad dine