‘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 st. bac. con; tI [2 , scl, pd, el mg: sl. pigs rin; scl pd. cl. ptg: st. ng: mb. lim. ex.-- rin.- 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 > ---- TENs--__ : vgs AS: See ——_ scl: st. [tyz-- Cp aa oF st.bac. con. oe Deere wp <2 eee ene SER rin; be ue dk ~, a md, ee ‘ <% x scl; ral aaah ae BAL, sl. hac. con: . ? . r) cra 19 292 . zi mb. lim. ex. Sab Tes OX 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’ ; cll. con, ell. con.- --con, acc, ell. bacs.. a ’ u 7. . . ; : mb. lim. ex. ci " . 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. The results here are definite and conclusive. Ji. / CEM ele Bia Apia viv a ou we Se sb Se Sage eye RR RIEN As to 213 Rae UCOL VUNG! DEODUAMING coh v ocap a oh wie sae «a v's 6s oie ody emia ot EEE Weta eS 215 PECUSEICL RG CCORHICHL TUBUMOUEs ac .n5 osc c css cecsaccc tenes Se aaMEME Es 84 sie 217 TU CeRPEATIOILURLET TITLE Us cy bc eC K MEE ae ovis doe o's ob ves 6 vivid tla We 0d En an Oe 218 SEeE SHELIMIETILALLON: WOM AINOLUINUS) oes isi6s cai 5 0k Sib wwe intels Clem ReRINIS s ere 218 DE SES DM TASU RDN BFA OTE Sheree gcse veo Nliesa salip iel-s'y 4.» vik, av Kate Adin eee Tor 219 OSSR CLT A ae ee eee re re 8 PR 230 b. Experimentation upon Abramis and Fundulus........................ 233 PERONOTAUEL GOUBICGPAUIONE. oi loys Wc oie va Ves ou oss ove stds cn ee nee eR 234 a Sy a eee ee tee 239 Oh a a ie. i re se 240 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 | : Mh oe SPN gt DPE MT 2 ; vf Wak iS staan ae i ye mai) oh | ea ti mtn Ps / ve « ; ‘ i e ox, oe nee eae , ; _ bs ; 7 | , 5 ¥ ) i ‘, + 4 a Oe : Vee ft) fe i) . Os *) 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 . . UIN]]9qQe.100 *9 BIpsuvs poyjeul s ple Ivquin] puv [BoTAIe. *q ; an]{q eue,Aqyour pIng s,pleH BIjsuvs Iequint [[upeery emoiyoA[Odg |} pmp- s,zoyuez | puv [ewotaseo ‘Jessop ‘pv | ‘say ¢ pus 2z ‘T ssop | ul suruund yoyoo - urlBiq °9 [oom an]q xUIpINjO], “q |-]e yued s9d CG queuIesi1B[Ue IequIny *q ® UL SulUUNI APIATIOR S.Pp[PH “Y | pue ping s.pley | JUeUeSIe[Ue ,woTAIOD “D ‘say ZI “BO sjB1 | puv SurmuIMs | poo010 qF (q) joyoo UIBIG OTOYM *9 onjq auIpNjOL “q | -[e yued aed cg BISuUBS [BIyOVAq “q S,Pp[PH “Y | pue ping s,pjex | UeWIESIv]UD [VIyORAG ‘Dp ‘say ZT “Bo | suoosid yoyoo UIBIG BJOYM *9 an[q IUIPINjOT, “gq | -[e yueo sed GG juUSUIESIv[US [BIyoRIq *q S.pPeH “P| pue ping s.pjeH BI[SUBS [BIYOVIg “Dd ‘say ZT “Bo | smorieds sulAy ISSIN jO UOl}RBOyIpoUl SN4IAS 9}RIONIO *9 S.PlPH 19}je joyoo wIn[[9qa1e0 *¢. auIsOIyyAII puB -[8 quad aed GG pioo jo JUuSUes1e[Ua suluuni AYIATIOB enjq osuejAyJoyy | puw ping s,ppey{ | [BorAte. puv azaequiny *p ‘say Ze ssop |pue SuUly[VM | [VUION (¥V) NIVLS NOILVXI4 aqaidows AOSSiL os eoreae IVWINV NOILVTOWILS 40 WHO T ATaVL 345 EFFECT OF ACTIVITY ON NERVE CELLS 8 anyiq sue,Aqyyour aMoIyoALO,g anjq sue, Aqjour aemorgoAl[og [ABIN, $0: GOR -BoyIpou SPOT] ISSIN JO WOT} “BOyIpour §,Pl°H eny{q eue|Aqyour aumor1yoAyTog Ureqs 8 PlPH uIsod pu anjq euIpmnjoy, anyq ouIpmyoy, ulyeur -10j jus Jed QT be -BULIOJ pu [OY -Oo]8 PING 8,PIPH PINY 8,PIOH yoyoo -[8 pus Ul[VULIO,T X9}100 IB] [9qQ9I109 pue sniAd ayeronso p4109 iequn, pue [BorAreo BIlsuvD rvquiny “q JUSWIOZIB[Ue LequIN] “Dp p4ioo requiny “q BYZUBS O1YBIOS “D eysues [vsiop pus sequmy *¢ p1oo jeurds jo lOyooTy | quowlesie[ue Lequiny, “Dp a a ee ee ee ee) ee ee eee 980} BUI0) ‘sry ¢ “SI OI-T sry 9 "sil ff (Yo “908 ch pues uo ‘008 CT) “sIy ¢ (‘oes aad “UIT}S) “SI TF ssop sd01J sdo1y syeo uossid sop ‘DY Pp ‘poaour -91 poojq “00 00% ‘“Brutevue yooyug #()—U01yN]OS 4/88 Jo uoIsny -1od Y4IM “13 uoly f-} ourayoAys | -vjnurnys Snug AAIOU OTYBIOS jo woryeyn -UITYS OIpBIBy Se] Jo uolRN u0l}B[NUITYS -UIIYS OIUBATVS | [VITIZIOITY 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 ‘Opl[s OURS OY} UO Pa}yUNOU Udaq DABY YOIYA\ SUOTIOIS [OIQUOD ONFIYBy oY} WOIy opeUl S}UDWIOINSBOUT VsoYy ATWO a1VdUIOD 0} 93BINIDB BIOUI PasOpISUOd SI 41 ‘A]JUOIOYIP poxY 10 ssouyory} JUdANyTp Jo oq yYyB1uT puw uorwser ewes oY} AT[ROTYMOpPT YBno1y} 4nd you 919M SUOTJOIS 9Y} BOUIg ‘“sepl[s JUsJOyIP UO epBUI syUNOD oyBavdes yueserdod OSOTLT, , 02 LT OF Le | OS Po2NSBOU S{[9d JO LaqUIN NY "16890 O18TS0"OZ68-0)226-010G2 GieeeB OF Se a eee “PAIOU OTPBIOS JO S][99 Uol[sues 101104s0g i lk eo | 72 | 0F | Og Sh | ZF | $e pe poinsvoul s]jeo0 Jo 1aquinNy G66 0/8280) 169° 0) F660 GHG OTE OBST O)ZST'O) , | FP *"* UOT|sUBS [BI O6L' 016610 -yoRiq JO s[j9o uolsuery oe | 8), Og SF LT | 0€& | 62 | 62 | 6¢ re of c% Gr poinsBaut s[[e0 JO aquInNN 260° 0\€920°0 GTE O/TPE'O) TE O/E9EO}ETS O/S6T'O | Fal'0) ‘er 0 ooce Ofc 0) umy[eq 92Z'O\S8T'O f -a109 jo sjjeo ofurpaing 8z | 9% |° °°‘ podnsvoul s[[oo Jo “ON l'0 ren olee Ol “paoo jo JUeUIESIB Ws IBquIN] jo s]j9o uwloy JOWeyuy” oni 8 6 | OL FI 6 8 Of €f | °°“ paansBoul sj[[90 Jo “ON GOT TEPG6°O)220° 12a 1 88% O/SCE 0 OfF GRP OOP” ee ae ge yusulesIB[Ua [BdTAII jo sjjeo uloy JOlo}UYy rr PN ey Sc ad ee Raa Re a a Ba a Pk fo ae ae u a ea spo S1140 AAMAN JO AGNI aL sNoapId spoa SLvu bag SMOUUVAS es INGWINGdX a 9 UNANINGdxXa ¢ INGWINGdXE f INANINGdXS i ae @ INAWINGdxXa ee sapere (pap.09a. jas yova fo woau ay}) sayour apnbs ur passaidxa auinjo A @ AIAVL 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 A. R. « 352 ec 00€ L 9 al &@ 00€ L L g OT 00€ 9 ) al Vv él O& 8G 6 96 IT GG 0Z 0 9€ 9 ifs GG 0 SI ‘pezld Used SvY YOIYM 0} sdUEIEJoI oY} ‘AaTIOG JO WOTJBOYISSB]D oY} WOrz Uye} oe “OYo “g ‘Z “T 9BVYS JOpUN poJBUTISep dsoY] 8B [[IM SB S]]90 poyuNooUN ay} “gE UUINOD “TTT eqz I, I ai IT Or 85BIg | 951g &G 0& 1G ¥G 6 8 ZL 9 g a5RYG | oBBIg | o5RIG | OBRIG | ERIC I100 ONILSAY LI LG cI VG 9G O€ O€ 02 0G Ji! &@ 0G (G0 GG SE OL OT VG LI GG cE ST cE TV 9T 61 al 9T €& VG 61 83 GE SI rot FI t (5 G 95819 | 25g | aBRIg 66 G9 | °* p4o0d Jo yuUeUT -951B[U9 [BOTAIID Soran . Banos 6F gg pi00 requinyT ze 06 |occctcctt UILIO AA 0g 90T |°° pao Jo yuour | -oB1B[Ud [BOIAIOD || sunoy 0S Sia ee P2109 1EQuuy 1G GI 7 ULIO AA 0g 88 |°*paxoo Jo yuour -99.1G[U9 [BOIAIOD see ; IOUT: se 09 pioo requin'yT 0z GOT Ge oe eae ULIO MA. J Ze OOM oa p41oo Jo yueur ye | eige oP SF pekee -m0 > ) [euids jo yuour -ds.1B[Ue IBqQuUIN'T Ly LT JG. \Egcaee erase ULIO MA J ke = zZ ee eg aD eShe aastouaxa : 4 aWIL yaa $7199 00g fo sjunod pouuasaffip Buinoysy € HTaVL 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. BIBLIOGRAPHY (1) Hopes, C. F. 1892 Journal of Morphology, vol. 7, p. 95. 1894 vol. 9. 1887 American Jour. of Psychiatry, vol. I, p. 471. (2) Van Durme, Pav. 1901 Nevraxe, p. 115. (3) Luxemsura, J. 1899 Neurol. Zentralblatt, vol. 8, p. 629. (4) LeGenpre, ReN#, et Pifron, H. 1908 Comptes Rendus de la Soe. de Biol. p. 1102. (5) Luearo, E. 1895 Archives Italiennes de Biologie, Nr. 24, p. 258. (6) Nisst 1895 Neurol. Zentralblatt, Nr. 15, p.39. 1896 Zeitschr. f. Psychi- atrie, vol. 48. (7) Dottey, D. H. 1909 American Jour. of Physiology, vol. 25, p. 151; Crip, 1911 Jour. of Med. Research, p. 309; 1909, p. 95. (8) Mann, G. 1895 Jour. of Anatomy and Physiology, vol. 29, p. 100. (9) Vas, F., Archiv f. mikr. Anat., 1892, Bd. 40, p. 375. (10) Lampert, M. M. 1893 Comptes Rendus. (11% Maaini, G. 1895 Archives Italiennes de Biologie, Nr. 22. (12) Evn, F. C. (13) ,Verworn, Max 1900 Archiv f. Anatomie u. Physiologie, supp!. p. 152. 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 «5.6 cieh eles Regen OA Sie ly 369 Be Pneteph Of itu BHO CATATIONS.....0dcsesis ei cnscciesas Rab eeNeNe book eae 369 fPoeeCr Olt ORMIAL GOIUOGIS. . 5 oa... cca he awh Od aE Es 6 sues 370 Di OCL OT ORDIBGO OV OMe soi ok Sida dio ds ae cach s andein een kha «bs 371 by Determination of adaption times..::..5.. 6.05. ccs0cendeeemeen venieme 374 ees Ue OG PAIRING be ci 530 5 0 cam bap on Dap ¥ >< here's wae ee ale owe 376 em Teu OT POTIiAl GIMNEIG, ... ais > ctcre s'v.ais a 5 <<'9 sala p ete RE eee ees 376 METER OL OXCISG. CYOH: « s ). All investigations relating to the physiology of pigment cells may be divided into two general categories, depending on whether body chromatophores or retinal pigment cells serve as the material for experimentation. The general tendency, however, to keep these fields of research sharply separated is wrong, inasmuch as the behavior of the two types of cells has much in common, and it is only by summating our knowledge, after the comparative method, that a thorough understanding of either may be gained. Light is the commonest and most potent of the natural stimu- lating agents concerned in pigment migration. In general, it may be said that light induces an expansion, and the absence of light a contraction, of pigment cells. Photomechanical changes have been demonstrated in tie body chromatophores of crustaceans, cephalopods, fishes, am- phibians, and lizards. The earliest experimentation showing the effect of light upon the retinal pigment of vertebrates was performed by Kiihne (’77) and by Boll (’78), who worked upon the frog. Their results have since been extended upon repre- sentatives of the remaining vertebrate classes, although strik- ing movements of the retinal pigment of reptiles and mammals have not as yet been demonstrated. Among invertebrates, photomechanical changes have been found in the compound eyes of insects (Exner, ’89), crustaceans (Exner, ’91), arachnids (Szezawinska, 91), and in the eye of cephalopods (Rawitz, ’91). Of the molluses, the cephalopods constitute the only group in which a response of the retinal pigment to photic stimula- tion has hitherto been demonstrated. Hensen (65), upon theoretical grounds, hazarded the guess that the pigment in these animals possessed a certain mobility, for Babuchin (764) had previously referred to finding the visual rods entirely free from pigment in some specimens of Sepia and Octopus. It was left for Rawitz (91) to show that in the light the visual rods of Sepia officinalis are pigmented along their whole length, with an especial accumulation at the lens border, whereas in the RETINAL PIGMENT OF PLANORBIS 361 dark the pigment is limited to the base of the rod. Chun (’03) confirmed these general conclusions on deep-sea cephalopods, as did Hess (05) by making a comparison of the pigment re- sponses in pelagic and littoral forms. Temperature is likewise a controlling factor in determining the ultimate distribution of pigment in many body melanophores. In general (Parker, ’06), low temperature has an influence similar to that of light, inasmuch as it favors pigment expansion; high temperature, on the contrary, like darkness, induces pigment contraction. A similar condition holds for the retinal pigment of certain animals. Congdon (’07, p. 547) found that: “In both Palae- monetes and Cambarus the proximal retinal pigment migrates distally when the temperature is lowered and proximally when it is raised.”” The writer (Arey, 16*) found an identical ten- dency in the retinal pigment of fishes. Working with the frog’s retina, Gradenigro (’85) first showed that at a tempera- ture of 30°C. the pigment expands, thereby closely simulating the distribution characteristic of light. Later Herzog (’05) confirmed this discovery, and further stated that maximal expansion likewise ensues when the temperature approaches the freezing point, the contracted condition typical of darkness being obtainable only between the temperatures of 14° and 18°C. A reinvestigation of this matter recently made by the writer (Arey, 16") has convinced him that Herzog’s results are sub- stantially correct and that the behavior of the retinal pigment of the frog to temperature must indeed be considered exceptional ; the explanation for this is presumably to be found in the exist- ence of a superimposed nervous control, resulting in the oblitera- tion of the more primitive response. Positional changes of the retinal pigment of gasteropods in response to definite physiological stimuli have never been re- corded. Smith (’06), in his work on the structure of the eyes of pulmonate gasteropods, noticed that the retinal pigment of Planorbis trivolvis Say showed processes of variable length, and accordingly he performed a few experiments to determine whether the pigment distribution could be correlated with 362 LESLIE B. AREY corresponding conditions of light and darkness. His results concerning a photomechanical influence were entirely negative, although he states his belief (p. 255) in the existence of an appre- ciable pigment migration caused by certain unknown factors. Assuming that the retinal pigment of these animals does undergo migratory movements, it is reasonable to expect that the behavior of pigment cells, having no demonstrable nervous connections (Smith, ’06), is dependent upon definite environ- mental stimuli, hence I decided to reinvestigate the influences that simple stimulating: agents such as light and temperature might have upon the retinal pigment of these snails. MATERIAL AND METHODS Planorbis was obtained in abundance at certain localities on the bank of the Charles River, Cambridge, Mass. During warm weather it is easily procurable within arm’s reach of the shore, but as the water grows colder a gradual withdrawal of animals occurs toward the deeper bed of the river. My experi- ence in collecting during the fall of 1914 will illustrate this behavior. In the second week of October a great wealth of material was discovered at a location where the previous June no specimens had been seen; at the time of this collection the snails were distributed as far up as the water’s edge. The next collecting trip was made on November 21, and, in the same spot, where several weeks previously hundreds of animals were obtainable, there now remained a few stragglers only, and these were withdrawn from the bank almost beyond reach. Ten days later scarcely a specimen was left within sight. I was interested to discover whether this withdrawal was due to an active migration of individuals, or to the effect of the low temperature which might cause the snails to become inac- tive, whence they would be washed by the current down the sloping bank into deeper water. Mr. W. F. Clapp of the Mu- seum of Comparative Zodlogy informs me that the former of the alternatives is undoubtedly correct. Moreover, low tem- perature does not necessarily cause these animals to become inactive, since he reports having often broken through the ice RETINAL PIGMENT OF PLANORBIS 363 in mid-winter and dredged Planorbis from the deeper water to which they resort; under these conditions he has always found them to be in an exceedingly active condition. This interesting migratory behavior to what is presumably the stimulus of low temperature has a definite ‘protective’ value to the snail, since an animal thereby removes itself to a depth at which ice is not formed. It is probable that the situation is not one in which an annual rhythm, independent of temperature, has been impressed upon the species, for when Planorbis, during its retreat to deeper water, is removed to laboratory aquaria and kept for some time at room temperature it neither manifests a tendency to seek the deepest water, nor does it exhibit negative geotropism. On the other hand, I do not recall that animals introduced into jars of ice water showed any marked tendency to seek the bottom of the dish. It is possible that there is, in fact, a temperature response which, however, comes on but slowly, as evinced by the gradual with- drawal under natural conditions. Except for minor changes, the following is quoted directly from an enlightening statement made by Mr. Clapp during a corre- spondence in which I sought his aid in solving the migratory behavior of this animal: What you write in regard to the migration of Planorbis is my idea also. There is little doubt that Planorbis has a rest period comparable to that of nearly all other gasteropods—the effect of inactive phases in the life cycle is often clearly marked on the shell. In many species of land shells there are two annual rest periods, the shorter occurring in the summer, the longer in the winter. I have never observed any general inactivity or burrowing during the summer on the part of our New England species of Planorbis, and therefore have thought it probable that their rest period occurs during the winter months. The winter rest period of the land shells in this section of the coun- try is governed entirely by the temperature. When spring arrives early the shells appear early—the hardier species first, the more deli- cate species later—but all appear earlier than in other years when the warm weather comes on tardily. It seems probable that Plan- orbis, Physa, Lymnaea and similar shells should follow the same prin- ciple, in other words, become inactive and bury themselves when the temperature of the water falls below a definite point. To a certain extent this may be true, but if so, the rest period must be much shorter than in land shells, and, I think, not compulsory as with the latter 364 LESLIE: B. AREY forms. My reason for believing this is that I have frequently obtained Planorbis, Lymnaea and Physa, in mid-winter, by breaking through the ice and using a small dredge or net. Some believe that the fresh water pulmonates do not hibernate at all. That appears to me to be based on negative evidence, since they have not been observed under natural conditions, or rather have been observed under unnatural conditions (vivaria). At the conclusion of all experiments, to be described subse- quently, the animals were beheaded, fixation of the head and the contained eyes occurring under conditions of light and tem- perature identical with those at which each experiment had been conducted. Perenyi’s fixing fluid gave very satisfactory preservation. Sections of 8 » thickness were cut parallel to the long axis of the eye and were stained with Heidenhain’s iron-haematoxylin and with orange-G. Whenever the experimentation involved the employment of light, strongly diffused daylight, such as is obtainable at north windows, was used. The microscopical preparations were studied and measure- ments were made at a magnification of 1400 diameters. In expressing the extent of pigment distribution in numerical terms, two measurements were used. One, which I shall call the ‘zonal measurement,’ represents the breadth of the band of dense pigment, exclusive of the finger-like processes extend- ing toward the bases of the constituent cells (figs. A, 4 and 5). The second, or ‘process measurement,’ consists of the zonal measurement plus the average length of the pigmented proc- esses just referred to. The sense in which a few descriptive terms will be consistently employed needs explanation. ‘Peripheral’ and ‘central’ will designate those parts of the eye which are respectively farthest from and nearest to its center (fig. B). ‘Distal’ and ‘proximal,’ used in describing retinal cells, are retained in a sense similar to that employed in descriptions of uninvaginated ectodermal cells—hence distal indicates the portion nearest the lens or the lumen of the optic sac, while proximal refers to that part of a cell nearer the connective-tissue sheath at the periphery of the eye (figs. B and C). RETINAL PIGMENT OF PLANORBIS 365 DESCRIPTION OF THE EYE OF PLANORBIS Planorbis is a representative of the group of pulmonate gas- teropods which is characterized by the possession of but one pair of non-retractile tentacles, at the bases of which the eyes are located. Although the tentacles are not retractile after the manner of an introvert, they nevertheless are capable of exhibiting a high degree of muscular shortening whereby their surface becomes thrown into transverse ridges (fig. A, ta.). =e = bes ieee for.mus. n.opt. Fig. A An axial section of both the eye and the tentacle of Planorbis, show- ing the position and mutual relation of these structures (< 60). fbr. mus., mus- cle fibers; n. opt., optic nerve; oc., eye; ta., tentacle. The eye (fig. A, oc.), which lies just beneath the surface epithelium, has the shape of a cone or pear whose base, cor- responding to the corneal portion, points outward. The base has a diameter of about 150 u, whereas the axial measurement is 200 MK. An axial section of the eye (fig. B) shows the following parts: (1) optic capsule; (2) cornea; (3) retina; (4) optie nerve; (5) lens; (6) vitreous humor. The optic capsule (fig. 3, cps. opt.) is a thin, connective- tissue sheath surrounding the eye, to which the retinal and cor- neal elements are attached. It is continuous with the sheath of the optic nerve. 366 LESLIE B. AREY The cornea and retina represent differentiated portions of an original sac-like epithelial invagination (figs. A and B). The wall of this closed ‘optic sac,’ as it is called, never becomes more than one cell thick. The pigment-free corneal cells are only slightly columnar in shape, hence this portion of the optic sac remains as a thin membrane, which is strikingly in con- crn.. Fig. B- An axial section (semi-diagrammatic) of an entire eye of Planorbis (X 275). ern., cornea; hu. vit., vitreous humor; lns., lens; nl. cl. rin., nucleus of retinal cell; n. opt., optic nerve; pig., pigment; rin., retina. trast with the thick cornea of some other pulmonates. Its extent is limited to the broad base of the conical eye (fig. B). The retinal portion of the optic sac is easily distinguishable from the cornea by the presence, in the former, of pigmented cells and by the great elongation of all its elements (figs. B and C). In general, the constituent cells may be said to have a radial arrangement with respect to the optic sac. Notwith- standing the fact that the retina is only one cell thick, it is con- RETINAL PIGMENT OF PLANORBIS 367 venient to follow the suggestion of Smith (’06) and distinguish three concentric zones, which, although of an arbitrary nature, are on the whole rather clearly defined. The peripheral zone (fig. C, rtn. ex.) is in contact with the optic capsule and is quite free from pigment; it contains the cell nuclei. The pigment of the non-sensory cells is aggregated in a middle or intermediate zone (fig. C, rtn. m.); peripherally its limits are ill defined be- cause of irregular processes, which extend down into the cells, whereas centrally a sharp line of demarcation separates the pigment from a third or central zone (rin. i). The position of the boundary common to the peripheral and intermediate zones is somewhat dependent on the degree of pigment migration under various environmental conditions, which will be dis- cussed in the main part of this paper. The internal or central zone (fig. C, rin. t.) is of constant width and comprises the por- tion of the retina between the pigmented zone and the lens. It contains the rods (bac.)—the photoreceptive elements. Two kinds of cells form the retinal epithelium, the non-sen- sory pigmented cells (fig. C, nl. cl. pig.) and the unpigmented sensory cells (nl. cl. sns.). The pigmented cells, which are the more numerous of the two, are grouped about the sensory cells thereby isolating the latter from each other. These pigmented cells are of two kinds. One set has its nuclei situated close to the optic capsule, while the nuclei of the other set are near the center of the cells and slightly distal to those of the sen- sory elements. Both types of pigment cells are exceedingly slender, although those with more distally placed nuclei gener- ally have an enlargement just distal to the nucleus; according to Smith (06, p. 255): ““When this part of the cell is free from pigment it appears to contain a vacuole.” The distribution of pigment is variable under different cir- cumstances; the limit of proximal migration in either type of cell, however, is conditioned by the position of the nucleus (fig. C), for pigment is never found proximal to it. The length of the pigment cells, and therefore the thickness of the retina as a whole, varies at different levels in the eye. The thickest portion of the retina is on the sides at a distance of about 60 u 368 LESLIE B. AREY from the apex of the optic sac. At this thickest region, the distance from the optic capsule to the central zone, that-is, the length of the pigment cells, is approximately 35 u. The unpigmented or sensory cells (fig. C, nl. cl. sns.) are much more robust and have larger nuclei than either kind of pigment cell just described. These cells are of an elongated spindle shape, thickest in the region of the nucleus. The cell body ivin.bace. ax.bac:----- : \rtn.exs 3 o o o ' ! Fig. C A portion of an axial section of a Planorbis eye, showing the com- ponent retinal elements (< 1000). az. bac., axis of rod; cps. opt., optic capsule; fobrl’., fibrillae of rod-axis; fbrl’’., fibrillae of rod-mantle; zvlr. bac., mantle (in- volucrum) of rod; nl. cl. pig., nucleus of pigment cell; nl. cl. sns., nucleus of sen- sory cell; pig., pigment; pre. n’t., neurite-process of sensory cell; rin. ex., periph- eral zone of retina; rin. 7., central zone of retina; rin. m., middle or pigmented zone of retina. continues through the pigment zone and ends, in the so-called central zone, in a structure known as the ‘rod’—this being the photoreceptive portion of the cell. Each rod consists of two parts, centrally a core or ‘axis’ (az. bac.), and peripherally a radially striate “mantle’ (ivlr. bac.). Proximal to the nucleus. each sensory cell gives off a neurite (pre. 7’t.), which courses along the inner face of the capsule and is ultimately gathered up with similar neurites to form the optic nerve (figs. A and B, RETINAL PIGMENT OF PLANORBIS 369 n. opt.). The optic nerve is covered by a sheath of connective tissue continuous with that of the optic sac. The work of Babuchin (’65), Henchman (’97), and particularly that of Smith (’06) has proved that the sensory cells of pulmonate gasteropods are of fibrillar composition, the terminal brush- like neurofibrils of the rod mantle (fig. C, fbrl.’’) being continuous with the fibers of the rod axis (fbrl.’) of the cell body, and of the neurite. The length of the rod is 6 uw or 7 u. The lens (fig. B, lms.) is a large pear-shaped body occupying almost the whole interior of the optic sac. It isa non-cellular secreted mass, the outer portion of which forms a denser cap- sule or rind. The vitreous humor (fig. B, hu. vit.) fills whatever spaces inter- vene between the central zone of the retina and the lens, as well as the spaces between the individual rods. EXPERIMENTAL PART a. Effect of light and darkness The initial experimentation consisted in determining whether or not the retinal pigment of Planorbis undergoes positional changes, whereby a characteristic distribution is assumed in light and in darkness. The citation of Smith’s statement (’06, p. 255) relative to the tests performed by him on this animal will serve to show both his methods and his results: In spite of the fact that the rods, lying, as they do, distal to the pigment zone, cannot be protected by pigment migration, as can the rods in cephalopods, the variable position of the proximal region of the pigment suggests the possibility of pigment migration in the eye of Planorbis. I therefore attempted to determine by a few experiments whether differing light conditions would produce corresponding changes in the position of the pigment. Several specimens of Planorbis were placed for an hour or more in water in a white, porcelain dish which was set in a sunny window. Their heads were then cut off with scissors and fixed in Perenyi’s fluid. Sections were made in the ordinary way and stained in Heidenhain’s iron-haematoxylin, followed by orange-G. Similar preparations were made from specimens which had been kept in darkness for an hour or more before killing. Comparisons were 370 LESLIE B. AREY then made between the two kinds of preparations in order to learn whether the position of the pigment was different in the two cases. More cases in the ‘light’ eyes had the pigment reaching quite to the nuclei than in the ‘dark’ eyes; but there was such a lack of uniformity in different animals, and even in the same retina, that the evidence was not at all conclusive. Repeated experiments did not lead to more definite results. I am satisfied that the pigment does travel up and down in the pigment cells under the influence of some stimulus, but just what is the exciting factor I have not determined. I have not been able to get any evidence of pigment migration in the eyes of either Helix or Limax. In another part of the same paper Smith again returns to a consideration of these results and becomes more bold in his inferences. Selected excerpts (pp. 268-269) make plain his final attitude toward the situation in Planorbis. It is probable that pigment movement is a direct response to light- stimulation in Planorbis . . . . Not having been able as yet to determine the exact conditions under which it occurs, I can only suppose from analogy that the migration is a response to light. The shape of the cells, the position of the pigment in some cells as com- pared with that in others, and the apparent need of pigment migration in the eye of Planorbis, all point to a probable responsiveness of its pigment cells to light; seit A personal experience (Arey, 16) in determining the lengths of time needed to complete light and dark adaption of the highly mobile retinal pigment of fishes led me to suspect that the ‘hour or more’ which Smith allowed for the execution of his experiments might be wholly insufficient to permit the photo- mechanical response to proceed to completion. Hence in my experimentation ample time allowances were made for both light and dark adaption. ; 1. Effect on normal animals. Five animals were placed in a battery jar containing water and were kept in a dark-chamber for 24 hours. In the same manner, an equal number of animals were subjected to daylight for 10 hours. A microscopical examination of the resulting preparations gave data as in table 1.! 1 The final measurements recorded for each eye represent the mean of the values obtained from several measurements on each side of the retina at adis- tance of about 50 u from the entrance of the optic nerve. Experience showed that RETINAL PIGMENT OF PLANORBIS 371 TABLE 1 Measurements showing the relative distribution of the retinal pigment of Planorbis in darkness and in light at room temperature. The values are mean values ex- pressed in micra, and indicate: (1) the thickness of the main pigment mass (zonal measurement) ; (2) the zonal measurement plus the length of the pigmented processes (process measurement); (3) the length of the pigment processes obtained by sub- tracting (1) from (2). The percentage change in each mean measurement in darkness as compared with that in light is also computed PER- PER- PER- NUMBER | MEAN | CENTAGE] MEAN | CENTAGE Breall peepee CONDITION OF RET- | ZONAL | CHANGE | PRocESS | CHANGE | Gp pig. lin LENGTH OF ILLUMINATION INAS _| MEASURE-| IN ZONAL | MEASURE-| IN PROC- | “Uo |/NUnNGt MEASURED| MENT |MEASURE-| MENT |ESS MEAS- : pa UREMENT (PROCESSES| MENT sr PROCESSES Bb ad OS Le Se 10 11.0 64 5 52 5.5 97 2 2 Darkness.......... 10 18.0 25.0 7.0 Since the distal margin of the pigment zone—the boundary between central zone (fig. C, rtn. 7.) and middle zone (rin. m.)— is fixed in relation to each pigment cell, these values show quite decisively that in the light (fig. 2) the pigment migrates distally in the cell, that is, toward the source of illumination, whereas in the dark (fig. 5) the reverse is true. An appreciable thinning out of the pigment in darkness due to its being spread over a greater area, is not demonstrable. It may be that the pigment involved in migration is only that which lies most proximally in the middle zone, or there may occur a general proximal move- ment, that is,a readjustment of the relative quantitative amounts, throughout the whole mass. Whichever alternative is true, it is certain that the edge of the pigment zone which forms the boundary between the middle and central zones (fig. C) main- tains a constant position in relation to the retinal cells. 2. Effect on excised eyes. Since light exercises a photome- - chanical influence on the retinal pigment, the interesting query arises as to whether this effect is direct, upon the cell itself, restricting the measurements to this most favorable region afforded the fairest numerical representation of the pigment distribution. In these, as well as all experiments to be described subsequently, individuals of Planorbis were selected of a more or less uniform size—this precaution will, I think, serve to obviate criticism, if not actual error. 372 LESLIE B. AREY or whether it is accomplished through some nervous mechanism related to the cerebral ganglion. It is true that the probability is against the existence of nervous connections with the pigment cells, since all previous researches have failed to demonstrate structures of this kind, yet it is entirely conceivable that such neuro-fibrils may exist and have hitherto escaped detection; indeed, the extreme difficulty with which the neuro-fibrils of the sensory cells are demonstrated, makes this possibility all the more real. Even if a direct influence of light can be proved, the existence of such neuro-fibrils is not precluded, nor yet their codperation in effecting the migration of pigment; but it does render this event less likely, and definitely shows, more- over, that whatever may happen in the normal animal, a direct action of light on the pigment cell is a demonstrable phenomenon. If the direct action of light can not be shown to induce migra- tory movements of the pigment in excised eyes, three possibilities exist: (1) fibers in connection with the pigment cell perform double conduction, transferring afferent impulses to the cere- bral ganglion and efferent impulses back to the retina; (2) affer- ent impulses travel in the optic nerve to the cerebral ganglion while efferent impulses return by hypothetical nerves to the pigment cells, or (3) the optic nerve is of a mixed nature, possess- ing both afferent and efferent components. Since, however, the existence of double conduction in nerves is a mere postula- tion, the first of the three possibilities may be safely eliminated. There is, nevertheless, still another way of viewing the situa- tion, and one that demands serious consideration. The metabo- lism of an organ isolated from the body to which it belongs is, of necessity, fundamentally altered. Not only is its supply of nutriment cut off, but what is more serious in a short experi- ment, the elimination of waste is not adequately provided for. ~ If isolated pigment cells do not respond to the direct action of light, the reason may easily be ascribable to an autoanaes- thetization caused by the accumulation of the cell’s own catabolic products. The probability of this course of events was suggested in some experiments upon the eyes of fishes (Arey, 16°; 16>). Moreover, it will be shown in another part of this paper (p. 378) RETINAL PIGMENT OF PLANORBIS 373 that anaesthetics are probably capable of exerting an inhibition on the migration of pigment, even in normal snails. Spaeth (713) found the melanophores on the isolated scale of Fundulus to be responsive to ultra-violet rays but not to those of the visible spectrum. Parker (’97), working upon Palaemonetes, obtained responses from all three types of pig- ment cells when excised eyes were brought from light into dark- ness, or the reverse. This result was observed equally well when stalks containing ganglia were used or when merely retinas, exclusive of ganglia were employed. Hamburger (’89) reported that the pigment of enucleated frog’s eyes exhibits migratory movements both in darkness and in light. In another paper (Arey, ’16*) I have described the occurrence of a pigment migra- tion when the previously dark-adapted excised eye of the com- mon horned pout, Ameiurus, is brought into the light; in the reverse exposure (light to dark), however, no response ensues; furthermore, in several other fishes which were studied, even the direct influence of light could not be shown. Experiments on Planorbis were made upon animals that had been adapted to light or to darkness for 6 and 24 hours, respective- ly. Such animals were beheaded and small pieces of tissue, bearing a tentacle, were then placed in a watch glass containing an abundance of Ringer’s solution and subjected to light or to darkness according to desire. The exposure to light lasted 4 hours, whereas in the dark, eyes were left for 5 hours. Table 2 summarizes the results of these determinations. The results of both these sets agree very closely with the values given previously for normal light-adapted eyes (11.0 u and 16.5 uw for zonal and process measurement respectively). The obvious conclusion to be drawn from these experiments is that light has a direct influence on a previously dark-adapted pigment cell, but that light-adapted retinas do not change in darkness. The direct action of light on the retinal pigment of this animal, therefore, is identical with that of Ameiurus. I am inclined to interpret these results in the following way. The conditions under which an isolated eye is placed undoubtedly favor the accumulation of catabolic products. This unremoved 374 LESLIE B. AREY TABLE 2 Measurements showing the relative distribution of pigment in the excised eyes of Planorbis in darkness and in light. The values are mean values expressed in micra and indicate: (1) the thickness of the main pigment mass (zonal measure- ment); (2) the zonal measurement plus the mean length of the pigmented pro- cesses (process measurement); (3) the length of the pigmented processes obtained by subtracting (1) from (2). The percentage change in the mean zonal measure- ment in darkness as compared with that in light is also computed PERCENTAGE MEAN MEAN NUMBER MEAN ZONAL| CHANGE IN CONDITION OF RETINAS MEASURE- ZONAL PROCESS LENGTH OF OF ILLUMINATION MEASURED MENT MBEASURE- MEASURE- PIGMENT MENT MENT PROCESSES. MB M M Dark-adapted eyes sub- jected to light.......... 6 11.0 14 14 3.0 Light-adapted eyes sub- > jected to darkness...... 6 12:5 14 1.5 waste exerts an inhibitory or anaesthetic influence upon the normal activity of the protoplasm of the pigment cells. Since in the case of the retina it is even probable that light favors catabolism, it follows that the movement of the retinal pigment in the light only, must be due to the greater efficiency of light as a stimulating agent. The strong stimulatory effect of light, therefore, is able to break through the protoplasmic inhibition caused by accumulated wastes, whereas darkness is ineffective in this respect. If this reasoning is tenable, one may infer that movements of the pigment would also be realized in dark- ness, provided artificial circulation could be maintained. That light actually is a more efficient stimulus than darkness, is further suggested from the determinations of adaption times (vide infra). (b) Determination of adaption times It is evident from the foregoing observations that photo- mechanical changes do occur in the pigment of the Planorbis retina, and furthermore, one is led to suspect that the indecisive results obtained by Smith (06) are assignable to the short duration of his experiments, which, in his own words, lasted but ‘“‘an hour or more.”’ Hence the pertinent inquiry as to RETINAL PIGMENT OF PLANORBIS ay Ais the exact periods of time necessary to complete light- and dark- adaption becomes the next object of consideration. Snails which had previously been thoroughly adapted to light (fig. 2) or to darkness (fig. 5), were subjected to opposite conditions of illumination. Individuals were removed and killed at half-hour intervals until the experiments had con- tinued for 5 hours. Microscopical examination of the retinas gave the following results: Dark-adapted Planorbis subjected to light: Incomplete light-adaption occurred in 3 hours Complete light-adaption occurred in 4 hours Light-adapted Planorbis subjected to darkness: Incomplete dark-adaption occurred in 4 hours Complete dark-adaption occurred in 5 hours Our suspicion as to the cause of Smith’s failure to procure satisfactory and consistent results is corroborated, since the time alloted for his experimentation was quite inadequate. From the comparative standpoint, it is interesting to see how the rapidity of pigment migration in Planorbis agrees with corresponding movements in the eyes of other animals. In the compound eyes of the prawn, Palaemonetes, Parker (97) gives the following data for the time consumed in the adaption of the proximal, or retinal, pigment: WIATENENS UOMO ts sche a ce ciekic co oe aoe ne s Saint seal ane 30 to 45 minutes ATAU CUOP OME RTIOSS tec © Sy, e's whatt ofe o.c:alaco eave eas eee ere 45 to 60 minutes The writer (Arey, 16°), working upon several fishes, has shown that the complete adaption of the retinal pigment requires longer periods of time than have generally been supposed. These determinations may be combined in the following sum- marization : IDI SS: GO nT Gib hie as ci fine tee sic on aye a cis ee ee 45 to 60 minutes WIIG TOSUATENERSin wc earese nce na os ke tec oe eee ee 30 to 60 minutes An interesting comparison with the cephalopod type of eye is made possible through the values determined by Hess (’05), from whose work it appears that many hours (48 or more) are THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4 376 LESLIE B. AREY necessary for dark-adaption, whereas a much shorter time (approximately 13 hours) suffices for light-adaption. Hess’s results upon Octopus defilippi (one of the several species studied by him) are especially astonishing, since he states that dark- adaption occurs in 48 hours, whereas light-adaption may be practically completed in 15 minutes. (c) Effect of temperature Next to light, temperature is the commonest environmental stimulating agent that controls the movement of retinal pig- ment. Accordingly, a series of tests were made, both on nor- mal and excised eyes, to discover whether a correlative tempera- ture response is demonstrable. TABLE 3 Measurements showing the relative distribution of pigment in the eyes of Planorbis at 8° and 32°C. both in darkness and in light. The values are mean values expressed in micra and indicate both the thickness of the main pigment mass (zonal meas- urement), and the zonal measurement plus the length of the pigmented processes (process measurement). Corresponding values at room temperature, copied from Table 1, are given in parenthesis NUMBER MEAN ZONAL MEAN CONDITION OF ILLUMINATION me ete TEMPERATURE MEASUREMENT dana deg. C. Me iL 1 bilfea OU Hates oreo MVNO ERD PLACA cytes 6 3 10.0 14.0 (11.0) (16.5) 1 Gifts IVF 58g he ia ea ee ae 6 30 25 19.0 Darkivess puter. cyte sees 4 3 13.0 19.0 (18.0) (25.0) Danknessey ac soe. fee 6 32 14.0 24.0 1. Effect on normal animals. Fully light- and dark-adapted snails, retained in jars of water, were each subjected to tem- peratures of approximately 3° and 32° C. At the end of 4 hours the eyes were fixed at temperatures identical with those at which the respective experiments had been conducted. The data from these determinations are presented in table 3. These data show that temperature is extremely efficacious in evoking positional changes in the retinal pigment. Since, RETINAL PIGMENT OF PLANORBIS ah both in the light and the dark, the pigment shows greater distal migration at low (figs. 1 and 4) than at high temperatures (figs. 3 and 6), it follows that low temperature and light on the one hand, and high temperature and darkness on the other, tend to influence pigment migration similarly. As the data show (this being a logical corollary of the proposition just stated), extreme distal migration is favored by the codperation of light and low temperature (fig. 1), whereas an extreme proximal migration is best obtained at high temperatures in the dark (fig. 6). It is pertinent to mention in this connection that the pigmented processes at the higher temperatures in the dark (figs. 5 and 6) were very thick as well as long. It will be noticed that in the light the values given in paren- thesis (representing the results at room temperature copied from table 1) are intermediate between the measurements at the extreme temperatures; in the dark, on the contrary, this progressive relation does not hold. TABLE 4 Measurements showing the relative distribution of pigment in the excised eyes of Planorbis at 2° and 30°C. in the light. The values are mean values expressed in micra and indicate both the thickness of the main pigment mass (zonal meas- urement), and the zonal measurement plus the length of the pigmented processes (process measurement) NUMBER Maas : MEAN CONDITION OF ILLUMINATION OF RETINAS | TEMPERATURE beepers PROCESS MEASURED nae, oS ae | MEASUREMENT deg. C. u uM | Ly (210 ee ace as Wy, See Gn ana 3 2 9.6 10.2 MOUs he oh cr vere oa, Giants 3 30 10.3 Leo 2. Effect on excised eyes. A few experiments were performed to determine whether the temperature response is obtainable in light-adapted eyes which are isolated from the body. In all respects the method of experimentation was identical with that employed when normal animals were used. Table 4 gives the results from the small number of retinas measured, an accident having destroyed the remainder of the material. 378 LESLIE B. AREY The results thus tabulated are so similar to those just discussed in the case of normal animals that comment is scarcely neces- sary. The specimens used were rather undersized and _ this probably accounts for the low values obtained in each set. Unfortunately no material was available when temperature experiments upon dark-adapted excised eyes might have been performed. Had an influence of temperature been proven under these conditions it would have been of considerable in- terest, inasmuch as darkness was found (p. 371) to be ineffective upon excised eyes which had previously been subjected to light. This result was indeed realized in my work (Arey, ’16*) upon the isolated eyes of fishes, thereby suggesting that temperature is a more efficient stimulating agent than is light. (d) Effect of anaesthetics In a few instances only, have clear cut observations been recorded as to the anaesthetic action of definite substances upon retinal pigment cells. Ovio (95) and Lodato (’95) agreed that co- caine arrested pigment migration in the frog. The writer (Arey, 16) has found that carbon dioxide or ether, without perma- nently injuring the retinal pigment cells of fishes, is capable of arresting completely the movements of the pigmeut in light as well as in darkness. Chioretone and urethane, on the con- trary, although of sufficient strength to kill the animals as or- ganisms, did not prohibit pigment migration. Since 5 per cent solutions? of carbon dioxide effectively controlled these migra- tions, 16 was suggested that this, the commonest of catabolic products, might well be the effective agent in preventing mi- eratory movements of the pigment in excised eyes.? It is pos- sible that the results, already presented, in which it was found that the pigment of the isolated eyes of Planorbis migrated in light but not in darkness, may be interpreted in a similar manner. 2 No attempt was made to ascertain the minimal concentrations which would accomplish this end. 3 Of four fishes used, Ameiurus, Abramis, Carassius, and Fundulus, the retinal pigment of the excised eyes of Ameiurus changed its position in the light, where- as in none of these species did a change occur in darkness. RETINAL PIGMENT OF PLANORBIS 379 A few experiments seemed to indicate that both ether and carbon dioxide did completely check the pigment movements when Planorbis was introduced from darsness into light, yet the snails were very susceptible to the anaesthetics and in no case survived. By careful experimentation it might be possible to find concentrations at which movements of the pigment would be prevented, yet the pigment cells and the animals (as deter- mined by controls) survive. After several trials, in all of which the snails did not outlive the 3 to 4 hours subjection to the anaesthetics necessary for light adaption, the work was dis- continued. It is evident that the chief value of the few determinations made lies in their suggestiveness and not in the actual results gained, which, because of the absence of appropriate controls, are properly open to criticism. Hence one may be allowed merely to suggest that the pigment in the excised eyes of Planor- bis, similarly to that in Ameiurus, is able to overcome the prob- able anaesthetic effect of accumulated wastes in the light only, whereas the weaker response in the dark fails to appear at all because of the presence of the same catabolic products. DISCUSSION In 1906 Parker analyzed the results of many workers con- cerning the influence of light and temperature upon melanophores and stated his conclusions in the following generalization (p. 413): “It is probable that in all melanophores in which there is a migration of pigment, light or low temperature will induce a migration toward the source of illumination and the absence of light or a high temperature a migration in the reverse direc- tion.”’ Certain cases, however, may be cited which do not conform to Parker’s dictum. Such reversed behavior to light is exhibited by the melanophores of the frog (Harless, ’54), the eel (Steinach, ’91) and Triton (Hertel, 07). A lack of conform- ity is likewise shown in the temperature responses of the frog’s retinal pigment (Herzog, ’05; Arey, ’16*). It is not improbable that, in these animals, a more or less active nervous control 380 LESLIE B. AREY has been superimposed upon the more primitive direct melano- phore response, thereby making the behavior appear anomalous. It is evident, however, that the results enumerated in this paper relative to the influence of light and temperature upon the retinal pigment of Planorbis, are conformable with the previously quoted generalization of Parker. The existence of a similar agreement in the behavior of the retinal pigment to temperature was found by Congdon (’07) upon the prawn, Palaemonetes, and by myself (Arey, 716°) upon several fishes. The role played by body chromatophores in the economy of an animal is supposedly adaptational with respect to its en- vironmental coloration, and perhaps the regulation of its body temperature as well. The significanace of movements of the retinal pigment, on the contrary, are by no means patent, and the agreement of its responses to light and temperature with those of melanophores in general is of considerable speculative interest. Is the reason for this unaminity of response a phylo- genetic one, retinal pigment cells retaining a primitive behavior? Or is it merely the similar but discontinuous expression of com- mon physiological needs? Or is the agreement purely fortuitous, not involving the fulfilment of amy common need? Or is the influence of light and temperature upon the pigment-containing protoplasm necessarily similar wherever mobile pigment. cells are found, the pigment migration under any condition (when not controlled by the nervous system), therefore, being always predictable? By allowing one’s fancy free rein, numerous other possibilities may be conceived. Since we know many more instances of immobile than of mobile pigment cells in the tissues of animals, it is reasonable to suppose, and, moreover, evolutionary doctrines compel us to assume, that the existence and perpetuation of the latter type of cells is not without significance. Thus, ignoring all ques- tions as to the reason for this or that behavior of iat cells, we at least can safely assume with Parker (’06, p. 411):“" .. it might be supposed that if a case arose in which a revere migration of pigment would be of service to the organism, such form of migration would be evolved and a set of pigment cells RETINAL PIGMENT OF PLANORBIS 381 in which the pigment granules under illumination would migrate away from the source of light instead of toward it would be produced.” It has already been pointed out that the exceptional behav- ior of the body chromatophores of the frog, eel, and Triton probably is the result of a nervous control. As far as direct responses are concerned, Parker’s further assertion (’06, pp. 411-412), for all we know to the contrary, is true: ‘Hence it seems probable that the melanophores, retinal pigment cells, and other like structures in which dark pigment granules ex- hibit migratory movements, are restricted as to these possibili- ties, and that in light they always transport their pigment toward the source and never in the reverse direction.” Since the pigmented cells of the gasteropod eye have no demonstrable nervous connections, this condition, if true, ren- ders these cells wholly indifferent in the process of light per- ception. It follows, therefore, that such cells are not comparable to the pigment-bearing retinular cells of certain arthropods in which the pigment granules, contained within the sensory cells themselves, change position in light and in darkness. The situation in the gasteropod, however, is quite similar to that in the vertebrate eye in this respect. In several crustaceans (e.g., Parker, 99, upon the amphipod, Gammarus) it has been shown that in darkness the pigment of the retinular cells moves proximally, thereby leaving part of the rhabdome devoid of pigment, whereas in light the rhab- dome again receives a pigment sheath. These responses were interpreted as having the following significance. In the light, the rhabdome, surrounded by pigment, is protected from over- stimulation by light reflected internally from the white pigment; in dim light, on the contrary, the efficiency of the visual apparatus is increased by the withdrawal of pigment, whereby the reflect- ing mechanism enables the eye to make the best use of the available diffuse light. Theories involving the principles of over-stimulation as well as of optical isolation have also found many supporters among those who attempt to explain the phenomenon of pigment migration in the vertebrate eye (Arey, 715). 382 LESLIE B. AREY The interpretation of migratory movements in the retinal pigment of gasteropods, or to go further, the interpretation of the presence of such pigment at all, from the standpoint of preventing overstimulation or of securing optical isolation, involves greater difficulties than is the case in arthropods. In the snail, the brush-like rods, which have been described as the photo-receptive portions of the sensory cells, are entirely distal to the pigment zone, hence these elements are at all times ex- posed to the full strength of light. It has been pointed out, however, that the pigment may serve as a background to pre- vent reflection and thus it indirectly produces a limited ‘opti- cal isolation.’ The accumulation of pigment far from the cell bases, and the slight extent to which it can be induced to move proximally, suggests that its chief utility may exist in a relationship with that portion of the sensory cell which it immediately surrounds. In this connection, a statement by Smith (06, p. 270) is suggestive: Aside from preventing internal reflections within the rod zone, it is possible that the pigment is directly protective to that part of the visual cell which is surrounded by it. We do not know that the middle part of the sensory cell is not sensitive to light. Neither do we know how or where light vibrations are transformed into nervous impulse. If the transformation takes place in the middle zone, the pigment may serve some purpose there. Having thus seen that the presence of retinal pigment of gasteropods is only to be interpreted with difficulty, how much greater is the task of assigning explanations to the feeble move- ments exhibited by this pigment. It may be said that explana- tions of the presence of retinal pigment and of its movements are self-inclusive, an explanation of one necessarily involving the other. That presence and mobility represent two more or less distinct factors, follows, however, from the fact that in some, and perhaps in most gasteropods (e.g., in Helix and Limax, Smith, ’06) the retinal pigment is non-motile. Theorists who have viewed the migratory movements of retinal pigment in those eyes where striking changes occur as indicative of the prevention of overstimulation or the procural RETINAL PIGMENT OF PLANORBIS 383 of optical isolation, have likewise associated a retreat of the pig- ment in dim light with the presence of a more diffuse and weaker photic stimulation, but, withal, a stimulation which, under the circumstances, thereby rises to its highest efficiency. An assump- tion, however, is involved in this reasoning, for it must be shown that the pigment really does retreat in dim light, as, in truth, it does in darkness. This essential point, however, has too often been ignored. Furthermore, if the distribution of pigment in twilight were essentially similar to that in bright light, it follows that ex- planations which attempt to solve the meaning of the position assumed by the pigment in light, only answer half of the ques- tions involved, for it may well be asked—Why should there be an extensive movement in total darkness, or why, since a permanently expanded condition would seem to be all that is required, any movement at all? These queries merely show that easily devised explanations, as for example those based upon the theory of optical isolation, may not touch the primary reason at all—such obvious relations may have a secondary importance or may even be purely fortuitous. Returning to the case under consideration, the movements of the retinal pigment of Planorbis, through the influence of light, are very limited in comparison with those in many animals. The dispersion in the compacted pigment mass, and the forma- tion of sparse granular processes, do not visibly alter the den- sity of the main pigmented zone. It is difficult to see (assuming that a proximal migration does occur in dim light as well as in darkness) how these changes would be of any great value to the animal in the ways in which the pigment commonly has been supposed to act. Even the vague assumptions of a nutritional relation of the pigment to adjacent sensory cells is discrepant with certain phases of the photo- and thermo-mechanical changes. In the body chromatophores of certain animals, e.g., lizards, the response of the pigment to light and temperature may be of use in regulating the body temperature of the animal. Thus (quoting from Parker, ’06, p. 411): ‘The dark color of the lizard’s skin in moderate illumination at a moderate temperature 384 LESLIE B. AREY insures, possibly, among other things, a certain degree of warmth which would be superfluous, if not dangerous, at a higher tem- perature, and in consequence the skin becomes light-colored in hot sunlight.” The significance of temperature responses of the retinal pig- ment of Planorbis is even more obscure than the meaning of photic influences. It is easy to construct several groundless hypotheses to explain why low temperature, acting similar to light, and high temperature, acting similar to darkness, could be of advantage to the snail in the regulation of its retinal econ- omy. Such interpretations, however, possess even less value than the speculations concerning the role played by light. To devise an explanation from the adaptional standpoint that will account for the temperature responses occurring 1n total darkness would seem a hard task. It may be, however, that the cell thus responds to temperature stimulation because the change is of use in the light-adapted eye only, whereas in darkness the similar response to similar stimulation is gone through with perfunctorily, as it were, even though it is of no use to the organism. The cautiousness of the following position, with the formula- tion of which this discussion will be closed, may be its only com- mendation; yet when our absolute ignorance of fundamental facts is considered, caution may well qualify as a cardinal virtue. An analysis of the conditions of pigment migration in the various vertebrate classes has led the writer (Arey, 715; 716) to an iden- tical conclusion for these animals as well. Recognizing, therefore, that the movements of the retinal pigment of Planorbis, as well as of other animals, to light and to temperature may have an adaptive significance, and, further- more, realizing that (with the possible exception of arthropods) - we at present are quite unaware of the meaning of these move- ments, it would seem that we are permitted to indulge only in interpretations formulated in terms of protoplasmic re- sponses to definite stimulating agents and that the questions of utility thereby involved must await the establishment of a more thorough knowledge of the co-existing factors. RETINAL PIGMENT OF PLANORBIS 385 SUMMARY 1. Light causes the retinal pigment of normal Planorbis to migrate distally (toward the source of illumination). Dark- ness causes a proximal migration. 2. About 4 hours is necessary for the pigment to assume the distribution characteristic of light-adaption, whereas about 5 hours is demanded in accomplishing the reverse process of dark-adaption. 3. The pigment of dark-adapted, excised eyes exhibits mi- gratory movements when exposed to light, and is, therefore, capable of direct stimulation. In the converse treatment (light to dark) of excised eyes, on the contrary, no change in the posi- tion of the pigment occurs. It is possible that the absence of positional change in darkness is indicative of an anaesthetic effect, produced by the unremoved products of catabolism, the more vigorous influence of light, however, being able to over- come this inhibitory tendency. 4. High temperature (30°C.) induces proximal migration of the normal retinal pigment both in darkness and in light. Low temperature (3°C.) induces distal migration both in darkness and in light. 5. Upon light-adapted excised eyes, low temperature likewise favors proximal migration and high temperature favors distal pigment migration. 6. Planorbis affords the first case among gasteropods in which the occurrence of positional changes in the retinal pigment has been correlated with the presence and absence of light. For the first time among molluscs, a thermo-mechanical in- fluence upon the retinal pigment has been demonstrated. 7. It is probable that low concentrations of anaesthetics, such as carbon dioxide or ether, are capable of completely arresting migratory movements of the Planorbis retinal pigment. This suggests that carbon dioxide, as a product of catabolism, may be the inhibiting factor that prevents migratory movements of the pigment when excised eyes are brought from light into darkness. 386 LESLIE B. AREY 8. The adaptional significance of the existence of mobile pigment in the retina of Planorbis is at present quite obscure. These movements can only be correlated with the presence of known environmental conditions, and interpreted in terms of protoplasmic responses to definite stimulating agents. Anatomical Laboratory, Northwestern University Medical School December 6, 1915 BIBLIOGRAPHY (Papers marked with an asterisk have not been accessible in the original Arey, L. B. 1915 The occurrence and the significance of photomechanical changes in the vertebrate retina—an historical survey. Jour. Comp. Neur., vol. 25, no. 6, pp. 535-554. 19162 The movements in the visual cells and retinal pigment of the lower vertebrates. Jour. Comp. Neur., vol. 26, no. 2, pp. 121-201. 1916 The function of the efferent fibers in the optic nerve of fishes. Jour. Comp. Neur., vol. 26, no. 3, pp. 213-245. Basucuin, A. 1864 Vergleichend histologische Studien. Wirzburger Natur- wiss. Zeits., Bd. 5, pp. 125-143. 1865 Ueber den Bau der Netzhaut einiger Lungenschnecken. Sitz- ungsb. Akad. Wiss. zu Wien., math.-naturw. K1., Bd. 52, Abth. 1, pp. 16-27. Bou, F. 1878 Zusatz zur Mitteil. vom 11. Januar, mitgeteilt in der Sitzung vom 19. Februar. Monatsber. preuss. Akad. Wissensch. zu Berlin aus dem Jahre 1877, pp. 72-74. Cuun, C. 1903 Ueber Leuchtorgane und Augen von Tiefsee-Cephalopoden. Verhandl. deutsch. Zool. Gesell., Bd. 13, pp. 67-91. Conepon, BE. D. 1907 The effect of temperature on the migration of retinal pigment in decapod crustaceans. Jour. Exp. Zodl., vol. 4, no. 4, pp. 5389-548. Exner, 8S. 1889 Durch Licht bedingte Verschiebungen des Pigmentes im In- sectenauge und deren physiologische Bedeutung. Sitzungsb. Akad. Wiss. zu Wien., math.-naturw., Kl., Bd. 98, Abth, 3, pp. 143-151. 1891 Die Physiologie der facettirten Augen von Krebsen und In- secten. Deuticke, Leipzig u. Wien., vi+ 206 pp. GRADENIGRO, G. 1885 Ueber den Einfluss des Lichtes und der Warme auf die Retina des Frosches. Allg. Wiener med. Zeitung, Bd. 30, No. 29 u. 30, pp. 348-344, u. 353. *HamBurGER, D. J. 1889 De Doorsnijding van den nervus opticus bij Kik- vorschen, in verband met de Beweging van Pigment en Kegels in het Netolies, onder den Invloed van Licht en Duister. Onderzoekingen d. Utrechtsche Hoogeschol, Reeks 3, Deel 11, pp. 58-67. Harurss, E. 1854 Ueber die Chromatophoren des Frosches. Zeitsch. f. wissensch. Zool., Bd. 5, Heft 4, pp. 372-879. RETINAL PIGMENT OF PLANORBIS 387 Hencuman, A. P. 1897 The eyes of Limax maximus. Science, n.s., vol. 5, no. 115, pp. 428-429. Hensen, V. 1865 Ueber das Auge einiger Cephalopoden. Zeitsch. f. wiss. Zool., Bd. 15, Heft 2, pp. 155-242. Hertet, E. 1907 Einiges iiber Bedeutung des Pigmentes fiir die physiologische Wirkung der Licht-strahlen. Zeitsch. f. allg. Physiol., Bd. 6, Heft 1, pp. 44-70. Herzoc, H. 1905 Experimentelle Untersuchung zur Physiologie der Beweg- ungsvorginge in der Netzhaut. Arch. f. Anat. u. Physiol., Physiol. Abth., Jahrg. 1905, Heft 5 u. 6, pp. 413-464. Hess, C. 1905 Beitrige zur Physiologie und Anatomie des Cephalopodauges. Arch. f. ges. Physiol., Bd. 109, Heft 9 u. 10, pp. 393-439. Kitune, W. 1877 Ueber den Sehpurpur. Untersuch. Physiol. Inst. Univ. Heidelberg, Bd. 1, pp. 15-104. *Lopato, G. 1895 Ricerche sulla fisiologia dello strato neuroepitheliale della retina. Arch. di Ottalmologia, vol. 3, pp. 141-148. Ovio,G. 1895 Diun speciale azione della cocain sulla funzione visiva. Annali di Ottalmologia, Anno 24, Suppl. al Fasc. 4, p. 23. Parker, G. H. 1897 Photomechanical changes in the retinal pigment cells of Palaemonetes, and their relation to the central nervous system. Bull. Mus. Comp. Zoél., Harvard Coll., vol. 30, no. 6, pp. 273-300. 1899 The photomechanical changes in the retinal pigment of Gam- marus. Bull. Mus. Comp. Zodl., Harvard Coll., vol. 35, no. 6, pp. 141-148. 1906 The influence of light and heat on the movement of the melan- ophore pigment, especially in lizards. Jour. Exp. Zoél., vol. 3, no. 3, pp. 401-414. Rawitz, B. 1891 Zur Physiologie der Cephalopodenretina. Arch. f. Anat. u. Physiol., Physiol. Abt., Jahrgang 1891, Heft 5 u. 6, pp. 367-372. Smitn, G. 1906 The eyes of certain pulmonate gasteropods, with special reference to the neurofibrillae of Limax maximus. Bull. Mus. Comp. Zool., Harvard Coll., vol. 48, no. 3, pp. 233-283. Spartu, R. A. 1913° The mechanism of the contraction in the melanophores of fishes. Anat. Anz., Bd. 44, No. 20-21, pp. 520-524. 1913 The physiology of the chromotaphores of fishes. Jour. Exp. Zool., vol. 15, no. 4, pp. 527-585. Sremnacu, E. 1891 Ueber den Farbenwechsel bei niederen Wirbelthieren, bedingt durch direkte Wirkung des Lichtes auf die Pigmentzellen. Centralbl. f. Physiol., Bd. 5, No. 12, pp. 326-330. Szczawinska, W. 1891 Contribution A l'étude des yeux de quelques Crustacés et recherches expérimentales sur les mouvements du pigment granu- leux et des cellules pigmentaires sous l’influence de la lumiére et l’ob- scurite dans les yeux des Crustacés et des Arachnides. Arch. de Biol., Tome 10, pp. 523-566. 38S LESLIE B. AREY PLATE 1 EXPLANATION OF FIGURES eps.opt., optic capsule rtn.ex., peripheral zone of retina pig., pigment rtn.i., central zone of retina The figures of this plate, taken from axial sections of the Planorbis eye, are photographs at a uniform magnification of 540 diameters and show the distribu- tion of retinal pigment at various temperatures in light and in darkness. 1 At 3°C. in the light. 2 At room temperature (21°C. +=) in the light. 3 At 30°C. in the light. 4 At 30°C. in the dark. At room temperature (21°C. +) in the dark. At 32°C. in the dark. mS or PLATE 1 RETINAL PIGMENT OF PLANORBIS LESLIE B. AREY Light Dark 10) gears pig. -pig. , cps.opt. rtn.ex.----- 3°C. | 4 pig. pig.- rtn ex rin.ex. cps.opt ca2rC pig pig rtn.ex rin.ex cps.opt ca.3I°C. L. B. A. Photo 389 ABSENCE OF CHROMATOLYTIC CHANGE IN THE CENTRAL NERVOUS SYSTEM OF THE WOODCHUCK (MARMOTA MONAX) DURING HIBERNATION A. T. RASMUSSEN AND J. A. MYERS From the Physiological Laboratory, Medical College, Cornell University, Ithaca, New York SIX FIGURES (TWO PLATES) INTRODUCTION Since the discovery by Nissl in 1885 that the chromophilous granules in the cytoplasm of nerve cells—granules which had already been described by Flemming (’82) and which von Len- hossék later termed tigroides, though now more generally spoken of as Nissl granules—are intensely stained by basic aniline dyes, especially by methylene blue, the disposition of these granules, under numerous physiological and pathological conditions, has been the subject of much study. From the extensive functional alterations that numerous authors (Valentin, Dubois, Merz- bacher, ete.) have reported to occur in the nervous system during hibernation, and from the morphological variations which are said to take place in the nerve cells in certain other conditions, such as sleep and starvation, one would naturally expect to find marked changes, especially in the Nissl granules, during hibernation. This state, as is well known, is attended in some animals by almost continuous sleep and profound torpor for four months and even longer, during which time no food whatever may have been eaten and the body temperature has been reduced to but a few degrees above the freezing point. HISTORICAL An examination of the literature revealed the fact that some observers have reported marked structural changes in the nerve cells during hibernation. Levi (’98) in the toad (Bufo vulgaris) 391 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4 392 A. T. RASMUSSEN AND J. A. MYERS found that the Nissl bodies had greatly diminished in size and the acidophil granules had become basophilic during winter- sleep; but in hibernating mammals no changes were observed. A more detailed study by Legge (99), however, led this author to conclude that the cells of the cerebro-spinal axis of hibernating bats do undergo visible changes. In the cells of the cerebral cortex he found the Nissl granules to have elongated, being more fusiform in shape, and to be displaced towards the periphery, forming a sort of envelope to the cytoplasm. Baronecini and Beretta (00) also reported morphological changes in the spinal cord and especially in the cerebral cortex of mammals (mus- cardin and bat) during winter-sleep. They found that the Nissl granules had greatly decreased and stained more diffusely. The chromatic substance of the nucleus was also more diffuse than in active specimens and the nucleolus seemed to have disappeared in many cases. Essentially the same changes in the Niss! granules in the cells of the cerebro-spinal axis and in the Purkinje cells of the cerebellum of hibernating hedgehogs, were reported by Marinesco (’05) who found a distinct decrease in these granules during the torpid state. Those that remained were reduced to fine granules or were diffused in the cytoplasm. A more recent study by Zalla (10) agrees with the older results obtained by Levi in regard to mammals. Zalla found no ap- preciable morphological difference in the Nissl substance in the dormouse (Myoxus glis) during hibernation as compared with the active state. His results in amphibia were not constant, but in reptiles he found a distinct decrease in this substance in the motor cells of the cord and pons during winter-sleep. The question of changes in the chromophilous substance of the nerve cells during hibernation in mammals thus seems to be unsettled. Legge, Baroncini and Beretta, and Marinesco apparently have observed marked changes, while Levi and Zalla could not establish any such chromatolysis. However, these authors did not all work on the same species of mammal. In amphibia there is also some disagreement. Levi found a dis- tinct decrease in the Nissl granules during hibernation, while Zalla, whose results were not very constant, believes that there CHROMATOLYTIC CHANGES DURING HIBERNATION 393 are no changes. In reptiles, however, Zalla did find a marked decrease during torpor. Some other morphological changes in the nerve cells during hibernation may be briefly mentioned. Querton (’98) by means of the Golgi method on cerebral neurones found that the pro- toplasmic prolongations retract and assume a moniliform ap- pearance during the dormant period. This, however, could not be substantiated by Baroncini and Beretta (’00).. Some work has been done on the neurofibrillae. Tello (’03) reported that in the motor cells of the spinal cord of lizards the neurofibrillae are much thicker than usually found. Tello and Cajal (’04) found that this was true only during the cold season when the animal is dormant, and that when the animal is warmed up and made active the fibrillae become more numerous and much finer. Cajal thus believes that the hypertrophy of the neuro- fibrillae is due to the cold and resulting diminished spinal reflexes, because exposure of the animal to a low temperature brought about this gigntism of the neurofibrillae while warming it up caused a return to the normal, and because this change is not seen in the telencephalon and mesencephalon whose cells retain their activity to a much greater extent during the lethargy. Marineseco (’05) has repeated these experiments on young cats and dogs and Dustin (’06) has done the same on young rabbits with essentially similar results. A temperature below 10°C., however, was found to be less effective than 10°C. in bringing about these changes in the neurofibrillae, according to Marinesco. This may be related to the fact that a temperature too low excites hibernating mammals and finally wakes them up since the body temperature rises as a result of increased activity. The latter author found no such modification of the fibrillae in hibernating hedgehogs, which fact he interprets as indicating that the activity of the nervous system of hibernating mammals is not reduced to the extent that it is in the lizard and other cold blooded animals. Zalla (10), on the other hand, found that the neurofibrillae were fewer and farther apart in the dormouse during hibernation, 394 A. T. RASMUSSEN AND J. A. MYERS PRESENT INVESTIGATION In view of the conflicting reports as just reviewed and the more recent observations by Crile on changes in the brain cells under various emotional and other conditions, much less strik- ing than the phenomenon of hibernation, the work we have done on the woodechuck in this regard, seems worthy of a brief note. Woodchucks, or ground hogs, which represent the American marmots, are some of the best examples of hibernating mammals in this country. All species remain dormant for four to six months each year, and hence constitute good material for a study of hibernation. This work was commenced early in January 1913 by J. A. Myers, who fixed and imbedded the central nervous system of six woodchucks, four of which were killed at various intervals while hibernating (January 18, Febru- ary 6, March 15 and April). One was killed shortly after waking up (Mareh 15) and another, during the following summer. In addition to studying the above series by means of the Nissl stain, the other co-author prepared another series consist- ing of the brain and spinal cord of fifteen woodchucks killed during the autumn, winter and spring of 1913-1914. This series includes one animal killed about a month before hibernation (October 25), one just before hibernation (November 22), five during hibernation (February and March)—one of these was partly awake when killed on February 16, but was sluggish and had a rectal temperature of 19°C.—one within two days after waking up (March 16) and seven others which had been awake from three days to more than a month. Three of this last group had been fed for one, two and three weeks respectively. These animals were kept in the artificial burrows which were designed by Professor Simpson of this laboratory and which have already been described elsewhere.! The rectal temper- ature of the dormant animals varied from 8°C. to 12°C., whereas, the temperature of the active animals ranged from 32°C. to 30 ©. 1 Rasmussen, A. T., Amer, Jour. Physiol., 1915, vol. 39, p. 20. CHROMATOLYTIC CHANGES DURING HIBERNATION 395 All the animals of both series were killed quickly by trans- fixing the heart through the chest wall. No anaesthetic was used, except in five cases where only sufficient ether was given to keep the animal quiet. These five animals were all killed after hibernation while awake and active. The amount of ether given did not seem to have any noticeable effect on the Nissl granules. If, however, these five cases are excluded from con- sideration because of the introduction of this additional factor, the two series involve as strictly comparable cases two before hibernation, nine during hibernation and five after hibernation. The blood was washed out immediately after death with normal saline solution by injection through the aorta. The saline was followed by a saturated aqueous solution of bichloride of mer- cury to which had been added 10 per cent of formalin. Thus the central nervous system was fixed very quickly in situ. The whole brain and cord were then removed and cut into transverse sections a few millimeters thick. The desired levels were further fixed in a saturated aqueous solution of bichloride of mercury for 48 hours, washed in running water 36 hours, and dehydrated in graded alcohols containing iodine in the usual manner. The tissue was cleared in xylol and imbedded in paraffin melting at 54°C. The levels thus imbedded were: olfactory bulb, motor cortex, mid-thalamus, midbrain at the level of the superior colliculus, mid-cerebellum and pons, medulla oblongata at ex- treme inferior border of fourth ventricle, first cervical, sixth cervical, sixth thoracic, second lumbar and lower lumbar seg- ments of the spinal cord. Sections were cut five microns in thickness, except in the case of the spinal cord where the sections were six microns thick, and stained on the slide for ten minutes in a large quantity of hot (70°C.) solution of 1 per cent methylene blue in water saturated with aniline oil. The excess stain was washed off rapidly in water and decolorization carried on by transferring the slides directly to 95 per cent alcohol for two to ten minutes. Dehy- dration was completed in absolute alcohol and clearing in caju- put oil followed by xylol. The sections were mounted perma- nently in Canada balsam dissolved in xylol. Where the size 396 A. T. RASMUSSEN AND J. A. MYERS permitted, corresponding sections from all animals of a series were fixed on the same slide to insure equal staining. In other vases several sections from the same block were placed on one slide and all the corresponding slides stained together by carry- ing them through the reagents by means of a basket, or rack, which would contain the entire lot. RESULTS The nerve cells of the woodchuck are essentially typical, containing the usual large round nucleus, with one nucleolus. The chromophilous substance in the cytoplasm has the usual appearance and arrangement so well known that no description will be necessary here. In spite of all precautions there are notice- able variations in the size, distinctness and arrangement of the Nissl bodies in homologous cells of animals in the same state and even in the cells of the same group in a particular section. These variations are found in both dormant and active animals. We can detect no modification in the Nissl granules characteristic of the hibernating as compared with the non-hibernating state. Certainly in these woodchucks there is not the difference indi- cated by the figures given by Marinesco in the case of the hedge- hog. The chromophilous substance is present during hibernation in at least as great a quantity as at other times and presents the usual appearance when stained with methylene blue. The arrangement of the granules varies somewhat even in cells of the same group, being more abundant in the periphery of the cells in some cases and in others being grouped more densely around the nucleus. The size and shape of the bodies vary from fine irregular granules to larger elongated ones; but when a large number of cells are examined the extreme variations may be found in animals in the same state and often in the same section. A predominance of a particular variation in either state can not be established. The accompanying figures and explanations will suffice to indicate the general cell picture before, during and after hibernation. The larger types of nerve cells were selected as illustrations because in them the Nissl bodies are more distinct and make better photographs. CHROMATOLYTIC CHANGES DURING HIBERNATION 397 We wish to thank Prof. Sutherland Simpson for his helpful guidance in this research. We are also indebted to Prof. B. F. Kingsbury for assistance in taking the photomicrographs.* LITERATURE CITED BaRoncini, L., AND Beretra, A. 1900 Ricerche istologiche sulle modif. degli organi nei mammiferi ibernanti. Riforma med., vol. 16, p. 206. CasaAL, RamM6N y 1904 Variaciones morphologicas, normales et pathologicas del reticulo neurofibrillar. Trabajos del lab. de invest. biolog. de la Univ. de Madrid, vol. 3, p. 9. Compt. Rend. Soe. Biol., vol. 56, p. 372. Dustin, A. P. 1906 Contribution 4 l’étude de l’influence de l’Age et de |’ activ- ité fonctionnelle sur le neurone. Ann. Soe. Roy. d. Se. méd. et natur. de Bruxelles, vol. 15. Lecce, F. 1899 Sulle variazioni della fina struttura che presantano durante V’hibernazione le cellule cerebrali dei pipistrelli. Monit. zool., vol. 10, p. 152. Levi, G. 1898 Sulle modificazioni morfologiche delle cellule nervose di animali a sangue freddo durante l’hibernazione. Riv. d. patol. nervos. e. med., p. 443. Marinesco, G. 1905 La sensibilité de la cellule nerveuse aux variations de température. Revue Neurolog., No. 14 (July 30, 1905), p. 784. Re- cherches sur les changements de structure que les variations de tem- p¢érature impriment 4 lacellulenerveuse. Revista Stiintelor Medicale, No. 3, Bucarest. 1906 Recherches sur les changements des neurofibrilles consécutifs aux différents troubles de nutrition. Le Névraxe, vol. 8, p. 149. QuERTON 1898 Lesommeil hibernal et les modifications des neurones cérébraux. Ann. Roy. d. Se. med. et natur. de Bruxelles, vol. 7, p. 147. TrELLo 1903 Sombre la existencia de neurofibrillas gigantes en la medula espinal de los reptiles. Trabajos del lab. de invest. biolog. de la Univ. de Madrid, vol. 2, p. 223. Zauua, M. 1910 Recherches expérimentales sur les modifications morpholo- giques des cellules nerveuses chez les animaux hibernants. (Résumé). Arch. ital. de biol., vol. 54, p. 116. Riv. d. patol. nervos. e. ment., ann. 15, p. 211. 2In regard to the general problem of the correlation of structural changes to activity in nerve cells, attention should be called to the experiments carried out on six different species of animals by R. A. Kocher, reported in this journal (vol. 26, No. 3, p. 341) since this article was submitted for publication. This author could find no correspondence between the size or structural character- istics of nerve cells and various grades of activity. PLATE 1 EXPLANATION OF FIGURES The three photographs in this plate were taken from the nucleus hypoglossus. The sections from the various animals were mounted on the same slide and hence stained together. > 320. 1 Before hibernation. Woodchuck killed October 25, 1918, without any anaesthetic. Animal active. Rectal temperature 37.6°C. 2 During hibernation. Woodchuck killed March 7, 1914, without any anaes- thetic. Animal very dormant and had been so nearly all the time for at least three months. Rectal temperature 9°C. 3 After hibernation. Woodchuck killed April 11, 1914, without any anaes- thetic. Animal active. Had been fed for two weeks. Rectal temperature 37°C. 398 CHROMATOLYTIC CHANGES DURING HIBERNATION PLATE 1 A. T. RASMUSSEN AND J. A. MYERS se 7 | yp , s a > > ell a . 2 ss ... > ’ * ‘ : ps . 1 CP. e . - => < ’ sd i ' M v. i i og \ at ’ Y ; ae 2 \ : ti 399 RGAE, 22 EXPLANATION OF FIGURES Photographs of motor cells from the antero-lateral group in the sixth cervical segment of the spinal cord. The sections were mounted on the same slide and hence stained together. > 320. 4 Before hibernation. Same animal as in figure 1. 5 During hibernation. Same animal as in figure 2. 6 After hibernation. Same animal as in figure 3. 400 CHROMATOLYTIC CHANGES DURING HIBERNATION PLATE 2 A. T. RASMUSSEN AND J. A. MYERS 401 A STUDY OF A PLAINS INDIAN BRAIN J. J. KEEGAN From the Anatomical Department, University of Nebraska, Omaha, Nebraska EIGHT FIGURES INTRODUCTION The primary object of this paper is to furnish an account of the morphology of the cerebral hemispheres of a North American Indian brain, thereby filling a gap in the literature of racial cerebral anatomy. Although a large amount of work has been done upon the Indian tribes from an anthropological stand- point, no description of an Indian brain has, to my knowledge, ever been given. The study of cerebral morphology has not yet furnished characters distinctive of race, but the studies of Elliot Smith (04), Duekworth (’07), and others have revived an interest in this work and advanced greatly the interpretation of sulei and gyri. The secondary but more important aim of this study,from which the conclusions are largely deduced, is the application of the principles of fissure relation to cortical areas, both in the in- terpretation of sulci and in the comparison of cortical areas and sulci of the two hemispheres and of other cerebra. This is especially valuable in this case on account of the completeness of the history and the exceptional mental abilities evidenced. The study of cortical localization has been conducted by two methods, cortical stimulation and histological differentiation. The most important names connected with the former method are those of Sherrington and Griinbaum (’01) in their experi- mental work upon the ape brain, supplemented by a few similar observations upon the human cortex and numerous clinical ob- servations proved by operation or autopsy. This work neces- sarily gave results in only a few regions of the cortex, mainly 403 404 J. J. KEEGAN the somatic motor and sensory areas of the central region. The histological field was first entered by Flechsig (’96) who de- limited cortical areas by the order of the myelination of their fibers. Campbell (05) and Brodmann (’07) made purely his- tological surveys of the cortex, but were not able by this tedious method to examine enough material to establish the relation of many cortical areas to fissures. KE. Smith (07) was the first to make such a topographical survey of the human cortex and, while in general the areas plotted agreed with the areas of Campbell and Brodmann, the added value of their interpretation in relation to fissures facilitated greatly the study of the fissures of the cerebrum. The value of E. Smith’s interpretation is evidenced by its general acceptance for the occipital region by most of the recent writers, as Duckworth (07), Appleton (10), Cole (11), Schuster (08), and many others. The application of this method to other regions, however, has been neglected, chiefly on account of the difficulty in obtaining fresh material and the inexpertness in distinguishing the lamination in macroscopic sections. The boundaries of the area striata are so easily identified that this area can be plotted even in imperfectly preserved material, which accounts for the general application in this region. In this study an attempt has been made to apply by compari- son the cortical area plan of E. Smith (14) to a valuable cerebrum in which the preservation prevented a knife-section analysis of the cortical areas and their relation to sulci. The accuracy of this method may well be questioned for undoubtedly many small errors are present, but it was found that such a plotting aided greatly in the interpretation of fissures and perhaps made possible a more intelligent comparison of similar cortical areas of the two hemispheres and of other cerebra. It at least furnishes the most expressive figures of the sulci and gyri that present knowledge makes possible and might be applied profitably for comparison to the figures accompanying studies of other brains, AN AMERICAN INDIAN BRAIN 405 HISTORY The brain which is the subject of this account was obtained at autopsy from the body of a female Indian of the Omaha tribe, and thanks are due Dr. A. A. Johnson of the Department of Pathology for the opportunity of the use of the specimen for study. The individual was fifty years of age, about 125 pounds in weight and possessed rather typical Indian features although not extreme. There was united in her person the blood of the Indian, the French, and the English settlers of Nebraska, her grandfathers carrying the half French and the half English admixture and the grandmothers of pure Indian descent. It is thus seen that the Indian blood was carried down entirely on the female side of the family, which might give rise to specula- tion upon the formerly much discussed question of the relation of sex to cerebral morphology. The intellectual status was far above the average as super- ficially judged of Indian people, and above the average of the white race as judged from the high quality of her attainments. The educational training consisted of mission school and gov- ernment Indian school as a child, five years of general education in preparatory schools, and a three year medical course which she completed in two years, graduating at the head of her class. A year’s hospital training completed the medical work. Her life was spent in medical service with her own people. She was generally recognized by the profession and laymen alike as ¢ woman of very superior personality whose intellectual endow- ments and qualities of character placed her’ quite above the ordinary level of humanity. DESCRIPTION The brain, after standing several weeks in a formaldehyde solution, weighed 1353 grams with the dura mater removed. A comparison of the weights of the two hemispheres showed the right considerably heavier than the left, 605 grams and 575 grams, 406 J. J. KEEGAN respectively, but these figures are unreliable on account of the pathological condition of the left hemisphere, which had de- stroyed a part of the temporal lobe. This hemisphere also was distorted in preservation, due to the softened condition of the cerebral tissue, consequently no measurements of any value of lengths or indices could be made. Such measurements were established by Cunningham (’92) but have not proven of com- parative value even in perfectly preserved brains. The general type of fissuration presented nothing very unusual, giving the impression of a fairly well and evenly fissured cere- brum. There was no undue prominence of any region, perhaps a greater tendency to a vertical course of the sulci of the central region of the right hemisphere and a tendency to irregular fissuration in the posterior parietal region of the left hemisphere. There was no indication of an exposure of the insula. The following brief account of the sulci and gyri is not intended as a description of all points observed, for many of these of no recognized significance or variation can be determined equally as well by an examination of the accompanying figures. These illustrations were made by tracing from photographs and subse- quently inking in the fissures by careful examination of course, depth and bridging gyri. The depth of the more important fissures is entered in the drawing. In general the heavier lines indicate the deeper fissures. A bridging gyrus is indicated by an interruption of the line and an intervening dot. The cortical areas are filled in with different symbols, the plan being to place no two of similar type adjoining. These areas were determined by placing those first in which there could be little doubt of their boundaries. The area striata was delimited by macroscopic examination of knife sections. The remaining areas generally could be filled in by a process of elimi- nation, but in some cases they were determined by arbitrary judg- ment. This latter is very evident in cases where the boundary does not correspond to a fissure, but it is surprising how little is left to the judgment and how easily the area conformation can be made to agree with the plan of E. Smith without violating the rule of the deeper fissures being in the main bounding fis- AN AMERICAN INDIAN BRAIN 407 sures. With this scheme it is hardly necessary to label the dif- ferent fissures, for a comparison with E. Smith’s figures in Cun- ningham’s Text-book of Anatomy will show the interpretation. The fissura cerebri lateralis is sharply upturned and _ bifur- cated in the left hemisphere at its posterior extremity, but hori- zontal and single in the right. Since this bifurcation is caused by an overgrowth of the posterior inferior parietal region, as has been determined in the Negro brain by Poynter and Keegan (15), the two hemispheres are peculiarly contrasted in their centers of greatest growth in the inferior parietal lobule, the left being in the posterior region and the right in the anterior region. This is further evidenced in the conformation of other sulci in these areas, and will be discussed at a later point. In both hemispheres there is a distinct operculation by the superior lip in the region of the area subcentralis. The anterior rami of the fissure are completely separate on both sides, enclosing a prominent pars triangularis. The sulcus centralis is unusual in its upper third in the left hemisphere. It lacks several millimeters of reaching the mesial border and in the superior third is displaced anteriorly in an arcuate form, apparently due to an overgrowth of the gyrus centralis posterior opposite the motor area of the lower limb. According to Cole (’10), interruption of the suleus by a sub- merged or bridging gyrus occurs at the posterior arch of the superior genu in poorly convoluted brains and just above in well developed brains. This brain shows the gyrus exactly at the apex of the superior genu in both hemispheres, narrower and more prominent in the left. An examination of a large number of Negro and Caucasian brains failed to corroborate Cole’s conclusion. The gyrus is always at the same point, varying only in depth and width. The maximum depth of the sulcus cen- tralis is about the same as the sulcus interparietalis. The greater depth of the latter is stated by Appleton (710) to be a distinctive feature of Australian and simian brains. It is a question whether this would not simply indicate an overgrowth of the parietal lobe in a human brain and not an undergrowth of any region. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4 408 J. J. KEEGAN The sulei precentrales are typical of Cunningham’s (’92) de- scription in the right hemisphere, but in the left hemisphere the superficial interrupting gyrus occurs lower, apparently between the two rami of the sulcus precentralis inferior. The sulcus diagonalis is incorporated in the ramus verticalis in both hemi- spheres, thus reducing the posterior area of the gyrus frontalis inferior to a small area in the floor of the sulcus precentralis inferior. The factor which would account for this might be an overgrowth of the motor area of the mouth region in the gyrus precentralis inferior or of the motor speech center in the gyrus frontalis inferior. Cole (10) has attempted to relate the sub- mergence of this area or gyrus to a simian condition and quotes Kohlbriigge and Bolk as supporting his contention that the sulcus diagonalis represents a detached portion of the simian sulcus precentralis inferior. The sulcus frontalis superior is well defined in both hemispheres and superficially continuous from the s. precentralis superior to the s. fronto-marginalis, maintaining about an even distance from the mesial border throughout. A prominent bridging gyrus near the posterior extremity permits the interpretation of the continuity of the posterior middle frontal region with the ante- rior superior frontal region as illustrated in E. Smith’s chart. Appleton (10) considers the tendency towards extra segmenta- tion of the s. frontalis superior as a lowly character. The sulcus fronto-marginalis is better defined in the left hemi- sphere than in the right but is too irregular in both to permit of much comparison. The sulcus frontalis inferior is a high arcuate fissure on both sides in full communication with the s. precentralis inferior. Its length and its distance from the fissura cerebri lateralis are each about a centimeter greater in the right hemisphere. This gives rise to a larger gyrus frontalis inferior on the right side which would be contrary to expectation if the speech center had any relation to the size of this gyrus. The sulcus frontalis medius is a very subordinate system of shallow sulci, according to the interpretation of the s. frontalis superior. The greater width of the gyrus frontalis medius of AN AMERICAN INDIAN BRAIN 409 the left hemisphere does not seem to increase the regularity of these elements. The greater tendency is towards a transverse direction. The sulcus frontalis mesialis is seen as an irregular system of alternating transverse and longitudinal elements extending to the frontal pole. The more posterior of these were chosen arbi- trarily as a guide between the superior frontal area and the anterior frontal area. The orbital surface has the usual type of fissuration, the only unusual feature being the extension of the mesial limb of the s. orbitalis transversus into the fossa Sylvii of both hemispheres. The sulcus cinguli corresponding to E. Smith’s chart is very poorly developed in its anterior two-thirds, the more prominent sulcus being the s. paracinguli. This might indicate a disap- pearance of the s. cinguli due to an increased growth of the mesial region, but was called to attention by Cole (10) in a micro- cephalic brain in which he suggested that the supracingulate suleus was the older. The sulcus postcentralis is a very prominent fissure in both hemispheres, extending from the mesial border to within a few millimeters of the fissura cerebri lateralis. In the right hemi- sphere there is no communication with the s. interparietalis, thus giving rise to a fissure very similar to the s. centralis. In the left hemisphere the posterior reflection of the two extremi- ties forms an arcuate sulcus and a wide gyrus centralis posterior in the corresponding regions. The sulcus interparietalis is contrasted on the two sides. In the right hemisphere it appears as a fourth vertical sulcus in series with the precentral, central and postcentral sulci. It communi- cates across an almost superficial bridging gyrus with the s. paroccipitalis. The tendency in the left hemisphere is towards a sagittal and more posterior arrangement. The sulcus is repre- sented by two horizontal elements in intercommunication with the s. postcentralis and the body of the s. paroccipitalis across bridging gyri. The suleus paroccipitalis is more lateral and more prominent in the right hemisphere. The independence of this sulcus is 410 J. J. KEEGAN well represented in both hemispheres. Appleton (’10) has classi- fied this as a fetal tendency. The narrow antero-posterior width of the gyrus arcuatus posterior is a noticeable feature and might be indicative of an unusual growth in the superior parietal area. The suleus parietalis superior is poorly represented, perhaps due to its partial incorporation in surrounding sulci. The lobulus parietalis inferior gives evidence of a prominent growth. This is more noticeable in the left hemisphere where the prominent sulcus interparietalis extends into the anterior region as an accessory fissure. The s. angularis, which to some extent is an index to the growth of the inferior part of this lobule, is not independent from the s. temporalis superior as found in the majority of Negro brains. It appears as the sharply upturned extremity of this fissure, more anterior in the left hemisphere. The sulcus temporalis superior of the right hemisphere was injured by the tumor growth. The absence of the anterior trans- verse element may be associated with an increased growth of the acoustic area of the superior temporal gyrus. This was further evidenced by very prominent transverse temporal gyri of Heschl and the extension to the lateral surface of the fissure separating the two larger of these gyri. The sulcus temporalis medius and the sulcus temporalis inferior have never been interpreted well enough to permit mor- phological comparison. The ascending ramus of the former, suleus occipitalis anterior of some authors, is typical in the right hemisphere of an arcuate communication with the s. occipitalis inferior, classified by Appleton (10) as a simian character. The fissura rhinalis is present in both hemispheres as a shallow groove connecting the Sylvian fossa with the s. collateralis. The interpretation of these two fissures, applied as in the Negro brain, indicates an earlier or less developed condition in the right hemisphere but in neither is the simian or fetal type ap- proached, regardless of the greater depth than usual. The sulcus collateralis is superficially continuous to the occipi- tal region in both hemispheres. The posterior portion lies con- siderably more lateral in the right, which, as a limiting sulcus for the area peristriata, would indicate a greater extent of this AN AMERICAN INDIAN BRAIN All area on this side. The rhinal element of the sulcus is separated in both hemispheres by a bridging gyrus near the anterior extremity. The sulcus lunatus can be very plainly identified in the left hemisphere. It is 35 millimeters in length and lies nearer the lateral border about 3 centimeters from the occipital pole. It has an arcuate or rather angular form and a slightly operculated posterior lip. The area striata, delimited by knife sections, _ lacked about 2 millimeters of reaching this lip but followed the course of the fissure quite regularly. In the right hemisphere the tendency is towards a longitudinal disposition of the fissures in this region, to which the boundary of the area striata does not bear any definite relation. The most prominent sulcus is inter- preted as a modified s. prelunatus or s. occipitalis lateralis of some authors. Comparison of the extent of-the area striata in the two hemispheres shows very plainly a greater lateral extent in the left hemisphere. The sulcus occipitalis paramesialis lies more upon the lateral surface in the left hemisphere but is very prominent in both hemispheres. This lateral position indicates that it bears a relation to the lateral extension of the area striata. The sulcus occipitalis inferior courses on the tentorial surface along the lateral border. It is very similar on the two sides, a deep, slightly operculated fissure about 40 millimeters in length, communicating at the two extremities with the sulci of the corresponding regions. The sulcus calearinus in both hemispheres is separated from the fossa parieto-occipitalis by a small bridging gyrus cuneus and from the sulcus retrocalearinus by a larger bridging gyrus. The sulcus retrocalearinus terminates in both hemispheres in a deep operculated polar element but separated by a prominent bridging gyrus. Landau (’15) interprets this polar element (s. extremus, s. occipitalis triradiatus, s. calearinus externus of E. Smith, s. occipitalis polaris), as the true posterior bifurcation as described by Cunningham (’92), separated by the gyrus cuneo- lingualis posterior and homologous to the sulcus triradiatus of ape brains. The prominence of this suleus was noted by the writer (’15) in the Negro brain, in which it was described as an 412 J. J. KEEGAN independent suleus occipitalis polaris. If the interpretation of Landau is correct, its prominence would be an indication of a greater proportionate development of the area striata in the same manner as the sulcus lunatus. The fossa parieto-occipitalis is quite typical of E. Smith’s description in the left hemisphere. The gyrus intercuneatus is about 5 mm. in height and separates widely the s. paracalearinus and the s. limitans praecunel. It is traversed by an independent sulcus incisura which appears superficially on the lateral surface, . separate from the termination of the sulcus limitans praecunei in the gyrus arcuatus posterior. The s. paracalearinus under- mines the posterior wall of the fossa, becoming superficial along the border of the hemisphere posterior to the gyrus arcuatus. The fossa of the right hemisphere is less typical. The gyrus intercuneatus is faintly indicated, the deepest part of the incisura being formed by the sulcus paracalearinus. The s. limitans praecunei incises the lobulus praecuenus. The: sulci limitantes areae striatae are easily identified by the delimitation of the area striata. The insula, as far as could be determined, presented nothing unusual in its fissuration. The operculation was complete in both hemispheres. SUMMARY The detailed study of the fissures and convolutions brings a number of points to attention. First is noted the striking differ- ence between the two hemispheres in almost every fissure or region examined. This variation is so great that a comparison of hemispheres is of very little value in the interpretation of sulci, which method proved so valuable in the interpretation in the Negro brain. A similar fact of asymmetry is stated by Appleton (10) to represent. an agreement with European cere- bra in general and a contrast with supposedly lower types of cerebra. Many writers have called attention to the asymmetry of the cranium, brain, and intracranial venous sinuses sepa- rately, but comparatively little work has yet been done to study the co-relation of these asymmetrical conditions the one to: the other or to explain their origin. E. Smith (’07), in a note upon AN AMERICAN INDIAN BRAIN 413 the asymmetry of the brain and skull, contrasts the asymmetry of the cranium of the higher races of man with the symmetry of the apes and to a less degree of the black races. This lack of symmetry of the cranium in man is attributed to the unequal development of homologous parts of the two cerebral hemi- spheres, especially the great parietal and frontal association areas. The greater size of the right parietal association area is given as an explanation of the greater prominence of the correspond- ing parietal bone and a relative shifting backward of the right parietal boss, of the usually greater extent of the lateral part of the left visual cortex and the retention of a more pithecoid form in the left hemisphere. This asymmetry is suggestive of the predominance of functional areas upon one side or the other of the brain. This has been generally accepted in the predominance of the speech center upon the left side in the inferior frontal gyrus, Broca’s convolu- tion, and of the visual cortex in the more extensive area striata and more prominent sulcus lunatus in the left hemisphere of the majority of brains. Cunningham (’02) attributed right-handed- ness to a functional pre-eminence of the left hemisphere, al- though not supported by any constant observation of greater weight or greater prominence of the so-called motor arm center, of appreciable histological difference or of any regular plan of asymmetry. The asymmetry, although noticeable also in the lower animals, ‘‘never attains the same degree as in man.” * Fig. 1 Right hemisphere, lateral view Fig. 2 Left hemisphere, lateral view EGAN J. KE J. 418 oo So o=o ° C) X69, o%o 2264 Chreby 95° bis ° a 20 99 fe) ©\9 5 y Og (-) 0195/09 @° 6° eke) NG on WP OS 00 Wie) aod So\2 2090 |” 09 Ey 0% 6 09 19 99 ° iew 1 view © mesial v ht hemisphere, here 1g 3 Fig. 1a mes d isp Fig. 4 Left hem AN AMERICAN INDIAN BRAIN 419 Fig. 5 Both hemispheres, basal view Fig. 6 Both hemispheres, superior view SS 985 SS $tPerO? 90.99 +t aeeser ec? : ete GAN 4 - 4 KEI J. J. 420 ital view , occip heres Smith’s (’14) figure isp of E hem 7 Both 8 Copy 1g. ig. F F AN EXPERIMENTAL STUDY OF THE VAGUS NERVE MARTIN R. CHASE From the Anatomical Laboratory of the Northwestern University Medical School' FOUR FIGURES In a previous paper a study was made of the structure of the roots, trunk and branches of the vagus nerve (Chase and Ran- son 714). It was shown, in conformity with the work of Molhant (’10) and Van Gehuchten and Molhant (’11) that the vagus nerve in the dog contains myelinated fibers which can be classified as large, medium and small. In addition there were found enor- mous.numbers of unmyelinated fibers. These are present in certain of the vagus rootlets, and throughout the trunk of the vagus. In the cervical and thoracic trunk they constantly increase in proportion to the myelinated fibers, so that at the level of the diaphragm the vagus is almost a pure unmyelinated nerve. The most of the large myelinated fibers, and many of medium size, are given off in the cervical branches of the vagus. The bronchial and esophageal rami receive nearly all of the remain- ing myelinated fibers. Relatively few unmyelinated fibers are found in the cervical branches. The increased proportion of unmyelinated fibers in the lower vagus is due in part at least to the withdrawal from the trunk of the myelinated fibers by the upper branches. Essentially the same histological picture was seen in sections from the vagus of man (Ranson ’14), the rat and the rabbit. Gaskell ’86 observed unmyelinated fibers in the vagus nerve, and found the lower thoracic vagus to contain few myelinated fibers. He interpreted the unmyelinated fibers in the vagus 1Contribution No. 37. 421 422 MARTIN R. CHASE as being post-ganglionic fibers arising from sympathetic cells in the vagus ganglia. Molhant 710 working with the Cajal reduced silver method, saw masses of unmyelinated fibers in the vagus nerve and thought they: were of sympathetic origin, being presumably post-gan- glionic fibers arising from cells in the sympathetic ganglia. Nei- ther Gaskell nor Molhant demonstrated unmyelinated fibers in the roots of the vagus. Langley has suggested the possibility of preganglionic vis- ceral efferent fibers losing their myelin sheaths during their course down the vagus. Ranson (’15) has recently studied the structure of the vagus nerve in the turtle, and has made observations of importance in clearing up the origin of the unmyelinated fibers in the vagus nerve. The vagus nerve in the turtle divides high in the neck into a cervical and a thoraco-abdominal ramus. The cervical ramus is composed almost entirely of myelinated fibers and the cells of the cervical ganglion of the vagus are associated: only with the fibers of this ramus. The thoraco-abdominal ramus presents the same structure as the thoracic vagus in mammals. It has a ganglion in the upper part of the thoraco-abdominal cavity. It consists largely of unmyelinated fibers, with scattered myelinated fibers, and maintains the same structure from its origin to the origin of the recurrent nerve. Ranson shows that the unmyelinated fibers are not post-ganglionic fibers arising from cells in the cer- vical ganglion, since the thoraco-abdominal trunk is not asso- ciated with this ganglion. Neither do preganglionic visceral efferent fibers lose their myelin sheaths, since the structure of the thoraco-abdominal ramus does not change. There is no association with sympathetic ganglia, and the proportion of unmyelinated fibers does not change below the thoraco-abdomi- nal ganglion, so there are probably no sympathetic elements in this ganglion. In the dog there is a very close association of the vagus and sympathetic trunks in the neck, and it seemed possible that some of the unmyelinated fibers of the thoracic vagus in this animal THE VAGUS NERVE 423 might be of sympathetic origin. The present paper presents the results of some experiments performed on dogs to determine by degeneration methods to what extent sympathetic fibers entered into the composition of the vagus. Figure 1 is a diagram of the right vagus nerve in the dog, showing the relations with the sympathetic, and the origin of the branches. Note that the two trunks are closely united from high in the neck to the upper thoracic region. Note also that just below the bronchial rami, 7.b., each vagus gives off a branch which goes to unite with the nerve of the opposite side. In addition each nerve is also joined by smaller twigs from the contra-lateral vagus. Sections of either vagus below the level of the bronchial rami may include fibers which originate in the opposite vagus, a fact of importance in interpreting the histology of the degenerated nerves. It would be possible for post-ganglionic fibers to enter the vagus from communications with the superior cervical ganglion of the sympathetic. In the preparation of the former paper (Chase and Ranson) we were unable, in a study of serial sections, to trace any considerable number of such fibers into the vagus. Throughout their course in the neck, although the two nerves are contained in a common sheath, no interchange of fibers eould be traced. At the level of the inferior cervieal (@.c.7.) ganglion of the sympathetic, however, the association of the vagus and sympathetic is very close, and numerous large bun- dles of sympathetic unmyelinated fibers can be seen in serial sections entering and leaving the vagus trunk. If, then, any considerable proportion of the unmyelinated fibers in the thoracic vagus of the dog have their origin in the sympathetic trunk, they must arise from cells in the inferior cervical ganglion g.c.7., or ganglion stellatum, g.s. METHODS The right vagus nerve was severed in a number of dogs. In some cases it was separated from the sympathetic trunk high in the neck and about an inch of the vagus nerve was removed (fig. 1, A). There would then be no interference with fibers THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4 MARTIN R. CHASE Stherine Hill, THE VAGUS NERVE 425 from the superior cervical ganglion, the sympathetic trunk or inferior cervical ganglion. In other cases about an inch of combined vagus and sym- pathetic trunk was removed in the midcervical region (fig. 1, B). Such a procedure would cut off any fibers to the vagus from the superior cervical ganglion, but would in no way interfere with fibers from the inferior ganglion. The dogs were autopsied after 2 to 8 weeks and portions of the nerves studied after preparation by the pyridine silver method (Ranson 711, 712) and the osmie acid method. CHANGES AFTER SECTION OF THE HIGH IN THE NECK VAGUS ALONE Sections from the right vagus of a dog autopsied five weeks after removal of a portion of the upper cervical vagus, the sym- pathetic being left intact, show definite degenerative changes. A eross section stained by the pyridine silver method, taken Fig. 1 Diagram of the right vagus nerve in the dog Fig. 2. From a section of right vagus nerve at level indicated (fig. 1, 2, 3), following removal of a portion of the vagus and sympathetic trunks at level B, figure 1. Pyridine silver. X 1140. Fig. 3 From a section of the same nerve at same level as figure 2. Osmic acid. xX 1140. Fig. 4a From a section of a normal vagus nerve just below pulmonary plex- uses (fig. 1, 4a). Pyridine silver. X 1140. Fig. 4b From a section of the same nerve as figure 2, below the pulmonary plexuses (fig. 1, 4b). Pyridine silver. X 1140. a, vagus rootlets A,B., indicate levels at which portions of the vagus trunk were removed art.s., arteria subclavia a.s., ansa subclavia b, bulbar rootlets of the accessory nerve c, spinal root of the accessory nerve g.c.v., ganglion cervicale inferior g.c.s., ganglion cervicale superius g.j-, ganglion jugulare g-n., ganglion nodosum g.s., ganglion stellatum l.v., left vagus n.c.i., hervus caroticus internus n.l.s., nervus laryngeus superior n.r., hervus recurrens n.v., hervus vagus n.v.t.s., nervus vagus and truncus sym- pathicus p.o., plexus esophageus r.b., rami bronchiales r.e., ramus externus n. accessoril r.p., ramus pharyngeus i.s., trunkus sympathicus 2, 3, 4a, 4b, indicate levels at which the corresponding figures were taken 426 MARTIN R. CHASE through the thoracic vagus just above the bronchial rami, shows no normal myelinated fibers and only a few scattered unmyelinated axones. There are present, however, a few bun- dles of unmyelinated fibers which are clearly of sympathetic origin. They cling in groups and stain in a characteristic man- ner. Similar groups of unmyelinated fibers, present at this level in the normal vagus nerve, were readily identified as of sym- pathetic origin by their contrast with the vagus fibers. They occupy only a small part of the total cross section area. Sections taken below the pulmonary plexuses show the right vagus to have retained its degenerated character. It is joined early by twigs from the normal left vagus, whose deeply stained fibers contrast sharply with the degenerated areas. It is im- possible to locate in these sections the sympathetic fibers seen at a higher level. They are not present in bundles, and it would be impossible to differentiate individual sympathetic fibers from normal fibers from the left vagus. CHANGES PRODUCED BY REMOVAL OF A PORTION OF THE CERVICAL VAGO-SYMPATHETIC TRUNK In the animal from which the illustrations are taken, a por- tion of the right sympathetic vagus trunk was removed as in- dicated (fig. 1, B) and the dog was allowed to live four weeks. Figure 2 is a drawing from a cross section of the right vagus trunk just proximal to the bronchial rami (fig. 1, 2, 3) stained by the pyridine silver method. There are seen no normal myel- inated or unmyelinated nerve fibers. The aggregations of granules, surrounded by definite zones, are nuclei of neurilemma cells, as is proven by longitudinal section. The entire cross section shows the same structure, there being only an occasional nakedaxone. Thereareno bundles of unmyelinated sympathetic fibers as was seen at the same level in the specimen previously described. Figure 3 is a drawing of a small part of a cross section of the same nerve at about the same level, stained with osmic acid. Note the presence of a number of deeply stained globules, which are the remains of degenerated myelin sheaths. There is pres- THE VAGUS NERVE 427 ent in the entire cross section only an occasional myelin ring, and most of these clearly represent degenerating fibers. Figure 4 is a composite drawing. Figure 4a, the upper half of the figure, is taken from a cross section of a normal right vagus nerve just below the origin of the bronchial rami (fig. 1, 4a). There are present in this field nine small myelinated axones. The remainder of the field is packed with unmyelinated axones. Figure 4a may be taken to represent the condition in the normal thoracic vagus, it being remembered that above the bronchial rami there are more myelinated axones, while near the dia- phragm there are even fewer than in the figure. Figure 4b is a drawing from a cross section of the same nerve as figures 2 and 3, taken at a level just proximal to the union with the branch from the left vagus nerve (fig. 1, 4b). There are in the field no normal unmyelinated or myelinated axones, and the entire cross section shows the same structure, there being only an occasional naked axone. In both pyridine silver and osmic acid preparations of the nerve below the level described sections, both of the right nerve after its union with the branch from the left vagus, and of the left nerve after union with the branch from the right, show a mingling of the degenerated nerve with the normal, but the degeneration can be clearly traced to the diaphragm. In the second case, where both the vagus and sympathetic were cut in the mid-cervical region, no bundles of fibers of sym- pathetic origin could be found, the entire nerve being degenerated. We conclude, then, that following section of the cervical vagus nerve in the dog, with or without section of the sympathetic trunk, all fibers of the vagus, myelinated and unmyelinated alike, undergo complete degeneration. There are to be found in the degenerated thoracic trunk, above the level of the pul- monary plexuses, in some individuals bundles of unmyelinated nerve fibers having their origin in the cells of the sympathetic, presumably in the inferior cervical ganglion. In some individuals no bundles of this sort are found. In no ease does the total number of fibers of sympathetic origin present amount to more than an insignificant fraction of the total number of unmyelinated 428 MARTIN R. CHASE fibers normally present. In the material studied it was possible to demonstrate more groups of unmyelinated fibers of sympa- thetic origin in the thoracic vagus in the animals in which only the vagus was severed high in the neck, than in those operated by division of both vagus and sympathetic trunks. It is thought that this finding is the result of individual variation in the ani- mals, but it is possible that the difference in operative procedure has something to do with it. The unmyelinated character of the thoracic vagus nerve in the dog is not due to the presence of fibers derived from the sympathetic trunk, during the close association of the vagus and sympathetic nerves in the neck. BIBLIOGRAPHY CHASE AND Ranson 1914 The structure of the roots, trunk and branches of the vagus nerve. Jour. Comp. Neur., vol., 24, no. 1, pp. 31. GaAsKELL, W. H. 1886 On the structure, distribution and function of the nerves which innervate the visceral and vascular systems. Jour. Phys., London, vol. 7, p. 19. 1889 On the relation between the structure, function and origin of the cranial nerves. Jour. Phys., London, vol. 10, p. 153. Lanecuery, J. N. 1900 The sympathetic and other related systems of nerves. Schifer’s text-book of Physiology, vol. 2, p. 665. 1903 Die Kranialen autonomen Nerven und ihre Ganglien. Ergeb. der Phy she Bde pone: Motuant, M. 1910 Le nerf vague. Le Névraxe, vol. 11, p. 187. Ranson, 8. W. 1911 Non-medullated fibers in the spinal nerves. Am. Jour. ANN, WO, WAY jo), Me 1912 The structure of the spinal ganglia and of the spinal nerves. Jour. Comp. Neur., vol. 22, p. 159. 1914 The structure of the vagus nerve of man as demonstrated by a differential axon stain. Anat. Anz., Bd. 46, S. 522. 1915 The vagus nerve of the snapping turtle (Chelydra serpentina). Jour. Comp. Neur., vol. 25, p. 301. CHANGES IN THE ROD-VISUAL CELLS OF THE FROG DUE TO THE ACTION OF LIGHT LESLIE B. AREY From the Anatomical Laboratory of the Northwes ern University Medical School! TWO FIGURES CONTENTS ee EU STANA LSE Wis 5 gil get ak aia oe VF o's) Gk a Coe. Dy prs. dst. bac. vr. _ . | ----prs, dst. bac. rb, prs. dst. con,----f mb, lim. exc” (fo: SEF: Fig. 1 From a dark-adapted retina of the frog, Rana pipiens, showing the positions assumed by the rods and cones. X 1130. ell.bac.rb., ellipsoid of red rod; ell.bac.vr., ellipsoid of green rod; ell.con., cone ellipsoid; gtt.ol., oil globule; mb.lim.ex., externa limiting membrane; my.bac.rb., myoid of red rod; my.bac.or., myoid of green rod; my.con., cone myoid; prs.dst.bac.rb., outer member of red rod; prs.dst.bac.vr., outer member of green rod; prs.dst.con., outer member of cone; st.nl.ex. external nuclear layer. results, the exact opposite of those reported by Ewald and Kihne, is assumed from the context of the previously cited quotation (p. 431), and from his illustrations. Angeluecci (84) measured the length of the outer member of the frog’s rod and found it shortens in the light; later (90) he confirmed this result by measurements of the large rods of the CHANGES IN THE VISUAL CELLS OF THE FROG 433 salamander where the differences in length were more striking. Arcoleo (90) and Garten (’07) reported similar conditions for the toad and frog respectively. In most accounts of the changes occurring in the anuran rod, due to the action of light, reference is not made to the number of individuals experimented upon, and in no case are definite measurements of length given, judgment of the eye apparently being the only criterion adopted. = | Be = ae. prs. dst. bac. vr. rs. dst. bac. rb: prs. dst. bac. r ----ell. bac. vr. j +---my. bac. vr. & ig dst. con. f mb. lim. exc Fig. 2. From a light-adapted retina of the frog, Rana pipiens, showing the positions assumed by the rods and cones. X 1130. In the experimentation which forms the basis of this paper, an attempt has been made to correlate an influence of light with the following possible changes in the frog’s visual rod: 1) changes in the position of the red rod nucleus with respect to the external limiting membrane; 2) changes in the length of the red and green rod myoids; 3) relative changes in the length of the red rod myoid at the center and at the periphery of the retina; 4) changes in the diameter of the various component parts of the red rod. 434 LESLIE B. AREY EXPERIMENTATION Individuals of Rana pipiens, approximately uniform in size, were exposed to bright diffuse daylight for eight hours, or to total darkness for a minimum length of twenty-eight hours. At the expiration of these periods of light and dark-adaption, the cranium of each animal was first split sagittally, and then cut transversely just caudad to the eyes. The resulting moieties of the cranium, with the contained eyes, were dropped into Perenyi’s chromo-nitric fixative; in this solution they underwent fixation, the condition of illumination being identical with that to which they had been experimentally subjected. The oper- ation on dark-adapted animals was accomplished by the light from a photographic red lamp and demanded but a few seconds time. After dehydration and the removal of the lens, the eyes were imbedded in paraffine, sectioned 8 thick, and stained with Ehrlich-Biondi’s acid fuchsin-orange G-methyl green mixture. In light-adapted retinas the expanded pigment masked the visual cells to a greater or less degree, hence, nascent oxygen was used as a bleaching agent in these preparations. The results obtained with the Ehrlich-Biondi stain were very satis- factory, yet it is of interest to note that the staining reaction of several definite structures was often variable. For example, the outer member of the red rod in most cases stained red with the acid fuchsin, yet in some retinas from the same series they were colored an intense orange from the orange G, although the treat- ment in both cases had been, so far as is known, identical, the series having been carried along simultaneously step by step. Moreover, in one preparation, at least, it was observed that the outer members in one half of the retina were stained orange, whereas in the other half they were stained red. ‘This selective stainability is doubtless indicative of a variable physiological cytoplasmic state. Preparations in which the outer members of red rods selected the orange G, showed the green rods stained red with the acid fuchsin. In retinas that had been bleached (potassium chlorate and hydrochloric acid being the reagents CHANGES IN THE VISUAL CELLS OF THE FROG 435 used), the rod outer member varied in color from blue-violet to red-violet. Cases involving a somewhat similar variability might be cited with respect to several other structures in the retina. Experimentation was made upon sixty retinas, from which twenty-three light-adapted and twenty-three dark-adapted prep- arations were selected for measurement. Preparations, in which the external limiting membrane was not apparent, or in which wrinkles caused oblique sections, were rejected. All Measurements were made with a Leitz 1/12 homogeneous im- mersion objective and a Zeiss No. 2 micrometer eyepiece. A. Influence of light on the position of the red rod nucleus The object of this series of measurements was to determine the effect of light and darkness upon the position of the nuclei of the red rods with respect to the external limiting membrane. The nuclei of the rod- and cone-visual cells comprise the external nuclear layer (figs. 1 and 2, st.nl.ex.) of the retina. According to Greeff (00, p. 133), these two kinds of nuclei in amphibians can be distinguished, morphologically, only with great difficulty; hence it is essential to inquire whether any criterion exists where- by the rod nuclei can be identified with a tolerable degree of certainty. The illustrations of the frog’s retina given by Greeff (00, pp. 96, 102) represent the nuclei of the red rod-visual cells as lying considerably nearer the external limiting membrane than the nuclei of either the green rod-visual cells or of the cone- visual cells. If, therefore, in any preparation attention be di- rected to the nuclei which protrude farthest beyond the external limiting membrane, one may be reasonably sure that the nuclei of the red rods only are under consideration. Ten light-adapted and ten dark-adapted retinas were selected at random for measure- ment. Not only was considerable regional variability in the position of nuclei found in individual retinas, but also the varia- bility in any limited area was extensive—ranging from nuclei whose edges were at the same level as the external limiting membrane to nuclei which were 3.0 « above that membrane. Partly for this reason, and partly to be certain that only the 436 LESLIE B. AREY nuclei of red rods were under observation, attention was directed solely to the mean distance to which the maximally extended nuelei protruded beyond the external limiting membrane. The following values (grand means) were obtained: in hght (10 retinas), 2.94; in darkness (10 retinas), 2.2n. On account of the degree of variability observed, I do not re- gard this slight difference, which, incidentally, is not in agree- ment with the results of Stort (87) and Garten (07) on Triton, or of Angelueci (90) on the salamander, as indicative of a photic influence: Since the condition recorded in Triton and the salamander is not clearly demonstrable in my preparations of the frog, it follows that whatever changes are found in the rod myoid will be due chiefly to the activity of the myoid itself. B. Influence of light on the length of the rod myoid. Relative changes at center and periphery of retina a. Red Rods. In every retina, at least ten measurements were made, on each side of, and not far from, the optic nerve. The averages of these twenty or more values are given in tables 1 and 2 as central measurements. Similarly the means of at least twenty measurements (ten on each side), made well toward the periphery of the retina, are recorded as peripheral measurements. In order to avoid unconscious selection all measurements were made on consecutively-placed rods. The myoid length in the tables constitutes the distance from the external limiting mem- brane to the nearer edge of the rod ellipsoid (figs. 1 and 2, my. bac.rb.). The tables show that there is considerable variation in indi- vidual retinas, yet the rod myoid unmistakably elongates in the light and shortens in darkness (figs. 1 and 2). Although the mean values, at the center and periphery of individual retinas, may also vary greatly in either set, the grand means are practi- cally identical. The measurements of individual light- and dark-adapted rods overlap in many instances; furthermore, the mean for certain groups of ten deviates considerably from the average for both groups of ten, which constitutes the central or peripheral values CHANGES IN THE VISUAL CELLS OF THE FROG 437 of the tables, as the case may be. In such instances the process of averaging two groups of ten serves to mask this condition, hence the tables, for the sake of compactness, are faulty in this respect. The conclusion follows, therefore, that the photomechanical responses of the frog’s red visual-rod myoid are in agreement with those of other vertebrates in which changes have been demonstrated. The older workers (Angelucci ’84; ’90; Gradenigro, °85; and Arcoleo ’90) perhaps erred, either in not making actual measure- TABLE 1 Measurements from twenty-three dark-adapted retinas of Rana pipiens. The values are in micra and represent measurements taken along axes coinciding with radii of the eyeball. Each value for the length of the red rod myoid is the mean obtained from twenty consecutively-placed elements | | NERVE FIBER CHORIOID TO LENGTH OF LENGTH OF NUMBER OF LAYER TO ee ine woh RED ROD MYOID RED ROD MYOID en a ere a oe 1 | 107 64 5.0 4.9 2 136 70 4.0 5.7 3 114 72 4.6 3.0 1 114 64 4.4 4.9 5 122 69 6.6 7.0 6 110 69 5.3 6.7 7 100 64 4.0 6.4 8 106 64 6.7 6.6 9 129 67 6.4 | 6.4 10 100 69 9.3 7.6 11 114 69 6.9 6.9 12 129 69 5.9 5.7 13 114 73 6.2 5.7 14 97 62 3.6 4.3 15 97 63 4.7 5.0 16 111 69 4.6 9.1 17 107 63 5.4 6.0 18 107 64 6.4 6.2 19 114 63 6.6 6.4 20 103 60 7.2 6.9 21 89 57 (Or 6.3 22 93 62 5.7 (hers 23 93 60 6.4 6.2 1) TN 7 109 66 5.8 6.0 438 LESLIE B. AREY ments, or were influenced by their observations on the more strikingly mobile cone (figs. 1 and 2; my.con.), the movements of which are the reverse of those exhibited (such is my belief) by mobile rods in general. In recent papers (715; 716) the writer favored the anomalous photomechanical responses of the frog’s rod, reported by the several older workers, as being more trustworthy than the some- what confusing account of Lederer? (08). The writer (15), TABLE 2 Measurements from twenty-three light-adapted retinas of Rana pipiens. The values are in micra and represent measurements taken along axes coinciding with radii of the eyeball. Each value for the length of the rod rod myoid is the mean obtained from twenty consecutively-placed elements NUMBER OF ; Bia = oe ‘i E resend e a NG eee ne bee D R a Sone D ANIMAL EXT pee ae NG MEMBRANE AT ee OF et 1 100 67 9.9 10.2 2 111 73 13.6 14.9 3 93 74 11.9 12.0 4 129 62 ml 7 12.6 5 ILL? CZ 11.9 12.6 6 114 72 12.6 11.9 a 114 64 13.33 12.2) 8 127 64 9.2 eZ 9 127 69 8.6 10.3 10 119 We, 16.9 16.2 11 114 72 13.9 14.7 12 120 72 143 AG 13 92 62 9.9 9.2 14 102 69 13)4 13.0 15 107 60 13.0 11.9 16 125 63 9.6 9.2 17 100 62 11.4 12.4 18 106 62 9.9 9.6 19 93 61 8.6 7.9 20 96 69 127 9.6 21 107 64 7.9 ee, 22 97 64 8.6 9.7 23 129 69 11.9 12.9 Miéeantic erie 100 67 1S | 11.6 3 Lederer s figures of isolated rods do not necessarily show how long he rod myoid really was, either in darkness or in light. Presumably, the myoid, as the result of teasing, broke approximately at the level of the external limiting CHANGES IN THE VISUAL CELLS OF THE FROG 439 furthermore, made use of these data in an argument against the feasibility of attempting to advance (in the light of our present knowledge) a single rational explanation for the diverse photo- mechanical movements of the visual rods. It is evident, how- ever, that the conclusion reached in the present investigation renders this particular objection invalid. It is reasonable to expect, although material is not available at present to put the matter to an experimental test, that the photomechanical behavior of the toad’s visual rod will be found to vary in no essential detail from that herein described for the frog. b. Green rods. From the sixty retinas experimented upon, the ten light-adapted and ten dark-adapted preparations which showed the most perfect histological preservation were selected. for measurement. In each retina, measurements were made of the myoid length (figs. 1 and 2, my.bac.vr.) of ten consecutively- placed green rods; the results are recorded in tables 3 and 4. TABLE 3 Measurements from ten dark-adapted retinas of Rana pipiens. The values are in micra and represent measurements taken along axes coinciding with radii of the eyeball. Each value for the length of the green rod myoid is the mean obtained from ten consecutively-placed elements wowsrn oF | qoexrmawat uiwriva | exremvatuimimina | gggtt"iTH OF 1 95 66 21.4 2 94 65 23.0 3 98 75 28.6 4 90 69 26.2 5 112 68 22.5 6 135 $2 27.7 ¥ 102 63 24.4 8 109 70 24.0 9 105 70 24.1 10 115 72 22.5 Meant. 2:3 2%’. 106 70 24.4 membrane, although nothing to this effect is stated. Furthermore, the varicose and atypical appearing rods naturally increases one’s caution in accepting his conclusions. Although Lederer gave but little emphasis to his observations on sectioned material (p. 431), I believe that those observations constitute the strongest evidence in support of his thesis. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, No. 4 440 LESLIE B. AREY TABLE 4 Measurements from ten light-adapled retinas of Rana pipiens. The values are in micra and represent measurements taken along axes coinciding with radii of the eyeball. Each value for the length of the green rod myoid is the mean obtained from ten consecutively-placed elements NERVE FIBER LAYER CHORIOID TO pistes TO ayant cea pr ieg ne Re Ge Piette iors pit ss 1 105 68 25.0 2, 102 P 7A). 1 3 127 5 28.2 4 120 69 28.2 5 97 68 31.0 6 112 63 28 .2 7 Pe 87 PAS 8 120 75 Dea 9 127 72 32.4 10 | 123 66 : 24.9 Mean sacs. | 116 2, Pil 7 The tables show that although the difference in the length of the rod myoid, in darkness and in light, is small, the length of the light-adapted element is quite consistently the greater (figs. 1 and 2); hence it seems probable that this difference is significant and the photomechanical responses. of the red and green rods are analogous. This conclusion is not in accord with the results of Angelucci (94). Angelucci believed that the red and green rod myoids responded similarly to photic stimulation, yet in both cases a shortening was said to take place. C. Influence of light on the diameter of the red rod From my preparations many rods were measured, yet no con- stant or significant differences were found in the diameters of the outer members (figs. 1 and 2, prs.dst.bac.rb.), or of the ellipsoids (figs. 1 and 2, ell.bac.rb.) of the inner members, which could be attributed to the influence of light and darkness. The myoid of the inner member (figs. 1 and 2, my.bac.rb.) naturally becomes tenuous when elongated by the action of light (p. 436). One is inclined to question the morphological normality of the teased rods figured by Lederer (’08), especially since the appear- CHANGES IN THE VISUAL CELLS OF THE FROG 441 ance of these elements in his own sectioned material was dis- similar. In the figures corresponding to the text descriptions of dark-adapted rods (the figures and their appended descriptions are apparently interchanged in his paper), the varicose outer members are twice, and the ellipsoids three to four times, as broad as the corresponding parts of the light-adapted rods. My observations, therefore, are opposed both to those of ‘Lederer (’08) who believed that darkness causes the rod to swell, and to those of Ewald and Kiihne (’78), who recorded that light acts in this manner. SUMMARY 1. Distinct movements of the nuclei of the red rod-visual cells, due to photic stimulation, are not demonstrable. Hence move- ments of the rods are not produced indirectly in this way. 2. The myoid of the rod-visual cell elongates in light and shortens in darkness. Therefore, contrary to the conclusions of the older workers, the photomechanical response of the frog’s rod myoid is found to be similar to that occurring in the retinas of all other investigated vertebrates. The mean length of approximately 1000 myoids from 23 light-adapted retinas is 11.6 u. The mean length of approximately 1000 myoids from 23 dark- adapted retinas is 5.9 uy. 3. A significant regional difference in the length of the red- visual rod myoid is not apparent from comparisons of approxi- mately 2000 measurements made at the center and the periphery of light- and dark-adapted retinas. 4. The myoid of the green visual rod probably elongates slightly in the light and shortens in darkness. The mean length of 100 myoids from 10 light-adapted retinas ea ag M. The mean length of 100 myoids from 10 dark-adapted retinas is 24.4 M. 5. Definite changes in the diameter of the outer member, or of the ellipsoid, of the red visual-rod can not be correlated with photic influences. The rod myoid, however, does become tenuous in the light. . 442 LESLIE B. AREY REFERENCES Papers marked with an asterisk have not been accessible in the original. Ance.uccr, A. *1884a Una nuova teoria sulla visione. Communic. preven- tiva presentata all’ Accad. med. di Roma, 14 Luglio. 1884 b Una nuova teoria sulla visione. Gazetta Medica di Roma, 1884, pp. 205-210; 217-223. 1890 Untersuchungen iiber die Sehthitigkeit der Netzhaut und der Gehirns. Untersuch. zur Naturlehre d. Menschen u. d. Thiere (Mole- schott), Bd. 14, Heft 3, pp. 231-357. ArcoLeo, E. 1890 Osservazioni sperimentali sugli elementi contrattili della retina negli animali a sangue freddo. Annali d’Ottalmologia, Anno 19, Fasc. 3 e 4, pp. 253-262. Argy, L. B. 1915 The occurrence and the significance of photomechanical changes in the vertebrate retina—An historical survey. Jour. Comp. Neur., vol. 25, no. 6, pp. 535-554. . 1916 The movements in the visual cells and retinal pigment of the lower vertebrates. Jour. Comp. Neur., vol. 26, no. 2, pp. 121-202. Bout, F. 1877 Zur Anatomie und Physiologie der Retina. Arch. f. Anat. u. Physiol., Physiol. Abth., pp. 4-86. Ewaup, A., unp KiiHne, W. 1878 Untersuchungen iiber den Sehpurpur. Teil 3. Verinderungen des Sehpurpurs und der Retina im Leben. Untersuchung. aus d. Physiol. Inst. d. Univ. Heidelberg, Bd. 1, Heft 4, pp. 370-422. GarRTEN, S. 1907 Die Verinderungen der Netzhaut dureh Licht. Graefe- Saemisch, Handbuch der gesammten Augenheilkunde. Leipzig, Aufl. 2, Bd. 3, Kap. 12, Anhang; 130 pp. GRADENIGRO, G. 1885 Uber den Einfluss des Lichtes und der Wirme auf die Retina des Frosches. Allz. Wiener med. Zeitung., Bd. 30, No. 29 u. 30, pp. 343-344, u. 353. GREEFF, R. 1900 Die mikroskopische Anatomie des Sehnerven und der Netz- haut. Graefe-Saemisch, Handbuch der gesam. Augenheilkunde. Leipzig, Aufl. 2, Bd. 1, Kap. 5, 212 pp. LEDERER, R. 1908 Verinderungen an den Stiibchen der Froschnetzhaut unter Kinwirkung von Licht und Dunkelheit. Centralbl. f. Physiol., Bd. 22, No. 24, pp. 762-764. Miituer, H. 1856 Anatomisch-physiologische Untersuchungen iiber die Retina bei Menschen und Wirbelthieren. Zeitschr. f. wissensch. Zool., Bd. 8, Heft 1, pp. 1-122. ScuowaLBeE, J. 1874 Microscopische Anatomie des Sehnerven, der Netzhaut und der Glasskérpers. Graefe-Saemisch, Handbuch der gesam. Augen- heilkunde, Leipzig, Aufl. 1, Bd. 1, Teil 1, pp. 321-456. Srort, A. G. H. van GenpeREN 1886 Uber Form und Ortsveriinderungen der Elemente in der Sehzellenschicht nach Beleuchtung. Bericht uber d. 18. Versamm. d. Ophthal. Gesell. zu Heidelberg., pp. 43-49. 1887 Mouvements des éléments de la rétine sous l’influence de la lumiére. Arch. néerlandaises des Sciences exact et naturelles, publ. p. l. Soc. holland. des Sciences, Tom. 21, Livr. 4, pp. 316-386. A PRELIMINARY DETERMINATION OF THE PART PLAYED BY MYELIN IN REDUCING THE WATER CONTENT OF THE MAMMALIAN NERVOUS SYSTEM (ALBINO RAT) H. H. DONALDSON From The Wistar Institute of Anatomy and Biology ONE CHART It is a familiar fact that there is a progressive loss of water in the brain and spinal cord with advancing age. This is illus- trated for the albino rat in Chart 1. SSSGSSEsseeesssceesssesssseeerizs wae at CEE ECE EEELE La SESSSER ARF outa CTT ere Te SB? 2_maee 200 250 300 360 ° . Chart 1 Showing the percentage of water on age in the central nervous system of the albino rat. The upper graph gives the values for the water in the brain as determined by the formulas (Hatai, in ‘The Rat,’ Donaldson, ’15). The lower graph gives the corresponding values for the spinal cord, determined in the same way. The small black dots indicate the observed values for the several age groups for the brain, and form the data for the formulas. The small black triangles have a like value in relation to the spinal cord. : 443 444 H. H. DONALDSON It is also well known that in some mammals all the axons in the central nervous system are unmyelinated at birth, while in other species a greater or smaller number of them may already have their sheaths. In all eases, however, an active formation of myelin occurs during the period of rapid growth (Koch and Koch 713). In a previous study on the percentage of water in the albino rat I was misled by certain graphs (Donaldson 710) to the con- clusion that probably both the axons and their myelin sheaths changed in their water content so as to produce the well known reduction of water which is observed, but further study has shown that this is an incorrect view, and it is the object of this paper to present the evidence of my revised conclusion. Since it has already been shown that the loss of water in the human brain follows the same course as in the brain of the albino rat—and has similar limits (Donaldson ’10)—it is permissible to use in the argument certain observations on the human brain. From the data for the human brain already in the literature I have selected those published by de Regibus (84), because this author evidently examined only the outer layers of the cor- tex when making his determinations for the water in the gray substance and because he was able also to obtain remarkably uniform results for all of his determinations. De Regibus tested four male brains, 25 to 76 years of age and three female brains, 30 to 60 years of age. In Table 1 there appear also determinations of the water content in the human cortex and in the fibers at birth. These are based on the records of Weisbach (’68). This enables us to contrast in Table 1 the conditions at birth with those at maturity. TABLE 1 Percentage of water in the gray and white substance of the human brain at birth and at maturity CALLOSUM | CORTEX (GRAY) (WHITE) per cent per cent AC ibintha (Weisbach) ie .cuae. > cite alee cee epee eeaereaetae 88 88 At Me puniby Ge: Ee2TDUS) \.4:05.5., so gmlsutetait at eee ae coed 86 70.4 WATER CONTENT—MAMMALIAN NERVOUS SYSTEM 445 According to this table the (gray) cortex has lost 2 points and the (white) callosum 17.6 points in the process of maturing. It is never possible at maturity to obtain the cortex or any other gray mass without some admixture of myelinated fibers and I have therefore, provisionally, credited one point of the joss, noted by de Regibus in the water content of the cortex, to the presence of myelin. This implies but a small proportion of myelin since if we assume that myelin has 48 per cent of water the reduction of 1 per cent would mean that about one thirty- ninth of the mass was represented by myelin. According to this assumption the mature gray substance (cortex)—when the myelin is excluded—contains 87 per cent of water, and in the computations which follow the neurons without myelin are assumed to have 87 per cent of water, except at ten days, when they are credited with 88 per cent. The fact that the fibers without myelin have at birth a high percentage of water (88°) while at maturity, after myelination, they have lost 17.6 points is the sort of evidence which fur- nishes the basis for the current, but unsupported view, that the loss of water is to be associated with the formation of the myelin. It is our object to present more precise information bearing on this point. To obtain a notion of the approximate distribution of the water between the myelin and neurons proper, it is necessary to have data on the relative abundance of these two constituents of the brain. In 1913 W. Koch and M. L. Koch made a study of the chemical composition of the brain of the albino rat at six ages between birth and maturity, and of the spinal cord at one age. The data thus obtained are those which will be utilized here. The authors determined seven fractions: Proteins, organic extractives and inorganic constituents, which three taken together, we shall designate protein (or non-lipoid), and phosphatides, cerebro- sides, sulphatides and cholesterol, which four taken together, we shall designate lipoid. These data give us at each age, therefore, the protein and the lipoid present in the brain, or to be a little more exact, we should 446 H. H. DONALDSON say the lipoid and the non-lipoid fractions. The lipoid (in part) represents the myelin sheaths, while the protein, with the remainder of the lipoid, represents the cell bodies and their unsheathed axons. With the exception of the one day group, the ages for which analyses were made, are given in table 2. TABLE 2 To show, for the brain of the albino rat at five ages and for the spinal cord at one age the percentage of water in the myelin as computed according to the method described. The protein and lipoid are given in percentages of the total dry sub- stance. (Based on table 2. Koch and Koch ’13) Brain (1) (2) (3) (4) (5) (6) (7) PERCENTAGE OF Ga ikee ron Weree WATER. AGE IN CORRECTED CORRECTED DIFFERENT IN MYELIN = DAYS PROTEIN LIPOID AGES. ; In Neurons} Liporps (C) LIPOID aT 20 Pere = protein (COMPUTED) DAYS = 1 b a a (C) (See p. 447) EOE (assumed) per cent per cent 10 93.80 6.2 86.5 88 63.8} 20 88.88 il 1.0 82.5 87 46.5? 40 82.86 IA te! 145) 79.4 87 42.7 120 (aye Ml 24 84 Pe Pe 78.4 87 52.4 210 76.36 23:08 Paya \t Toil 87 47.4 ANenage;oih20 to 2h ais aercs eiaahelnc atetvale Gait enna store eee 47.8 Spinal cord 120 | 52.92 | 47.08 | 4.2 | 70.4 | 87 | AL) 1 First traces of myelin. ? Myelin well shown. At one day—or practically birth—it is found that the lipoid is present to the extent of 0.31 or nearly one-third of the weight of the protein. There is, however, no visible myelin at this age, so it is concluded that this proportion of the total lipoid is nor- mally associated with the protein and is not to be included in the lipoid which forms the myelin sheaths. We have treated the data for the later age groups in accordance with this relation, and in each case have taken from the total lipoid found an WATER CONTENT—MAMMALIAN NERVOUS SYSTEM 447 amount equal to 0.31 of the protein found. The remaining amount of lipoid is assumed to be that used for the sheaths. In table 2, the column (2) headed Corrected Protein gives the observed protein (non-lipoid) plus 0.31 of itself and the column (3) headed Corrected Lipoid gives the observed 5 less the amount of lipoid added to the protein. _ In table 2 the data are given in five age groups for the bos and in one age group for the spinal cord. It is to be noted that the 10 day brain group—which stands just at the beginning of the myelin formation—is for the moment excluded from the discussion and we begin the comparisons which are to be made, with the 20 day brain group. In the brain series (with one exception) the corrected protein diminishes and the corrected lipoid increases with advancing age. Between 20 and 210 days the proportion of the lipoid doubles—column (4). We have in column (5) the observed percentage of water in the brain as a whole. It is assumed, as previously noted, that the corrected protein (neurons in the strict sense =both cell bodies and axons) have 87 per cent of water. From these several data we can compute the percentage of water to be assigned to the corrected lipoid, which represents the myelin.! The method of computation may be illustrated by the data for the 20 day group. Reference to table 2 shows that at this age there is 1 part of lipoid (11.12°%) to 8 parts of protein (88.88%). This gives 9 parts, representing the entire brain and having 82.5 per cent of water. The product, 9 X 82.5 = 742.5. We assume that the 8 parts of protein have 87 per cent of water. The product, 8 X 87 = 696. The 1 part of lipoid, representing the myelin, will then have a percentage of water equal to the dif- ference of these products = 742.5 — 696 = 46.5 per cent. It is hardly necessary to point out that a division of the brain into myelin on the one hand and neurons on the other fails to enumerate several structural elements which are also present though representing a relatively small mass. There are, in addition to the neurons, glia and ependymal cells; blood and lymph vessels; blood and lymph and a slight amount of connective tissue. Over against the neurons plus this group of elements we put the myelin, but for the purposes of the present discussion it is convenient to speak of the rest—the non-myelin portion—of the brain as if represented by the neurons alone. 448 H. H. DONALDSON As table 2 shows, similar computations give percentages of water for the myelin in the brain, which range from 42.7 per cent to 52.4 per cent and which yield a mean value of 47.8 per cent. The computation for the spinal cord gives 51 per cent. The significance of these results lies not in the particular percentage of water here determined for the myelin—as that depends some- what on the percentage of water assumed for the protein—but on the similarity of the values found in all the five cases examined. However, it is found on trial that one cannot depart far from the value of 87 per cent for the proteins without obtaining rather improbable percentages for the myelin, so that this value is probably nearly right. DISCUSSION Those familiar with the published data for the percentage of water in the cortex will at once perceive that the value given by de Regibus (86%) seems high. The various water records for the cortex run down as low as 83.5 per cent. The differences between the various determinations are, however, almost cer- tainly due to the varying amounts of white substance included in the sample, and as has already been stated the value chosen is probably close to the true value. In connection with the computations there are, however, two conditions which have been assumed to be constant but which in all probability, are subject to variation. I refer to the density of the myelin and to the fraction of the lipoid to be assigned to the protein. As to this last condition, it would be plausible to think of a larger fraction of lipoid in the axons than in the cell bodies. If this were true it would be necessary to increase this fraction in the case of the spinal cord or the callosum. It is also possible that aside from this method of distribution the fraction of lipoid in the neuron may increase with age. The slight loss of water during the first ten days of (rat) life is possi- bly due to such an increase. Finally, the density of the myelin may change with age, as its chemical composition certainly does, and it is conceivable that it has a higher water content when first formed, as is suggested by the 63.8 per cent given for the ten day record in table 2. WATER CONTENT—MAMMALIAN NERVOUS SYSTEM 449 If we compare the drawings of Watson (’03), showing the in- crease of the visible myelin in the cerebral hemispheres and in the spinal cord of the rat, with the chemical results here used, we see that the histological pictures show a more gradual ap- pearance of myelin than the chemical results, or the water determinations, would suggest. This probably depends on the fact that it is only a fraction of the lipoids forming the sheaths, which takes the haematoxylin stain, and-this stainable fraction forms at first a smaller, but later a larger portion of the entire sheath (Koch and Koch 713; Smith and Mair ’08). There is still one more modification in the formation of myelin. Tribot (05) has contrasted in terms of dry substance the relative amounts of albuminoides and the fats in the nerve tissue of the guinea-pig at different ages. The percentage value of the fats increases from 11 days (his first observation) up to 120 days— after which it begins to fall. The fats of Tribot are the lipoids of Koch’s analysis and it is of interest to note that the 120 day record for the brain in table 2, column (4), also shows the highest proportion of corrected lipoids. The observations of Dunn (’12) which show in the myelinated fibers of the second cervical nerve of the rat the highest relative areas for the myelin sheaths at 75 days and 132 days—seem to fit with these other observations and to suggest that the formation of lipoids with advancing age fluctuates in such a way as to show a maximum about the end of the active growing period of the central nervous system. This discussion of possible factors modifying these determi- nations has been introduced to clear the way for further work on the main question, but so far as one can foresee the effect of taking them into consideration, it would tend to make more uniform the values thus far obtained. CONCLUSIONS We conclude from these results that there is no evidence that the cell bodies.and their unsheathed axons suffer more than a slight loss of water between birth and maturity, and that the progressive diminution in the water content of the entire brain and spinal cord is mainly due to the accumulation of myelin, 450 H. H. DONALDSON with a water content of about 48 per cent. Moreover, the myelin must be regarded as a more or less extraneous substance, having but little significance for the characteristic activities of the neurons. If we compare the loss of water in the case of the nervous system with that in the muscular system, which also contains a large proportion of fat (Tribot ’05), we find that while the two systems lose about the same percentage of water between birth and maturity (Lowrey 713), yet in the case of the nervous system alone is this lipoid (or non-protein substance) accumulated out- side of the cell. From this it is seen that the neuron is peculiarly able to maintain its early water-solids composition and that it accomplishes this by throwing out the material, which in the muscles is retained within the cells. As the diminution in the percentage of water in the central nervous system is preéminently a function of age, and as it appears to be due almost entirely to the formation of the myelin, it follows that the myelin formation is also a function of age (Donaldson 711). A glance at the graphs in Chart 1 shows that the most active production of myelin, as indicated by the rapid loss in the percentage of water, occurs early, i.e., during the first forty days of rat life, in the brain, and during the first hundred days, in the spinal cord. WATER CONTENT—MAMMALIAN NERVOUS SYSTEM 451 LITERATURE CITED Donautpson, H. H. 1910 On the percentage of water in the brain and in the spinal cord of the albino rat. Jour. Comp. Neur., vol. 20, no. 2, pp. 119-144. 1911 The effect of underfeeding on the percentage of water, on the ether-alcohol extract, and on medullation in the central nervous system of the albinorat. Jour. Comp. Neur., vol. 21, no. 2, pp. 139-145. 1915 The Rat. Memoirs of the Wistar Institute of Anatomy and Biology, no. 6, pp. 1-278. Dunn, E. H. 1912 The influence of age, sex, weight and relationship upon the number of medullated nerve fibers and on the size of the largest fibers in the ventral root of the second cervical nerve of the albino rat. Jour. Comp. Neur., vol. 22, no. 2, pp. 131-157. Kocu, W. anp Kocu, M. L. 1913 Contributions to the chemical differentiation of the central nervous system. III. The chemical differentiation of the brain of the albino rat during growth. J. of Biol. Chemistry, vol. 15, no. 3, pp. 423-48. Lowrey, L. G. 1913 The growth of dry substance in the albino rat. Anat. Rec., vol. 7, pp. 143-168. DE Rearisus, C. 1884 Determinazione dell’ acqua contenuata nelle sostanze grigia e bianca del cervello umano. Atti. d. reale Accad. di Med. di Torino, vol. 6, pp. 323-328. Smit, J. L. anp Marr, W. 1908 An investigation of the principles underlying Weigert’s method of staining medullated nerve. J. of Pathology and Bacteriology, vol. 13, pp. 14-27. Trisot, J. 1905 Sur les chaleurs de combustion et composition chimique des tissus nerveux et musculaire chez le cobaye, en fonction de l’age. C. R. Acad. Se. Paris, T. 140, pp. 1565-1566. Watson, J. B. 1903 Animal education. Contributions from the Psychol. Lab. of the University of Chicago, vol. 4, no. 2. Univ. of Chicago Press, Chicago, Ill. Werssacu, A. 1868 Der Wassergehalt des Gehirns nach Alter, Geschlecht und Krankheiten. Med. Jahrbiicher, vol. 16, nos. 4 and 5, pp. 1-76. x ry" 4 sie oy f' ht a bust ve Drea i, A wi was Yuk: ‘3 +a sam Wey ay vow Tah i eau) fo Mame ac prey fie, jy aha * : it gta Meee, hs, ti j / io mer <. y HE te rt “ge ys ie 5% Wier tal aa rh SN oe a ~ j if; ae ‘4 tage sd MY) pes Sb at aeay von ee of ay in yh i he f 4 y i? ches y A “te ‘ y oe zi 4 PPA IW ery Stan + | F ani x Wie. mt a, | ' ¥ Vf j . shear Be q 4 Bis Shh Bod \ i ”, Pe Ce aan ay 7 f ae eat 4)" 4 nO! { u g4A ties , . . nh ih f 7 C ‘> j i, Ps a a y h - Bb | ee | Abeta inte A Rae ) ‘ina ' . Ly : WAS a . ; Je My ’ RED gh h lbp! a ‘ Lz oe Oe ea er Vane) wn hd Teo aly r \ ’ a ain : Man ; P ir oL ae! Ke i. ey A iF fo , ‘ Pah bone Oe bt 13 4 Ve arent é oe id : a tS L } ‘ + a’ 7 Hal mp | rie ene a Ty re meee ete GOA be. A yi Aa na a ' * , a 4 . ‘| g Se i ' a i y * +h) { ( ee aha: | to a te ' DAF * . 4 ; id ton ae . oe vey ae ri B \ * ; er M i} ~ yy P , . A s : a Pe dad 0 ie wf y i , ea ik ca i [ cy _ ‘ “ x ‘ = 7 ’ wv , ‘ ah ‘ / > a . ui uf cS : ci ‘ Be 5 é ; ‘ x rn j THE TASTE OF ACIDS! W. J. CROZIER TWO FIGURES I. The problem of irritability is essentially a matter con- cerning sense organs; in these structures we find cells ‘‘as nearly as possible unifunctional”’ (Lucas, ’09, p. 328) with respect to the capacity for being stimulated. Hence the analysis of irri- tability should properly be founded upon the study of receptor physiology in higher animals, rather than upon an examination of the properties of protoplasm in deceptively simple protozoans. Two contrasted methods have in the past been used in the study of stimulation. The first of these deals with the responses of unicellular organisms, together with the (usually vague) appli- cation of principles derived from cells of more or less generalized type. The second method has consisted in the analysis of sensations. Yet, if we consider such questions as those involved in taste stimulation, we find that no definite conclusions have been reached. The very complexity of organization in ‘simple’ | forms is probably in the main responsible for the results obtained in the first field, results which we find stated in such weak generali- zations as occur in Verworn’s book (’13, pp. 41, 86, 94). ‘The second procedure, the study of human sense organs, has inevit- ably come to be clouded by psychological interpretations. It appears that the possibility of arriving at some understand- ing of what occurs in stimulation has not yet been sufficiently tested out from the standpoint of sense organ irritability as revealed in animal reactions. This paper is a contribution toward that end; it deals with the problem presented by the taste of acids. I am indebted to Prof. G. H. Parker for his criticism of the manuscript. 1 Contributions from the Bermuda Biological Station for Research, No. 46. 453 454 W. J. CROZIER II. Practically the whole of the difficulty is contained in the often considered case of acetic and hydrochloric acids. A compar- ative study of the reactions to these and other acids as given by the earthworm (Hurwitz, ’10; Crozier, ’16), leads to a preliminary simplification of the matter, namely to the formulation of two separate problems: these acids stimulate, they also result in a sour taste. These two problems must be considered inde- pendently; we must distinguish between (a) that property of an acid which causes it to be efficient in stimulation and (b) that which determines the sourness of an acid solution. The relative efficiency of an acid as an agent of stimulation may be measured by the liminal effective dilution. The point I wish to make is that the property of hydrochloric acid which causes it to be tasted in solutions about five times as dilute as that limiting the tastability of acetic acid may have no immedi- ate connection with the common cause of the sourness perceived when these acids act upon the tongue. This point of view contains the possibility of an explanation for (and is, reciprocally, in part justified by) the fact that acids exhibit a characteristic astringency in solutions so dilute that they are no longer sour. There is some evidence, also, that the sourness and astringency may be separated experimentally, as by the use of cocaine. There is therefore ground for the opinion that in taste excitation by acids two processes occur, and that the production of a sour taste is the secondary one. The points which require solution with respect to human taste excitation by acetic and hydrochloric acids are: (a) Both these substances stimulate. (b) They stimulate in different degrees, in dilute solutions the stimulus from hydrochloric acid being the more intense. (c) They both result in a sour taste. (d) They fail to stimulate when too greatly diluted. (e) The dilutions which limit the capacity of the two acids to stimulate are very different (HCl n/900 =; acetic, n/200 =). Each of these points—except (c)—is closely paralleled by the details of earthworm reactions to acids (Crozier, 716), and also THE TASTE OF ACIDS 455 by the reactions of AZolosoma, a freshwater oligochaet (Kribs, “AD ): | It adds somewhat to the clearness of the discussion if, as I have previously proposed (Crozier, ’14, p. 16), we restrict the word ‘stimulus’ to mean the change induced in a receptor by the action of a stimulating agent. The explanation of the acid taste is made easier by the fact that one is not called upon to account for a sour taste resulting from heterologous stimula- tion; there is no good evidence for the existence of a sour taste not directly produced by acid. The extreme specialization of the acid taste makes it a very favorable case for analysis. III. It is sufficiently obvious that only the surface of the receptor is immediately concerned in stimulation. This view is reasonable upon purely morphological grounds, such as the modifications of the exposed ends of sensory cells? and the rela- tions of nerve fibrils to the surface of secondary sense cells. The way in which this conception of the cell surface as the organ of irritability has been elaborated by R. 8. Lillie and others need not be discussed here. But it may be pointed out that the method of interpreting mechanical excitation has been indicated by Osterhout (15) and by such observations as that of Evans and Winternitz (Evans and Schulemann, ‘14, p. 453). Un- equivocal evidence in this direction is afforded by Harvey’s work on the penetration of cells by alkalies (Harvey, ’14 c), by his experiments with acids (Harvey, ’14 ¢), and by my own study of this latter subject (Crozier, °15, °16); the evidence - referred to is, in brief, to the effect that penetration of the body of the cell substance is altogether too slow a process.to account for the rapidity of taste stimulation. The slowness of acid penetration is due to the resistance offered by the cell surface, a resistance which practically disappears with the death of the cell. An examination of cell penetration by various acids, employing a wide range of dilutions, should therefore yield valuable information regarding the composition and behavior of the resistant cell surface. In several cases a study of this 2 The propable chemoreceptors of the earthworm have been described by Miss Langdon (’95) and Bovard (’04). THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 4 456 W. J. CROZIER kind has been made possible by the discovery of intracellular indicators sensitive toward acids (Harvey, ’14b, Crozier, 715). Measurements of cell penetration by acids show, so far as concerns the present problem: 1) that both acetic and hydro- chlorie acids are able to penetrate the cell; 2) other things being /60 140 /20 /00 gO 60 Penetration time, minutes 40 Zot 6 0 100 200 300 400 500 £600 Dilution (Liters containing one equivalent) Fig. 1 The average time of penetration of cells by hydrochloric, butyric, and acetic acids. 1, Acetic acid ) 8, Butyrie acid i integument of Chromodoris zebra, a nudibranch. §, Hydrochloric acid : 2, Butyric acid 4. Hydrocloric¢ acid iO manoma@rozter (2116). ‘ab 2 ie 2, 4, from Harvey (14¢c), at 28°. Butyric acid is introduced for comparison, since Harvey (loc. cit.) gives no data for dilutions of acetic; the principle is the same for both these acids. It should be noted that in Harvey’s experiments the acid was dissolved in a balanced salt solution, in mine in distilled water. f testis epithelium of Stichopus ananus, a holothurian. THE TASTE OF ACIDS 457 equal, the speed of this penetration varies with the acid and its concentration; 3) the curves relating penetration time to con- centration are different for the two acids, dilute solutions of hydro- chloric penetrating much more easily than acetic. Points of re- semblance, in fact of identity, are found among the characteris- tics of stimulation by these acids, and have been enumerated as a, b, e, in section II. The closeness of the parallelism indicates that the stimulus is due to the penetration of the surface layer of the cell. In stimulation by these acids the essential step is the union of the stimulating agent with some constituent of the ceptor surface. According to Becker und Herzog (’07), the decreasing order of intensity of taste sensation due to the acids they tested was HCl, HNOs, trichloroacetic, formic, lactic, acetic, butyric. These acids penetrate the indicator-containing cells of Chromodoris zebra in the order HCl, HNQOs;, formic, monochloroacetic, lactic, butyric, acetic (considering a range of concentrations; Crozier, 16). There is reason to believe that trichloroacetic penetrates more speedily than monochloroacetic; and with this in mind, the parallelism between these two series becomes surprisingly close. This particular case is quoted with the purpose of point- ing out that a small series of acids, such as that just cited, may to all appearances support the view that the intensity of taste excitation is proportional to the ionization strength of the acids; similarly, a penetration series may be chosen which seems to prove a corresponding relation. Yet, further examination of a large number of acids shows that ionization is but one of the factors determining cell penetrating power, and the existence of influences in addition to ionization has also been suspected with reference to acid stimulation; direct evidence upon this point has hitherto been lacking. IV. Analysis of penetration data obtained with eighteen acids has shown (Crozier, 716) that in any given case ability to pene- trate the cell appears to depend upon two (or more) factors: one is the ionization strength of the acid; the second depends upon some other aspect of the acid molecule. To account for these results it is further necessary to assume that the cell surface is 458 W. J. CROZIER of heterogeneous constitution. This conclusion may be extended to include the surface of chemoreceptors, since it enables us to account not only for the reactions of the earthworm to dilutions of acids (Crozier, 716), but also clears up a well known anomaly in regard to human taste. It was shown by Richards (’98) and by Par ibe: (98) that the hydrogen ion must in some way be responsible for the sour taste. Yet acetic acid, which ceases to be tasted at dilutions 60 © Penetration time, minutes A os P SD 10 5) di = = 96) s5c100 Per cent ionization Fig. 2 The relation between percentage ionization and time of cell pene- tration from solutions of hydrochloric, butyric, and acetic acids. 1, Acetic acid 3, Butyrie acid integument of Chromodoris zebra. §, Hydrochloric acid 2, Butyric acid 4, Hydrochloric acid 1, 3, 5, from Crozier (’16). 2, 4. from Harvey (’14c). } testis epithelium of Stichopus ananus. THE TASTE OF ACIDS 459 below about n/200 is only 6 per cent dissociated at that con- centration, whereas hydrochloric acid, fully dissociated (99 per cent), is tasted down to about n/900. The actual hydrogen ion concentrations in these solutions limiting tastability are: HCl (n/900), C,, = 0.00119 N; acetic (n/200), C, = 0.00035 N. Acetic acid is more stimulating than would be calculated from its dissociation. Exactly similar conditions are found in studying the pene- tration of cells by acids, as will be clear from an inspection of the two sets of curves in figure 2. In table 1 acid solutions giving TABLE 1 The ionization of hydrochloric, acetic, and butyric acids, in solutions which pene- trate cells within equal times. For sources of data see corresponding reference numbers in figure 2. IONIZATION, PER CENT TIME OF PENETRATION, Hydrochloric Acetic Butyric MINUTES (5) (4) [1] (3) [2] 5 95.0 94.5 2.0 10 98.5 96.7 2.0 2.1 15 98.8 97.4 2.1 3.3 2.5 20 99.0 97.5 3.0 4.0 2.6 50 4.0 5.5 3.5 the same penetration time appear in the same horizontal row; it is seen that the penetration of cells by acetic acid is much more efficient than if its dissociation were the deciding influence. It follows that the stimulus produced by acetic acid is due, in the first place, to its union with the cell surface; this pene- tration of the plasma membrane is more efficient than if dissocia- tion strength were the determining factor. The indication is that this acid dissolves in a fatty substance located at the cell surface (for further evidence, see Crozier, ’16). The stimulus due to hydrochloric acid also depends upon its penetration of the cell surface, but there is some evidence that it involves proteins. It is not clear to what extent stimulation by acetic and hy- drochlorie acids may be interpreted in terms of the effects which 460 W. J. CROZIER these substances have upon surface colloidal conditions, and thus upon cell permeability (Spaeth, 716). Herlitzka (’10) has discussed this question with reference to the taste of salts, but measurements of the effect of acid on permeability (Oster- hout, 714) have not considered the possibility that characteristi- eally different results may follow from the action of diverse acids; Osterhout’s (14) measurements deal only with hydrochloric acid. V. There remains to be accounted for the production of a unitary taste quality, the sourness of acid solutions. This must be related to ionizable hydrogen (Kahlenberg, ’98, Richards, 98). But it has just been shown that hydrogen ions outside the cell surface cannot be the effective agents, since stimulation by these acids parallels so closely the peculiarities of their pene- tration of the cell. The hydrogen ion must therefore act after the acid has united with the ceptor surface. At least two possibilities are obvious: (1) the presence of potentially ionizable hydrogen within sufficiently concentrated undissociated mole- cules is enough to produce the sour taste, or (2) the acids ionize secondarily after entering. The first suggestion is not so far fetched as it may at first seem, though it is not in accord with current teaching. Numerous parallels could be drawn from the taste of organic substances (Francis and Fortescue-Brick- dale, ’08, pp. 331 et seq.). I believe that this view is possibly correct, although something could also be said for the hypothesis of secondary ionization following some reaction involved in gaining admission to the outer layer of the cell. It is in this latter direction that the explanation (Beutner, ’14) must be sought for the source of electromotive effects accompanying stimulation in general, which presumably occur also in taste excitation.’ This interpretation disposes of the difficulty (very conspicuous in the case of taste) which is encountered by those who would make the process of stimulation and the production of electromotive effects completely identical in every case. 3 A taste cell bathed by a stimulating solution is in a condition very similar indeed to that of the various tissues experimented upon by Loeb and Beutner (Loeb, 715). THE TASTE OF ACIDS 461 SUMMARY An attempt has been made to account for the action of acetic and hydrochloric acids upon the sense of taste. It is shown that the stimulus due to these substances probably depends upon their union with diverse constituents of the ceptor surface. It is thus possible to explain, by comparison with the penetration of cells by these acids: how they stimulate at all, why they stimulate to different degrees, and why acetic acid gives a more powerful stimulus than if it acted by dissociation alone. Why these acids produce a unitary (sour) taste quality is a problem of a different order; it is possible that this depends upon the presence of potentially ionizable hydrogen within undissociated acid molecules, though secondary ionization may also play a part. AGAR’S ISLAND BERMUDA. LITERATURE CITED Becker, C. T. unp Herzoa, R.O. 1907 Zur Kenntnis des Geschmackes. I. Mit- teilung. Zeits. f. physiol. Chem., Bd. 52, p. 496-505. BeEuTNER, R. 1914 Studies on a new kind of E. M. F. Jour. Amer. Chem. Soc., vol. 36, p. 2040-2045. Bovarp, J. F. 1904 The distribution of the sense organs in Microscolex elegans. Univ. Calif. Publ., Zodl., vol. 1, p. 269-286. Crozimr, W. J. 1914 The orientation of a holothurian by light. Amer. Jour. Physiol., vol. 36, p. 8-20. 1915 On cell penetration by acids. Science, N.S8., vol. 42, p. 735-736. 1916 Cell penetration by acids. Jour. Biol. Chem., vol. 24, p. 255-279. Evans, H. M., AND ScHULEMANN, W. 1914 The action of vital stains belong- ing to the benzidine group. Science, N. 8., vol. 39, p. 443-454. Francis, F., AND Fortescur-BrICKDALE, J. M. 1908 The chemical basis of pharmacology. London, 8°, xii + 372 pp. Harvey, E. N. 1914a The relation between the rate of penetration of marine tissues by alkali and the change in functional activity induced by the alkali. Publ. Carnegie Instn. Wash., No. 183, p. 131-146. 1914b Cell permeability for acids. Science, N.S., vol. 39, p. 947-949. 1914¢ The permeability of cells for acids. Intern. Zeits. Physik.- chem. Biol., Bd. 1, p. 463-478. HeruitzkKa, A. 1910 Contributo all’analisi fisico-chimica del sapore dei sali. Archivo d. fisol., vol. 7, p. 557-578. Hurwitz, 8.H. 1910 The reactions of earthworms to acids. Proc. Amer. Acad. Arts and Sci., vol. 44, p. 67-81. 462 W. J. CROZIER KAHLENBERG, L. 1898 The action of solutions on the sense of taste. Bull. Univ. Wisconsin, Sci. Ser., vol. 2, p. 1-31. Kriss, H. G. 1910 The reactions of Molosoma to chemical stimuli. Jour. Exp. Zodl., vol. 8, p. 43-74. Lanapon, Fanny 1895 The sense-organs of Lumbricus agricola, Hoffm., Jour. Morph., vol. 11, p. 193-234, pl. 13. LARGUIER DES BANCELS, J- 1912 Le godt et l’odorat. Paris, 8°, vii + 94 pp. Lors, J. 1915 Electromotive phenomena and membrane permeability. Science, N. S., vol. 42, p. 643-646. Lucas, K. 1909 The evolution of animal function, II. Science Progress, vol. 3, p. 321-331. OsterHouT, W. J. V. 1914 The effect of acid on permeability. Jour. Biol. Chem., vol. 19, p. 493-501. 1915 The nature of mechanical stimulation. Science, N. §., vol. 41, p. 175. Ricuarps, T. W. 1898 The relation of the taste of acids to their degree of dissociation. Amer. Chem. Jour., vol. 20, p. 121-126. Spartu, R. A. 1916 The vital equilibrium. Science, N. S., vol. 438, p. 502- 509. Verworn, M. 1913 Irritability. New Haven, 8°, xii + 264 pp. THE NERVOUS SYSTEM OF PYCNOGONIDS WILLIAM A. HILTON Zoological Laboratory of Pomona College, Claremont, Cal. TWENTY-ONE FIGURES The nervous system of pyenogonids presents many peculiari- ties. It is rather difficult to find the counterpart of this sys- tem in other arthropods. The nervous system of some Crus- tacea suggests it, especially in those forms with an elongated thoracic region and reduced abdomen. ‘The general arrange- ment of the ganglia is totally unlike the central nervous system of arachnids although the general form of the body of ‘sea spiders’ strongly suggesis arachnid relationships. The rather small supraesophageal ganglion and the well developed chain of ven- tral ganglia suggest a rather primitive type of nervous system, but the innervation of the pharynx and proboscis presents com- plex and apparently unique conditions. Although there is an extensive_literature on the classification, structure and development of pyenogonids, there is little or nothing on the structure of the nervous system. The general form of the ganglia with their chief branches is quite well known, for nearly every paper on the classifica- tion of the group contains a more or less detailed sketch of the animals deseribed with the nervous system shown in place. The supraesophageal ganglion seems to contain but two pairs of ganglia recognized by early authors in other arthropods as the protocerebrum and deutocerebrum, the tritocerebrum found in some arthropods being absent. This is but one of several structures that point to a closer relationship with arachnids than with Crustacea. However, without going into further reasons at this time, I am inclined to side with Dohrn and con- sider Pyenogonida a separate class. 463 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 OCTOBER, 1916 464 WILLIAM A. HILTON As the tendency has been to regard these animals as arachnids, it may be worth while to glance through the neurological litera- ture on this group. Among the earliest work on the nervous system of arachnids was that of Treviranus in 1812. No hint of pyenogonids is given in this paper, nor is there any mention of these animals in the work of B. Haller just a century later. There is no refer- ence to pyenogonids in the extensive work of Saint Remy, ’90. Dahl, in 19138, gives a brief summary of the work of Dohrn in connection with various types of arachnids. If we go through the extensive literature on the pyenogonids as a group we find, it is true, little of the structure of the nervous system, but much about the arrangement of the ganglia composing it. From the works of Hoek, ’78, ’81, Dohrn ’70, ’81, Sars 791, Meinert ’98, and a number of others, as well as from the study of Pacific coast forms, we learn that the central nervous sys- tem consists of a supraesophageal ganglion and a ventral chain of from four to five chief ganglia. The smaller number of gan- glia we find when the body is less elongate. The supraesopha- geal ganglion has a ventral median nerve to the proboscis, nerves to the eyes and a pair to the chelifori. Each ventral ganglion has at least one main branch. Three branches from the first ventral ganglion are as follows: 1) A small pair or two pairs to the proboscis; 2) a pair to the palps; 3) a pair to the ovigers; 4) if the first ganglion is fused with the second as it is in those with four ganglia, then there is also a pair to the first pair of walking legs. Figures 1 to 7 show different types of nervous systems from Pacific species of pyenogonids. The method by which the nervous system was studied by some observers was simply to determine the position of the ganglia through the transparent body-wall. This was tried with a number of specimens after the animals had been fixed in mercuric fluids. In some cases the whole animal was stained and mounted in such a way as to show the internal ganglia. In some cases the animals to be studied were placed for a short time in caustic or acid and by one or the other of these methods the internal parts were NERVOUS SYSTZM OF PYCNOGONIDS 465 cleared so that the ganglia might be seen. Serial sections of the whole animals were also made for study, but the chitin often makes perfect series impossible. Hoek and_ possibly } 4 x. 7 - Re = Y. ~eae oR YY Pe os 24 sa Sane j \ { ~ Ge ‘ \ \ a i 8 / w° 62 « f \ SS JIS ¥ t } Qe 1 / fi ( | ie Ses Pi 17 18 { ya Nee ) eee 4 16 Fig. 14 Outline of ventral view of larva and ganglia from below A. erectus, third larval stage. X 85. Fig. 15 Outline of a ventral view of a later stage larva than figure 14 of A. erectus. X 85. Fig. 16 Outline of a dorsal view of a larva of A. erectus about the same stage as figure 14. The brainisshown. X 80. Fig. 17 Fourth stage larva of A. erectus from below. X 35. Fig. 18 Central ganglia of a larva of A. erectus with three pairs of walking legs. The drawing is from below. The upper area without nerves in the figure is the supraesophageal ganglion. X 36. 470 WILLIAM A. HILTON such a period that new ganglionic material seems to be developed. Figures 9, 11 and 14 are drawn from such early stages. At a later moult more ganglia are evident, as in figures 10, 15, and 16. The ventral ganglia at first are mere groups of cells, as is Shown in the frontal section from which figure 10 was taken. As may be seen from the figures 10 and 15, ganglia are devel- oped in each segment, a pair for each appendage and several for the cephalic region and a common mass of cells for the ab- DO, eto 08 es) ats Shut oan SOL OO? OSI ey a), 2) eee oe 2! porn. a 3.58 Fig. 19 A longitudinal section through the central ganglia of Lecythorhynchus marginatus, Cole. Two small abdominal ganglia show at the end of the last thoracic ganglion. 35. Fig. 20 A longitudinal section through the supraesophageal ganglion of L. marginatus. The dorsal side is up, the cephalic side to the right. X 210. dominal. In a stage just before this there are two pairs of ganglia on the dorsal side of the larva; these are shown in fig- ure 16. They represent the brain. At about the third moult, as shown in figures 12 and 17, the ganglia have developed central fibers, but still show their paired nature. There seems to be some indication of more ganglia than there are appendages, some of the caudal elements may not be evident in later stages, and the first ventral ganglion seems composed of two small pairs of elements. In the proboscis Fig. 21. Drawing of the nerves and ganglia of the proboscis of L. marginatus. Slightly diagrammatic. No structures shown in the proboscis but nerves and ganglia. The drawing was made by Miss M. L. Moles from the first sketch taken from the dissection. Much enlarged. 471 472 WILLIAM A. HILTON of this stage there seem to be two small pairs of ganglia. The dorsal ganglia are not shown in figure 17. When the larvae moult again and leave the cavity of the hydroids they have all but one pair of legs. Figure 18 shows the whole central nervous system from below at such a stage. The brain above the esophagus is at the upper end of the figure, then follow the ventral ganglia, seven paired masses and a small unpaired caudal ganglion. There is a gradual fusion of these ganglia until the adult condition shown in figure 7 is attained. The structure of the adult nervous system of pycnogonids is quite simple. There is the same general arrangement of cells that we find in other arthropods. The ventral ganglia have few cells on the dorsal side, but many on the lateral and ventral sides. The supraesophageal ganglion is sheathed in cells on the lateral and dorsal sides. Nerve fibers connect the ganglia and certain regions but in no place is there a concen- tration of the fibers. The fibrous mass is not particularly dense at any point. There do not seem to be many long tracts and the supraesophageal ganglion is not more complicated than other parts so far as could be determined. There are no marked decussations of nerve fibers and the nerve cells pre- sent a uniform appearance. Among the nerve cells are many nuclei of neuroglia networks which form the framework of the ganglia especially in the area of the cells. Although there are indications of special groups of cells and fibers, there was no indications of mushroom bodies. The animals do not seem to have a special brain. The su- praesophageal ganglion is not a very special center. The move- ments of the animals agree with this; they move sideways, forwards or backwards when stimulated. No part of the body seems to lead in the locomotion. NERVOUS SYSTEM OF PYCNOGONIDS 473 BIBLIOGRAPHY ALDERZ, G. 1888 Bidrag till Pantopodernas Morphologi och Utvecklings historia. Bihang till k. Svenska Vetenskap Akad. Hand., Bd. 13, afd 4, no 11, Stockholm. Dau, F. 1913 Vergleich. Phys. und Morph. der Spinnentiere, Erster Teil, Jena. Dourn, A. 1870 Untersuch. tiber Bau und Entw. der Arthropoden. 2. Pycno- goniden. Jen. Zeit. f. Nat., Bd. 5. 1881 Die Pantopoden des Golfes von Neapel. Fauna und Flora des Golfes von Neapel. Monog. 3, Leipzig. Hatter, B. 1912 Uber das Zentralnervensystem des Scorpions und der Spinnen. Arch. f. Micr. Anat.. Bd. 79, Abt. 1. Hauwez, P. 1905 Observations sue le parasitisme des larves de Phoxichilidium chez Bouganvilla. Arch. zool. exp. et gen., 4me serie, t. 3. Hittron, W. A. 1916 The life-history of Anoplodactylus erectus Cole. Jour. ent. and zool., vol. 8, no 1, March. Hoek, P. P.C. 1881 Report on the pyenogonida. Voyage of H. M. 8. Challen- ger, Zoology, vol. 3. 1881 Nouvelles études sur les pyenogonids. Arch. Zool. exp., t. 9, Paris. Hopae, G. 1862 Observations on a species of pyenogon (Phoxichilidium coc- cineum) with an attempt to explain the order of its development. Ann. mag. nat. hist. (3), vol. 9. Kroyer, H. 1842 Notes sur les metamorphoses des pyenogonides. Ann. sc. nat: Ze ser., t. 17. LENDENFELD, R. von 1883 Die Larvenentwicklung von Phoxichilidium plumu- larie. Zeit. Wiss. Zool., Bd. 38. MEISENHEIMER, J. 1902 Beitrage zur Entwick. der Pantopoden. Zeit. f. Wiss. Zool., Bd. 72. Mertens, H. 1906 Eine auf Tethys leporina, parasitisch lebende Pantopoden larva (Nymphon parasitica n. sp.). Mitt. aus der Zool. Stat. Neapel, Bd. 18. Meinert, Fr. 1898 Pyecnogonida den Daniske Ingolf-expedition. Morgan, T. H. 1891 Contribution to the embryology and phylogeny of pycno- gonids. Stud. biol. lab. Johns Hopkins Univ., vol. 5. EVIDENCE OF A MOTOR PALLIUM IN THE FORE- BRAIN OF REPTILES! J. B. JOHNSTON University of Minnesota ONE FIGURE At the title indicates, the purpose of this note is not to describe the structure of the reptilian motor cortex nor to discuss the localization of function within this area. ‘The purpose is only to give such evidence as is now at hand that a specialized area comparable to the mammalian motor cortex probably exists in the reptilian brain and to indicate the general position and extent of this area. The writer has indicated in a previous paper (‘15 b) that the rostral portion of the dorsal pallium in the turtle differs struetur- ally from the rest and has suggested the possibility that this area may correspond to the motor cortical area in the mammalian brain. This hypothesis is being tested by degeneration methods and by cortical stimulation. Sufficiently definite results have been obtained by the latter method to indicate that motor and sensory fields can be distinguished in the reptilian pallium. Three species of turtle (Chelydra serpentina, Cistudo carolina, and Chrysemys marginata) and one lizard (Gerrhonotus) have been studied. Two methods of stimulation were used: induction shocks with two-point electrodes, the two points closely approxi- mated; induction shocks with one point electrode, the second electrode being formed by a copper plate covered with moist cloth on which the body of the animal rested. ; An important factor in the experiments is the degree of anaes- thesia employed. The clearest results have been obtained with very deep anaesthesia. Some of the turtles were given a dose of morphine by hypodermic injection, and all were anaesthet- 1 Neurological Studies, University of Minnesota, no. 22, June 29, 1916. 475 476 J. B. JOHNSTON ized by means of chloroform placed on cotton in the mouth or injected directly into the trachea or both. Although the resist- ance of these animals was well known, in several cases the anaesthesia was not carried far enough and the reactions to cortical stimulation were prolonged contractions of neck muscles or struggling movements of the limbs indicative of pain. In these cases occasional responses, as of the eye muscles or temporal muscle, appeared to be due to local excitation of the cortex but the more extensive and prolonged movements were elicited from any part of the cortex and were not distinguishable from the responses to stimulation of the dura mater, the lower brain regions or even the tissues exposed in the head. Under con- ditions of deep anaesthesia the responses consisted of contraction of a small set of muscles, and of short duration, and these were obtained from a certain region of the pallium only. The lizards are not so difficult to anaesthetize, but long continued appli- cation of chloroform is necessary (one and a half to two hours in a closed dish containing a wad of cotton wet in chloroform). Another factor presenting difficulty is the small size of the brain in these animals. This makes it necessary to use a single point electrode or to have the two points very close together. It is of the greatest importance that the meninges be carefully removed from the whole hemisphere and that the brain and surrounding tissues be kept as dry as possible without injury to the brain tissue. If the electrode comes too close to the dura mater or if fluid facilitates the spread of the current to the dura, the muscles, thalamus or midbrain, the results are vitiated because of responses coming from two or more sources of stimu- lation. The responses which follow stimulation of the dura or brain stem are very different from those produced by cortical excitation, being usually more vigorous and prolonged as well as more extensive. In the lizards and in several turtles, after the dura was laid back and the olfactory and optic connections were cut, the entire forebrain was raised out of the skull so as to be free from contact with other tissues. Under these con- ditions the entire surface of the hemisphere was explored with the electrodes. MOTOR PALLIUM IN REPTILES 477 With the induced current minimal stimuli were applied at first and increased later in the experiment when it became necessary in order to obtain responses. In many cases the brain fatigued quickly and in some it recovered with equal promptness. Individuals differed greatly as to the recovery. Often when the strength of stimulus was increased struggling movements resulted and the experiment had to be discontinued. In some eases also, the animals gave definitely localized responses to the first stimulation and later, probably because of recovery from the anaesthesia, gave irregular and prolonged reactions. CORTICAL REACTIONS IN TURTLES If we take into account only the short contraction of restricted groups of muscles, the following parts of the hemisphere have been found to give muscular responses: dorsal surface of the olfactory bulb, retraction of the neck, extension of the legs, movements of eyeball and eyelid; dorsal surface of pallium near olfactory peduncle and lateral border of pallium in the anterior one-half or two-thirds of hemisphere, movements of eyes, jaw, neck, legs and tail; striatal area, movements of all parts. No responses were obtained from any other part of the dorsal sur- face or the medial wall or from the tuberculum olfactorium, the amygdaloid eminence or elsewhere, except that contractions typical of thalamic stimulation were sometimes obtained when the caudal pole or amygdaloid eminence was being explored. These were probably due to spread of current to the thalamus. The responses from the striatal area were presumably due to the direct stimulation of descending fibers in the crus. ‘The responses from the olfactory bulb may be due to the close proxim- ity of the ‘motor area’ which is indeed overlapped by the caudal border of the olfactory formation. Thus it appears that a somewhat comma-shaped area involving the rostral and lateral borders of the general pallium (fig. 1) may be regarded as a motor area in the turtle’s pallium. This area corresponds roughly to the ‘pallial thickening’ described in a previous paper (715 b). Not enough experiments have been conducted under uniform conditions to furnish a basis for the discussion of localization 478 J. B. JOHNSTON within this area. As the whole area is only a few millimeters in extent, it is obvious that great care must be exercised in accepting as conclusive any apparent indications of special function of particular parts of this area. For this reason I mention only tentatively and with reserve that leg movements have been obtained most often from the anterior part of this area and eye, jaw and neck movements from the lateral portion. Further experiments will be made to determine whether a localization within the motor area clearly exists. In certain experiments Fig. 1 Sketches of the dorsal surface of the right hemisphere of the turtle (left) and lizard (right) on which the motor area is shaded. the eye and jaw movements were homolateral only or more frequently, while the neck and leg movements were mostly heterolateral. Excitation of the dorsal ventricular ridge in the turtle was earried out by cutting away the whole dorsal pallium and apply- ing the electrodes to the ventricular surface of the ridge. _Muscu- lar responses were often obtained, such as retraction of the eye- ball, contraction of temporal muscle and twitching of fore leg, but there is the double danger of spread of stimulus to the closely adjacent pallial thickening and of stimulation of the underlying crus. Lo) MOTOR PALLIUM IN REPTILES 47 MOTOR RESPONSES IN THE LIZARD Only four animals have been examined, two of which gave much clearer and more definite results than those obtained from most of the turtles. From the area shown in the accompany- ing sketch one animal gave movements of the fore leg, jaw and eye muscles, neck and throat muscles, and anterior body muscula- ture. Stimulation of the olfactory tubercle also caused contrac- tion of small muscles in the throat, which was felt as the head was held in the fingers. Stimulation of the crus in the striatal area produced movements of legs and body muscles. Move- ments of the fore leg were regularly heterolateral, although homolateral movements occurred occasionally. Just the op- posite was true of the eye and jaw muscles. The other animal responded to stimulation of the anterior part of this area by strong contraction of pelvic muscles and movements of the hind legs and by weak movements of the fore legs. Stimulation of the lateral border of the right pallium in this animal produced a definite torsion of the fore part of the body. Gerrhonotus is a rather large lizard and its brain is fairly satisfactory to work with. The results in these two cases were so definite that I regard them as strong evidence that a motor area exists in the lizard pallium essentially similar in position and extent to that in the turtle. Early in my studies I examined the brain of a single specimen of Alligator about a foot and a half in length, and obtained motor responses from the anterior part of the dorsal pallium, but I have no written record of this experiment. From these experiments, together with the study of the struc- ture of the hemisphere already reported, I am strongly inclined to believe not only that reptiles possess a general or somatic pallium which has been in dispute until recently, but also that in that pallium are to be distinguished definite sensory and motor areas in the sense in which those terms are commonly used of the mammalian pallium. Localization within these areas and the significance of these areas in the evolution of the mammalian pallium are subjects for further study. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 nay ek jee Spe eg,” THE DEVELOPMENT OF THE DORSAL VENTRIC- ULAR RIDGE IN TURTLES! J. B. JOHNSTON Universily of Minnesota TWENTY-SEVEN FIGURES In the adult turtle (Johnston, 15 b) the large ridge project- ing into the ventricle from the lateral wall of the hemisphere has strong fiber connections with the cerebral peduncle and has obvious close relations with the cortical layers of the pallium in the caudal pole. Indeed, in its caudal part this ridge appears to be a fold of the whole thickness of the brain wall and is marked externally by the ‘amygdaloid fissure.” Here the cell layers of the general pallium turn in to become continuous with the cell masses of the ridge. It is evident from these facts that the relations of the dorsal ventricular ridge are chiefly with the general pallium. Its position lateral and dorsal to both the caudate and lentiform nuclei shows that it does not belong to the corpus striatum. Its independence from other cell masses in its rostral part gives one the impression (from the study of adult material alone), that the ridge has been formed by infolding from the caudal region (15 b, p. 417). The origin of this ridge in the embryo should throw light not only on its relations in the turtle but also on its significance in the evolution of the mammalian brain. I am able to give the main outlines of the development on the basis of a series of early embryos of Chelydra serpentina, for which I am in- debted to Dr. C. E. Johnson of this University; and of sec- tions of 17, 20, and 28 mm. embryos of Chrysemys, belonging to the Anatomical Laboratory of Washington University for the loan of which I am indebted to Dr. Edwin A. Baumgartner. ' Neurological Studies, University of Minnesota. No. 23, July 1, 1916. 481 482 J. B. JOHNSTON The material in both cases was beautifully sectioned and stained. The brains of freshwater turtles are so similar that the younger stages of Chelydra can be readily compared with the later stages of Chrysemys for the rather general purposes of this study. AREAS OF PROLIFERATION IN THE EARLY EMBRYO IN RELATION TO CELL MASSES OF THE ADULT BRAIN Broadly speaking, three periods are to be recognized in the development of cell masses in the telencephalon: a period of evagination of the hemispheres and formation of indifferent cells by mitosis of the germinal cells; a period of form changes and further proliferation of cells in which the successive forma- tion of functionally ind pendent cell masses is noticeable; and a period during which the growth of fiber bundles and other factors bring about the definitive form of the adult cell masses. In the first period the wall of the telencephalon consists of a deep layer of spongioblasts and dividing germinal cells, a thick layer of indifferent cells and a superficial clear layer or marginal veil of very varying thickness. The thickest part of the marginal veil forms the floor of the groove along the lower border of the hemisphere, at its junction with the brain stem. This is the course of the future lateral forebrain bundle or crus cerebri. The indifferent cells form a continuous layer with no indication of individual masses. The second period begins with the appearance of independent cell masses separated by cell-free zones which show in sections as clear lines or areas. Such cell-free zones in adult brains have been commonly regarded as boundary lines between individual cell masses which are presumed to have different functions; i1.e., centers or nuclei. Similar cell-free zones in the brains of lower vertebrates have been used in recent years as landmarks between important morphological regions, as the pallial and basal areas. The writer has pointed out the need of caution in interpreting these zones in this connection (713 b, p. 382). It is now to be noted that the formation of individual masses of cells is dependent upon the proliferation of cells from differ- ent regions of the germinal cell layer and upon the proliferation DORSAL VENTRICULAR RIDGE 483 of cells at successive periods of development. Thus the pro- liferation of cells in the dorsal wall gives rise to pallium, that in the basal wall gives rise to various olfactory centers, corpus striatum, ete. Also, certain centers owe their origin to the proliferation of cells in an early stage, other centers to prolifera- tion in later stages. The former come to lie superficial to the latter and the two are separated by cell-free zones. A further factor is the shifting or spreading of cell masses, whether due to cell-migration or to mechanical forces. These several fac- tors must be taken into account in any attempt to use the em- bryological method in the study of the significance of cell-group- ing in the brain. CHELYDRA SERPENTINA In the oldest Chelydra embryo studied, it is possible to recog- nize several of the important cell masses and fiber bundles of the telencephalon and in part the boundary lines between pal- lial and basal areas. Examination of the model (figs. 1, 2) and of sections (figs. 6 to 14) shows that the telencephalon and dien- cephalon are narrow from side to side and high dorso-ventrally. The hemisphere is a simple sac which projects much farther rostrad than caudad. The interventricular foramen is. still very large. Behind it, the thin wall which represents the choroid plexus extends nearly half way to the caudal pole. This stage illustrates very clearly, both in its general form and its inter- nal structure, how much the rostral pole of the hemisphere precedes the caudal pole in development. The outer surface of the brain shows few of the landmarks which are seen in later embryos and adults. The stem-hemisphere sulcus is of course the most prominent. The olfactory peduncle is not yet formed, but the broad depression on the lateral surface may represent the beginning of constriction. The fissura prima and diagonal band are recognizable at the medio-basal angle in front of the preoptic recess. There is a shallow furrow over the rostral part of the foramen which may be the beginning of the sulcus fimbrio-dentatus. 484 J. B. JOHNSTON The ventricle shows a very sharp ventral groove leading forward from the foramen and much deepened in the region of the future tuberculum olfactorium. The middle ventricular groove is clearly marked in the region rostral to the foramen, while the dorsal groove has not yet appeared. In spite of the simple form of this brain, the internal structure shows considerable advance in differentiation. The sections illustrated in figures 6 to 14 can be understood best by beginning with that which passes through the rostral border of the foramen interventriculare (fig. 9). In the medial wall above the foramen are recognized the primordium hippo- campi and the fimbria, one of the early fiber tracts to be formed in the embryo. The dorsal wall is occupied by pallium, the medial portion by hippocampal, the lateral portion by general pallium. In the lateral wall the pallium seems to be limited by a ventricular groove and by a cell-free space. This bound- ary line strongly reminds one of the lateral zona limitans of the selachian and freg brains (Johnston, ’lla). The later embryos show this groove to be the middle ventricular groove of the adult brain and the clear area becomes the cell-free zone separating the dorsal ventricular ridge and the nucleus lenti- formis in the adult. In the lower half of the lateral wall the following structures are seen in the figure: an active prolifera- tion which gives rise to the nucleus lentiformis, the nucleus caudatus, the lateral and medial forebrain bundles, the anterior commissure bundle, the fiber layer of the diagonal band, and the lateral olfactory area. Although I have not sufficient stages to enable me to follow the history of these structures, there is evidence that the superficial cell layer which represents the lateral olfactory nucleus has been derived from the layers of indifferent cells in the early embryo and that the mass of the caudate nucleus represents a later proliferation. ‘The embryo shows clearly that the lentiform nucleus is beginning to form when the caudate is already completely formed or nearly so. As the other sections are studied it will become evident that the lensiform nucleus is formed by a proliferation distinctly dorsal to the caudate and that the changes in later development bring DORSAL VENTRICULAR RIDGE 485 it into a position lateral to the caudate. There is some indi- cation of the superficial layer of the olfactory area spreading upward beyond the middle ventricular groove (at the point marked l.pyr). Passing caudad from this level, in the next section drawn (fig. 8) the same structures are to be seen in the lateral wall. The middle ventricular groove has almost disappeared, however, and the proliferating area for the lentiform nucleus is much smaller. The next section drawn (fig. 7) is the last one in which the lentiform proliferation can be recognized and with it the locus of the pallial border. In the lower part of the hemisphere the lateral olfactory area is represented by a large collection of cells closely related to the caudate nucleus and the diagonal band. This is the nucleus of the lateral olfactory tract and the next section shows the close relation of both this and the diago- nal band to the stria medullaris. Caudal to this the nucleus of the lateral olfactory tract soon disappears and the hemisphere wall presents the appearance of an undifferentiated pallium, except for the fimbria and the thin choroidal area in the medial wall. The next section rostrad from the one first described passes through the anterior commissure (fig. 10). The lentiform pro- liferation appears larger, the diagonal band fibers approach those of the olfacto-hypothalamic system. In figures 9, 10, and those of sections farther rostrad, one of the most noteworthy features is the mass of cells proliferating from the deep layers of the pallum. The medial border of the pallium does not show active proliferation but in the lateral three-fourths many new cells are forming and these appear to be streaming laterad into the thick wall just dorsal to the middle ventricular groove. Indeed the proliferation and streaming of these cells is the active cause for the thickening of this part of the wall and the forma- tion of the middle ventricular groove. Further thickening of this part of the wall deepens this groove and produces the dor- sal ventricular groove at the point indicated in figure 9. In figures 9 to 12 appear important relations of the dorsal border of the olfactory area to the pallium. Here it is quite 486 J. B. JOHNSTON clear that the superficial layer of cells of the olfactory area ex- tends up on the outer surface of the pallium and that it is ac- companied by a special bundle of fibers. The crowding of cells in this stage is such that 1t is impossible to determine whether the pyriform lobe cells have spread or migrated up from the lateral surface in the basal region or have been formed here by the proliferation which at this stage is giving rise to the pal- lium. Farther caudad and in later stages the relations are such as to make the former appear more probable. The cells of the pyriform lobe probably all come from the layer of indifferent cells seen in earlier stages and formed by proliferation in the lateral and basal region before the pallial proliferation seen in this stage began. Pallium and pyriform lobe are still visible in a section (fig. 14) through the peduncle in which the olfactory formation appears in the dorsal wall. The olfactory formation extends far back on the dorsal surface, as is already well known. CHRYSEMYS Models were made of the 17 mm. and 28 mm. stages. Most of the chief landmarks of the adult brain are already visible in the 17 mm. embryo (figs. 3, 4, 15, 16, 17). The caudal pole still lags behind the rostral portion in development. ‘The cau- dal pole projects much farther back than in the oldest Chelydra embryo and in the 28 mm. stage the olfactory area (lobus pyri- formis) extends relatively farther back than in the 17 mm. stage. The tuberculum olfactorium is well developed and the ventral groove of the ventricle dips deep into it. The mid- dle ventricular groove (fig. 4 and sections) is very sharp and deep in the middle part of the brain and stops abruptly just caudal to the level of the foramen interventriculare. The dor- sal ventricular groove appears in the 17 mm. embryo (figs. 4, 17) as a slight groove in the lateral wall some distance from the medio-dorsal angle of the ventricle. Three sections caudal to the foramen are drawn from the 17 mm. embryo and nine sections from various leve's of the 28 mm. embryo. The internal differentiation has already pro- gressed so far in the 17 mm. embryo that it will be unnecessary DORSAL VENTRICULAR RIDGE 487 to speak of it separately. In both stages many features of the sections can readily be compared with the structures seen in corresponding sections of the adult brain. For this pur- pose the reader should consult the description of the cell masses in the brain of Cistudo in this Journal for October, 1915. The necessary description of individual sections is given in connec- tion with the figures. Here I shall take up the relationships of the lateral margin of the pallium and the dorsal ventricular ridge. Corpus striatum and lateral olfactory area. In the younger embryos the loosely arranged cells of the lateral olfactory cen- ters cover the outer surface of the caudate and lentiform nuclei and form a continuous layer over the lateral surface of the hemisphere (except dorso-caudally). This is shown in figures 7 to 12, and this interpretation is given to the models. In the 28 mm. embryo there is a small area opposite the foramen in which the striatum and the crus bundle contained in it come to the surface (fig. 21) and the olfactory centers form the pyri- form lobe above and the diagonal band below. This small area is painted blue in the model and appears white in figure 5. Sections show that it is the elbow of the crus, with certain large cells contained in it ('15 b, p. 405), which comes to the surface here. This is presumably due to the rapid growth of both ascending and descending fibers in the crus. The crowding thus produced leads to the shifting of the cells of the olfactory center upward and downward. This process must go much further in later development until the large striatal area is left exposed in the adult. The embryonic history thus seems to support very clearly the hypothesis (15 b, p. 428, 429) that the exposed striatal area in the turtle had been covered at an earlier stage of evolution by the lateral olfactory area. Relations of pyriform lobe and pallial margin. In the caudal half of the hemisphere the pyriform lobe forms a layer external to the corpus striatum and extends up to a thin edge superficial to the dorsal ventricular ridge and the margin of the pallium. This overlapping of the pallial margin by the pyriform lobe persists in the adult. At about the level of the interventricular 488 J. B. JOHNSTON foramen the pyriform lobe begins to extend higher on the lateral surface. At the same time thickening of the lateral border of the pallium moves dorsally, becomes crowded and folded on itself and sinks in, forming a projection into the ventricle (figs. 22 to 25). These changes take place rapidly from be- hind forward so that in thirty or forty sections of 10 microns the place occupied by the thick border of the pallium comes to be taken by the pyriform lobe. From this point forward the pyriform lobe forms a characteristic ridge filled with a dense layer of cells, very much as in the adult. Origin of dorsal ventricular ridge. In the Chelydra embryo above described the dorsal part of the lateral wall is thickened by cells coming from a proliferating area in the dorso-lateral wall. The groove which bounds this thickening was identified with the middle ventricular groove of the adult. In the older embryos of Chysemys the groove is readily identified but has been very greatly deepened by further thickening of the mass just above it. Likewise the thickening of this mass has pro- duced a groove above it, the dorsal ventricular groove. The thick mass, then, is the dorsal ventricular ridge and in earlier stages it is indistinguishable from the dorsal pallium. In the caudal part of the hemisphere of the older embryos the relations of the dorsal ventricular ridge are not essentially changed from those seen in the Chelydra embryo. Caudal to the level of the stria medullaris (figs. 18, 19) this ridge bulges into the ven- tricle and is covered outside and below by the thick mass of the nucleus of the lateral olfactory tract. In the caudal pole the ridge merges gradually with the general pallium caudal to the level at which the pyriform lobe ends In the rostral part of the hemisphere (figs. 22, 23) the dorsal ridge has become prac- tically independent of the pallial thickening, very much as it is in the adult. From these embryos it is quite clear that the dorsal ventricular ridge arises as a thickening of the lateral border of the pallium and that its continued growth causes is to project into the ventricle and produces the middle and dor- sal ventricular grooves. In the 28 mm. embryo the mass of cells which in the adult was called the core-nucleus of the ridge DORSAL VENTRICULAR RIDGE 489 is clearly recognizable in the rostral part, back to the level of the caudal border of the foramen interventriculare. Between the pallial thickening and the cells of the dorsal ventricular ridge appears in the sections a clear space filled by fibers. In the caudal part of the hemisphere, where the dorsal ventricular groove is already formed, this bundle of fibers is seen to hold the same position as an important bundle in the adult. In the adult many fibers from the crus enter this bundle or, more probably, pass through it on their way to the pallium. Here in the embryo the crus is not yet sufficiently developed to enable one to trace it up through the striatum to this level. The strong development at this stage of the bundle here men- tioned indicates that it is composed chiefly of fibers connecting parts of the hemisphere; an association bundle, in other words. Relations of dorsal ventricular ridge to basal structures. In the rostral part of the hemisphere the ridge is from the first clearly marked off from the striatum below by the middle groove and by a prominent cell-free zone. In later embryos this cell- free zone becomes compressed into a narrower line as seen in section and scattering cells are found in it. There is, however, always a clear boundary between the ridge and the developing lentiform nucleus. In section the cell-free zone inclines dorsad toward the lateral surface and passes above the lateral olfac- tory area. In the rostral part, where the pyriform lobe crowds far dorsal this limiting zone is bent into a semicircular or U- shaped line (figs. 28 to 27). When the writer first studied the adult turtle brain he found it impossible to establish a direct comparison between the lateral zona limitans of the selachian, frog and other simpler vertebrates, and either of the cell-free zones seen in the lateral wall of the turtle brain. It is now evident that the clear zone which in the adult brain passes from a point near the middle ventricular groove toward the lateral surface and then bends dorsad to reach the surface in the sulcus rhinalis above the lobus pyriformis, is the zona limitans of the embryo and corresponds to the zona limitans lateralis in the selachian and frog. (See Johnston, 715 b, figs. 19 to 22 and compare with ’1la, fig. 75). Since the relations of the pyri- 490 J. B. JOHNSTON form lobe, corpus striatum and pallium in the turtle are readily comparable with those in the mammal, the identification of the lateral zona limitans in the turtle completes the history of this important landmark throughout the series of vertebrates. The boundary lines between the pallial and basal portions of the hemisphere are now clear in both medial and lateral walls in fishes, amphibians, reptiles and mammals (Johnston, ’11 a, Altovlon Ilkley The relation of the dorsal ventricular ridge to the amyg- daloid complex requires further comparative study. In the adult, it will be remembered, the caudal part of this ridge is separated from the rest by a broad shallow groove which con- tinues in the general direction of the deep middle ventricular groove (15), figs. 4 and 10). This caudal portion of the ridge is made up of the medial amygdaloid nucleus and of an apparent infolding of the general pallium. Even in the oldest of these embryos the dorsal ventricular ridge extends into the tip of the caudal pole as a simple thickening of the general pallium. It seems probable, although the evidence is not sufficient for a definite conclusion, that the caudal portion of the ridge in- cluding the large-celled medial amygdaloid nucleus is formed from this thickening of general pallium. For the photographs of the models I am indebted to Dr. W. F. Allen of the University of Oregon. LITERATURE CITED JounsTon, J. B. 191l1a The telencephalon of selachians. Jour. Comp. Neur., vol. 21. 1913 b The morphology of the septum, hippocampus, and _ pallial commissures in reptiles and mammals. Jour. Comp. Neur., vol. 23. 1915 b The cell masses in the forebrain of the turtle, Cistudo carolina. Jour. Comp. Neur., vol. 25. DORSAI VENTRICULAR RIDGE 491 FIGURES ABBREVIATIONS a.p., area parolfactoria b.0., bulbus olfactorius c.a., commissura anterior c.h., commissura hippocampi ch. op., chiasma opticum c.p.a., commissura pallii anterior crus, crus cerebri or lateral forebrain bundle c.st., corpus striatum d.b., diagonal band of Broca d.v.r., dorsal ventricular ridge fi., fimbria f.o., formatio olfactoria f.p., fissura prima for.i., foramen interventriculare f.rh., fissura rhinalis g.p., general pallium g.s., gyrus subcallosus h., hippocampus hy., hypothalamus l. pyr., lobus pyriformis l.t., lamina terminalis m.fb.bdl., medial forebrain bundle n.c., nucleus caudatus n.d.b., nucleus of the diagonal band n.l., nucleus lentiformis n.o., nervus olfactorius n.rot., nucleus rotundus n.tr.olf.lat., nucleus of the lateral ol- factory tract pa., pallium pa.th., pallial thickening p.h., primordium hippocampi p.o., pedunculus olfactorius r.p., recessus praeopticus r.s., recessus superior s.f-d., sulcus fimbrio-dentatus s.m., stria medullaris s.v.d., dorsal ventricular sulcus s.v.m., middle ventricular sulcus s.v.v., ventral ventricular sulcus thal., thalamus t.o., tuberculum olfactorium tr.olf-hy., tractus olfacto-hypothalam- icus tr.op., tractus opticus 492 J. B. JOHNSTON Fig. 1 Chelydra serpentina. The size of this embryo is indicated by the owner by the words ‘8.5 mm. carapace.’ Lateral view of a model of-the right hemisphere. Description in the text. This and the following models have been merely rubbed with a blunt instru- ment to smooth the surface. The external surface has been painted so that the olfactory centers and hippocampus appear dark and the general pallium light. The diencepha'on has been left with the gray color of the modelling paper and the ventricu‘ar surface of the hemisphere has had no treatment whatever. The lamina terminalis and line of attachment of the choroid plexus have been painted white. All the models were made at a magnification of 50 diameters and are reduced one-third in reproduction. DORSAL VENTRICULAR RIDGE 493 2 Fig. 2. Medial view of the model shown in figure 1. Description in the text 494 J. B. JOHNSTON DORSAL VENTRICULAR RIDGE 495 Fig. 3 Chrysemys embryo of 17mm. Medial view of a model of the right hemisphere. The narrowing of the interventricular foramen seems to have taken place from above downward on account of the expansion of the hippo- campus and general pallium. The white area labelled ¢.h., is the cut surface of the primordium hippocampi in the median plane. The thin portion of the medial hemisphere wall which will form the choroid fissure and plexus is bounded by a white line. Fig. 4 Same model as figure 3, with the thalamus, hippocampus and primor- dium hippocampi removed. The lateral wall of the ventricle shows the dorsal ridge bounded below by the deep middle ventricular groove as described in the text. The dorsal groove is shallow. There is no evident boundary between the dorsal ridge and the pallial thickening at the rostral end. Fig. 5 Chrysemys, 28mm. Lateral view of a model of the right hemisphere. Note the great elongation of the pyriform lobe in the caudal pole as compared with the condition in figure 1. With regard to the exposure of the corpus striatum on the lateral surface, compare figures 1 and 5 with the figures in ’15 b. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 496 J. B. JOHNSTON Figs. 6 to 14 Transverse sections through the right hemisphere of the Chelydra embryo illustrated in figures 1 and 2. Fig. 6 Section just caudal to the foramen interventriculare. This is the most caudal section in which the nucleus caudatus can be recognized and here it practically fuses with the nucleus of the lateral olfactory tract. Both are undoubtedly parts of the primitive basal olfactory centers which have been car- ried out into the caudal pole of the hemisphere evagination. There is in this section no apparent boundary between these nuclei which enter into the amygda- loid complex and the general pallium. Fig. 7 Section through the caudal part of the foramen, 120 microns rostral to figure 6. The nucleus caudatus is much more distinct and the proliferation of cells for the nucleus lentiformis begins to appear. There is now a slight indi- cation of the latera. boundary of the pallium. The letters s.v.m. in this and figure 8 indicate the position in which the middle ventricular sulcus is to be formed, although it is not actually present in these sections. Fig.8 Section through rostral part of the foramen, 100 microns rostral to igure 7. The lateral olfactory area is represented chiefly by cell masses adjacent to the diagonal band. The nucleus caudatus begins to be separated from this area by the crus entering the hemisphere. The lentiform proliferation is larger and the clear space just above it, which corresponds to the zona limitans lateralis in lower vertebrates, is present from this level forward. Fig.9 Section through the rostral wall of the foramen. The position of the foramen is represented by the light space at the medio-ventral angle of the ventricle. The crus has now separated the nucleus caudatus from the nucleus of the diagonal band and both are somewhat removed from the larger mass of the lateral oifactory area which is here labeled /.pyr. The middle ventricular sulcus which is present in this section extends farther caudally in order embryos and adult. 497 498 J. B. JOHNSTON Fig. 10 Section through the anterior commissure. Although the lateral ol- factory area is continuous in these sections and even the nucleus caudatus is nowhere wholly separated from it, one can clearly see the beginnings of the segregation of the pyriform lobe, diagonal band and caudate nucleus by reason of the enlargement of the lateral forebrain bundle (crus). This process will be completed in later stages by the development of the lentiform nucleus and the further enlargement of the crus. Fig. 11 Section just rostral to the preoptic recess where the diagonal band turns up in the medial wa.l. The dark mass on the medial surface of the area parolfactoria represents the beginning of the nucleus of the diagonal band, which corresponds to the gyrus subcallosus of the mammalian brain. The nucleus caudatus is connected beneath the ventricle with the area parolfactoria. From this level rostrad the olfactory area or lobe is quite continuous from its lateral border at l.pyr. to its medial border near fi. Fig. 12 Section 60 microns rostral to figure 11, where the diagonal band fibers rise up in the medial wall to join the fimbria. Although the cells of the pyri- form lobe and pallium lie immediately in contact, there appears in many sec- tions a very sharp line as if it were caused by a delicate septum running dorsad from the small fiber bundle beneath l.pyr. Fig. 13 Section through the tuberculum olfactorium. Note that the ventral ventricular suleus here descends nearer to the surface than elsewhere so that the somewhat bulging tuberculum contains a large pouch of the ventricle. Fig. 14 Section through the olfactory peduncle. The ventral part of the section cuts the rostral wall of the tuberculum while its dorsal part cuts the olfactory formation. DORSAL VENTRICULAR RIDGE 499 500 J. B. JOHNSTON Figs. 15, 16, 17 Three transverse sections of the right hemisphere of the 17 mm. Chrysemys. Figure 17 falls in the caudal part of the foramen and figure 15 represents next to the last section in which the connection of the hemisphere with the thalamus is seen. Figure 16 is between these two. In this embryo the mass formed by the pallial proliferation of the earlier stage has become divided into a general pallium and the dorsal ventricular ridge. The ridge projects into the ventricle forming a deep middle ventricular suleus and a shallow dorsal suleus. Note that the dorsal sulcus is quite independent of the dorso-medial angle of the ventricle. The dorsal ventricular ridge therefore is clearly a thickening of the lateral wall above the lateral zona limitans. This zona limitans is the ight streak in the drawings running diagonally between d.v.r. above and n.l. and l.pyr. below. The nucleus of the diagonal band is very large and definite in figure 17, while in figure 15 and 16 its substance is fused with the nucleus of the lateral olfactory tract. The lentiform proliferation is still in progress, especially from the dorsal lip of the middle sulcus. The peculiar arrangement of ependyma cells seen at this point in the adult already begins to be apparent here. Figs. 18 to 27. Transverse sections of the right hemisphere of the 28 mm. embryo of Chrysemys. Fig. 18 Section at about the point where the fimbria curves down behind the choroid area. Caudal to this the nucleus of the lateral olfactory tract soon disappears and the dorsal ridge continues on into the caudal pole as a simple thickening of the lateral walls. At g.p.? is a partly segregated mass of cells which probably represents general pallium extending from the caudal pole for- ward to this point in the basal wall as it does in the adult. DORSAL VENTRICULAR RIDGE 501 502 J. B. JOHNSTON Fig. 19 Section 120 microns rostral to figure 18. In both these sections the connection of the dorsal ridge with the pallium is clear and they illustrate the more embryonic conditions which prevail in the caudal pole than in the rostral part of the hemisphere. Fig. 20 Section at the point of connection of the hemisphere with the thala- mus where the stria medullaris passes into the thalamus. In this and the next two figures the lateral zona limitans appears as a diagonal light line running dorsolaterally from s.v.m. to the outer surface. Kig. 21 Section through the foramen interventriculare. The lentiform and caudate nuclei, neither of which was distinguishable in figure 20, are here quite distinct. The crus in this figure separates the caudate from the nucleus of the diagonal band. From this point rostrad for thirty to forty sections the crus lies practically exposed on the lateral surface. This condition is represented by a small striatal area in the model of this brain (fig. 5). Fig. 22 Section through the anterior commissure. Here the general pallium begins to be decidedly thickened. The motor area of the adult includes this region and extends farther caudad. Forward from this level the olfactory area is again continuous on the lateral, basal and medial surfaces. DORSAL VENTRICULAR RIDGE 503 504 J. B. JOHNSTON Fig. 23 Section through the rostral wall of the preoptic recess and the nucleus of the diagonal band (gyrus subeallosus). Between the level of figure 22 and this the pyriform lobe has rapidly pushed up over the outer surface of the pallial thickening as described in the text. The dorsal ridge is decreasing in size and the arrangement of its cells in this and in figure 22 suggests the core-nucleus of the adult. The dorsal ridge and the pallial thickening form a common large ridge, while in the adult they are separated by a groove (see 715 b, fig. 10). Fig. 24 Section through the rostral end of the dorsal ridge and through the fissura prima, in which the fibers of the diagonal band are turning up into the medial wall. Both pallial thickening and pyriform lobe are massive. Figs. 25, 26 and 27. Sections through the anterior part or motor area of the general pallium. The whole of what is called general pallium here becomes more massive in the adult and is included in the pallial thickening. An important feature of these sections is that the general pallium especially at the dorso-medial angle is composed of conspicuously large cells. If the results of experiments re- ported elsewhere in this journal are to be credited, this is certainly a part of the motor area and probably is concerned with the control of the limbs. The pres- ence of the large cells is readily understood if this is true. DORSAL VENTRICULAR RIDGE 505 THE MORPHOLOGY AND MORPHOGENESIS OF THE CHOROID PLEXUSES WITH ESPECIAL REFER- ENCE TO THE DEVELOPMENT OF THE LATERAL TELENCEPHALIC PLEXUS IN CHRYSEMYS MARGINATA PERCIVAL BAILEY From the Anatomical Laboratory of the Northwestern University Medical School TWENTY-SEVEN FIGURES CONTENTS MET RUCHOULOULOTI one tap 6 ice a akbar b ninth wee wise ides iwletahe eee eer ae 507 ANS UOE Ye 26 osa/5, abs stevials wah b & >, Sea etane nb v'S der via a) Ah ce eee > 508 Material and Methods........... ate ar Sasin ha ta SRO RC ee a 519 IDEROTIPULON:.« >. 02 ss'< > « Pe idle OE aR hoa caren Ue ahaun b jars St RRR nectar aeee e 520 MDIACUESION: aces 2 ec0s Lei eer PO na ee Pe 525 GONG IBLOUS ee is ol vans v's wp un ey ea Wy) -0) - Fal e 528 INTRODUCTION In a recent communication, the author (Bailey, 715) presented an interpretation of the lateral telencephalic choroid plexus in the human embryo based on ontogenetic and phylogenetic evi- dence. The phylogenetic evidence presented consisted of the most accurate descriptions available of the development of this structure in the lower vertebrates, viz., Warren’s (’05) account of Necturus maculatus and Tandler and Kantor’s (’07) account of Platydactylus mauritanicus. With a view to confirming, and strengthening if possible, the phylogenetic evidence it was deter- mined to investigate more definitely the development of the prosencephalic choroid plexuses in others of the lower verte- brates. The following scheme taken from Wilder was accepted 1 Contribution No. 42. Submitted for publication July 20, 1916. 507 50S PERCIVAL BAILEY to represent as accurately as possible the phylogenetic stages of greatest value: Amphioxus Cyclostomes Selachians Ganoids Urodeles (for Stegocephali) Chelonia (for Theromorphs) Monotremes (for Pantatheria) Marsupials (for primitive Insectivora) Insectivora Lemurs (modern Mesodonta) Cercopithecoidae (tailed monkeys of Old World) Tailless Apes (Gorilla, ete.) Pithecanthropus (extinct) Homo primigenius (extinct) Homo sapiens Below the Urodeles, the lateral telencephalic choroid plexus is very rudimentary if it appears at all. Its condition in the Urodeles has been thoroughly elucidated by Warren’s (’05) ac- count of Necturus maculatus. For the Chelonia, Chrysemys marginata was selected because it was readily accessible and also because a great part of the work had already been admirably done by Warren (711) in the course of his investigation of the ‘“‘Paraphysis and Pineal Region in Reptilia.”’ It stands a little closer to the line of ascent of the mammals than the lizard, Platydactylus mauritanicus, described by Tandler and Kantor. Of the forms above the Chelonia, only Didelphys virginiana representing the Marsupials, was available. The present com- munication is concerned with Chrysemys marginata and a review of the literature, leaving Didelphys for a later paper. HISTORY There are no choroid plexuses in Amphioxus (Burckhardt, ’94). Ahlborn (’83) describes well developed plexuses in the roof of the hindbrain of Petromyzon planeri, and also in the roof of the midbrain. The plexus of the diencephalic roof is not so well developed and no reference is made to any telencephalic plexuses. Burckhardt (94) writes of Petromyzon fluviatilis: MORPHOGENESIS OF THE CHOROID PLEXUSES 509 Auch hier wie bei Teleostiern bleiben die Plexus inferioris (median telencephalic choroid plexus) nur in Gestalt einer Querfalte nach- weisbar, die Paraphyse ist eine blosse Kuppel, der caudalen Rand wir als rudimentidres Velum auffassen. There are no lateral telencephalic choroid plexuses in Cyclo- stomes. Among the Selachians, however, there is a well formed plexus inferioris in Notidanus according to Burckhardt (94), but no lateral telencephalic plexuses appear. On the other hand, I infer from Kappers and Carpenter (711) that in Chimaera monstrosa the lateral plexuses are present. Der ependymale Theil der Schluszplatte w6lbt sich in ihrem frontal- sten Abschnitt etwas iiber dem Niveau des Gehirnes hinaus eine Art paraphyse darstellend um sich dann pl6étzlich wieder einzufalten und in den unparen Ventrikel eindringend den plexus chorioideus ventriculi imparis zu bilden, wovon auch geringe Ausliufer in den schmalen Seitenventrikeln des Gehirns eindringen. And Minot (’01) in deseribing Aecanthias remarks: The velum has now distinctly the character of a choroid plexus, being very irregular in the form of its surface, rich in blood vessels, covered by a thin ependyma and projecting far into the cavity of the brain. Laterally the projections from its surface are much more developed and as the organ has grown forward alongside the median paraphysal arch, it has produced what we can now easily identify as the plexus of the lateral ventricle. These plexuses are therefore to be interpreted morphologically as secondary modifications or appendages of the pri- mary velum transversum. . . . . Attention should be paid to the two lateral projections, L.ch., of the ependyma on the anterior surface of the velum, because these projections not only fix the lateral bound- aries of the paraphysal arch but also are the anlages of the choroid plexuses of the lateral ventricles. These anlages from this stage (28.0 mm.) on rapidly increase both in size and in complication of form. D’Erchia (96) shows in Torpedo the velum transformed into a plexus and the tela chorioidea diencephali practically non- existent. In fact, this seems to be the tendency of these two structures in the entire Selachian group. There is a plexus in the roof of the fourth ventricle in all Selachians. No plexus develops in the tela chorioidea telencephali medi in Ganoids, but in Acipenser, von Kupffer (’93) figures a plexus formation arising from the anterior wall of the velum trans- 510 PERCIVAL BAILEY versum, and Terry (10) describes such a formation in Amia, as well as folds which appear on the caudal wall of the velum. The tela chorioidea diencephali itself forms a large thin-walled dome with no plexus formation. ‘There is a plexus in the roof of the fourth ventricle. Lateral telencephalic choroid plexuses according to Burckhardt (’94) are absent, but Hill (94) writing of Amia says: “It (the paraphysis) may be thought of as an iso- lated portion of the roof of the fore-brain which owes its existence to the formation of the folds marked Pl.chr. in figure 20, and which are themselves the representatives of the choroid plexuses of the lateral ventricles.” Of the Urodeles, Burckhardt (’91) has described extensive plexuses developing both from the tela chorioidea telencephali medii and from the tela chorioidea diencephali in Ichthyophis. Warren (’05) shows the enormous diencephalic plexus of Necturus absorbing also the entire caudal wall of the velum transversum. There is a plexus in the roof of the fourth ventricle. The lateral telencephalic plexuses are present and arise from the base of the median telencephalic plexus (plexus inferioris) as has been at- tested by Mrs. Gage (93), Studnicka (’93), Warren (’05) and Burckhardt (91). Burekhardt (91) says of Ichthyophis: ** Die Plexus der Hirnhemisphiren aber spalten sich je in zwei Stimme von denen der eine sich gegen das Zwischenhirn ausstreckt, und in der Folge zuerst sich in Zweige spaltet, indess der andere in den Hemisphirenventrikel eindringt und sich sodann in zwei Zweige spaltet, einen nach riickwarts umbiegenden, welcher den Ventrikeltheil des Temporallappens und einen, welcher das iibrige Vorderhirn versorgt.’”’ And Warren (’05) writes con- cerning Necturus: “The telencephalic plexus develops from the paraphysal arch. .’ “The plexuses of the hemi- spheres arise on either side from the origin of the telencephalic plexus and pass into the lateral ventricles.” Concerning the Chelonia, Humphrey (’94) shows a plexus aris- ing from the tela chorioidea telencephali medi in Chelydra ser- pentina, as does also C. L. Herrick (91) in Cistudo. Warren (11) states that the plexus is not present in Chrysemys marginata and with this statement my observations agree. The “two ‘MORPHOGENESIS OF THE CHOROID PLEXUSES 511 paired masses growing backward from the origin of the lateral plexus into the diencephalon” of which Warren writes do not seem to me to be at all homologous with the median telencephalic plexus. They arise too far posterior on the plexus; are at no time connected with the roof plate; and are merely prolongations of the lateral plexus. The diencephalic plexus is not so well devel- oped as in Urodeles but is present in all forms, as is also the plexus of the fourth ventricle. Humphrey (94) shows in Chelydra serpentina the lateral telencephalic plexus arising from the base of the median telencephalic plexus and passing into the lateral ventricle. According to Warren (11), in Chrysemys marginata, “The plexus chorioideus lateralis springs from the paraphysal arch immediately in front and lateral to the mouth of the para- physis, figure 25, and invaginates the dorso-mesial wall of the hemisphere.” In considering the Mammalia, considerable space will be given to interpreting accurately the velum and puraphysis. This is rendered necessary if one is profitably to homologize the lateral telencephalic plexus of Mammalia with the same structure in the lower vertebrates. Since, as we shall see later, the paraphy- sis never appears in Mammalia except as an arch of the roof plate of the telencephalon in early stages of development, it will be referred to as the paraphysal arch. Minot (’01) used the term ‘to apply to the entire roof plate of the telencephalon between the velum transversum and the lamina terminalis, a sense in which it is no longer used. The only observations of value dealing with the Monotremes are those of Th. Ziehen (’05) on Echidna hystrix and G. Elliot Smith (97) on Ornithorhynchus, and these leave much to be desired. Smith describes the origin of the lateral telencephalic plexuses and a structure which he says ‘“‘constitutes the para- physis of Selenka.”” I do not know what evidence he had for stating that the structure he describes is the paraphysis of Selenka for so far as I have been able to determine, Selenka has nowhere a description of the paraphysis but merely the bald statement that it is present in Marsupials. Smith’s statement probably implied no more than that he interpreted this structure as the THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 512 PERCIVAL BAILEY paraphysis. Extracts from this paper follow and will be dis- cussed later. The lamina supraneuroporica takes a sudden bend backwards (fig. 3) to form a horizontal band, which gives origin in many lowly verte- brates to the plexus inferioris, and in higher animals to the plexus lateralis as well, or exclusively. In the specimen under consideration, however, although the plexus laterales do not actually spring from this lamina, they are formed from the caudal prolongation of its lateral parts. . . . . In describing the structures met with in a medial sagittal section it was mentioned that the dorsal part of the (actual) anterior wall of the median cavity of the forebrain was bulged out to form a large sac. The corresponding structure is well seen in the early embryo of Parameles. . . . . In the Platypus embryo, however (fig. 2), a well developed choroidal fold extends from the superior commissure to the lamina from which the lateral plexus arises, com- pletely invaginating the paraphysis (figs. 7, 9 and 15) in the middle line. In Platypus the transition from optic thalamus to paraphysis is a very gradual one, so that in examining a series of coronal sections the lateral walls of the diverticulum would seem to be merely the forward continuation of the ependymal layer of the Fligelplatten (fig. 15). As a matter of fact, the appearances in this series of coronal sections are not deceptive at all, the sac being undoubtedly. just what it appears to be, an anterior pouch of the choroid plexus of the diencephalon, the lateral walls of the pouch being actually the forward continuation of the Fliigelp’atten. A glance at figures 1, 2 and 3 will make this quite apparent. The similarity would be much more obvious if figure 3 were from a coronal instead of a transverse section. In front of this pouch lies the velum transversum and then the roof plate of the telencephalon (lamina supraneuroporica as he calls it), from the caudal prolongations of the lateral parts of which arise the lateral telencephalic plex- uses. In a later article, Smith (03) reaffirms his belief that the anterior extremity of the lateral telencephalic plexus arises from the roof plate. The embryo is doubtless too far advanced to show the true paraphysal arch. Ziehen’s work on Echidna is eminently unsatisfactory from the standpoint of this discussion, because the sections he shows invariably skip the anterior end of the lateral telencephalic plexus. He has but one suggestive statement referring to this region: MORPHOGENESIS OF THE CHOROID PLEXUSES 513 . Im Bereich des Sulcus hemisphaericus ist die mediale Hemisphiren- wand verdiinnt und taschwartig in das Hemisphérenlumen eingestiilpt. Diese Tasche entspricht dem Plexus chorioideus ventriculi lateralis. Sie 6ffnet sich also in den Sulcus hemisphaericus (und zwar in seine laterale Wand) in der Decke des Foramen Monroi und communicirt sowohl mit der Sichelspalte wie mit dem hinten obsteigenden zwischen Zwischenhirn und Hemisphirenhirn gelegenen Abschnitt des Sulcus hemisphaericus. Fig. 1 Coronal section from the forebrain of an embryo of Parameles nasuta. Copied from Smith. Labels mine. Fig. 2 Coronal section from the forebrain of a foetal Ornithorhynchus. Copied from Smith. Labels mine. Fig. 3 Transverse section through the forebrain of a 19 mm. human embryo. H 173, Chicago Embryological Collection. Slide 21, Section 11. X 13}. REFERENCE LETTERS a.a.c.t.l., anterior area chorioidea tel- encephali lateralis c.o., chiasma opticum c.p., commissura posterior c.s., corpus striatum d-t.gr., di-telencephalic groove ep., epiphysis f.c., fissura chorioidea f.M., foramen interventriculare hem., hemisphere wall hy., hypothalamus l.t., lamina terminalis mes., mesencephalon n.a.t., nucleus anterior thalami n.0., nervus opticus p.a.c.t.l., posterior area chorioidea tel- encephali lateralis p.a., p.t.l., pars anterior plexus telen- cephali lateralis par., paraphysis p.p.,p.t.l., pars posterior plexus telen- cephali lateralis r.inf., recessus infundibuli r.m., recessus mamillaris r.post., recessus postopticus r.pre., recessus preopticus t.c.d., tela chorioidea diencephali t.c.t.m., tela chorioidea telencephali medii ' t.f., taenia fornicis th., thalamus t.r-p., telencephalic roof-p!ate t.t., taenia thalami v.t., velum transversum 514 PERCIVAL BAILEY The ‘Sichelspalte’ I understand to be the cleft between the hemispheres in front of the diencephalic roof. The lateral telen- cephalic plexus, then, must extend anteriorly beyond the velum transversum into the roof plate of the telencephalon. Ziehen also is inclined to interpret the anterior extremity of the diencephalic roof in Echidna as the paraphysis. He remarks: Das ganze Bild erinnert an die Paraphyse mancher Reptilien. Ich stehe auch nicht an, diese leichte fordere Zuspitzung des hinteren Kuppelgebietes der Paraphyse homolog zu sitzen, wie im vergleichenden Abschnitt specieller erértert werden soll. Eine tiefere Einsenkung hinter der Zuspitzung—etwa im Sinne eines Velum transversum—fehlt. Vor der Zuspitzung beginnt sofort die Kinsenkung der Fossa interhemisphaerica. Fig. 4 Section from the forebrain of a 63 mm. embryo of Echidna hystrix. xX 30. Copied from Ziehen. Labels mine. Fig. 5 Transverse section from the forebrain of a 14 mm. pig. no. 18, Chicago Embryological Collection. Slide 5, section 19. x 25. In an older embryo of which he says, “‘Sehr bemerkenswerth is wiederum das Verhalten des Kuppelgebietes des Zwischenhirns gegen die Fossa praediencephalica hin,” he describes nothing resembling a velum transversum and a comparison of figures 4 and 5 will show the resemblance of his ‘Kuppelgebietes’ to the anterior pouch of the tela chorioidea diencephali of a pig embryo. If the plane of section in figure 5 were homologous with that of figure 4 the resemblance would be much more striking. If it be really the paraphysis, it forms a good transition stage to the condition found in higher Mammalia. MORPHOGENESIS OF THE CHOROID PLEXUSES 5a Observations on the Marsupial brain are remarkably meagre. I have been able to find but two statements bearing on the sub- ject. Broom (’97) says of a 14.8 mm. embryo of Trichosurus vulpecula: ‘‘ The choroid folds into the lateral ventricles, is partly formed, and the paraphysis well marked.’ He has no figures of sections. Selenka (’91) has a simple statement that the paraphy- sis is present in Marsupials, presumably in the opossum. “Bis jetzt habe ich die Paraphyse bei embryonen von Haifischen, Reptilien, und Beuteltieren beobachtet, zweifle jedoch nicht, dass sie allen Wirbeltieren gemeinsam ist.’’ That is all, but I hope soon to fill this gap. Fig. 6 Sagittal section of the forebrain of a 15 mm. pig. Copied from John- ston. XX 10. Labels mine. Fig. 7 Sagittal section of the forebrain of a 11 mm. embryo of Erinaceus europaeus. Copied from Grénberg. X 10. Labels mine. Thanks mainly to Grénberg, the brain of the Insectivora is better known. His work was done with Erinaceus europaeus. Ziehen (’06) says of Tupaja, ‘‘ Die Verhiiltnisse gleichen den fiir den Igel beschriebenen in hohem Masse.” Groénberg’s figures 25, 26, and 27 show clearly the velum trans- versum and paraphysal arch although he is loath to call them so. A comparison of figures 6 and 7 will make this plain. He describes also plexuses in the diencephalon and fourth ventricle. Of the lateral telencephalic plexus he writes: 516 PERCIVAL BAILEY Die Adergeflechtsfurche entsteht am frithesten. Sie ist schon bei meinem Stadium C (11 mm.) vorhanden und sowohl ausgebildet, dass ihre erste Entstehung sicher auf einem bedeutend jiingern Stadium zu suchen ist. Doch findet sich auf Stadium B (8 mm.) noch keine Spur einer Faltenbildung. Die Form und das Aussehen der Falte ergeben sich aus den Figg. 52 und 53. Man sieht, dass sie in ihrem vordern Theil weiter in die Hemisphirenhohle hineinreicht, als es mehr caudal- wirts der Fall ist. Es zeigt sich auch, wenn man eine Schnittserie durchmustert, dass die Falte nach hinten allmahlch kleiner wird und schliesslich nur eine leichte Einbuchtung darstellt, welche nach hinten ganz allmiihlich verstreicht. Der vordere Theil dagegen verhallt sich ganz anders. Die Falte ist hier tief und erstreckt sich mit ihrem freien Rand weiter nach vorn als die mit der itibrigen Hirnwand in Verbindung stehende basis. . . . . Auf den folgenden Stadien vergréssert sich die Adergeflechtsfalte bedeutend, besonders in ihr vorderer, freier Theil, : Fig. 8 Cross section through the forebrain of a 15 mm. embryo of Erinaceus europaeus. Copied from Grénberg. X 10. Labels mine. Fig. 9 Cross section through the forebrain of a 32mm. humanembryo. H41, Chicago Embryological Collection. X 25. Slide 29, Section 6. One would conclude from this description that the anterior end of the lateral plexus had arisen the earlier, and that it arises from the roof plate of the telencephalon is clearly shown in his figure 54, which is here reproduced with a section from a human embryo for comparison (figs. 8 and 9). Again if the section of the human embryo were a coronal instead of a transverse section, the similarity would be more obvious. MORPHOGENESIS OF THE CHOROID PLEXUSES 517 Of the forms between Insectivora and Man nothing of value is known. Ziehen (’06) shows sections of the lateral telencephalic plexus of Tarsius spectrum, a prosimian, but not in the proper plane to be of value, and again describes the anterior pouch of the diencephalic roof as the paraphysis. Sehr beachtenswert ist auch, dass unmittelbar hinter der Fossa prae- diencephalica die epitheliale Decke des Zwischenhirns sich zu einer stulen Falte, welche an die Paraphyse erinnert, erhebt und dass erst einige Schnitte weiter occipitalwiirts diese steile Falte durch den mediane Plexus des 3. Ventrikels eingestiilpt wird. In an earlier paper, the author (Bailey, ’15) called attention to the true homologue of the paraphysis in the human embryo and insisted upon its position in the telencephalic roof plate just anterior to the velum transversum. Previously, Streeter (12), Francotte (’94) and D’Erchia (’96) had written of the paraphysis in the human embryo. Streeter homologizes the anterior pouch of the diencephalic roof with the paraphysis, and from a com- parison of Francotte’s figures with sections of embryos of approxi- mately the same stage, I am convinced that he mistook the same structure for the paraphysis. D’Erchia shows a section from a 30 mm. human embryo in which he labels a structure ‘paraphy- sis’ which seems to me to be merely an oblique section of the lateral telencephalic plexus. In his model of a 13.6 mm. human embryo, His shows clearly the lateral telencephalic plexus coming off lateral to the paraphy- sal arch, but his statement (’04) of the origin takes no account of this fact. ‘‘Sein dem Thalamus angehefteter Randstreifen bleibt ependymal und in ihm bildet sich die Fissura chorioidea, von der aus die Epithelfaltungen des Corpus chorioideum in den Seitenventrikel sich einstiilpen.”’ Hochstetter (13) in his account of the development of the lateral telencephalic plexus in the human embryo shows one figure (fig. 6) of a section through the plexus which passes also through the telencephalic roof plate, but his description contains nothing concerning the origin of this part of the plexus. D’Erchia (’96) considers the lateral -telencephalic plexus to be derived from the velum transversum: “‘Per questa parte volga 518 PERCIVAL BAILEY tutto quello che si e detto per la cavia. Dal velum per diverti- coli laterali di formano i plessi emisferici inferiori destro e sinistro, dei quali uno e disegnato nella fig. 28.” Finally the author (Bailey, 715) has presented an account of the development of the lateral telencephalic plexus which it is the purpose of the present paper to supplement. Concerning these structures in other groups of vertebrates this discussion is not so much concerned, but a few words at least about the lateral plexus in other vertebrates will not be out of place. ‘This plexus is not present in Teleosits (Burckhardt, ’94) nor Anura (Herrick, 710). In Lacertilia 1ts development has been described by Tandler and Kantor (07) and Warren (11) and agrees in all essential respects with that hereinafter detailed for Chrysemys marginata. In examining some young alligators in the collection of Dr. Elizabeth Crosby, the posterior part of the lateral telencephalic plexus was found to be present, but very small and poorly developed. ‘The brain of an adult pigeon was also examined and the lateral telencephalic plexus was seen arising from a plexus formation in the roof of the telen- eephalon, but of the posterior part of the plexus there was no sign except a plexus of blood vessels along a thin but uninvaginated medial hemisphere wall. Dr. C. Judson Herrick suggested that this may represent a stage in the phylogenetic development of the plexus. Mrs. Gage (’95) figures the brain of the embryo of the English sparrow. Both the figures and the context show that the’ lateral telencephalic plexuses arise from the median telencephalic plexus and just lateral to the paraphysis, appar- ently not invaginating the medial hemisphere wall. She writes: “In the young sparrow ( g. 2) the paraphysis occurs in the midst of a mass which gives off the paraplexuses, and it opens directly dorsad of the portas, i.e., into the aula. . . . It is notice- able in the older embryo that the union of the auliplexus with the paraplexuses lies dorsad of the porta (fig. 14).’’ The works of Neumayer (’99) and Hochstetter (98) contain nothing of value for the present discussion. The two invaginations which they describe are temporary and unimportant. A similar con- dition is shown in my figure 19 (Bailey, ’15). D’Erchia (96) MORPHOGENESIS OF THE CHOROID PLEXUSES 519 considers the lateral plexus in the guinea pig to be derived from the velum transversum. “E dal velum che si origina il plesso del III ventricolo e i plessi coroidei emisferici destro e sinistro.’’ It is interesting also to note that although Tilney (’15) correctly labels the paraphysal arch in models of the brains of young cat embryos, in later stages he invariably transfers the label to the anterior pouch of the diencephalic roof. Johnston (09) has correctly interpreted the paraphysis in pig embryos, but his statement that the lateral telencephalic plexus appears as a folding of the anterior wall of the velum transversum is misleading for he immediately follows with the statement that it is separated from the velum by the paraphysal arch. In the angle between the vesicle and the diencephalon appears the chorioid plexus pushing into the lateral ventricle. It appears as a fold- ing of the anterior wall or limb of the velum transversum and its lateral prolongation. In this way appears the chorioid fissure whose further history need not be traced. Near the median line the plexus appears as a fold projecting into the interventricular foramen and separated from the velum transversum by the paraphysal arch. MATERIAL AND METHODS The material on which this study is based consists of a series of twelve embryos of Chrysemys marginata from my own col- lection, ranging in size from 5.1 mm. greatest length to one having a carapace 10.6 mm. long, and an 8.8 mm. embryo from the Harvard Embryological Collection, very kindly loaned to me by the Harvard Laboratory. All of my material was fixed in Zenker; stained in bulk with borax carmine; embedded by the celloidin-paraffin method; cut at 10. in transverse series; and counterstained on the slide with orange G. The excellent pres- ervation of the form relations of the delicate membranous por- tions of the brain may be attributed to the method of embedding. The embryos were all passed from 95 per cent alcohol to ether- alcohol; then through 0.5, 1 and 2 per cent celloidin; hardened in chloroform alcohol, and cleared in benzol, before embedding in paraffin. The Harvard embryo is cut in 10 » sagittal sections and stained with borax carmine and eosin. : 520 PERCIVAL BAILEY The forebrains of four embryos—l1 a, greatest length 5.1 mm.; H. E. C. no. 1433, greatest length 8.8 mm.; 5b, carapace 8.6 mm.; and 4b, carapace 10.6 mm.—were reconstructed by the Born method at a magnification of 100 diameters. Millimeter plates were used and every section drawn. It was necessary to dissect the models rather extensively to expose: the fissura chorioidea. The models were stacked from a side view of the head drawn from a photograph, with the exception of the Har- vard embryo. This latter embryo was cut sagittally and the stacking was guided by the epiphysis and paraphysis. Since the embryo was not cut in an exactly sagittal plane, after the paraphysis and epiphysis had passed out of the plane of section, most of the lateral telencephalic plexus had been stacked and the remaining sections were added with no other guide except comparison with other models. DESCRIPTION This description will be confined to the lateral telencephalic plexus, since concerning the other plexuses I find no reason to differ from Warren’s account, with the exception noted in the history. The main landmarks of the region in which the lateral telen- cephalic plexus develops are already laid down in an embryo of 5.1 mm. greatest length. Figure 26 shows the roof plate of the forebrain in such an embryo and figure 10 shows the same region schematically represented. The roof plate of the telencephalon, back of the lamina terminalis, appears as a triangular area (only half of it shown in figure 26) its base formed by the velum trans- versum and its apex lying at the posterior end of the lamina terminalis while from its center arises the paraphysis. The lateral sides of the triangle are formed by the taeniae fornices. At the lateral angles of the triangle, taenia fornicis, velum trans- versum, taenia thalami and di-telencephalic groove meet. It is not possible accurately to determine this point in the embryo under consideration because the roof-plate has been but imper- fectly differentiated histologically, but in later stages these angles of the triangle may be easily located. | MORPHOGENESIS OF THE CHOROID PLEXUSES 521 The earliest unquestionable appearance of the lateral telen- cephalic plexus is in the Harvard embryo, 8.8 mm. in length. It appears here as a crescent-shaped ridge projecting into the lateral ventricle, with two more strongly developed points . showing as small elevations (fig. 22). The anterior extremity of the plexus lies clearly in the roof of the telencephalon lateral to the paraphysis and medial to the taenia fornicia. The taenia fornicis appears in figure 22 as a ridge apparently in the medial hemisphere wall. It bears here a very close superficial resem- blance to the hippocampal ridge in a young human embryo, but its later development and internal structure show plainly that Fig. 10 Diagram of the region around the paraphysis in the roof of the fore- brain of a 5.1 mm. embryo of Chrysemys marginata. Fig. 11 Diagram of the same region in an embryo of 8.8 mm. Fig. 12 Diagram of the same region in an embryo having a carapace of 8.6 mm. it is the taenia fornicis. Its apparent position in the medial hemisphere wall is an illusion produced by the invagination of the plexus between it and the paraphysis. The triangular area of the telencephalic roof plate is well marked and the lateral angles can be determined. A sagittal section through the tri- angle is practically a straight line, except for the evagination of the paraphysis (figs. 13 and 21) as are also parasagittal sec- tions (fig. 14). Nevertheless the lateral angles are depressed and the sides of the triangle are concave outward. Transverse sections through this region are therefore curved and convex upward (fig. 21). The velum transversum sharply delimits the telencephalic roof plate at the base of the triangle (fig. 14). 522 PERCIVAL BAILEY Figure 15 shows a parasagittal section still farther laterally through the triangle. The velum transversum is obvious, the plexus invaginating the roof plate medial to the taenia fornicis and tilting the portion of the roof between itself and the taenia. Above and lateral to the taenia fornicis the brain wall is his- tologically differentiated; medial it is ependymal. This differ- ence is much more apparent in later stages (fig. 17). Poste- riorly the plexus is less well developed and as we approach the lateral angles of the triangle, plexus, velum, and taenia fornicis tend to merge into one groove which becomes continuous with Fig. 13 Sagittal section no. 135 from an 8.8 mm. embryo of Chrysemys mar- ginata. Harvard Embryological Collection, no. 1433. X 33}. Hig. 14 Parasagittal section no. 130 from the same embryo. Fig. 15 Parasagittal section no. 124 from the same embryo. the di-telencephalic groove. Lateral and anterior to the point where the taenia thalami meets the di-telencephalic groove and velum transversum the hemisphere wall is massive and unin- vaginated. It would seem, then, that in this embryo, the lateral telencephalic plexus lies entirely in the roof plate of the telencephalon. The condition is diagrammatically represented in figure 11. Figures 23 and 24 depict the region around the foramen of Monro in an embryo with a carapace 8.6 mm. long, about 14 mm. greatest length. Figure 23 looks at the foramen of the right half of the brain from the medial side. The cephalic end of the MORPHOGENESIS OF THE CHOROID PLEXUSES 523 model is, consequently to the left. The paraphysis which had been removed is represented by a line of white dashes. Figure 24 looks at the same foramen from the lateral side. The model is therefore reversed and the cephalic end points to the right. The condition of the roof-plate is diagrammatically represented in figure 12. The differentiation between hemisphere wall and Fig. 16 Transverse section from the forebrain of an embryo of Chrysemys marginata having a carapace 8.6 mm. in length. Embryo 5 b, slide 13, sect. 11. X 33}. Fig. 17 Transverse section of the same embryo; section 16, slide 13. X 33}. roof plate is clear and the taenia fornicis obvious (figs. 16 and 17). The anterior extremity of the lateral telencephalic plexus arises plainly from the telencephalic roof plate lateral to the paraphysis and medial to the taenia fornicis (fig. 16). Figure 17 shows a section posterior to figure 16 through the main body of the plexus, the plexus still lying in the roof plate. Figure 18 is of a section still farther posteriorly. The plexus is here shown crossing the taenia fornicis into the medial hemisphere wall. The taenia fornicis is now medial to the plexus and drop- 524 PERCIVAL BAILEY ping down to meet the anterior nucleus of the thalamus, which it does in a few sections and becomes continuous with the taenia thalami. In figure 19, much farther posteriorly, the taenia thalami is present, the plexus being entirely in the medial hemi- sphere wall. The portion of the plexus arising from the medial hemisphere wall is very poorly developed (fig. 19) but its area of invagination is extensive (fig. 24). In later stages this posterior part of the plexus develops more rapidly and overshadows the other. Figure 25 shows a lateral %, & a, Fig. 18 Transverse section of the same embryo as figure 16. Slide 13, sec- HONEA DOOR. Fig. 19 Transverse section of the same embryo; section 5, slide 14. X 333. view of the region around the foramen of Monro in an embryo with a carapace of 10.6 mm. A pen sketch of the entire model is appended (fig. 27) showing the region represen ed in figure 25. (Figure 24 is of a homologous region in a younger embryo.), The fissura chorioidea appears merely as a big hole in the medial hemisphere wall; all the landmarks are lost. In still later stages, this hole becomes reduced to a long narrow slit. The development of the plexus in size and shape is so well discussed and figured by Warren (711) that it will not be con- sidered here. MORPHOGENESIS OF THE CHOROID PLEXUSES 525 DISCUSSION From the foregoing history and description, and from an analysis of the remaining literature, for in the history are in- cluded only the most important papers and especially those dealing with the lateral telencephalic plexuses, it will be seen that there are certain definite regions of the brain wall wherein choroid plexuses develop. These regions and the plexuses which develop from them may be tabulated as follows: Tela chorioidea telencephali medii—Plexus telencephali medius Anterior area chorioidea lateralis telencephali—Plexus telencephali lateralis (below Chelonia) Anterior area chorioidea lateralis telencephali—Plexus telencephali lateralis (Chelonia and above) Posterior area chorioidea lateralis telencephali (hemispherici)—Plexus telencephali lateralis (Chelonia and above) Velum transversum—Plexus velares Tela chorioidea diencephali—Plexus diencephali Tela chorioidea mesencephali—Plexus mesencephali Tela chorioidea myelencephali—Plexus myelencephali Tilney (’15) has suggested that the saccus vasculosus should be reckoned with the choroid plexuses in the forms where it is present and in this opinion I concur. It is best developed in those forms in which the diencephalic plexus is rudimentary or absent, i.e., in Cyclostomes, Selachians and Ganoids. The plexus formation is very poorly developed in Urodeles and never again present. There should be added to the above therefore: Recessus infundibularis (posterior wall)—Saccus vasculosus. The myelencephalic plexus arises in the roof of the fourth ventricle, tela chorioidea myelencephali, in every known verte- brate above Amphioxus. The mesencephalic plexus is found only in Petromyzon, where it arises from the mesencephalic roof, tela chorioidea mesen- cephali. The diencephalic plexus arises from the tela chorioidea dien- cephali. Appearing first in Cyclostomes, but very poorly developed, it disappears almost entirely in Selachians, where the tela chorioidea diencephali is almost completely absorbed 526 PERCIVAL BAILEY by, the overgrowth of the velum transversum. In Ganoids, the tela chorioidea diencephali begins to emerge, forming a thin walled sac, and in Urodeles is again invaginated by an enormous diencephalic plexus. It may be that the diencephalic plexus in Selachians and Ganoids is represented by the choroidal folds on the posterior limb of the velum, developing from the anterior portion of the tela chorioidea diencephali which has been drawn down into the velum transversum by the overgrowth of the latter. The diencephalic plexus is present in all forms above Urodeles but is never again so well developed. The velar plexuses are present only in Selachians, Ganoids, and Urodeles. They may involve either the diencephalic limb or telencephalic limb, or the entire velum transversum. The choroidal folds on the diencephalic limb have been homologized, in Selachians and Ganoids, with the diencephalic plexus of other forms, and with some reason. The choroidal folds on the ante- rior limb are not homologous with either the median or lateral telencephalic plexuses. The median telencephalic plexus arises from the tela chorioidia telencephali medu, just in front of the paraphysis (paraphysal arch of Mammalia), from the Selachians to the Chelonia, in- clusive, with the apparent exception of the Ganoids. It is not constantly present in Chelonia and is never found in Mam- malia. Its development seems to be in inverse ratio to the degree of development of the lateral telencephalic plexus and the latter in direct ratio to the size of the hemispheres. The lateral telencephalic plexus is found in all groups of verte- brates from the lowest to the highest in the line of ascent of Mammals with the exception of the very lowest, Amphioxus and the Cyclostomes. Apparently sporadically and imperfectly developed in Selachians and Ganoids, it is present constantly thereafter. Wehave stated previously that it is absent in Teleosts and Anura, with which we are not concerned. In all forms below Chelonia, it develops in what I have called elsewhere (Bailey, 715) the anterior lateral telencephalic chorioidal area, in the roof . plate of the telencephalon between the paraphysis and the taenia fornicis of the medial hemisphere wall. Where the median MORPHOGENESIS OF THE CHOROID PLEXUSES Hoe telencephalic plexus is strongly developed the lateral plexus appears to be an appendage of it (Necturus, Warren); where the velum is involved in an extensive plexus formation, the lateral plexus appears to be an appendage of it (Acanthias, Minot); but always arises from the region of the telencephalic roof plate described above, and medial to the taenia fornicis. With the Chelonia comes a change. The lateral plexus arises in the anterior area chorioidea lateralis telencephali as has been previously described but in its later development crosses the taenia fornicis and invaginates also the posterior area chorioidea lateralis telencephali in the medial hemisphere wall. Such a condition is found also in the Gecko brain (Tandler and Kantor). This involvement of the medial hemisphere wall comes more and more to predominate in the development of the lateral plexus as the hemispheres come more and more to dominate the develop- ment of the telencephalon, but even in the highest Mammalia the anterior extremity of the lateral choroid plexus develops from the roof plate of the telencephalon and the evidence may briefly be summarized here: (a) Elliot Smith (97) in deseribing the brain of a foetal Orni- thorhyneus described the lateral choroidal plexus as arising from the continuation backwards of the horizontal part of the lamina supraneuroporica, which term he used to inelude all the roof plate of the telencephalon between the velum transversum and the lamina terminalis. Again in 1903, he reiterated his belief that the anterior extremity of the lateral choroidal plexus arises from the roof plate. (b) Th. Ziehen’s figures of Echidna do not show the anterior extremity of the lateral plexus, but he states that it opens into the Sichelspalte, that is, into the cleft between the hemispheres anterior to the diencephalon, as well as the suleus hemisphaericus (di-telencephalic groove). (c) Observations on the Marsupials up to present reveal nothing. (d) Grénberg states of Erinaceus europaeus that the anterior extremity of the lateral plexus is better developed in his earliest stage than the posterior extremity and apparently arises first. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 528 PERCIVAL BAILEY This holds true of all mammals and is what one would expect from the phylogenetic history of the plexus. His figure 42 of a section anterior to the velum shows clearly the anterior extrem- ity arising from the roof plate of the telencephalon. (e) The earliest appearance of the plexus in the human embryo occurs in His’ embryo of 13.6 mm. and his model shows clearly the plexus arising just lateral to the paraphysal arch. (f) Hochstetter (13) figures a section of a human embryo showing the anterior extremity of the plexus, and the author recently (Bailey, 715) has described three human embryos show- ing the plexus extending into the roof plate alongside the para- physal arch anterior to the velum transversum. (g) Collateral evidence is derived from the development of the lateral plexus in other mammals, for example, Johnston (’09) states that in the pig the plexus arises from the anterior limb of the di-telencephalic groove but is separated medially from the velum transversum by the paraphysal arch. CONCLUSIONS 1. The lateral telencephalic plexus of Chrysemys marginata arises from the anterior area chorioidea lateralis telencephali in the roof plate of the telencephalon between the paraphysis and the taenia fornicis of the medial hemisphere wall and in its later development oversteps the taenia fornicis just anterior to the point where taenia fornicis, taenia thalami, velum transversum and di-telencephalic groove meet, and invaginates also the pos- terior area chorioidea lateralis telencephali in the medial hemi- sphere wall, just anterior to the di-telencephalic groove. The pars anterior plexus telencephali lateralis which develops from the anterior area chorioidea lateralis telencephali in early stages is much larger and better developed than the pars posterior plexus telencephali lateralis, but in later stages develops much less rapidly than the pars posterior. 2. All the data at present available support the conclusion that the lateral telencephalic plexus arises from the anterior area chorioidea lateralis telencephali in the roof plate of the telen- cephalon between the paraphysis (or paraphysal arch) and the MORPHOGENESIS OF THE CHOROID PLEXUSES 529 taenia fornicis in all vertebrates where it is present, but that in Chelonia and the forms above, it oversteps the taenia fornicis and invaginates the posterior area chorioidea lateralis telencephali in the medial hemisphere wall and that this latter portion comes more and more to dominate its development as we ascend the vertebrate scale. I wish here to express my gratitude to Dr. 8. Walter Ranson for his unvarying kindness and interest throughout the progress of this work. 530 PERCIVAL BAILEY LITERATURE CITED Aunxtporn, F. 1883 Untersuchungen tiber das Gehirn der Petromyzonten, Zeits. wiss. Zool., Bd. 39. BaiLtey, P. 1915 The morphology of the roof plate of the forebrain and the lateral choroid plexuses in the human embryo. Jour. Comp. Neur., vol. 26. Buake, J. A. 1900 The roof and lateral recesses of the fourth ventricle, con- sidered morphologically and embryologically. Jour. Comp. Neur., vol. 10. Broom, J. 1897 The embryology of Trichosurus vulpecula. Proc. Roy. Soe. of N. S. Wales, vol. 27. Burckuarpt, R. 1891 Untersuchungen am Gehirn und Geruchsorgan yon Triton und Ichthyophis. Zeitschr. f. wiss. Zool., Bd. 52. 1894. Der Bauplan des Wirbeltiergehirns. Morph. Arbeiten von G. Schwalbe, 4. D’Ercuia, Fu. 1896 Contributo allo studio della volta del cervello intermedio e della regione parafisaria In embrioni di pesci e di Mammiferi. Moni- tore Zoologico, vol. 7, pp. 118 and 201. FRANcoTTE, P. 1894 Note sur l’oeil parietal, ’epiphyse, la paraphyse et les plexus choroides du troisieme ventricule. Bull. de lAcad. roy. de Belg., no. 1. Gaae, Mrs. 8. P. 1893 The brain of Diemyctylus viridescens. Wilder Quarter- Cent. Book. - 1898. 1895 Comparative morphology of the brain of the soft-shelled turtle (Amyda mutica) and the English sparrow (Passer domesticus). Proc. Am. Mier. Soe., vol. 17. GRONBERG, GOstTa. 1901 Die Ontogenese eines niedern Sdiugergehirns nach Untersuchungen an Erinaceus europaeus. Zoolog. Jahrb., Abth. Anat., Bas 15s» 26s Herrick, C. J. 1910 The morphology of the forebrain in Amphibia and Reptilia. Jour. Comp. Neur., vol. 20. Herrick, C. L. 1891 and 1893 Topography and histology of the brain of certain reptiles. Jour. Comp. Neur., vols. 1 and 3. Hitt, C. 1894 The epiphysis in teleosts and Amia. Jour. Morph., vol. 9, p. 237. His, Wituetm. 1904 Die Entwicklung des menschlichen Gehirns. Leipzig. Hocusterrer, F. 1898 Beitriige zur Entwickelungsgeschichte des Gehirns. Bibliotheca Medica., Abthg. A., Heft. 2. 1913 Uber die Entwickelung der Plexus Chorioidei der Seitenkammern des menschlichen Gehirns. Anat. Anz., Bd. 45, pp. 225-238. Humpnrey, O. D. 1894 On the brain of the snapping turtle. Jour. Comp. Neur., vol. 4. Jounston, J. B. 1909 On the morphology of the forebrain vesicle in vertebrates. Jour. Comp. Neur., vol. 19. KAPPERS AND CARPENTER 1911 Das Gehirn von Chimaera monstrosa. Folia Neuro-Biologica, Bd. 5. Kineassury, B. F. 1897 The encephalic evaginations in Ganoids. Jour. Comp. Neur., vol. 7. MORPHOGENESIS OF THE CHOROID PLEXUSES 531 Kuprrer, C. von 1893 Die Entwicklung des Kopfes von Acipenser sturio. Studien zur vergleichenden Entwicklungsgeschichte des Kopfes der Kranioten. Heft. I., Miinchen. 1903 Die Morphogenie des Nervensystems. Oskar Hertwig’s Hand- buch. Minot, C. S. 1901 On the morphology of the pineal region based upon its development in Acanthias. Am. Jour. Anat., vol. 1. Neumayer, L. 1899 Studien zur Entwicklungsgeschichte des Gehirns der Siugetiere. Festschr. z. 70 Geburtstag von C. von Kupffer. Jena. Fischer. Sevenka, E. 1891 Das Stirnorgan der Wirbeltiere. Biol. Centralbl., Bd. 10. Smitu, G. Exvuiort 1897 The brain of a foetal ornithorhynchus. Quart. Jour. Micros. Sci., n. s., vol. 39. 1903. On the morphology of the cerebral commissures. Trans. of the Linnaean Soc., London., vol. 8, part 12. Srerzi1,G. 1907 Il sistema nervoso centrale dei vertebrate. Vol. 1, Ciclostomi. Srreeter, G. L. 1912 The development of the central nervous system. Keibel and Mall’s Human Embryology. Srupnicka, F. K. 1893 Zur Losung einiger Fragen aus der Morphologie des Vorderhirns der Cranioten. Anat. Anz., Bd. 7. TANDLER, J. AND Kantor, H. 1907 Beitrige zur Entwickelung des Vertebrat- engehirns. I. Die Entwickelungsgeschichte des Geckohirnes. Anat. Hefte, Bd. 33, Heft 101. Terry, R. J. 1910 The morphology of the pineal region in teleosts. Jour. Morph., vol. 21. Tinney, Frepertck 1915 The morphology of the diencephalic floor. Jour. Comp. Neur., vol. 25, no. 3, pp. 213-282. WarrREN, Jonn 1905 The development of the paraphysis and pineal region in Necturus maculatus. Am. Jour. Anat., vol. 5, pp. 1-29. 1911 The development of the paraphysis and pineal region in reptilia. Am. Jour. Anat., vol. 11, pp. 313-392. ZinHEN, Tu. 1904 Verhand. Kon. Ak. van Wetensch. te Amsterdam. 26 Noy. (pp. 331-340). 1905 Zur Entwickelungsgeschichte des Centralnervensystems von Echidna hystrix. Jenaische Denkschriften, Bd. VI, 2. Theil. 1906 Morphogenie des Centralnervensystems der Siugetiere. Oskar Hertwig’s Handbuch. PLATE 1 EXPLANATION OF FIGURES The reference letters for all figures are found on page 513. It is impossible to portray all the topography of the region around the foramen interventriculare by means of drawings. For aid in orientation, the position of the principal landmarks is indicated on the models as follows: the taenia fornicis by a line of dashes; the velum transversum by a line of crosses; the taenia thalami by dots; and the di-telencephalic groove by dots and dashes alternating. The cross sections will also aid. On figures 22 and 23 the positions of the sections are indicated by arrows. Since the models are tilted, the arrows indicate accurately only the points where the sections cross the lateral telencephalic plexus. 20 Median view of the forebrain of a 5.1 mm. embryo of Chrysemys marginata. Embryola. X 50. 532 MORPHOGENESIS OF THE CHOROID PLEXUSES PLATE ft PERCiVAL BAILEY 533 PLATE ‘2 EXPLANATION OF FIGURES 21 Median view of the forebrain of an 8.8 mm. embryo of Chrysemys margi- nata. Embryo 1433. H.E.C. X 40. 22 Lateral view of the forebrain of an 8.8 mm. embryo of Chrysemys mar- ginata. Embryo 1438. H.E.C. X 40. Lateral hemisphere wall removed. 5384 MORPHOGENESIS OF THE CHOROID PLEXUSES PLATE 2 PERCIVAL BAILEY te.t.m. a hem ‘inf 992 539 MORPHOGENBSIS OF THE CHOROID PLEXUSES PLATE 3 PERCIVAL BAILEY Lelim = 3 soe he ge 2 Bia alo tic oo\td at ISS1O, 17. 18 19 ae i 23 EXPLANATION OF FIGURES 23 Median view of the region around the foramen interventriculare in an embryo of Chrysemys marginata having a carapace 8.6 mm. in length. Embryo 5b. > 100. In order to show the fissura chorioidea it was necessary to remove the paraphysis and represent the fissure slightly higher than it really is. 536 MORPHOGENESIS OF THE CHOROID PLEXUSES PLATE 4 PERCIVAL BAILEY EXPLANATION OF FIGURES Lateral wall of the hemi- 24 Lateral view of the same region as in figure 23. horioidea. sphere and plexus telencephali lateralis removed, exposing the fissura ¢ Embryo 5b. X 100. 537 PLATE 5 EXPLANATION OF FIGURES 25 Lateral view of the region around the foramen interventriculare in an embryo of Chrysemys marginata having a carapace 10.6 mm. in length. Embryo 4b. & 662. Lateral hemisphere wall and lateral telencephalic plexus removed, exposing the fissura chorioidea. 26 Dorsal view of the forebrain of a 5.1 mm. embryo of Chrysemys marginata. Embryola. X 80. 27 Pen sketch of model of entire forebrain of embryo 4b, showing the area ~ from which figure 25 was taken. XX 25. 538 PLATE 5 MORPHOGENESIS OF THE CHOROID PLEXUSES PERCIVAL BAILEY THE STRUCTURE OF THE THIRD, FOURTH, FIFTH, SIXTH, NINTH, ELEVENTH AND TWELFTH CRANIAL NERVES SUMNER L. KOCH From the Anatomical Laboratory of the Northwestern University Medical School FIVE FIGURES Following the demonstration of unmyelinated fibers in the spinal nerves and in the vagus nerve by means of the pyridine- silver technique (Ranson, ’11 and ’12; Chase and Ranson ’14), Professor Ranson suggested the application of the same method to the study of certain of the cranial nerves, with especial refer- ence to the presence or absence of unmyelinated fibers. The nerves studied were the oculomotor, trochlear, trigem‘nal, and abducens of the dog and of man; and the glossopharyngeal, accessory, and hypoglossal nerves of the dog, the cat and the rabbit. The nerves were obtained by lifting off the skull cap and following them distally from their cerebral origin by chip- ping away the base of the skull about their foramina of exit. The dissected specimens were laid on glass slides and prepared by different methods. Some were stained by the pyridine-silver method; others were placed in 50 per cent pyridine solution for seven days, washed, and then treated with silver nitrate, water, and pyrogallic acid as in the pyridine-silver method; others were stained by the Pa’-Weigert method and the osmic acid method. All were cut and mounted serially. The oculomotor, trochlear, and abducent nerves form a natu- ral group formerly described as purely motor and consisting of large and small myelinated axons, but now recognized as con- taining somatic afferent as well as efferent fibers. The nerves are described as communicating with the ophthalmic division of the trigeminal nerve and with the cavernous plexus of the sympathetic system. 1 Contribution no. 40. 541 542 SUMNER L. KOCH Gaskell (89) studied the cranial nerves with especial reference to their relation to a typical spinal nerve. The oculomotor, trochlear and abducent nerves he described as pure efferent nerves. The oculomotor was composed of large and small mye- linated fibers, in the dog 14.4 to 18 micra and 38 to 5 micra in diameter. The smaller fibers passed to the ciliary ganglion, the larger to the extrinsic muscles of the eye. The trochlear nerve consisted of large myelinated fibers, 14.4 to 18 micra in diameter, supplying the external oblique muscle, and small myelinated fibers 3.6 to 5.4 micra in diameter, of unknown function. The abducens consisted of large myelinated fibers 14.4 to 18 micra in diameter, and a few smaller ones, but contained no distinct group of small fibers as did the IIIrd and IVth nerves. In the roots of these nerves he saw fibrillar structures which he inter- preted as the remains of ganglion cells present at an earlier stage of their development, and representing the afferent portion of the nerves. Barratt (98, 799, and ’01) described the oculomotor nerve as composed of large and small myelinated fibers, 11 to 15 micra and 3 to 5 micra in diameter, in about the ratio of three to one. He found unmyelinated fibers also, ‘‘both in the fibrous sheath and in the main trunk; in the latter situation usually at the periphery.”’ He did not find any communication with the cav- ernous plexus nor with the ophthalmic division of the trigeminal nerve. The trochlear nerve wes composed of large and small myelinated fibers 12 to 19 micra and 4 micra in diameter, in the ratio of three to one. There were no unmyelinated fibers pres- ent. The abducent nerve he described as composed of large and small myelinated fibers, 11 to 17 and 3 to 6 micra in diame- ter. A few small twigs composed of small myelinated and un- myelinated fibers joined it 25 mm. from its superficial origin, and left it again 10 mm. distalward. Carpenter (06) studied the development of the oculomotor and abducent nerves in the chick. He found that the oculo- motor nerve was composed of myelinated axons 3 to 15 micra in diameter. The trunk was composed of comparatively large axons, with a few scattered small axons, and a zone of small STRUCTURE OF THE CRANIAL NERVES 543 axons at the periphery which passed into the ciliary ganglion. He found a communicating branch extending from the ophthal- mic division of the trigem‘nal nerve to the ciliary nerve, but none to the undivided oculomotor nerve. He did not speak of a direct communication between the nerves studied and the sympathetic system, but referred to Jegorow’s suggestion that a distribution of abducent fibers to the eyeball in birds might be accounted for by sympathetie fibers joining the abducens as it passed through the cavernous sinus. Boughton (’06) found an almost regular increase in the number and size of myelinated fibers in the oculomotor nerve of the white rat and the cat at different ages. In rats of 730 days weighing 414 grams the large fibers averaged 8 1 micra in diame- ter, the small 4.3 micra. In cats of 2893 grams the large fibers averaged 13.5 micra in diameter, the small 7.2 micra. Kopsch (’07) described the ceulomotor nerve in man as com- posed of about 15,000 mostly large myelinated fibers grouped in a number of secondary bundles. In the roots between the fibers were isolated, branched, spherical nerve cells. The trochlear nerve he described as ecmposed of about 1200 myelinated fibers, the abducens of about 2600 myelinated fibers. All three nerves received communicating branches from the carotid plexus of the sympathetic system, and from the ophthalmic nerve. THE OCULOMOTOR, TROCHLEAR AND ABDUCENT NERVES Specimens of the oculomotor nerve of the dog and of three humen adults were studied in serial sections. Upon dissection at Jeast two fine branches from the sympathetic system could be seen joining the nerve. Microscopically sections showed in each case clear pictures of a typical motor nerve (fig. 1) com- posed of large and small myelina ed axons. In the human the ratio of large to small axons was about three to one; in the dog it varied between two to one and three to one. In osmie acid preparations the large myelinated fibers of the dog averaged 12 to 16 micra, the small 3 to 6 micra. Close to its peripheral origin the fibers of the nerve were grouped in one large bundle, with incomplete septa extending toward the center of the nerve. As THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 n44 SUMNER L. KOCH the nerve approached the cavernous sinus the septa became complete, and divided it into from five to eight fasicles of vary- ing size. Nowhere along the course of the undivided nerve were unmyelinated fibers seen within its substance. In sections of the intra-cavernous portion of the nerve a few clusters of un- myelinated fibers were seen approaching it, but could not be followed into its substance. The characteristic grouping of cy ag sas (7 SS ans IA S 22, En S is} ee ny) 07, Seo 2 Soe (Se & CSS SI Mose, . lo, oo soe 1S, ok Ye) EY , ee Doe ee ee een, INE SS ED MO ee SOS meOC mesa Des Cp OUl SOUR RODS, esccodO/o E09 OOOO 000 DDG 0, pioroge Barat ep caus npeeva acon y OO, MsOscd2 0020) pees avasicce SO: Le? e Se AS O} o a Jos is) On 0; eae COUaaTG Beare Sosreoa G0sI009. easter SPSS Ps Gee CR soe Eats EN she Ss Seo; Con: = oe Coos: 'O' iS ts) IS ea ao: iS; oor epee aes (adow PISS {0 aS rs 1 Sal LEG Se Sass 30% sa aaao agate OEIC i=] S26) St co ch = Asks Sass Qc Sx 500 {2} Ca) eat Ps: ae Ose OF Dig7cg Doses enesom Ura trecoos| y CTPA Oooo topo cer cote ed | O a 5 i SEO RSD 9 NOG eosaeee NO DONC OCOsUOMO LY \GhessePoreys Hak rs c Ros SOSOe Que 7, OC 00S OLS OO OO O59 0525 CACAO U MO OC aN a0S Oo) LA Neto R TOG / Nate obo OO ASO OR ORO nem ACC Ue Nan Re cee OO DOITS BODO nse eH NOS OG MO we Oe A DSO COD OER DOD COS eater Oe O es 0622 O DLAC DLS COR opr O Osos OM No: Cn OO LOLOOO CO as COONS COs se Orne Re DOO CRD), paces Bon eRaGrcanerbes Cap \eocoaa arate is OIG: OSS Oo con Wo gsce ara Toul, Sacog acoso Cosa Coe roe OCaeOSBOOacrepotanun pen GSU outa ey an pepe 16) OHO a ~OAO, x Rese soon ) rs? a Lh, Xe) co ae i) 1 o Sl SS Oo: ae ey ee es 3 al OIeaaeasscs ets = i) 30) 6 Co ATG ey ea O; 7 5 (2) ga LC 1S nO 5 Se Ne) SS Xe) =a Ler / Forno! (TEX te {ok elp) Pear AO SOLt Oe OC sx Z {) 4 0, } BCU OUT EOS eR CEOS OSE AN se eco Sob eee Fig. 1 Section of the oculomotor nerve of the dog. Pyridine-silver. Ocu. 0, Obj. 6. small fibers at the periphery of the nerve, noted by Gaskell and Carpenter, could be seen most clearly in osmic acid preparations; but not all of the small fibers were grouped at the periphery, nor did they all leave the nerve to pass to the ciliary ganglion. The cellular structures in the roots of the nerves, mentioned by Gaskell and other observers, were not observed in our prepara- tions, as even in the most proximal sections the roots had already united to form a common trunk. STRUCTURE OF THE CRANIAL NERVES 545 Microscopic sections of the trochlear nerves of the dog closely resembled those of the oculomotor nerve; large and small mye- linated fibers, 11 to 15 and 2 to 6 micra. in diameter, in about the ratio of three to one, with a few fibers of intermediate size. No communicating branches from the cavernous plexus or the ophthalmic nerve were seen. The abducent nerve of the dog near the brain stem consisted of a single group of large and small myelinated fibers, 11 to 15 and 3 to 6 micra in diameter, in about the ratio of three to one. Within the cavernous sinus the nerve was joined by a large bundle of sympathetic fibers, the majority of which formed an intimate union with three of the six or seven fasicles which made up the nerve at this point (fig. 2)... More distally the main group of sympathetic fibers left the nerve to pursue an independent course. The striking characteristic of these three nerves is their simi- larity in structure, both as to the size of the fibers and the propor- tion of large and small fibers. Gaskell and Carpenter noted the presence of small fibers in the oculomotor nerve, and considered the ciliary ganglion as their destination, though they did not say that all the small fibers entered the ganglion. Gaskell further noted the similarity in structure between the IlIrd and IVth nerves, but said the destination of the small fibers of the latter was as yet unknown. The VIth nerve he described as composed of large myelinated fibers ‘‘with a few smaller ones; with no sign of any distinet group of small fibers as in the IIIrd and IVth nerves.”’ In our preparations all three nerves showed strikingly similar characteristics as to the size of the fibers and the ratio of the small and large fibers. The VIth nerve could not be distinguished from the other two by a diminished number of small myelinated fibers, but was characterized by the pres- ence of unmyelinated fibers from the sympathetic system which came into intimate contact with the myelinated fibers and travelled distalward with them as far as the nerve was followed. The only clue to an explanation of this fact is Jegorow’s sugges- tion that a distribution of fibers of the VIth nerve to the eyeball in the birds might be accounted for by the presence of sympa- 546 SUMNER L. KOCH thetic fibers which joined the nerve as it passed through the cavernous sinus. No such communications from the sympa- thetic system were seen in connection with the [lIrd and [Vth nerves. Branches of the cavernous plexus, which on gross dis- section could be seen to join the IIIrd nerve, on microscopic Fig. 2. Section of the abducent nerve of the dog, showing bundles of sympa- thetic fibers, a, Pyridine-silver. Ocu. 0, Obj. 6. examination were seen to accompany the nerve for a short dis- tance and again separate from it farther distalward, without entering into intimate contact with its fibers. No communica- tion whatever between the IVth nerve and the sympathetic system was seen. STRUCTURE OF THE CRANIAL NERVES 547 The unmyelinated fibers seen in connection with the IlIIrd and VIth nerves nowhere showed the characteristic arrange- ment of the unmyelinated fibers seen in the spinal nerves and the vagus. In the IIIrd nerve the unmyelinated fibers remained grouped in the nerve sheath and did not enter its substance. More distally they separated from the nerve completely. The unmyelinated fibers which joined the VIth nerve and entered into intimate association with the myelinated fibers were not uniformly distributed throughout the nerve substance, but re- mained at the periphery, definitely grouped in two or three of the fasicles of the nerve. In no sections did these unmyelinated fibers from the sympathetic system appear evenly and uniformly distributed throughout the nerve substance as do the unmye- linated fibers in the spinal nerves and the vagus. THE TRIGEMINAL NERVE The intracranial portion of the trigeminal nerve of the dog and the cat, and of one human specimen was studied. Pyridine- silver sections showed large and small myelinated fibers, and in addition small numbers of unmyelinated fibers, appearing in largest numbers in two fasicles of the sensory portion of the nerve. No sympathetic fibers were seen joining the nerve within the cranium. In osmie acid preparations of the Vth nerve of the dog the large myelinated fibers measured 12 to 16 micra in diameter, the small 3 to 6 micra. In the cat the small fibers measured 4 to 7 micra, the large 12 to 16 micra, with occasional fibers 18 micra in diameter. THE GLOSSOPHARYNGEAL, ACCESSORY AND HYPOGLOSSAL NERVES The glossopharyngeal nerve is usually described as a mixed nerve, and is recognized, according to Herrick’s classification, as containing both general and special vsceral efferent fibers, and general and special visceral sensory fibers, as well as somatic fibers with sensory function. The accessory and hypoglossal nerves are pure motor nerves, the former containing general and special visceral fibers, the latter special somatic fibers. d48 SUMNER L. KOCH Gaskell described the small myelinated fibers in the glosso- pharyngeal nerve as 1.8 to 3.6 micra in diameter, the large as not exceeding 10.8 micra. He found that large myelinated fibers were present in all the roots of the accessory nerve, but that the small fibers were confined to the bulbar and upper cervical roots. Barratt described the [Xth nerve as composed chiefly of small myelinated fibers 4 micra in diameter. Kopsch described the IXth nerve as consisting of a motor and sensory portion, and Fig. 3. From a section of the accessory nerve of the dog, showing a bundle of sympathetic fibers joining the nerve. Pyridine-silver. Ocu. 0, Obj. 8. recelving sympathetic fibers from the superior cervical ganglion. He described the accessory nerve as arising in two parts, the accessorius vagi, which joined the vagus as the ramus internus; and the accessorius spinalis, which as the ramus externus received fibers from the jugular ganglion of the vagus. Chase and Ran- son found numerous unmyelinated fibers in the roots of the vagus but thought the bulbar rootlets of the accessory nerve contained few if any unmyelinated fibers. Kopsch described the hypoglossal nerve as arising in 10 to 15 root bundles, and STRUCTURE OF THE CRANIAL NERVES 549 forming anastomoses with the vagus, the upper three cervical nerves, and the superior cervical ganglion of the sympathetic system. Pyridine-silver sections of the roots of the glossopharyngeal nerve showed large and small myelinated fibers, and a few Fig. 4 Section of the accessory nerve of the dog, showing bundles of darkly stained unmyelinated fibers. Pyridine-silver. Ocu. 0, Obj. 8. unmyelinated fibers. Sympathetic fibers could be followed into the trunk of the [Xth nerve between its two ganglia. In the trunk of the nerve the small myelinated fibers outnumbered the large fibers nine to one. In osmie acid preparations the large fibers measured 9 to 12 micra in diameter, the small 3 to 550 SUMNER L. KOCH 5 micra. Sections of a pyridine-silver preparation of the [Xth nerve of the cat, just proximal to the superior ganglion, showed few large myelinated fibers 12 to 15 micra in diameter, many small myelinated fibers, 3 to 6 micra, and a few unmyelinated fibers. Sections of the IXth nerve of the rabbit showed a similar picture. No connections with the sympathetic system distal to the petrous ganglion were seen in any preparations. The bulbar rootlets of the accessory nerve are composed chiefly of small myelinated fibers 2 to 5 micra in diameter. The spinal Fig. 5 From a section of the hypoglossal nerve of the dog, showing bundles of darkly stained unmyelinated fibers joining the nerve. Pyridine-silver. Ocu. O2Obj2 8: portion was composed of large and small myelinated fibers in about the ratio of five to one. The majority of the large fibers were 9 to 12 micra in diameter, with a few larger fibers of 14 to 16 micra; the small fibers 2 to 5 micra. In several preparations at the point of its separation from the vagus, a few unmyelinated fibers could be seen in the ramus externus, arranged in three or four groups. Bundles of sympathetic fibers could be traced for some distance in the sheath of the XIth nerve, finally entering it (fig. 3) and forming well marked clusters of fibers within its STRUCTURE OF THE CRANIAL NERVES 551 substance (fig. 4). In Pal-Weigert preparations the bluish-black myelin rings were separated in places by small unstained areas, corresponding closely with the location of the unmyelinated fibers seen in pyridine-silver preparations. No essential differ- ences were seen in the XIth nerve of the cat and rabbit. The large myelinated fibers of the spinal root of the accessory of the cat were the largest of all the fibers measured, some of them having a diameter of 18 micra. The hypoglossal nerve of the dog showed a picture closely resembling that of the oculomotor nerve, large and small mye- linated fibers in about the ratio of three to one, measuring 11 to 15 micra and 3 to 6 micra in diameter. No unmyelinated fibers were seen within the roots of the nerve, but slender bun- dles of sympathetic fibers could be followed as they approached the nerve, entered its sheath, and finally joined the nerve sub- stance (fig. 5). The hypoglossal of the cat and rabbit showed similar pictures. Nowhere did the unmyelinated fibers seen in sections of the XIth and XIIth appear evenly distributed among the mye- linated fibers, as in the spinal nerves and the vagus; but always grouped in clusters, as in the VIth nerve, and in largest numbers near the periphery. SUMMARY 1. Unmyelinated fibers are present in the Vth, VIth, [Xth, XIth and XIIth eranial nerves. Those in the VIth, XIth and XIIth are probably all derived from the sympathetic system. In these nerves they have an arrangement characteristic of sympathetic fibers; they are grouped in clusters, and are most numerous near the periphery of the nerve. 2. The oculomotor and trochlear nerves are strikingly similar in composition; they are composed of large and small myelinated fibers, without any accessions or unmyelinated fibers from the sympathetic system. 3. The abducens is similar to the IIIrd and [Vth nerves as regards its myelinated fibers, but in addition receives a large number of unmyelinated fibers from the sympathetic system, 552 SUMNER L. KOCH some of which enter the nerve sheath and travel distalward with the myelinated fibers. 4. The accessory and hypoglossal nerves are composed of large and small myelinated fibers, and are similar in appearance and structure to the IlIrd and IVth nerves. Like the VIth nerve, they receive considerable numbers of unmyelinated fibers from the sympathetic system which can be followed to the ter- mination of the nerves in the musc'es which they supply. LITERATURE CITED Barrarr, J.O. W. 1899 Observations on the normal anatomy of the 9th, 10th, 11th, and 12th cranial nerves. Arch. of Neur. from the Path. Lab. of the London County Asylums, vol. 1, p. 537. 1899 On the anatomical structure of the 9th, 10th, llth, and 12th cranial nerves. Brit. Med. Jour. 1899, part II, p. 837. 1901 Observations on the structure of the third, fourth, and sixth cranial nerves. Jour. Anat. and Phys., vol. 35, n. s. 15, p. 214. Bouauton, T. H. 1906 The increase in the number and size of the medullated fibers in the oculomotor nerve of the white rat and of the cat at differ- ent ages. Jour. Comp. Neur., vol. 16, p. 153. CarRPENTER, F. W. 1906 The development of the oculomotor nerve, the ciliary ganglion and the abducent nerve of the chick. Bull. Mus. Comp. Zool. Harv. Coll., vol. 48, no. 2. Cuases, M. R. anp Ranson, S. W. 1914 The structure of the roots, trunk, and branches of the vagus nerve. Jour. Comp. Neur., vol. 24, p. 31. GASKELL, W. H. 1889 On the relation between the structure, function, dis- tribution and origin of the cranial nerves; together with a theory of the origin of the nervous system of vertebrates. Jour. Phys., London, vol. 10, p. 153. Koprscu, Fr. Rauber’s Lehrbuch der Anatomie des Menschens, Abt 5, Leipzig. Ranson, 8. W. 1911 Non-medullated fibers in the spinal nerves. Am. Jour. AmiaitenvOle IQ ips Oie 1912 The structure of the spinal ganglia and of the spinal nerves. Jour. Comp. Neur., vol. 22, p. 159. THE BRAIN AND THE. FRONTAL GLAND OF THE CASTES OF THE ‘WHITE ANT,’ LEUCO- TERMES FLAVIPES, KOLLAR CAROLINE BURLING THOMPSON Department of Zoology, Wellesley College TWENTY-SIX FIGURES CONTENTS MU OCULC DLO IN erate tain ete eee carehoke the fete a ein od x 8S oe he TERM aS ks ex 353 Material and methods...... Pe Me Sicha se iciwis al GEC TCT © ees ODE The members of the colony of L. flavipes ie heraciilin CAStes ANGE tA. COUNT. ccs 5 cs.cu. Wek ee ae tes ww ace 555 2. Distinguishing characters of the five adult castes and of three types OF nymphs.:..7,.. PORE ORR CE Lice erg Ss se a 558 3. Means of distinguishing the sexes... ee res ae ee 560 Generalssnatomy: ofthe termite’ Drain. 5....-.- vais... sons binlgn sien EMER nes fe oss 561 The finer structure of the brain...... fa NE ee UR EMS Sc 50's % 564 ieerlhe DYSIn SHEA. o.<.06.2+<<~ ss PE rt set a ee 564 2 aL NES MTOCOCETE DIAL LOWES W7 os a oases a ace ave daw a Oe eee ests viola 564 3. The mushroom bodies...........-..... PP er 7 dt, eee 566 Bee NBs COMUTAL DOC Vacs shasien Weld > kin uvihis: da su vids ia SC ROR emt SIs Pers a eM) DauOcellu ama, OCEUEY NEXVER,: vin cawais sso vce e's 5.nse > 0 lle eRe ie as as os 570 65) Dhe optic-lobes:<..<........ Ee ERE ho i ee 572 Met NCTANDEN NEL MODE. sc. kk ith vcs & soit en woe oa Re Ogee eos sok 574 8. The tritocerebral lobes and the tritocerebral commissure............ 574 Gre NearrOnUal: SANGO. 5 oa.co25 a8 ve ec ao x.k 2 ile cyte EROS cle ie eos wae 574 10. The ventral connectives and the subesophageal ganglion............ 576 11. The frontal or fontanel gland and the fontanel nerve................ 576 PIERO SARBUT IAN PRS UIAE Va. gh 59d ain. Ls PAs Finn Ee eno hy CR eR tbs cuss vo 23 589 Obs WEEDS DINE cert co each deni Roca ah dco e Gots Sh ee neg ERIN MOMMIES Lt, ow Ne 592 INTRODUCTION This paper was undertaken with the idea of studying the finer structure of the brains of the different castes of the common termite or ‘white ant,’ and to compare them with the brains of the castes of true ants, the chief point of interest being that both termites and ants are social insects, living in communities 553 5A CAROLINE B. THOMPSON composed of many types of individuals, but differing greatly in their degree of specialization and in intelligence. As the work progressed it was found necessary to study the frontal gland in detail, and it is now my intention to follow this paper with a second, dealing with the development of the frontal gland and the differentiation of the different types of brains in the recently hatched nymphs. The only form to be discussed in the present paper is the common termite, Leucotermes flavipes, Kol. As is well known, this insect is not an ant, but belongs to the order Isoptera, family Mesotermitidae, Holmgren (’11). The species, flavipes, was de- seribed by Kollar (°37) ; the genus Leucotermes, formerly included in the Linnaean genus Termes, was established by Silvestri (01). In the northeastern United States there is only this one genus and species of termites; in the southern states there is a second species, L. virginicus, with three different genera in the Gulf states, Snyder, and several other genera occur in California and the southwest, Heath (’03). MATERIAL AND METHODS My material was collected in Wellesley, Mass., in May, 1915, and June, 1916, beneath and within some old planks of wood. The fixatives used were Bouin’s Fluid, and Gilson’s Fluid. The former, however, is decidedly better for termite nervous tissue, and three hours in the fluid gives a good fixation. Whole mounts of all the heads were made by staining a long time, twenty-four to thirty-six hours, in Conklin’s picro-haematoxylin, and then destaining for a day or longer in acid alcohol, clearing in cedar oil and mounting with the frontal side up. The heads of workers and of both types of nymphs, were embedded in hard paraffin, three to four hours, and were sectioned at a thickness of 6u. The frontal or horizontal plane proved the most satis- factory for sections of the head. In the ease of the soldier, where the chitin of the head is very thick, the brain was first dis- sected out, under a dissecting lens, stained, embedded, and then sectioned. This was also done for the worker, in addition to sections of the entire head, the great hardness of the chitin making BRAIN OF THE ‘WHITE ANT’ 555 the sections of this caste frequently imperfect. Whole mounts of the true adult and other brains were also made. It should be stated here that on account of the internal chitin of the tentorium, in addition to the chitin in the cuticula of the skin, the termite head is a difficult object to section. Cnly the pro- longed time in paraffin has given good results. The sections were stained on the slide with (1) Ehrlich’s acid haematoxylin and eosin, or (2) iron haematoxylin and orange G. For the adult sexual forms, I am indebted to Mr. A. D. Hop- kins and Mr. T. E. Snyder of the division of forest entomology of the U. 8. Department of Agriculture, who have kindly fur- nished me with young and old adults of both the ‘true’ and the neoteinic type with short wing pads, and with much other termite material that will be utilized later. I wish here to express my thanks to both Mr. Hopkins and Mr. Snyder for their kind- ness in sending me this material. THE MEMBERS OF THE COLONY OF L. FLAVIPES I. The adult castes and the young The colony of Leucotermes flavipes is composed of many different kinds of individuals, some of which are adult or final stages, the castes, while others are merely the young, or the developmental stages of the castes, their presence, in some degree, depending upon the season of the year Each caste contains both males and females, caste not corresponding with sex, as in the bees and ants. The term adult is here used in the general and _ biological sense to denote an insect which has undergone its last molt, and which has, in general, acquired its final definitive form and structure. The phrase “‘in general’ is inserted to cover the case of those older females whose abdomens have become great- ly enlarged in the course of egg-laying. I am aware that some biologists would exclude from the category of adult stages the neoteinic reproductive forms and the sterile workers and soldiers, on the ground that the former do not possess the outer bodily structure characteristic of the species, as seen in the ‘true’ adult, THOMPSON B. . 4 CAROLINE (SSoydUIM) (0 & “UU J 0} 9 ‘SLIIP[OR °c (SSopsurIM) 9 & “UU Q 0} C ‘STOYIOAL “Ff pedojoaep JOU SUBSIO XOQ (sped Sut ou) “uu § 09 4 ‘uavenb SUIAR]-380 posIR[Uy “¢ (sped SuiM 410Ys) “UU § 0} Gy ‘uaenb SUIAR]-550 posIvVlUy °“Z (SSUIAL jo sqnjs) “urlUr GFL 0} Gy ‘usenb BUIAR]-3589 posIe[U *T (sped SUL ou) “wu ¢ 9 6 “SUIIOF OINATSGNS IU1d}O9} (sped BULM JLOYS) “UU Gy 03 G9 (8 6 ‘SuTIO] OINIYSGNS OIUTOIOIN °G (SULM con Bondy, ‘T SuOy) “Wu Cc ¢79 ‘PS ‘SzN] 0 }é “sydUIAU TdIP[ON “G ‘ulut 4 ‘sped BUIM OU YQIM syduAN “¢ (sped Sura 4.10ys) “uu G*y 07 G°9 ‘ULIOF puodos oy} JO SYduUIAN *Z (Sped suIM suo) “UU GY OF @'9 “ULIOF qsdy oy} jo syduiAN ‘Ty \ piieice, aie) ets sm impr (dojeaep | jou | IA SUvsIO Xos) ‘sydutrdu soysoyy “fF | sydurdu poproy osaery eee me enone (CLOTON -op ][[IM suBsI0 xaos) syduxu poproy yypeurg | “sss sydurAu poe -1,Ud.LO FJ IPU/]—So95] pedoyaAep suvs10 xog SaDViIS IVNIA HO LINGY SUDVLS IVINGUNdOTSAAGG HO DNOOXK “IOM SHUdIAV THA SUNUALOOVAT IsSsDLy WOLf pajdopy T WIaVo BRAIN OF THE ‘WHITE ANT’ a iF and that the latter have undeveloped sex organs. One disad- vantage of excluding these forms is that we are then left with no general term to denote the full grown stage, which after all is the original sense of the word adult. The adult stages may be divided into two groups: (1) forms with functional and mature sex organs, the ‘kings’ and ‘queens,’ of which there are three different types, (a) kings and queens ‘with long functional wings, the ‘true’ royal pair, (b) kings and queens with short wing pads, the neoteinic substitute pair with short wing pads; (c) kings and queens with no wing pads, the neoteinic substitute wingless pair, sometimes known as ergatoid or worker- like; (2) forms with undeveloped sex organs, (d) the ‘workers,’ (e) the ‘soldiers,’ making in all five adult castes. To these five types of adults, three more, representing phases of a later devel- opment, may be added, namely: the enormously distended fer- tilized egg-laying queens of the three queen types (table 1). The young, or the developmental stages of the five adult castes, occur In many stages, the number of these differing with the caste. Irom the egg hatch forms which are said to be all alike and undifferentiated, Grassi, Snyder, and from these undif- ferentiated nymphs, ‘larvae’ of Snyder,!develop nymphs with large heads and nymphs with small heads. The large-headed nymphs develop ‘‘after a series of molts and quiescent stages of com- paratively short duration,” the whole process requiring less than one year, Snyder ('16), into the sterile workers and soldiers. The small-headed nymphs undergo a more complex development, which ‘‘apparently requires two seasons,’’ and develop into: (1) nymphs of the ‘first form,’ with long wing pads, which become the true royal pair, the sexual adults with long wings, (2) nymphs of the ‘second form,’ with short wing pads, which become the substitute sexual forms with short wing pads, (3) nymphs with- out wing pads, whose development is not fully understood, and which become the substitute wingless sexual adults. 'The term nymph is used in this paper to denote any developmental stage of an insect with incomplete metamorphosis, whether the form possesses wing pads or not. Snyder, in accordance with the older authors, applies the term ‘larva’ to the younger nymphs which possess rudiments of wing pads that can only be distinguished with magnification. He uses the term nymph to designate forms with wing pads that can be distinguished with the naked eye. 558 CAROLINE B. THOMPSON 2. Distinguishing characters of the five adult castes and of three types of nymphs Before beginning the discussion and comparison of the brains of the different forms of L. flavipes some descriptions of the types themselves and the means of distinguishing them may be of interest. Forms with sex organs developed in the adult. 1. Nympus. Nymphs of the first form, with long wing pads. These nymphs are most numerous in the early spring before the transformation into the adult has taken place. According to Snyder they attain a length of 7.5 mm. before the last molt, but they may be dis- tinguished while much shorter. The color is creamy white, with a pale pink or rose pigmentation of the compound eyes. The hind wing pads extend back as far as the fourth abdominal segment. Nymphs of the second form, with short wing pads. These nymphs are found at the same time and in about equal numbers with the long-winged nymphs. They likewise attain a length of 7.5 mm. before the last molt. They may be readily distin- guished from the ‘first form’ by the short rudimentary wing pads which hardly reach the second abdominal segment. The color is creamy white and the compound eyes show no trace of pig- ment. Nymphs with no wing pads. I have never found these nymphs and therefore quote from Snyder, ‘‘ Another type of substitute or neoteinic reproductive form, which greatly resembles the worker (‘ergatoid’) is developed from young larvae of the sexed forms.” 2. Aputts. The true royal pair, with long wings. The true winged adults are found in the nest or in the air in late spring and early summer. The head and body are dark brown, of about the same size as the nymphs of the first form, the long filmy wings are nearly twice the length of the body. The compound eyes are black surrounded by a lighter rim of unpigmented skin. The two lateral ocelli are visible as small light spots in front of the compound eyes and behind the antennae. The opening of the frontal or fontanel gland lies in the median line of the frontal surface of the head. BRAIN OF THE ‘WHITE ANT’ 559 The egg-laying true queen. The old egg-laying true queens are always found within the nest, often in large ‘queen cells’ or chambers, and they may attain a length of 14.5 mm., Snyder (16). The head and thorax are of normal size and brown in color, the abdomen is enormously distended, and is straw-colored with intersegmental bars of brown. The broken off stubs of the long wings remain attached to the thorax. The neoteinic substitute pair with short wing pads. This pair is found within the nest and is similar in length to the nymphs of the second form. The body color is light brownish and the “compound eyes are brown. The wing pads are now merely short, transparent chitinous plates. The egg-laying substitute queen with short wing pads. This old substitute queen with short wing pads attains a length of 9 mm., Snyder (716). The abdomen is distended and is creamy white with darker chitinous bars, the head and thorax are of normal size. The compound eyes are brown.? The neoteinie substitute wingless pair. As I have never seen this form, I will again quote from Snyder (’16, p. 7), ‘‘Of a pale yellowish or grayish color, and having no wing pads (fig. 3; fig. 4c), individuals of this larval supplementary reproductive form are apparently blind and never leave the parent colony, except by underground tunnels.” The egg-laying substitute wingless queen. The old egg-lay- ing substitute wingless queen attains a length of 9 mm. and has a distended abdomen, Snyder (16). Forms with sex organs not developed in the adult. 1. The work- ers. Workers are found in termite colonies throughout the year. The smallest of all the castes, the body measures only 5 mm. in length. The head is considerably broader from side to side than that of the nymphs. The abdomen is shorter, softer, and covered with a more transparent skin. The color of the abdo- men js usually grayish, owing to particles of wood in the ali- mentary canal seen through the skin. No eyes are visible in 2 The above descriptions of the enlarged egg-laying queens are based upon the alcoholic specimens sent to me by Mr. A. D. Hopkins and Mr. T. E. Si yder of the U. S. Dept. of Agriculture. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 560 CAROLINE B. THOMPSON the worker until after staining, when small rudimentary com- pound eyes may be distinguished. No ocelli are present. 2. The soldiers. Soldiers are found in the colony throughout the year. The entire body measures 6 to 7 mm. in length, but the head is much longer and the abdomen shorter than that of the worker. No eyes can be seen until after the head is stained, when rudimentary compound eyes even smaller than those of the worker become visible. The opening of the frontal gland is in the median line of the frontal surface of the head. Fig. 1 The abdomens of three individuals seen from the ventral surface. A, adult male; B, adult female; C, female nymph with long wing pads; 2 to 10, 2nd to 10th abdominal segments. Obj. 32, oc. 6, reduced one-half. ITI. Means of distinguishing the sexes The only differentiation between the two sexes in any caste of L. flavipes is in the size of the abdomen, and in the relative size and arrangement of the sternites, i.e. the ventral parts, of the posterior abdominal segments. The sex of any termite may be distinguished by the size and the shape of the seventh and eighth abdominal sternites.® In the male viewed from the ventral surface (fig. 1, A) the seventh and eighth abdominal segments measure about the same in length, i.e., in an antero-posterior direction, and the seventh 3 The ventral surface of the first abdominal segment is entirely covered by the metathorax, so that the actual seventh segment is apparently the sixth. BRAIN OF THE ‘WHITE ANT’ 561 segment is shorter than in the female (B). The posterior mar- gin of the ninth segment is concave and two slender cerci are present on the lateral surfaces of the tenth segment. In the adult female (fig. 1, B) the seventh segment is very long, with a rounded posterior edge, and completely covers the eighth and part of the ninth segment. Jointed cerci are borne on the sides of the tenth segment. In all female nymphs (fig. 1, C) and also in the female workers and soldiers, the seventh segment, although long, does not com- pletely cover the small indented eighth segment. Cerci similar to those of the male are borne on the median surface of the ninth segment and also on the lateral surfaces of the tenth segment. GENERAL ANATOMY OF THE TERMITE BRAIN The present paper will discuss the brains of the nymphs of the first and second forms, the true sexual adults, and the workers and soldiers. It is generally thought today that the insect brain includes six regions, corresponding to the embryonic segments of the head, three of which are supraesophageal, namely: the proto- cerebrum, the deutocerebrum, and the tritocerebrum, and three subesophageal, the mandibular, the maxillary, and the labial ganglia. Holmgren (’09) finds these six regions in the termite brain and discusses at some length the origin of the labrofrontal nerves, which he decides is in the tritocerebrum. It need only be remarked here that the same six regions may be distinguished in Leucotermes, and that in agreement with Holmgren I find the labrofrontal nerves arising from the tritocerebrum. Table 2 names the regions and the parts that are present in the brain of L. flavipes. Figure 2 is a diagram of the brain of L. flavipes, as seen from the frontal surface. The supraesophageal ganglion is drawn from an entire mount of a brain dissected out from the head of a nymph with long wing pads; the ventral connectives, v.c., and 562 CAROLINE B. THOMPSON TABLE 2 The parts of the brain I. The supraesophageal ganglion. A. Protocerebrum _ The protocerebral lobes f anterior, dorsal \ posterior, dorsal (anterior roots The mushroom bodies eee body roots porterior roots The protocerebral commissures The central body The optic lobes B. Deutocerebrum The antennal lobes C. Tritocerebrum The tritocerebral lobes The tritocerebral commissure (subesophageal) II. The subesophageal ganglion The ventral connectives The mandibular The maxillary ganglion The labial The nerves of the head I. A. The optic nerves The ocellar nerves The fontanel nerve B. The antennal nerves C. The labrofrontal nerves to {the nerve to the protocerebrum the frontal ganglion Pe labral nerves the recurrent nerve II. The mandibular nerves The maxillary nerves The labial nerves the subesophageal ganglion, sb.g., are drawn from sections of the same form.* 4 The heads of all termites are held in a somewhat slanting position, making a large obtuse angle with the long axis of the body, so that the morphologically dorsal surface of the head has become frontal or anterior. The slant of the head is much less in termites than in ants, and in the termite soldier the head is almost horizontal, the so-called frontal surface being practically dorsal, but for the sake of clearness the same terms of direction will be applied to all the castes of termites. In describing the entire heads the terms anterior and posterior imply toward and away from the frontal surface, in the same sense that Hesse (1901 b) uses the terms rostral and caudal; in like manner dorsal and ventral imply toward or away from the vertex of the head. BRAIN OF THE ‘WHITE ANT’ 563 The insect brain froms a ring of nerve tissue which encircles the esophagus and which is composed of the supraesophageal gan- glion,. the ventral connectives, and the subesophageal ganglion. The three parts of the supraesophageal ganglion, the proto- cerebrum, the deutocerebrum, and the tritocerebrum, are merged into a single mass which constitutes the principal part of the - brain. To the protocerebrum belong the protocerebral lobes, p.l., the optic lobes, o.l., connected by the fibers of the optic oc. Ock- 2.3. 7 Bo. Se Fig. 2 Diagram of the brain of a nymph with long wing pads, as if seen in frontal optical section. A.l., antennary lobe; f.g., frontal gland; f.n., fontanel nerve; fr.gn., frontal ganglion; l/.n., ‘abrofrontal nerve; lb.n., labial nerve; m.b., mushroom body; md.n., mandibular nerve; mz.n., maxillary nerve; oc.n., oc llar nerve; 0.l., optic lobe; p.!., protocerebral lobe; sb.g., subesophageal ganglion; tr.cm., tritocerebral commissure; v.c., ventral connective. nerves with the compound eyes, the two mushroom bodies, m.b., the ocellar nerves, oc.n., connected with the two lateral ocelli, and the fontanel nerve, f.n., connected with the frontal gland, f.g. ‘To the deutocerebrum belong the antennal lobes, a.l., and the antennal nerves of the antennae. The tritocerebrum con- sists of the tritocerebral lobes, and the tritocerebral commissure, tr.cm., beneath the esophagus. The labrofrontal nerves, l.f.n., or the tritocerebral nerves, arise on the inner, median surfaces of the tritocerebral lobes, and running upward and forward unite in the frontal ganglion, fr.gn., which lies anterior to the main 564 CAROLINE B. THOMPSON part of the brain and directly above the mouth opening. The connection of the frontal ganglion and the labrofrontal nerves is not shown in the diagram nor is the recurrent nerve which runs backward from the frontal ganglion. The delicate unpaired nerve from the dorsal surface of the frontal ganglion to the protocerebral lobes, and the labral nerves from the ventral surface of the frontal ganglion, are figured but not labeled. Posterior to the tritocerebral commissure the two slender ventral connectives, v.c., run first backward (which can- not be shown in this diagram), then downward, and unite to form the large subesophageal ganglion, a single mass, consisting of the fused mandibular, maxillary, and labial ganglia, and from which arise the mandibular, maxillary, and labial nerves. Pos- terior to this ganglion the thoracic connectives, not shown in the figure, pass upward and then backward into the thorax. THE FINER STRUCTURE OF THE BRAIN I. The brain sheath The entire brain is surrounded by a membranous sheath com- posed of a single layer of cells resembling mesenchym cells, their fibrous processes making a continuous double walled membrane between which the cell bodies and the nuclei are situated. ITI. The protocerebral lobes The protocerebral lobes (fig. 2, p.l.) form the central part of the supraesophageal ganglion; they are continuous on their dor- sal surface with the mushroom bodies, on their lateral surfaces with the optic lobes, and on their ventral surface with the an- tennal lobes, and are also connected by a slender nerve with the frontal ganglion, fr.gn., which lies anterior and somewhat ven- tral to the protocerebrum. ‘The ocellar nerves, from the lateral ocelli, and the fontanel nerve, from the frontal gland, also enter the protccerebral lobes, and will be considered in more detail under those headings. Like other parts of the brain the proto- cerebral lobes consist of a central fibrous core and an outer in- vesting layer of nerve cells. Most of these nerve cells are small BRAIN OF THE ‘WHITE ANT’ 565 with round nuclei and a round cell body which appears as a mere rim of cytoplasm (fig. 3, A,B), but in the median dorsal region, the intercerebral region of Haller, and here and there on the ventral surface, the cells are larger with pear-shaped cell bodies (fig. 3, C, D). The smallest nerve cells of the proto- cerebral lobes are, however, almost twice as large as the adja- cent nerve cells belonging to the mushroom bodies (fig. 3, £) and to the optic lobes. In a series of frontal sections, beginning with the frontal or anterior surface (figs. 13 to 20) it will be seen that the proto- B Fig. 3 Nerve cells from the protocerebral lobes and the mushroom body. A, small cells, from the lateral region of the protocerebral lobes; B, medium sized cells, from the same region; C, large cells, from the median ventral region of the protocerebral lobes; D, large cells, from the intercerebral region; EF, cells from the mushroom body. Homog. immers. 1.8 mm., oc. 6. cerebral lobes are at first entire (fig. 13), then, farther back in the series (fig. 14) the two great anterior roots of the mushroom bodies penetrate deep into the fibrous core of the protocerebral lobes, dividing them into a median and two lateral portions, and this division is further continued by the stalks of the mushroom bodies (figs. 15, 16). a. The protocerebral commissures. About the middle of the protocerebral lobes, in the plane of the central body (fig. 15) the lateral and median parts of the lobes are again connected by a stout fiber tract passing from side to side beneath the central body and connected also with the antennal lobes. This tract or commissure is homologous with the so called ‘ventral com- 566 CAROLINE B. THOMPSON missure’ of the hymenoptera and other insects. The lateral portions of the protocerebral Icbes are also connected by two dorsal protocerebral commissures, the anterior, a delicate strand of fibers, lying above the central body (figs. 15, 16, a.cm.) the posterior, on the same level as the first, lying immediately pos- terior to the central body, fig. 18, p.cm. These two commissures are homologous with the two dorsal commissures found in ants, and it is interesting to note that the posterior one, the ‘Bricke’ of the older authors, has the same characteristic wide inverted N shevpe. In figures 15 to 18, the connection of the optic lobes with the protccerebral Jcbes may be seen. Figures 19 to 20 show the relation between the protocerebral lobes, the deutocerebrum or antennary lobes, and the trito- cerebrum. ITI. The mushroom bodies The two mushroom bodies, or the globuli, as Holmgren terms them, are now generally considered the chief centers of intelli- gence in insects. They appear, in frontal views of the head (figs. 8 to 12, m.b.), as two slightly grooved projections from the dorsal surface of the brain, the frontal gland occupying the space between them. Each mushroom body consists typically of two lobes, which in the termites are not distinctly separated but form cone continuous mass of tissue, the lobes being indi- cated on the exterior of the brain merely by a slight groove or depression of the surface. In sections (figs. 15 to 18) it may be seen that the mushroom bodies are composed of an outer nerve cell layer and an inner fibrous portion which forms the cups or calyces, the stalks, and the three pairs of roots. a. The nerve cells of the mushroom bodies. The nerve cells of the mushroom bodies are all of the same size and are among the smallest cells of the brain (fig. 3, #). They have a round or oval nucleus and so small an enveloping layer of cytoplasm that it cannot be distinguished, even with the immersion lens, except at the distal end where the axon is given off. The chromatin masses of the nucleus are all about the same size, forming a BRAIN OF THE ‘WHITE ANT’ 567 peculiar characteristic pattern by which these cells may be recognized. Although the nerve cells are of similar size throughout the mushroom bodies, they are differentiated into groups, or zones, according to their position. In the center of each cup or calyx, as seen in section (fig. 4), ies an oval mass of cells, 7, which is homologous in position with the central oval mass of large cells found in the bees, Jonescu (09), and in the ants, Pietschker (’11), Thompson (712), and which I have termed Group I. On each side of Group I, are broad wedge-shaped masses of cells (fig. 4, Fig. 4 Diagram of a mushroom body, showing the nerve cell layer divided into groups, the calyces, and the beginning of the stalk. J, cell group I; JJ, cell group II; JJ/, cell group IIT; gl., glia cells. II), which occupy most of the dorsal surface of each lobe and whose inner margins overlap and enclose the central group. I. These masses, which appear separate in sections, form a con- tinuous zone if seen in surface view, and are homologous with Group II of ants. Again, in each lobe, on each side of Group II lie smaller masses of cells which form the lateral surfaces of the lobes (fig. 4, J7Z). There are only three, instead of four, of these cell masses, because the inner lateral surfaces of the two lobes are in contact and their cells are continuous. These groups are equivalent in position to Groups III and IV of the ants. b. The fibrous core of the mushroom bodies. The cups or calyces of the inner and outer lobes of the mushroom bodies are com- posed (1) of converging bundles of fibers, the axons of the three 568 CAROLINE B. THOMPSON zones of nerve cells just described, and (2) of masses of glia cells (fig. 4, gl.) resembling the ‘glomeru'i’ of the antennal Jobes, which envelop the fiber bundles-on all sides and unite the calyces of the two lobes into one continuous whole. The stalks of the mushroom bodies are the continuations downward and inward of the fiber bundles of each lobe. These lie side by side, the fibers from the inner lobe on the median side of those from the outer lobe, and for a short distance they remain distinct. The two stalks, surrounded by a delicate sheath, pene- trate deep down into the fibrous core of the protocerebral lobes and then run forward. There is no ‘decussation’ of fibers such as is seen in the hymenoptera. In the same frontal plane with the central body (fig. 15) the distal ends of the stalks lie beneath the central body, and at this point each stalk gives rise to three roots, the anterior, the central body, and the posterior roots. The central body roots, which are the shortest, pass upward and directly into the central body (fig. 16, c.b.r.). The anterior roots which are the longest, bend sharply upward and forward, mak- ing an elbow with the stalks from which they have arisen (fig. 14, a.r.m.b., m.b.s.). In sections that are farther forward in the head, the anterior roots may be seen as two great bundles of fibers curving latero-dorsally and dividing the protocerebral lobes in the manner already described. On reaching the dorso-lateral margins of the protocerebral core, the anterior roots curve in a posterior direction and are seen in sections as two detached masses on the outer sides of the mushroom bodies (figs. 15, 16, a.r.m.b.), then (fig. 14) returning again forward, the narrow dis- tal ends turn downward and terminate among the nerve cells of the anterior part of the protocerebral lobes. The posterior roots branch off from the stalks together with the central body roots, and, after the latter have passed into the ventral surface of the central body, pass dorsalward, posterior to the central body and to the posterior dorsal commissure, there expanding into two large and very prominent lobes which nearly fill the intercerebral region (figs. 19, 20, 28, 26, p.r.m.b.). The ventral part of these lobes or roots is connected with the proto- cerebral fibrous core, the dorsal part, however, projects back- BRAIN OF THE ‘WHITE ANT’ 569 ward for some distance, evidently giving fibers to and receiving fibers from the posterior cells of the mushroom bodies, and then ending in about the same plane with the beginning of the trito- cerebral lobes. c. Comparison of the mushroom bodies of the different castes of L. flavipes. The mushroom bodies differ little in the different castes except in size, and even in this respect there is not a very great dissimilarity (figs. 8 to 12). The worker has the largest mushroom bodies, largest by actual] measurement and in the estimated number of nerve cells; the nymphs of the first and second forms have mushroom bodies about sitnilar in size, but slightly smaller than those of the worker; the soldier has the smallest mushroom bodies of any easte. The cells of the mushroom bodies of the worker and soldier penetrate into the intercerebral region (figs. 22, 25), but merely border upon it in those of the nymphs of the first and second forms. My only true adult material, as already stated, was alcoholic and much of it seemed to have shrunk. It will be noted that in the true adult brain outlined in figure 9 the mushroom bodies are farther apart than in the nymph of the first form from which this adult has developed (fig. 8) and the frontal gland has in- creased in size. Some other better preserved true adult brains are considerably larger than the brain of a nymph of the first form, and the mushroom bodies are likewise larger. Holmgren (’09) emphasizes the great deterioration of the brain tissue which he observes in the older enlarged and egg-laying queens. My material has been preserved in alcohol and is not the best for finer study, but the examination of several old and enlarged queens leads me to believe that in L. flavipes this deterioration of the brain has not taken place. d. Comparison of the mushroom bodies of termites and hymenop- tera. A comparison of the Leucotermes mushroom body with those of ants and bees shows, as one would naturally expect, that the termite mushroom body is much more simple and primitive. This primitive condition is apparent in the small and uniform size of all the nerve cells, especially in the cell 570 CAROLINE B. THOMPSON group I; in the presence of three zones of cells instead of the four found in ants; in the incomplete differentiation of the two lobes, whose cells are not separated by a deep furrow, as in ants, and whose two cups or calyces are completely fused by inter- vening masses of glia cells; in the shallowness of the cups; finally, in the smaller size of the entire mushroom bodies and their slight differentiation in the different castes. IV. The central body The central body is situated in the central frontal plane of the protocerebral lobes, embedded in their fibrous core, directly beneath the intercerebral region. In form as well as in’ position the central body of L. flavipes resembles that of bees and ants. It has two parts (fig. 15, c.b.), a curved dorsal portion, composed of fiber bundles that are radially arranged with intervening spaces, and a flatter ventral part, also fibrous. No nerve cells are found within the central body, as in ants, where small nerve cells cecupy the spaces between the radial bundles of fibers, but a few scattered nerve cells are occasionally found along the outer surface. The structure of the central body is the same in all the castes, but the size varies with the size of the different brains (figs. 15, BOIS): Lying beneath the central body are two small round bodies which were formerly known as the ‘“‘tubercles of the central body” and also as ‘ocellar glomeruli.’ I have shown, Thompson (12, ’14), that in ants and in the bumble bee these previously misinterpreted bodies are the posterior roots of the mushroom bodies. In L. flavipes these rounded bodies are in connection with the central body roots of the mushroom bodies (figs. 15, 16, c.b.r.), which, it will be remembered, arise from the distal ends of the stalks in close proximity to the posterior roots. V. Ocelli and ocellar nerves Two simple eyes or ocelli are present in the adults and nymphs of the sexual forms, and are situated on the lateral surfaces of the head, in front of the compound eyes and behind the antennae. BRAIN OF THE ‘WHITE ANT’ 571 In the head of the true adult (fig. 9, 0.c.) the ocelli may be readily seen as small colorless spots which stand out sharply from the surrounding brown skin, but in the heads of nymphs (figs. 8, 10), whose skin is not pigmented, the ocelli are not visible except in sections. The ocelli are of a very simple and primitive type, Hesse (01 6), without a lens and with little or no pigment. The outer surface of the hypodermis above the ocelli is slightly convex, the inner surface is very concave, the latter caused by the sudden thinning of the inner cuticula, and in this concavity the bulb- like ocelli are situated. In general contour the ocelli resemble the tactile buds found in the skin of Amblystoma punctatum. The visual cells are Jong slender and curving, with spaces between their bases. As to the finer intracellular structure, I am not prepared to make a statement at this time. I shall also leave for further study the question whether the distal ends of the visual cells lie between the hypodermal cells, a primitive position, according to Hesse, or wholly, and secondarily, beneath them; although the first view would seem to be upheld by my present material. . In a frontal section of the anterior part of the brain of a nymph with long wing pads, the ocelli (fig. 13, oc.) may be seen on each side of the section, just beneath the hypodermis, and covered by a thinner layer of cuticula, the inner cuticula, 7.cw., being absent at this point. From the ocelli the slender ocellar nerves, lying just outside the brain sheath, run in toward the median line and enter the nerve cell layer of the protocerebral lobes just above the anterior roots of the mushroom bodies. Passing down into the intercerebral region, the ocellar nerves (figs. 15 to 18, oc.n.) run backward and finally enter the dorsal surface of the protocerebral lobes just behind the posterior dorsal commissure and in the same frontal section as the fontanel nerve from the frontal gland (fig. 19, oc.n., f.n.). At no point along their entire Jength do the ocellar nerves expand into ocellar lobes such as are present in the bees and ants. After my first cursory examination of the brain sections of L. flavipes, I was inclined to assign the réle of ocellar lobes to the large lobes, 572 CAROLINE B. THOMPSON p.r.m.b. seen in figures 19, 23, 26, on the ground that these lobes occupy a position similar to that of the ocellar lobes of ants and are in the neighborhood of the entering ocellar nerves. A very careful examination of a large number of sections, however, proved that there is no connection between the ocellar nerves and these adjacent lobes, and that there is direct connection be- tween the mushroom body stalks, the protocerebral lobes, and the lobes in question, which are, as shown in a preceding section, the posterior roots of the mushroom bodies. VI. The optic lobes The optic lobes are situated on the lateral surfaces of the proto- cerebrum and are present in all the castes of L. flavipes, although they are well developed only in the sexual forms (figs. 8 to 12, o.l.). They are continuous with the protocerebral lobes and consist of an outer layer of very small nerve cells, similar in size to those of the mushroom bodies, and an inner fibrous portion that is subdivided into the outer, middle, and inner fiber masses, and the outer and inner crossings of fibers found in the typical insect brain, Berlese (09). The optic lobes of the nymph with long wing pads are the largest and most highly developed of any caste studied. The outer fiber masses. (fig. 17, 0.f.) are slender and elongated in a dorso-ventral direction. Nerve fibers pass in to them from the compound eyes, and continue inward, as the outer crossing, to the middle fiber mass, mf., the largest of the three masses. Fibers again cross between the middle and the inner fiber masses, i.f., making the inner crossing. The small inner fiber masses are directly continuous with the fibrous core of the protocerebral lobes. In the nymph with short wing pads (fig. 10) the optic lobes and the compound eyes are slightly smaller than in the nymph just described, but the relative size and arrangement of the parts is the same. In the worker, although the optic lobes are very much reduced in size, they are readily seen in surface views (fig. 11) as small BRAIN OF THE ‘WHITE ANT’ 573 projections from the lateral surfaces of the brain, and a slender optic nerve passes from each greatly reduced compound eye to the optic lobe. In a frontal section of the worker brain (fig. 25) the small inner, middle, and outer fiber masses may be distin- guished in the optic lobes, and also the fibers of the distal part of the optic nerve. By carefully following the optic nerve in subsequent sections it may be traced to the vestigeal compound eye. The condition of the compound eyes evidently varies con- siderably in different termites. Holmgren (’09), describing the optic lobes in the worker of Eutermes, writes as follows: Die Reduktion der Facettaugen hat eine entsprechende Reduktion der Sehganglien mitgefiihrt, Die Sehnerven sind vielleicht vorhanden aber enthalten nur wenige Nervenfiidchen und von den bei den Ge- schlechtstieren, besonders den jungen, so wohlentwickelten Sehganglien kann man gar nichts entdecken. Die Seiten des Protocerebralganglions sind somit kreisférmig abgerundet. The optic lobes of the soldier, like those of the worker, appear as small rounded projections on the lateral surfaces of the proto- cerebrum (fig. 12, 0.l.), with optic nerves extending to the still smaller vestigeal compound eyes, c.e. On account of the very hard chitin of the soldier’s head no attempt was made to sec- tion the entire head. The brain was dissected out, under a dis- secting microscope, stained, embedded, and then cut in frontal sections. A section through the optic lobes (fig. 22, 0.1.) shows that the inner, middle, and outer fiber masses, although small, are clearly distinguishable, and that fibers enter the outer fiber mass from the optic nerve. Holmgren (’09) states of the soldiers of EKutermes: Die Soldaten sind ja blind und deshalb sind die Sehganglien voll- stiindig verschwunden. Man kann von denselben kein Spur ent- decken, obwohl ein sehr dinner n. opticus meistens vorhanden ist. Dieser fungiert aber als Hautnerv. Zufolge des Fehlens der Sehganglien sind die Seiten des Protocerebrums beinahe kreisrund. The optic lobes of the true adult have not been studied in sections. 574 CAROLINE B. THOMPSON VII. The antennal lobes The antennal lobes compose the deutocerebrum, or the second brain segment, and are continuous with the anterior and ventral part of the protocerebrum (fig. 2, a.l.). In form they are elon- gated masses that continue into the antennae as the antennal or olfactory nerves. On their inner lateral surfaces the antennal lobes are continuous with the third brain segment, the trito- cerebrum. In sections it may be seen that the antennal lobes have an outer nerve cell layer containing both large and small cells, and an inner fibrous core that contains scattered masses of glia cells, the so-called ‘glomeruli.’ The relative size of the antennal lobes differs very slightly in the different castes of L. flavipes (figs. 8 to 12). The antennal lobes are largest in the nymphs with long wing pads and in the worker, but are smaller in the nymph with short wing pads and in the soldier. Holmgren states that these lobes are much larger in the worker of Eutermes than in the other castes: ‘‘die Deuto- cerebralganglien der Arbeiter sind verhiltnissmiassig grésser und enthalten eine auch absolut bedeutend gréssere Zahl Ganglien- zellen als bei den Geschlechtsindividuen.”’ VII. The tritocerebral lobes and the tritocerebral commissure The tritocerebral lobes are small lobes extending ventrally along the sides of the esophagus and continuous with the inner, lateral, surfaces of the antennal lobes (fig. 20). From the inner median surfaces of the tritocerebral lobes arise a pair of nerves that run forward alongside of the esophagus and unite in the frontal ganglion; these are the tritocerebral or labrofrontal nerves (fig. 2, l.f.n.). Beneath the esophagus the tritocerebral lobes unite in the slender tritccerebral commissure (fig. 2, ér.cm.), which is almost devoid of any investing nerve cells. IX. The frontal ganglion The frontal ganglion is a small mass of nerve tissue situated just beneath the clypeus, the sclerite of the head to which the labrum is attached, and above the mouth opening, somewhat antero-ventral to the brain (figs. 2, 8, fr.gn.). BRAIN OF THE ‘WHITE ANT’ 575 In sections (figs. 13, 21, 24) the frontal ganglion is rather tri- angular in appearance, the apex composed of nerve cells and the base of nerve fibers. The nerves connected with the frontal ganglion are (1) the paired tritocerebral or labrofrontal nerves, l.f.n., (2) the paired labral nerves, la.n., (3) an unpaired nerve to the protocerebrum, (4) the unpaired recurrent nerve, 7.7. The tritocerebral or labrofrontal nerves, l.f.n., arise from the tritocerebrum as described under that heading, and their anterior ends are connected with the latero-ventral surfaces of the frontal ganglion. From the median ventral surface of the frontal ganglion a bundle of nerve fibers emerges that runs first ventralward for a short distance and then divides into delicate right and left branches that pass to right and Jeft through some muscle fibers in the clypeus, continuing down and forward into the labrum as two delicate but distinct nerves. Those branches are distributed throughout the labrum. I have termed these nerves the labral nerves. In this I am apparently not in agreement with Holm- gren (’09) who does not describe the labral nerves as arising from the frontal ganglion but speaks as though the labrum were inner- vated directly from branches of the labrofrontal nerves, which is not the case in L: flavipes. In all the sections which I have examined the nerves of the labrum have no direct connection with the labrofrontal nerves but arise indirectly, from the base of the frontal ganglion as described above. ‘To quote Holmgren: Ganglion frontale liegt unmittelbar vor dem oberen Schlundganglion und ist mit diesem durch einen unpaaren Nervenfaser, der im Proto- cerebrum eintritt, verbunden. Vor dem Ganglion frontale gehen nach vorn Nerven aus, welche einige clypealen Muskeln innervieren. An den Seiten des Ganglion frontalekommen die tritocerebralen Kon- nective ein, und hinten lauft der nervus recurrens aus, um oberhalb des Darmes sich nach hinten zu begeben. Aus Tritocerebrum werden folgende Teile innérviert: 1) Die ganze oben beschriebene Labralmuskulatur von n. labro- frontalis 2) Die Labraldriisen von n. labrofrontalis. In agreement with Holmgren I find a very delicate unpaired nerve running between the dorsal apex of the frontal ganglion THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 26, NO. 5 576 CAROLINE B. THOMPSON and the ventral surface of the protocerebral lobes, also the un- paired recurrent nerve which arises from the posterior surface of the frontal ganglion. This latter nerve (figs. 14 to 26, r.n.) runs backward above the esophagus into the thorax and at a short distance posterior to the supraesophageal ganglion expands into the socalled ‘esophageal ganglion.’ X. The-ventral connectives and the subesophageal ganglion The two slender ventral connectives (fig. 2, v.c.) arise from the posterior surface of the tritocer.bral commissure. They con- tinue backward side by side, their inner surfaces touching, beneath the esophagus and above the brcad tentorial band of © chitin that is present in the interior of the head. They become still smaller, and pass down through the tentorial aperture, finally merging into the dorsal surface of the subesophageal ganglion. The subesophageal ganglion (fig. 2, sb.g.) is large and consists of a fibrous core and a thick investing layer of nerve cells on the ventral surface. From the anterior end arise the mandibu- lar nerves, md.n., from the posterior end, the maxillary, mz.n., and the labial, Jb.n., nerves. The branches of these nerves, described by Holmgren, have not been traced. Immediately behind the point of exit of the Jabial nerves the subesophageal ganglion ends, or rather is continued into the thoracic connec- tives, which pass upward and out of the head through the great tentorial aperture. XI. The frontal or fontanel gland and the fontanel nerve The frontal or fontanel gland of the termites has long been an object of much interest and speculation. It is situated in the median Jine of the frontal surface of the head beneath the so-called ‘fontanel,’ which is defined by Brues (715) as ‘“‘a small, depressed pale spot on the front of the head between the eyes (isoptera).”’ In surface views of the heads of the castes of L. flavipes, (figs. 8 to 12) the frontal gland appears as a rather triangular or rounded mass lying in the space between the two mushroom bodies. In the adult sexual forms and in the soldier a tiny round BRAIN OF THE ‘WHITE ANT’ 577 opening may be observed in the cuticula above the anterior end of the gland, but I have not been able to distinguish any such opening in the worker or in the two nymphs. The opening is situated in a shallow depression of the surface, the fontanel, which is paler than the surrounding skin. The frontal gland differs greatly in size in the various castes. It is smallest of all in the worker (fig. 11) and greatly elongated in the soldier (fig. 12). In neither of these castes does the frontal gland completely fill the space between the mushroom bodies. In the sexual forms the true adult (fig. 9) has the largest frontal gland; the nymph with long wing pads (fig. 8) has a gland of similar proportions but smaller; the nymphs with short wing pads (fig. 10) has an even smaller gland. In all the sexual forms the frontal gland completely fills the space between the mushroom bodies. Looking down into the frontal gland as it is seen in surface views is like looking into a shallow crater. The edges rise into folds which approach each other and nearly meet at the antero- ventral end and enclose the central lumen. Sections show that the marginal cells of the gland are directly continuous with the hypodermal cells and that the cuticula above the center of the gland is slightly depressed and thin, consisting only of the pri- mary or outer cuticula, the secondary, inner, cuticula ending at the gland margin. This depression and thinning of the cutic- ula is the structural explanation of the pale ‘fontanel’ spot seen on the outer surface of the head. In the series of sections seen in figures 13 to 20, the first indi- cation of the frontal gland is seen in a group of elongated hypo- dermal cells (fig. 13, f.g.) lying in the median line directly beneath the cuticula, which is here of normal thickness. This group of cells forms the anterior margin of the frontal gland and it should be noted that it occurs in the same frontal section as the simple eyes, or ocelli, oc., a point which will be further discussed later. Figure 14, which is six sections farther back in the series, includes a section of the anterior part of the frontal gland, f.g. The high columnar cells of the gland are continuous with the low cuboid hypodermal cells; the cuticula is slightly depressed and 578 CAROLINE B. THOMPSON the secondary, inner, layer is lacking above the lumen of the gland. In figures 15, 16, 17, the deeper, central, part of the gland is shown; figures 18, 19, contain the posterior outpocketing of the gland, not connected with the margin in the figures, and since these sections are considerably farther back in the series, in the longest part of the head, the cuticula and hypodermal Fig. 5 Frontal section of the frontal or fontanel gland of a nymph with long wing pads. B.m., basement membrane; hyp., hypodermis; 7.cu., inner cuticula; o.cu., outer cuticula; 1, long slender cells, probably not yet secretory in function; 2, swollen cells, probably glandular. Homog. immers. 1.8 mm., oc. 6, reduced one fifth. cells are now some distance from the brain, and, for consider- ations of space, are no longer included in the figures. Figure 20 shows the posterior wall of the frontal gland which ends in the next section. a. The finer structure of the frontal gland. « , \ . . ts i =< ‘ AP ak a en ; oer ve ty Ae Hy i 1 - | 5 14 a / Oe may ' J * . « ® ' i . as! ¢ a . ; SUBJECT AND AUTHOR INDEX 7 eccnee RMREIAEEE ON Caden 8553 cau A tbe accom AOS Action of light. Changes in the rod-visual cells of the frog due to the................ 429 Activity on the histological structure of nerve (OVERS Dd BUTS | AC) A 341 Afferent system of the head ‘of Amblystoma. Correlated anatomical and physiological studies of the growth of the nervous sys- fern oD Ampnipins Ul. Phe... ............ 247 (Albino rat). A preliminary determination of the part played by myelin in reducing the water content of the mammalian nerv- UMMM Gs Vette esis ite seem ae ese 443 ALLEN, Witt1aAM F. Studies on the spinal cord and medulla of cyclostomes with special reference to the formation and ex- pansion of the roof plate and the flatten- WUE Of LHS SPINK OOKG steve. ves cssces. ese 9 Amblystoma. I. The forebrain. Regenera- Pere re eyieys SOM in SON GR eee 203 Amphibia. II. The afferent system of the read of Amblystoma. Correlated ana- tomical and physiological studies of the growth of the nervous system of.......... 247 Ant, Leucotermes flavipes, Kollar. The brain and the frontal gland of the castes Ply Geer NEES 2 exces tates bans bbw A os vce « 553 Arey, Lesuie B. Changes in the rod-visual cells of the frog due to the action of light.. 429 Arey, Lestiz B. The function of the effer- ent fibers of the optic nerve of fishes. . 213 Arey, Lestre B. The influence of light and temperature upon the migration of the retinal pigment of Planorbis trivolvis..... 359 Arey, Lestre B. The movements in the visual cells and retinal pigment of the JOWOr VEriODTAtAas. << os .c;.0. icc Taree 79 Forebrain of reptiles. Evidence of a motor pallitimbinithes.. eke cease ce sees 475 Forebrain. Regeneration in the brain of Amblystoma-. cl. Phexeeresseeceee ne. 203 Frog due to the action of light. Changes in the rod-visual cells of the............. ... 429 Frontal gland of the castes of the ‘white ant,’ Leucotermes flavipes, Kollar. The brain Piste Mulole co eee oc deo coder raodacoro kc 553 LAND lf the castes of the ‘white ant,’ Leucotermes flavipes, Kollar. The braimvand theiirontaltes seeceeeeeceees 553 EAD of Amblystoma. Correlated ana- tomical and physiological studies of the nervous system of Amphibia. II. The afferent system of the............... 247 Hibernation. Absence of chromatolytic change in the central nervous system of the woodchuck (Marmota monax) during 291 Hiton, Wruutam A. The nervous system of DY. CHOZONIGS 44... sae eee eee 453 | goes brain. A study of a Plains....... 403 OHNSTON, J. B. Evidence of a motor pallium in the forebrain of reptiles...... 475 Jounston, J. B. The development of the dorsal ventricular ridge in turtles......... 481 EEGAN, J.J. Astudy of a Plains Indian DVRINM Petes oc tinea ts ean eee 403 Kocu,Somner L. Thestructure of the third, fourth, fifth, sixth, ninth, eleventh and twelfthveranial) nerves): 2/2 .4..)5..0.05.00. 541 Kocuer, R. A. The effect of activity on the histological structure of nerve cells....... 341 | Bary and temperature upon the migra- tion of the retinal pigment of Planorbis trivolis. The influence of............... 359 Light. Changes in the rod-visual cells of the frog due to the action of........... Meroe 429 Leucotermes flavipes, Kollar. The brain and the frontal gland of the castes of the Swi tevanty weer arch tlie a rns 553 AMMALIAN nervous system (albino rat). A preliminary determination of the part played by myelin in reducing the water content of the................. 443 Marginata. The morphology and morphogen- esis of the choroid plexuses with especial reference to the development of the lateral telencephalic plexuses in Chrysemys..... 507 ' Nerve of fishes. INDEX (Marmota monax) during hibernation. Ab- scence of chromatolytic change in the cen- tral nervous system of the woodchuck.... 391 Medulla of eyclostomes with special reference to the formation and expansion of the roof plate and the-flattening of the spinal cord. Studies on the spinal cord and.......... Migration of the retinal pigment of Planorbis trivolvis. The influence of light and temperature upon the.................... 359 Monax) during hibernation. Absence of chromatolytie change in the central neryv- ous system of the woodchuck (Marmota.. 391 Morphogenesis of the choroid plexuses with es- pecial reference to the development of the lateral telencephalic plexus in Chrysemys marginata. The morphology and........ 507 Morphology and morphogenesis of the choroid plexuses with especial reference to the de- velopment of the lateral telencephalic plexusin Chrysemys marginata. The.... 507 Motor pallium in the forebrain of reptiles. Hividence Of'8 ct... Ronse eee 475 Movements in the visual cells and retinal pig- ment of the lower vertebrates. The...... 121 Myelin in reducing the water content of the mammalian nervous system (albino rat). A preliminary determination of the part played) DY-.iscewaksx oe ae eee 443 Myers, J. A., Rasmussen, A. T. and. Ab- sence of chromatolytic changein the cen- tral nervous system of the woodchuck (Marmota monax) during hibernation.... 391 ERVE. An experimental study of the VAP USI erceeiiem issn ©) aa ea a ee 421 Nerve cells. The effect of activity on the his- toloricalistructureloh.. +. 052. see eee 341 The function of the efferent fhibersomtheropticus. cree cee nan 213 Nerves. The structure of the third, fourth, fifth, sixth, ninth, eleventh and twelfth Cranial? op ees kee eee eee 541 Nervous system (alhino rat). A preliminary determination of the part played by mye- lin in reducing the water content of the Mammalian) cece eee eee eee Eee 443 Nervous system of Amphibia. II. The af- ferent system of the head of Amblystoma. Correlated anatomical and physiological studies of the growth of the.............. 247 Nervous system of pyenogonids The....... 453 Nervous system of the woodchuck (Marmota monax) during hibernation. Absence of chromatolytic change in the central...... 391 PTIC nerve of fishes. The function of the efferent fibers of the................ 213 ALLIUM in the forebrain of reptiles. Bividence lof a motores. ccc cine ee Pigment of Planorbistrivolvis. The influence of light and temperature upon the migra- tion/of theiretinalass.aca-e-6 pare eee 359 Pigment of the lower vertebrates. The move- ments in the visual cells and retinal...... 121 Plains Indian brain. A study of a........... 403 Planorhis trivolyis. The influence of light and temperature upon the migration of theiretinal pigment ole sss ote 359 Plate and the flattening of the spinal cord. Studies on the spinal cord and medulla of cyclostomes with special reference to the formation and expansion of the roof.. 9 Plate of the forebrain and the lateral choroid plexuses in the human embryo. Mor- phology, of the roof 32. cles e son's Rat). A preliminary determination of the part played by myelin in reducing the water content of the mammalian nervous BYBLCII GIO yt de ee actor ssc ea dee a's 44 Regeneration in the brain of Amblystoma. I. PNM OVEDURI 20; te oeena sn ota aes acs. eb Reptiles. Hvidence of a motor pallium in the TOLEDO ny AURA NS eae eo Sie aes eed « 47 Retinal pigment of Planorbis trivolvis. influence of light and temperature upon Lhe mictation OF CHO): says cbseee dba scek : Retinal pigment of the lower vertebrates. The movements in the visual cells and... Ridge in turtles. The development of the DKSHLVERULICIIAT...vavebaenns esse cs... 5 Rod-visual cells of the frog due to the action of light. (Changes in the...............5. Roof plate and the flattening of the spinal cord. Studies on the spinal cord and medulla of cyclostomes with special ref- erence to the formation and expansion of ENSE’ in vertebrates. Regarding the existence of the ‘common chemical..... Spinal cord and medulla of cyclostomes with special reference to the formation and ex- pansion of the roof plate and the flattening of the spinal cord. Studies on the....... INDEX 507 79 7 453 79 607 during hibernation. Absence of chromato- lytic change in the central nervous....... co OlROGE: VEHO Meme. =o. o.sdguces 453 Telencephalic plexus in Chrysemys margi- nata. The morphology and morphogene- sis of the choroid plexuses with especi«l reference to the development of the lateral 507 Temperature upon the migration of the retinal pigment of Planorbis -trivolvis. The in- Hance of light aiid) sesms. ss... .0.-08 THompson, CAROLINE Beritne. The brain and the frontal gland of the castes of the ‘white ant,’ Leucotermes flavipes, Kollar 553 Trivolvis. The influence of light and tem- perature upon the migration of the reti- nal pigment of Planorbis................. 359 Turtles. The development of the dorsal ven- tricular ridge in............ yo pes AGUS nerve. An experimental study OF CRA ila coda: « «tc vc vlc c ale bate 421 Ventricular ridge in turtles. The develop- ment of the dorsal. /5Vi sme. «2... .c008 Vertebrates. Regarding the existence of the ‘common chemical sense’ in Vertebrates. The movements in the visual cells and retinal pigment of the lower.... Visual cells and retinal pigment of the lower vertebrates. The movements in the..... ATER content of the mammalian nerv- ous system (albino rat). A prelimi- nary determination of the part played by myelin in reducing the;.............. Woodchuck (Marmota ‘monax) during hiber- nation. Absence of chromatolytic change in the central nervous system of the...... 391 ~~ en = NUTT TET TE © ©O ep) ae! Ls) LL UAT TT 5 WHS ee ee Cate ee ee) + +_2_¢ 2 2 0 2 A 070 4525", 63-49 e* ee ee ee 9555 °=*. 8 ae Ais resets tse 8) 5 a @ > ’ i 6 % > ee eae Ca) Ce? 9 3 Feis tage 8168 © le tes tats" ° 4 eg ta % ft sth stat ele: