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He e534 “ peas i Peg : ae ult i pete a Citte i te Peteretote sii Heaniet i be $s eH Ser ites: * “ . + Le etate rotary, v4 pete Tbstiter i? im t6 ieetite sie! ae 4M ae tit sPars: yinct Nett tafe ae seta tteae LY Sa oee i, ¥ ritercbttatse SMa % # ates F fA siybteth ik Ssiase te tittath te Sanit a a if us as eias : . oe an Sit t Rees . $ihiy a of i tf se oe aN ; Ate 5 eat stetitites ritetse rote a ts oe : Het ot pets 2S f At it, ste Hehe viet PLSLPLS LY OL ERPS furs is Patsy rs ¥ifate Byte} si ee cite Rett GBH Yeraracatidetbtadesslalegebe tatititararotreterer ter tatcnd SR eRe TET aS Tae eM a tata ante a*, ety oA raz el i i tt i ae ries % ee br ea sits ofa fy we shee ibis ie ut Lee arte) wane eae att te Ta 33 settee aie } taht rise Tenth bistetetester ; ne sit Sa pil i wistergrectees misc iach ateg tenn sis sem sitet yn cect ee RATS ene a tebe ete se 4? ‘ Cal z yy: : uteeels “fe oh is £5, Sore 2 it THE JOURNAL OF COMPARATIVE NEUROLOGY EDITORIAL BOARD Henry H. DoNALDSON ApoutF MEYER The Wistar Institute Johns Hopkins University J. B. JoHNsTON OLIVER S. STRONG University of Minnesota Columbia University C. Jupson HERRICK, University of Chicago Managing Editor VOLUME 23 1915 PHILADELPHIA, PA. THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY OD 2a COMPOSED AND PRINTED AT THE WAVERLY PRESS By THE WivuraMs & WILKINS CoMPANY Bautimore, U.S. A. CONTENTS 1918 No. 1. FEBRUARY §. B. Vincent. The tactile hair of the white rat. Thirteen figures........ Max Mayo Mitter. Prenatal growth of the human spinal cord. Twelve AT OUTS Me eww emM NC se oe A INE MT RES Pot Si VIOTA AR eee nee SIRE ir On ret Noz2.. APRRKE: ALBERT Kuntz. The development of the cranial sympathetic ganglia in the PLE PRUE LCO My HOURS tA atl Want woe ee te RR fee eRe atk et aye, J. B. JoHnston. Nervus terminalis in reptiles and mammals. Twelve PUPA OS ers ee ce OS ANI 3 OER aE etc EER ARE Toe Nee Tc, Oe G. E. Coauitu. The primary ventral roots and somatic motor column of ANN Hbysrorne, AL eminyeeriey oh VOIDS Goons oo eoo cand bo bononecaccesedoocE. Rouyio BE. McCorrrr. The nervus terminalis in the adult dog and cat. Four PE OUT CS Ap ear ae Src eh Se Penta. oes a YOR nts eh Ne A No. 3. JUNE PauLt 8. McKissen. The eye-muscle nerves in Necturus. Eight figures.... AuBerRT Kuntz. On the innervation of the digestive tube. Five figures. D. Davipson Boack. The central nervous system in a case of cyclopia in homes ahatty=one apes i ashe actin o CA. cele 8 cae aerate Ste are No. 4. AUGUST S. Watrer Ranson. The course within the spinal cord of the non-medul- lated fibers of the dorsal roots: A study of Lissauer’s tract in the cat. 39 145 REV Cnehlounesry er: een hee aed et eae oe grat, 2h ctr. ogee oe ae ee net 259 HeLen Dean Kina. The effects of formaldehyde on the brain of the albino UAC TIE MDE Ste = ctu mene ts ects tan Bele oot el-'s is Kings a! are asia Cyeerane sche igen Cee 283 Tuomas J. Hetpt. . Méllgaard’s reticulum. Six figures....................- ill lV CONTENTS No. 5. OCTOBER D. Davrpson Buack. The study of an atypical cerebral cortex. Nine IT eqU Les ear aera near RMA aT UE oe Mn epee Ae Daeg reer mney oc Bete ely, ara 3 is ois Goo oeston ae J. B. Jounston. The morphology of the septum, hippocampus, and pallial commissures in reptiles and mammals. Ninety-three figures .. get M. J. GREENMAN. Studies on the regeneration of the peroneal nerve oo; fhe albino rat: number and sectional areas of fibers: area relation of axis to sheabhan “Shree ieuness... a2 lee oe =. nae 5 Se CYA ae are ee ee No. 6. DECEMBER CAROLINE BuRLING THoMPsoN. A comparative study of the brains of three genera of ants, with special reference to the mushroom bodies. Forty- LUN ADY- 101101 c( 1S) ea aa RR Sir Me we de telly Mn Ga core ih aie, Oe ena cine o/S < F. L. LanpAcrE and A.C. Concer. The origin of the lateral line primordia im shepideseusosseus.. Uhirty-tour feures./-2. 442 sakes oe we ee ie C. Jupson Herrick and JEANETTE B. OpENCHAIN. Notes on the anatomy of a eyclostome brain: Ichthyomyzon concolor. Twelve figures ......... 479 515 575 635 THE TACTILE HAIR OF THE WHITE RAT Ss. B. VINCENT From the Otological Laboratory of the Northwestern University Medical School THIRTEEN FIGURES CONTENTS feugoreraldesprtphion se 25 side = 25 ke. saheierte > ee Sapa aa PO He ee ee 1 TASS a Sty ore Vers | eee 1 a ME. OF ie aioe min. RUN Sc ee oS A 6 ilesIVMeHN GAS! OLB bUON ohare scclsicce 5.5 oe SEM Se ao aS od te ee eae remake meeiete 8 LOWES anit C01 hs he eo eo 5 ME Bea ty SOM SAO. &.4tpaec o co de 8 VenComparativeanatomy. sass deete rs. Adams aod pickers tarl2 inert icteric 16 Wile ERD b ACL O La Mean Se eM ae a aA REE RIE Leite een a eS Aine eRe MEMI « SUNT yD bea Che 20 SUN SCL TTAENY AT ior ps Scrip fc steve. vd Sys io oe Beets nd See wine hete GHRLW anadtow shes int Oe eer as . 24 Bt lta emalp lines ateee oaks Sites rh ee ceed eine eco oraane eine echo eee ole lente oie ccvennetanatats 26 I. GENERAL DESCRIPTION The principal tactile hairs of the white rat are found on the upper lip arranged in rows on either side of the nasal fossae. The longer and larger of these hairs are the most lateral ones of the second, third and fourth rows counting from below. Besides these there are a few scattered hairs on the lower lip, on the cheeks, above the eyes and on the fore limbs at the wrist joint. We are chiefly concerned with the vibrissae of the upper lip which in an active animal are in constant motion. By the term ‘hair’ we usually mean the shaft which projects from the surface of the skin, but considered as a sense organ the important part is the follicle beneath the surface which encloses the base of the shaft. We may think of it as an invagination of the epidermis and see in it the usual skin layers somewhat modi- fied under the different conditions of growth. The follicle is a long oval in shape varying in length from 1 to 5 mm. and in width from 0.5 to 2 mm., and it is surrounded by two sheaths, a dermal and an epidermal. As we look at the folli- cle in a longitudinal section it appears like two pockets, one within 1 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 1 FEBRUARY, 1913 2 Ss. B. VINCENT the other, which are separated by clear spaces or spaces crossed by connective tissue bridges—fine trabeculae (fig. 1). These cavities lie in a diverticulum of the fibrous, dermal, sheath and constitute the blood sinuses. The upper clear portion is known as the ring sinus (e), the lower as the venous or cavernous sinus (7). In the lower part of the follicle may be seen an ingrowth of connective tissue which pushes the epithelial layers back and forms a central core at this place where the blood and lymph vessels can come into intimate contact with the growing portion of the hair. This is the papilla. In the upper third of the follicle are ine sebaceous glands which arise from the outer root sheath and whose ducts open upon the shaft of the hair. These glands lie above the ring sinus while below it or rather extending into it is an outgrowth from the root sheath named variously as the ringwulst, kissen, bourrelet annular, or pulvinus. The follicle shows in a longitudinal section two distinct enlarge- ments which are known as the superior and inferior swellings and the thickened portion of the root sheath above the sebaceous glands has been called the conical body (0). Having looked at the salient features of this organ, we may now examine its structure more in detail. As has been said, the epidermal covering consists of two sheaths, an inner and an outer (fig. 2). The inner sheath corresponds to the stratum corneum of the epidermis and in good sections exhibits three layers of cells known respectively as the cuticle, Huxley’s and Henle’s layers. The latter, a continuation of the stratum lucidum, is frequently lacking. These cells differ somewhat from the usual epidermal cell layers, the two outer ones being nucleated while the cuticle is imbricated in such a way as to interlock with the plates of the cuticle of the hair shaft. For, as the tip of the invaginating hair column grows downward, an indentation is formed in it by the developing hair papilla ‘‘which is just suffi- cient to redirect the growth of the central hair column toward the cutaneous surface” which it reaches through a central canal formed by the degeneration of the central epidermal layer of the original hair column. According to this theory the cuticle of TACTILE HAIR OF THE WHITE RAT 3 the hair shaft is continuous with one of the layers of the inner root sheath and as its plates are turned in an opposite direction they interlock with the cuticle plates of the sheath. The cortical substance is a continuation of the stratum spinosum or middle Malpighian layer, while the cells of the medulla are formed by the proliferation of the cylindrical cells of the outer root sheath. The outer root sheath is a continuation of the stratum germi- nativum and consists of three layers, a basement layer of cylin- drical cells, the Malpighian layer of large prickle cells, and a granular layer of flattened cells which are often lacking. It is the cells of the second layer which increase so greatly in some parts of the follicle and are associated with the nerve endings. The dermal sheath also has three layers. Naming these from without inward they are a layer of longitudinal connective tissue fibers, a layer of circular fibers and the glassy layer. The last of these corresponds to the basement membrane of the derma. It is a clear, thick, highly refractive layer and the inner portion is said to be an exoplasmic product of the adjacent epithelium (Kolliker ’02). The papilla has a great growth in the rat and often reaches the neck of the follicle. In its lower part it has a rich plexus of nerves and blood vessels. While the above account of the sheath layers is generally true, they have a far greater thickness in some places than in others and merge into indis- tinctness both in the region of the papilla and in the conical body. From this description of its development it will be seen that the tactile hair of mammals is similar to the ordinary hair, from which it differs only in its greater development and higher spe- cialization. It is a cell structure arising by differentiation from the epidermal cutaneous cell layers and thus is essentially unlike the invertebrate organs which resemble it but which are formed by a chitinous secretion. The superior and inferior enlargements in the follicle have hitherto attracted much attention. They are the result of a thick- ening in the Malpighian layer of the outer root sheath. These cells not only multiply so as to form a greater number of layers but the cells themselves increase greatly in size. This growth 4 S. B. VINCENT may be due to the augmented vascular supply to this place or to the stimulating effect of the many nerves which have their terminations here. The outer layers of these cells lie upon the leaf-like terminal expansions of the nerves and are connected in some intimate way with their functioning. The ringwulst grows out of the root sheath. It appears as a somewhat oval shaped body projecting into the ring sinus. In prepared sections it is always much shrunken, but in its expanded state it must nearly fill the space between the walls of the diver- ticulum in which it lies (fig. 6). It 1s composed of connective tissue fibers (fig. 7)—fibro-hyalin in the rat—which enclose in their meshes great, clear, round, transparent cells with pale nuclei. It is penetrated in every part by loops of capillaries and by delicate varicose nerves. The nerves are not only distributed to the organ itself but also many of the larger nerve trunks which terminate in the superior enlargement of the root sheath perforate the substance of this body on their way thither. The conical body is the name given to an enlargement of the root sheaths above the sebaceous gland. It is really no separate structure, but here the follicle layers are fused and it is simply that portion of the follicle walls nearest the surface of the skin. Many nerves and blood vessels pass through it on their way to the lower parts of the follicle. It has a muscular formation which probably indicates its chief function (fig. 1). The lower sinus is sometimes called the cavernous sinusor sometimes this part of the follicle is named from the tissue which fills it, the spongiose body. The space is crossed by delicate cordons of connective tissue which enclose lacunal cavities. These are found filled with blood if the animal be killed without bleed- ing. The amount of blood is so great that good stained sec- tions are impossible to obtain unless the blood has been drawn. The connective tissue network extends as far as the ringwulst and upon it and among it are found fine nerve fibers and small blood vessels. It is so tender of fiber that in injecting or in sectioning it is inevitably destroyed and one sees, usually, only broken fragments with here and there a few intact interlacing strands from which one must imagine the whole (fig. 8). The TACTILE HAIR OF THE WHITE RAT 5 upper part of this sinus when filled is practically closed by the ringwulst which separates it from the entirely free space above, the ring sinus. Arteries and veins. The fibrous sheath is supplied with the ordinary nutritive blood vessels. These are particularly numer- ous in the middle layer of the sheath. The large follicle artery enters with the main nerve at about the lower third of the sheath. It here divides and sends a branch to the lower part of the follicle and several branches upward which in turn divide and encircle the follicle longitudinally as far as the ring sinus into which they empty from below. ‘To do so they run close to the walls of the follicle between them and the ringwulst to which they give many capillary branches. Besides this main artery there are other smaller ones which come from the subcutaneous plexus and penetrating the walls of the dermal sheath debouch into the sinus at about the same place as the others. More numerous than these vessels from below, however, are those which come down from the upper vascular plexus which lies just below the corium papillae. They accompany the nerves for the nerve ring about the neck of the follicle and open into the roof of the sinus just below the ring. In good preparations there may be seen a series of such perforations encircling the follicle in the constriction of the walls which form this roof. I never saw any large veins in the follicle and think the venous outlets are, as Bonnet (’78) and Dietl (’73) describe them, through the outer coats of the bulb emptying into the skin veins. Muscles. There is a veritable network of muscles surrounding the follicle. For the most part these are skin muscles and they help to obscure the real muscular attachments of the bulb. There are longitudinal muscle fibers running down from the surface of the skin which have a membranous attachment to the walls of the follicle. It is also enclosed by horizontal bands of fibers so that it shares in the general skin musculature. Besides these there are long tendinous cords apparently forming part of the walls of the follicle which run deep down into the subcutaneous tissue and firmly anchor the follicle below. From the upper part 6 S. B. VINCENT of the walls of one follicle muscle fibers run to the lower part of another in the same series so that any movement is a general movement. These may lay the hair back. They are all striped fibers (fig. 5). Besides this connection of the follicles, there is a flat muscle band of fibers which surrounds the follicle on three sides. It originates in the wall of one follicle, runs around it and is inserted at the same level in the walls of another follicle. ‘This muscle is well seen in a horizontal section (fig. 4). The nerve enters, as Bonnet (’78) says, on the muscle free side. These muscles prob- ably have to do with the constant quivering movement of the hairs. About the neck of the follicle are both longitudinal and hori- zontal contractile fibers and also about the neck running horizon- tally are plain muscle fibers which lie just below the nerve ring. The conical body also has a muscular structure. The con- traction of this organ opens the mouth of the follicle and permits a free vibration of the hair for a considerable depth in the follicle just as the contraction of the smooth fibers about the neck closes the walls about the hair and dampens the vibrations. Il. HISTORICAL Although the tactile hair has been the object of many and extended studies, there are still histological and functional ques- tions unsolved. The great size of its follicle tempts the unwary investigator; those who study the skin upon which it is found can not ignore it; and to the complexity of its structure each new histological method and stain must be applied. One is struck by the contradictory statements made in these reports, particularly in the earlier ones; but the differences have several explanations. These hairs have such a general similarity to the body hairs and to the hairs of the head that the facts found true of the one have been carried over, wrongly, to the other. Then there is the false assumption that the tactile hairs of all animals agree in structure. Dietl (’73) has shown, and his state- ments have been confirmed by others, that there are two kinds TACTILE HAIR OF THE WHITE RAT ¢ of sinus hairs, those with a ringwulst and a ring sinus, as in the eat and rat, and those without the ringwulst with only one sinus and that filled with trabeculae, as in the horse, cow, etc. Still another cause of the apparent difference between authors is the matter of terminology, of inexact definitions of particular histo- logical parts. This causes considerable confusion, for example in determining in which of the layers of the follicle the nerve end- ings are found. Some who speak of the outer sheath mean merely the fibrous part beyond the blood sinus, but others mean the coats up to and including the glassy layer within the blood sinus. The glassy layer has been called a dermal and an epider- mal structure; it has been thought by some to consist of one homogenous layer and by others to be made up of two layers; and still another view recognizes in it two layers of different origin, a dermal and an epidermal. For this reason when a writer says that the nerves go through the outer root sheath or through the dermal sheath one must stop to inquire what is meant. , The literature of this form goes back to Haller in the eighteenth century and the subject has been a most fruitful one ever since. Bonnet made an exhaustive study of the literature in 1878, Botezat in 1897, Szymonowicz (’09) gives some of the more recent references, and a rather extensive bibliography will be found in connection with the general bibliography on hair given by Frieden- thal (08). This paper therefore will not attempt to deal with the historical side of the question in any thorough way. Gegenbaur (’51) should be mentioned, who studied the tactile hairs of nine mammals, and Leydig (’59) who secured specimens of skin from every mammalian family and gives a wealth of detail considering the methods and means at his disposal, and Odenius (’66) who thought that the development of the ringwulst had to do with nocturnal habits. Ten years later Merkel (’76) found the touch cells in this organ and about the same time Ranvier (’75) described the menisques. Bonnet made one of the most complete studies. He devotes twenty-eight pages to a description of the innervation of this hair. Retzius published a series of articles in 1892, 1893 and 1894 on the subject and 8 Ss. B. VINCENT Ostroumow a good paper in 1895. The same year Messenger had a very brief article. Botezat put out two careful studies in 1897 and 1902, while some of the more recent contributions are those of Szymonowicz (’09), and Tello (’05). In the limits of this paper it is impossible to do more than mention a few of the many studies, but some of them will be referred to for matters of detail in the discussion which follows. III. METHODS OF STUDY The grosser structure of the nerve supply of the tactile hair may all be seen in a careful dissection. After hardening the head in 10 per cent formaline for a few days, if the skin be cut posterior to the vibrissae and slipped down over the snout until the hairs are reached, the follicles appear, as they are torn away from the subcutaneous tissue, projecting from the epidermis. The branches of the facial nerve are in sight lying over the strong masseter muscle. If the anterior portion of this muscle be now carefully cut away, the sensory nerve will be exposed as it lies directly upon the bone. This nerve may now be followed - even to the divisions entering each separate follicle. The microscopic studies in this work were made from sections of degenerated nerves stained by the Marchi method, normal tissue stained with osmic acid, by Cajal’s silver nitrate method and by Bielschowsky’s variation of the same; but the intra-vitam methylen blue method proved the most satisfactory of all. The process adopted with this stain was that described by J. G. Wilson in The Anatomical Record, 1910 (vol. 4, no. 7). IV. INNERVATION General description. The large sensory portion of the fifth nerve emerges from the Gasserian ganglion in three divisions, of which we are here only concerned with the second, the superior maxillary. This passes through the fissura spheno-orbitalis and then runs along in the infra-orbital groove and while there branches are given off to various parts of the mouth, pharynx and nostrils; but the infra-orbital branch in which we are inter- TACTILE HAIR OF THE WHITE RAT 9 ested is the terminal one. It passes through the infra-orbital foramen and lying close against the maxilla and pre-maxilla bones runs forward until just beneath the tactile hairs. In sections stained with osmic acid the nerve is seen to be a typical cutaneous nerve consisting of many bundles of very dif- ferent sizes and containing large and small fibers, fibers which stain very black, paler fibers and fibers which are not stained at all. It is a very large nerve in comparison with the branch of the seventh which lies superficial to it. Even as it emerges from the ganglion it has the form of a large flattened band and its branches look like mere threads beside the main trunk. It is estimated that this trunk as it leaves the infra-orbital foramen contains from 15,000 to 20,000 fibers. Its great size corresponds to the functional relations of the mouth parts which it serves. In animals like the elephant, tapir, etc., or animals with strong tactile hairs, its proportions are increased enormously. It often divides into ten or a dozen branches before passing through the foramen, which is very much enlarged in many rodents. The inferior portion of the anterior maxillary part of the zygomatic arch is thin and flattened in rats, forming a verti- cal fissure through which the infra-orbital nerve passes. The superior part of the fissure is a rounded depression which at first glance might be taken for the orbit itself. Soon after leaving the foramen this nerve anastomoses with the infra-orbital branch of the facial, which is the motor nerve to the same region. The fibers of the trunk thus formed are dis- tributed to the skin of the upper lip and nose, but about half of them go to the tactile hairs of which there are between forty and fifty on each upper lip (fig. 3). These are arranged in six rows with from five to nine hairs in each row, and there are often 150 or more large medullated fibers in the nerve bundle entering a single follicle of a large tactile hair. Usually, just before reaching the root of the hair the nerve divides in two parts which pene- trate the dermal sheath at the level of the lower third. One part turns back, both divide many times and together they encircle the follicle in a sort of palisade of longitudinally running fibers. The further course of these fibers will be followed later (fig. 9). 10 S. B. VINCENT The dermal plexus and the plexus of the outer root sheath. All over the dermal sheath of the follicle is a plexus of fine varicose fibers and a very similar one is seen on the surface of the outer root sheath. As one looks down on the follicle it appears to be covered with a fine network of nerves. It is difficult to say whether the fibers really anastomose or not, but in many cases where they can be followed they seem to lie over or under one another. They grow finer and finer with repeated divisions and when the end can be seen it is usually a simple fiber, or what is more common the fibril ends with one of the varicosities with which, as has been said before, it is studded. The fibers have very much the appearance of sympathetic fibers and some of them can be seen branching from nerves which accompany arte- ries. Botezat’s opinion (’07) that these plexuses have to do with the nourishment of the follicle seems the true one. Since our interest is in the sensory nerve, these will not be discussed further and no attempt will be made to describe the motor nerves which may be seen going to the muscles of the follicle. Nerves on trabeculae. Of the same nature are the nerves in the connective tissue strands of the lower sinus. The trabeculae which fill the large cavity of the blood sinus below the ringwulst furnish bridges for crossing fibers (fig. 8). Some of these sur- round the walls of the arteries which they accompany and others resemble these so much that, though their immediate connection with any blood vessel cannot be seen, one may infer them to be from the sympathetic system. Still others detach themselves from the trunk of the main nerve coming from below and cross singly on these pathways. The latter are for the most part sensory nerves destined for the lower part of the follicle and immediately turn back in that direction. Nerves in the papilla. There are nerves forming a rich plexus within the papilla. They are usually varicose and sometimes can be seen running with the arteries. They often reach a con- siderable height in the medulla of the hair (fig. 10). Bonnet was not the only one who thought the papilla with- out nerves (’78, p. 390), but Retzius -(92-’96), Orru (94) and others described nerves in this place. Ostroumow thought TACTILE HAIR OF THE WHITE RAT 11 them all vaso-motor but Botezat (97) denied this. They looked to him like the intra-gemmal fibers of the touch cells and he also described thickenings not unlike the knobs of the intra-epithelial nerves. He thought, therefore, some of them might be sensory. From the position of the papilla and the little likelihood that the movements of the hair can be very effective here, as well as from the appearance of the fibers themselves, we believe these nerves to be comparable to those which may be found in any vascular structure and to be non-sensory in character. They come to the papilla in many little fibers from the subcutaneous tissue below and no connection can be seen with the large sensory trunk which enters the side of the follicle. ; Sensory nerve. ‘The great nerve which we have described before pierces the dermal sheath together with the main artery at about the lower third of the follicle (figs. 1 and 9). Here a few branches which serve the lower part of the organ break away from the rest, but the main portion of the nerve divides in two, one part turns to the opposite side, both divide and redivide and when they have crossed the sinus, surround the follicle with rows of parallel, ascending bundles of medullated fibers many of which may be distinctly traced to the constriction at the neck of the follicle. ‘These bundles are not entirely separate, for fibers can constantly be seen detaching themselves from one bundle to join another so that it looks as though the follicle rested in a coarse cup-like meshwork of bundles of heavily medullated fibers. This is the outer plexus of the follicle proper and is most pronounced over ° the superior swelling of the root-sheath. It must not be for- gotten that all along the course of these bundles, fibers which terminate at different levels are continually separating off; but the main trunks are of such size and stain so heavily that they are the most noticeable feature of the whole structure. As they come to the follicle and rest thus upon it they seem to lie embedded in a colorless substance which is probably the gelatin- ous endoneurium of the nerve trunk. Fibers which serve the upper part of the follicle bend outward at the region of the ring- wulst and pass -up through it. It is the bending of these nerves outward which emphasizes the swelling of the root sheath here. 12 Ss. B. VINCENT For the most part these nerves terminate in a one-layer mantle of touch cells all over the follicle in the Malpighian layer of the outer root sheath, but the endings are the largest and most characteristically developed over the superior swelling of the sheath (a, fig. 9). The large nerves preserve their myelin almost to the very end, when they suddenly go over into disc-like expan- sions of various sizes—leaf-like endings with thread-like stalks. The whole appearance is as if the sheath were flattened out very thin to furnish a support for the intertwined neuro-fibrils (fig. 11). At times there is just one of these leaf-like endings, again there may betwo, three or four. When there is more than one, each member is connected with the preceding by a deeply stained fiber which arises from the corner or tip of the expansion and the whole series has a somewhat definite arrangement. I have often seen follicles which looked as if they were surrounded by horizontal or oblique running bands of these menisques. They have been well described by Cajal (09, p. 474): They show in the interior a fine network of neurofibrils separated by an abundant uncolored neuroplasm. This network is of the same com- position as that of the body of the nerve cells. We find first, the large neurofibrils frequently bending and forming the framework of the ter- mination and second, pale fine fibrils which bind together all the others. A large number of menisques are supplied by one nerve fiber. The cells between which or under which these menisques lie are large ovoid cells, probably modified Malpighian cells of the “outer root sheath. There is no connection to be seen between the cell and the fibrillar plexus of the menisques. What has happened is this: The nerve has lost its myelin and one of the fine fibers described before has pierced the glassy layer and expanded within under one of the large cells found here. The glassy layer is very thin over the superior swelling and disappears above it. As it is very dense and hard to perforate elsewhere, the greater number and size of the endings found at this place is accounted for. Yet fibers do push through at lower levels also. There has been a great deal of discussion as to the relative position of these menisques with regard to the cell body and to the axis of the hair. In the rat’ there seems to be no one char- TACTILE HAIR OF THE WHITE RAT 13 acteristic position. In the superior root swelling where they attain their greatest size they usually lie vertical to the long axis of the hair and beneath the cell body. The flattened surface of the menisque is somewhat parallel to the surface of the skin. Near the glassy layer, however, the position is almost at right angles to the surface of the skin and parallel to the long axis of the hair. This is due no doubt to the resisting power of this layer. In the lower portions of the follicle—for these endings cover the whole of the follicle contrary to the opinion of many of those who made the earlier studies of the endings—they are more varied in position but may often be seen parallel to the axis of the hair shaft. Ranvier (’75) first described these struc- tures truly and recognized them as the touch cells which Merkel (76) had previously found in other parts of the skin. Subse- quent investigations have shown that the fiber does not end in the cells, as Merkel first thought. It is generally believed now that this cell is simply a modified epithelial cell which may serve as a protective cushion for the nerve expansion and possibly help to increase or modify the pressure stimulus. Besides the nerves which end in this way, fibers leave the main bundle the whole length of the follicle, run to the glassy layer and arborize upon its surface or end in free or flattened spgtulate endings, so that the entire outer surface of the glassy layer is covered with a fine nerve plexus. Among these fibers are some which penetrate the glassy layer and end between the epithelial cells of the outer root sheath. Tello (05) and Ostroumow (95, p. 914) say that these fibers are of a different order from those which end in menisques within the glassy layer. That they usu- ally appear smaller and not so heavily medulated, at least not so deeply stained, is true, but that they are different in origin or function seems doubtful. Among those fibers which end in menisques occasionally one is found which forms several small menisques before piercing the glassy layer; again from menisques within the glassy layer go off little fibrils which run between the cells of the outermost layer of the outer root sheath and end intra-epithelially or arborize about these cells, as Merkel first described; but besides these one ,may see at times nerves which 14 S. B. VINCENT cross the glassy layer from within and form a small plexus without the layer in the same fashion and one then finds the leaf-like expansions both within and without the layer from the same fiber. Thus there can be discerned little difference save in devel- opment between the two kinds of fibers (fig. 12). The nerve ring. ‘To the neck of the follicle come also nerves which take part in the formation of the dermal plexus (fig. 13 and fig. 1). Some of them are diverted to the follicle before joining to form the plexus, some send a long branch downward to the follicle and another up to the plexus, others come out from: the plexus itself, often in groups of from three to a half dozen fibers among which may be seen deeply stained medullated fibers, pale and vari- cose fibers. ‘These nerves are much smaller than those of the large sensory trunk which enters the lower part of the follicle but on the other hand there are many of them and they approach the mouth of the follicle from all sides. With them are blood vessels from the vascular plexus and many both of the nerves and blood vessels go to serve the very large sebaceous glands of this struc- ture. The rest run to the region just above the ringwulst, that is just below the sebaceous glands, and here most of them lose their myelin and encircle the hair at the level of the glassy layer. The nerve ring is deeper than the longitudinal ascending fibers, many of which pass over it. The nerves which form it often divide once before beginning their circuit and the two branches sometimes take opposite directions, but there is no further divi- sion until they have nearly or quite completed the circle; then they ascend and break up into what looks like an arborization in the conical body. From this brush of fibers whose ends are cut across in a longitudinal section may often be seen fibers running up to the surface of the skin again. I have never seen the longitudinal fibers take any part in the formation of the nerve ring. The course here described is for the strong deeply medullated fibers. As these run down from the surface they are accom- panied by paler fibers and varicose fibers, as has been said, but their course I could not follow after they were lost in the intri- cate ring plexus. TACTILE HAIR OF THE WHITE RAT 15 A brief review of some of the positions taken as to this nerve ring band may be useful. Leydig (’59, p. 390) saw the ring but others after him denied its existence. Bonnet (’78, p. 366) said the fibers came from the outer plexus about the neck of the follicle and reached the ring in some unknown way, but Messenger (’90, p. 401) thought it was composed of fibers from the nerve trunk entering from below and Botezat declared that he saw fibers in the ring from both sources (’03). Most authors agree that these fibers end on the outer surface of the glassy layer, but Ranvier says that they go through this layer (75). As to their mode of termination, few have been able to describe them. Ranvier said they end in spatules (’75, p. 915); Botezat (02) described them as simple endings, thickened or flattened forms and Szymonowicz (09, p. 622) as forked endings. It is very easy to confuse the terminations of these fibers with those of the branches of the longitudinal fibers which end many of them here on the glassy layer. The fibers of the ring are very fine and very easily broken in the preparations, so that it is exceedingly difficult to be sure as to free endings or endings in varicosities. Sometimes I have thought that I saw connections between these delicate fibers and some plain muscle fibers which surround the neck of the follicle but farther than this I can say nothing about the endings here. I think the significance of this ring has been over-estimated and for reasons which follow do not agree with Messenger who says, ‘‘The annular nerve band is so situated that when the pulvinus is not turgid tactile impulses are little felt but when it-is turgid the slightest impact produces a marked effect on the nerves surrounding the hair” (’05, p. 401). Neither do I agree with Bonnet who thinks that the hypothesis of a real intensification of power of perception through this nerve ring is perhaps justified. Odenius’ (’66) assertion that the ring is confined to nocturnal animals has been refuted by others. Ringwulst. The structure of the ringwulst has been described before. It springs out just beneath the superior swelling so that all the large sensory fibers which terminate above it must pass through it. Many of these nerves run close to the walls of the follicle but others bend outward and pass through the ringwulst 16.94 Ss. B. VINCENT midway striking the walls of the superior enlargement above. It has been said by Botezat, Szymonowicz and others that the ringwulst only surrounds the follicle from two-thirds to three- fourths of the circumference, but in the white rat this is not so. The tissue is very fragile and in most of the methods of prep- aration it is badly torn, but with silver nitrate and osmic acid the structure is often whole and entirely surrounds the follicle. It shrinks much in staining, in length as well as in width, which may account for the gap one often sees in the circumference. This very tender organ consists of almost transparent connec- tive tissue fibers which enclose large pale nucleated cells. In the rat, in a longitudinal section, the structure has somewhat the shape of a lung which comes out by a stalk from the follicle walls and hangs suspended in the cavity of the sinus. It is traversed in all parts by loops of capillaries which are unusually large for the size of the organ (fig. 7). As one looks down upon it in well prepared sections it seems covered with fine varicose fibers which run from the base out to - the periphery and end with a few fine branches or with small varicosities (fig. 6). Whether these are sensory or vaso-motor, I have no way of knowing. The ringwulst is not found in all animals according to Bonnet and others. The horse, cattle and swine are without it while in carnivora and rodents it is fully developed. Botezat (’97, p. 144) shows, however, that some animals, as the swine, have two kinds of tactile hairs; those with and those without a ringwulst and a ring sinus. Where there is no ringwulst as in the horse, there are often, Bonnet (’78, p. 348) says, similar thickenings on the walls of the trabeculae near the conical body or else where several such walls come together. These thickenings may serve the same purpose as the ringwulst, whatever that may be. V. COMPARATIVE ANATOMY Hair is a mammalian characteristic, but the tactile hair is probably phylogenetically the first to appear and when all other hair is lost, as in the whale, a few of these still persist about the TACTILE HAIR OF THE WHITE RAT ib7E mouth parts. Human hair begins to develop around the cuta- neous orifices between the first and third months. Krause saw tactile hairs earlier than any other hairs in the mole foetus of 9.5 mm., and in rabbits in the second half of the embryological period they were completely keratinized, the follicle showed the two swellings and contained many blood vessels which corre- sponded to the blood sinuses. A number of investigators have said that the structure and innervation of the hair differed in young and adult animals but tissue which I have stained from the rat of a few days contained perfectly formed follicles with the usual mantle of touch cells and a nerve ring as well. The course of development of this mantle and nerve ring is worth our further consideration. ° Merkel (’76) discovered some peculiar cells in the snout of pigs, round glistening cells in the inter-papillary spaces, the ‘epithelial Einsenkungen’ of the Germans, to which came terminal fibers ending in flattened dises about the cell or, as he then thought, within it. He concluded that these were ganglion cells. Later studies showed that there was no real connection of the fiber with the cell and also that these cells were not confined to the mouth parts of animals but were found on the cutaneous epithe- lial border of human skin, in tactile hairs and in other places. Szymonowicz, in a long series of articles, has shown that these are merely the usual epithelial cells whose differentiation has been caused by the coming of a nerve fiber and the formation of a fibrillar plexus about the cell. Eimer (’94) studied in the snouts of animals a peculiar arrange- ment of cells in a eylindrical or hour-glass form. These have since been studied by Jobert (72), Krause, Szymonowicz (’95), Botezat (02) and others. The structure, which has been named Eimer’s organ, lies in the same skin layers as the touch cell and to it come medullated nerves which lose their sheaths and run up between the cells as a central bundle of fibers whose lateral branches lie between the cells or as a cup-shaped plexus about them. These are evidently of the same nature as the touch cells of Merkel in the inter-papillary spaces and also of the touch cells of the tactile hair. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 1 18 S. B. VINCENT The embryological studies of Szymonowicz (’95) prove that these cells originally lay in a horizontal layer between the cutis and the epidermis but were pushed by the formation of the cutis papilla into the inter-papillary spaces and finally lay in groups over each other. Botezat (97, p. 103) tells how in the invagi- nation of the hair follicles these cells and the nerve endings are carried down and le in the root sheath of the follicle in the same position which they originally occupied on the cutis border. He calls attention to the fact that if one were to fix a hair in one of these groups of cells, particularly in Eimer’s organ, one would have a structure very similar to the hair follicle without the fibrous sheath. He mentions the likeness of the touch cells to the cells of Grandry’s corpuscles and also to the platelets which Bethe describes in the tongue of the frog. Merkel found the touch cells in the region of the mouth or nasal openings of many mammals and apart from this part of the body chiefly in the unhaired portion of the skin. He also found them on the hard palate and the bills of birds, on the tail of the hedgehog and the vulva of swine. In man he said they are situated for the most part where there are no Meissner’s corpuscles. There are many over the abdomen. Ranvier (’60) says there are more menisques in the finger tips than there are cells. : Enough has been said I think to show the very general distri- bution of these simple endings; of the functional importance as well as the particular distribution we will speak later. The nerve ring may be explained as the position of the touch cells has been explained, as arising as a result of the process of invagination. In the infolding of the follicular layers the nerve plexus is carried down too for a short distance. The follicle finally breaks through it but in the subsequent enlargement of this organ, and great increase in diameter, the fibers of the plexus are stretched out in the so-called ring. The brush-like ends which are seen cut across in the conical body are a part of this same plexus and from these fibers may often be seen running up to the surface. These nerves in the hair then have ‘no special function but are simply a distorted part of the common skin plexus. TACTILE HAIR OF THE WHITE RAT 19 All comparative study has shown that neither the size and development of the tactile hair follicle nor the nerve endings are proportional to the size of the animal. The rat, for instance, possesses a far more highly developed tactile hair than the horse or the ox or even the rabbit among the rodents. Leydig (’59) fifty years ago, noted that in the horse the follicle was smaller than in the dog or ox and the walls were thinner. In the polar bear he found only a slightly developed tactile hair with a small follicle and a sinus much more tender than a dog. The weasel and otter, on the contrary, have longer follicles with a greater ring sinus than the dog. The porcupine’s follicle is egg shaped and unusually large, but the greatest development of all the trabecular filled sinuses is in the seal where it may be seen with the naked eye. Of the investigations of the tactile hairs of the apes we shall mention only one, that of Frederick (05). He says that in some apes the tactile hairs are so strong that they can be seen as such by the eye alone. On the upper lip are many longer hairs which are the longest and strongest laterally. As a rule the hairs are weaker on the under lip. On microscopic study it is seen that these hairs on the lips and in the supra-orbital regions are real tactile hairs. The follicle is enclosed from without by a fibrous capsule of connective tissue and between the external and inter- nal lamina are cavernous spaces filled with blood. These extend from the mouths of the sebaceous glands to the papilla. Fre- quently they extend beneath the papilla, between it and the base of the follicle, the whole space being filled with a close mass of interpolated cavernous tissue. In none of the apes studied was there any differentiation between the ring sinus and the spongiform body; instead the whole cavernous space in its whole length was crossed by numerous closely radiating connective tissue bundles which connected the lamina externa with the lamina interna. He found the ring sinus lacking in apes, which in this respect are like hoofed animals. Szymonowicz (’09) finds the endings deseribed in the tactile hairs of other animals on the beard hairs of men. The nerve ring contains more medullated fibers than is usually found in 20 S. B. VINCENT other animals and forms a rich plexus of medullated, unmedul- lated, pale and varicose fibers. The touch cells are similar to those of other animals and in a similar position. He thinks the beard hairs of men lie structurally between the usual body hairs without the root sheath swelling and the tactile hairs of other animals. We will neglect entirely the discussion as to whether hairs originally developed from the scales of reptiles or fishes or from the Becher organs of amphibia, but there is one view of the course of their development that is not without interest for us. Many believe the theory which Botezat advances that the com- mon body hairs are retrogressive, that the tactile hairs were primary in development but that as functional need changed the ereater part lost their root sheaths and high degree of innervation and came to serve more and more as a mere body covering. As to the higher development of these tactual organs in some animals than in others, Edinger (’08) calls attention to the large fiber tract leading from the nucleus of the trigeminus to the tuberculum olfactorium in birds and some other animals. He thinks the increase in size of this lobe in some instances due to the ‘“‘importance of the beak which is innervated by the trigemi- nus” and “‘the extraordinary rich trigeminal supply about the mouth and in the tongue.” Probably other fibers find a center in or about the anterior perforated space but Edinger’s emphasis of the ‘oral sense’ has not been without value. We must recog- nize that the mouth parts of animals have a much more varied functional significance and are of far greater relative importance in them than in man. VI. FUNCTION The generally recognized character of the tactile hair is implied in its name. The purpose of such sense organs about the mouth has never been satisfactorily explained. They are not confined to nocturnal animals, as Odenius thought (’66), nor do they have chiefly to do with the size of openings. In a long series of experiments with animals in open, elevated mazes and in problem boxes, some facts were established as to TACTILE HAIR OF THE WHITE RAT PAl| the use of these tactile organs. The vibrissae were turned down and trailed along the edges of narrow supports or they brushed the sides of vertical walls or the path beneath as the animal ran. Animals deprived of them learned such problems less easily and had many more slips and falls. The inference was drawn that they furnished guiding sensations, sense of support, in locomotion, that they were intimately connected with equilibration and that their extreme mobility and sensitivity in rats was a partial com- pensation for poor vision. This was confirmed in part by obser- vations of rats whose labyrinths had been destroyed. The tactile hairs seemed also to assist in determining the exact position of openings or turns and in the discrimination of inequalities of surface. Animals in which the sensory nerve to this region had been cut, and whose noses as well as vibrissae were insensitive, were unable to make this tactual discrimination. ........| 36.75 31.52 27.09 27.82 27.66 36.33 31.33 Thoracic region (%).......... 32.19 | 34.32 34.94 39.12 41.82 | 45.34 | 50.00 Lumbar region (%)......... | Aesth ti) 20293 19.43 id os3o5 5 eas Sacral region (%)........... aoe Heo 13.86 ele 11.59 GOL en s109 TABLE 2 Areas of cross-sections of the spinal cord in an 11 mm. human embryo, showing the absolute and relative amounts of gray matter, of white matter, and of ependyma with canal | AREA AREA AREA AREA OF | %oOF % or % oF SEGMENT OF CROSS- | OF GRAY | OF WHITE |EPENDYMA! GRAY WHITE EPENDYMA | SECTION | MATTER MATTER | AND CANAL| MATTER MATTER | AND CANAL | sq. mm. | sq.mm. | sq. mm. $9. mm. Cervical I 0.600 0.244 0.120 0.236 40.67 20.00 39.33 Cervical II 0.560 0.232 | 0.112 0.216 41.43 20.00 38.57 Cervical IIT 0.540 0.224 0.112 0.204 | 41.48 20.74 37.78 Cervical IV 0.528 0.220 0.108 0.200 41.67 20.45 37.88 Cervical Vv 0.544 0.204 0.104 0.236 37.50 19.12 43.38 Cervical VI 0.532 0.208 | 0.084 0.240 39.10 15.79 45.11 Cervical VII 0.508 0.224 | 0.072 0.212 44.10 14.17 41.73 Cervieal VIII 0.464 0.168 | 0.068 0.228 36.21 14.65 49.14 Thoracic I 0.436 0.160 | 0.064 0.212 36.70 14.86 48.62 Thoracic II 0.416 0.148 0.060 0.208 35.58 14.42 50.00 Thoracic III 0.392 0.136 0.056 0.200 34.69 14.29 51.02 Thoracic IV 0.384 0.148 0.044 0.192 38.54 11.46 50.00 Thoracic V 0.364 | 0.144 0.040 0.180 39.56 10.99 49.45 Thoracic VI 0.380 | 0.1382 | 0.056 0.192 34.73 | 14.74 50.53 Thoracic VII 0.384 0.148 | 0.052 0.184 | 38.54 13.54 47.92 Thoracie VIII 0.376 0.144 0.044 0.188 38.30 11.70 50.00 Thoracic IX 0.372 | 0.136 | 0.052 0.184 36.56 13.98 49.46 Thoracic xX 0.344 | 0.136 0.040 0.168 | 39.53 11.63 48.84 Thoracic XI 0.364 O32). || 0.044 0.188 | 36.26 | 12.00 51.65 Thoracic XIT 0.396 | 0.144 | 0.044 0.208 =| 36.36 11.11 52.53 TABLE 3 Areas of cross-sections of the spinal cord in a 17 mm. human embryo, showing the absolute and relative amounts of gray matter, of white matter, and of ependyma with canal AREA AREA | AREA | AREA OF | % or COR eoOr SEGMENT OF CROSS- | OF GRAY | OF WHITE |EPENDYMA| GRAY WHITE | EPENDYMA SECTION MATTER | MATTER |ANDCANAL| MATTER MATTER | AND CANAL sq. mm. sq. mm. sq. mm. sq.mm. | | Cervical I 1.300 0.644 0.376 0.280 } 49.54 28.92 | 21.54 Cervical II 1.188 0.596 0.356 0.236 | 50.17 29.97 | 19.86 Cervical IIT 1.152 0.576 0.348 «| Ou228) am) 50.00 30.21 | 19.79 Cervical IV 1.180 0.576 0.372 0.232 48.81 31.53 | 19.66 Cervical V 1.264 0.652 0.372 0.240 51.58 29.43 | 18.99 Cervical VI 1.256 0.636 0.376 0.244 | 50. 64 29.93 19.43 Cervical VII 1.232 0.636 0.340 0.256 51.62 27.60 20.78 Cervical VIII 1.128 0.560 0.340 | 0.228 49.64 30.14 20.22 Thoracic I 1.036 0.524 0.280 0.232 50.58 27.03 22.39 Thoracic Ir 0.932 0.444 0.272 0.216 47.64 29.18 23.18 Thoracie III 0.924 0.448 0.256 0.220 48.49 27.70 23.81 Thoracic IV 0.880 0.424 0.248 0.208 48.18 28.18 23.64 Thoracic V 0.920 0.464 0.240 0.216 50.44 26.08 23.48 Thoracic VI 0.932 0.484 0.244 0.204 51.93 26.18 21.89 Thoracic VII 0.908 0.460 0.232 0.216 50.66 25.55 23.79 Thoracis VIII 0.900 0.444 0.232 0.224 49.33 25.78 24.89 Thoracic IX 0.916 * 0.456 0.232 0.228 49.78 25.33 24.89 Thoracic x 0.896 | 0.428 0.232 0.236 47.77 25.89 26.34 Thoracic D. 0.872 0.420 0.220 0.232 48.16 25.23 26.61 Thoracic XII 0.956 | 0.448 0.248 0.266 46.86 25.94 27.20 Lumbar I 1.044 0.492 0.268 0.284 47.13 25.67 27.20 Lumbar II 1.076 0.516 0.276 0.284 47.96 25.65 26.39 Lumbar Il 1.128 0.540 0.292 0.296 47.87 25.89 26.24 58 PRENATAL GROWTH OF SPINAL CORD 59 TABLE 4 Areas of cross-sections of the spinal cord in a 31 mm. human embryo, showing the absolute and relative amounts of gray matter, of white matter, and of ependyma with canal AREA AREA AREA | AREA OF % oF % OF | % oF SEGMENT OF CROSS- | OF GRAY | OF WHITE | EPENDYMA GRAY WHITE EPENDYMA SECTION MATTER MATTER |ANDCANAL| MATTER MATTER | AND CANAL sq. mm. sq. mm, sq. mm. sq. mm. Cervical I 2.166 1.175 0.701 0.290 54.25 32.36 13.39 Cervical II 2.142 1.182 0.688 0.272 55.18 32.12 12.70 Cervical III 2.080 1.160 0.670 0.250 55.77 32.21 12.02 Cervical IV 2.112 1.204 0.672 0.236 57.01 31.82 iby Cervical V 1.984 1.120 0.668 0.196 56.45 33.67 9.88 Cervical VI 1.780 0.992 0.596 0.192 55.73 33.38 10.89 Cervical VII 1.496 0.808 0.532 0.156 54.01 35.56 10.43 Cervical VIII 1.268 0.652 0.488 0.128 51.42 38.49 10.09 Thoracic I 1.100 0.564 0.424 0.112 51.27 38.55 10.18 Thoracic II 0.904 0.460 0.352 0.092 54.88 38.94 10.18 Thoracic III 0.852 0.416 0.348 | 0.088 48.83 40.84 10.33 Thoracic IV 0.816 0.400 0.328 | 0.088 49.02 40.20 | 10.78 Thoracic V 0.764 0.384 0.296 | 0.084 | 50.26 38.74 11.00 Thoracic VI 0.812 0.404 0.320 0.088 =| 49.75 39.41 | 10.84 Thoracic VII 0.808 0.400 0.324 | 0.084 | 49.50 40.10 | 10.40 Thoracic VIII 0.848 0.436 0.308 | 0.104 | 51.42 36.32 | 12.26 Thoracic IX 0.860 0.436 0.312 0.112 | 50.70 36.28 13.02 Thoracic x 0.944 0.492 0.320 0.132) | 52.12 33.90 13.98 Thoracic XI 0.992 0.536 0.332 On | 54.03 33.47 12.50 Thoracic XII 1.206 0.708 0.368 0.140 58.22 30.26 11.52 Lumbar I 1.248 0.672 0.412 0.164 | 53.85 33-01 | 13.14 Lumbar Il 1.428 0.768 0.432 0.228 53.84 | 33.02 13.14 Lumbar III 1.488 0.832 0.436 0.220 53.78 30.25 15.97 Lumbar IV 1.540 0.848 0.476 0.216 55.91 | 29.30 14.79 Lumbar Vv 1.528 0.832 0.460 0.236 55.45 SOnL Le 15.44 Sacral I 1.392 0.764 0.408 0.220 54.89 29.31 | 15.80 Sacral II 1.372 0.748 0.400 0.224 54.52 29.15 | 16.33 60 MAX MAYO MILLER TABLE 5 Areas of cross-sections of the spinal cord in a 65 mm. human embryo, showing absolute and relative amounts of gray matter, of white matter, and of ependyma with canal AREA AREA AREA | AREA OF % OF % OF % OF SEGMENT OF CROSS- | OF GRAY | OF WHITE | EPENDYMA GRAY WHITE EPENDYMA SECTION | MATTER MATTER | AND CANAL| MATTER MATTER | AND CANAL sq.mm. | sq.mm. sq. mm. | sq. mm. Cervical I (1.936) | (1.016) | (0.880) (0.040) (52.48) (45.45) (2.07) Cervical II (1.832) (1.000) (0.796) | (0.036) (54.59) (43.45) (1.96) Cervical Ill 1.772 0.976 0.764 | 0.032 55.08 43.12 1.80 Cervical IV 1808, | 0.992 0.784 | 0.032 54.87 43.36 ei Cervical Vv | 1.896 1.028 0.832 | 0.036 54.22 43.88 1.90 Cervical VI Weritie | 1.008 On 7G 0.032 57.40~ 1 40.78 1.82 Cervical VII 1.592 | 0.904 0. 664 0.024 56.78 | 41.71 1.51 Cervical VIII 1.376 0.756 | 0.592 | 0.028 54.94 | 43.02 2.04 Thoracic I 1.120 0.612 0.484 0.024 54.64 | 43.21 yal iy Thoracic II 1.020 | 0.588 0.408 | 0.024 55.65 | 40.00 Desh Thoracic II 1.044 | 0.604 0.420 | 0.020 57.85 40.23 1.92 Thoracic IV 0.960 | 0.556 0.384 0.020 57.92 40.00 2.08 Thoracic Vv 0.988 0.548 0.420 0.020 55.46 42.51 2.03 Thoracic VI 0.980 0.564 0.892 0.024 57.55 40.00 2.45 Thoracic VII 1.040 0.576 0.440 | 0.024 55.38 42.31 Pai Thoracic VIII 1,024 0.572 0.424 0.028 55.86 41.41 2.73 Thoracic IX 1.096 0.612 0.456 0.028 55.84 41.61 2.55 Thoracic xe 1.208 | 0.680 0.500 0.028 56.29 41.39 2.32 Thoracic XI) 1.244 | 0.724 0.492 0.028 58.20 39.55 2.25 Thoracic XII 1.508 0.860 0.616 0.032 57.03 40.85 ele Lumbar I 1.820 1.036 0.748 0.036 56.92 41.10 1.98 Lumbar Il 2.184 1.224 0.912 | 0.048 56.04 41.76 2.20 Lumbar Ill 2.416 1.404 0.972 0.040 58.11 40.23 1.66 Lumbar IV 2.560 1.516 1.000 0.044 59.22 39.06 1.72 Lumbar V 2.412 1.448 0.920 0.044 60.03 38.14 1.83 Sacral I 2.344 1.420 0.872 0.052 60.58 37.20 2.22 Sacral II 1.828 1.120 0.660 0.048 61.27 36.10 2.63 Sacral Il 1.416 0.892 0.492 0.032 §2.99 34.75 2.26 Sacral IV 1.032 0.616 0.384 0.032 59 69 37.21 3.10 Sacral Vv 0.560 — 0.288 0.252 0.020 51.48 45.00 3.57 Coccygeal...... 0.360 0.216 0.124 0.020 60.00 34.45 5.55 Conus med..... 0.244 0.100 0.132 0.012 40.98 54.10 4.92 Filum term..... 0.168 0.016 0.128 0.024 9.52 76.19 14.29 PRENATAL GROWTH OF SPINAL CORD 61 TABLE 6 Areas of cross-sections of the spinal cord in a 150 mm. human embryo, showing abso- lute and relative amounts of gray matter, of white matter, and of ependyma with canal AREA AREA AREA AREA OF % OF % or % or SEGMENT OF CROSS- | OF GRAY | OF WHITE | EPENDYMA GRAY WHITE EPENDYMA SECTION MATTER MATTER |ANDCANAL| MATTER MATTER AND CANAL sq. mm. sq. mm. sq. mm. sq. mm. | Cervical TG ene 28) (4.452) (3.220) (0.056) (57.61) (41.67) (0.72) Cervical II (7.460) (4.160) (3.248) (0.052) (55.82) (43.54) (0.64) Cervical III (7.360) (4.022) (3.280) (0.048) (54.78) (44.57) (0.65) cervical IV 7.386 4.040 3.304 0.044 54.68 44.72 0.60 Cervical V 7.560 4.280 3.232 0.048 56.61 42.75 0.64 Cervical VI 7.748 4.476 3.224 0.048 57.77 41.61 0.62 Cervical VII 7.164 4.036 3.080 0.048 56.34 42.99 0.67 Cervieal VIII 5.256 2.676 2.540 0.040 50.91 48 33 0.76 Thoracic I 3.640 1.816 1.800 0.024 49.89 49.45 0.66 Thoracic II 2.812 1.496 1.296 0.020 53.20 46.09 0.71 Thoracie III 2.412 1.256 1.140 0.016 52.07 47.26 0.67 Thoracic IV 2.524 1.308 1.204 0.012 51.82 47.70 0.48 Thoracic V 2.404 1.236 1.156 0.012 51.41 48.09 0.50 Thoracic VI 2.500 1.260 1.288 0.012 50.40 49.12 0.48 Thoracic VII 2.464 15252 1.196 0.016 50.81 48.51 0.65 Thoracie VIII 2.420 1.216 1.188 0.016 50.25 49.09 0.66 Thoracic IX 2.652 1.384 1,252 0.016 52.19 47.21 0.60 Thoracic x 2.832 1.464 1.352 0.016 51.69 47.74 0.57 Thoracic XI 3.132 1.632 1.480 0.020 52.11 47.25 0. 64 Thoracic XII 3.636 1.812 1.804 0.020 49.84 49.62 0.54 Lumbar I| 4.228 2.264 1.944 0.020 53.55 45.98 0.47 Lumbar II 5.268 3.028 2.220 0.020 57.48 42.14 0.38 Lumbar Ill 5.796 3.328 2.444 0.024 57.42 42.17 0.41 Lumbar IV 6.140 3.584 2.536 0.020 58.37 41.30 0.33 Lumbar V 6.080 3.620 2.440 0.020 59.54 40.13 0.33 Sacral 1 5.792 3.396 2.372 0.024 58.63 40.95 0.42 Sacral II 4.948 2.644 2.284 0.020 53.44 46.16 0.40 Sacral Ill 4.204 2.272 1.196 0.016 54.04 45.58 0.38 Sacral IV 3.540 2.040 1.480 0.020 57.63 41.81 0.56 Sacral Vv 3.104 1.772 1.304 0.028 57.09 42.01 0.90 Coccygeal..... 2.296 1.220 1.048 0.028 53.14 45.64 e272) Conus med.... 1.644 0.884 0.732 0.028 538.07 44.53 1.70 Filum term..... 0.424 0.032 0.280 0.112 7.55 66.04 26.41 TABLE 7 Showing the average cross-sectional area of gray matter in the various regions, and the relative amounts of the anterior and posterior horns EMBRYO CHILD | ADULT REGION 7 1imm./17mm.|31mm.)65mm.|150mm.|__ (Stilling) Area of gray matter ie Cervical V, V1, (SORMMINS) Saset ace oe 0.201 | 0.621} 0.893 | 0.924| 3.867) 15.91] 17.89 WADI WANE SE aaa bot lols ters eee 74.63 | 69.40 | 58.45 | 62.45 | 54.69 58.80 ((%) IRoste Norneace = ser 25.37 | 30.60 | 41.55 | 37.55 | 45.31 41.20 Area of gray matter Thorseie (Gq. mm:)..0-oo0.0-| 0.142 OL454 OLS GON OL b2a7|aMense 6.45 5.36 on eameeeeel(%G) ANG. NOL... 2.025 O200) |2s0n al POScOOn Oden osaGe 49.16 (%) Post. horn..........} 20.42 | 27.97 | 41.31 | 35.52 | 46.38 50.84 Area of gray matter : asec | (Cee atid pesnegenne: 0.515 | 0.790] 1.326) 3.165| 14.55) 14.41 7 aie (9G) Amitahornieecesen ce: 73.59 | 56.58 | 57.16 | 05,04 52.80 (%) Post. horn.......... 26.41 | 43.42 | 42.84 |. 44.96 | 47.20 TABLE 8& Showing the average cross-sectional area of white matter in the various regions, and the relative amounts of the anterior, lateral, and posterior columns EMBRYO CHILD | ADULT REGION 1lmm.|}17mm.| 31mm.|/65mm.|150mm,| — (Stilling) Area of white matter (sq)emond.) hase ee 0.082) 0.357) 0.571 | 0.701 | 3.019 42.24 | 37.64 Cervical V, VI, 4 (%) Post. column....... 36.59 | 20.45 | 35.20 | 28.10 25.55 33.00 Vine AVIND. 2 52: (%) Lat. column....:... j stay tat 51.14 | 48.08 | 41.75 36.40 (%) Ant. column....... } 20.17 | 13.66 | 23.82 32.70 30.60 Area of white matter (Sqeamimns) bee oe eres 0.050; 0.245) 0.336 | 0.453 1.387 | 23:47] 24.22 SRhorsete crete cc (%) Post. column.......| 38.00 | 20.41 | 35.71 | 26.27 32.30 27.60 %) Lat. column........ box a (hae 51.49 | 46.14 | 45.93 54.05 %) ant. column........ ' 10.59 | 12.80 | 27.59 21.77 18.35 Area of white matter (Sani) eee 0.279| 0.444) 0.910 2.321 | 21.88} 20.80 TA Dae eee sae (%) Post. column. ..... 20.48 | 26.80 | 31.65 34.38 30.20 (%) Lat. column........ 56.99 | 53.40 | 47.47 40.84 39.80 (%) Ant. column....... 22.58 | 19.80 | 20.88 24.78 31.00 TABLE 9 Absolute and relative volumes of white matter, of gray matter, and of ependyma with the canal in various regions of the cord in the 11 mm. and 17? mm. embryos 11 MM. EMBRYO 17 MM. EMBRYO REGION | pe eyroyi % of % of % of Volume repion total Volume pemion total ce, | ce. White matter......... 0.000269 18.18 50.69 | 0.001094 29.07 35.54 Gervicali=..:... 4 Gray umattersa..s-- ee 0.009643 43.48 42.14 | 0.001919 51.00 32.31 Canal and ependyma.)| 0.000567 38.34 28.81 | 0.000750 19.93 25.69 White matter.........| 0.000166 12.82 31.38 | 0.001073 26.20 34.86 Thoracic .......- Gray matter... 22..3.. 0.000485 37.45 31.77 | 0.002005 48.95 33.76 | Canal and ependyma | 0.000644 49.73 32.73 | 0.001018 24.85 34.88 White matter......... 0.000095 7.60 17.92 | 0.000911 22.35 29.60 Lumbo-Sacral .. ; Gray matter..........| 0.000398 31.84 26.09 | 0.002015 49.42 33.93 | Canal and ependyma | 0.000757 | 60.56 38.46 | 0.001151 28.23 39.43 62 TABLE 10 Absolute and relative volumes of white matter, of gray matter, and of ependyma with the canal in various regions of the cord in the 31 mm. and 65 mm. embryos 31 MM. EMBRYO 65 MM. EMBRYO |= = : : REGION Volume ae Ha Volume aes Ft cc. ce. White matter.........| 0.00187 32.64 27.26 0.01625 38.80 26.97 @ervicalacs. ss. [em mMabvers.ss.c0... || OLO0826 56.89 28.08 0.02480 59.22 28.49 Canal and ependyma | 0.00060 10.47 22.39 0.00083 1.98 25.70 { White matter.........| 0.00276 37.35 40.23 0.02644 44.90 43.88 Thoracic........ Gray matter..........| 0.00427 55.07 35.06 0.03181 54.02 36.55 loa and ependyma | 0.00056 7.58 20.89 0.00064 1.08 19.81 White matter:........| 0.00157 30.78 22.89 0.01182 37.52 19.61 Em bare Gray matter..........| 0.00284 55.64 24.46 0.01860 59.05 21.37 Canal and ependyma | 0.00069 13.58 Pt fi 0.00108 3.43 33.44 White matter......... 0.00066 22.52 9.62 0.00575 31.49 9.54 SACral eee Gray matter..........| 0.00144 49.15 12.40 0.01183 64.79 13.59 Canal and. ependyma | 0.00083 28.33 30.97 0.00068 si) 21.05 = ~ TABLE 11 Absolute and relative volumes of white matter, of gray matter, and of ependymawith the canal in various regions of the cord in the 150 mm. embryo. REGION VOLUME inom os i cc. Wht bermdatters ae). Jue. helinte dee ees 0.04848 | 44.16 26.47 Cervical... sy TSOUZH RE) Povey Oke ck te RAE nS otis Eilean, Se ee 0.06059 | 55.19 28 .65 Canal-and ependymaas:.. es. eee. ac: 0.00071 0.65 30.60 Wihiitelma ter: says ys eee aeyS fe 0.08087 | 49.31 44.16 Thoracic. . ss Ia eT es res eee a ee ee 0.08213 | 50.09 38.82 Canaltandiependivimas eee eee 0.00100 0.60 43.10 White matter: . 225) 5..45 5rd OROS oo. in Ase 18.22 Lumbar... Las matter: Ngee ese eee bs 0.04345 | 56.33 20.57 Canalvandependymanee eee eee 0.00032 0.42 13.80 Wihite matter 4: eee aoe eae ae 0.02043 | 44.41 We SiS) Sacral... te matter... opeeenoeare ee se ..| 0.02528 | 54.96 11.96 Canal-and\ependyimamas sae) 2 0.00029 0.63 12.50 TABLE 12 Relative volumes of white matter and of gray matter in the various regions of the spinal cord in a child of two years (Stilling) and in a composite adult (Donaldson and Davis). CHILD ADULT REGION = = = % of region| % of total |% of region] % of total Coen Wie THOR. oc, oboe on oes ee 75 .60 37.46 80.35 31.27 Gay HAG GOR 20. 2s errlnyaten ane: 24.97 33.23 19.65 31.41 Tienes Wihitemmat tensa aan aeese 78.26 | 48 .29 85.58 53.20 NGietny TOMER Heo Baeiee ea bile see 21.74 | 36.87 14.42 36.89 ambes Wihttesmatten....:40+s2.. 4 63.04 | 10.59 70.49 12.99 ee Guavemat tener. sees 36.94 | 17.06 29.53 22.14 Saal Wihiitemnoatitense sre.) .hcc ae 43 .90 3.66 53.28 2.54 imme (Gor anainiat tennessee aes 56.10 | 12.84 46.72 | 9.56 64 MAX MAYO MILLER EXPLANATION OF FIGURES Figures 1 to 5 represent outline drawings of actual cross-sections of different regions in the various spinal cords studied. X12. Where the sections drawn did not show any nerve roots, the lines of separation for the various columns of white matter were approximated. C, central canal; E, ependyma; P, posterior horns of gray matter; A, anterior horns of gray matter; /, lateral columns of white matter; p, posterior columns of white matter; a, anterior columns of white matter. Fig. 1 a 5th cervical segment, 11 mm. embryo. Fig. 1 b 5th thoracic segment, 11 mm. embryo. Fig. 2a 5th cervical segment, 17 mm. embryo. Fig. 2b 5th thoracic segment, 17 mm. embryo. Fig. 3a 5th cervical segment, 31 mm. embryo. Fig. 3b 5th thoracic segment, 31 mm. embryo. Fig. 3c 4th lumbar segment, 31 mm. embryo. Fig. 4a 5th cervical segment, 65 mm. embryo. Fig. 4b 6th thoracic segment, 65 mm. embryo. Fig.4c 4th lumbar segment, 65 mm. embryo. Fig.5 a 5th cervical segment, 150 mm. embryo. Fig.5 b 6th thoracic segment, 150 mm. embryo. Fig.5 ¢ 4th lumbar segment, 150 mm. embryo. PRENATAL GROWTH OF SPINAL CORD 65 65 ; MAX MAYO MILLER Area in sq. mm. PRENATAL GROWTH OF SPINAL CORD 67 EXPLANATION OF FIGURES Figures 6 to 12 represent by curves the cross-sectional areas in each segment of several embryonic and adult human spinal cords, as well as the corresponding areas of gray and white matter (also the ependyma with the canal in figures 6 to 9). The curves are so plotted that the areas enclosed between the base-lines and curves represent the total volumes of the cords and of their component parts, respectively. The figures are so drawn that the areas representing the total vol- umes of the cords are approximately the same. The lengths of the segments are represented on the abscissa and so calculated that the total lengths of the various cords are represented by the same length of abscissa. In any given figure, the changes in the height of the curves therefore represent changes in the caliber of the cord as a whole (or in the relative amounts of its component parts) at different levels. A comparison of the different figures shows for the various stages the changes in the form of the cord as a whole, and in the relative amounts of the component parts. The following points must be held in mind to avoid error in comparing the various curves: 1. Curves of figures 6, 7 and 8 are incomplete at the lower end. 2. Curves of figures 9 and 10 are estimated at the upper end (dotted lines) as explained in the text. 3. The apparent increase at the upper end (all of the cervical region) of figure 6 is mostly due to the obliquity of the sections corresponding to the curvature of the spinal cord. This also applies to the lower six thoracic segments of figure 6, to the lumbar segments of figure 7, and to the upper four cervical, to some extent, in figure 8. In figure 9, all the cervical segments are thus slightly enlarged, although not enough to require dotted lines. i Total cord eee we _Ependyma with canal aiserran eee SS oe ms a aS Semel White matter | i} Cervical! Vill l Thoracic Xu Segments of spinal cord Fig. 6 Spinal cord of human embryo of 11 mm. 4.5 Area in sq. mm. a fe) 2.0 a Area in sq. mm. ° 68 MAX MAYO MILLER Total cord Gray matter Ependyma with canal White matter SS ——— ti} Cervical Vi Thoracic xil Segments of spinal cord Cervical Fig. 7 Spinal cord of human embryo of 17 mm. Total cord Gray matter White matter Ependyma with canal vill Thoracic XII Segments of spinal cord Fig. 8 Spinal cord of human embryo of 31 mm. _--- | Lumbar Lumbar V ! Sac-Cce. PRENATAL GROWTH OF SPINAL CORD 69 2.5 2.0 e Total cord 3 Gray matter gs White_matter c + Ependyma with canal ara Cervical Vile Thoracic Xl tLumbar VY 'Sac-Ce. Segments of spina! cord Fig. 9 Spinal cord of human embryo of 65 mm. ==" Total cord Gray matter White matter Area in sq. mm. _! "Cervical vil 4 Thoracic Xl! !Lumbar VY ! Sac-Ce. Segments of spina! cord Fig. 10 Spinal cord of human embryo of 150 mm. 70 MAX MAYO MILLER 60 50 40 Total cord 30 White matter - 20 E € 3 10 = : \ to : Vit Cervical vi | Thoracic xi tLumbarV! Sac-Ce. Segments of spinal cord Fig. 11 Spinal cord of a two-year-old child; data taken from Stilling’s observations. 400 75 Total cord s White matte E Eas fey 12) is Gray matter © \ ac ' ay, % S / N SZ / . pel EeASN i 4 1 "a my AS 1 t ii Lan ‘ ! (tir esh VN U 1 roe etl ‘ 1 ‘(enn Sse ete 1 \ \ Cp peed i ‘ Neto el r . \ as ‘ SS \ 4 1 x \ U SK 4 ¢ n.opticus --- “a eS 1 === =t=--08 parietale n. oculomotorius --- fi “Sx ° . ‘\ pn. trigeminus SA f \ mn facialis“N 5 et acusticus, \ ee ear ' \ . 1 n. abducens -.--SS j 1 / / 4 i / 4 Zz 4 “N a G = ¢ —---- Fig. 3 Dorsal aspect of the brain of Necturus showing relations of the nasal KGAS sac, eye and ear and the course of the trochlear nerve. 169 \ 1 \ \ ¢ U \ 4 }} \ 4 J / nasal sac / \ 7 4 i\ a Di aN 4 a \ ' / goa \ aaa t i ai 5 , \ i \ (/ Oo ~ ine hen ‘ Z \ f Ne ' / \ 1 1 | Choana } a : eh © va \ Ns 2 So Sscescs =” np -- --- \ Se N nob U6 inte “\ foramen S ee f 7 aN EL SO olfactortum-|---- N 4 “XN PG Sey) x Mn. rectus ae N = a . -\-os m.rectus inferior” parictale m. rectus lateralis” zee rdorsalis rventralis n.oculomotorii-------- n. oculomotorii n.abducens ------- . n. oculomotorius - nopticus --- n.trochlearis --- Saccus vasculosus _ hypophysis sm aa WY nb “ A) .ntrigeminus Os ; i Toes : | or 4) ug / au Aas Yn tacialis i Hh V -) etacusticus A A : ear \ \ \ \ \ \ \ vn. glosopharyngers ~~ et vagus Fig. 4 Ventral aspect of the brain of Necturus showing relations of the nasal ac, eye and ear and the cour x 4.5. se of the optic, oculomotor and abducent nerves. 170 Se nasal sac Pe ® \ “=--=—-— - _- a ~-- s SAS aN ‘m.obliguus superior YY", ‘A bf V4 y 2 Ze ZS aS - EZ i ~ _-=- -— [| F--~r-dorsalis n. oculomotorii /*/:=frophthalmicus protundus n. trigemint ete Saas Z / nasal sac i 4 tm obliquus inferior | Th-tr ophithalmicus protundus s t . alee | n.trigemint ~------------- SS ~ ~ Fig. 5 Dorsal view of the right orbit showing termination of the trochlear and abducent nerves and of the dorsal ramus of the oculomotor nerve. X 10. Fig. 6 Ventral view of the right orbit showing termination of the ventral ramus of the oculomotor nerve. X 10. 171 / branch for m. obl iquus =m. rectus interior Tae! i “~~-yp, abducens , j ----#: ventralis n. oculomotorii DP ae a Pers n.ciléaris n.trigemini ¢ mee “a ¢ ges i re ae ‘ rode. | We A i ae es iF vel if Seales r-ophthalmicus protundus n. trigemin« ‘ Vth Vj i ee ae deg} ------b - r dorsalis n. oculomotorii ’ a s A \ ies } / FO soa ee n.oculomotorius ts v / n.opticus ------~ n.trochlearis---~ -n.trigeminus ‘syn. facialis etacusticus Fig. 7 Dorsal view of the right orbit. The superior rectus and the superior oblique muscles have been cut. The optic nerve has been cut close to the eye- ball and turned medially so as to show the ciliary nerves on its caudal surface. This drawing shows also a case in which the ventral ramus of the oculomotor nerve is dorsal to the inferior rectus muscle (compare fig. 6). X 15. Fig. 8 Dorsal view of the mid-brain and medulla oblongata showing relations of the trochlear nerve. On the left side the most usual relations are shown. the right side appear the relations in a case in which the nerve was split into two fascicles, the main trunk of the nerve passing caudad over the cerebellar com- missure to the choroid plexus of the fourth ventricle. the edges of the choroid plexus. The dotted lines indicate The connections of the fibers which appear coming from the trochlear nerve to the choroid plexus have not been determined. KO: 172 ON THE INNERVATION OF THE. DIGESTIVE TUBE ALBERT KUNTZ The Laboratories of Animal Biology of the State University of Towa FIVE FIGURES CONTENTS LEO CU CULOMS vee yore Seats otek oe icles anes othe, 2 oie fas RCPS teas tei 173 Maternal tandsmethod Starrett tes scrote soe aac eel ae ee eer ae 175 OBSeR Vat OM Sree nye sen ye ieee cetera yan Berets oe) Same tie inet ah Pees ee ee ee ge 176 My Cmte rie yp lems feee es id eal Ayes as gus snide A we hea = Ries Wee a 176 SUMP MUMCOUS OER Seer akc ote ke UO Ie te ee 179 Miber-termimahlOnsyer asthe ce ele oe terse anos che case oe UE co ee 182 DIS CMSSLOMEE ere ieee Neh eu ry ae SR rE rca ata ee A Re gee 186 PS UMRRINN ATV oy. 5 shes char al nyt, Sram te tu aes eee ncn Wiash ARPS BAe ik Gia le keyias OMAN Sa Ae ae ae 190 IBS Tp luo par Bp yes Prevesti teaehe ees HOE a ceria set hee ir olinyat attention A 191 INTRODUCTION A review of the literature bearing on the morphology of the sympathetic nervous system shows that the histological char- acters of the sympathetic neurones are quite definitely known. Ever since the introduction of the Golgi method, sympathetic neurones have been described. More recently our knowledge of the sympathetic neurones has been furthered by the use of the methylen blue method of Dogiel, the silver reduction method of Cajal and the modified silver reduction method of Bielschowsky. Recent investigators, notably Cajal, Dogiel, Michailow and Miller, have described the neurones in the various parts of the sympathetic nervous system in the Mammalia, including man, in great detail. Each of these investigators has attempted a more or less complete classification of sympathetic neurones according to morphological type. Thus Cajal (’05, ’06), whose 173 174 ALBERT KUNTZ classification is accepted by Miller (712), recognizes three dis- tinct types. Dogiel and Michailow both recognize a greater diversity of sympathetic neurones, the latter (11) claiming to be able to recognize as many as nine distinct types. In general it may be said that the neurones in the various parts of the sympathetic nervous system show certain distinc- tive histological characters. Miller (12) expressed the opinion, however, that all sympathetic neurones are fundamentally of the same morphological type, but differ somewhat in the struc- ture and disposition of their dendrites according to the demands of the functions of the organs innervated by them. To quote: ‘““Zusammenfassend glaube ich annehmen zu diirfen dass schlies- lich der Grundtypus aller Zellen des vegetativen Nerven- systems derselbe ist, dass sich aber die Zellen beziehungsweise ihre Dendriten unter den verschiedenen Anspriichen welche die Function des betreffenden Organs an sie stellt, verschieden gestalten.’’ This opinion is of interest in view of the fact that, as pointed out by the writer in an earlier paper (’10), all sympa- thetic neurones arise from cells which are the descendants of the ‘germinal’ cells (Keimzellen) of His, that is, they all have a common origin. The myenteric and the submucous plexuses with their com- ponent elements were described by Cajal as early as 1893. By the use of his own modification of the Golgi method, he was able to determine that all the neurones in these plexuses are multipolar and that the fibers are of the non-medullated variety. He believed, however, that all the protoplasmic processes of these neurones are essentially axones. Cajal’s observations were substantially corroborated by the work of Kolliker (’94) who suggested, furthermore, that these mul- tipolar neurones might provide the apparatus for local reflexes. He says: “‘So kénnten beispielsweise Zellen des Meissner’schen Ge- flechts mit oberflichlichen Ausliufern in den Darmzotten gewisse Erregungen aufnehmen und mit andern Auslaufern auf die Muskelfasern der Zotten oder der Muscularis mucosa einwirken. In einem solchen einfachsten Falle. wiirde schon eine einfache multipolare Zelle einen volstiindigen Reflexapparat darstellen.”’ INNERVATION OF THE DIGESTIVE TUBE 175 Dogiel (’95) also described the neurones in the myenteric and the submucous plexuses in great detail. So successful was this investigator with his own methylene blue method that the accu- racy and detail of his figures has scarcely been equaled by the work of later investigators. Among the more recent investigators, Miller (11) has given us a more or less detailed description of the myenteric and the submucous plexuses in various regions of the digestive tube. His figures and microphotographs indicate a wide range of varia- tion among the neurones in these plexuses. His studies show, furthermore, that with some differences in detail the myenteric and the submucous plexuses are similarly constructed throughout the length of the digestive tube. In spite of our knowledge of the general morphology of the sympathetic nervous system and the histological characters of its component neurones, little is known concerning the physiological relationships of the sympathetic neurones and the distribution of their axones and dendrites, especially in the peripheral sympa- thetic plexuses. The present paper is primarily an attempt to point out some of the morphological relationships of the neurones in the myenteric and the submucous plexuses to each other and to the muscle, gland and epithelial cells of the organs innervated by them; thus to advance our knowledge of the morphological basis for the physiological activities of the sympathetic nervous mechanism in the walls of the digestive tube. I desire to express my indebtedness to Prof. G. L. Houser for suggestions during the progress of this investigation and for reading the manuscript. I am also indebted to Prof. F. A. Stromsten for suggestions in technique. , MATERIAL AND METHODS \ The present series of observations is baSed primarily on prep- arations of the stomach and the small intestine of the cat and the dog. Good preparations for the study of the myenteric and the submucous plexuses are not easily obtained. The silver reduction method of Cajal and the method of Bielschowsky, both of which were employed more or less successfully for the — 176 ALBERT KUNTZ study of other parts of the sympathetic nervous system, failed utterly in the hands of the writer when applied to the digestive tube of the cat and the dog. After numerous unsuccessful attempts at intra-vitam staining with methylene blue, prepara- tions of the stomach and the small intestine of the cat were obtained by the use of this method in which some of the neurones in the myenteric plexus and many of the fiber-tracts involving both the myenteric and the submucous plexuses were well stained and could be studied quite satisfactorily. Methylene blue prep- arations in which the cell-bodies of neurones in the submucous plexus were well stained were not secured. The pyridine-silver method as employed by Ranson! was found very useful for the study of the neurones in both the myenteric and the submucous plexuses. In sections of the small intestine of the dog successfully prepared by this method, sympathetic neurones and fibers are well stained and may be satisfactorily studied. In these preparations, however, it is rarely possible to trace sympathetic fibers to their terminations on gland or epithelial cells. Even this method does not yield as uniformly good results when applied to the sympathetic plexuses in the walls of the digestive tube as when applied to other parts of the sympathetic nervous system or to the cerebro-spinal nervous system. OBSERVATIONS Myenteric plexus The ganglia of the myenteric plexus are somewhat irregular flattened or less-shaped aggregates of neurones interposed be- tween the longitudinal and the circular muscle-layers of the digestive tube. These ganglia are variously connected with each other by commissures of non-medullated fibers arranged either in distinct bundles or in broad flattened bands. In sections of the stomach or the small intestine taken in the plane of the myenteric plexus, these commissures may be traced from one ganglion into another. In many instances four or more commissures may be traced in as many directions from a single 1Amer. Jour. Anat., vol. 12. p. 69. INNERVATION OF THE DIGESTIVE TUBE | WAP ganglion. Within the ganglia the paths of the fibers composing these commissures intersect each other at various angles, while in the commissures the fibers run more or less parallel with each other. In pyridine-silver preparations the slender fibers running in these commissures are stained somewhat more intensely than the surrounding tissue and present a slightly wavy and vari- cose appearance. In those commissures which are composed of a distinct fiber-bundle the fibers are more or less compactly aggregated, while in the more flattened commissures fibers running parallel with each other may often be observed distrib- uted more or less uniformly over areas of considerable width. In transverse or longitudinal sections numerous commissures may be traced between the bundles of circular muscles from the ganglia of the myenteric plexus into the submucous plexus. The ganglia of the myenteric plexus vary greatly in size, being composed of relatively few or of relatively many neurones. The neurones are not compactly aggregated. In good pyridine- silver preparations, however, little tissue may be observed in the ganglia except the fibers which pass through them at various angles and the neurones with their processes. Pericellular cap- sules were not observed in these ganglia either in pyridine-silver or in methylene blue preparations. The writer is, therefore, inclined to agree with Miiller (11) that such capsules do not occur in this plexus. The neurones in these ganglia may be studied most satisfactor- ily in sections taken in the plane of the plexus because many of their processes lie approximately in this plane. In good prepara- tions of this kind axones and dendrites may often be traced for a considerable distance from the cell-body. The neurones in these ganglia vary greatly in size as well as in their general character and form. Neurones of distinct types may be observed, but the deviations from such types are so numerous and so varied that one finds little satisfaction in attempting any rigid classification. Perhaps the most significant morphological difference between neurones in these ganglia consists in the length and the distribu- tion of their dendrites. The dendrites of some are long and slender while those of others are too short to be traced out of 178 ALBERT KUNTZ the ganglion in which the cell-body is located. ‘These neurones have been so well described and illustrated by earlier observers that any attempt to illustrate their diversity of form in this paper would be superfluous. A few representative neurones taken from the ganglia of the myenteric plexus in the small intestine are illustrated in the accompanying figure (fig. 1, A, B, C). Fig. 1 Sympathetic neurones. A, in myenteric plexus, ileum of cat; B and GC, in myenteric plexus, ileum of dog; D, E, F, in submucous plexus, ileum of dog. a, axone. Spencer, obj. 1.8, oc. 8. The axone is usually a slender, somewhat varicose fiber which arises either from the base of a protoplasmic process or directly from the cell-body. Not infrequently axones may be traced from their origin into the commissures connecting the ganglia of the myenteric plexus or into those leading from this plexus into the submucous plexus. In some instances axones may be traced INNERVATION OF THE DIGESTIVE TUBE 179 directly into the muscle-layers where they terminate on smooth muscle-fibers. Dendrites may also be traced from their cells of origin into the commissures. Although in many instances it is impossible to distinguish between axone and dendrites, there can be no doubt that dendrites are present in the commissures. Not infrequently several processes arising from the same cell may be traced into a commissure leading from the ganglion in one direc- tion while other processes arising from the same cell may be traced into commissures leading from the ganglion in other directions. In transverse or longitudinal sections axones as well as many of the longer dendrites may be traced directly into the commissures leading from the myenteric plexus into the submucous plexus. In figure 2, A, which is taken from a longitudinal section of the ileum of a kitten prepared by the methylene blue method, is illustrated a commissure connecting a ganglion of the myenteric plexus with a ganglion of the submucous plexus. This commis- sure lies in the connective tissue between the muscle-bundles. Individual nerve-fibers can not be traced from one ganglion into the other in this section. However, nerve-fibers are present in the commissure throughout its entire length. Figure 2, B, is taken from a section through the posterior region of the oesopha- gus of an embryo of the chick of ten days incubation prepared by the pyridine-silver method. Neurones in the myenteric and the submucous plexuses do not appear in these preparations. Individual nerve-fibers may, however, be traced from the myen- teric plexus to the level of the submucous plexus. There can be no doubt, therefore, that individual nerve-fibers extend from one of these plexuses into the other. Submucous plexus The ganglia of the submucous plexus, like those of the myenteric plexus, are variously connected by commissures of non-medullated fibers in which both axones and dendrites may be traced. These commissures, in which the fibers are usually more or less com- pactly aggregated, with the ganglia interposed at their nodal points form a network which in general is confined to the sub- 180 ALBERT KUNTZ mucous layer. As indicated above, the submucous plexus is connected with the myenteric plexus by fibrous commissures. Fibers which have their origin in the ganglia of the submucous plexus may be traced into these commissures. Furthermore, nerve-fibers may be traced from the ganglia of the submucous plexus into proximity with the digestive glands where many of them terminate on gland-cells and into the gastric folds and Fig. 2 A, from longitudinal section of small intestine of kitten, showing ganglia of myenteric and submucous plexuses with connecting commissures; B, from section through posterior region of oesophagus of ten day embryo of chick; C, from section through cardiac region of stomach of cat, showing nerve- fibers extending from submucous plexus into gastric fold. 1, tunica propria; 2, muscularis mucosae; 3, submucosa; 4, muscularis; c, commissure connecting ganglia of myenteric and submucous plexuses; cm, circular muscles; cs, commissure connecting ganglia of submucous plexus; ep, epithelium; f, nerve-fibers; ft, nerve- fibers extending from submucous plexus into gastric fold; /m, longitudinal muscles; M, ganglion of myenteric plexus; Mf, fibers in myenteric plexus; pc, parietal cells; S, gangha of submucous plexus. INNERVATION OF THE DIGESTIVE TUBE 181 plicae and the intestinal villi where many of them terminate on cells of the digestive epithelium. Such fibers are illustrated in the accompanying figure (fig. 2, C, ft) which is taken from a section through the cardiac region of the stomach of the cat prepared by the methylene blue method. The ganglia of the submucous plexus show a wider range in size and form than do the ganglia of the myenteric plexus. The latter, being interposed between the longitudinal and the circular muscle-layers, are more or less regular in form. The ganglia of the submucous plexus, on the’ other hand, are surrounded by loose connective tissue which apparently exercises little influence in determining their form. Some of these ganglia appear as small rounded or elongated cell-groups. Others are more orless irregular in form or even T- or Y-shaped according to the angles between the commissures which radiate from them. ‘These gan- glia may be relatively small, containing relatively few neurones, or relatively large, containing relatively many neurones. In all of them, however, the neurones are more compactly aggregated than are the neurones in the ganglia of the myenteric plexus. It becomes correspondingly more difficult, therefore, to trace out the processes of these cells. In sections of material successfully prepared by the pyridine-silver method taken in the plane of the plexus, however, this may be done quite satisfactorily. In good preparations of this kind little tissue appears in these ganglia except the fibers which pass through them at various angles and the neurones with their processes. In these ganglia also, as Miller (11) has suggested, pericellular capsules are probably not present. The neurones in the ganglia of the submucous plexus present quite as wide a range of variation in size and form as do the neurones in the ganglia of the myenteric plexus. They possess certain distinctive characters, however, by which the experienced observer may without difficulty recognize them as belonging to the submucous plexus. They are also relatively smaller than the neurones in the myenteric plexus. A few of these neurones taken from pyridine-silver preparations of the small intestine of the dog are illustrated in figure 1, D, E, F. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 3 182 ALBERT KUNTZ The great majority of the neurones in the ganglia of the sub- mucous plexus are more or less regular in outline and possess relatively few dendrites which are usually long and slender and give rise to relatively few slender branches. The slender proc- esses of these neurones may frequently be traced into the com- missures connecting the ganglia of the submucous plexus, into the commissures connecting this plexus with the myenteric plexus or into the fiber-tracts leading from the ganglia of the submucous plexus into proximity with the digestive glands and into the gastric folds and plicae or the intestinal villi. F'iber-terminations In sections of the stomach and the small intestine of both the cat and the dog prepared by either the methylene blue or the pyridine-silver method, nerve-fibers may not infrequently be traced from the ganglia of the myenteric plexus quite directly into the longitudinal or the circular muscle-layer. Although individual fibers could not be traced from their origin to their termination on smooth muscle-cells, terminations of nerve-fibers on muscle-cells could frequently be observed. In good methylene blue preparations, slender varicose fibers may be observed in the inter-cellular cement between the muscle-cells. The terminal portions of these fibers are usually exceedingly varicose and fre- quently give rise to very slender lateral branches which terminate in minute terminal enlargements on the same muscle-cell on which the terminal portion itself ends or on another muscle-cell lying parallel with it. Figure 3, A, illustrates the terminal portion of a nerve-fiber with several lateral branches ending on the same muscle cell. Terminations of sympathetic nerve-fibers on smooth muscle-cells have been similarly described by Erik Miiller (92), Retzius (92) and Huber (’97). As indicated in an earlier section of this paper, sympathetic nerve-fibers may be traced from the ganglia of the submucous plexus into proximity with the digestive glands and into the gastric folds and plicae and the intestinal villi. In methylene blue preparations of the stomach of the cat, fibers could not infrequently be observed terminating on parietal cells (fig. 3, D). INNERVATION OF THE DIGESTIVE TUBE 183 In similar preparations of the small intestine sympathetic fibers may be traced into close proximity with the digestive glands and not infrequently such fibers could be observed among the gland- cells. There can be little doubt, therefore, that some of these fibers form physiological contact with cells of the digestive glands. Many of the sympathetic fibers reaching down from the sub- mucous plexus may be traced beyond the level of the digestive glands into the gastric folds and plicae or the intestinal villi where many of them terminate on cells of the digestive epithelium. Fig. 3. Terminations of sympathetic nerve-fibers. A, on smooth muscle- cell; B, on cells of digestive epithelium, ileum of cat; C, on cells of digestive epithelium, stomach of cat; D, on parietal cell, stomach of cat. A and B, Leitz, obj. 1/16, oc. 4; © and D} Leitz, obj. 1/12, oc. 4. These terminations are very delicate, but in good methylene blue preparations they may be observed without difficulty under high magnification. In methylene blue preparations of the stomach and the small intestine of the cat, terminations of this kind could not infrequently be observed on several adjacent epithelial cells. The sympathetic fibers extending into the gastric folds and plicae and the intestinal villi lie in the tunica propria close to the diges- tive epithelium. The terminal portions of these fibers or lateral branches given off by them deviate from the course of the fiber- 184 ALBERT KUNTZ tracts toward the epithelial cells. On approaching the basal part of an epithelial cell the fiber breaks up into two or more branches which continue along the surface of the cell, giving off short branches along their course. These terminal branches may in some instances be traced along the surface of an epithelial cell for nearly half its length. Figure 3, B, illustrates the terminations of sympathetic nerve-fibers on epithelial cells in the ileum of the eat. In this instance the epithelial cells were but slightly stained. The fibers terminating on them could, therefore, be traced to their distal extremities. Figure 3, C, illustrates similar sympa- thetic fiber-terminations on epithelial cells located at the free end of a gastric fold in the cardiac region of the stomach of the cat. In this instance the epithelial cells were stained more intensely. Consequently, the terminal portions of the fibers could be traced but for a short distance on their surfaces. Whether the sympathetic nerve-fibers terminating on cells of the digestive epithelium are axones or dendrites can not be deter- mined by their microscopic appearance. The fact that they ter- minate on epithelial cells, however, seems to warrant the conclu- sion that they are receptive fibers. Furthermore, in view of the preponderance in the ganglia of the submucous plexus of neurones with long slender dendrites, it is highly probable that they are the dendrites of these neurones. Dogiel (96) was led to believe, by observations of his pupil, Sakusseff, on the digestive tube of fishes, that sympathetic nerve- fibers actually terminate on cells of the digestive epithelium. The opinion, based on both anatomical and physiological consid- erations, that sympathetic nerve-fibers terminate on epithelial cells in the digestive tube has been repeatedly expressed by recent investigators. As far as the writer is aware, however, termina- tions of sympathetic nerve-fibers on cells of the digestive epithe- lium in higher vertebrates have not previously been described. In both the myenteric and the submucous plexus, terminations of sympathetic nerve-fibers on sympathetic neurones may occa- sionally be observed. In methylene blue preparations such fiber- terminations may be studied most satisfactorily on neurones which are stained but lightly. Terminations of nerve-fibers on INNERVATION OF THE DIGESTIVE TUBE 185 neurones which are well stained may readily be overlooked because there is little difference in the intensity of the stain of the fiber and the cell on which it terminates. In the myenteric plexus in the small intestine of the kitten prepared by the meth- ylene blue method, terminal nets could be observed on many of the smaller neurones which were stained so lightly that none of their protoplasmic processes could be traced and the cell-bodies appeared only in faint outline. A terminal net of this type is illustrated in figure 4, A. As the fiber approaches the cell-body on which the terminal net is formed it breaks up into two or more ~ branches which twine about the cell, giving off numerous smaller branches along their course. These slender fibers interlace with Fig. 4 Terminations of sympathetic nerve fibers on sympathetic neurones. Spencer, obj. 1.8, oc. 8. each other until a network is formed which more or less completely encloses the cell-body. Terminations of this type have been repeatedly described in various parts of the sympathetic nervous system. Another type of sympathetic fiber-termination on a sympa- thetic neurone which occurs also in the myenteric plexus is illus- trated in figure 4, B. In this instance the fiber terminates, not on the cell-body, but on the proximal part of a large protoplasmic process in a flattened terminal enlargement from which several small protoplasmic processes reach out like slender pseudopodia. Fibers terminating on the proximal portions of large dendrites were observed also in the submucous plexus in pyridine-silver prepara- 186 ALBERT KUNTZ tions of the ileum of the dog (fig. 4, C). In these instances, however, no distinct terminal enlargement is apparent, but the terminal part of the fiber breaks up into short delicate branches which spread out over the surface of the proximal part of the dendrite. Sympathetic fiber-terminations similar to those here described were described by Huber (97) in other parts of the sympathetic nervous system. In the myenteric plexus of the ileum of the dog prepared by the pyridine-silver method, sympathetic fibers were observed which terminate directly on the cell-bodies of large neurones (fig. 4. D). A sympathetic fiber terminating in this manner may without difficulty be distinguished from the processes which arise from the neurone because it is more slender than the latter and stains more intensely. Furthermore, the terminal enlargement ean not be confused with the cone of origin of any of the proto- plasmic processes. Terminations of sympathetic nerve-fibers on sympathetic neurones similar to those here described were described by von Lenhossék (’94) in Golgi preparations of embryos of the chick. Whether the sympathetic fibers whose terminations on sympa- thetic neurones in the myenteric and the submucous plexuses are here described are the axones of neurones within these plexuses or whether they are fibers whose origin is in some center outside the walls of the digestive tube could not be determined. That some of them may represent fibers which arise in more centrally located centers is highly probable. On the other hand, it is highly probable that axones of neurones in either the myenteric or the submucous plexus may terminate on neurones in the same or in the other of these plexuses. DISCUSSION According to the doctrine of Langley which is still more or less prevalent, the neurones composing the sympathetic ganglia are all excitatory in character. These neurones, whether located in the ganglia of the sympathetic trunks, the prevertebral or the peripheral sympathetic plexuses, are interpreted as links in effer- INNERVATION OF THE DIGESTIVE TUBE 187 ent chains. According to this doctrine, the only sensory neurones associated with the sympathetic nervous system are the visceral afferent neurones whose cell-bodies are located in the cerebro- spinal ganglia. According to this scheme, all sympathetic reflexes involving sensory neurones must involve an entire afferent and an entire efferent chain. Local reflexes involving only sympa- thetic elements, if they occur at all, can occur only as axone- reflexes involving only excitatory neurones. This conception of Langley regarding the physiological relation- ships of the sympathetic neurones is based largely on the results of experimental methods which are, doubtless, better adapted to determine the constitution of the visceral afferent and the visceral efferent chains than to determine whether or not local reflexes involving sensory and motor elements occur in the peripheral sympathetic plexuses. The facts presented in this paper strongly suggest that the sym- pathetic system, like the other functional divisions of the nervous system, is essentially a system of reflex arcs, involving both sen- sory and motor neurones, some of which are strictly local in char- acter, while others are less local or even involve centers in the cerebro-spinal nervous system. That local reflexes occur in the peripheral sympathetic plexuses is shown by experimental observations. Recent experimental in- vestigations, notably those of Bayliss and Starling (99), Cannon (06) and Auer (’10), have shown conclusively that the motor activities of the digestive tube may be carried on more or less normally for a considerable period after the nerves connecting the digestive tube with the cerebro-spinal nervous system have been severed. The possibility that axone-reflexes may occur under such circumstances is not precluded. Furthermore, it is well known that the effect of adrenalin on involuntary muscles is the same as the effect of sympathetic stimulation. Nevertheless; in view of the fact that, as shown in this paper, some of the neurones in the submucous plexus send their dendrites into the gastric folds and plicae and the intestinal villi where many of them terminate on cells of the digestive epithelium, it is highly probable that these neurones are stimulated either directly or indirectly by the 188 ALBERT KUNTZ presence of food in the digestive tube and that their impulses are transmitted to motor neurones with which they form physiolog- ical contact. The axones of some of these ‘receptive’ neurones in the submucous plexus may be traced into the commissures connecting the ganglia of the submucous plexus or into those connecting this plexus with the myenteric plexus. Furthermore, dendrites as well as axones extend from the myenteric plexus into or through the submucous plexus. The anatomical relationships of the sympathetic neurones in the myenteric and the submucous plexuses, doubtless, are such as to provide both shorter and longer reflex arcs involving both sensory and motor neurones. We may, therefore, conceive of sympathetic reflexes which are strictly local in character while others pass through several or even many gan- glia and thus transmit impulses from one level of the digestive tube to another separated from it by an appreciable interval. Still other reflexes stimulated in the same manner may involve centers in the prevertebral sympathetic plexuses or in the sympa- thetic trunks. Besides these types of reflexes, doubtless, sympa- thetic reflexes occur which involve centers in the spinal cord and the brain. The fact that the motor activities of the digestive - tube may be carried on more or less normally when the paths for these longer reflexes are severed, however, seems to. indicate that the normal nervous control of the digestive functions is exercised primarily by the local sympathetic mechanism. The schematic diagram in the accompanying figure (fig. 5) is introduced to illustrate some of the probable relationships of the sympathetic neurones in the myenteric and the submucous plex- uses. The motor neurones in the diagram are stippled while those which are supposedly sensory appear solid. That all the types of reflexes described above actually occur in the sympathetic nervous system has not been demonstrated experimentally. As has been pointed out, however, the orienta- tion of the sympathetic neurones in the myenteric and the sub- mucous plexuses and the peripheral distribution of their axones and dendrites is obviously such as would be required by a system of shorter and longer reflex ares. Therefore, the conclusion that all the types of sympathetic reflexes above described are possible INNERVATION OF THE DIGESTIVE TUBE 189 in the nervous control of the digestive functions can hardly be avoided. The nervous mechanism in the walls of the digestive tube is connected with the cerebro-spinal nervous system primarily by the vagi and the splanchnics. In general the vagi act in an excit- J AL] SK 1A AD AIA A PR HIF BARA Bad AA BRE HW Ad Al AVA AIA OIA EE HH AW AA AIA BIA BBE HAHA aA ea BI BIA yon AHA es Be HA BY ee Be alae ewe HH a SHA ail BGHe HH 4 AA AH BI BW AGHA EAE BW BB 4764 BY E'S AY BY BL IT wa WY ww we ap Fig. 5 Schematic diagram illustrating probable relationships of sympathetic neurones in myenteric and submucous Bee Motor neurones, stippled; sen- sory neurones, solid. 1, tunica propria; 2, muscularis mucosae; 3, submucosa; 4, muscularis; M, myenteric plexus; S, submucous plexus; a, axones; d, dendrites. atory and the splanchnies in an inhibitory manner on the diges- tive organs. The recent work of Cannon (’06) and Auer (’10) shows that the vagi possess both excitatory and inhibitory fibers for the digestive tube. The work of these investigators shows, furthermore, that while the splanchnics are not necessary for the 190 ALBERT KUNTZ quite normal control of the digestive functions in the presence of vagus influences, these functions are carried on more nearly nor- mally with both the splanchnics and the vagi severed than with the splanchnics alone intact. These facts seem to indicate the fundamental importance of the vagi in the extrinsic nervous con- trol of the digestive organs. ‘This is suggested, also, by the facts of evolution. The writer has presented evidence in an earlier paper (711) in support of the theory that the peripheral sympa- thetic plexuses which are genetically related to the vagi represent those parts of the sympathetic nervous system which arose earli- est in the process of evolution and that the vagi constitute the primary connection between these plexuses and the cerebro-spinal nervous system. We should expect, therefore, that the vagi constitute also the primary functional connection between the nervous mechanism in the walls of the internal organs and the cerebro-spinal nervous system. The normal nervous control of the digestive organs is, doubtless, exercised more or less directly by the local sympathetic mechan- ism, the general control which is normally exercised by extrinsic nerves being largely tonic in character. Inasmuch as the vagi form the primary connection between the cerebro-spinal nervous system and the sympathetic plexuses in the walls of the digestive organs, it is highly probable that the major part of such tonic control is exercised by the vagi. SUMMARY 1. The ganglia of the myenteric plexus are interposed between the longitudinal and the circular muscle-layers of the digestive tube. The ganglia of the submucous plexus are imbedded in the submucous layer. The ganglia of each of these plexuses are vari- ously connected by commissures of non-medullated fibers among which may be traced both axones and dendrites. 2. The myentric and the submucous plexuses are connected with each other by fibrous commissures. Nerve-fibers also extend from the submucous plexus into proximity with the digestive glands where many of them terminate on gland-cells and into the INNERVATION OF THE DIGESTIVE TUBE 191 gastric folds and plicae and the intestinal villi where many of them terminate on cells of the digestive epithelium. The fibers which terminate on cells of the digestive epithelium are, doubtless, the dendrites of ‘receptive’ or sensory neurones. 3. The orientation of the neurones in the ganglia of the myen- teric and the submucous plexuses and the distribution of their axones and dendrites strongly suggest that the sympathetic sys- tem, like the other functional divisions of the nervous system, is essentially a system of reflex ares involving both sensory and motor neurones, some of which are strictly local while others are less local or even involve centers in the cerebro-spinal nervous system. 4. The normal nervous control of the digestive functions is probably exercised primarily by the local sympathetic mechan- ism, the general control which is exercised by extrinsic nerves being largely tonic in character. The major portion of such tonic control is probably exercised by the vag. BIBLIOGRAPHY Aver, Joun 1910 The effect of severing the vagi or the splanchnies or both upon gastric motility in rabbits. Amer. Jour. Physiol., vol. 25, pp. 334-344. Bayuiss, W. M. anp Staruina, E. H. 1899 The movements and innervation of the small intestine. Jour. Physiol., vol. 24, pp. 99-148. Casa, 8. R. y. 1905 Tipos celulares de los ganglios sensitivos del hombre y mamiferos. Trabajos de Lab. de Invest. Biolog. de la Univers. de eMadrid, T. 4. Cannon, W. B. 1906 The motor activities of the stomach and small intestine after splanchnic and vagus section. Amer. Jour. Physiol., vol. 17, pp. 429-442. 1912 Peristalsis, segmentation, and the myenteric reflex. Amer. Jour. Physiol., vol. 30, pp. 114-128. Doatgnr, A. S. 1895 Zur Frage iiber die Ganglien der Darmgeflechte dei den Siugetieren. Anat. Anz., vol. 10. pp. 517-528. 1896 Zwei Arten sympathischer Nervenzellen. Anat. Anz., vol. 11, pp. 679-687. Huser, G.C. 1897 Lectures on the sympathetic nervous system. Jour. Comp. Neur., vol. 7, pp. 73-145. 192 ALBERT KUNTZ Kuntz, A. 1910 The development of the sympathetic nervous system in mam- mals. Jour. Comp. Neur., vol. 20, pp. 211-258. 1911 The evolution of the sympathetic nervous system in vertebrates. Jour. Comp. Neur., vol. 21, pp. 215-236. Lanatey, J. N., aNp AnpERSON, H. K. 1894 On reflex action from sympathetic ganglia. Jour. Physiol., vol. 16, pp. 410-440. Lanetey, J. N. 1903 The autonomic nervous system. Brain, vol. 26, pp. 1-26. Mititer, Erik 1892 Zur Kenntnis der Ausbreitung und Endigungsweise der Magen- Darm- und Pancreas-Nerven. Archiv f. mikr. Anat., vol. 40. Miter, L. R. 1911 Die Darminnervation. Deutsch. Archiv f. klin. Med., vol. 105, pp. 1-48. 1912 Stand und Lehre vom Sympathicus. Deutsche Zeitschrift f. Nervenheilkunde, vol. 45, pp. 1-18. Retzius, G. 1892 Zur Kenntnis der motorischen Nervenendigungen. Biolo- gische Untersuchungen, N. F., Bd. 3. THE CENTRAL NERVOUS SYSTEM IN A CASE OF CYCLOPIA IN HOMO D. DAVIDSON BLACK From the Anatomical Laboratories of the University of Chicago and Western Reserve University FIFTY-ONE FIGURES CONTENTS LNG ROCICET OMS otra n se hence AE al ae oe oh ee ee ee Sr 193 GeneraledescriptionvorabherCaser.nrcce.3 56 ose tis ene eee reer te eae 195 Extermalttieatunest asin eae eee ecco os spec Te ed ne eae 195 Cranial cavity, dura in Sits) io. 22.03. «och ne a ee re seats 198 Cranial cavdityscdurasemovedaa. ...5ci- | enn ee eae ee eer rer arr: 198 Mascularranomaliege ae t.tet a te he voc eo ae ee crete Tees 201 Contentsrotvhevorbitbaleiossan sense aoe ee eee eee 204 Macroscopic deseription-ol the the brain, 2470-4 eee ee ee ee 210 MIS GOH ATA GIDE 23cm ss ee Pea eae rw aaa ta gear Oc ec 215 Microscopic description. oi the brain... k..5 see ee oe ee ee 215 Braim-stem and :cerebellum=,- 49-445 ene ee eee ree eee 215 aialannie mass 36 ee. oe tise ce tee nae i eee 220 @erebralicvesicles gia isi. aes neat ee Se ee See ee eae 222 C@ortexiCene brie. Ase occa es ee ee ee 222 Cerebral himibitie: 3 oe 2. sheen oe oe ae rey ee a 231 Wentricular anomalies,.-.. 2.0% 2. ele ee ee ae ee eee 233 IMechamicalkconstdleraitlOls ea. cette eae ee eee 235 Reviewsol case: reported: by:O:Nacgeliz .. 2 j3oaesae es See as a eee 239 (OM CMIST OME eh a See ees oo oc bass teoke Wits Oe ees Ae eee 241 TEV ERATUMEKCILCGe Mts lee dies Secs Sette nS ee ne nn ane a a 243 INTRODUCTION Although cyclopia in man is a somewhat rare condition, vet among the cases reported few deal with other than the external features of the malformation. Up to the present, the only in- vestigation into the finer structure of the central nervous system in a case of cyclopia in homo has been made by O. Naegeli in 193 194 D. DAVIDSON BLACK v. Monakow’s laboratory (17). In the case reported by him, how- ever, the condition of cyclopia was much complicated by the presence of a very extensive malformation in connection with the brain stem and cord, which without doubt was quite independent of the cyclopian condition. With the exception of a moderate degree of hydrocephalus, the present case presents no malformations other than those which may safely be considered as due to the cyclopic condition. Such being the case, this material is well adapted to determine among other things, whether the cyclopian brain can in any way be regarded as an arrest of development at an early phylogenetic stage, as Naegeli suggests. The research was begun in the hope of clearing up this point, and as will be shown, all the evidence in this case is against the assumption of such a reversion. The central nervous system is of paramount interest in this case, and the necessarily incomplete general description is only warranted in so far as it records certain data which may be of use in any future complete investigations. In the course of the work, the condition of development in the pallium has proved to be of the greatest interest, as it tends to throw additional light on the subject of the evolution of the normal cortex. This phase of the research has not been as fully dealt with here as is desired, and will be the subject of a future communication. The cyclopian foetus, upon which the following observations have been made, was obtained in Chicago through the courtesy of Dr. Warren H. Hunter, Cook County Coroner’s Physician, and Prof. E. R. Le Count of Rush Medical College, Chicago. The clinical details of the case have already been reported by Dr. Harry Jackson (11). I am indebted to Prof. C. J. Herrick for the opportunity of taking up this work and for much helpful criticism throughout the investigation. The work was begun in the Anatomical De- partment of the University of Chicago in the spring quarter of 1910. The major portion of the technique in connection with the microscopic study of the central nervous system and also the examination of this material has been done in the Anatomical Department of Western Reserve University. A CASE OF CYCLOPIA 195 The specimen was preserved immediately after death, under Dr. Le Count’s direction, by an injection through the carotid artery of 10 per cent formalin (4 per cent formaldehyde) after which the whole body was immersed in a solution of the same. On account of this careful fixation, the histological preservation was excellent. A research had been begun upon this specimen before it came into my possession, and on this account I have been unable to determine the exact attachments of the cerebral roof over the tha'amic mass. I am indebted to Miss Katherine Hill, medical artist at the Hull Laboratory of Anatomy, for the four drawings of the entire brain made shortly after its removal from the skull cavity and before the relations referred to above had been disturbed. GENERAL DESCRIPTION OF THE CASE External features The body is that of a well developed male infant weighing seven and one-half pounds. The head is enlarged and shows a condition of moderate hydrocephalus so that the cranium cere- brale is considerably more developed than the cranium viscerale. The skull is dolichocephalic, though not markedly so, as is usu- ally the case in hydrocephalus (18) It measures 95 mm. in greatest transverse diameter, and 130 mm. in greatest longitudinal diameter, the cephalic index being 73. The sagittal suture is widely open, the parietal bones being separated from one another by a considerable space (2 to 3 cm.) throughout. The metopic suture is also widely open, its lateral margins being separated in the upper part by a distance of 10 mm. At the base of the regio frontalis, which is high and prominent, in the mid-line is a finger-like process of about 2 cm. in length. The walls are firm and it presents at its distal extremity a single orifice which leads into a blind passage extending inward as far as the attached end of the organ. This appendage, which proba- bly represents an abortive naso-frontal process, overhangs the single median eye (fig. 1). 196 D. DAVIDSON BLACK The eyeball protrudes from the orbital fossa and is somewhat larger in its transverse than in its vertical diameter. The cornea is ‘dumb-bell’ in shape—the long axis of the dumb-bell, which is slightly asymmetrical, being transverse. The condition of the pupil could not be accurately determined on account of the opacity of the cornea. The exposed superior surface of the bul- bus is of dark brownish black color, due to the thinness of the Fig. 1 Photograph of the specimen sclera overlying the uveal pigment. The superior palpebrae are represented by two thickened ridges sparsely beset with cilia and separated from one another by a notch immediately below the base of the overhanging proboscis. In either side a notch representing the outer canthus limits these ridges laterally. The inferior palpebrae are represented on either side by short blunt tubercles bearing a few cilia and situated immediately below A CASE OF CYCLOPIA 197 and to the inner side of the external canthi. The remainder of the inferior margin of the orbit for the short distance medial to the tubercles representing the lower lids is formed on either side by the palatine processes of the maxillary elements. Owing to a condition of hare lip and partial cleft palate, a boundary in the mid-line ventrally is lacking between the mouth cavity and the conjunctival sac above. It thus happens that the mucous membrane lining the roof of the mouth becomes continuous with that lining the conjunc- tival sac, around the margins of the cleft palate (fig. 2). The Fig. 2 Schematic coronal section through face region, showing relations of oral cavity and conjunctival sac. B.O., bulbus oculi; C.O., oral cavity; O.C., ocular conjunctiva, continuous with the parietal layer (P.C.), at the fornix con- junctivae (F’), which is indicated in dotted lines as being on a deeper plane; O.M., oral mucosa which becomes continuous with the parietal layer of the conjunctiva (P.C.), through the cleft (X) between the maxillary elements (Maz). Fig. 8 Diagram of anterior surface of bulbus. F., fornix conjuntivae indicated in dotted lines; P., pupil; S.C., sclero-corneal junction; x., small pigmented area’ at margin of cornea. condition of cleft palate ceases about 3 cm. from the red margin of the upper lip. In the median line at the point of closure of the palatine cleft the parietal layer of the conjunctiva becomes reflected on to the eyeball. On both sides lateral to this point of attachment, the conjunctival sac extends for some distance backward before the inferior fornix is reached. A similar con- dition obtains above, the two lateral pouches being separated from one another in the mid line dorsally at the notch situated at the base of the proboscis. These relations will be made clear by reference to the diagram (fig. 3), where the line of reflection of the bulbar conjunctiva is indicated in dotted lines. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 3 198 D. DAVIDSON BLACK There is thus every indication of bilateral symmetry in the cornea, the conjunctival sac, the palpebrae, and, as will be subse- quently seen, in the contents of the orbital fossa. The uvula and soft palate are present but the posterior nares are represented only by a slight recess above these structures. Cranial cavity, dura wm situ As it was desired to preserve the external form of the specimen for museum purposes, many of the following descriptions are necessarily incomplete. The cranial cavity presents a somewhat elongated appearance, as 1s indicated in figure 4, and is divided into anterior, middle and posterior fossae. Posterior fossa. ‘The posterior fossa and its boundaries are approximately normal in appearance. The dural foramina for the exit of the nerves normally leaving the cranial cavity in this region may be made out with the exception of the foramen for the trochlear nerve. The vertebral arteries and the acces- sory nerves may be made out in the foramen magnum. The internal occipital protuberance is well marked. The attach- ment and extent of the tentorium are normal. Middle fossa. The middle fossa is subdivided into two lateral portions by a much elongated, sharp, median ridge. Slightly more than 1 em. from the anterior end of this ridge, a single large artery opens on its dorsal surface. At its anterior extremity the median ridge becomes continuous with the median process of the posterior boundary of the anterior fossa. Anterior fossa. The anterior fossa, which is quite extensive, is bounded on either side posteriorly by curved ridges whose concavity is directed backward. Cranial cavity, dura removed Anterior fossa. No ethmoid element can be made out. A wide metopic suture is present (fig. 5). In the mid-line, slightly in front of the junction of the posterior boundary of the anterior fossa with the median ridge of the middle fossa, there is seen a slight prolongation of the dura into a small pit in the bone. A CASE OF CYCLOPIA 199 This subsequently was found to mark the point of attachment of the fibrous remnants of the optic nerves. Middle fossa. The two carotid arteries enter the skull on either side of the median ridge postero-laterally. The left carotid is a mere fibrous thread, while the right carotid is somewhat larger than normal. The left carotid canal is proportionately reduced. These vessels approach one another, become united on the crest of the median ridge and pass forward as a single vessel. At the Ant. Fossa 5 Fig. 4 Diagram of floor of skull cavity, durain situ. A.c., carotid artery A.v., vertebral artery; F.M., foramen magnum; z., a slight depression in dura which marks the site of attachment below of the fibrous remnant of the optic nerve. The cranial nerves in their dural foramina are numbered in Roman numerals. Fig. 5 Diagram of floor of skull cavity, dura removed. /F'.M., foramen mag- num; H., pit in basi-sphenoid into which were prolonged fine processes from dura; M.C., median carotid artery; M.S., metopic suture; O.p.A., ophthalmic artery; 0.T.S., occipito-temporal suture; P.O.S., parieto-occipital suture; P.7.S., pari- eto-temporal suture; R.i.c., right internal carotid artery; S., suture between great wing of sphenoid and frontal element; X., marks the cut surface of bone removed to expose the course of right internal carotid artery; cranial nerves in- dicated in Roman numerals. The remnant of the left internal carotid artery is indicated beneath the cut ends of the third and sixth nerves. 200 D. DAVIDSON BLACK anterior extremity of the ridge a small vessel is given off and - passes downwards through a median foramen to the orbit. This vessel is an azygos ophthamic artery. Immediately after the ophthalmic branch is given off, the main vessel turns backward upon itself and courses a short distance in this direction before passing upward through the dura as the single median artery already noted. There is no appearance whatever of anterior or posterior clinoid processes. On either side of the median ridge the third and sixth nerves pass forward beneath the dura to the median foramen mentioned above to gain the orbit. Slightly more laterad the ophthalmic division of the fifth nerve passes forward to the same destina- tion. Accompanying the latter is a small nerve which may (?) represent the trochlear though subsequent dissection failed to identify it definitely. The spheno-temporal suture may be clearly made out, extend- ing outward from the region of the carotid canal. Anteriorly another suture may be made out passing parallel to the posterior boundary of the anterior fossa, and marking the line of union between the frontal element and the great wing of the sphenoid. Below the posterior end of the median crest, between the enter- ing carotid arteries and behind the point of their junction, there is found a small pit in the basi-sphenoid into which are prolonged a few fine processes from the dura. This depression was not apparent before removal of the dura. Further examination showed that the fornix pharyngis was situated immediately be- low this pocket. In fact it marks the site where normally the pituitary body should be lodged. Although no pituitary tissue could be identified, I am inclined to view this pit as a remnant of Rathke’s pouch. Posterior fossa. In the posterior fossa the occipito-temporal, the parieto-occipital and the parieto-temporal sutures can readily be made out. The foramina for the exit of the nerves in this region presented no peculiarities. A CASE OF CYCLOPIA 201 Vascular anomalies As has been already noted, the left internal carotid artery within the skull was reduced to a mere thread-like vessel. Exam- ination also showed that the left carotid canal was proportionately reduced. The right internal carotid is somewhat larger than normal, as might be expected. In examining the origin of these vessels in the neck region it was found that the relations on the right side were normal. On the left side the internal carotid was so much reduced as to be distinguished only with difficulty. It arises from the posterior aspect of the common carotid at about the level of origin of the lingual artery. The left facial artery is somewhat larger than the right (diagram, fig. 6). Tr. Fig. 6 Diagram of arrangement of arteries in neck region. A.c.e., external carotid artery; A.c.7., internal carotid artery; A.f., facial artery; A.l., lingual artery; A.o., occipital artery; A.t.s., superior thyroid artery; Md., lower jaw; T’r., trachea. The course of the internal carotid arteries within the skull to the point where a single median vessel pierces the dura, has already been described. Encephalic arteries. The vessels of this region are represented in figure 7 as pinned out and seen from above. Postreiorly the two vertebral arteries unite in a normal fashion to form the main basilar trunk. The origin of the anterior spinal artery is normal. The posterior inferior cerebellar arteries are small and arise from the basilar. A similar anomaly has been reported by 202 D. DAVIDSON BLACK Blackburn (1) in a few of his cases. The basilar artery is long and gives off numerous irregular branches. Six pairs of these branches arise in the pontine region. The superior and anterior inferior cerebellar arteries arise from a common branch of the basilar. Of the two, the superior cerebellar is much the smaller. Fig. 7 Diagram of encephalic arteries pinned out and seen from above. A.b., basilar artery; A.J.C., anterior inferior cerebellar artery; A.s., spinal artery; A.v., vertebral artery; L.P.C., left posterior cerebral artery; MW.C.A., median carotid artery; P.C., posterior communicating artery; P.J.C., posterior inferior cere- bellar artery; Swp.C., superior cerebellar artery. X >. Blackburn (q.v.) has noted that when the anterior inferior cerebellar artery is ill developed, the superior cerebellar artery occasionally sends branches to reinforce it. He also noted that when the posterior inferior cerebellar arteries are absent or small, and arising from the basilar, the anterior inferior cerebellar arter- ies send down compensating branches. In this case, both supe- rior and posterior inferior cerebellar arteries are small and the anterior inferior cerebellar arteries are proportionately enlarged and send branches in both directions. A CASE OF CYCLOPIA 203 Anteriorly the basilar artery becomes continuous with a large left posterior cerebral vessel. No right posterior cerebral artery is present. A small median branch passes forward at the point of origin of the left posterior cerebral artery. This vessel is to be considered as a single posterior communicating artery. It is represented in figure 50 as being much larger than is actually the case. From the median carotid trunk the cerebral vessels radiate out in such a manner that they become distributed to the right side and anterior portion of the left side of the cerebral vesicle. The posterior portion of the cerebral vesicle on the left side is supplied wholly by the posterior cerebral branch of the basilar artery. From the vertebral arteries posteriorly to the point of origin of the common trunk of the superior and anterior inferior cere- bellar arteries, the encephalic vessels have shown but little vari- ation from the normal condition and may be identified with a reasonable degree of certainty. Beyond this point there has been a very marked disturbance in the origin and relations of the various vessels supplying the cerebral vesicle. Mall (14) has shown that under normal circumstances the arrangement of the encephalic vessels arising as branches from the future circle of Willis varies remarkably at different stages in the growth of the embryo. Anomalies of the encephalic vessels are very commonly met with among the insane (1), more so than among normal individ- uals. The above fact implies that the growth and arrangement of these vessels is influenced in no small degree by the growth of the cerebral tissue. Very slight anatomical variations in the latter increase the tendency toward vascular anomalies. When, as in the case in point, there has been a profound dis- turbance in the development of the primary forebrain vesicle a correspondingly wide deviation from the normal arterial arrange- ment may be looked for. The anomalies will be partly due to the mechanical difficulties encountered in growth, but mostly to the absence of certain parts of the cerebral tissue itself. 204 D. DAVIDSON BLACK Thus the conditions of these vessels may be looked upon as being to some extent an indication of the degree of perfection of cerebral growth. The two disturbances cannot be considered as being mutually dependent upon one another, for the cerebral condition in cases such as this is certainly the prime factor. There is a large blood supply from the median carotid to the right half of the cerebral vesicle, while there is no component to this side from the basilar artery. On the other hand, the left side of the cerebral vesicle receives a large component from the basilar artery which effectually compensates for the small supply derived from the branches of the median carotid trunk on this side. So, although the vessels are asymmetrically arranged, it is to be noted that the blood supply is approximately equal on ‘both sides. The sharp bend which is seen anteriorly in the median carotid before it pierces the dura is similar to the bending of the normal bilaterally symmetrical vessels before their division into anterior and middle cerebral arteries. It is to be noted in this connection that the single ophthalmic artery arises at this ‘genu’ as is also the case with the normal ophthalmic arteries. Contents of the orbital fossa The following incomplete dissections were made by removing the roof of the orbital fossa. Only those structures which could be dissected without disturbing the external relations of the bul- bus are described. The diagrams represent the structures in this region as seen from above. All the structures examined were essentially symmetrical. First stratum (fig. 8). On removing the bony roof of the orbital fossa and dissecting away the peribulbar fat and connec- tive tissue, the muscles of this region were found to radiate out- wards to their insertions from a central fibrous mass in which are imbedded the remnants of the unpaired optic nerve. In the region of the central tendon the third nerve divides into a number of small branches and freely communicates with its fellow of the opposite side. A CASE OF CYCLOPIA 9205 The sixth nerve comes to lie ventral to the oculomotor and in the region of the central tendon turns laterad and passes to the inferior surface of a muscular band which, from its nerve supply and relations to the bulbus, represents the rectus lateralis muscle. Fig. 8 Diagram of structures exposed on removing the bony roof of orbit. The metopic suture (M/.S.) and an outline of the skull fossa are indicated, though not in proportion to the dissection of the orbit. B.O., bulbus oculi; Md.V., mandibular division of the fifth nerve; M.M., Miiller’s muscle overlying the max- illary division of the fifth nerve (Mz.V.); Oph.V., oplithalmic division of the fifth nerve; P.F., squama frontalis; R.S., rectus superior muscle; 7’., central ten- dinous mass; z., represents cut surface of bony ridge separating anterior and middle skull fossae; /77, oculomotor nerve: V/., abducent nerve. Lateral to the nerves mentioned, the ophthalmic division of the fifth nerve passes into the orbital fossa. Upon its dorso-medial surface there lies a small nerve which has already been alluded to as the possible representative of the trochlear. The relations of this nerve could not be accurately followed after reaching the region of the central tendon. Here it becomes lost in the median mass of fibrous tissue. 206 D. DAVIDSON BLACK The ophthalmic division of the fifth nerve is the most dorsally placed nerve trunk in the orbital fossa and at the level of the central tendon it divides into two branches of about equal size. The most mesial branch divides into two parts upon the dorsal surface of a flat muscular band whose origin is from the common central tendon and whose insertion is into the deep fascia of the skin lateral to the notch representing the inner canthus. The branches of the nerve become related to the mesial and lateral borders of this muscle. The more lateral branch courses laterad almost at right angles to the parent trunk upon the anterior border of a second flat mus- cular band arising from the central tendon and being inserted into the fascia in the region of the external canthus. All the branches of the ophthalmic division of the fifth nerve enter the subcutaneous tissue at the bony margin of the orbit. Although considerable disturbance occurred in this case in the areas usually supplied by the supratrochlear, frontal, and supraorbital nerves, it is possible that the branches of the fifth nerve here described may correspond to these. The flattened bands of muscular tissue described in connec- tion with the branches of the fifth nerve are present in essentially similar relations on both sides and probably represent an anom- alous arrangement of the levator palpebrae superioris muscle. Both foramen ovale and foramen rotundum, transmitting re- spectively the mandibular and maxillary divisions of the fifth nerve, are illustrated in figure 8, and the course of the maxillary division is indicated in dotted lines till it reaches the space opened for the dissection of the eye. The posterior boundary of the ‘orbital fossa is here seen to be formed by two rounded bony emi- nences having their convex anterior borders inclined to one an- other in such a fashion as to form a V-shaped notch in the mid- line. Examination shows that these apparently represent the bony roofs of the tooth erypts. It is to be noted that the pos- terior boundary of the orbital fossa is a considerable distance behind the region of the central tendon. The maxillary division of the fifth nerve passes over the roof of the crypt and is lost sight of at about the middle of the an- A CASE OF CYCLOPIA 207 terior convex margin where it dips down beneath the bulbus oculi. An anomalous band of muscular tissue passes obliquely across the upper aspect of the maxillary element and over the 2nd division of the fifth nerve. From its relation to the nerve this muscle may (?) represent the rudimentary bundle described as Muller’s muscle in the normal orbital fossa. Second stratum (fig. 9). The third and sixth nerves, and the ophthalmic division of the fifth nerve on each side, together with Fig.9 Diagram of structures exposed in dissection of orbital fossa from above; second stratum. C., small opening in fornix conjunctivae; R.L., rectus lateralis muscle; S.P.M., superior postero-median muscular band; other lettering as in figure 8. the median carotid artery were cut posteriorly and reflected forward to expose the muscular mass situated beneath (they are not represented in the figure). From the median notch formed by the junction of the two maxillary elements there arises a thin muscular band which anteriorly becomes divided into two symmetrical halves as indicated in the diagram. It becomes inserted into the central tendinous mass. On reflecting this muscle forwards, another similar, though slightly larger, muscular band is seen lying im- mediately beneath it. (For the sake of convenience in de- scription these muscles will be termed the superior and inferior 208 D. DAVIDSON BLACK postero-median muscular bands respectively). Its origin and insertion are similar to the first mentioned muscle and are shown in the next dissection. Lateral to these muscular bands and situated on a lower plane, there is seen a broad sheet of muscular tissue whose fibers are more or less parallel to the anterior convex border of the max- illary element. The relations of this mass are more clearly brought out in the next dissection. Fig. 10 Diagram of structures exposed in dissection of orbital fossa from above; third stratum. J.P.M., inferior postero-median muscular band; Mz., maxillary element; Ps., point of attachment of proboscis; Pt.M., muscular mass probably belonging to the pterygoid group; R./., rectus inferior muscle; S, deep fossa be- hind maxillary element. Note: the arrangement of the muscle7.P.M. and the one overlying it is such as to suggest the use of the term ‘retractor bulbi.’ Other let- tering as in figure 8. Third stratum (fig. 10). The entire muscular mass represent- ing the levator palpebrae superioris and the superior portion of the postero-median muscular band were removed. The vessels and nerves’ were also removed with these. It was noted that a small branch of the ophthalmic artery entered the bulbus oculi in the mid-line at the lower margin of the fibrous optic nerve. With the exception of the rectus lateralis the muscular mass in relation to the eye received its innervation in a very irregular fashion from branches of the third nerve. The sixth nerve, as A CASE OF CYCLOPIA 209 has already been noted, pee directly to the inferior surface of the latter muscle. On the left side, the opening in the bony roof of the orbit was ‘continued posteriorly and the floor of the middle fossa of the skull was removed to the level of the foramen ovale. It will be here noted that the whole extent of the upper surface of the left maxillary element is exposed, together with the maxillary division of the fifth nerve and its overlying muscular band. Pos- teriorly is seen a well marked deep fossa. The mandibular di- vision of the fifth nerve divides into a number of branches which pass into a thick muscular mass which, as far as it can be judged from its location, probably represents some part of the pterygoid group. Posteriorly in the orbital fossa is seen the inferior portion of the postero-median muscular band. Also at this level from the region of the central fibrous mass two muscular bands pass lat- erad to be inserted into the fibrous tissue on the anterior surface of the bony wall of the maxillary element. At a lower level in this region is seen a layer of muscle whose fibers have already been noted as coursing laterad parallel to the anterior convex bor- der of the crypt. It is seen to have a somewhat wide origin in the mid-line below the central tendinous mass. As the fibers pass outwards they converge and are inserted into the deep fascia at the lateral margin of the orbital fossa. Two muscles, which from their relations represent the inferior recti, pass almost directly downwards from the central tendon to their insertions on the bulbus. The insertions of the rectus superior and lateralis muscles are also indicated. In the mid-line in front, the roof of the orbit has been dissected away to show the point of attachment of the proboscis and its relation to the eye. The arrangement of the orbital contents throughout is very similar to that obtaining in the cyclopian eyes dissected by Wilder (28). As in his cases, the mesial recti muscles are absent owing to the complete suppression of the area in which they normally develop. The superior oblique muscle was not identified in this case. 210 D. DAVIDSON BLACK MACROSCOPIC DESCRIPTION OF THE BRAIN For the sake of convenience in description the following terms have been made use of. Those structures which together form that portion of the brain anterior to the pineal region are col-— lectively spoken of as the primary forebrain vesicle. The pri- mary forebrain vesicle is further subdivided into an anterior cere- bral vesicle or cerebrum, and a posterior thalamus or thalamic mass. Primary forebrain vesicle From above: As seen in figure 48, this region appears as a large unpaired vesicle having a smooth arched roof which extends caudad to a point immediately in front of the corpus pineale. At no point does it arch over the posterior brain segments. From the side: In the lateral line is here seen a very distinct sulcus passing in a circumferential manner around the cerebral vesicle. Its origin is hidden posteriorly in a deep fissure which exists between the basal portion of the primary forebrain vesicle and the brain stem. The relation of this sulcus may be seen by comparing figures 48, 49 and 50. It sharply marks off the smooth bulging roof from the thickened and somewhat furrowed base. From below: The general configuration of the inferior surface of the primary forebrain vesicle is well illustrated in figure 50. It is seen to be divided by a quite marked Y-shaped furrow into two paired posterior lobes and a single azygos anterior lobe. This furrow cannot be compared with any sulci appearing on the surface of the normal cerebral hemisphere. It is apparently only the result of a mutual adaptation between the cerebral vesicle and the floor of the skull cavity. Smaller secondary fur- rows are seen on each of the lobes but the direction in each case is always at an angle to the main Y-shaped furrow. Apparently these very shallow sulci are due mainly to the presence of blood vessels. In the azygos anterior lobe a small fossa is seen directly in front of the diverging limbs of the Y-shaped principal furrow. There is no appearance whatever of olfactory lobes, optic nerves or tract, infundibulum, or in fact of any structure nor- mally appearing in a basal view of this portion of the brain. A CASE OF CYCLOPIA 211 As has been noted, the basal portion of the cerebrum, which posteriorly is more or less bilaterally symmetrical, is separated from the cerebellum by a very deep fossa. From above on removal of the roof: On laying back the smooth, thin, arched roof, a cavity is brought to view which repre- sents the dilated ventricular cavity of the primary forebrain. The floor of this cavity, which is quite vascular, is seen in figure 51 to be marked by a Y-shaped ridge which corresponds to the external furrow before mentioned. Overhanging somewhat the basal limb of this Y-shaped ridge posteriorly there is seen a smooth rounded protuberance of more or less pyriform outline. Sub- sequent examination has shown this to represent the only per- sisting portions of the thalamus. At its base this thalamic mass becomes flattened from within outward, and is continuous with the thickened basal portion of the cerebrum as seen in the dia- gram of the brain in sagittal section (fig. 11). Cor.pin, C.hb. Vent.” ee C.p. 4 / *s Cor.cer. Fig.13 Fig. 12 Fig. 11 Diagrammatic medial sagittal section through the entire brain. The levels at which the following coronal sections (figs. 12 and 13) were taken are indicated. Ag.c., iter; C.hb., habenular commissure; Cor.cer., cortex cerebri; Cor.pin., pineal body; C.p., posterior commissure; N.IV., trochlear nerve; Nw.o.1,. inferior olive; Nu.r., red nucleus; P., pons; Rad.th., ventral thalamic radiations; T.c., taenia cerebri, or point of attachment of thin cerebral roof (Epy) to inner pillar of cerebral margin; Tec.Mes., midbrain roof; Th.D., dorsal thalamic mass; T.thtI., attachment of cerebral roof to thalamus; 7.th.J/I., attachment of thin roof of third ventricle to thalamus; Vent.I., cerebral ventrizle; Vent.JIII., third ventricle; Vent.JV., fourth ventricle; X., ependymal diverticulum fromiter. 3, pal Be D. DAVIDSON BLACK The roof, which is made up of ependyma, pia and fibrous tis- sue fused together, is everywhere quite thin. Its relations to the thickened base are best brought out in the diagrams (figs. 11, 12 and 13). These represent respectively a sagittal and two transverse sections of the brain. Vent. x. 12 Sed oe hee as aon Vent.I. X. Rdg. Bas.F. 13 Fig. 12 Coronal section through fore-part of cerebral vesicle. Vent.J., cav- ity of cerebral vesicle; X., marks point of attachment of thin roof to inner pillar of thickened recurved cerebral margin. X 3. Fig. 13 Coronal section through mid-part of cerebral vesicle. Rdg., ridge in ventricular cavity corresponding to external furrow (Bas.F.). The relations of this ridge and furrow are further illustrated in figures 50 and 51. Other letters as in figure 12. xX 3. It will be seen in these diagrams that the thickened basal por- tion of the cerebral vesicle presents an arched margin, and the roof is not attached to the apex of this arch but to the base of the inner pillar. This recurved margin subsequently has been identified as a modified hippocampal formation. Thus the point of attachment of the thin roof will represent the fimbria. A CASE OF CYCLOPIA Dp ba} The relation of this roof to the thalamic mass as subsequently determined may be seen by comparing a sagittal section (fig: 11) with a surface view from above and in front (fig. 14). It will be seen that there is a discontinuity of the ventricular system in this region so that the large cavity of the primary fore brain vesicle is not in connection with the iter. The line of attachment of the thin roof to the surface of the thalamic mass Fig. 14 Diagram of posterior portion of forebrain seen from above and in front. The thin roof has been removed and the point of its attachment to the cerebrum and thalamic mass is indicated in dotted lines. Cor.cer., cortex cere- bri; Cor.pin., pineal body; 7'.c., taenia cerebri; Th.ex., extraventricular portion of the thalamus; Jh.in., intraventricular portion of the thalamus; Vent./., cav- ity of cerebral vesicle. corresponds to the taenia thalami. The taeniae become contin- uous with one another some distance in front of the pineal body. At the point of junction between the thalamic mass and the cerebrum, the roof is attached to the margin of the latter struc- ture and the taenia thalami becomes continuous with the fimbria. In the diagrams (figs. 11, 12 and 13), the basal plate of the cerebrum is represented as split into two laminae. This split- ting apparently occurred during fixation and the line of cleavage passes in most cases through the portion repesenting the medul- lary center. THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 3 214 D. DAVIDSON BLACK It is to be noted that the posterior poles of the so-called basal lobes of the cerebral vesicle project backward for a short distance on either side of the thalamic mass and peduncular region. In connection with the foregoing description it must be borne in mind that beside the malformations which are the direct out- come of the cyclopic condition, others are present which are consequent on the internal hydrocephalus. It is to this factor that we must look for the cause of the marked dilatation of the forebrain vesicle with the bulging of its thin walled roof, and probably also for the lack of continuity in the ventricular sys- tem. These points will be more fully dealt with later. Brain stem and cerebellum The midbrain, as may be seen in figures 48 and 49, is plainly visible from above and on account of its thick pial covering shows no external indications of division into corpora quadrigemina. The marked flexure which obtains between the primary fore- brain vesicle and the brain stem causes the peduncular region to be completely hidden in a ventral view of the brain. No cerebral peduncles are present. Both vermis and lateral lobes of the cerebellum are well devel- oped. Their various subdivisions show but little departure from the normal. The pons, which cannot be distinctly seen in the figures on account of the marked flexure of the brain stem, is also well developed. The absence of the pyramids and the great prominence of the olives are the only external features in which the medulla appears to vary from the normal. The attachments of the cranial nerves could not be made out in the gross as they had been removed, together with the vessels and pia, before the material came into my hands but subsequent to the drawings shown in figures 48 to dl. It is to be noted that the marked abnormal flexure of the brain stem in the region of the midbrain is apparently due to the non- development of the cerebral peduncles. on A CASE OF CYCLOPIA ZN TECHNIQUE Without further examination, the brain stem and thalamic mass were imbedded in celloidin. When this had been hardened suffi- ciently it was subdivided into blocks of convenient size and serial sections made 45 microns in thickness. Sections were selected at suitable intervals (about every tenth, but in places every fifth section) and stained by a modified Weigert method and counterstained with Upson’s carmine. For comparison, a sim- ilar series was prepared from the brain stem and left cerebral hemisphere of a normal full term foetus. Blocks also were cut from selected areas of the cortex of the cerebral vesicle and sections of various thicknesses were exam- ined. For comparison, sections were studied from the cortex of the right cerebral hemisphere of the normal foetus mentioned above. In each ease the following stains were used: modified Weigert, modified Nissl, and a simple hematoxylin-eosin stain. The drawings of the microscopic preparations from the brain stem and cerebral cortex have been made with the aid of a Leitz projectoscope. MICROSCOPIC DESCRIPTION OF THE BRAIN Brain stem and cerebellum (figs. 24 to 47) On comparison with sections taken from the normal brain stem at birth, this series shows several outstanding points of difference. There is a complete absence of the crura cerebri in the midbrain, the fasciculi longitudinales pyramidales in the pons and the pyramids in the medulla. The marked S-shaped flexure of the brain stem causes sections, which are transverse to the long axes of the medulla and midbrain, to be oblique and horizontal in the intervening regions. There is also a marked deficiency in the number of well medullated fibers present throughout. The development of the myelin sheaths has evi- dently been retarded. In the region of the midbrain the pia has been greatly thickened and forms a thick capsule which entirely masked the surface markings over this area in the recent state. 216 D. DAVIDSON BLACK Cranial nerves. As has already been mentioned in the gross description, the olfactory bulbs and tracts are entirely wanting. The optic nerve is represented by a few fibrous strands which pass from the bulbus oculi at the region of entrance of the central artery into the surrounding connective tissue of the orbit. As no microscopic examination was made of these, it is impossible to say whether any true nerve fibers were present. No connection existed between the eye and the central nervous system. The deep connections of the cranial nerves from the oculomotor eaudad are practically normal and will be but briefly noted here. The oculomotor nerve (N.IJT, figs. 36 to 39) is essentially nor- mal in its origin. Cephalad, the oculomotor nuclei (Nu.N.IIT) are in relation with a well marked nucleus of the posterior longi- tudinal bundle or nucleus of Darkschewitsch (Nu.f.l.m.). Some of the more caudal fibers of this nerve have a crossed relation. The emergent fascicles do not pierce the red nucleus but curve around its caudal and mesial surfaces. The trochlear nerve (N.IV. figs. 32 to 35), although very small, can be made out readily. Its nucleus (Nu.N.IV) is in rela- tion to the posterior longitudinal bundle and its root fibers pass towards the superior medullary velum on the median aspect of the mesencephalic root of the trigeminal nerve. The decussa- tion in the velum is quite normal. The trigeminal nerve (N.V) is normal and its relations are well brought out in figures 24 to 37. The abducent nerve (N.V/J, figs. 32 to 36) shows no departure from the normal. The connection between its nucleus (Nu.N.V 1) and the superior olive (Nu.o.s.) is also to be made out. The facial nerve (N.VJJ, figs. 32 to 39) is normal in all its relations. The emergent fascicles on the right side are arranged around a small artery. The vestibular nerve (N.VIII, vest., figs. 32 to 39) is normal in its relations and connections. The cochlear nerve (N.VJIJJ, coch) is also normal and the dorsal and ventral cochlear nuclei are quite evident (Nu.coch.d. and Nu.N.coch.). The corpus trap- ezoideum (c.t.), superior olive (Nuw.o.s.) and lateral Jemniscus (Z.1.) are well shown, and the continuity of the superior olive with the A CASE OF CYCLOPIA 217 nucleus of the lateral lemniscus (Nu.l.l.) is also apparent. The striae acusticae (s.a.) are but poorly medullated. The glossopharyngeus and vagus nerves (N.JX and X) to- gether with the tractus solitarius (7.s.), nucleus alae cinereae (Nu.a.c.) and nucleus ambiguus (Nu.amb.), are shown in figures 2EAtO 32: The accessory nerve (N.XJ) shows a somewhat anomalous condition of development. But few fibers of its medullary por- tion could be distinguished en the left side. On the right, these fibers are grouped peripherally in a compact bundle which is well shown in figure 24, lying just within the ventral border of the substantia gelatinosa (s.g.). These fibers apparently take their origin from the central gray matter lateral to the central canal. No definite nucleus could be distinguished in this, region but these fibers are clearly not related to the nucleus ambiguus; vide Cajal (3). The hypoglossal nerve (N.XJI) and its nucleus (Nu.N.XIT) are normal in their relations. There is no sharp line of demar- cation between the hypoglossal nucleus and the motor nuclei of the anterior horn in the upper cervical cord. ‘The accessory nucleus of Roller is also to be made out. Arcuate nuclei (Nu.a., figs. 25 to 34). These bodies are prom- inent in the lower medulla and are here somewhat larger than normal. On the appearance of the inferior olive, however, they diminish in size and at the level of the pons become continuous - through the nucleus of the raphe with the nuclei pontis. Inferior olive (Nu.o.t., figs. 27 to 35). These form very promi- nent projections on the ventral aspect of the medulla. They are somewhat larger than normal. , As the series in this region was not absolutely complete, the exact antero-posterior diameter could not be determined but it would not be much less than 8mm. The greatest dorso-ventral diameter is 5.6 mm and the transverse diameter is 3.8 mm. ‘These dimensions in Sabin’s model (20) were 7.5 mm., 4.48 mm., and 6.5 mm., respectively. There has been, therefore, an increase of the dorso-ventral at the expense of the transverse diameter. This change of form is probably due to the absence of the pyramids. In transverse 218 D. DAVIDSON BLACK section it is seen that the olives differ from the normal in showing three very definite outpouchings—a dorsal, a ventral and a lateral. The fibers arising from the olive are quite lacking in myelin. The mesial and dorsal accessory olives are present in practically normal relations. Lemniscus system. The decussatio lemniscorum (Dec.l.), the stratum interolivare lemnisci (S.2.l.) and the mesial lemniscus (L.m.) up to the level of the anterior portion of the red nucleus (Nu.r.) are essentially normal. At about the caudal end of the nucleus ruber, a well marked strand of fibers is given off from the lateral part of the main sheet of the fillet. This has been identi- fied as the superior lemniscus (Z.s., fig. 39). It passes up to end in the region of the superior colliculus. Beyond the nucleus ruber the lemniscus cannot be traced as a definite fiber system. Here its fibers become scattered and are lost in the dorso-lateral portions of the thalamic mass. Fasciculus longitudinalis medialis (F.l.m.). This fiber system is prominent throughout the series caudad to the posterior com- missure. It is the most completely medullated tract in the brain stem and on this account can be easily separated from the lem- niscus in the stratum interolivare. Its relations throughout are normal and it is traceable, together with fibers from the stratum album profundum of the midbrain, into the posterior commissure. Above the oculomotor nucleus (Nu.N.II/) it is related to a well developed nucleus (Ny,f.l.m., figs. 38 to 40) having the same rela- tions as the nucleus of the posterior longitudinal bundle in normal sections (Darkschewitsch). Corpus trapezoideum (C.t., figs. 35 to 37). The trapezoid body and the lateral lemniscus (L.l. figs. 33 to 37) together with their associated nuclei are quite normal in their relations and course. The lateral lemniscus terminates in the well marked nucleus of the inferior colliculus (Nu.c.2., figs. 33 to 36). No brachium of the inferior colliculus is present. Cerebellum and peduncles. The cerebellum shows a well de- veloped dentate nucleus (Nu.d.) together with globose (Nuw.g., figs. 31 to 32) emboliform (Nu.emb., figs. 32 to 34) and roof nu- clei. There are almost no medullated fibers to be found in the A CASE OF CYCLOPIA 219 cerebellum other than those entering by way of the corpus resti- forme (C.r., figs. 27 to 37) and those arising in the dentate nucleus. Even the medullary center of the flocculus is lacking in myelin, whereas in the normal term foetus this area is usually well medullated. The relations of the cerebellar peduncles are essentially nor- mal. The corpus restiforme is but slightly medullated and shows a very distinct and circumscribed nucleus (Nu.c.r., figs. 34 to 35) ‘after its entrance into the cerebellum. The pons, which is well developed, contains no medullated fibers. The brachia conjunc- tiva (Br.c., figs. 32 to 37) are readily distinguished, arising in the nucleus dentatus and coursing ventrad and cephalad to decussate caudad to the red nucleus. Their fibers are poorly medullated. Nucleus ruber (Nu.r., figs. 37 to 42). The red nucleus is well formed and prominent in the midbrain region. Owing to the absence of the crusta and also of a well marked substantia nigra, this nucleus is only separated from the periphery by a very short distance. It is to be noted that the emergent fibers of the oculo- motor nerve do not at any point pierce the substance of the red nucleus as they do in the majority of cases normally. The ce- phalic end of this nucleus is in relation with the lateral nucleus of the thalamic mass. Superior colliculi (C.s., figs. 35 to 39). These form prominent projections which are completely covered over by a thickened layer of pia (P., figs. 31 to 46) which surrounds the midbrain region. The cellular elements are but poorly differentiated. A few medullated commissural fibers pass across the mid-dorsal line above the stratum album profundum (s.a.p.).. In the mid- line dorsal to the aqueductus (Aq.c.) in this region there is a well marked oval mass of embryonal cells (Vg., figs. 36 to 39). The dorsal tegmental decussation of Meynert (D.t.d.M., fig. 37) is well shown ventral to the oculomotor nuclei. The ven- tral tegmental decussation was also distinguished -but is not shown in the figures. Fasciculus retroflecus of Meynert (F.r.M., figs. 38 to 46). This bundle forms a prominent landmark in the region of junc- 220 D. DAVIDSON BLACK tion between the midbrain and diencephalon. The corpora habe- nulae are not well developed, but on each side their site is marked by the beginning of the fasciculus retroflexus. The bundle passes down and comes into relation with the dorso-mesial sur- face of the red nucleus. From this point onwards it is applied to the mesial surface of this nucleus in its course to the ganglion interpedunculare. At no point does the fasciculus pierce the red nucleus. Throughout the major part of its course this bun- dle is accompanied by a small collection of gray matter, as is - usually the case, normally. The ganglion interpedunculare con- sists of a somewhat diffuse collection of cells between and ven- tral to the red nuclei toward their caudal ends. Thalamic mass Cephalad to the red nucleus there is found a large irregularly arranged nuclear mass, in the lateral portions of which the lem- niscus medialis becomes lost. This area may be roughly divided into a dorsal cellular portion and a ventral fibrillar area. The dorsal cellular area. Posteriorly, the habenular bodies (Nu.hb., figs. 44 to 46) may be distinguished, together with the fibers of the fasciculus retroflexus of Meynert which arise in these nuclel. The cells making up the thalamic nuclei are of two varieties: a. Both large and medium sized, well developed multipolar cells whose cytoplasm takes the carmine stain deeply and which do not differ in any marked degree from those found in the pul- vinar of the normal thalamus. b. Scattered between these large cells are numerous small em- bryonic or neuroglial elements, having a small amount of cyto- plasm which does not take the carmine stain deeply. These cells are far more numerous than the large multipolar variety, and are found both in the dorsal nuclear portion of the thalamus and in the ventral field, though far more abundant in the former area. The large cells, on the contrary, are almost entirely con- fined to the dorsal nuclear area. These cellular elements are arranged in irregular groups so that it is possible to distinguish certain irregularly arranged nuclei in the dorsal area. A CASE OF CYCLOPIA A | In the anterior portion, the dorsal area is divided into two large irregular lateral nuclei (Nu.lat.Th., figs. 41 to 46) and a smaller mesial nucleus (Nwu.med.Th.). Caudally the lateral nuclei are further subdivided so that it is possible to distinguish three nuclei in this region. ‘These may be termed for descriptive purposes, dorsal (Nuw.lat./), lateral (Nu.lat.2), and central (Nu.lat.3) nuclei of the lateral mass. The mesial nucleus (Nu.Med.Th.) is present in the caudal portion in essentially similar relations. It is to be noted that these thalamic nuclei do not come into relation with the cortex cerebri at the junction of the thalamic mass and the cerebral vesicle, but are separated from it by the ventral thalamic radiation. The ventral fibrillar area. This area occupies but a small space in sections through the more caudal part of this region, but increases in size as one passes forward by the addition of fibers arising in the dorsally placed nuclei. It is made up of a complex of both medullated and nonmedullated nerve fibers, which, in the caudal portion, are twisted into irregular whorls and tangles. It is remarkable that, in this region, irregular strands of poorly medullated fibers may be seen in numerous places piercing the outer limiting layer of neuroglia mthe ven- tral region and ramifying within the thickened pia (8, figs. 42 to 44). Passing forward, the thalamic mass becomes united to the cerebral vesicle by a narrow peduncle. This peduncle is seen to be made up almost entirely of ventrally coursing fibers con- tinuous posteriorly with those of the ventral area of the thalamic mass. These fibers must be taken to represent an atypically developed thalamic radiation (Rad.Th., figs. 41 to 47). The major portion of the fibers of the thalamic radiation are applied to the ventral surface of the cerebral vesicle on which they rapidly spread out and come to an end. ‘The ventral sur- face of the cerebral vesicle represents the free surface of the cor- tex. Thus most of the fibers ar:sing in the thalamic mass (fad. Th.) pass directly into the zonal or plexiform layer of the cere- bral cortex (St.z., figs. 46 to 47). 222 D. DAVIDSON BLACK Cerebral vesicle There is no appearance whatever of corpus striatum, rhinen- cephalon, or in fact of any of the structures developed ventral to the recessus neuroporicus in this region under normal circum- stances. The term ‘rhinencephalon’ is here used to indicate the basal structures of the forebrain in most intimate connection with the olfactory nerve and does not include pallial olfactory centers. It is only for the sake of convenience in the present description that I consider myself justified in employing the term rhinencephalon in this fashion; for when used in the above sense it effectually defines the limits of those areas which are quite absent from this brain. Cortex cerebri. 'The basal portion of the cerebral vesicle varies in thickness from about 10 mm. at its thickest at the point of entrance of the thalamic radiations, to about 5 mm. at its thin- nest. The cortex shows well developed cell lamination, and the types of lamination vary in different regions. It is impossible, however, to identify these areas as representing any of the his- tologically differentiated regions to be found in the normal cor- tex at birth. There is practically no medullated tissue to be found through- out the cortex. Indeed, the only area in which it is prominent at all is at the point of entrance of the thalamic radiations. In this region in figures 46 and 47, the cortex of the left posterior pole is shown cut tangentially. In the second layer of the cortex here, numerous groups of cells (C.zsl.) disposed in irregular, more or less circumscribed areas, are to be noted. These islands are made up of two kinds of cells; (a) very numerous, small, embry- onic elements, and (b) large polymorphic multipolar cells. In figure 15 this arrangement is brought out in transverse section. Only over this area of the cortex in this specimen were these large polymorphic elements to be found predominating in the stratum which is normally the layer of small pyramids. It would thus appear that the presence of the fibers of the atypical tha- lamic radiation has exerted an influence upon the growth of these neurones. Just what connection, if any, exists between these cells and the thalamic fibers could not be ascertained. The tis- A CASE OF CYCLOPIA 2a sue was not in a favorable condition for metallic impregnation and, although numerous attempts were made, no successful prepa- rations were obtained. Everywhere in this case the cortex is markedly thickened. The thickest cortex at birth (at least in the case I have used as control) is over the central area. Here the cortex is about 2 mm. in thickness, while the thinnest cortex in the cyclopian foetus meas- ures 3 mm. or more, the line of demarcation between cortex and medulla being very indefinite. The thickening is mostly the result of an increase in the deep polymorphic layer of cells, although all layers show an increased thickness. When examined at a low magnification the wall of the cerebrum is everywhere seen to be divisible roughly into five strata. This lamination is illustrated somewhat diagramatically in figure 18. The strata appear as follows: (1) an outer layer comparatively free from cells and varying in thickness in different areas, (2) a stratum rich in cells but whose elements tend to become arranged in arge irregular groups (3) a layer of densely packed cells, (4) a layer whose cellular elements resemble somewhat in arrange- ment those of the second stratum, and (5) a layer representing the medullary center. For a more detailed description, sections have been selected from two histologically distinct areas of the cortex (figs. 15 and 16). Figure 17 shows a section through the precentral area in a normal full term foetus for comparison as to thickness and gen- eral arrangement of elements. These drawings were made with the aid of a Leitz projectoscope and are each at a magnifi- cation of 65 diameters. Figure 15 has already been referred to and is a section taken through the cortex near the junction of the thalamus and cere- bral vesicle. The plexiform layer (I) at its periphery shows an irregular layer of embryonic, or more probably neuroglia, ele- ments. Into this plexiform layer stream thalamic fibers. The boundary between layer (I) and the next stratum (II) is not sharp. In the second stratum (II) occur the groups of large polymorphic elements mentioned above. The third layer (III) is made up of medium and small pyramidal cells and embryonic elements. It is slightly thicker than layers I and II combined 224 D. DAVIDSON BLACK and the average size of its cell elements become progressively smaller from without inwards. The fourth or polymorphic layer (IV) in the figure appears to be further subdivided into a more superficial zone of scattered small cells and a deeper more compact zone ‘This is not the case, however, and this appear- ance is due to some of the elements in this layer being arranged in groups of irregular size, the cells in which are more closely packed than in the intervening spaces. Only a portion of one of these groups appears in the drawing and included in it are shown several very large well developed pyramidal cells. These large pyramidal cells occur singly or in groups over this area and are situated at about the level of the middle trisection of the poly- morphic layer. The whole depth of this layer is not shown in the figure. The cells referred to exceed in size the largest cells found n the normal foetal cortex used as control. Figure 16 is taken from the so-called anter or lobe of the cere- bral vesicle at a point about an inch from the arched margin of the same in the mid-line. The plexiform layer (I) is here quite sharply marked off from the subjacent cell layer. In this layer (II) the elements are somewhat closely packed and arranged in irregular groups. It varies considerably in thickness at differ- . ent points at the expense of layer III. In the latter layer the arrangement of cells is somewhat looser and it apparently cor- responds to Bolton’s fourth layer or ‘nner fiber lamina (2). Layer IV is made of closely packed small cells having a quite character- istic embryonic arrangement in the form of irregular rows at right angles to the surface of the cortex. The line of demarca- tion between this layer and the preceding one is very easily made out and in places is a’most as sharp as that between the plexi- form layer and layer II. Layer V, the whole thickness of which is not shown in the figure, is the thickest of the cortical laminae, and combined with layer [TV makes up more than one-half of the total thickness of the cortex. Its cel’s are arranged in a Fig. 15 Cyclopian cortex cerebri. Section taken from the region of junction of the cerebral vesicle and the thalamus. Explanation in text (page 223). X65. Fig. 16 Cyclopian cortex cerebri. Section taken from the ‘anterior lobe.’ Explanation in text. X 65. means M4 n"s* =, o _ ~ ~ Eicras — Bae nm oO Rod ota) a ‘4 3 bate FO, a2 3 — as} = a) fe o 5) o Lal joy} 3 o H =< small pyramid layer, the cells here’ are small mal term foetus. IIT., layer of medium and large pyr I Cortex cerebri of nor I., plexiform layer; JJ. Fig. 17 and many are quite embryonic; Smith). and indistinct lamina; V, Betz cell granule layer which is a very thin polymorphic layer. x 65. 226 A CASE OF CYCLOPIA 227 scattered fashion and occasionally form irregular groups of various sizes. There are no giant cells in this stratum over this area of the cortex. The blood supply of the cyclopian cortex, as evidenced by the size and number of blood vessels, seems to be quite as rich as is the case normally. | With regard to the atypical course of the thalamic projection fibers it is to be noted that their presence in the plexiform layer ~ , is only the result of the altered relations between the pallium and the thalamus. Normally in mammals the thalamic fibers, to reach the pyramidal dendrites, must traverse the cortex from within outwards. Harrison (10) has shown that nerve processes wil develop readily even when the neurone is situated in an entirely strange environment. Under the altered form rela- tions, then, in this case it is but natural that these fibers should still retain their growth energy and pass ventrad along the only course open to them to gain the cerebrum (figs. 46 and 47). On reaching the latter, they mostly take the shortest way available by which they can reach the dendritic processes of the pyramidal elements, namely, by coursing in the zonal layer. The apparent influence of this contact has already been noted. It is of interest to note here that the passage of both efferent and afferent projection fibers in the plexiform or zonal layer of the cortex is the normal condition obtaining in Amphibia. In these forms the zonal layer represents the only white layer of the cortex visible in transverse section (4). The cortical neu- rones occupy the space between the zonal layer and the epen- dyma. Their axones curve outwards to reach the peripheral white matter, while their dendrites come into contact with the afferent projection fibers in this layer also. In the more caudal portions of the thalamus are found numerous fibers which in view of crowding have been unable to reach the cerebrum. Even under such adverse conditions the growth of these fibers was not arrested. They are here woven together into knots and tangles, and numbers of them have already been described as even piercing the limiting layer of neuroglia and ramifying in the pia (6 figs. 42 to 44). 228 D. DAVIDSON BLACK Owing to the great modification of cell lamination brought about by the presence of thalamic projection fibers in the plexi- form layer of the cortex in certain regions, it is not considered advisable to attempt to compare in detail the type of lamination found in such regions with that normally occurring in the devel- oping cortex. In areas remote from such disturbing influences, however, such comparison may, I think, be safely made. In figure 16 it was noted that layer III apparently corresponded to Bolton’s inner fiber lamina (2). The inner fiber lamina in the normal developing cortex is a layer which develops as the result of a separation of the cortical neuroblasts into an outer and an inner cell lamina. The polymorphic layer of the normal adult cortex is derived from the inner cell lamina, while practi- cally the whole cortex above the inner line of Baillarger is derived from the laminae superficial to this. If, then, layer III repre- sents the inner fiber lamina, it would appear that the outer layers of the cortex in this case are in a state of sub-evolution. For the total thickness of layers I to III inclusive is less than that of the much thickened polymorphic layer (IV and V). The somewhat closely packed lamina of cells constituting layer IV, which is well developed over most areas of this cortex, appears to be peculiar to this case and not comparable to any layer nor- mally present at term. In the case reported by Naegeli (17) however, a layer of apparently similar nature is shown in his figure 42. It would thus seem that the cortex had begun its development from within outwards in this region, as is normally the case. . At first sight it appears difficult to account for the greatly increased thickness of the cortex in a cerebrum in which cell differentiation has been described as subnormal. This difficulty is only an apparent one for, as will be subsequently pointed out, there is evidence in this brain to show that at least the greater part of the undifferentiated normal pallial anlagen are present. The surface area of the cortex, however, has not been increased by the formation of cerebral convolutions. Thus a large num- ber of cell elements have to accommodate themselves over a lim- ited area, resulting in the increased thickness of the cortex which A CASE OF CYCLOPIA 229 otherwise is characterized by subnormal development. As will be subsequently noted in a future communication, there are reasons for regarding this thickening in some areas as being also partly due to hyperplasia. Under normal circumstances Bolton (2) has pointed out that the cells in the cortex of the term child are less crowded than are those in the cortex of the developing foetus. As an important factor in reducing this aggregation of cells he points to the in- creased superficial area of the cortex at term due to the maturing convolutional pattern, ‘‘and the consequent smaller number of » cells in a section of the same thickness.” Bolton and Moyes (3) have shown that the first large well developed cells in the cortex are the Betz cells and that these are prominent as early as the eighteenth week of foetal life. They are situated in the basal portion of the inner fiber layer. These authors also express an opinion that the sensory or afferent fibers to the cortex are in all probability developed before the motor or efferent fibers from the cortex. In this case it has been shown that in those areas reached by the thalamic fibers, there is a marked tendency toward atypical overgrowth in certain neurones of the superficial layers with which these fibers come in contact. The presence of afferent fibers thus influences the growth of cortical neurones. Ordinarily these afferent fibers must enter through the basal portion of the cortex. It is in this basal portion of the cortex that the first well differentiated neurones appear and it is the basal laminae of the cortex that are the first to be evolved. It would be interesting to determine how far the tardy ontogenetic devel- opment obtaining over some areas of the normal cortex were dependent upon the late appearance in these areas of afferent projection fibers. Naegeli (17) has described numerous well developed pyramidal cells in the cortex in his ease, which have attained a considerable degree of differentiation and are possessed of short axone pro- cesses but which do not come into relation with any projection fibers from the thalamus, none of which, he says, gained the cerebrum. He has termed this growth process ‘self-differentia- THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL, 23, NO. 3 230 D. DAVIDSON BLACK tion,’ as opposed to ‘dependent differentiation,’ using the terms © suggested, as.he says, by Roux (19). In this case, in some of the more basal portions of the cortex which are lacking in medullated fibers of thalamic origin, there are found in the deeper strata numerous giant pyramidal ele- ments. These cells are much larger than any similar elements to be found in the normal new-born cortex, and each possesses a large vesicular nucleus with well developed karyosomes, while in the cytoplasm Nissl bodies are well formed and prominent. Although no medullated fibers of thalamic origin could be made out in such sections, it is very doubtful if these large elements can be considered as cells showing only ‘self-differentiation;’ they are probably influenced by non-medullated afferent fibers. On the other hand, in areas quite removed from possible thalamic in- fluence as for example, the anterior portion of the rim of the cere- bral cup, the smaller, more numerous and generally under-devel- oped elements, making up the thickness of the cortex may, I think, be classed as ‘self-differentiated,’ in the sense of Roux. The giant cells referred to in the preceding paragraph, which are located in the deep cell lamina of the cortex, are strikingly like the large efferent perikaryons described as Betz cells in the normal cortex of mammals. It has been shown by Bolton (3) that in the evolution of the cortex, those laminae situated above the level of the Betz cells, are the site of greatest differentiation and growth in the higher mammals and man. It thus happens that the increased cortical thickness causes these large efferent cells to lie more deeply in the human cortex than in that of lower forms. If then the giant cells in this cyclopian cortex represent the cell bodies of potential efferent neurones of the Betz type, the location at such a cortical level would be a further argument against any theory of phylogenetic reversion. An accurate knowledge of the number, arrangement and distribution of these cells would aid in determining how far such a comparison as the above is justified. In the event of the presence of sufficiently differentiated cortical tissue in any of the other cases of cyclopia at present in my possession, a complete topographical survey of | such will be made. It would also be of interest to note what A CASE OF CYCLOPIA 231 relation exists between the development of the corpus striatum when present, and such efferent neurones as thase under dis- cussion. j One other point may here be noted in connection with the examination of this cortex. It has been found that the character of the lamination, the thickness of the cortex and the condition of cell development, vary in different regions. Two of these histologically distinct areas are illustrated in the figures. It would seem that these areas are not sharply marked off from each other but are separated by transitional zones. However, as no complete topographical survey was made, this point was not definitely determined. Thus, histologically distinct areas are present but, with the exception of the dentate and fimbrio- dentate fissures, there is a complete absence of true sulci. This, then, is further evidence, it would seem, of the truth which Bolton has pointed out, namely, that the development of the convolu- tional pattern is secondary to the differentiation of the cortex into histologically distinct areas. Cerebral limbi. At the base of the inner pillar of the thick- ~ ened recurved margin of the cerebral vesicle, the thin roof be- comes attached to the edge of the cortex. The relations of the roof in this region have already been noted in the gross descrip- tion (figs. 11 to 13). Sections were made at intervals along this margin and the relations of the cerebral limbi studied. Drawings of three of these sections are here reproduced. Figure 18 is a section taken in the mid-sagittal plane; figure 19 is a section taken at the junction of the anteror and middle thirds, and figure 20 a section at the junction of the middle and posterior thirds of the left cerebral margin. The outlines of these drawings were made with the aid of a Leitz projectoscope and at the same time lines were drawn marking off the various cell laminae of the cortex. The spaces between these lines were subsequently shaded in free hand, so the details of cell arrangement are only indicated in a somewhat diagrammatic fashion. However, the relative thickness of the cortex and the arrangement of its vari- ous strata, as seen under low magnification, are fairly accurately shown. Fig. 18 Section through cerebral margin in mid-sagittal plane. Hp.R., epen- dymal roof; Lb.c., limbus corticalis; Lb.m., limbus medularis; 7’.c., taenia cere- bri; Vent.I., cavity of cerebral vesicle. Nos. 1 to 5, cortical lamina2, explanation in text (page 231). X 3.5. Fig. 19 Section through cerebral margin in plane of junction of anterior and middle thirds of lateral rim. F.d., fascia dentata: Pl.Ch.L., choroid plexus. Other letters as in figure 18. X 3.5. Fig. 20 Section through cerebral margin in plane junction of middle and pos- terior thirds of lateral rim. F.d., fascia dentata; F'.h., hippocampal fissure; F'm., fimbria; S.F.d., fimbrio-dentate sulcus; 7'.f., taenia fimbriae. Other letters as in figure 18. X 3.5. 232 A CASE OF CYCLOPIA 2a0 The major portion of the posterior two-thirds of the cerebral rim on both sides has been found to represent a but slightly modified hippocampal formation (fig. 20). The line of attach- ment of the thin roof (Hp.R.) corresponds to the edge of the lim- bus medullaris or fimbria (/’m.). A well marked fascia dentata (F.d.) is present, bounded laterally by the fissura hippocampi (F.h.), and mesially by the fimbrio-dentate sulcus (S.F.d.). Above the fimbria, the thin roof is invaginated by vascular tissue to form a band of choroid plexus (PI.Ch.L.). This ‘choriogenous zone’ extends forward only as far as the extreme anterior tip of the fascia dentata (F.d., fig. 19). It is to be noted that if the normal lateral plexus could be straightened out it would repre- sent an area such as is found here and bearing precisely similar relations to the fimbria. Throughout the major portion of the anterior third of the cere- bral rim, the fascia dentata can not be made out. Here the limbus corticalis has not become rolled upon itself (Lb.C., fig. 18). The limbus medullaris (£b.M/.) becomes on this account more extensive. No choroid invaginations are present over this area. The identity of the limbus corticalis between the anterior ex- tremities of the two definitely identified hippocampal areas is as yet obscure. It is hoped that further study in comparison with the other cases in my hands may clear up this point. As has already been mentioned, no medullated fibers are pres- ent in this area of the cortex. In this connection it is to be noted that there was a complete absence of medullated tissue through- out the hippocampal area in the normal term foetus used as control. Ventricular anomalies The cavity of the fourth ventricle (Vent. IV) is essentially normal in its relations. It is lined throughout by a well devel- oped layer of ependyma. Turning now to the aqueductus (Aq.C., figs. 34 to 42), it is to be noted that numerous irregular ependymal diverticula arise from its ventral and lateral walls in the region just caudal to the posterior commissure. One of these evaginations passes 234 D. DAVIDSON BLACK ventrad and cephalad for a considerable distance and may be traced into the mesial thalamic nucleus (CX, figs. 42 to 47). Just cephalad to the posterior commissure the pineal recess may be distinguished, and in front of this a few fine medullated fibers cross in the thin roof and constitute the habenular com- missure (C.Ab., fig. 44). Immediately cephalad to the habenular commissure, the tae- nia thalami (7’.th./IT, fig. 45) is to be seen and two small areas of choroid plexus (PI.Ch.IIT, figs. 44 to 45) project into the small ventricular cavity (Vent. III, figs. 44 to 46). Separating these two plexuses is a slightly thickened area in the roof repre- senting the attachment of the anterior limb (dorsal) of the pineal stalk. In front of this the thin roof of the ventricular cavity has been torn away for a short distance and its relations cannot be accurately followed. However, the roof appears again in the sections somewhat more cephalad and is now seen to be thickened and lacking in choroidal invaginations. Traced forward from this region, the ventricle is seen to end blindly in the dorsal part of the thalamic mass. It was noted in the gross description that the thalamic mass projected into the cavity of the primary forebrain vesicle and was covered with ependyma. From a study of the sections in this region it now appears that the line of attachment of this ependyma, or rather the line of its reflexion from the surface of the thalamus, approximately coincides with the line of demarca- tion between the dorsal nuclear mass and the ventral fibrillar area. Thus the greater part of the thalamic mass is extraven- tricular and is covered by a thick layer of pia and fibrous tissue. The taenia thalami (7.th.JII), noted just in front of the pineal region, is not continuous with the taenia (7.th. I, figs. 45 to 47) over the anterior part of the thalamus to which the thin roof of the forebrain vesicle is attached. These relations are best seen in the diagram of a mesial sagittal section of the brain (fig v1): It is thus seen that there is a discontinuity in the ventricular system of the brain and that this interruption occurs in the region A CASE OF CYCLOPIA 235 of the third. ventricle (figs. 11 and 23). This obliteration of a part of the ventricular cavity is not necessarily the result of the growth conditions producing cyclopia; but probably these growth conditions rendered such an SOE ae more liable to oecur in this region than e!sewhere. Mechanical considerations Stockard has shown that the condition of cyclopia may be produced at will in fish embryos in a high percentage of cases by treating the eggs with MgCl, or Mg(NO;)2 solutions (24 and 26). He was thus able to study histologically a great number of cases in fish embryos otherwise perfectly normal. His observations showed that “‘the cyclopian defect is present from the first in the same condition that it will continue throughout develop- ment”’ (25). This statement was subsequently (27) somewhat modified by ‘further experimental work, for it was found that cyclopia could be produced in a small percentage of cases by the action of magnesium chloride, even after segmentation had gone as far as the periblast stage (15 hours). Eggs older than the fifteen-hour stage were not affected by the Mg solutions. Thus, although cyclopia is not necessarily of germinal origin, the growth inhibition begins at so early a stage of embryonic life that it practically amounts to absence of certain areas dur- ing development. In other words, cyclopia is not the result of fusion of parts originally separate, but is due to absence during development of certain parts normally separating the eye anlagen. Cyclopian monsters belong to that class of bilaterally symmet- rical beings which have been termed by Wilder (28) ‘cosmobia.’ Development in these cases proceeds in a orderly fashion until some mechanical difficulty arises which cannot be overcome by the individual and death results. In fish the development in eyclopian forms goes on until the yolk supply is entirely used up, when the animal dies of starvation unless suitable food is artifi- cially provided. In this connection it is also of interest to note that the absence of tissue anlagen does not postulate a non- functional nervous system, for individuals which have been kept alive and observed, reacted to stimuli in a quite normal fashion. 236 D. DAVIDSON BLACK In cyelopian mammals no mechanical difficulties incompatible with life are met until after birth. The animal then usually dies in a short time from interference with feeding and respiratory functions. Thus, bearing in mind that the cosmobion is governed in all its growth changes by quite definite mechanical laws, an attempt may be made to interpret the form relations in this case. Certain areas have been practically absent during development. If one removed such areas from a model of a very young normal brain and approximated the cut edges, would the resulting malforma- tion be similar to the case in hand? If one examine one of His’ models of the brain at the end of the fourth week, it will be seen that the telencephalon is rep- resented by an expanded, thin walled unpaired vesicle, for at this stage the median furrow between the two pallial expansions is not developed. Looking at a mesial sagittal section through such a model, it is possible to mark out on the ventricular surface, with a considerable degree of accuracy, the areas which will later be developed into the corpus striatum and rhinen- cephalon, the pars optica and the pars mammillaris hypothalami, and the pallium (fig. 21). ; It has been shown that in the present case, the pallium alone of these parts is present. Thus, if one cuts out fromboth sides of a clay model of the brain at the end of the fourth week, these areas which are not developed, and places the two halves remain- . ing in apposition, then by simply pressing the cut surfaces of each half together one has reproduced in all its essentials the form relations obtaining in this cyclopian brain (fig. 22). It must be borne in mind, however, that this is reversing the true sequence of events. We are left with a cup whose thickened walls are formed by all the pallium, and whose rim is formed by the two original me- sial edges, to which of course is attached the much stretched thin roof. These mesial edges in the normal pallium for the most part are the areas in which the hippocampal formations are laid down. It is also along these mesial edges that the lateral choroid plexuses are normally invaginated. So in this case, we A CASE OF CYCLOPIA Dal Met. Fig. 21 A Medial sagittal section of a His’ model of the brain at the end of the fourth week (modified from Spalteholz). C.st., corpus striatum; Jst., isthmus; Mes., mesencephalon; Met., metencephalon; Myel., myelencephaion; Pai., pal- lium; P.m.th., pars mamillaris hypothalami: P.o.th., pars optica hypothalami; Rh., rhinencephalon; S.Lim., sulcus limitans; Th., thalamus. Fig. 21 B Right half of a His’ model of the brain at end of fourth week, from which the anlagen of the rhinencephalon, corpus striatum, pars optica and pars mamillaris hypothalami have been removed. The cut edges are lined. Fig. 22 Model resulting from the approximation of the cut surfaces as indi- cated in the text. -T7'.c., taenia cerebri; 7'.th., taenia thalami; X, marks the line of union between the original ventro-mesial edges of pallial anlagen. Other jetters as in figure 21A. 238 D. DAVIDSON BLACK find the hippocampal formation developed in the edges of the pallial cup posteriorly, and above the fimbria we find choroidal invaginations of the ependyma. Turning to the thalamus, it will be seen that the absence of the pars optica and pars mammillaris hypothalami would mate- rially reduce both the volume of the thalamus and the size of its ventricular cavity. The use of such a model is justified only in so far as it helps to demonstrate the mechanical tendencies that would arise in a brain in which these areas are missing from the start. The brain at this stage of development was taken because, while the relations are simple, it is yet possible to outline the areas occupied by the corpus striatum, and so forth, fairly accurately. Cor. pin. Cor.pin. F —— Ep. R- Sore Vent. lil Vent.IH Vent. Ill Eo Th. Th. Th. Xx. Vent. | Vent. | Wental A? Cor.c. B Cor.c. C Cor.c. Fig. 23 A B and C. Diagrams of medial sagittal sections through the brain illustrating the hypothetical closure of the connection between the cavity of the cerebral vesicle and the third ventricle. Cor.c., cortex cerebri; Cor.pin., pineal body; Ep.R., ependymal roof; Th., thalamus; Vent.I., cavity of cerebral vesicle; Vent.I/1., third ventricle; X, in figure 23 C. indicates the final point of attachment of the much expanded thin roof to the front of the thalamus. Sometime during the development of this case the condition of hydrocephalus set in and, as a result, the roof of the cerebral vesicle bulged upward. If one refer now to the diagrams (fig. 23), it can be readily seen how the discontinuity in the cavity of the already reduced third ventricle might be brought about through pressure of the expanding roof of the cerebral vesicle. This thin cerebral roof being confined above by the skull, ex- tended backward as a pocket over the thalamus. Pressure of the fluid contents being transmitted equally in all directions would then tend to close the fore part of the third ventricle. This closure might subsequently be followed by adhesion be- A CASE OF CYCLOPIA 239 tween the approximated walls and finally by complete obliter- ation of the ependyma. Such an explanation is however purely theoretical, as there is no way of determining the time relation between the onset of hydrocephalus and the occlusion of the anterior portion of the third ventricle. REVIEW OF CASE REPORTED BY O. NAEGELI In the case reported by Naegeli (17) the cerebrum was repre- sented by an unpaired, thick walled vesicle, having a very slight attachment to the massive thalamus. The basal ganglia were quite defective and could not be defi- nitely identified. He described, however, a thin plate of embryonic cells in the region of junction between the thalamus and cerebrum which he concludes may represent these structures. No olfactory bulb or stalk was present but he describes the hippocampal formation as being well developed, and the fimbria as containing medullated fibers. In figure 29 of his report he shows the cornu ammonis in transverse section and it is strikingly similar to sections through this region in the present case. Un- fortunately, he has not described the relations of the hippocam- pus except to mention that it is a distinctly paired formation, so further comparison is impossible. The fornix is also mentioned as an unpaired bundle which divides into symmetrical halves to end in the region of the corpora mammillaria. The relations of this fornix to the fimbriae are not clear. The cortex of the cerebral vesicle showed a distinctly laminated arrangement of its cells and was quite markedly thicker than normal—the greatest increase in thickness being in the deep or polymorphic stratum. In figure 42 of his report he shows a carmine stained section through the cerebral cortex, in which the cell lamination is almost precisely the same as that described as appearing under low magnifications in the present case. He also found a few poorly medullated radial fibers in the layer of large pyramidal cells. No tangential fibers were present. The thalamus was massive and of nearly normal form, but the two halves were strongly fused in the mid-line ventrally. 240 D. DAVIDSON BLACK He does not describe any connection between the remains of the third ventricle and the cavity of the cerebral vesicle. From his figures I am led to believe that this connection was inter- rupted. The attachment of the thalamus to the cerebrum was very slight and wholly basal. Into this region, projection fibers converge from the lateral nuclei of the thalamus, constituting an atypical thalamic radiation. Ventrally the fibers of the two sides decussate and end blindly in the basal region of the cere- brum, which before has been alluded to as the probable repre- sentative of the corpus striatum. It is thus seen that, as in the present case, the third ventricle has been considerably reduced in volume and its connection with the cavity of the cerebral vesicle has also apparently been entirely interrupted. A single median optic nerve was present which divided pos- teriorly at a partial decussation into paired optic tracts. In- fundibulum, corpora mammillaria and lateral geniculate bodies could be distinguished and a commissure of Meynert is described crossing in the tuber cinereum. Dorsally the habenular bodies were prominent, together with the posterior commissure and fas- ciculus retroflexus of Meynert. Medullated fibers were found in the taenia thalami (stria medullaris thalami). The thalamic nuclei were not definitely marked out, but the whole thalamic mass was relatively rich in well formed ganglion cells together with more numerous embryonic elements and neuro- blasts, being thus similar in these respects to the present case. From the region of the interpeduncular ganglion caudad, the brain stem was much distorted by a malformation quite inde- pendent of the cyclopian condition. A split had occurred in the mid-line, resulting in a more or less complete separation of the brain stem and cerebellum into right and left halves. Rela- tions were further complicated by the presence of an additional flexure in the brain stem and by the bending dorsally of a con- siderable portion of the split cord so that it came to lie upon the malformed brain stem halves within the skull cavity. A con- siderable amount of fusion between the cord and underlying brain stem halves was also present. A CASE OF CYCLOPIA 241 Notwithstanding these malformations, the cranial nerves from the oculomotor caudad and most of the fiber systems with their nuclei could be identified in the brain stem. There was a com- plete absence, however, of the pyramidal system throughout. Naegeli calls attention to von Monakow’s (16) classification of fiber systems and nuclei into phylogenetically old and phylo- genetically young groups, and points out that only the former are to be identified throughout in his case. He concludes from the striking resemblance between the ar- rangement of structures in the forebrain region in his case and those obtaining in the forebrain of teleosts, and also from the complete absence of so-called phylogenetically young fiber sys- tems, that the condition of the brain in cyclopia may represent an arrest of development at a phylogenetically early stage. It is evident from the above that the prosencephalic disturb- ance here noted, dependent upon the cyclopian condition, was not so extensive as in the present case. CONCLUSION In the case I have reported, there is a very marked superficial resemblance between the forebrain vesicle and the telencephalon in teleosts. The resemblance, however, is only superficial, for the thickened basal structures present in this case are altogether pallial. In teleosts the basal nuclei form the bulk of the thick- ened base of the telencephalon, while in this case these basal structures are absent. The absence of the so-called phylogenetically young fiber sys- tems is sufficiently explained by the lack of complete develop- ment of the suprasegmental neurones whose processes make up the bulk of such systems normally. There can be no doubt that the very slight attachment of the thalamic mass to the cerebral vesicle had much to do with the growth inhibition of the cortical neurones. The presence of a well marked hippocampal formation while the rhinencephalon, as before defined, is entirely wanting, may be explained on mechanical grounds. Such a finding offers the 242 D. DAVIDSON BLACK strongest evidence that the condition of the brain in ecyclopia cannot represent an arrest of development at a phylogenetically early stage. The observations in connection with the histological structure of the cortex, which will be more fully recorded elsewhere, have, I think, shown the value of a close investigation into the finer anatomy of the abnormal nervous system. It is in such cases as this that the key to many of the problems concerning the devel- opment of the normal brain may be found. My conclusions can best be summarized as follows: 1. That the central nervous system in this case of cyclopia does not show any evidence that may be taken to indicate an arrest of its development at an early phylogenetic stage. From a study of Naegeli’s paper, and from what I have seen in other cyclopian brains in my possession, I am led to the conclusion that any apparent indication of such a reversion is purely super- ficial in character and may be explained on mechanical grounds. 2. That the persistence of so-called phylogenetically old neu- rone systems in the brain, and the absence of phylogenetically young systems, is due solely to the absence during development of certain portions of the forebrain in the ventral region (mid- line), and to the interference with the mechanics of growth caused thereby. 3. That the condition of development of the primary fore- brain will present a new mechanical problem in each case of cyclopia, depending upon the extent of the primary absence of tissue anlagen, and that the extent of this ‘lesion’ cannot always be judged by the condition of the cyclopian eye. In contrast to this, the condition of development in the brain stem in each case, provided no other malformations are present, will be very similar; and such abnormalities as are present will be due to the absence of fiber systems of the suprasegmental type. 4. That the condition of cortical development is such as to confirm the opinion already expressed by Bolton and Moyes (8), namely: that the sensory or afferent fibers to the cortex develop in all probability before the motor or efferent fibers from the cortex. A CASE OF CYCLOPIA 243 5. That the initial stimulus resulting in the differentiation of specialized efferent cortical neurones is probably dependent upon the arrival of these afferent fibers in the cortex. 6. That the condition of development in the cerebrum of this case offers further proof that the differentiation of the cortex into histologically distinct areas precedes the development of the convolutional pattern. LITERATURE CITED! (1) Buacksurn, I. W. 1907 Anomalies of the encephalic arteries among the insane. Jour. Comp. Neur., vol. 17., no. 6. (2) Bouton, J. S. 1910 A contihibution to the localization of cerebral func- tion based on eclinico-pathological study of mental disease. Brain, vol. 33. (3) Bouton, J. S. and Moyss, J. M. 1912 The cytoarchitecture of the cere- bral cortex of a human foetus of eighteen weeks. Brain, vol. 35. (4) Bonne, Cu. 1906 L’écorce cérébrale. Revue Générale d’Histologie, Renaut et Regaud, no. 2 (5) Casau, S. Ram6n 1909 Histologie du systéme nerveux de Vhomme et des vertébrés. Trad. par Azoulay, tome 1. (6) 1911 Idem, tome 2. (7) CAMPBELL, ALFRED W. 1905 Histological studies on the localization of cerebral function. Cambridge. : (8) Darestre, C. 1891 Recherches sur la production artificielle des monstres. II Ed. Paris. (9) GRAvELoTTE, E. 1905 Contribution A l'étude des anomalies de développe- ment de l’extremité céphalique—un cas de cyclopie. Thése. (10) Harrison, R.G. 1906 Further experiments on the development of periph- eral nerves. Am. Jour. Anat., vol. 5. 1No attempt has been made here to prepare a complete bibliography of the literature dealing with cyclopia, for the great majority of the cases reported have no bearing upon the subject of this paper. A complete critical review of the lit- erature up to 1872 has been made by Kundrat (12). For a short review of the subject of cyclopia up to 1897 and a statement of the various theories that have been held regarding the nature and cause of this malformation, reference may be made to Naegeli’s paper (17). A somewhat more extensive historical survey of the subject, with a bibliography may be found in the thesis by Gravelotte (9). Most of the important contributions of recent date on this subject have been made by the application of experimental methods in the production of this mal- formation in lower forms. Some of the more important papers in this connection are cited, as well as some of the literature bearing upon the general subject of teratology. Papers dealing with the histological arrangement of the elements in the normal cortex, which have been consulted in connection with the study of the cortex in this case, are also referred to in this list. D. DAVIDSON BLACK Jackson, Harry 1909 Cyclopian monsters: Some general observations with report of a case. Jour. Am. Med. Assoc., vol. 53, no. 18. V. Kunpratr 1882 Arhinencephalie als typische Art von Missbildung. Graz. Lewis, WARREN H. 1909 Experimental production of cyclopia in the fish embryo (Fundulus heteroclitus). Anat. Rec., vol. 3, no. 4. Matt, F. P. 1905 On the development of the blood-vessels of the brain in the human embryo. Am. Jour. Anat., vol. 4. 1908 A study of the causes underlying the origin of human monsters. Philadelphia. von Monakow, ©. 1895 Experimentelle und pathologische-anatomische untersuchungen tiber die Haubenregion, den Sehhiigel und die Regio _ subthalamica, nebst Beitrigen zur Kenntniss friih erworbener Gross- und Kleinhirndefecte. Arch. f. Psychiat., Bd. 27. Nance, O. 1897 Ueber eine neue mit Cyclopie verkniipfte Missbildung des Centralnervensystems. Archiv f. Entw. Mech., Bd. 5, H. 1. OpreNHEIM, H. 1911 Text-book of nervous diseases. Vol. 2. Trans. by Alex: Bruce. Edinburgh. Rovux, W. 1895 Gesammelte Abhandlungen iiber Entwickelungsmechanik. Bd. 1, pp. 348, 804. Bd. 2, pp. 281. 909. SaBin, FLroreNcE R. 1901 An atlas of the medulla and midbrain. Bal- timore. 1911 Description of a model showing the tracts of fibers medullated in a new-born baby’s brain. Am. Jour. Anat., vol. 11, no. 2. Scuwause, E. 1906 Die Morphologie der Missbildungen des Menschen und ‘der Tiere. Teil 1. SmityH, G. Exuior. 1907 A new topographical survey of the human cere- bral cortex. Jour. Anat. and Phys., vol. 41. StockarD, C. R. 1907 The artificial production of a single median cyclo- pian eye in the fish embryo by means of sea water solutions of magne- sium chloride. Arch. f. Entw. Mech., Bd. 23. 1908 The question of cyclopia: one-eyed monsters. Science, vol. 28. 1909 The development of artificially produced cyclopian fish, “The magnesium embryo.’ Jour. Exp. Zool., vol. 6. 1910 The influence of alcohol and other anaesthetics on embryonic development. Am. Jour. Anat., vol. 10. no. 3. Witprer, H.H. 1908 The morphology of cosmobia. Am. Jour. Anat., vol. 8. A CASE OF CYCLOPIA 245 ABBREVIATIONS FIGURES 24 To 47 Figures 24 to 47 are drawn from a series of transverse sections through the cyclopian brain stem, stained by a modified Weigert method followed by Upson’s carmine. The drawings which are here reproduced at a magnification of X 2.5, are all so arranged that the right and left sides of the sections correspond to the right and left sides of the page respectively. In examining this series it is well to bear in mind that in this brain stem two marked flexures persist. Thus for example, in figure 34, the midbrain and medulla appear in almost transverse section while the intervening pons is cut horizontally for the most part. An explanation of the abbreviations used in these figures will be found in the accompanying list. Aq.c., aquaeductus cerebri NEVES N. trigemini B., thalamic fibers that have wandered N.V.m., N. trigemini (motor) ventrad into the pia IN Wal N. abducentis Br.c., brachium conjunctivum NeValilien N. facialis C.c., canalis centralis N.VIITI., coch.N. cochleae C.hb., commissura habenularum N.VIII., vest.,N. vestibuli C.isl., cell islands in second layer of N.IX,X., N. glossopharyngei et cortex cerebri vagi ‘ C.i., colliculus inferior NEG. N. glossopharyngei et C.L., continuation into the medulla of vagi the lateral column of the cord INEST N. accessorii Cor.cer., cortex cerebri INXS. N. hypoglossi Cor.pin., corpus pineale C.p., commissura posterior cerebri C.s., colliculus superior C.t., corpus trapezoideum D.Br.c., decussatio brachii conjunctivi Dec.l., decussatio lemniscorum D.N.V., decussating tract of N. tri- geminus D.t.d.M., decussatio tegmenti dorsalis Meynerti F.a.e., fibrae arcuatae externae F.a.i., fibrae arcuatae internae F.c., funiculus cuneatus F.l.m., fasciculus longitudinalis medi- alis F.r.M., fasciculus retroflexus Meynerti L.l., lemniscus lateralis L.m., lemniscus medialis L.s., lemniscus superior N.C.I., first cervical nerve NEI. N. oculomotiru INET N. trochlearis THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL, Nu.a., nucleus arcuatus Nu.a.c., nucleus alae cinerae Nu.amb., nucleus ambiguus Nu.c.a., nucleus of the anterior horn Nu.c.i., nucleus colliculi inferioris Nu.coch., nucleus N. cochleae ventralis Nu.coch.d., nucleus N. cochleae dorsalis Nu.c.r., nucleus of restiform body Nu.d., nucleus dentatus Nu.emb., nucleus emboliformis Nu.f.c., nucleus funiculi cuneati Nu.f.g., nucleus fyuniculi gracilis Nu.f.l.m., nucleus faciculi longitudi- nalis medialis (Darkschewitsch) Nu.g., nucleus globosus Nu.h.b., nucleus habenulae Nu.l.l., nucleus lemnisci lateralis Nu.lat.1,2,3, nuclei distinguished in the lateral portions of the thalamic mass Nu.lat.th., lateral thalamic nucleus Nu.med.th., medial thalamic nucleus 23, NO. 3 246 Nu.m.N.V., nucleus motorius N. tri- gemini Nu.N.coch., nucleus N. cochleae ven- tralis Nu.N.coch.d., nucleus N cochleae dor- salis Nu.N.vest., nucleus N. vestibuli lat- eralis Nu.N.IIT., nucleus N. oculomotorii Nu.N.IV., nucleus N. trochlearis Nu.N.VI., nucleus N. abducentis Nu.N.VITI., nucleus N. facialis Nu.N.XII., nucleus N. hypoglossi Nu.XII., nucleus N. hypoglossi Nu.o.a.d., nucleus olivaris accessorius dorsalis Nu.o.a.m., nucleus olivaris accessorius medialis Nu.o.i., nucleus olivaris inferior Nu.o.s., nucleus olivaris superior Nu.Pon., nuclei pontis Nu.r., nucleus ruber P., thickened layer of pia enclosing midbrain and extraventricular por- tion of thalamus D. DAVIDSON BLACK Pl.ch.JIIT., choroid plexus of third ven- tricle Rad.th., thalamic radiations. fibrillar area) R.d.N.vest., radix descendens N. vis- tibuli R.d.N.V., radix descendens (mesence- phalica) N. trigemini S.s., striae acusticae S.a.p., stratum album profundum S.g., substantia gelatinosa Rolandi S.i.l., stratum interolivare lemnisci Si.z., stratum superficiale of cortex cer- ebri T.s., tractus solitarius T.thlI., attachment of the ependymal roof or cerebral vesicle over the an- terior aspect of thalamus T.th.IIT., taenia thalami T.v.q., taenia ventriculi quarti T.s.N.V., tractus spinalis N. trigemini Vent.I., cavity of cerebral vesicle Vent.I/1., ventriculus tertius V.q., ventriculus quartus X., ependymal diverticulum from aqua- ductus cerebri (Ventral A CASE OF CYCLOPIA 247 248 D. DAVIDSON BLACK ~ Nu.N. vest / fOr eS iy .. \\ Nu.N.VILb, e = wi A CASE OF CYCLOPIA Nu.m.N. Ve .D.N.V. N.Vil-cZ a.N.vest.1/ — u.N. VII Md V | iN ve) i 8 Vill. vest. ¢ .VIIl.coch 35 249 = 2 ; 7 S h 2 N. VIII. vest. A} 1 Vil 250 A CASE OF CYCLOPIA 251 nmen SD. is Pees N.VII N.VIII. vest. N.VI e—N.VI 39 252 D. DAVIDSON BLACK N. Ill & Oi GE NN. - 4! Cor.cer. Cor. pin A : Nu. lat.1 30 { ip a“ / F.r.m ‘ we Nu. med.th P= ASRS ! Nu. lat.3 EM ESE Nu. lat. Oval 43 Cor.cer. 253 > Nu.lat.3 i Nu. lat.2-11 — { ént.| Seek C.hb- SS —— Nu. lat.1 are eA ene oe: Fy z Nu es Nu, lat.2 PAN Lie eee St) Pea i he fe pnt ai fps Ni jou Vs! AN Nu. lat. ae 1A) ee Wh Cor.cer. 44 Cor.cer. 45 =< = < Nu. lat. th Nu. med.th. Rad.th Cor.cer. Cor.cer. Rad.th, Rad.th, 256 D. DAVIDSON BLACK ~-— Mid brain _ Medulla oblongata ) — Cerebellum ~ __ _ _ Roof of cere- 5 bral vesicle Limiting sulcus’ Base of cere- bral vesicle __ Roof of cere- bral vesicle -— Mid brain Limiting sulcus - Cerebellum Base of cere- ‘bral vesicle ~—Medulla ~ oblongata” Median °~—="basal furrow’ we 7 AO EXPLANATION OF FIGURES 48 Dorso-lateral view of entire brain. X 3. 49 Posterior view of brain. X 3. 257 A CASE OF CYCLOPIA Anterior limb of -_ basal furrow Roof of cere- bral vesicle Limiting sulcus Anterior basal ~lobe of cerebral vesicle a Olive 4 ‘ ps Vertebral artery 1 i 1 Basilar artery } Median basal furrow oO (e) Left posterior 1 cerebral artety 4 1 Median carotid artery Posterior communicating artery Thalamic mass Rim of thickened cerebral . base Ridge corre- i : "sponding to basal furrow 3 3 | = 2 Ventricular : cavity ' Thin roof of oe ae j cerebral vesicle laid back to expose cavity EXPLANATION OF FIGURES 50 Ventral view of brain. X 3. 51 Dorsal view of brain in front, with the thin roof of the forebrain vesicle opened up. X 3. The Expressions of Emotion in the Pigeons. I. The Blond Ring-Dove (Tur- | tur risorius). By Wallace Craig. Journal of Comparative Neurology and Psychology, vol. xix, no. 1, April, 1909, pp. 29-82, with one plate. ERRATA I. In the musical records Nos. 1, 3, and 31, and the score on page 33, the eighth-note with a line across its hook should have been made much smaller by the engraver; it is an acciac- catura. ; “8ve,’’ wherever it occurs, applies to all the notes in that record. No. 12, the first note is mezzo forte. Nos. 10, 12, and 24, the sharps and flats represent a degree of sharpness or flatness less than a semitone. The runs are continuous, with very small inter- vals. In No. 12, the double-flatted e is but slightly lower than e flat. All this should have been explained in “‘Prefatory Remarks,”’ p. 32-33. Nos. 7, 8, 9, 10, 11, 14, and 31 should have no time signature and no division into bars. No. 14, the ¢ in the last bar is natural. No. 17, the a in the last bar is natural. ‘ No. 21, the crescendo sign over the second bar should be a diminuendo; in the last bar, the half-rest should be a whole-rest. Nos. 22 and 23, omit the rest at end of each; also, in each, the syllables “go o”’ should be placed under the last two notes, and the “Sve” should be placed above the score. Nos. 25, 26, 27, the single slur should be a double slur. No. 33B, last bar, and No. 33C, last bar, the a is natural. II. In the text Page 438, line 5 from bottom, for aspects read respects. Page 49, lines 12 and 8 from bottom, for “goo o’’ read “go o.”’ Page 51. line 7, for 16 read 46. Page 56, line 18, for legs read leg. Page 57, line 2 from bottom, for fifteenth read sixteenth. line 1 from bottom, for sixteenth read seventeenth. Page 58, line 1, for twenty-fourth read twenty-fifth. line 2, for twenty-third read twenty-fourth. line 4, for thirtieth read thirty-first. Page 59, line 15, for twelfth read thirteenth. Page 66, in the table, first column, fifth item, for 9th to 14th read 10th to 14th sixth item, for 15th to 17th read 16th to 18th. Page 73, line 4 from bottom, for goes read coos. Page 77, line 3 to 2 from bottom, for indiffer-cut read indiffer-ent. This table of ERRATA is printed and published in 1913. ‘ Please insert this leaf at the beginning of the article referred to. Oe Ee a i a ia ee 1K, i THE COURSE WITHIN THE SPINAL CORD OF THE NON-MEDULLATED FIBERS OF THE DORSAL ROOTS: A STUDY OF LISSAUER’S TRACT IN: THE, CAT S. WALTER RANSON From the Anatomical Laboratory of the Northwestern University Medical School ELEVEN FIGURES It has been shown that the small nerve cells of the spinal gan- glia give rise to non-medullated axons, each of which divides dichotomously into a small non-medullated fiber running toward the periphery and an even smaller one centrally directed in the dorsal root. ‘These non-medullated fibers can be followed for long distance$ in the nerve on the one hand, and through the dorsal roots to the spinal cord on the other. Just as the small cells of the ganglion far outnumber the large cells, so the non-medullated fibers of the nerve and the dorsal root far outnumber those which are medullated. Since these facts with the related literature have recently been discussed in detail (Ranson ’11, 712) it will be unnec- essary to repeat them at length in this place. Partly with reference to this problem and partly with reference . to the variations in the pyramidal tracts, we have recently been studying the spinal cord in a number of mammals. In looking over the accumulated material it became evident that the sec- tions of the spinal cord of the cat were especially suited for the solution of the problem of the central course of the afferent spinal non-medullated fibers. In fact, the course of these fibers stands out with diagrammatic clearness in the pyridine-silver prepara- tions of the spinal cord of the cat. 259 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 4 auGustT, 1913 260 S. WALTER RANSON TECHNIQUE The spinal cord of the cat forms very favorable material to which to apply the pyridine-silver technique. Preparations ob- tained from the cords of other laboratory animals (dogs, rabbits, guinea-pigs and rats) are not so perfect. Under anaesthesia the animals were exsanguinated. The spinal cord was exposed and the ganglia on the C’, C’, T4, Ts, T”, L, and S! spinal nerves were dissected out and left attached to their respective segments of the spinal cord. These segments with their associated roots and ganglia were then removed and freed from dura. Preparations were made by the Pal-Weigert as well as the pyridine-silver method. For the latter the pieces were not more than 5 mm. long, and after the usual treatment (Ranson 711), were imbedded in paraffin and cut into sections varying from 8 to 124 in thickness. Serial sections of a number of levels were mounted, to trace more in detail the entering bundles from the roots. The Pal-Weigert material was cut in celloidin into sections of 12 » and 24 u thickness. Usually sections of both thicknesses were cut from each segment. The technique of the silver stain has already been published (Ranson: ’11 and 12) and the Pal-Weigert method needs no explanation. Structure of the substantia alba of the spinal cord Cajal’s silver method and its various modifications are known to give instructive preparations of the gray substance of the nervous system. In the preparations of the spinal cord of the cat it is superbly stained, showing the greatest detail in the peri- cellular fiber plexuses, and the endocellular fibrillar reticulum. It is, however, in the fiber columns of the cord that the pyridine- silver preparations present the most interesting differentiation. As in the peripheral nerves, the axons of the medullated fibers stain a light yellow and are surrounded by unstained rings of myelin; while the non-medullated axons are dark brown or black. Areas consisting chiefly of medullated fibers appear light yellow under low magnification because of the predominance of light yellow axons and unstained myelin. Other areas are lighter or LISSAUER’S TRACT IN THE CAT 261 darker brown according to the number of dark brown non-medul- lated axons which they contain. Under higher magnification the non-medullated axons are sharply differentiated from the faintly yellow, granular neuroglia. The neuroglia fibers are not differ-_ entiated. We wish briefly to call attention to those tracts which imme- diately surround the posterior cornu. The cuneate and gracile fasciculi are composed very largely of medullated fibers (fig. 1). But in the neighborhood of the pos- terior commissure and posterior cornu there are numerous non- medullated fibers probably of endogenous origin. On the whole, however, these two fasciculi contain fewer non-medullated fibers than any other portion of the substantia alba, with one exception to be mentioned later. They are for this reason more lightly stained than is the anterior and most of the lateral funiculus. The number of non-medullated fibers in the fasciculus cuneatus and fasciculus gracilis is not sufficient to account for an upward continuation in these columns of the non-medullated fibers of the dorsal roots, and indicates that one must look elsewhere for their course within the cord. The dorsal spino-cerebellar tract is also composed chiefly of medullated fibers, and is the most lightly stained of all the fiber columns of the cord. It is indicated in figure 1, c, as a light area contrasting sharply with the darker pyramidal tract (d) and the still darker tract of Lissauer (6). It is broadest near its posterior medial extremity where it is sharply marked off from the latter tract by a rather thick pial septum. It becomes gradually less distinct in an antero-lateral direction. Ventro-medially from this fasciculus and separated from it by no very sharp line is the pyramidal tract, d. The area occupied by the latter is somewhat rounded in outline and lies just lateral to the cervix and caput of the posterior cornu. ‘Sharply outlined by its dark staining from the dorsal spino-cerebellar fasciculus, it is not so clearly separated from the remainder of the lateral funiculus. Next to Lissauer’s tract the pyramidal tract is the darkest part of the substantia alba, and contains a very large number of non-medul- lated fibers. In comparison with the tract in the rat (Ranson 713), 262 S. WALTER RANSON the cat’s pyramidal tract is much better medullated and contains in addition to non-medullated and small medullated axons also large axons with thick myelin sheaths. This is not the place, however, to enter into a discussion of the comparative histology of the pyramidal tract. ; Lissauer’s tract Pal-Weigert preparations. Lissauer’s tract occupies the apex of the posterior cornu, lying just dorsal to the substantia gelatinosa. It occupies the entire apex, reaching to the surface of the cord, except in the thoracic segments. Here the substantia gelatinosa, capped by the tract of Lissauer is at some distance from the sur- face; and the posterior part of the apex consists of a thin pial septum. Because of the light stain which the tract takes in Weigert preparations it is usually considered as a part of the posterior cornu, although it is admitted by all that it properly belongs with the longitudinal fiber columns of the cord. In Pal-Weigert preparations it is seen to consist of rather sparsely arranged and uniformly fine medullated fibers. The spacing is uniform, each fiber being separated from its neighbors by a considerable interval. There is no grouping of these fibers into bundles nor are there any large spaces devoid of fibers. The fibers for the most part run vertically in the bundle, but some have an inclination forward toward the substantia gelatinosa. The obliquity of the fibers is most pronounced at the posterior extremity of the tract and in the neighborhood of entering root bundles. Starting in the region of an entering root bundle and passing forward toward the substantia gelatinosa, one can often see all gradations between horizontal fibers, and oblique fibers and again between oblique and vertical fibers. This would be in harmony with the generally accepted view that the medullated fibers of this tract are derived from the dorsal root, a view which has recently been called into question. In addition to the verti- eal and oblique fibers, just mentioned, one sees horizontal fibers of fine caliber, a few in each section running long distances through the tract to enter the stubstantia gelatinosa. These are prob- LISSAUER’S TRACT IN THE CAT 263 ably in part fibers directly out of the dorsal roots which have not had a previous vertical course in the cord, and in part the hori- zontal continuation of the vertical fibers. Although there is no septum separating the fasciculus cuneatus and entering root zone from Lissauer’s tract, the border between the two is represented by a very sharp almost straight line; and the contrast between the closely packed large medullated fibers of the one and the more scattered fine medullated fibers of the other is very striking. On the lateral side the tract is separated from the lateral funiculus by a rather thick and very constant pial septum (not present in man). This pial septum never quite reaches the substantia gelatinosa; and ventral to this septum there is no sharp line of separation between this tract and the adjacent portion of the lateral funiculus. Lissauer’s tract seems to extend laterally upon the dorsal surface of the substantia gelatinosa, its fine scattered medullated fibers gradually giving place to more thickly packed large ones. This gradual transition between the ventral part of Lissauer’s tract and the lateral funiculus attracted the attention of Lesz- lényi (12) who made a very elaborate comparative study of Pal- Weigert preparations of the spinal cord of mammals, birds and reptiles. He saw many oblique fibers in the transition zone between the tract of Lissauer and the lateral funiculus; and con- cluded that many of the vertical fibers of Lissauer’s tract come from the lateral funiculus through this transition zone. But the evidence which he presents in his paper, and the observations which I have been able to make on the cord of the cat, do not seem to me to show anything more than that there is an inter- mingling of the fibers of Lissauer’s tract with those of the lateral funiculus ventral to the pial septum. Leszlényi states that in all the cords he examined, with the possible exception of the human cord, the dorsal root fibers enter the posterior funiculus in such a way that one may be sure that no considerable number find their way into the tract of Lissauer. This seems to agree with ‘the findings of Waldeyer (’88) on the cord of the gorilla, where few, if any, fibers could be traced from the dorsal roots into Lissauer’s tract. Leszlényi finds, in all, four 264 S. WALTER RANSON kinds of medullated fibers in the tract of Lissauer: (1) fibers from Flechsig’s ground bundle; (2) fibers which unite the posterior and lateral funiculi at the same level; (3) fibers out of the substantia gelatinosa which after a short course enter the gray substance again; (4) in man and many animals there are horizontal fibers which come from the dorsal roots and cross the tract of Lissauer to enter the substantia gelatinosa. In his opinion the dorsal roots contribute practically nothing to the vertical fibers of the tract. This seems to be borne out, in part at least, by experimental and pathological data. There are a large number of papers, deal- ing with the course of the dorsal root fibers within the spinal cord, based on a study of Marchi preparations of the cord after lesions of the dorsal roots. Human cords, in which extensive degeneration of the dorsal roots has resulted from tumors, syphi- lis and other causes, have been studied as well as the cords of animals in which the roots have been divided. Most of these papers, although describing extensive degeneration in the pos- terior funiculus, make no mention of any degeneration in the tract of Lissauer. (See the papers of Darkschewitsch ’96, Frolich ’04, Kopezynski ’06, Marguliés ’96, Orr ’06, Wallenberg ’98 and Zappert 798.) Nageotte (’03) states that the tract of Lissauer is composed of fine medullated fibers of endogenous origin. He maintains that they can not be derived from the dorsal roots, because in a case reported by him, in which a tumor involved all the spinal roots in the cauda equina up to and including the fourth lumbar, the fine fibers of the tract of Lissauer were intact. The presence of these intact fibers in this case shows conclusively that many, perhaps a majority, of the fine vertical medullated fibers of this tract are of endogenous origin. It can not be taken as conclu- sive proof that none of the fibers in this tract are derived from the dorsal roots. | . Laignel-Lavastine (’08) studied the spinal cord in a case of syphilis involving the cauda equina. The Marchi stain showed a very few degenerating fibers in Lissauer’s tract, and the Weigert stain showed the vast majority of the fibers in this tract to be LISSAUER’S TRACT IN THE CAT 265 intact. He admits the contention of Nageotte that such degen- eration as he finds may be tertiary, but insists that one cannot be certain that the tract does not contain some fibers from the posterior roots. Sibelius ('05), who studied three cases In which the cauda equina was involved, found some degeneration in Lissauer’s tract which he considers as the direct result of the root lesion. He explains the negative findings of Nageotte by assuming that the fine horizontal fibers of Lissauer’s tract had disappeared and that the author had failed to notice their absence. This explanation is tantamount to an admission that the proportion of dorsal root fibers in the tract is small and that they are chiefly horizontal. Sottas (93) and Collier and Buzzard (’03) find a limited amount of degeneration in Lissauer’s tract after dorsal root lesion. The evidence seems to show that the medullated fibers in the tract of Lissauer are in part endogenous and in part exogenous and that the endogenous fibers predominate. We shall return to this question again in discussing the entrance of the dorsal root fibers into the spinal cord. he Pyridine-silver preparations. In pyridine-silver preparations the tract of Lissauer is stained very dark and is even more sharply outlined from the rest of the cord than in the Pal-Weigert prep- arations. It is seen to consist of vertically or obliquely coursing axons of the smallest diameter (fig. 2,b). These are stained a brownish black and are very sharply differentiated from the almost colorless background. They are very closely set together, although there are scattered among them a few medium sized yellowish brown axons which correspond in number and arrange- ment to the medullated fibers seen in the Pal-Weigert prepara- tions. When compared with Pal-Weigert preparations of the same segment of the cord the contrast is very striking. In the latter the tract of Lissauer is very lightly stained and is seen to consist of rather sparsely scattered fine medullated fibers. A glance at the two preparations is sufficient to show that the number of axons in the one is several times greater than the number of myelin sheaths in the other. Lissauer’s tract consists then in part of small medullated fibers but its chief and charac- 266 S. WALTER RANSON teristic content is an enormous number of fine vertically coursing non-medullated fibers. Before taking up the account of the course taken by the dorsal root fibers as they enter the cord it will be desirable to call atten- tion to the variations in shape and topography of the tract of Lissauer at different levels in the cat’s cord. In the cervical region as illustrated by the seventh cervical segment (fig. 1) the tract is long and narrow, since the substantia gelatinosa is at some distance from the surface of the cord and the tract fills the entire apex of the posterior cornu. Throughout the entire tho- racic region of the cord (fig. 3, T. 8) the posterior cornu is not well developed; the substantia gelatinosa is placed a long dis- tance from the surface of the cord; and the cone-shaped Lissauer’s tract which caps it is also some distance removed from the sur- face. The apex of the cone is connected with the surface by a septum containing few fibers; occasionally isolated portions of Lissauer’s tract are seen along the medial side of this septum. In the lumbar cord (fig. 4, L. 5) the substantia gelatinosa has approached the surface; and the tract of Lissauer, which fills the interval between it and the surface has become short and wide. In the sacral region (fig. 5, S. 1) the substantia gelatinosa has flattened out laterally and the tract of Lissauer occupies a short but wide interval between it and the periphery of the cord. The size of the tract varies somewhat from level to level and seems to be roughly in proportion to the size of the nerve roots entering at that and adjacent levels. The first sacral segment seems to be an exception to this rule. Although its entering rootlets are larger, it presents a Lissauer’s tract somewhat smaller than that in the fifth lumbar segment. These facts would be easily explained if we assume that the majority of the non-medul- lated fibers in the tract are short ascending fibers from the dorsal roots. The small rootlets of the lower sacral segments would then contribute fewer ascending fibers to the tract in the first sacral segment, than would be contributed by the large rootlets of the upper sacral and last two lumbar nerves to the tract in the fifth lumbar segment. The ascending fibers must be rela- tively short, however. Otherwise there would be a steady increase LISSAUER’S TRACT IN THE CAT 267 in size from lower to high levels in the cord. But the tract seems rather to be proportional in size to the entering rootlets; and instead of a steady increase in size in an ascending direction, there is a marked decrease in going from the lower lumbar into the thoracic cord and again in passing from the lower cervical to the upper cervical segments. These facts indicate that the majority of the non-medullated fibers are short ascending fibers and in the next section we will show that many, probably a great majority, are derived from the dorsal roots. Entrance of the-dorsal roots into the spinal cord Pal-Weigert preparations. In 1885 Lissauer observed that fine medullated fibers grouped themselves on the lateral side of an entering rootlet and turning lateralward separated themselves from the remainder of the rootlet to enter the apex of the pos- terior horn, where they turned to run vertically in the tract which now bears his name. Similar observations were made by Bech- terew (86), and have formed the basis of the standard text-book accounts of this tract. It is an easy matter to confirm these observations in Pal-Weigert preparations of the cat’s cord, espe- cially in the case of the larger cervical and sacral roots. There ean be no doubt that in the cat medullated fibers enter the tract of Lissauer from the dorsal roots in considerable number, and in exactly the manner described by Lissauer and Bechterew. There can also be no doubt, on the basis of the pathological and experi- mental evidence presented in preceding paragraphs, that these medullated fibers from the dorsal root do not constitute all or even the majority of the medullated fibers in this tract. Pyridine-silver preparations. 'To the medial side of the tract of Lissauer is the entering root zone. In this region the medullated fibers, which have just entered the cord, can be seen running more or less obliquely (fig. 10, a). The fibers seen here are for the most part large medullated ones; and, when one remembers the large number of non-medullated fibers seen in the dorsal root, one is impressed with the scarcity of these fibers in the entering root zone. Somewhat further medialward large numbers of fine 268 S. WALTER RANSON collaterals are given off from the medullated fibers and take a more or less oblique course toward the posterior horn. But these, for the most part, take a much lighter stain than the non-medul- lated fibers. Occasionally medium sized bundles of non-medul- lated fibers are seen in the entering root zone as at e in figure 10, but such bundles usually have a direction nearly at right angles to the medullated fibers, and are making their way toward the tract of Lissauer. We shall see that the majority of non-medul- lated fibers separate themselves from the medullated just before the entrance into the cord. The scattered non-medullated fibers as well as the bundles of such fibers in the entering root zone, were delayed in their separation from the medullated fibers; but most of them finally find their way into the tract of Lissauer. It is possible, however, that a few of the non-medullated fibers pass medially to the tract of Lissauer and enter the fasciculus cuneatus. A dorsal root as it enters the cord is broken up into a large number of fila radicularia or rootlets. As each rootlet enters the cord it is surrounded and constricted by an encircling band of pia. Shortly before the rootlet reaches this constricting band, the non-medullated fibers, which nearer the ganglion have been distributed quite uniformly throughout the root, separate out from the medullated ones and come to lie either at the periphery of the radicles or along septa which divide the radicles into smaller bundles. In this way large flat bundles of non-medullated fibers are formed; often a thin layer of such fibers is seen making a complete tubular sheath at the periphery of the radicle. In serial sections it is possible to trace these compact bundles into the cord and see that they enter Lissauer’s tract. It is this early separation of these fibers from the main mass of the radicle that causes the entering root zone to be composed almost entirely of medullated fibers. We will now take several typical-instances and show how these fibers can be traced into Lissauer’s tract. In the first sacral segment (figs. 5, 6 and 7) the radicles are of good size and are divided by connective tissue septa, running in a general antero- posterior direction, into smaller fascicles which are displaced LISSAUER’S TRACT IN THE CAT 269 medially as one after another enters the cord. Figure 5 is a diagrammatic representation of the level from which figures 6 and 7 were taken. At a is indicated the tract of Lissauer; b and c represent connective tissue septa. In the high power drawings the same lettering has been used. In figure 6 one sees that most of the non-medullated fibers have separated out from among the medullated and arranged themselves along the septa b and c. At d and d’ are seen bundles of non-medullated fibers arranged along the inner border and the medial part of the posterior border of the radicle. The fibers in bundle d can be traced at this level into Lissauer’s tract. The fibers in d’ can be traced along the surface of the cord for a short distance and then turn ventrally into the same tract. Bundle c is composed of fibers which have separated out from the two root fascicles between which it lies. It is composed of two layers of fibers arranged one on each side of a connective tissue septum. If one follow this bundle upward in the serial sections its fibers are seen to turn ventrally and run into the tract of Lissauer (fig. 7, c). These fibers along the con- nective tissue septa of the roots are in size and staining reaction exactly like the non-medullated fibers which farther distally are scattered uniformly through the roots. They are also identical in size and staining reaction with the non-medullated fibers of the tract of Lissauer into which they have just been followed. It seems that a clearer demonstration of the fate of the non- medullated fibers of the dorsal roots could scarcely be desired. In the fifth lumbar segment of the cat’s cord the entering radicles are smaller. Only when they are covered by a thick coat of connective tissue and bound down tightly to the surface of the cord are the physical conditions satisfactory for the impreg- nation of the non-medullated fibers. Figures 4 and 8 show such a rootlet entering the cord. Notice that its fibers pass through Lissauer’s tract and separate off a small dorso-median portion from the main tract. This unites again with the rest of the tract above and below the entering root bundle. The constrict- ing ring at the entrance of a radicle into the cord is well seen in figure 8. Points a and ¢ are joined in the thickness of this sec- tion and the next by an arched band of connective tissue. The 270 S. WALTER RANSON projecting points and this band form part of the constricting ring. Upon the surface of this band and separating it from the medul- lated fibers of the bundle can be seen a layer of closely packed non-medullated fibers, b. They can be followed for only a short distance at b as they arch over the constricting band but between b and ¢ they can be traced over this band into Lissauer’s tract. Along the ventral surface (d) of the extra-medullary part of the bundle and along the connective tissue septum (e) separating this from the next root bundle the non-medullated fibers have accumu- lated. Along the line a, ), c, d, e, there is indicated a peripheral layer of non-medullated fibers which a little more than half sur- rounds the bundle of entering medullated fibers. Whether a similar layer is present on the dorso-medial surface of the radicle it is impossible to say, since it is not likely that the fibers would take the stain in this superficial position if they were present. The general principles which govern the entrance of the non- medullated ‘fibers into the cord are well illustrated by the radicle from the seventh cervical dorsal root which is shown at three levels in figures 9,10 and 11. The lettering in these three figures is the same as in figure 1 from the same segment and to which reference should be made for orientation. Figure 9 is from a section just above the level of entrance of the radicle in ques- tion. The medullated fibers of this rootlet are seen cut obliquely at a, and at b is indicated the tract of Lissauer. At 1 is seen a bundle of non-medullated fibers which are arching ventro-laterally over the obliquely coursing medullated fibers, a, of the entering rootlet. Dorso-laterally bundle 1 is centinuous.with bundle 2 of more vertically running fibers. A study of serial sections shows that bundle 1 is derived from two sources. First there are upon the upper surface of bundle a, as it enters the cord, a large number of non-medullated fibers which turn upward and then ventro-laterally to enter into the formation of bundle 1. A few of the uppermost of these are seen at 3. The second group of fibers entering into bundle 1 are derived from bundle 2. Tracing the same structures downward through the series we find them arranged as in figure 10. Here bundle a, composed LISSAUER’S TRACT IN THE CAT D7 chiefly of medullated fibers, is seen entering the cord. At 4a bundle of non-medullated fibers can be seen running over the constricting ring to pass directly into the tract of Lissauer on entering the cord. At 5 are seen a few non-medullated fibers gathering along a line which represents the beginning of a line of separation between two fascicles of the root. Bundle 2 is larger here than in the preceding section and is placed between bundle a and another bundle more dorsally situated. It is composed of fibers which have separated out from these two bundles and especially from bundle a, and, instead of running directly across the bundle of medullated fibers to reach Lissauer’s tract, have turned upward or downward on the dorsal surface of bundle a. In this way a vertical bundle of large size is formed, which traced upward is seen to arch ventro-laterally over the upper surface of bundle a to form part of bundle 1, figure 9, and so to reach the tract of Lissauer. Traced downward, the descending fibers which enter into its composition are seen to turn ventro-laterally below bundle a to run into Lissauer’s tract (fig. 11). Here they form part of a rather wide band of fibers on the medial and under surface of bundle a. A part of this band is seen at 6, figure 11. But the band is much wider than is indicated in the figure. In the succeeding sections the band is seen to underlie bundle a to the very edge of the cord. , This band is composed in part of fibers derived from bundle 2, figure 10, and in part from fibers entering the cord as a layer upon the under surface of bundle a. It thus appears that the non-medullated fibers separate out from the bundles of entering root fibers. They occupy the pe- riphery of the bundles as these enter the cord and then take the route of least resistance to reach Lissauer’s tract. In the case of the fibers on the dorso-medial surface of such entering root bundles this path of least resistance is usually around the bundle of entering medullated fibers rather than through it. For this reason these fibers form bundles arching over or under the bundle of medullated fibers. For those non-medullated fibers occupying the ventro-lateral portion of the surface of the entering dorsal root bundle the path to Lissauer’s tract is direct (fig. 10,4). In 272 S. WALTER RANSON the case of the first sacral segment the non-medullated fibers sepa- rate out along the septa separating the fascicles of the rootlet, and run forward along these septa into the underlying Lissauer’s tract before the rest of the bundle has entered the cord. The non-medullated fibers of the dorsal root, then, enter the tract of Lissauer, of which they form the chief and characteristic part. They run for short distances in this tract chiefly in an ascending direction and then probably pass forward into the substantia gelatinosa. The close relation of Lissauer’s tract to this peculiar substance which caps the posterior horn, the fact that fibers can be seen passing from one into the other, and the fact that there is no other apparent outlet for the fibers of Lissauer’s tract indicate that the substantia gelatinosa is the probable nucleus of reception of these non-medullated fibers. The fact that the substantia gelatinosa contains a large number of very small nerve cells and many non-medullated nerve fibers is of interest in connection with the probable relation of the two structures. Experimental evidence is needed, however, to prove this relation conclusively. So far as the function of the non-medullated fibers is concerned, their course within the cord shows that they can have little or nothing to do with the afferent impulses received from muscles and joints which travel up the posterior funiculus. This does not necessarily include muscle and joint pain. Their early ter- mination within the gray substance would agree with the course of the sensations of pain and temperature and probably also with that of touch. But there are, of course, no data on which one would care to hazard a guess as to their function, beyond the statement that they can have little or nothing to do with those sensations which are known to travel directly upward in the posterior funiculus. LISSAUER’S TRACT IN THE CAT 273 SUMMARY 1. The small cells of the spinal ganglion give rise to non- medullated fibers whose centrally directed branches form the non- medullated fibers of the dorsal roots. 2. The non-medullated fibers of the dorsal roots can be traced with diagrammatic clearness into Lissauer’s tract. 3. The tract of Lissauer contains rather sparsely arranged fine medullated fibers which are in part derived from the dorsal roots but are in greater part of endogenous origin. 4. The tract of Lissauer contains a very great number of fine non-medullated axons, at least the great majority of which are derived from the non-medullated fibers of the dorsal roots. 5. It is probable that the substantia gelatinosa is the nucleus of reception for the non-medullated fibers. 6. It seems clear that the non-medullated fibers have little or nothing to do with the transmission of the afferent impulses from the muscles and joints, at least with such part of these impulses as are transmitted upward in the posterior funicull. BIBLIOGRAPHY BrecuTEREW, W. 1886 Uber einen besonderen Bestandtheil der Seitenstriinge des Riickenmarks und iiber den Faserursprung der grossen aufsteigen- den Trigeminuswurzel. Arch. f. Anat. u. Physiol., Anat. Abt., p. 1. Couuier, JAMES, AND Buzzarp, E. F. 1903 The degenerations resulting from lesions of posterior nerve roots and from transverse lesions of the spinal cord in man. Brain, vol. 26, p. 559. DarkscHEwitscH, L. O. 1896 Zur Frage von den secondiiren Veriinderungen der weissen Substanz des Riickenmarks bei Erkrankung der Cauda equina. Neurol. Centralbl., vol. 15, p. 5. Frouicu, A. 1904 Beitrag zur Kenntniss des intraspinalen Faserverlaufes einzel- ner hinterer Riickenmarkswurzeln. Arb. a. d. Neur. Inst., Wien., Bd. ILE 4 oy Gitfters GoLpsTEIN, K. 1903 Die Zusammensetzung der Riickenmarkshinterstringe. Monatschr. f. Psych. u. Neur., Bd. 14, p. 401. Jacospsoun, L. 1907 Beitrige zum intramedulliren Verlaufe von hinteren Wur- zeln des Cornus medullaris. Neurol. Centralbl., Bd. 26, p. 386. Kopcyznsk1, 8. 1906 Experimentelle Untersuchungen aus dem Gebiete der Anatomie und Physiologie der hinteren Spinalwurzeln. Neurol. Cen- tralbl., Bd. 25, p. 297. LAIGNEL-LAVASTINE 1908 Le systéme des fibres endogénes des cordons pos- térieurs dans la dégénérescence ascendante des racines de la queue de cheval. Compt. rend. Soc. de biol. T. 64, p. 223. 274. S. WALTER RANSON LeszLenyI, O. 1912 Vergleichend-anatomische Studie iiber die Lissauersche Randzone des Hinterhorns. Arbeiten a. d. Neurologischen Inst., Wien, Bd. 19, p. 252. Lissaver, H. 1885 Beitrag zur pathologischen Anatomie der Tabes dorsalis’ und zum Faserverlauf in menschhchen Riieckenmark. Neurol. Cen- tralbl., Bd. 4, p. 245. Mareuuins, A. 1896 Zur Lehre vom Verlaufe der hinteren Wurzeln beim Men- * schen. Neurol. Centralbl., Bd. 15, p. 347. Naceotre, M. J. 1903 Note sur les fibres endogénes grosses et fines des cordons postérieurs et sur la nature endogéne des zones de Lissauer. Compt. rend. Soc. biol. T. 55, p. 1651. Orr, D. 1906 The descending degenerations of the posterior columns in trans- verse myelitis and after compression of the dorsal posterior roots by tumors. Rev. of Neurol., and Psychiat., vol. 4, p. 488. Ranson, &. W. 1911 Non-medullated nerve fibers in the spinal nerves. Am. Jour. Anat., vol. 12, p. 67. 1912 The structure of the spinal ganglia and of the spinal nerves. Jour. Comp. Neur., vol. 22, p. 159. 1913 The fasciculus cerebro-spinalis in the albino rat. Am. Jour. Anat., vol. 14, p. 411. Stpeuius, C. 1905 Drei Falle von Caudaaffektionen nebst Beitrigen zur topo- graphischen Analyse der Hinterstrangserkrankungen. Arbeiten a. d. Path. Inst. Univ. Helsingfors., Bd., 1; p. 79 Sorras, J. 1893 Des Dégénérescences de la Moelle Consécutives aux Lésions des Racines Postérieures. Rev. de Médicine, T. 13, p. 290. WaupEyerR, W. 1888 Das Gorillariickenmark. Abhandlungen der Berliner Akademie; cited after Leszlényi. WaLLENBERG, A. 1898 Beitriige Topographie der Hinterstriinge des Menschen. Deut. Zeitschr. f. Nervenheil., Bd. 13, p. 441. ZAprERT, J. 1898 Beitriige zur absteigenden Hinterstrangsdegeneration. Neurol. Centralbl., Bd. 17, p. 102. PLATE 1 . EXPLANATION OF FIGURE The drawings were made with the aid of a camera lucida from pyridine-silver preparations of the spinal cord of the eat. 1 From the seventh cervical segment. The substantia grisea presents a dense mass of interlacing fibers. The nerve cells are indicated in solid black. Capping the posterior cornu is the lightly staining substantia gelatinosa. More dorsally is seen the darkly staining tract of Lissauer, b. At a, is seen the obliquely cut medullated fibers of the entering dorsal root in the fasciculus cuneatus. At c, is represented the dorsal spino-cerebellar tract, and at d, the pyramidal tract. The differentiation of the fiber columns of the cord shown in the drawing is due to the varying proportion of non-medullated fibers which they contain. These are most abundant in the tract of Lissauer. The pyramidal tract contains the next greatest number, and the dorsal spino-cerebellar tract the fewest. > 23. LISSAUER’S TRACT IN THE CAT PLATE 1 Ss. WALTER RANSON ie! Al f . ' th Vie Bh WY Nil NAO THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 4 276 S. WALTER RANSON PLATE 2 EXPLANATION OF FIGURES 2 From the seventh cervical segment. A narrow strip at right angles to the apex of the posterior cornu: a, fasciculus cuneatus; b, Lissauer’s tract; c, the dor sal spino-cerebellar tract. The medium sized and large lightly stained axons are medullated, the fine darkly stained ones non-medullated. The latter are very numerous and closely packed together in the tract of Lissauer. 648. 3 Diagrammatic representation of the eighth thoracic segment, showing the shape and position of Lissauer’s tract. X 17. 4 Diagrammatic representation of the fifth lumbar segment, showing the shape and position of Lissauer’s tract. X 17. 5 Diagrammatic representation of the first sacral segment, showing the shape and position of Lissauer’s tract. a, Lissauer’s tract, b and c, connective tissue septa dividing the entering root bundle into smaller fascicles. Bundles of non- medullated fibers are grouped along these septa. Other bundles of non-medul- lated fibers are seen at d andd’. X 17. PLATE 2 LISSAUER’S TRACT IN THE CAT S. WALTER RANSON e Co z » x v = . 4 s 2 ¢ ae KAU. 1903. KA W9- 278 S. WALTER RANSON PLATE 3 EXPLANATION OF FIGURES 6 Lissauer’s tract and entering dorsal root in the first sacral segment. Letter- ing is the same as in figure 5, which gives the topography of the high power drawing. Non-medullated fibers are separating out along the borders of the entering radicle and along the connective tissue septa which separate it into fascicles. At d non- medullated fibers are seen running forward into Lissauer’s tract. > 100. 7 Same area as represented in figure 6 but about 504 farther cephalad. Letter- ing the same as in figures 5 and 6. The non-medullated fibers of the dorsal root, which have separated out along the connective tissue septum c, are seen running forward into the tract of Lissauer, a. X 100. 8 Lissauer’s tract and entering dorsal root in the fifth lumbar segment. For to- pography see figure 4. At a, b, c, is seen a part of an encircling band of pia which surrounds and constricts the entering radicle. Upon the surface of this band a layer of non-medullated fibers is seen entering the cord. At d, and e, are seen thin layers of non-medullated fibers at the periphery of the radicle and along the sep- tum separating the radicle into two fascicles. > 100. PLATE 3 LISSAUER’S TRACT IN THE CAT 8. WALTER RANSON 279 280 S. WALTER RANSON PLATE 4 EXPLANATION OF FIGURES 9,10 and 11 Three sections from a series through an entering radicle of the seventh cervical dorsal root. For topography see figure 1. Figure 9 represents a level just above the entrance of the radicle (a) into the cord, figure 10, a level through the middle of the entering radicle, and figure 11, a level near its lower mar- gin. The lettering is the same as that in figure 1. At 1, 2, 3, 4, 5 and 6 are indi- cated bundles of non-medullated fibers which can all be traced into Lissauer’s tract. XX 82. PLATE 4 LISSAUER’S TRACT IN THE CAT S. WALTER RANSON ty “Say : a ry "903 ‘ty thers th ae he Wit. Kothari 281 iy - 7 A ¥ ‘ni NA y Vie” Seb Bu, " het al WEP ' i ad 7 nay a - fa ; . on es . if : y = - alas = => xe an | " ‘ 4 . } a | c be ' , - > ‘ ‘ A nt ae ’ nya ~~ 7 q 7 : ane THE EFFECTS OF FORMALDEHYDE ON THE BRAIN OF THE ALBINO RAT HELEN DEAN KING The Wistar Institute of Anatomy and Biology TEN CHARTS Although formaldehyde was discovered in 1863, it was not until thirty years later that Blum (93) and Hermann (’93), working independently, found that an aqueous solution of this substance is an excellent medium for preserving and hardening various ani- mal tissues. Owing to its many admirable properties and to its relative cheapness, ‘formalin’ (a commercial 40 per cent solution of formaldehyde) soon became extensively employed as a fixing and preserving reagent for entire brains as well as for other large pieces of tissue, being used either in a 5 to 15 per cent aqueous solution or combined with other substances such as aleohol, alum or salt. In an extensive series of experiments dealing with the effects of various formalin solutions on the weight of the brains of man, of sheep and of various other mammals, Hrdlicka (’06) showed that the size of the brain, the age of the animal from which the brain was taken and the strength of the solution used were all factors that influenced the weight and volume changes in the brain to a very considerable extent. Hrdlicka did not, however, ascertain the relative importance of these various factors, nor did he make any study of the histological effects produced in the brain tissue by formaldehyde solutions. At the present time formalin is commonly used in laboratories and museums as a fixative and also as a preservative for the brains of man and of other mammals. It has seemed worth while, therefore, to make a careful study of the changes produced by this substance, act- ing under different conditions, on a series of brains from animals of known ages from birth to maturity. Experiments of this kind 283 284 HELEN DEAN KING ought to define the limits of the use of formalin as a brain pre- servative and to indicate when and how this substance can be used to the greatest advantage in neurological work. The pres- ent paper records the results of such a series of experiments made on brains of the albino rat (Mus norvegicus albinus). As a 4 per cent solution of formaldehyde (10 per cent formalin) is the one that the experience of many investigators has shown is the best for brain preservation as well as for general histological work, a solution of this strength was the only one used in this series — of investigations. The technique employed in all the experiments was as follows: Animals of known ages were killed with ether, and their body lengths and body weights recorded. The brain, with its menin- ges intact, was removed as soon as possible after the death of the animal, being cut from the cord at the tip of the calamus seriptorius. Each brain was then weighed to a tenth of a milli- gram in a closed weighing bottle and placed on absorbent cotton in a definite amount of 4 per cent formaldehyde. The glass stoppered bottles in which the brains were kept were of uniform size, and they were inclosed in black covered cases to exclude light as, according to Fish (’95), this precaution will prevent the decomposition of the solution and the subsequent formation of paraformaldehyde. The brains were weighed at definite times which varied somewhat in different series of experiments. On . removal from the solution the brain was placed for a moment on filter paper to remove the superfluous liquid, it was then weighed as quickly as possible in a closed weighing bottle and returned to the solution. After a final weighing at the end of a stated period, the brains were dried for one week in a water bath which had a temperature of about 95°C. They were then cooled in a desiccator and reweighed in order to determine the effects of the solution on the percentage of solids in the brains. In some few cases, after the final weighing, brains were trans- ferred into alcohol, imbedded by the celloidin-paraffine method of Bédecker (’08), and then sectioned and stained with thionin in order to ascertain the histological effects that had been pro- duced. EFFECTS OF FORMALDEHYDE ON BRAIN OF RAT 285 THE WEIGHT CHANGES PRODUCED BY FORMALDEHYDE IN BRAINS OF ALBINO RATS OF KNOWN AGES Series 1. This was a preliminary set of experiments made to discover the general effects of a 4 per cent solution of formalde- hyde on the brains of rats of different ages. In these experi- ments brains were taken from animals of the following ages: new- born, 10, 20, 40, 50, 70 days and adults, approximately 200 days old. Three rats of each age were used, animals of a given age being taken from the same litter except in the case of adult rats which were of unknown parentage and therefore may or may not have belonged to the same litter. As it was not possible to obtain all the rats wanted for the experiments at one time, the initial weighing of the first lot of brains was made early in October, 1910, while the final lot of material was not obtained until February, 1911. Each brain was put into 40 ce. of a 4 per cent solution of formaldehyde that was neutralized with NaCQs. The solution of formaldehyde used was, in some cases, one that had been made up for some weeks; in other cases a fresh solution was made as wanted, either from formalin that had been in the laboratory for some time or from a newly purchased supply. The age of the solution or the condition of the formalin used in making the solution were factors that were not thought to be of importance and therefore no attention was paid to them. The different lots of brains were weighed at irregular intervals during the first week they were in the solution, then every seven days for nine weeks. At the end of this time each brain was transferred into a fresh solution, made at the time it was wanted for use. The bottles containing the brains were then sealed with paraffine and kept for two months at laboratory temperature. After eighteen weeks the brains received their final weighing and they were then dried to obtain the percentage of solids. In each set of brains of the same age the individual weighings were very uniform, as they showed a difference of only 2 or 3 per cent in the majority of cases. These differences can undoubtedly be ascribed to the fact that animals taken from the same litter often vary considerably in size even when they are of the same 286 HELEN DEAN KING sex, and with this difference in size is found a corresponding dif- ference in brain weight since, as shown by Donaldson (’09), the size of the brain is correlated with the size of the animal, not with its age. The times when the various weighings were made in this series of experiments, together with the average percentage weight increase for each group of three brains are given in table 1. TABLE 1 Percentage weight increase in rats’ brains kept for eighteen weeks in a stock solu- tion of 4 per cent formaldehyde neutralized with NaCO, (averages for three brains at each age) AGE OF RATS TIME SOLUTION ACTED l > New- 10 20 40a DO 70 200 | born | days | days | days days days days RNa ay: Pe Ea Be hc 21.6 | 30.0 | 28.5 | | 25.8 DG a ene a a DPR Oe es ho 24.3 | 29.6 | ROA VISe 1. Sere ee emer tera ome tee tee ors 26.7 | 715 |G fs ae aR rea een Bee 27.02 26.7 PMCS oy cou. di Mes Toe eee 31.5 76ST MO, a a a | 23.4 | 38.11] 29.91) 53.51] 27.81 40.9 | 35.8 BRCLEUNIS siecss 2 Seema: ote cute Slots 32 28.9 27.0 Obibas co cic.) 2 Set eek ae 28.3 25.4 Die elated 21.7 | 29.0 | 28.9 | 51.9.| 25.3 | 41.7 | 40.81 PUWEGKSEh, 1-2). «ae Aor oe he 20.9 | 27.2 | 28.4 | 48.0 | 25.2 | 42.9 | 40.7 Dwecse 20... 5, Me cake &, 18.9 | 30.1 | 28.0 | 47.8 | 24.9 | 44.2 | 39.6 RY WCOKSUeL. vs - 3/1 ae Onn 17242707) 28. % | 48:2)| Goal Aaa aes Biweckie,. a.s\.':.. 2a Aue ee He 15.4 | 29.4 | 27.9 | 46.7 | 24.3 | 44.9 | 38.0 EWC CRS ty! ... oa SR ee | 16.1 | 29.9 | 28.0 | 46.1 | 25.1 | 45.41) 39.8 Saweeksiee kin eee | 15.2 | 28.7 | 27.7 | 47.0 | 24.9 | 44.8 | 39.8 Ohwceksewr a0.) el eee 14.5 | 28.3 | 27.0 | 47.0 | 25.0 | 44.1 | 40.6 TDG) ee OR eee, 8 15.1 | 27.1 | 27.6 | 46.4 | 25.4 | 43.8 40.3 DS rwieckess. ges . SME 65.1 | 58.4 | 48.9 | 45.3 | 44.8 | 43.2 | 38.6 | 33.0 BUVRE GIS Seats. -New born. ‘60 10 days.- - -|- ~--} 10 days. 20 days... __|_ __- | 20 days. 50- 50 days. Seah ; : H3 50 days. 70 days. Za Pe || ‘70 days. 100 days. - “| ee: x “100 days. 200 days: 3o|- ; “200 days. 20 r 10 E Time in days. 4 { [es 1c ae NAS. ppaks 7 14 21 28 Chart 3 Showing the weight changes in brains of rats of various ages, each kept for four weeks in 40 cc. of a 4 per cent solution of formaldehyde neutralized with NaCO;. As presumably all the solutions used in this set of experiments had the same chemical composition, the variations in the amount of swelling in the different groups of brains can doubtless be attributed to a difference in the chemical composition of the brains at different ages. The size of the brain, as will be shown later, is not a factor that influences the amount of swelling to any considerable extent. 294 HELEN DEAN KING The data obtained when brains were kept in a non-neutralized solution of 4 per cent formaldehyde for four weeks are given in table 5. TABLE 5 Percentage weight increase in rats’ brains, each kept for four weeks in 40 cc. of a non-neutralized solution of 4 per cent formaldehyde made fresh for each lot of animals killed (averages for two brains at each age) AGE OF RATS TIME SOLUTION ACTED tak am | Pre Sa Oey : New- 10 20. |e 740 eeo0 70 | 100 206 born | days | days | days | days | days | days | days 1 day 5 0 Oc Rey he Fee 34.51 37.3 | 36.7 | 39.71 44.21 39.5 | 41.1! ee 533 (0 EVs 5 et ae ene Re ee eins a 18.6 | 45.14) 45.41 39.1 | 42.8 | 42.31) 39.4 | 35.4} i days Olt ee occ a ats MERA Ce AY 30 9.9) | 37-8") 88-2 | eorGr tos. 14 S470) |oore,|| oOLZ 2 weeks 3.5 | 30.4 | 34.6 | 31.5 | 32.6 | 31.5 | 29.0 | 26.7 ORWICE KS eee ial hone | 0240025295) 80afel 28he. | 00.6 |°:29.5 27.4 | 24.5 4 weeks......................J—1.5 PPT || PEASY PAD) || Parhte: | Piifasy |p mites) | ahs Average percentage gain..... 13.1 | 33.3 | 35.6 | 33.5 | 36.0 | 34.1 | 32.5 | 28.9 1 Maximum weight increase. As shown in the table, the effects of an acid solution of form- aldehyde on the brain of a new-born rat is most remarkable. The maximum weight increase amounts to only 34.5 per cent of the original brain weight, and it is attained at the end of the first day. There is then a rapid decrease in weight with each succeeding weighing until, at the end of four weeks, the brain actually weighs 1.5 per cent less than the original weight: this indicates that the solution has extracted some substance from the brain tissue. In both the brains of this age used the per- centage weight changes were practically the same, as there was a difference between them of less than 2 per cent at any weighing. Brains of ten-day-old rats do not show such remarkable weight changes as do those of new-born animals. In fact, an acid solu- tion of 4 per cent formaldehyde causes nearly the same amount of swelling in brains of all ages from ten days to maturity, there being a difference of less than 10 per cent between the maximum weight increase in any two sets of brains, and a difference of EFFECTS OF FORMALDEHYDE ON BRAIN OF RAT 295 less than 5 per cent between the final weights. The very great difference between the effects of the solution on the brains of new-born animals and those on the brains of other ages is brought out very clearly in the graphs in chart 4 which show the per- centage weight changes for each set of brains during the four weeks the experiments were continued. ° Percentage weight increase. Age of rat. 60 20 days.__ 50 days.- - - | - j SS 100 days:4y : : _| Age of rat 40 days. ~~ S _-20 days. 70 days’ 4 _|.-50 days. 30 ag 10 days ; teed 70 days. 200 days. ~ . ~ 40 days. » 100 days. 20- ‘ *. | 200 days. New born. Ss | 10 days. ig _| New born. - “Time in days. : i 14 21 28 Chart 4 Showing the weight changes in brains of rats of various ages, each kept for 4 weeks in 40 cc. of a non-neutralized solution of 4 per cent formaldehyde. This series of experiments shows that the neutralization or non- neutralization of a 4 per cent solution of formaldehyde has a marked effect on the amount of swelling of the brain tissue. The difference is shown in the graphs A and B of chart 5 which are plotted from the final percentage weight increase in the various sets of brains. As the weight of the brains of the new-born rats kept in the neutralized solution is some 62 per cent greater than that of the brains of the same age treated with the acid solu- tion, the two graphs are very far apart at their beginning. At the ten-day period the difference between the graphs is reduced nearly one-half. They then approach gradually, and at the end are comparatively close together as the final weighings of the two sets of adult brains differed by only 10 per cent. 296 HELEN DEAN KING Percentage weight increase. _ C. =| B. D. Age in days. | ee Bed Ee) | ees L 10 20 40 60 70 100 200 Chart 5 Showing the final percentage weight increase in series of rats’ brains kept for four weeks in different quantities of neutralized and of non-neutralized solutions of 4 per cent formaldehyde. A, 40 ec. of a neutralized solution used; B, 40 cc. of a non-neutralized solution used; C, 20 cc. of a neutralized solution used; D, 20 cc. of a non-neutralized solution used. Serzes 4. As it is known from the experiments of Donaldson (94), Hrdlicka and others, that the amount of liquid in which brains are kept has a decided effect on the weight increase, the present series of experiments was made to test this point with brains of rats of various ages. The experiments were made exactly like those in Series 3, except that the amount of solution used was reduced from 40 to 20 cc. in every case. The data obtained in the experiments in which the brains were kept for four weeks in 20 cc. of a neutralized solution are given in table 6. A comparison of the data given in this table with that in table 4 shows that the maximum, as well as the average, per- centage weight increase is much lower when brains are treated with 20 cc. of solution than when double this amount of solution is used. This is the result one would expect if the amount of swelling diminishes as the strength of the solution is increased; EFFECTS OF FORMALDEHYDE ON BRAIN OF RAT 297 for the dilution of the solution by the tissue fluids is greater when a small amount of solution is used, and the effect is then the same as if the brains were kept in a weaker solution. According to the observations of Hrdlicka, the weight increase in brains treated with unneutralized solutions of formaldehyde is “larger with the weakest solutions and decreases as the proportion of formalin increases.’’ It is evident, therefore, that in these exper- iments some factor, possibly the NaCO; used in neutralizing the solutions, has checked the swelling action of the weakened solu- TABLE 6 Percentage weight increase in rats’ brains, each kept for four weeks in 20 cc. of a neutralized solution of 4 per cent formaldehyde made fresh for each lot of animals killed (averages for two brains at each age) AGE OF RATS TIME SOLUTION ACTED Be i New- 10 20 40 50 70 100 200 born | days | days | days | days | days | days | days Ae AV ieee ete 3, Mee et ches 54.61 50.5 | 42.7 | 41.21) 46.611 39.9 | 40.11] 32.9 ICA Sips ae wees 53.3 | 51.81 46.41 40.8 | 44.3 | 42.41 38.5 | 35.6! UAVS Se gcion¥ 3 Aohoh oe eee 50.1 || 49.9 | 48.3 | 38.7 | 41.4 3725 | 35.3 | 32.3 PRWCE Key Mover. rae coc tet era SOFA TAON2 OO h on lose 408Sh eo OMmESae sn roles oie ananepeoamoapacaeaceall Zi/ce) Wy ho. || OG || S876 i CONG || SB. | S¥eS | Sle ALAS aedosodasuodonenoe son Cell era! 4a | SO STE) || SIRS 1) SUL |) Sie Average percentage gain..... 50.9 | 49.7 | 42.5 | 39.3 | 42.4 | 37.9 | 36.2 | 32.4 1 Maximum weight increase. tion. ‘This seems probable from the results obtained in the second set of experiments in this series which show that a weak acid solution of formaldehyde causes a greater amount of swelling than does a stronger one. The final weight changes for the various groups of brains are plotted in the graphs in chart 6. Table 7 gives the data obtained in the experiments in which brains of different ages remained for four weeks in 20 cc. of a non-neutralized solution of 4 per cent formaldehyde. In this instance brains of new-born rats do not show such striking weight changes as are shown in table 5. The initial rise 298 Age of rat. New born:-~ 50; 10 days. -- ~ 20 days.-- - 40 days- 40 50 days.” - HELEN DEAN KING Percentage weight increase. _ Age of rat _ New born. EST) days. =f _|- 20 days. ee aOR aye. : a, ~-50 days. 70 days.” \ Uf oe ~.70 days. 100 daver ie . | 100 days. 200 days.” ~-200 days. » 20 Ai Time in days. eee eee | = Eee I ins 7 14 21 28 Chart 6 Showi ng the weight changes in brains of rats of various ages, each kept for four weeks in 20 cc. of a neutralized solution of 4 per cent formaldehyde. Age of rat. 10 days. - - - |. 50 days.-40 is 40 days.- 100 days, 30/-" 200 days.-~ New born-~ 20;- Percentage weight increase. Age of rat. _10 days. -|--200 days. New born. Time in days. { EN eee) i) 3 7 14 2 28 Chart 7 Showing the weight changes in brains of rats of various ages, each kept for four weeks hyde. im 20 cc. of a non-neutralized solution of 4 per cent formalde- EFFECTS OF FORMALDEHYDE ON BRAIN OF RAT 299 TABLE 7 Percentage weight increase in rats’ brains, each kept for four weeks in 20 cc. of a non-neutralized solution of 4 per cent formaldehyde made fresh for each lot of animals killed (averages for two brains at each age) AGE OF RATS TIME SOLUTION ACTED | ee ae Tapas New- 10 20 40 50 70 100 200 bern | days | days | days | days | days | days | days ING yet etches tnceenen ee MeN nels 38.91 51.5 | 47.1 | 40.21) 44.54) 45.14) 37.74) 31.1 TOA Sees ach ae ee eee 31.2 | 52.14) 51.31) 89:9 | 41°38 | 44.8 | 36.9 | 33.12 MAAS take tee en, ia Semele Somes 24.2 | 46.5 | 44.9 | 35.2 | 37.4 | 38.6 | 31.4 | 29.2 DEWEEKSH Sree 1 cries ahi ae eel. 9) 4323) |. Sorllpo2eOnoomial seo ror | eo SEWECCKSHaee a a eae alone 41 O AOE. 30a 7a colon mooeiaite elle oon ADVICES rca ios eee aie ero eitys 13.4) 41.1 |) 40.0) | 2829) 32228319) 725-30) 2129 Average percentage gain..... 23.4 | 46.1 | 44.6 | 34.7 | 36.6 | 38.3 | 31.3 | 27.3 1Maximum weight increase. of 38.9 per cent, found at the end of the first day, is greater by 4 per,cent than that found in the previous set of experiments, but the subsequent fall in weight is not so rapid and at the end of four weeks the brains still weigh an average of 13.4 per cent above the original weight and therefore do not appear to have lost any of their substance. In these experiments the greatest percentage gain in weight occurs in the brains of the ten-day and twenty-day-old animals, the average gain for the entire period over which the weighings extended being in each case more than 40 per cent of the fresh weight: the data for the brains of animals from 40 to 200 days old differ but slightly from the corresponding data in table 5. The weight changes in the groups of brains of different ages are plotted in chart 7. The contrast between the results of this series of experiments and those of Series 3 is brought out sharply in chart 5. Graph C was plotted from the final weights of the brains kept in 20 ce. of a neutralized solution of 4 per cent formaldehyde; graph D was plotted from data obtained where 20 cc. of a non-neutralized solution was used. These graphs run, for the most part, between the graphs A and B, which were plotted from the final brain 300 HELEN DEAN KING weights given in tables 4 and 5. Where the solutions were neu- tralized the form of the graphs is practically the same whether 40 or 20 cc. of the solution was used, but graph C falls consid- erably below graph A at every point. Where the solutions were not neutralized the form of the graphs show more variation, but graph D, for most of its length, runs higher than graph B. Since the diluent action of the tissue fluids is undoubtedly greater when 20 instead of 40 cc. of solution is used, it is evident that a stronger neutralized solution of 4 per cent formaldehyde causes more swelling in brain tissue than does a weaker neutralized solu- tion, whereas the reverse is the case where the solutions are not neutralized. Series 5. Since the temperature at which a solution acts is known to have a marked effect on the rate at which the solution will be absorbed, a final series of experiments was made to ascer- tain how different temperatures would affect the swelling of rats’ brains in 4 per cent formaldehyde. In all of these experiments each brain was put into 40 cc. of a solution that was freshly made and neutralized when wanted for use. The bottles containing one set of brains were kept in a water bath at a constant temper- ature of 36°C. for four weeks: the corresponding set of brains remained at a temperature of 8 to 11°C. for the same length of time. The data for the brains kept at the higher temperature are given in table 8. In these experiments, as shown in the table, the maximum weight increase was reached in every case at the end of the first day. The decrease in weight at three days was practically the same for the brains of all ages, amounting to about 8 per cent. Subsequent weight changes were comparatively slight and, except in the very young brains, the final weighings differed but little from those noted for the third day. In this instance, also, there is a direct relation between the age of the animals and the per- centage increase in brain weight, but the average percentage gain for the entire set of brains is considerably less than that found in the experiments in which the brains were kept in a neutralized solution at room temperature (table 4). These results accord EFFECTS OF FORMALDEHYDE ON BRAIN OF RAT 301 TABLE 8 Percentage weight increase in rats’ brains, each left for four weeks in 40 cc. of a neutralized solution of 4 per cent formaldehyde kept at a constant temperature of 36°C. (averages for two brains at each age) AGE OF RATS TIME SOLUTION ACTED cara [RY ieee New- 10 20 40 50 70 100 200 bern | days | days | days | days | days | days | days ICL AN eek ee ebrtr tasty none Sekar ciacG 61.51) 59.53] 50.91) 51.81 45.51 44.31) 39.31) 38.8} DIC AVS wre dy oe he means Lisette. 53.6 | 51.6 | 42.97) 4379 37.8) 66.6 | 31-7 | 32.7 O-GIERYBRK Go Bartlet een tas cee AS" 3) "49.6 43.6") 4ae2ileor saoGro | oll aO le EW COCKS ere re nets Ac HeN an ote 50.4 | 48.4 | 43.7 | 44.7 | 38.1 ee 32.8 | 31.6 By Tel ch aunecse couse seounseodh fee) We ydatia eee Pee) BIS.G) | Stsi.t8) | aves || oillaty A weeks. :...:........--:--:-.| 40.6 | 48.3 | 45.0 | 44:8 | 38.2 ) 39.3 | 33.3 | 32.6 Average percentage gain..... 49.1 | 50.9 | 45.0 | 45.5 | 39.3 | 38.7 | 33.4 | 32.9 1 Maximum weight increase. with Donaldson’s (’94) observations that brains of sheep kept in a 2 per cent solution of bichromate of potassium at a tempera- ture of 38°C. attain their maximum weight at a very early period and gain relatively less than when kept at a temperature of 10 to 17°C. In the case of the rats’ brains kept at a temperature of 36°C. the decrease in the amount of swelling can be attributed, in part at least, to the fact that this temperature partially de- composes the solution of formaldehyde and liberates a consider- able amount of formaldehyde gas. This of course weakens the solution, and a weaker solution of formaldehyde that has been neutralized with NaCO; does not cause as much swelling in rats’ brains as does a stronger one, as was shown in the experiments in Series 4. Graphs for the weight changes in the various sets of brains kept at a temperature of 36°C. are shown in chart 8. All of these graphs, it may be noted, are grouped in pairs according to the age of the animals. While a paired arrangement of some of the graphs is to be found in other charts (6, 7 and 9), in no case is the phenomenon as marked as in chart 8. 302 HELEN DEAN KING Percentage weight increase. Age of rat. | New born.__ 10 Baysee r Age of rat. 40 days__ ey 10 days. 20 days. male es ico days. 50 days.. x a a _-40 days. 70 days.- _|_ __~ |--New born. 100 days. : a -}-70 days. 200 days-_ | \ ne "|-80 days ae ~~ _ 5400 days. ~)>-200 days. 20 Time in days. == a le 4 2\ 28 Chart 8 Showing the weight changes in brains of rats of various ages, each remaining for four weeks in 40 cc. of a neutralized solution of 4 per cent formalde- hyde kept at a constant temperature of 36°C. Table 9 gives the data for the weight changes in the brains kept in 4 per cent formaldehyde for four weeks at a temperature of 8 to. 11°C. As indicated in table 9, the maximum weight increase in all sets of brains was attained on the third day except in the case of the brains of the new-born rats where, as in most of the pre- vious experiments, the maximum increase comes at the end of the first day. The subsequent loss in weight is very slight and it does not amount in any case to more than 8 per cent of the original brain weight. While the brains of young rats (birth to 40 days) show a relatively greater weight increase than the brains of older animals, there is not the very uniform decrease with advancing age that was noted in the previous set of experiments (table 4) where the brains were kept in 40 cc. of a neutralized solution at ordinary room temperature (about 20°C.), neither is the average increase for the various groups of brains as high. EFFECTS OF FORMALDEHYDE ON BRAIN OF RAT 303 TABLE 9 Percentage weight increase in rats’ brains, each left for four weeks in 40 cc. of a neutralized solution of 4 per cent formaldehyde kept at a temperature of 8 to 11°C. (averages for two brains at each age) AGE OF RATS TIME SOLUTION ACTED Lae ; 5 it Bertie cs, _ New- 10 20 AO) = 750 70 100 200 | born | days | days | days | days | days | days | days [GEENA | anne nee 55.91) 41.6 | 37.3 | 38.0 | 31.3 | 36.7 | 30.9 | 30.8 SUAVS sere tie rere ees ae Sao: 51.6 | 53.81) 43.61 48.31] 34.01] 37.01 31.51) 33.41 1h GOW Sige mid hea enin 4 Rn ie Boe 49.3 | 48.8 | 42.9 | 40.4 | 31.1 | 34.3 | 28.4 | 31.1 PEWEE KS RR He enate eres ean | 50.7 | 48.0 | 38.7 | 40.1 | 30.3 | 33.8 | 28.7 | 29.6 SESS eat ne Mae ate ae | 47.9 | 47.7 | 39.2 | 40.4 | 29.9 |.33.7 | 27.4 | 29.2 AMWEEK Shae ac Soca cisdaeiales hefaials 47.9 | 45.7 | 38.9 | 40.4 | 30.0 | 33.5 | 27.9 | 28.4 | | Average percentage gain..... 50.6 | 47.6 | 40.1; 41.3 | 31.1 | 34.7 | 29.1 | 30.4 1 Maximum weight increase. This latter result is what might be expected, since a low tem- perature tends to lessen the amount of absorption of a liquid by brain tissue. This is the only set of experiments in which the average gain in the brains of adult rats is greater than that in the brains of 100-day-old animals. The increase, however, amounts to only about 1 per cent, so it is probably merely a chance variation. Chart 9 shows the graphs plotted for the weight changes in the brains kept at a low temperature. There is a tendency here also to a paired arrangement of the graphs according to age, but it is not as pronounced as in chart 8. The final percentage gain in weight for the two sets of brains used in this series of experiments is shown by graphs in chart 10. The form of the graphs is much the same, but the graph for the brains kept at relatively high temperature (A) runs some- what higher than that for the brains kept at a low temperature (B). It is with a feeling of deep obligation that the author expresses his indebt- edness to Messrs. M. D. Ott, H. L. Kearney, M. M. Miller, J. S. Homan, S. H. Snider, W. H. Taylor, T. K. Kruse, and Geo. Klinkerfuss, for the assistance so willingly and kindly granted. MOLLGAARD’S RETICULUM 323 neck obtained by the decapitation contains a portion of the spinal cord varying from the first to the fifth cervical segments according to the adjustment of the apparatus and as to the par- ticular place on the neck where the blow happens to fall. This neck segment should of course be obtained as soon as possible after the blow is struck. Then, with or without depriving the segment of its adherent musculature, a long slender knife is passed around the spinal cord just within its dural sheath. Simultane- . ously, one of the assistants carefully grasps an end of the cord and drawing it from the canal quickly hands it to the investi- gator. The isolated cord segment is now cleft longitudinally in the region of the anterior horns. From the exposed gray sub- stance (anterior horns) smears are made on glass slides which are dropped into Coplin jars containing the fixing fluids. When . the foregoing was carefully and successfully carried out it was found that the smears could be placed in,the fixing fluids twenty- five seconds after the moment of decapitation. Fixation was carried out both with and without freezing; that is, in the one case the jar containing the fixing fluid was sur- rounded by a freezing mixture while in the other case it was not. In general, 96 per cent alcohol was used as a fixative for Nissl’s bodies; and, when the smears were frozen, for the neuro- fibrillae also. For fixing the neurofibrillae of unfrozen smears the fluid (24 parts absolute alcohol, and 1 part ammonia) recom- mended by London (’05) was more frequently used. For com- parison and checking of results on the Nissl’s bodies, Ohlmacher’s fluid and formol-corrosive were used in a few cases. For a like purpose 12 per cent formol was used as an additional fixative for the neurofibrillae in a few instances. The length of time the smear preparations were left in the fixatives varied in the case of the Nissl’s bodies from one-half hour to three days, and for the neurofibrillae from five hours to four days. Fixation with freezing, to be more explicit, was carried out as follows: A medium sized vessel (capacity approximately one gallon) containing 95 per cent alcohol was placed within a larger container. In the smaller vessel with the alcohol were placed small Coplin jars containing 96 per cent alcohol and ten or twelve 324 THOMAS J. HELDT glass slides respectively. The larger container was now filled with ice and salt which was carefully packed about the smaller vessel. When the temperature of the alcohol and the fixative had been reduced to —8°C. or lower, carbon dioxide snow was added to the 95 per cent alcohol in the smaller of the containers until the temperature was further reduced to —20° to —50°C. At this time the dog was immediately killed and smears made on the cold slides upon which they froze instantly. Then they were quickly dropped into the cold fixative. ‘The time consumed in making the preparations was judged from the instant of decap- itation to the exact moment the fixative received the smear. The smears were left in the fixative for a time varying as above noted. When the time was prolonged the whole of course ac- quired room temperature in the meantime. In those cases where preparations were made at various inter- vals after death the cord segment was in all cases kept under the ordinary conditions of the laboratory and at ordinary room temperature. The majority of the long post-mortem interval preparations, however, were made during the winter months so it may be said that the temperature of the laboratory ranged irom 9, -to 23°C; The staining methods used are comparatively simple. For the staining of the Nissl’s bodies the method outlined by Dolley (11) was used. The method is in the main as follows: The smears fixed in 96 per cent alcohol are brought through a series of alcohols of decreasing strength to distilled water, then stained with warm (ca. 40°C.) erythrosin for three minutes, and well washed in water. They are then brought into a 1 per cent aqueous solution of toluidin-blue for five to eight minutes, again well washed in water, after which they are dipped in 95 per cent alcohol and differentiated, until the Nissl’s bodies and nuclear structures are clearly defined, in a mixture of 96 per cent alcohol 9 parts, anilin oil 1 part. The differentiation is stopped by bringing the slide into absolute alcohol from which it is brought into xylol and mounted in Canada-balsam or damar. To stain the neurofibrillae, London’s method was followed. The method is essentially the following: After fixation, the smears MOLLGAARD’S RETICULUM 325 are brought into a 1.5 per cent aqueous solution of silver nitrate, kept at a temperature of about 37°C. for three to seven days, if unfrozen, and for one to three weeks if frozen, after which time they are treated with a solution of pyrogallic acid, 2 grams, for- mol 5 ce., and distilled water 100 ec., for twenty-four hours. The smears are then placed in a 1 per cent aqueous solution of gold chloride for five to ten minutes, brought into a 5 per cent aqueous solution of sodium hyposulphite for ten minutes, carried through distilled water and a series of alcohols of increasing strength to xylol and mounted in damar or Canada-balsam. In the case of the neural tissue, the foregoing methods were used almost exclusively. Variations were made only occasion- ally and in fact so rarely that they need not be mentioned, ex- cepting that some of the smears were stained for Nissl’s bodies without any previous fixation, and that in staining for neuro- fibrillae a few control preparations were made according to Legendre’s (’06) modification of Bielsechowsky’s method. For control purposes too, smear preparations of hepatic and pancreatic tissues were subjected to the above technique, and the freezing of distilled water and egg-albumen was carefully studied under various experimental conditions. It is thought unnecessary to describe the various magnifica- tions employed in the study of the smears. An ordinary Leitz oil immersion lens was in all cases quite sufficient to determine the points in question. OBSERVATIONS The observations made may be conveniently assembled under the following headings: Nissl’s bodies -1. In unfrozen smears 2. In frozen smears a. Fresh, frozen, unfixed, and unstained smears b. Frozen, fixed, and stained smears considered as a whole c. Nerve cells of smears frozen at —5° to —10°C. d. Nerve cells of smears frozen at —10°C. and lower Additional observations 326 THOMAS J. HELDT NISSL’S BODIES 1. In unfrozen smears The observations on Nissl’s bodies in nerve cells of unfrozen tissue may be very briefly considered. In smears not subjected to freezing, but fixed in alcohol at ordinary room temperature the Nissl’s bodies are present, clearly and definitely defined in cells fixed twenty-five seconds after decapitation (fig. 2). They were also present at all intervals thereafter until they were finally lost in the complete. post-mor- tem disintegration of the tissue. Similarly Nissl’s bodies are present, distinctly defined, in the nerve cells of smears placed, with- out the usual fixation, in the toluidin-blue stain one and three- fourths minutes after death. Not only are the Nissl’s bodies distinctly present in the foregoing preparations, but also their form and their distribution in the cell body and the dendrites are clearly evident. In some of the best smears, and with good magnification, it is even possible to see that the Nissl’s bodies are composed of granules which lie embedded in a matrix, the ‘gerinnselartige Masse’ of Held (95). In Nissl’s bodies in the periphery of a well differentiated cell this matrix appears of slightly purplish hue while the Nissl’s granules are a deep blue. These observations on the matrix are in agreement with those of Held (95 and ’97) and Becker (’06). 2. In frozen smears In the case of the frozen tissue the picture is quite different from that of tissue that has not been frozen, and as an aid to its interpretation a few preliminary notes on the effect of freez- ing on some fluids and fluid-masses may first be mentioned. Since such a great percentage of all tissues and fluid masses is water and because so much separates out during the process of freezing, it is of interest to note the appearance of a frozen drop of water or of the ice as it fills the interstices of the tissue. The appearance is one of large and small prismatic and spherical foam-cells (Quincke) filled with clear pure or nearly pure con- gealed water with here and there small air bubbles between their MOLLGAARD’S RETICULUM oil adjacent walls. This appearance is observed of course only in freshly frozen preparations, not after their fixation, and is men- tioned only to insure a more correct association between the appearances of the aqueous and less aqueous portions of the preparations. The appearance of ice in animal tissues seems to have been quite neglected, but its appearance in plant tissues has been variously noted by Miiller-Thurgau (’80 and ’86), Fischer (11), Wiegand (’11), and others. The details of ice formation however need not be entered into here; for such details, with physico- chemical explanation, are given at length by Quincke (’05). Fig. 1 This figure is a reproduction of Molisch’s (’97) figure 6, with a reduc- tion of one-sixth. It represents the reticulum observed in a thin film of starch- paste, first frozen and then permitted to thaw out. The appearance of frozen egg-albumen is also instructive. Depending on the conditions of the freezing, many variations occur, but in general the appearance is one of a variable network or reticulum the meshes of which are filled with ice. Disregard- ing the variations, the network is not unlike that figured by Molisch (97) for frozen starch-paste with subsequent thawing (fig. 1). Small air-bubbles are usually present, and intermingled with the reticulum, if the temperature employed be low enough, many small clefts occur. It should be remarked that Molisch (’97 and ’11) has made extensive observations on the action of freezing on many sub- stances, solutions, and emulsions, colloidal and otherwise. From 328 THOMAS J. HELDT his studies Molisech concludes: Freezing causes a separation of water; this water at numerous points forms ice crystals which by molecular force continue to extract water from the substance in question and by their consequent enlargement push aside the substance, and in this way give rise to the network or reticulum observed. Several of his figures, one of which is reproduced here in figure 1, strikingly illustrate this point. The observations on various gums and other colloids all agree, in their essentials, with those of Molisch: Ambronn (’91); on a number of inorganic and organic colloids, at temperature reduc- tions of —10°, —70°, and —180°C. by Bobertag, Feist, and Fischer (’08); on starch-paste, various gums, and hemoglobin, at temperatures as low as —180°C., by Fischer (’11); and, on gela- tin by Liesegang (11). Likewise, except for some details, the observations of the author are in accord with those of the inves- tigators named. The observations on frozen neural tissue may be considered under the following divisions: (a) fresh, frozen, unfixed, and unstained smears; (b) frozen, fixed, and stained smears consid- ered as a whole; (c) nerve cells of smears frozen at —5° to —10°C.; (d) nerve cells of smears frozen at —10°C. and lower. a. Fresh, frozen, unfixed, and unstained smears. A smear of gray matter from the spinal cord of dog, frozen over the gas- escape of a carbon dioxide tank or by making the smear on a very cold slide, becomes of whitish opacity and at very low tem- peratures develops clefts not unlike those observed in ice and frozen egg-white, though in general it takes a much lower tem- perature to develop such clefts in neural tissue. The appearance presented under the microscope varies considerably according to the thickness of the smear, the rapidity of the freezing, the degree of temperature reduction, and undoubtedly too on the surface tension and other intrinsic forces of the individual neural ele- ments. In general, however, it may be said that small foam- cells resembling those of aqueous ice, variously arranged, are as a rule to be observed around the margin of the smear; the smear itself appearing as a coarse network or reticulum with large and small, irregular or multiangular meshes. Within the meshes is MOLLGAARD’S RETICULUM 329 the more liquid frozen matrix or ice, while the network itself consists of the more solid portions of the tissue. In many places the largest of the nerve fibers found in gray matter appear to form initial strands upon which the network is formed, in other places however they extend into or through the lacuna-like spaces quite unaccompanied by other neural elements. Regarding the various kinds of cells (nerve cells, blood cells, and ghia cells) nothing can be made out with certainty in these frozen and unstained preparations. Molisch (’97) has shown that the reticulum produced by freez- ing in such colloids as gelatin and starch-paste is more or less permanent after thawing out, others such as gum tragacanth, eum arabic, and egg-albumen the reticulum disappears quite completely. The author’s observations on the last named col- loid confirm this statement and so too for the non-fixed neural tissue described above no noticeable trace of the effects of the * previous freezing can be noted after thawing. b. Frozen, fixed, and stained smears considered as a whole. Smears frozen, fixed and stained according to the directions given under the heading ‘Material and technique’ show, when frozen at a temperature of —5° to —10°C., the first effects of freezing. Like the fresh, frozen, unfixed, and unstained smear, the prep- aration, when looked upon as a whole, appears to consist of a more or less coarse network or reticulum the interstices of which are of variable size and form. With greater temperature reduc- tions (—10° to —40°C.) this network becomes more definite and distinct, and presents a number of special features which will be discussed under other headings. The network observed is stained in part blue (by the toluidin-blue) and in part pink (by the erythrosin). Coarse and fine nerve fibers and blood capil- laries lie, apparently unaffected by the freezing (at —5° to 25°C.), surrounded by the network. The pink stained portion of the reticulum appears to be composed of the most delicate nerve fibers and condensations, as it were, of the tissue fluids; in fact, it may be regarded as being composed of all the acidophilic sub- stances of the tissue. The blue stained portion, on the other hand, may be looked upon as composed of the basophilic sub- 330 THOMAS J. HELDT stances of the tissue, especially the chromatin. The relation of the pink and blue portions to each other, and to the different conditions of the technique, will be considered more in detail under subsequent divisions. In these stained. preparations the various kinds of cells may be clearly distinguished. Red and white blood cells and glia cells appear, at temperatures above —25°C. quite unaffected by the freezing, excepting of course those that have been crushed or otherwise distorted in making the smear. Such crushed forms produce areas of coarser network. At lower temperatures (—25° to —40°C.) these cells, and the nerve cells as well, show a con- siderable variety of changes which will be discussed under the headings following. c. Nerve cells of smears frozen at —5° to —10°C. The multi- polar nerve cells of smears frozen at the temperature stated present appearances that vary somewhat depending, in addition to the reduction in temperature, on the thickness of the smear, the rapidity of the freezing, and no doubt also on*the surface tension and other intrinsic forces of the different cellular ele- ments. The size of the cell does not appear to play so great a part in these variations as one might expect. The change which is the most marked and characteristic of this temperature inter- val, and, in fact, the first change to become noticeable in the cells, is in the Nissl’s bodies which become minutely reticular or vacuolated, thus forming many small blue-staining networks, each network corresponding to a single Nissl’s body. These net- works are loosely connected together by finer trabeculae of the same blue-staining substance (fig. 3). The trabeculae appear to be direct extensions of the Nissl’s substance which has probably a lower freezing point. By virtue of this property these delicate processes have been squeezed out into the surrounding cytoplasm under the force of the displacement brought about by the enlarg- ing and expanding ice masses. None of the networks extends beyond the walls of the cells. Aside from the Nissl’s bodies, the cellular cytoplasm, the nucleolus, and the other nuclear struc- tures show as yet no change, unless the nuclear chromatin, exclu- sive of the nucleolus, may be said to be beginning to suggest MOLLGAARD’S RETICULUM 331 reticular arrangement. Though this description applies particu- larly to smears frozen at —5° to —10°C., it also applies in part to smears frozen at temperatures as low as —25°C. d. Nerve cells of smears frozen at —10°C. and lower. The changes next in occurrence to those described under the heading just preceding are to be observed in cells of smears frozen at —10° to—25°C. In such preparations some of the cells show many of the Nissl’s bodies still appearing as individual networks but with larger meshes and more closely interconnected, all how- ever being still confined within the cell wall (fig. 4). In the cellular cytoplasm no changes are yet to be noticed. The nuclear chromatin (and also the achromatic substance of the nucleus which stains pink with erythrosin) has become distinctly reticu- lar, apparently forming one continuous network limited to the area within the nuclear membrane; or, extending beyond the nuclear membrane and becoming continuous with the blue-stain- ing network derived from the Nissl’s bodies. The nucleolus may be unaffected, or may take part in the formation of the nuclear chromatin reticulum, forming as it were its starting point by sending out a varying number of processes. Some nerve cells show these changes much intensified with the cellular cytoplasm, exclusive of Nissl’s bodies, also becoming reticular. This cyto- plasmic reticulum, although continuous with the blue-staining network, is readily distinguished, when once fully formed, by the fact that it stains pink with the counterstain employed. In general, too, this pink-staining cytoplasmic reticulum has smaller meshes than the blue-staining network, and lies within the inter- stices of the latter. In order to avoid confusion, only the blue- staining portion of the reticulum is shown in the figures. Still other cells at this temperature (—25°C.) and at lower temper- atures (—25° to —50°C.) show a reticulum with much larger meshes. The elements composing this reticulum are none other than those already mentioned, namely, the blue-staining network derived from the Nissl’s bodies, nuclear chromatin, and nucleolus; and, the pink-staining network derived from the cellular cyto- plasm (exclusive of Nissl’s bodies) and the achromatic substance of the nucleus. The continuity of these two networks is one of Jon THOMAS J. HELDT intimate blending and so a single double-staining reticulum results. This single reticulum extends throughout the entire cell and even beyond (fig. 5), and thus becomes directly continuous with the pink-staining extracellular network previously described under the observations on the stained smear as a whole. In these cells there is of course a complete obliteration of the Nissl’s bodies as such. Thus, to repeat, it appears quite evident that the intracellular networks are directly continuous with each other, and again, are similarly continuous with the network formed in the cellular interstices. These last changes described may be called the final form the networks assume, for the appear- ances just described are quite constant and do not change to any noticeable extent in a temperature range of 20° to 30° (—40° to —60°C.). It cannot be said that any of the appearances described have an absolutely definite temperature at which they appear. For instance, the last form described may even be found to a certain extent in smears frozen at —15°C., none of the earliest forms, however, occurs as a rule at temperatures below — 25°C. The significance of the networks described above and their relation to Méllgaard’s reticulum or ‘glia-network’ will be dis- cussed later. ADDITIONAL OBSERVATIONS The perivascular network described by Mollgaard may also be satisfactorily studied in the foregoing preparations. Its origin is plainly from the chromatin of the nuclei of the endothelial cells of the blood and lymph capillary and precapillary vessels (fig. 6). The variations and various relations of the network merit no description. The chromatin of the nuclei of the glia cells also form networks but their relation to both the networks of the nerve cells and the perivascular networks seems to be merely one of accident. The rather constant appearance of what Mollgaard believes to be a glia cell at one pole of the nerve cell _ nucleus the author did not observe and it is probably explained as being a modification of a chromatin nuclear cap. All the observations on frozen neural tissue apply to smears frozen and fixed as soon as thirty-two seconds after decapitation MOLLGAARD’S RETICULUM 333 and at various intervals thereafter up to twenty-five hours post- mortem. After this time no further preparations were made, for it seemed quite evident that the networks were in some way related to the process of freezing rather than to the length of time after death. In this regard the author was entirely unable to confirm Mdllgaard’s statement that the earliest form of the reticulum (in nerve cells of tissue excised from the living animal) consists of a network of 3 to 4 meshes, and that the reticulum becomes finer and reaches its greatest development ten to twelve minutes post-mortem. The reticula of nerve cells in smears frozen and fixed thirty-two seconds after decapitation, and this time probably does not correspond very unfavorably with the time consumed by Mollgaard in excising and transferring the tissue from the living animal, were found as fine and as extensive as the reticula of cells in smears frozen and fixed at various later intervals up to twenty-five hours after death. It seems appar- ent, therefore, that very little, if any, connection exists between the fineness and extensiveness of the reticulum on the one hand and the time after death, up to at least twenty to twenty-four hours, on the other. Neither was the author able to note any disintegration of the reticulum in nerve cells of frozen smears left in 96 per cent alco- hol for a time varying from half-an-hour to three days. Even when Mollgaard’s modification of his technique, as previously explained under ‘Material and technique,’ was carefully followed out, as nearly as the technique and conditions of the present investigation would permit, no such changes as noted by Moll- gaard were observed in the nerve cells of the smear preparation. Alterations in the staining properties of the protoplasm, as observed by Modllgaard in frozen preparations made at various intervals after death, were given but little attention because smear preparations do not lend themselves readily to a reliable study of such details. Furthermore, very much depends on the thickness of the smear and its degree of differentiation after staining. It may be noted however that in smears frozen twenty- four to twenty-five hours after death there is a more diffuse staining with the basic stain in consequence of which the blue and 334 THOMAS J. HELDT pink staining networks become less distinct and in places ill- formed. That post-mortem changes have a marked influence on the cytological appearance of the tissue there can be no question. But it is questionable if the post-mortem changes produce alter- ations sufficiently great to be detected by our present technique until a considerable time after death, at least half-an-hour. The relationship between the acid reaction of the neural tissue and the post-mortem changes produced are not so strikingly notice- able as Moéllgaard’s emphasis on this point would lead one to believe. Mo6llgaard states that the cerebrum of dog shows a marked acid reaction to moist neutral litmus paper ten minutes after death. It was however fully twenty minutes before the author could be sure of such acid reaction. Nevertheless, it is quite possible that with a more delicate indicator an acid reac- tion might be observed somewhat earlier. To determine whether the network could be produced in cells that had first been fixed, some smears were made approximately seven minutes after death, fixed in 96 per cent alcohol at room temperature for one hour, and then subjected to atemperature of —15°C. At this temperature (which was possibly not low enough) no networks could be observed in the cells. The Nissl’s bodies had remained unchanged. This question of the possi- bility of structural change through freezing after fixation, how- ever, was insufficiently investigated to warrant any definite con- clusions; yet, it is a matter of common knowledge that in the use of various neurological methods where the tissue is frozen after fixation no marked changes at least have been reported. Smears of fresh hepatic and pancreatic tissues, prepared after the manner of the neural tissue preparations, show networks which are markedly similar to those of the neural tissue, the chromatin of the nuclei giving rise to a blue-staining network while the non-chromatic elements give a pink-staining network. Thus it may be said that Méllgaard’s blue-staining reticulum is not a characteristic of neural tissue alone but may be formed from the chromatin of the nuclei of hepatic and pancreatic cells as well. MOLLGAARD’S RETICULUM 335 The networks in the liver and pancreatic tissues, as well as those of frozen smears of egg-albumen, may also be stained with silver nitrate. In corroboration of the foregoing observations on animal tis- sues it may be mentioned that Molisch has observed very similar appearances in the freezing of amoebae, fungi, yeasts, and vari- ous algae. Molisch’s observations however apply principally to eytoplasmic changes. Equally instructive and showing chiefly the nuclear changes due to freezing are the numerous figures of Matruchot and Molliard (’02) in their work on the influence of freezing on plant cells. The observations on the neurofibrillae, like those on the Nissl’s bodies, may be considered under two headings; namely, their presence in the unfrozen and in the frozen cells. The neuro- fibrillae are unquestionably present in the unfrozen cells of smears fixed twenty-five seconds after decapitation and thereafter until lost in the total disintegration of the tissue. Although the peri nuclear network and other finer details of the endocellular neuro- fibrillar structure of the nerve cell (such as may be seen in thin sections) are not visible as such in smear preparations, the neuro- fibrillae are distinctly seen at the origin of the cell processes and an endocellular neurofibrillar network of varying richness is more or less discernible in the cell-body. In regard to neurofibrillae in frozen preparations it may be noted that smears frozen at —5° to —20°C. fixed in 96 per cent alcohol, and impregnated with 1.5 per cent silver nitrate for from nine to twenty-one days, show networks corresponding more or less closely to the networks stained with toluidin-blue and erythrosin. Large numbers of darkly stained nerve fibers render the appearance somewhat difficult of recognition at first sight, especially where the smears are thick. In the larger meshes of these networks lie the nerve cells whose individual reticular structure may or may not be continuous with the walls of the meshes about them. In these preparations, when not too darkly stained, the neurofibrillae are unmistakably present in the nerve cells and their processes as well as in the intercellular nerve fibers. They are well stained and quite distinct. The neuro- THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, No. 4 336 THOMAS J. HELDT fibrillae however are best observed in preparations frozen quite rapidly and at moderately low temperatures (—5° to—15°C.), for when thus treated the intracellular neurofibrillae do not seem to take part in the formation of the cellular reticulum corre- sponding to that stained with toluidin-blue and erythrosin. At temperatures below the foregoing, especially if frozen slowly, the intracellular neurofibrillae cannot be made out with any degree of certainty though they are still quite distinct in many of the cell processes and in the fine nerve fibers of the intercellular spaces. The above observations were made on smears frozen and fixed forty-two seconds after decapitation and at different times afterwards up to twenty-five hours after death. These observations on the neurofibrillae in frozen tissue are quite in agreement with Liesegang’s (’11) conception; but, on the other hand, quite opposed to that of Auerbach (’11). In the above study of neurofibrillae both in the unfrozen and the frozen preparations many variations were met with. In this regard, as Legendre (’06), Marinesco (’09), and many others have noted, it may be mentioned that the methods for the dem- onstration of neurofibrillae are not sufficiently adequate or relia- ble to permit of constant results. Only by extensive observation and many controls can definite conclusions be reached. So it may be questioned whether the above observations on the neuro- fibrillae in frozen nerve cells, in this one investigation, are suff- cient to determine the point at issue; yet, since this point is merely the presence of the neurofibrillae in such frozen cells, without reference to the details of their distribution, and so forth, the author believes the results may be regarded as quite reliable. It may be of interest to note that both the Nissl’s bodies and the neurofibrillae were unmistakably present in nerve cells from the cervical portion of the spinal cord of horse (cf. ‘Material and technique’), fixed three minutes afterthe instant of the shoot- ing of the animal. In imbedded tissue, that is, tissue fixed, dehydrated, imbedded in paraffin (or celloidin), cut, stained, and mounted; both the Nissl’s bodies and the neurofibrillae were clearly and distinctly MOLLGAARD’S RETICULUM 337 present in cells fixed twenty-two seconds after decapitation. Small pieces of the cervical portion of the spinal cord of dog were quickly dropped in the fixative at the time stated. Then in the cutting, staining, and mounting of the sections care was taken to use only those sections cut from the most superficial parts of the tissue block. Thus one may be reasonably sure that the fixation of the superficial cells was practically simul- taneous with the committal of the tissue to the fixative. No observations were made on imbedded specimens of frozen tissue. DISCUSSION It was one of MO6llgaard’s primary objects to produce a method which would be simpler than our present neurocytological meth- ods. His procedure however is not so strikingly simple as he would have us believe. In the first place, the production of a temperature as low as —40°C. and the necessity for maintaining it for a definite length of time requires a painstaking and rather cumbersome technique. Sectioning the tissue at a temperature of —20° to —15°C. at best, and that with a specially constructed microtome, is not easy. Even in the fixation, if the author has correctly interpreted Mollgaard’s following statement, compl- eating factors enter: ‘‘Die Fliissigkeit, in der man zu schneiden wiinscht, wird in den innersten Kasten des Kalorimeters gegossen und durch Zusatz von fester CO, auf eine Temperatur von —20° bis —25° heruntergebracht.”” Judging from the description of his apparatus this seems an unnecessary step, yet he so directs us. Bohr (Annalen der Physik, IV F, Bd 1, S. 244, 1900) states that at —20° and —40°C. (760 mm.?) 98.7 per cent (by weight, 15°, 760 mm.) ethyl alcohol will absorb 7.16 cc. and 13.89 cc., respectively, of CO, (0°, 760 mm.). In 96 per cent (the per- centage used by Mollgaard) alcohol the absorption is of course less. However, Mollgaard thus leaves without control the possible changes the tissue may suffer from the effects of the CO... At the temperature stated it may be questioned if there is any effect on the tissue, still it cannot be nil. The presence, and its liberation as the temperature rises, of the CO, in the fixative does not simplify the technique to say the least. 338 THOMAS J. HELDT Finally, his method requires the preparation of the animal, its anesthetization, the performance of a craniotomy or a laminec- tomy, and the waiting for the effects of the anesthetic and the shock of the operation to disappear. In connection with the latter features, it may be well to mention that Dolley (09 and 10) has conclusively shown that the effects of the anesthetic and the shock of the operation do not disappear in so short a time (seven to eight hours) as Moéllgaard assumes. So that for various reasons, in addition to that urged by Retzius, M6llgaard’s technique is by no means ideal. It was in order to avoid the foregoing objections that the technique previously described was decided upon for the present investigation. The decapitation avoids all preliminary prepara- tion of the animal, and likewise excludes the effects of an anes- thetic and the prolonged shock of the operation. The apparatus for the decapitation permits one to obtain practically instan- taneously a segment of the animal’s neck, after which the isola- tion of the cord, with a little experience, requires but a few seconds. By resorting to smear preparations, the freezing proc- ess, which the sectioning of an unimbedded tissue demands, is avoided. The smear preparations have the further advantage of being fixed the very instant they are consigned to the fixative. Thus it is evident that the entire procedure, namely, the taking of the tissue, putting the tissue in a form suitable for study, and the fixation, all occur in less than a half a minute of time, or at most a minute. It should be noted that the cumbersome freez- ing method of Modllgaard is designed merely to preserve the tissue structure in the ‘vital condition’ until thin sections can be made upon which the fixative can act. The smear method accomplishes this far more easily and quickly, and eliminates the production of artefacts by freezing. It is of course freely granted that the smear preparations do not show the minute details of structure as readily as do thin sections. Nevertheless, for the points in question the smear preparations are amply sufficient. In smear preparations not subjected to the freezing process the Nissl’s bodies and the neurofibrillae are unquestionably pres- MOLLGAARD’S RETICULUM 339 ent twenty-five seconds after decapitation. It has been previ- ously mentioned that in nerve cells of imbedded tissue the Nissl’s bodies and neurofibrillae were found in tissue fixed twenty-two seconds after death. It has been stated too that the smear prep- arations do not permit of the detailed study that sections do. It is therefore unnecessary to discuss the various details of obser- vation noted under this heading. ‘It is particularly the time element with which we are here concerned. Mo6llgaard found no Nissl’s bodies or neurofibrillae in freshly-frozen and fixed nerve cells. He considers at least seven minutes post-mortem change, followed by several hours.of slow alcohol fixation, neces- sary to produce the Nissl’s bodies even imperfectly. But the results submitted in the present paper prove that in unfrozen smear preparations (which have the advantage of excluding the production of artefacts by freezing and allow immediate fixation) both the Nissl’s bodies and the neurofibrillae are found in tissue fixed less than half a minute after decapitation, while the cells are still practically in a living condition. Hence Mollgaard’s contention that Nissl’s bodies are produced by post-mortem change and slow alcohol fixation is totally wrong. The freezing is responsible for his misleading results. Whether the sojourn in the fixative be comparatively long or short seems to have no noticeable influence on the Nissl’s bodies. In smears taken at various intervals after death the Nissl’s bodies show no appreciable change till about twelve to eighteen hours post-mortem, when they gradually begin to disintegrate. To determine whether the alcohol fixation could in any way be responsible for the presence of the Nissl’s bodies in the freshly- fixed cell, some smears, instead of being dropped into the fixa- tive, were immediately consigned to the toluidin-blue stain, after which they were carefully washed, mounted in water, and stud- ied. Such smears show the Nissl’s bodies to be undeniably present. They show distinctly, but it seems that they are some- what more granular and possibly a little more diffuse than those of cells fixed in aleohol. In this connection it may be mentioned that Dogiel (’96) stained the Nissl’s bodies in unfixed nerve cells, with dilute solution of methylene-blue in warm physio- 340 THOMAS J. HELDT logical salt solution five to ten minutes after death. It may be possible however, as Held (’95) has pointed out, that the tolu- idin-blue or the methylene-blue (Dogiel) may fix, or partially fix, the tissue put into it. Regarding the alterations which freezing produces in the smear preparations, very much depends upon the conditions under which the freezing occurs.’ The factors previously referred to may be repeated, namely, the composition of the substance or tissue in question, the thickness of the smear, the degree of temperature reduction, the rapidity of the freezing, the surface tension and other intrinsic forces of the individual elements of the substance or tissue investigated (e.g., the forces of molecular attraction, imbibition, osmosis, ete.), to say nothing of thawing and fixation. These factors have been wholly or partially recog- nized by all investigators of the phenomenon of freezing. The freezing of water has been sufficiently discussed. The physico-chemical explanation of ice-formation, though of funda- mental importance in the following considerations, are given in detail by Quincke and so will be referred to here only as occa- sion demands. The appearances observed in the freezing of egg-albumen are concisely expressed in Molisch’s following statement in which he describes his microscopical findings for the freezing of a 2 per cent aqueous solution of gelatin: An zahlreichen Punkten tauchen unter Abscheidung von Luftblassen rundliche Eismassen auf, die, der benachbarten Gelatinegallerte das Wasser entziehend, sich rasch vergréssern und dabei die immer wasser- armer werdende Gelatine ringsum zur Seite schieben, so das diese, wenn die Eisbildung ihr Ende erreicht hat, als ein héchst complicirtes Ma- schenwerk zwischen den Eiskliimpchen ausgespannt erscheint. Die urspriinglich homogene Gelatine ist nun in eine Art Schwamm unge- wandelt, in welchem das héchst complicirte Geriistwerke aus Gelatine, die Hohlriume aber aus Eis bestehen. Molisch’s observations on frozen egg-white are similar to his above observations on gelatin, excepting that in the case of the egg-albumen the network disappears on thawing, while that pro- duced in gelatin is quite permanent for some time. It is of inter- est here to note that Ambronn (’91) in his study of frozen gelatin and agar-agar states that the appearance of the fine network MOLLGAARD’S RETICULUM 341 due to the freezing of dilute solutions of these substances is exactly similar to the appearance of a section through a paren- chymatous plant tissue. Molisch casually remarks that his studies confirm this statement. Furthermore, Ambronn states that in an optical respect the walls of the meshwork in the frozen colloids named are entirely similar to the walls of plant cells, showing a strong double refraction and the same orientation of the optical elasticity ellipsoid as do the cell-walls. From the observations and references presented, it must be clear then that on freezing there is a separation of water from the substance in question. This fact has long been known to many observers (Miiller-Thurgau, Molisch, Fischer, Wiegand, and others). The water separating out under the reduced tem- perature forms ice, which, omitting for the present the details, by displacement of the substance produces the network or sponge- like structure described. The great part played by this water separating out during the freezing of the substances and tissues under consideration, may be further realized by a study of smears of egg-albumen, fresh neural, liver, and pancreatic tissues thor- oughly evaporated or desiccated in an oven. Such preparations show networks somewhat suggestive of those produced by freez- ing. So similar are the processes of freezing and desiccation, in producing a loss of water, says Fischer, that the curve for the loss of water by freezing may be calculated, for some colloids at least, from the curve for the lost of water by desiccation. Matruchot and Molliard have compared the separation of water during freezing to plasmolysis; and, wilting, or slow and rapid desiccation, as well. Noting the comparative uniformity of the results arrived at by many observers regarding the fundamental principles under- lying the freezing of numerous simple substances, colloids, and plant and animal tissues we may consider, as does Molisch, and with considerable support, that the cells of a plant or animal tissue may be regarded, as far as their behavior in freezing is concerned, as aggregate masses of solutions, emulsions, and col- loids. Thus, for explanatory purposes, we may consider that neural tissue with all its various elements consists of a complex aggregate of solutions, colloids, and emulsions, one within the 342 THOMAS J. HELDT other, most delicately interrelated and adjusted, specialized and differentiated, if such terms are permissible in this connection, to a high degree. There is however the one common element, water, whose proportional presence depends on the various con- ditions to which the tissue may be heir. If now this complex aggregate be subjected to a freezing temperature, there is, as is already evident from previous statements, a separation of water. This separation of water is strikingly dependent on the factors before noted. If the freezing is slow the water as a rule collects in the interstices of the tissue and thus in the subsequent ice formation there are comparatively few centers of crystalliza- tion. If however, the freezing is rapid the rapidity of the proc- ess does not permit the water to collect in a few, apparently the least resistant, interstices but forces it to crystallize in numerous places. It would seem that in the employment of low temper- atures, the more rapid the reduction the more numerous the centers of crystallization. Liesegang states that if the reduction in temperature be great enough the centers of crystallization become so numerous as to warrant the designation colloidal ice, the existence of which has been proven by Ostwald and Weimarn (cited by Liesegang). Since it is in the more slowly frozen tissue that the more typical reticular structure occurs, we must return to that con- dition. Just how and why the water of the various tissue ele- ments collects in the interstices of the tissue during the process of freezing is explained in considerable detail, in the case of plant tissue, by Wiegand (’06 b). Just what, at a freezing tem- perature, determines the formation and location of a center of ice crystallization Wiegand does not state. Many factors no doubt are involved, the more important ones however are prob- ably the following: the minimal amount of solute present in the water, and the relative molecular capillarity with which this water is held, together with the molecular distribution and ar- rangement, at that particular instant and position. With the formation of the ice crystals of course comes the molecular force of crystallization which continues to abstract water from the particular tissue element in question with a simultaneous increase ° MOLLGAARD’S RETICULUM 343 in the size of the ice crystal, or crystals. This process of abstrac- tion of water and enlargement of ice crystals continues until the force of crystallization is equal to the force of imbibition, or molecular capillarity, of the tissue element or cell, that is, until an equilibrium is reached. With a new reduction in temperature the process is again set up until the equilibrium is once more restored. Whatever the exact details of the process may be, it is evident that with the first formation of the center of ice crystallization the tissue elements, and cellular elements especially, are sub- jected to a displacement which is increased in extent both by the expansive force of the water changing to ice and the actual increase in size of the ice crystals under the force of crystalli- zation. With a moderate rate of freezing (such as making smears on slides cooled to —20° to —40°C., with fixation at the same temperature) and a like temperature reduction this displacement gives rise to the various networks or sponge-like reticula described for the egg-albumen, nerve cell, and so forth. These reticula are therefore the resultant of aqueous abstraction, and displace- ment of the subsequent tissue or cellular residue, as it were, by the formation and growth of ice crystals or ice masses within , the tissue or cell. The expansion and contraction of the ice, as the maximum and minimum temperatures for these phenomena are reached and surpassed, augment the displacement, while the force of imbibition is probably the main retarding force. With - very rapid freezing at very low temperatures (—50°C. and below) the formation of multiple centers of crystallization subjects the individual cellular elements to the contraction of the frozen mass of course much more than at higher temperatures. That the networks discussed arise in consequence of the displacement by the enlarging ice masses is particularly emphasized by Miiller- Thurgau, Molisch and Wiegand, as before stated. The blue and pink staining properties of the reticulum are explained as being due to the fact that some of the substances composing it are basophilic while others are acidophilic, that is, some of the composing substance stains with toluidin-blue while other portions of it stain with erythrosin. If now, we regard 344 THOMAS J. HELDT the Nissl’s bodies as composed of substances of the nature of chromatin, as is generally done, then we may say that the blue- staining (with toluidin-blue) networks or reticula are derived from the Nissl’s bodies and nuclear chromatin of the nerve cells as well as the nuclear chromatin of all other cells present in the preparation. The pink-staining (with erythrosin) networks, on the hand, may be regarded as arising from all the achromatic substances of cellular cytoplasm and tissue (blood, lymph, etc.) alike. That such is indeed the origin of the networks or reticula is confirmed by this investigation. Mdéllgaard’s reticulum there- fore arises from the Nissl’s bodies and nuclear chromatin. The reticulum is a product of the Nissl’s bodies rather than the converse as believed by Moligaard. The close relationship between the reticulum and the Nissl’s bodies is further supported by the many transition forms that may be noted in nerve cells of smears frozen at moderately low temperatures (—5° to — 25°C.) (figs. 3 and 4). Retzius in his study of the freezing of various tissues and fluid masses lays much stress on the formation of a system of clefts and lacunae due he says to the collection of the water at the moment of freezing at the points of lowest resistance. The for- mation of ice in this system produces distortion and laceration. With fixation the entire picture is preserved and is known to us as the artefacts due to freezing. Excepting for the undue stress laid on the formation of a system of clefts and lacunae and the production of real laceration, it is evident that Retzius’ results are in the main quite similar to those arrived at in this investigation. The cleft and lacunar system of Retzius, how- ever, must not be confused with the clefts spoken of here as due to the contraction of the ice present. CONCLUSIONS From the observations presented and the discussion made the following conclusions may be summarized: 1. With a simplified smear method, both the Nissl’s bodies and the neurofibrillae are found present in the spinal nerve cells of the dog, fixed twenty-five seconds after decapitation. MOLLGAARD’S RETICULUM 345 There is no evidence that they are artefacts due to post-mortem changes as described by Mollgaard. 2. Nissl’s bodies and neurofibrillae may also be demonstrated, in a more or less modified condition, in frozen neural tissue. The freezing causes the Nissl’s bodies and nuclear chromatin to as- sume the form of a reticulum. This reticulum is identical with M6llgaard’s reticulum, or ‘glia-network.’ 3. Mollgaard’s reticulum is produced during the process of freezing, and is due to the displacement incurred by the enlarg- ing and expanding ice-masses which form in the cell or tissue at the reduced temperature. BIBLIOGRAPHY AmBRONN, H. 1891 Einige Beobachtungen iiber das Gefrieren der Colloide Ber. d. Verhandl. d. Kénigl. Sachs. Ges. d. Wiss. (Mathem.-Phys. Klasse), Bd. 43. AverBacH, L. 1911 Mo6llgaards vitale Fixation und meine Kritik der Neuro- fibrillenlehre. Anat. Anz., Bd. 40. Brecker, H. .1906 Zur Physiologie der Nervenzelle. Neurol. Centralbl., Bd. 25. BospertaG, O., Feist, C., anp Fiscuer, H. W. 1908 Uber das Ausfrieren von Hydrosolen. Ber. d. Dtsch. chem. Gesell., Bd. 3. Doaiet, A. 8. 1896 Der Bau der Spinalganglien bei den Saéugetieren. Anat. Anz., Bd. 12. Doutiey, D. H. 1909 The pathological cytology of surgical shock: I. Jour. Med. Research, vol. 20. 1910 The pathological cytology of surgical shock: II. Jour. Med. Research, vol. 22. 1911 Studies on the recuperation of nerve cells after functional activ- ity from youth to senility: I. Jour. Med. Research, vol. 24. FiscHper, H. W. 1911 Gefrieren und Erfrieren, eine physicochemische Studie. Beitrige z. Biol. d. Pflanzen, Bd. 10, H. 2. Fiscuer, H. W., ann Bosertac, O. 1909 Uber das Ausfrieren von Gelen. Biochem. Ztschr., Bd. 18. Hep, Hans 1895 Beitriige zur Structur der Nervenzellen und ihrer Fortsatze: I. Archiv f. Anat. u. Physiol., Anat. Abth. 1897 Beitrige zur Structur der Nervenzellen und ihrer Fortsatze: II. Archiv f. Anat. u. Physiol., Anat. Abth. Key, A., AND Rerzius, G. 1874 Om fryningsmetodens anviindande vid his- tologisk teknik. Nordiskt Medicinskt Archiv, Bd. 6, no. 7. 1882 Uber die Anwendung der Gefrierungsmethode in der histolo- gischen Technik: Biol. Untersuchungen, I F., Bd. 2. LEGENDRE, R. 1906 Sur divers aspects de neurofibrilles intracellulaires obtenus par la méthode de Bielschowsky. Anat. Anz., Bd. 29. 346 THOMAS J. HELDT LigseGanc, R. 1911 Die Moellgaardsche vitale Fixation. Anat. Anz., Bd.39. Lonpon, E. 8. 1905 Zur Lehre von dem feineren Bau des Nervensystems. Archiv f. mikrosk. Anat., Bd. 66. Martnesco, G. 1909 La cellule nerveuse. Tome 1. Marrucuot, L., anp Mouiiarp, M. 1902 Modifications produites par le gel dans la structure des cellules végétales. Revue Gen. de Botanique, ae 14. M6uieGaarD, H. 1911a Die Vitale Fixation des Zentralnervensystems. Anat. hefte, Bd. 48, H. 3. 1911 b Uber die Verwendung der Gefriermethode fiir vitale Fixation des Zentralnervensystems. Anat. Anz., Bd. 39. Mo.utscu, Hans 1897 Untersuchungen iiber das Erfrieren der Pflanzen. Jena. (Fischer. ) 1911 Erfrieren der Pflanzen. Vortrige d. Vereines z. Verbreit. natur- wiss. Kenntnisse in Wien. 51 Jahrgang. Heft 6. Wien. (Wihl. Brau- muller). Mitter-Tuuraav, H. 1880 Uber das Gefrieren und das Erfrieren der Pflan- zen. Landwirtsch. Jahrb., Bd. 9. 1882 Landwirtsch. Jahrb., Bd. 11. 1886 Landwirtsch. Jahrb., Bd. 15. Quincke, G. 1905 Uber Eisbildung und Gletscherkorn. Annalen d. Physik, Bd. 323, IV F. (See also Nature, 1905, vol. 72, and Proc. R. Soc., Lon- don, Series A, vol. 76.) Retzius, G. 1874 Jahresber. d. Anat. u. Physiol., Bd. 3, 8S. 7. 1911 Uber die vitale Fixation des Nervensystems von H. Mdllgaard und iiber die Gefriermethode im allgemeinen. Anat. Anz., Bd. 39. Wisecanp, K. M. 1906a The occurrence of ice in plant tissue. Plant World, vol. 9. 1906 b The passage of water from the plant cell during freezing. Plant World, vol. 9. PLATE 1 EXPLANATION OF FIGURES Figures 2 to 6 were all drawn with the aid of a camera lucida. Figures 2 to 5 are anterior horn cells of spinal cord of the dog, from smears fixed in 96 per cent alcohol and stained with erythrosin and toluidin-blue. In the case of the frozen preparations only the blue-staining reticulum is represented, with this exception, that in figure 5 a small portion of the pink-staining extracellular reticulum is represented to show its continuity with the blue network. The individual dif- ferences will be noted in connection with the separate legend for each figure. 2 Anterior horn cell of an unfrozen smear, fixed twenty-five seconds after decapitation. The Nissl’s bodies are distinctly present. Magnification 750 diameters. 3 Anterior horn cell, frozen and fixed at about — 20°C., seventy-four seconds after decapitation. The figure represents one of the early transition forms, the Nissl’s bodies appearing as minute networks loosely joined together. Magnifi- cation 750 diameters. MOLLGAARD’S RETICULUM PLATE 1 THOMAS J. HELDT me 8: oR 3 ae ‘ “2 x) xt as ar LoS as aie i PLATE 2 EXPLANATION OF FIGURES 4 Anterior cell, frozen and fixed at about —25°C., one minute and forty-three seconds after decapitation. This is a more advanced stage than figure 3, but there are still evident traces of the Nissl’s bodies from which the network was derived. The nucleus is faintly discernible. Magnification 400 diameters. 5 Anterior horn cell, frozen and fixed at about —25°C., one minute and forty- three seconds after decapitation. From the same smear as figure 4. This repre- sents one of the extreme forms in which practically all evidence of the former Nissl’s bodies is lost. In the area marked X there is observed a small portion of the extracellular pink-staining network which is continuous with the intra- cellular blue-staining reticulum. This area in this figure is the only place where any of the pink-staining network is shown. The nucleus is represented by the area having but few meshes. The nucleolus is much distorted. Magnification 600 diameters. 6 A somewhat diagrammatic representation of Méllgaard’s ‘perivascular net- work’ in the walls of a precapillary vessel. Frozen and fixed at about — 40°C., forty-two seconds after decapitation. From a smear of neural tissue. Note that some of the networks extend beyond the nuclear walls while others are still confined within them. In those extending beyond the limits of the nucleus the nuclear wall is apparently lost in the network. Magnification 1000 diameters PLATE 2 MOLLGAARD’S RETICULUM THOMAS J. HELDT 5 349 THE STUDY OF AN ATYPICAL CEREBRAL CORTEX D. DAVIDSON BLACK The Anatomical Department, Western Reserve University Medical School NINE FIGURES CONTENTS Herr EOCM DION ASS LGR EEAD eicra did ae etN's asd a Sodas «9 cts sso tat ee eee a ee A 351 IDescripionvoteorticalvaneas? s.2s0.145 cos sorce «losaies RO EEE ane 354 DTS CUSST OME CG erat sete yest ola ace. ah Gaciei ste lass) ofc SG She las OCC Mera ence: 360 Coxnticalilaminatiome.cce co ca.s nes «cee ateo st 0 cll eco shoe Cree RPC POI eseyeisss 360 elationvoticortexsvoamenenusdberss- 45.4 eee eee eee eee eee 362 Projection fibers entering mainly via zonal layer.................-2..6-- 363 Projection fibers entering mainly via medullary center.................. 364 Regions destitute ofunalamice fibers). e eee acme e eee se teeeino ocr 365 Prophicactivitiesof aterent fibers: 242.02... .saac ons aee ee me 366 Results of absenceot trophic actions s. .././45 .0 45 i ae eee 367 SUTIN TINA reesei tla de ea eeie oe, alee tape ree es Ceseie Lec ois oe A Se nr 368 GORA GUNG [C1bEds.55.2)-15 5 s.5!imce sya oa. hae erontnistean Sirerovedeaes cna ik «ss Sy ae RN 369 INTRODUCTION In a previous paper (2) I have described briefly some of the histological findings in a quite atypical cerebral cortex from a cyclopian term foetus. In the present communication it is my in- tention to deal with the structure of various areas in the same cor- tex somewhat more fully, for it seems to me that these details are warranted in view of the manner in which the cortical develop- ment in this case illustrates several rather fundamental theories concerning the growth of nervous tissue. The cerebrum in this case was represented by a single unpaired vesicle, having an extensive expanded ependymal roof which was attached to the recurved margin of a cup-shaped thickened base representing the cortex proper. The attachment of the cerebral vesicle to the thalamus was very slight and wholly basal. On 351 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23. NO. 5 OCTOBER, 1913 34 D. DAVIDSON BLACK this account projection fibers of thalamic origin encountered con- siderable mechanical difficulty in gaining the cortex. Many of these fibers failed to reach the cortex and of those that did, the majority come to an end within a short distance of their entrance. These somewhat complicated relations are fully described i in the paper cited above. The thickened basal portion of the cerebrum representing the cortical area was divided by a Y-shaped median furrow into a median anterior and two lateral posterior regions or lobes. This furrow is not of morphological significance and represents only a mutual form adaptation between the cerebrum and skull base. However, for purposes of description I shall refer to these regions outlined by this furrow as lobes. The relations obtaining in this brain may be seen on reference to figures 1, 2 and 3. In these figures are indicated the approximate areas from which the sec- tions here illustrated were taken. | The drawings of these sections were all made with the aid of a Leitz projectoscope at a magnification of 130 and are reduced to < 65 in reproduction. This magnification was selected as being the lowest at which even the approximate shape of the smaller cells could be shown. The whole thickness of the inner cortical stratum is not indicated in any of these drawings. Technique Modified Weigert, modified Nissl and simple hematoxylin and eosin stains were used. The tissue was not in a favorable condi- tion to react to metallic impregnations and although numerous attempts were made, no successful preparations were obtained. The approximate distribution of the thalamic fibers, which were the only medulated fibers present in the cortex, was determined by a modified Weigert method. It was found that these fibers when present coursed in general, parallel to the surface of the cor- tex. No typically radial fibers were found. The fibers were poorly medullated and gave to the tissue a peculiar, coarse, reticu- lated character quite different from the almost homogeneous appearance of the fiber laminae of the cortex proper. A. Fig. 1 Outline drawing of ventral view of whole brain. Cor. cer., cortex cere- bri; Ep.R., ependymal roof; Met., cerebellum; M.F., Y-shaped median furrow; Myel., medulla; P, pons. The approximate areas from which the sections drawn were taken are indicated by their respective numbers. Fig. 2 Outline drawing of entire brain—posterior view. Lettering as in figure 1. The region on the posterior pole of the right posterior lobe from which the drawing for Area 4 was taken is indicated by the numeral. Fig. 3 Diagram of a median sagittal section of the entire brain. Agq.c., iter; Cor. pin., pineal body; Nw.o.7., inferior olive and Nu. r., red nucleus; the position of these nuclei are indicated by dotted lines; Tec. mes., midbrain roof; Th., thal- amic mass; Vent., cavity of forebrain vesicle; V.q., fourth ventricle; other letter- ing as in figurel. The approximate areas from which the sections drawn were taken are indicated by their respective numbers. 353 854 D. DAVIDSON BLACK DESCRIPTION OF CORTICAL AREAS Over the major portion of thecortex the cellular elements are arranged in such a way as to form five quite definite strata. These are: I, an outer or plexiform layer well marked over all areas; II, an irregularly arranged cell layer; III, a layer sparsely supplied with cells and which I have compared elsewhere with Bolton and Moyes’ (8) inner fiber lamina; IV, a layer of closely packed cells; and V, a very thick inner or polymorphic cell layer. In many regions this last layer tends to be subdivided into two strata described when present as layers V and VI. After this general statement as to cell lamination, a more de- tailed description of certain definite areas will be given. The regions selected are as follows: (1) two different areas from the anterior median lobe; (2) an area bordering on the right anterior limb of the Y-shaped median furrow; (3) from the central portion of the base of the left posterior lobe; (4) from the posterior pole of the right posterior lobe; and (5) from the region of junction of the thalamus and cerebral vesicle. Area 1A. Cortex over the anterior basal portion of the ante- rior lobe. Average thickness 6.7 or 6.8 mm. (fig. 4). Layer I. The zonal layer is thick, prominent and sharply marked off from the subjacent stratum. Occasionally there are found in the deeper parts of this layer irregular medium sized multipolar cells, but most of the cells found here are of embryonic character. Layer II. Aor ae og * = » (tee 2, tale ff. <--> ° oye pe fa% lo? 2? 2°, 22 2°40 & or02 300 356 D. DAVIDSON BLACK toward typical embryonic arrangement into rows at right angles to the surface of the cortex. Layer V. A stratum of less densely packed cells which varies in thickness at the expense of the subjacent lamina. Layer VI. An irregular stratum of varying thickness made up of poly- morphic and embryonic elements arranged in groups of various sizes and on the whole showing a denser arrangement of cells than Layer V. Area 1B. Cortex over the inner pillar of the anterior recurved margin of the cerebrum. Average thickness 2.5 or 3 mm. (fig. 5). The lamination occurring over this area is essentially similar in its arrangement to that already described for Area 1 A. The cortex is here however markedly thinner and cell differentiation is even less evident. In the zonal layer there are no medium sized multipolar elements to be found. At the cortical limbus all cell laminae blend as described in a previous paper (2). In the figure the inner or polymorphic layer is interrupted by a shrinkage cleft. In both these areas described Layers V and VI are easily to be distinguished as separate strata. Below, Layer VI gradually merges with the medullary center. Area 2. Cortex from the anterior part of the right posterior lobe. Average thickness 5.2 mm. (fig. 6). Layer I. Well developed but, as may be seen from the figures, it is much thinner than the corresponding layer in the sections described above. In the deeper portion of this stratum or in the upper parts of the subjacent layer there are found not infrequently quite large irregular multipolar elements. When seen they form a marked contrast to the small und fferentiated cells predominating at this level. Layer II. This layer shows no essential difference from Layer II as described in Area 1. Layer III. In some places in this region this layer is to be distin- guished only with difficulty. Its presence depends upon the size of the irregular cell groups of the superadjacent layer, which at times almost blends with Layer IV. The cells are small and are mostly of embryonic nature. Occasionally at the junction of Layers II and III there are found medium sized pyramids fairly well differentiated. Fig. 6 Section through the cortex bordering the right anterior limb of the Y- shaped median furrow. Area 2, region indicated in figure 3. X 65. Fig. 7 Section through the cortex from the central portion of the base of the left posterior lobe. Area 3, region indicated in figure 3. X 65. yur 7. a eral 357 358 D. DAVIDSON BLACK Layers IV and V. These two layers are so blended as to be indis- tinguishable in this region. Together they make up a well marked and densely packed cell stratum which varies in thickness considerably at different points. In its deeper portion there occur either singly or in groups, certain giant polymorphic cells averaging 36 by 43 microns in size. Layer VI. The polymorphic layer is not sharply marked off from the layer above and the line of demarcation is in places very indistinct. This layer and the superadjacent stratum make up a good two-thirds of the total cortical thickness in this area. Area 3. Cortex from the central portion of the base of the left posterior lobe. Average thickness 5 mm. (fig. 7). Layer I. The zonal layer is well developed but not very sharply marked off from Layer II. Irregular quite large multipolar elements are of frequent occurrence in this stratum, which is otherwise similar to the corresponding lamina in the areas described. Layer II. This layer is not so well developed as is the case in the more anterior portions of the same lobe. The crowding of the cells into irreg- ular islands is not so marked. The most characteristic feature of this layer is the occurrence here and there of giant polymorphic cells averag- ing 33 by 40 microns in size. Layer III. This layer is readily distinguished by the sparseness of its cell content but its boundaries are by no means definitely marked. Layer IV. This layer is thick and well marked. The majority of the elements in the more superficial portions of the stratum are small and poorly differentiated. The characteristic arrangement into rows or columns is well shown. In the deeper parts of the lamina, numerous well developed medium and large pyramidal elements occur. This part of the stratum is quite evidently in a more advanced state of differentiation. Layer V. Though seen in some places, as for instance in the area drawn in figure 7, this layer is to be made out only with difficulty in this region. Layer VI. There is a tendency toward grouping of the elements of this layer into irregular islands as was the case in this stratum over the anterior lobe. Otherwise this layer does not differ essentially from the polymorphic stratum of other areas. Area 4. Cortex from the posterior pole of the right posterior lobe. Average thickness 5.2 to 6 mm. (fig. 8). LayerI. The zonal layer is very thin, being little more than one-third as thick as over other areas of the cortex. Layer II. This layer is fairly well marked and is made up of medium and small sized polymorphic and pyramidal elements somewhat loosely arranged. In the outer portion of this stratum large polymorphic cells AN ATYPICAL CEREBRAL CORTEX 359 are of frequent occurrence. The tendency toward arrangement of ele- ments into island-like groups is not so marked as is the case in this stratum over other areas. Layer III. Though not well developed, this layer can for the most part be distinctly made out and presents no special peculiarities. LayerIV. This lamina is markedly developed and is somewhat thicker than all the strata superficial to it combined. It is made up of a dense collection of cells of embryonic, pyramidal and polymorphic types. The crowding of the elements here is not so great, however, as is the case in this stratum over the anterior lobe and there is a relative predominance of quite well differentiated cells. No lamina corresponding to Layer V in the areas already described can be made out in this region. Layer VI. The line of demarcation between this and the superad- jacent stratum is very indistinct. The polymorphic layer is here very thick, being thicker in comparison to the total depth of the cortex here than is the case in any other region except in Area 5. Large polymorphic cells—27 by 36 microns on the average—occur singly and in groups at different levels throughout this stratum. Area 5. Cortex from the region of junction of thalamus and cerebrum. Average thickness 6.6. to 7 mm. (fig. 9). Layer I. The zonal layer is well marked and shows a peculiar coarse reticulated appearance due to the presence here of great numbers of large thalamic projection fibers. At the periphery of this stratum there is a layer of small embryonic or neuroglial elements. Layer II. The line of demarcation between this layer and the pre- ceding is very indefinite. Numerous large and medium sized polymor- phic elements, together with small embryonal cells make up this stratum The embryonic elements, however, by no means predominate there as is the case in this stratum elsewhere in the cortex. No lamina corresponding to Layer III in other regions can be distin- guished here. Layer IV. This stratum is indistinctly marked off from the layers above and below it. It is a thick lamina whose elements show a decided tendency to become progressively smaller in size from without inward. There is no lamina corresponding to Layer V as described over the anterior lobe. Layer VI. This is a very thick stratum and is poorly marked off from Layer IV. The elements are occasionally arranged in groups of irregular size in which the cells are more closely packed than in the inter- vening spaces. At various levels in this stratum, but more especially in the middle third, there are found certain very large giant cells occurring either singly or in groups of two or three. Many of these cells are as large as 36 by 54 microns but the average size is about 34 by 50 microns. Most of these giant cells are of quite normal pyramidal type but not a few are of irregular polymorphic shapes. The nucleus is large, vesicular 360 D. DAVIDSON BLACK and usually centrally placed, and shows as a rule a well developed eosino- phile karyosome. - In the cytoplasm Nissl bodies are present, but show as distinct and sharply defined granules only in some cases; in others the tigroid substance is very variable in its appearance. In all cases there is a well defined circumnuclear zone practically free from Nissl substance. In a few cases the nucleus is eccentric and irregular in shape. In Weigert stained sections of these areas, the following dis- tribution of medullated tissue was noted: Area 5, many medul- lated fibers in both zonal layer and medulla, no radial fibers; Area 4, a few medullated fibers in the zonal layer and numerous medullated fibers in the medullary center; Area 3, occasional medullated fibers in the zonal layer and a few present in the medulla; Area 2, no medullated fibers in the zonal layer and a very few in the medullary center; Area 1, no medullated fibers anywhere. DISCUSSION Cortical lamination The cell lamination. in this case shows a number of points of resembance to that obtaining in the normal cortex. ‘Thus, there is everywhere to be distinguished a zonal or outer fiber lamina and the cortex in all those regions not subject. to marked modi- fication by the entering thalamic fibers may be divided into an outer and an innér cell stratum by the lamina described as Layer Ill This division, then, would correspond to that obtaining in the normal cortex where, as Bolton and Moyes (3) have shown, there is a very early splitting of the primary cortex into two layers by the formation of an inner fiber lamina. However, the lamination also shows several quite distinctive features not at. all comparable to the normal. The stratum Fig. 8! Section through cortex of posterior pole of the right posterior lobe. Area 4, region indicated in figure 2: X 65. Fig. 9. Section through the cortex at the region of junction of the thalamus and cerebrum. Area 5, position indicated in figure 3. X 65. 1 Tn figures8and/9, certain cortical layers, which are described in figures 4 to7, are not represented or numbered. It is not the intention, however, to imply that the laminae bearing the same numbers throughout these figures are necessarily altogether homologous. This point is made clear in the text. qs Pliny 2 9S a * 361 362 D. DAVIDSON BLACK described in the normal cortex as the inner fiber lamina has been shown to correspond to the inner line of Baillarger in the adult (3). This means that the greatest amount of cortical differentiation in man occurs normally in the outer cell lamina. If, then, the stratum described as Layer III correspond to the inner fiber lamina of Bolton and Moyes, it will readily be seen that practically all of the cortical differentiation has taken place below this level in this case except in those areas markedly modified by the presence of projection fibers in the zonal layer. Disregarding these areas then, this cortex has conformed to the general embryological rule and has begun its development from within outwards. Beyond this the condition here seems to pre- sent an exception to any normal—‘usual’ might be a better term —cortical lamination. The inner cell lamina has become modified in an atypical fashion to form three fairly distinct layers while the outer cell lamina has apparently suffered involution. It is also to be noted that any large pyramidal elements which simulate in form the cell bodies of efferent projection neurones are placed, not in relation to the homologue of the inner fiber lamina, where normally these cells are situated, but at much deeper levels in the much modified inner cell lamina. Relation of cortex to afferent projection fibers The atypical projection fibers of thalamic origin on gaining the » cortex are not distributed equally to all areas. It thus happens that the cortex may be divided into two major regions: (1) a region in which thalamic fibers end, and (2) one destitute of such fibers. Area 1, described above, may be taken as typical of the histological formation characteristic of the cortex in those regions devoid of thalamic projection fibers. Furthermore, the region supplied by thalamic fibers may be also subdivided according to the method of distribution of these into (a) a region in which the majority of the thalamic fibers enter by way of the outer or zonal layer, and (b) an area of greater extent containing fewer projection fibers which enter the cortex mainly by way of the medullary center. The histological arrange- AN ATYPICAL CEREBRAL CORTEX 363 ment of the cortex in the latter region is illustrated in the pre- ceding description by Areas 2 and 3, while that of the former which shows greatest variation from the general cortical pattern, is well shown in Areas 4 and 5. Projection fibers entering mainly via zonal layer Let us first consider the effect of the entering thalamic fibers when distributed mainly by way of the outer or zonal layer of the cortex. This is the case especially in Area 5, at the region of junction of the thalamus and cerebrum. The second layer of the cortex, which is, elsewhere, for the most part, made up of poorly differentiated pyramidal, polymorphic and embryonal cells, shows here a fewer number of elements on the whole and of these the greater number are quite large well differentiated pyramidal or polymorphic cells. It is of significance to note that the average size of the elements decreases while their number increases in passing more deeply into the cortex in this region. The various cortical laminae are much disturbed and difficult to make out. Certain very large giant cells occur in the deepest stratum and it is to be noted that this stratum bears the same relation to the projection fibers in the medullary layer as the second layer of the cortex bears to these fibers in the zonal layer. In Area 4, large polymorphic cells are present in the second layer of the cortex in considerable numbers, though this condition is not so marked asin Area 5. Sf rolt. lat. Gy SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 455 f vA. cL pyr. Nes Ss, e i Z i tv. off. . 51 Figs. 48 to 53 Mole (Scalops), six transverse sections from a Weigert series. Magn. 12 diam. : Fig. 48 Mole, transverse section through the rostral end of the hippocampus (line a in fig. 55). Note the enlarged and in-curved primordium containing large cells. Compare with figure 26 (Opossum). Fig. 49 Mole, transverse section between the foregoing and the genu (line b in fig. 55). Description in text. Fig. 50 Mole, transverse section at genu corporis callosi (line c in fig. 55). The medial parolfactory nucleus is coextensive with the precommissural bundle. The nucleus is very similar to that in the bat (fig. 42). Fig. 51 Mole, transverse section through neuroporic recess (line d in fig. 55). The mole is conspicuous for the large volume of primordium hippocampi which lies adjacent to the ventricle and lateral to the fornix and fimbria. fh, Ty f.vh. I pyr 53 Fig. 52 Mole, transverse section through the interventricular foramen (line e in fig. 55). Description in the text. Fig. 53 Mole, transverse section through point of junction of primordium hippocampi with the hippocampal flexure (line f in fig. 55). The right side of 456 bo at 0. === post ZE-_= Z ———S—$S[s—T—==_ /71a5SQ@ latermedia 5S the section is a little farther forward than the left, and here only the primordium is seen. The small mass beneath the commissures in the middle line is the nodu- lus marginalis. Fig. 54 Mole, transverse section through the peculiar plate of large cells by which the indusium is brought into connection with the hippocampus beneath the splenium. The fascia dentata nowhere else approaches so near the splenium. Fig. 55 Mole, median sagittal section reconstructed from several sections. Description in the text. 457 458 J. B. JOHNSTON 1.0.4.~ Figs. 56 to 62 Rat, seven transverse sections of the hippocampal region ros- tral to the interventricular foramen. Magn. 40 diam. Fig. 56 Section through the rostral end of the hippocampus in the olfactory peduncle. Fig. 57 Section through the rostral part of the olfactory tubercle showing the special medial hypertrophy of the deep cells of the tubercle. Between it and the hippocampal cortex the deep cells come out to the surface as the primordium hippocampi. In all the sections of the rat it is particularly noticeable how the primordium appears as a continuation of the deep or polymorphic layer of the hippocampus. Fig. 58 Section showing the indusium bending around the genu. Note the large cells of the polymorphic layer. Below the genu nearly all the cells, deep as well as superficial, are pyramidal in form and there is a distinct hippocampal fissure and in-curving of the cortex. Fig 59 Section caudal to genu. Note the extent of the hippocampal cortex and that its deep or polymorphic layer is now part of the primordium and will continue back over the foramen as the bed of the hippocampal commissure. SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 459 460 ae J..B. JOHNSTON . | !.. ta Figs. 60 and61 Sections farther caudad. : Here the hippocampal cortex below the corpus callosum becomes smaller (60) and then insensibly merges with the undifferentiated cells of the septum. In other words, we have here a hippo- campus without a cortical layer and this is the equivalent of the polymorphic layer and of the primordium hippocampi of the selachian. A well developed nucleus parolfactorius medialis appears in figure 61, quite comparable to that in the bat and mole but placed somewhat more caudally. Fig. 62 Section at the level of the neuroporic recess showing how the primor- dium hippocampi becomes the bed of the hippocampal commissure. On the left side where the cells are drawn, all the white space is filled by commissural fibers. Fig. 63 Rat, parasagittal section near the median plane. The hippocampal flexure and the upper end of the facia dentata are cut. The section euts length- wise of the fornix superior where that pushes between the hippocampal com- missure and the corpus callosum toward the splenium. From the septum (p.h.) to the right many cells extend back among the fornix fibers and blend with the polymorphic layer of the hippocampus. SEPTUM, HIPPOCAMPUS, .PALLIAL COMMISSURES 461 462 J. B. JOHNSTON * Fig. 64 Rat, sagittal section slightly inclined to the right at the dorsal edge. The section crosses the median plane at the point x. Above this point the sec- tion cuts the right fornix column, below it cuts the left. In front of the fornix columns (to the right) is the large-celled nucleus of the septum; behind, the small-celled nucleus in which the commissura hippocampi is imbedded. .The continuity of the two nuclei between the fornix columns (as well as lateral to them) is interesting just here because there is seen at the same time a continuity with the nodulus marginalis to which the tela is attached at m. It is clear that this nodule is composed simply of the cells nearest the dorsal margin of the lamina supraneuroporica and that the whole commissure system has developed in the cell-mass between this and the level of the neuroporic recess. Figs. 65 to 70 Rabbit, six transverse sections rostral to the interventricular foramen. Weigert stain. © Fig.65 Section through the rostral end of the hippocampus near the peduncle. On the right side the hippocampus and the cortical layer of the tuberculum are shown in black. Note the olfacto-cortical fibers rising close past the caudate nucleus and the olfactory commissure bundle. Compare figure 27. Fig. 66 Section just in front of the genu. On the right the indusium is shown (black) bending about the genu. Note the prominent band of precommissural fibers which mark the zona limitans. Compare figures 27 and 42. 4a SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 463 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 5 y | Bd TE LM GP ij a SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 465 Fig. 67 Section caudal to the genu. A small band of hippocampal cortex is seen beneath the genu as in the rat. The sharp separation of the hippocampal primordium from the parolfactory area is more clear in the rabbit than in most mammals. Fig. 68 Section a short distance in front of the anterior commissure. Note how the precommissural fibers in part contribute to the fornix superior and in part enter the hippocampal primordium. Fig. 69 Section at the level of the neuroporic recess. The fornix columns contribute to the fornix superior. The perforating fibers appear to enter the cingulum. It is possible that they go to the hippocampus farther back. 466 J. B. JOHNSTON Fig. 70 Section just in front of the foramen. Description in the text. SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 467 Figs. 71 to 75 Bear (Ursus americanus). Five transverse sections from a Weigert series. Magn. 2 diam. Fig. 71 Section through the interventricular foramen. The section cuts the caudal border of the anterior commissure. The base of the neuroporic recess is seen between the fornix columns. It extends forward through the whole thick- ness of the anterior commissure (see figs. 72 and 90). Note the prominent striae Lancisii. The fimbria is free from gray matter but a few cells accompany the fornix superior. 468 J. B. JOHNSTON Fig. 72 Section through the hippocampal commissure, fornix columns and neuroporic recess. Fig. 73 Section 2.15 mm. rostral to the last. The zona limitans is sharply marked by the precommissural fibers as in the opossum and rabbit. Islands of Calleja come up to this level as in the other forms. 469 SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES \\\ \ \\\ \ \\ \ Fig. 74 Section 1.1 mm. rostral to the last. Note that in the bear the primordium hippocampi has become almost thin enough to be called a septum pellucidum. Fig. 75 Section behind the genu and cutting the rostrum beneath. Note that here the indusium coming down around the genu as in all forms is continued It is diagrammatically down from the rostrum toward the olfactory peduncle. represented in black. This is essentially the condition of the hippocampus in the embryos of higher mammals and man. Fig. 76 Bear, a part of the section shown in figure 73, drawn at a higher mag- nification. Description in the text. Fig. 77 Bear, section through the genu showing that the indusium as it curves beneath the genu develops a complete, though small, hippocampal for- mation with all the typical parts present. Compare figures 29, 43 and 58. Fig. 78 Pig embryo, 23 mm. Medial surface of the right half of the head. The lamina terminalis contains the anterior commissure. The lamina supra- neuroporica is only slightly thickened. Between it and the velum transversum is the paraphysal arch, the angulus terminalis of His. 470 Fig. 79 Pig embryo, 28 mm. The lamina supraneuroporica is raised to a more vertical position by the growth of the hemisphere and the thalamus is rela- tively crowded. There is still no commissure visible in the lamina supraneuro- porica but it is somewhat thicker than in the 23 mm. stage. Fig. 80 Pig embryo, 40 mm. The lamina supraneuroporica is still more elevated and the paraphysal arch is crowded into a deep narrow sac, in front of the developing choroid plexus. Now the anterior pallial commissure is evident as a fibrous mass occupying the lamina supraneuroporica. There is up to this time no secondary fusion nor extensive thickening in the region of the commis- sures. 471 Ate, J. B. JOHNSTON Fig. 81 Pig embryo, 50mm. Rapid development of the anterior pallial com- missure has produced a condition very similar to that of the adult bat. The commissure still occupies the lamina supraneuroporica but also extends forward from its dorsal border to form a crescent. .Above the crescent is seen the indu- slum in a condition very much like that of the hippocampal formation in the opossum or bat. I can find no indication of secondary fusion. The rostral limb of the commissure is formed simply by the invasion and stretching of the dorsal border of the lamina supraneuroporica by additional corpus callosum fibers. SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 473 TTL Figs. 82 to 90 Diagrams to illustrate the relations of the hippocampus, hippo- campal primordium and paraterminal body. The medial surface of the right hemisphere is drawn in each case. The paraterminal body is shaded with hori- zontal lines, the hippocampal primordium is distinguished by means of spindle- shaped spots or flecks and the hippocampus is in solid black. The commissures are merely outlined with pen lines. All the diagrams except those of the bat and the bear are drawn from dissections. Fig. 82 Diagram of the brain of a reptile to illustrate the relations of hippo- campus and paraterminal body as defined by Elliot Smith. The outline is taken from the turtle’s brain. Herrick recognizes that the narrow ridge over the fora- men in the turtle is primordium hippocampi but describes a much larger supra- foraminal portion of the paraterminal body in lizards. Fig. 83 Diagram of the brain of the rat similar to the last. The whole commissural system is supposed to be imbedded in the paraterminal body. 474. J. B. JOHNSTON — ————=———— Fig. 84 Diagram of the forebrain of Scyllium to illustrate the author’s con- ception of the relations. The hippocampal primordium is separated from the paraterminal body by the neuroporic recess. This primordium gives rise to two structures in higher vertebrates: hippocampal formation and a residual body less highly organized which is represented in mammals by the septum pellucidum. This residual structure is represented in the following diagrams by the flecked or spotted area. : Fig. 85 Diagram of the brain of the turtle to illustrate the view set forth in this paper. Not only in the turtle but in lizards and other reptiles the body which runs along over the foramen, between it and the hippocampal cortex, is hippocampal primordium. > SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 475 Fig. 86 Diagram of the brain of the opossum. This type of brain has been made familiar by Elliot Smith’s work. The reasons for assigning a portion of Professor Smith’s paraterminal body to pallial area are set forth in the text. Fig. 87 Diagram of the brain of the bat. The commissures are poorly devel- oped and there is nearly as close connection between hippocampus and its primordium as in the opossum. 476 J. B. JOHNSTON i = — Fig. 88 Diagram of the brain of the mole. Although there is a large corpus callosum and a splenium is well formed, there is an unusually large primordium reaching almost the entire length of the corpus callosum and connecting with the hippocampal flexure. The boundary of the paraterminal body as it appears in the ventricular surface is represented here. Reference to figure 55 will show that the medial parolfactory nucleus rises much higher on the outer surface. The diagram of the rat brain following is drawn with reference to the boundary on the outer surface. Fig. 89 Diagram of the brain of the rat. The gradual merging of the indu- sium beneath the genu with the primordium is represented by tooth-like projec- tions. The paraterminal body rises higher on the medial surface than it does next the ventricle. On the ventricular surface the boundary line runs almost straight forward from the neuroporic recess. SEPTUM, HIPPOCAMPUS, PALLIAL COMMISSURES 477 Fig.90 Diagram of the septal region in the brain of the bear, based on serial transverse sections. The hippocampal commissure is a very thin band. The gray matter in the caudal part of the septum is reduced to a very slender strand accompanying the fibers of the fornix superior. Figs. 91, 92, 93 Diagrams of the brains of Scyllium, the bat and the rat to illustrate the relations of the fibers of the fornix system to the pallial commis- sures. See section on this subject in the text. The arabic numerals J, 2, 3, have the same significance as in figure 38. 478 bo. J. B. JOHNSTON STUDIES ON THE REGENERATION OF THE PERO- NEAL NERVE OF THE ALBINO RAT: NUMBER AND SECTIONAL AREAS OF FIBERS: AREA RELATION OF AXIS TO SHEATH M. J. GREENMAN The Wistar Institute of Anatomy and Biology THREE FIGURES The work here presented is part of a program of considerable magnitude, bearing upon the growth of the nervous system, now being carried out under the direction of Professor Donald- son. The experiments thus far made have already suggested so many new problems that it seems best to publish some of the results now in hand while further observations are being made. The primary object of this study was to determine the number and size of the medullated nerve fibers within a regenerated peripheral nerve of the albino rat and compare the conditions on the side of the operation with those found in the correspond- ing unoperated nerve of the opposite side in the same animal. The following series of animals furnished the data for this study: Series 1. Animals nos. 1 to 79 inclusive. This series was used in perfecting the technique of operation and no further record of it will appear. Series 2. Comprised 92 animals in all. From 18 of these, sections suitable for the purposes of the study were obtained. These 18 animals, of different known ages, were operated and after the lapse of varying periods of time were killed. The weight at the time of operation and at the time of killing is given in all cases except one. One specimen, no. 100, was excluded because although a young animal it did not gain in weight after the operation, and two specimens, nos. 93 and 116, were excluded because they were the only representatives of their respective age groups. The 479 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 5 480 M. J. GREENMAN remaining specimens fell into three different age groups and presented data upon the following points: A. Number of fibers in left or control nerve. B. Number of fibers in proximal end of operated nerve. C. Number of fibers in distal end of operated nerve. D. Effect of operation upon body weight. Series 3. Three adult unoperated animals, nos. 222, 223 and 224 of known weights were used to determine the following points: A. The number of fibers in the peroneal nerve of a normal animal. B. The difference in the number of fibers at the two ends of that portion of the peroneal nerve used in these experiments. C. The sectional area of peroneal nerve fibers of a normal animal and the area relation of axis to sheath. Series 4. This series included operated animals nos. 192, 193, 210, 211 and 220. Two were adults and three were young animals of known ages; all were of known weights. They furnished data upon the pro- gressive change in the number of fibers in operated nerves. Series 5. This series included animals nos. 167, 168, 169, 170, 171 and 172. The ages and weights were known. This series was used to determine when degeneration is complete. Series 6. Animals nos. 316 and 317, Norway rats, of known weights, were used to demonstrate the equality in the number of fibers in the peroneal nerve of each side. Series 7. Operated animals nos. 106, 114 and 154. This series was used to furnish data on the se¢tional area of fibers and the area rela- tion of axis to sheath. These animals were selected from Series no. 2. The nerve in the right leg was consistently selected for experi- ment and the corresponding nerve of the left leg was used as the control. The observations made in the course of these experiments comprise the determination of the number of fibers and the area relations of axis to sheath in the left or control nerve and similar determinations at different levels above and below the point of operation on the right nerve. The nerve selected for operation was the peroneal. This nerve is the anterior (or lateral) division of the sciatic and passes dis- tad beneath the biceps femoris muscle in close contact with the gastrocnemius; it curves over the lateral margin of the gas- trocnemius sometimes piercing the edge of this muscle to dip into the fleshy mass formed by the fused bodies of the upper extremities of the peroneal muscles (P. longus, P. brevis, P. tertius and P. quartus). REGENERATION OF PERIPHERAL NERVES 481 For about 10 mm. (in the adult rat) proximal to its entrance into the peroneal muscles, the peroneal nerve normally gives off no branches. This was the region selected for operation. Distal to the region of operation, the nerve gives off branches to the peroneal muscles, the tibialis anterior, the extensor digi- torum longus and also extends its fibers to the dorsum of the foot. TECHNIQUE OF OPERATION The method of operation was as follows: The animal was anesthetized, the nerve exposed by a cut about 1.5 cm. in length through the skin, fascia and biceps muscle. The nerve was lifted free from surrounding structures, crushed and wired or simply wired. The flaps of this wound were then closed by two or three stitches in the skin and the wound sealed by apply- ing a bit of sterilized cotton covered with collodion. Crushing the nerve was accomplished by means of a small pair of bone forceps, the blades of which had been polished off, leaving instead of sharp cutting edges, blunt smooth edges. By this means the bundles of white glistening nerve fibers were _ divided, leaving the perineurium intact as a tube connecting the divided ends of the nerve fibers. The space within the peri- neurium, between the divided nerve fibers, usually filled with serum or blood. This operation was followed in every case by loss of ability to extend the toes properly and to rotate the foot outward. Recovery was in most cases rapid and the repaired nerve was usually free from interfering masses of connective tissue. The difficulties in determining at the autopsy the exact point of injury to the nerve and the importance of having this infor- mation, later led to a modification of the simple crushing tech- nique, as follows: At the point of crushing, a silver wire hook was caught around the nerve and by means of a long narrow nosed forceps this hook was forced to close tightly on the nerve. The extended ends of the wire hook were then trimmed off, leaving a small bit of wire doubled upon itself and holding be- tween its two limbs the perineurium or a portion of it. The 482 M. J. GREENMAN forceps especially made for this operation have long slender jaws on the inner surface of each of which at the extreme end is a transverse groove so placed that when the jaws are brought to- gether they present opposed depressions between which the wire loop may be firmly clamped together without danger of slipping. In later operations the preliminary crushing of the nerve by the bone forceps was omitted and the crushing done by clamp- ing the wire hook on the nerve. No. 26 annealed silver wire was used. The wire clamp was left on the nerve. In most cases the wire clamp remained on the nerve and became embedded in new tissue deposited about it. This new tissue formed a bridge or capsule structure connecting the proximal end of the nerve to its distal segment. This bridge was usually best developed on one side of the ring while in some cases its development formed a more or less uniform enclosure for the ring. Through this connecting bridge the fibers of the regenerated nerve were found to pass from the proximal to the distal segment of the nerve. The animals selected varied in age from 31 days to 250 days (adults) at the time of operation and they were permitted to live from 3 to 105 days after operation. When the animal was killed, about 10 mm. of the operated nerve, including the region of operation, was removed. An effort was made to have an equal length of nerve on either side of the clamping wire. About 10 mm. of the corresponding nerve of the left side was also removed as a control. These specimens were fixed in 1 per cent osmic acid, embedded in paraffin and sectioned. All sections were cut seven micra thick. A few sam- ple sections of the control side were mounted. The operated (right) nerve was cut in continuous series and mounted, the series beginning in the nerve above the point of crushing where the nerve appeared to be normal, and continuing through the region of operation into the new or regenerated nerve. The sections of the operated or right nerve have been designated as proximal or distal according to their relation to the point of REGENERATION OF PERIPHERAL NERVES 483 crushing. Sections of the left side have been designated as left or control. Only such cases as presented proximal, distal and control sec- tions suitable for photographing and counting were selected. The photographs were made with a Zeiss 8 mm. apochromatic objective and a no. 4 apochromatic eyepiece. Hardesty’s (99) method of counting fibers was employed. This consists in re- cording each fiber automatically by pricking a hole in each fiber image of a photographic print. The original specimen is ob- served under the microscope during the process. The counting was done with a Zeiss 2 mm. immersion objective, a no. 4 eye- piece and a draw tube of 160 mm. All counts and measurements were made with the same optical combination. EFFECT OF OPERATION In order to understand the effects of destroying the continuity of the peroneal nerve, it should be borne in mind that the fibers of this nerve are supplied to the following muscles: the peroneus longus, which passes around the external maleolus across the plantar surface of the foot and is inserted into the base of the first metatarsal—its action is to extend and slightly rotate the foot outward; the peroneus brevis, which passes around the external maleolus and is inserted into the base of the fifth meta- tarsal—its action is to abduct and to extend the foot; the P. tertius and quadratus fused with the extensor longus digitorum —their action is to extend the toes; and the tibialis anterior which is inserted into the base of the first metatarsal; its action is to extend the foot at the ankle and rotate the foot inward. The immediate effect of the operation was in every case to cause a paralysis which resulted in a flexion of the toes and a rotation of the foot inward. Since no fibers of the peroneal are distributed to the gastrocnemius muscle and plantar muscle, it may be assumed that the deformity immediately following the operation is due to the action of these muscles. This deformity in many cases rapidly disappeared and in most cases after the lapse of six to ten days it was difficult to detect any abnormality in the movements of the animal. 484 M. J. GREENMAN Passing to the histological changes following the operation, we find on the fourth day after operation that all the medullated fibers distal to the lesion show appearances characteristic of degeneration. Crushing, therefore, interrupts the fibers com- pletely. This degeneration extends from the point of crushing distally as far as observations have been made—that is, from 5 to 8 mm.—and it was assumed that the degeneration had - extended to the termination of each fiber. In the other direc- tion, degeneration extended from the point of crushing proxi- mally from 2 to 3 mm.—according to actual measurements, from 2 to 3.2mm. Above this point the structure of the great ma- jority of the fibers appeared to be normal with here and there one modified in a way to suggest degenerative changes even as much as 8 mm. proximal from the point of lesion. Similar changes in structure have been figured by Boll (76) and described as due to histological methods. I am inclined to think that the changes here observed are preparatory to the regenerative proc- ess which is about to begin and which I have found by numeri- cal determinations to begin as much as 7 mm. proximal to the lesion. Ranson (712) finds in dogs that non-medullated fibers degenerate more than 10 mm. up the proximal stump where re- generation begins. Perroncito (’06) has shown that a sectioned axone begins to regenerate within three hours after cutting. In the present ex- periments no animals were killed earlier than three days after operation and from that period up to 105 days. No effort has been made here to trace the earliest development of the new fibers. To determine the time when degeneration in the rat was complete, six animals were killed as follows: One 3 days after operation, two 4 days after operation and three 6 days after operation. ‘Two of these animals were 89 days and four were 90 days of age at the time of operation. Series 5. The three-day and the four-day animals showed complete de- generation of all fibers and the clinical signs indicated complete loss of control of muscles supplied by the peroneal. The six- day animals showed complete degeneration, but the clinical signs REGENERATION OF PERIPHERAL NERVES 485 © were so modified as to suggest readjustment of muscular control so as to mask in a measure the paralysis produced by the operation. The evidence from the three-day and four-day specimens seems conclusive that by the fourth day degeneration is complete. NUMBER OF FIBERS IN PERONEAL NERVE OF NORMAL ANIMAL Series 3 was prepared to determine, among other facts, the number of fibers in the peroneal nerve of the normal animal. Three young unoperated adults were used. Of each nerve three counts were made, one for each end and one for the middle. The distances between counts were also determined. Referring TABLE 1 NO. 224 9 NO. 223 9 NO. 222 1 UNOPERATED UNOPERATED UNOPERATED RIGHT NERVE, RIGHT NERVE, LEFT NERVE, WEIGHT 104 WEIGHT 117 WEIGHT 182 Hirst or, proximal count..c..2 22/40 +. 3h. 2240 2430 2192 Distance in » from Ist to 2d count....... 3031 4746 3080 second or middle count? ew... eae. ees 2118 2292 2418 Distance in » from 2d to 3d count....... 4466 | 2345 | 3332 hirdroy distal: coumiti: 21.5.2 aceon. oes 2392 | 2213 2364 | IAVCLAG ONC OUI sats leis e siesncly hcl srl kTateene 2250 | 2312 2325 Combined average count, 2296 Average of the distal counts, 2323 Average of the proximal counts, 2288 Average body weight, 135 Average of the middle counts, 2276 to table 1, we note that the average number of fibers at the three levels in no. 224 is 2250, in no. 223 it is 2312 and in no. 222 it is 2325. The combined average counts of all three specimens is 2296 for an average body weight of 135 grams. Various studies on the peripheral nerves by others (Dunn ’00) indicate that substantial symmetry exists normally between the nerves of the two sides of the body in respect to their numerical composition. This view is supported by our own direct observations. A486 M. J. GREENMAN TABLE 2 NUMBER OF FIBERS UNOPERATED NORWAY RAT NO. Right nerve Left nerve 316 of 1990 2031 317 9 2055 2002 1930! ANVORD ECs ore ee oo a 26 of 1992 2017 1 Second and more distal count. Table 2 presents the results of the determinations of the number of fibers in the right and left peroneal nerves of two specimens of the Norway rat (Mus norvegicus) from which the albino strain has been derived. Two counts were made, at different levels, in the case of the right peroneal of no. 317; one count was made for each of the other nerves. The average number of fibers in the right peroneal nerves is 1992, in the left 2017—a difference of 25 fibers or about 1.5 per cent. Since this difference is within the probable limits of normal variation (2 per cent) we may conclude that the right and left peroneal nerves contain approximately the same number of fibers. These data on the Norway rat have been introduced here merely to show the similarity of the number of medullated fibers in the peroneal nerve on the two sides of the same animal. The difference in the absolute number of medullated fibers in this nerve in the Norway rat and in the albino ealls for a special study. Returning to table 1, we note that the average of the proximal counts of the three nerves is 2288 and that the average of the distal counts of the three nerves is 2323, showing an average excess of 35 fibers in the distal end of the nerve or an increase of about 1.5 per cent in 10 mm. of nerve. Taking the average of the two extremes 2288 and 2323 we get 2306 as the estimated number in the middle portion of the nerve. The average of the observed numbers of the middle counts is 2276, a difference of a little more than 1 per cent. We can, therefore, safely assume that 2306, the estimated number of the middle count is approxi- REGENERATION OF PERIPHERAL NERVES 487 mately the normal number of fibers for the middle zone of the peroneal nerve of the albino rat with a body weight of 135 grams and belonging to the strain here used. Referring again to table 1, we observe that no. 224 weighed 104 grams, no. 223 weighed 117 grams and no. 222 weighed 182 grams or 75 per cent more than no. 224. The average of the three fiber counts of no. 224 is 2250, the average of no. 223 is 2312 and the average of no. 222 is 2325 or an increase of about 3.5 per cent over the average of no. 224. In this series, there- fore, the heavier animal has the greater number of medullated fibers. 3 Taking body weight as a rough index of age, we note that there is a constant increase in the number of fibers as the ani- mal grows older. With an increase of 75 per cent in body weight we find an increase of about 3.5 per cent in the number of fibers in the peroneal nerve. This observation must be held to apply to young or developing animals for Dunn (711) has shown that old animals lose fibers. Summarizing the observations on the normal peroneal nerve we may state that: In an animal of 135 grams body weight the middle zone of the peroneal nerve contains about 2306 fibers; the number is the same for each side; in a developing animal an increase in the number of fibers accompanies increase in body weight or advancing age; in the 10 mm. of nerve used in this series there is an increase of about 1.5 per cent in the number of fibers as we pass from the proximal to the distal end. Dunn (02) has shown that in the frog there is an increase in the number of fibers between the sciatic trunk and its two distal divisions of about 5 per cent, due to branching. NUMBER OF FIBERS ON THE CONTROL SIDE OF OPERATED ANIMALS Having examined the numerical relations in the peroneal nerve of the normal animal we will next examine the control (left) side of our operated animals. Table 3 presents the data from Series 2, all of which are oper- ated animals. In this table the data are arranged according to the age of the animal at the time of killing. GREENMAN J. M. 488 *peqUNOd a19M SUOT}OIS ][B JON ¢ *AdoAOO0I YOojodumt ‘JT {AraAOOOI Arey ‘Yq fpoteaoda1 [euIUe ‘Ys ‘S10QY JO JoqUINU JO SoSvIOAB FUIYVUI UL poyyIMIO p10d9xy _ £962 G68Z ZO lenePalleieverere g Sc wMayiela\ionalbirehel| misteunlieNaian sie listaedelttaiellsite | oveleXerreteke 6? cilies arene Mie veravetale ‘OSBIOAY I 61 P16 | SSIS | OL— 0 FFT 0° TST 92 922 OGG LOI ‘ 81 a Tree | Ive | 810Z | o'zz—| O'sPT | O'sat | 92 92 | sz 901 “WLI I -t9¢ | cezz | 9202| o'2— | s'est | stot | 92 912 | 082 gol “W 91 U = =9F6T €S61 O89E | ISEh | SETS | Ghz GoST 0 82T £01 €61 06 TOLL, AP Sh a sore | zeoz | 8I |eset|seer| st | oge | sez | Got | got | 19 1001 “WW FI A e192 898¢ SSTF | S81z | 0202 | 8S 0° 09T ¢ TOT GOT v9T 6¢ cll ‘dH &1 U 2066 LLES LLVE | C€6Z | T86T | SSP 0° LST SMG SOT POT 6S VEE 2 eccr WU 968 016 | 29zh | gorz | Leet | s'9e | O'OPT | s'eor | gor | FOr | 69 ell “A I U chl e90P Tg0@ | 88T€ | OL0c | G's 0 €ST ¢ 6IT €01 TOT 8g Sit “a OL WU sod 6609 GCEE | €90Z | OOIS | GS 6F ¢ 6¢T 0°08 €01 6ST 9g Ill IN 6 a 28ot | se. | 20ze | coor | Se6T | O'86 | sest | oss | .eor | est | 98 * oll “W 8 U_ «e861 €68P ITZ | OOFZ | 8902 | 09S 0 €8T 0 LeT 68 9ST L9 ch. WN 2 aM SPOT | eager | eave | goes | zg6T| s¥zt | o'oLT | sar | 86 est | Ie lel “W 9 U = s68ST 9ECP P69 | 9GFE | P86T GCE 86 661 Té ost WS UU sbh0G PSLg G99G | L882 | EF6T | O TET ¢ COT cE 86 661 T€ Paes Ney Ue I8cT s9EPG G6LE | OLTP | S66T | 0 66 0 841 0 6F L6 821 1é Po ING te: A cole 66EL SOE | G80 | S66T | & TFT € sl oer | 26 8cT Tg » Ot... NG eu OPLGT LL8G S8TE | SE0E | 00cZ | FOL 0 €&T go9 LG g9 8€ 16 “WN I Te3sta jeurrxo1g ba aur aS a ie )maneredo | ode al 2utllet woryeiedG | ens ore tEN2O2 SNOMOUSOL | guaatasouanan | | gO gWInavaHOIEM 40 ant LY EDV : € ATaVL REGENERATION OF PERIPHERAL NERVES 489 If we divide these records into age groups, as is done in table 3, we find that five such groups are formed, namely: a 65-day group, a 128-129-day group, a 156-166-day group, a 193-day group and a 276-day group. For the 65-day and the 193-day groups only one entry is available, these, therefore, may be ex- cluded. Owing to the failure of the 166-day animal (no. 100) to gain in weight, this will be omitted in the discussion, since it may be safely assumed that whatever has interfered with the growth of the animal has also modified the regeneration process. This leaves for comparison the 128-129-day group with 5 entries, the 156-164-day group with 7 entries and the 276-day group with 3 entries. Bringing the averages of these groups together in tabular form we have the following: TABLE 4 AVERAGE NUMBER OF FIBERS IN AVERAGE WEIGHT enced ee Operated Nerve pe es mew’ | Proximal | Distal 128-129-day group.....| 129 40.3 | 166.2 1981 3188 3162 156-164-day group..... 161 104.1 | 158.0 2025 2793 3560 276-day-group......... 276 162.5 148.5 2083 2646 1241 The average number of fibers in the control nerves of each of these groups is respectively 1981, 2025 and 2083. ‘This indicates a steady increase in the number of medullated fibers with advanc- ing age, the interval being 147 days between 129 and 276 days of age. The difference in number of fibers between the extremes is 102 or about 5 per cent of the smallest number. Comparing the averages of these three groups with the records of normal unoperated animals, as presented in table 1, we ob- serve that the averages of operated animals fall below the counts of even our lightest unoperated animal no. 224. Taking 2306 as our estimated number of fibers for the middle zone of an unoperated animal of 135 grams body weight and comparing this number with the number of fibers (1937) found in the control nerve of operated animal no. 113 of 140 grams body 490 M. J. GREENMAN weight (table 3) we note that our operated animal has 369 less fibers than the normal animal, a difference of 16 per cent, the normal number being taken as the standard. It is, therefore, evident that the operation causes in this in- stance a loss of about 16 per cent of the medullated fibers in the control nerve. If further we compare the grand average of the number of fibers in the control nerve 2022 (table 3) with the estimated normal number 2306, we still find a deficiency of 284 fibers or 12.3 per cent. It thus appears that there is a substantial deficiency in the number of the medullated nerve fibers in the control nerve of the operated animals. Again, if we note the average weights of the different groups at the time of operation and at the time of killing as shown in table 4, we see that the 276-day or oldest animals lost weight, while the two younger groups gained weight between the time of operation and the time of killing. We also observe that at the time of operation the younger the group the less is its aver- age weight, while at the time of killing the younger the group the greater is its average weight, showing that the effects of operation were more profound on the older animals. Dunn (’09) found a less number of efferent fibers in the unoper- ated leg of an operated frog, but apparently did not attribute the loss to the treatment the frog had received. So far as I am aware, there are no records in the literature touching the loss of fibers in the intact nerve as a result of an operation. Appreciating the importance of these effects of operation upon the unoperated nerves, experiments have been extended along this line and the results will be presented in a later study. NUMBER OF FIBERS IN THE OPERATED NERVE We will now pass to the consideration of the relations to be found in the operated peroneal nerve. The data to be discussed are presented in table 3, which gives the number of fibers found on the proximal and the distal sides’ of the lesion; the position REGENERATION OF PERIPHERAL NERVES 491 of the counts as measured from the point of lesion; the time elapsed and the gain or loss in weight since the operation. We see from this table that the number of fibers found on the proximal side of the lesion in the regenerated nerve 27 to 105 days after operation is always equal to or greater than that of the corresponding control, since the one instance (no. 111) in which this number of proximal fibers is recorded as less than that in the control is quite within the limits of normal varia- tion, about 2 per cent. On the distal side, however, there are several instances in which the number of fibers is distinctly less than in the corresponding control nerve, although in most cases it is largely in excess. Considering first the nerve proximal to the lesion, we find in table 3 twelve instances (omitting no. 100) in which the distance TABLE 5 DISTANCE PROXIMAL FROM LESION NUMBER OF CASES i eee IDIRGaaL ts). HO) (GO) WHINE do asenoeowoRo de odeesodoa. 2 20741 TROMsG 1) Lamhe eae teat, 5 a oe es: 6 2866? Eionnaete tO Pe TIM me geese eee hoes «fede ed tere 1 30358 Homi 2 stor Ohmi meee a ee enlaces eta 3 37094 1 The control average at this level is 3 The control average at this level 2047 is 1938 2 The control average at this level is 4The control average at this level 1997 is 2023 from the lesion to the level of the section has been measured. A preliminary study of these enumerations arranged in the order of their distance from the lesion showed that there was an evi- dent though irregular increase in the number of fibers as the section approached the level of the lesion. When we take the average of these records in successive groups as they appear at intervals of 2 mm., we obtain the values given in table 5. This table shows an increase of 1635 in the number of fibers between the extremes or approximately 79 per cent of the smaller number or 80 per cent of the most proximal control average as we pass from a mean point 7 mm. proximal to the lesion toward the immediate neighborhood of the lesion. 492 M. J. GREENMAN Passing next to the distal side of the lesion and dividing the five available records into two groups, one group including the enumerations between the lesion and 1.5 mm. distal to the lesion, the other group including those between 1.5 mm. and 3.1 mm. distal to the lesion and omitting all those in which sections were lost, as well as the records of no. 100, we obtain the following: TABLE 6 AVERAGE NUMBER AL T L DISTANCE DIST O THE LESION OF FIBERS NUMBER OF CASES Krone 2030 1-5 nin. ea aoe eee 2 3725! ldixoren IG Ano ve whites ola odloaaceodomeduc se 6 3 3396 1 The control averages for these groups are 2069 and 2067 respectively. Table 6 shows that there is an 80 per cent increase: in the number of fibers over the control average in the first group located between O and 1.5 mm. distal to the lesion and a 64 per cent increase over the control average in the second group located between 1.5 and 3.1 mm. distal from the lesion. From these enumerations it is evident that, following the com- plete degeneration which extends from 2 to 3.2 mm. proximally from the lesion there occurs in the course of regeneration a branching growth of the axis cylinders which appears to take place at considerable distance above the point at which complete degeneration terminates. This branching results in an increase of about 80 per cent in the number of medullated fibers as the region of the lesion is approached; this increase diminishes as the distance distal to the lesion increases. This decrease in ex- cess fibers distal to the lesion is probably due to the failure of a portion of the fibers to continue their development for any considerable distance beyond the lesion. In specimen no. 100, age 61 days at the time of operation, it will be noted (see table 3) that when killed, 105 days after oper- ation, the animal had increased in weight only 1.5 grams. This failure to grow is also accompanied by a reduction in the number of regenerated fibers most marked on the distal side of the lesion where at a distance of 3.4 mm. from the lesion only 18 medul- REGENERATION OF PERIPHERAL NERVES 493 lated fibers could be found. At 2.6 mm. proximal to the lesion 1999 fibers were found the control for this nerve being 1938. Similar conditions exist for nos. 105 and 107. In these cases, however, the animals being adults, the results are not so indic- ative of profound disturbance in the growth processes. The data given in tables 5 and 6 have been used for the con- struction of figure 1. In this figure the intent is to show by the length of the transverse lines the number of fibers at the given sectional level of the nerve; the locatién of the count proximal or distal to the lesion is shown by the position of the transverse line and is measured in micra by the scale on the left, from zero, the point of the lesion. The solid portions of the transverse lines indicate the number of fibers in the corre- sponding left or control nerves—for the most ‘part taken from the middle zone of the control nerves—while the broken line prolongations complete the representation for the operated nerve. The values of these lines are given by the scale, ‘‘number of fibers” at bottom of the figure. In entering the data from tables 5 and 6, the distance from the lesion used in the figure is inter- mediate between the limiting values as given in the tables. CONFIRMING COMPOSITE RESULTS BY A NUMBER OF DETER- MINATIONS ON THE SAME INDIVIDUAL Since the foregoing results are composite or based upon single determinations made on the proximal and distal segments of the operated nerves of a series of different animals, it seemed advisable to verify them by a number of determinations made at different levels on the operated nerve of the same animal. For this purpose Series 4 was operated and prepared. The data relating to this series are presented in table 7 and arranged according to the weights of the animals. The table gives the counts made at different levels and indicates in micra the dis- tances between the successive counts and also between the lesion and the nearest proximal and the nearest distal counts. Referring to table 7, we observe that in each instance there is a large increase in the number of fibers as we pass from the first to the third count; the gain in number amounting to 249 494 M. J. GREENMAN per cent of the first count in the case of no. 211, while no. 193 shows the lowest increase of 106 per cent. The largest number of medullated fibers is found in each case just proximal to the lesion. Distal to the lesion the number of fibers found, while greatly in excess of the normal, is some- what less than the number found just proximal to the lesion, and still less in the most distal determinations. Thus it is seen that some of the regenerated fibers fail to find their way through the scar tissue in the region of the lesion. TABLE 7 No. 193 OPERATED AGE 127, WEIGHT 64 no. 220 OPERATED AGE 121, WEIGHT 84 no. 192 OPERATED AGE 127, WEIGHT 117 wo. 210 OPERATED ADULT, WEIGHT 184 no. 211 OPERATED ADULT, WEIGHT 198 First or proxi- mal count... Distance from first to second count, inp.. Second count.. Distance from second to third count, TNO He AR cy Third count... Distance from third count to lesion, in Point of lesion Distance from lesion to fourth count, a) Bos eee Fourth count.. Distance from HOMErMiGhe | FO fifth count,in Fifth count.... 2560 | 2982 2788 3115 5260 448 | 4272 2091 3045 | 5725 938 2478 3991 2485 1442 | | 36765 | 637 | 7611 2400 3969 3164 553 1232 5150 | 938 4390 | 4870 2144 3430 7497 095 1225 1981 3140 REGENERATION OF PERIPHERAL NERVES 495 PSiaaiaw microm #roximal | \ + _G000 | y : | | \ — | | | J S000 Ie Et [ 71 l | “ear it | | oo | Yo re 2 | | oo L. --4 'S | \ | | & \ Ney 222 | 2 | N % Caen oll ) ie | ee eh Y x a) “3 Lesion me | =e | Shy a ae = 8 | | & | I G & \ | iL 000 | y ‘2 Q ee _1 Lhstal 35000 Skee] j : : humber of fiters Figure 1 THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 5 496 M. J. GREENMAN To put the results into more condensed form, we have arranged table 8. TABLE 8 No. WEIGHT Ber ps tree ty FIRST | THIRD eOumecae GAIN IN | OPERATION COUNT COUNT echt FIBERS grams | micra iy 193 64 79 2560 5260 6545 2700 220 B43 76 2091 5725 | 6468 3634 192 117 sce ee 2280 | 4158 7590 1878 210 184 77 2400 | 7611 7686 5211 211 198 77 2144 7497 7686 5323 Average..| 129 | 97 2295 | 6050 7195 3749 If we deal with the averages of this table, we note that the first counts were 7195 micra proximal to the lesion or practi- cally 7 mm. and that the average of the first counts is 2295. Referring to table 3, which presents the data of an operated series and is, therefore, comparable with the data of table 8, we note that the average number of fibers in the control of this» series is 2022. Thus we see that the average number of fibers in the operated nerves 7 mm. above the lesions is more than 13 per cent greater than the average of the controls given in table 3. It should be borne in mind that the number of fibers increases with the body weight in growing animals. The average body weight in the series of table 8 is 129 grams, while the average weight of the series of table 3 is 158.3 grams. But, nevertheless, the series of table 8 has the greater average number of fibers. We conclude, therefore, that more than 13 per cent of the in- crease in number of fibers of the regenerated nerves takes pe at a point higher than 7 mm. proximal to the lesion. Referring to table 7, it will be observed in no. 220 that the fifth count (distal to the lesion), unlike that of no. 210 and 211, exceeds the fourth by 281, so that instead of a decrease in num- ber as*we pass distally from the lesion there is in this case a slight increase. This is probably due to the fact that medulla- tion has begun to disappear from some of the excess of new REGENERATION OF PERIPHERAL NERVES 497 fibers, and that this disappearance of myeline has taken place from above downward. This point will be dealt with in another study. Figure 2 presents in graphic form the increase in the number of fibers of the regenerated nerves of Series no. 4, the data for which is to be found in table 7. Nos. 192 and 193 are, however, not recorded in the figure since in each case no counts were made beyond the lesion. As in figure 1, the length of each transverse line indicates the number of fibers and its position, measured by the scale on the left from 0, the point of operation, tells the level at which the count was made. There are no data for the control nerves. The solid line, the broken line and the dotted line are used to connect the entries for nos. 220, 210 and 211 respectively. Figures 1 and 2 and the tables on which they are based show similar numerical relations within the regenerating nerve so that we may conclude that there is a progressive increase in the number of fibers from a point at least 7 mm. above the point of operation to the level of the lesion, followed by something of a loss in the next few millimeters distal to the lesion. SECTIONAL AREAS: AREA RELATION OF AXIS TO SHEATH Numerous methods have been used for determining the diameter, sectional areas and number of fibers in nerves. These methods were summarized by Vashkevitch (’89), since which time con- siderable improvement has been made in optical devices for this work. The method which appeals to me as the most accurate is to measure a drawing of the projected and highly magnified image by means of the planimeter. A Zeiss 2mm. apochromatic objec- tive with no. 4 ocular and tube length of 160 mm. was used in connection with a specially constructed camera of such rigidity as to permit the outlining of fibers on very finely ground glass plates mounted in the plate holder end of the camera. An electric arc consuming 25 amperes of current was used as an illuminant. The optical system was protected from heat by a 498 M. J. GREENMAN water jacketed condenser through which tap water flowed continuously. By this means we were able to focus upon any fiber and obtain a sharp image which was outlined with pencil on the ground glass at a magnification of 4000 diameters. Measurements were then taken by the planimeter directly from the glass plate and recorded in square centimeters. Each planimeter measurement was repeated five time to insure accu- racy and the result reduced to square micra. SECTIONAL AREAS OF NERVE FIBERS FROM THE PERONEAL NERVE OF NORMAL ANIMALS Series no. 3 consisting of three unoperated animals, nos. 222, 223 and 224, of known weights was utilized fer these measure- ments. To determine the sectional areas of normal nerve fibers, forty of the largest nerve fibers from the proximal end and forty of the largest nerve fibers from the distal end of each specimen were measured. 'These measurements were then tabulated in the order of their size beginning with the largest fibers. The ‘aver- ages of the first ten records were then taken, the percentage rela- tions of the axis computed and the data arranged as in table 9. It will be seen from this table that the average of the ten largest fibers in the proximal end of each nerve gives us a meas- urement of 109.8 square micra for no. 224, 137.7 square micra for no. 223 and 150.3 square micra for no. 222. The average TABLE 9 Normal rats: Sectional area of fibers; relation of axis to sheath PROXIMAL END DISTAL END NO. WEIGHT 5 Entire! Avis | Sheath Pe cory Wetee) Axis | Sheath Por come 224 104 109.8} 55.6 | 54.1 | 50.6 | 85.0 | 42.3 | 42.7 | 49.7 223 hey 137 .7| 75.2 | 62.5 | 54.6 | 85.8 | 42.6 | 43.2 | 49.6 222 182 150.3] 82.9 | 67.4 | 55.1 {113.0 | 56.7 | 56.2 | 50.1 Average..... 135 132.6] 71.2 | 61.3 | 53.7 | 94.6 | 47.2 | 47.3 | 49.9 Average sectional area of the proximal and distal ends, 113.6 square micra. REGENERATION OF PERIPHERAL NERVES 499 i Me TO kel eed tae Proximal ! 1 f t f q q q f q 1 It | | | | | | | | dinmicra n ‘4 ESSE = m lesto ty 2 SG ° j ko. 2/0 fro Listal i le R0a220 Number af fibers Figure 2 Distance _ 600 M. J. GREENMAN measurements for the ten largest fibers of the distal ends are 85 square micra, 85.8 square micra and 113 square micra re- spectively. We thus observe that the size of fibers diminishes from the proximal to the distal ends, this diminution averaging about 29 per cent of the proximal measurements, in a distance of 10 mm.—the length of nerve excised. Combining these aver- ages of the proximal and distal ends, we have 113.6 square micra as the average sectional area for the ten largest fibers in the middle zone of the nerve. We observe also from table 9, that the sectional area of fibers increases as the weight of the animal increases. Taking weight as an index of age we may infer that in young animals nerve fibers increase in sectional area with the age of the animal. SECTIONAL AREAS OF NERVE FIBERS FROM THE CONTROL SIDE OF OPERATED ANIMALS Series 7, including operated animals nos. 106, 114 and 154, was utilized for the determination of sectional areas of the fibers of the control nerves and the sectional areas of fibers of the proximal and distal ends of the operated nerves. Forty or more of the largest fibers from each control nerve and from each proximal and each distal end of each operated nerve were measured. The measurements were tabulated under their proper headings in the order of their size beginning with the largest fibers. From this tabulation the averages of each successive group of ten fibers, beginning with the largest, were taken, the percentage value of the axis was determined for each group and the data arranged in table 10. From the table we note that the average for the control fibers in group 1 is 65.7 square micra. The average weight of the three animals is 156 grams and their average age is 189 days. From table 9, we find that the average sectional area of the proximal and distal ends of the 10 largest fibers of three normal animals, of an average weight of 135 grams, is 113.6 square micra or an excess of 47.9 square micra over the controls of the REGENERATION OF PERIPHERAL NERVES operated animals. control nerve about 42 per cent. 501 We conclude, therefore, that in this instance the operation has reduced the sectional area of the fibers of the This result is all the more striking when we recall that the operated animals are markedly heavier than the unoperated and might be expected therefore to have larger nerve fibers. TABLE 10 Measurements in square micra no. 106; acu, 276; WEIGHT, 148; TIME ELAPSED, 26 DAYS no. 114; aan, 164; WEIGHT, 157; TIME ELAPSED, 105 DAYS no. 154; aan, 129; WEIGHT, 162.5; TIME ELAPSED, 98 DAYS Operated Operated Control nerve Control} nerve nerve nerve Prox. | Distal Prox. | Distal Group ls area... 2... 65.5 | 57.0 | 20.7 | 73.8 | 51.5 | 34.5 Per cent of axis......| 47.5 | 46.1 | 52.1 | 40.6 | 33.0 | 46.9 Group 2: area........| 53.7 | 31.4 | 16.0 | 63.9 | 33.0 | 25.5 Per cent of axis......| 44.8 | 52.8 | 50.6 | 41.4 | 28.0 | 48.6 (Group |: Aleas..2 70 .. 44.5 | 23.0 | 10.4 | 44.9 | 22.0 | 18.4 Per cent of axis......| 48.0 | 50.8 | 48.0 | 40.0 | 32.2 | 50.0 Group 4: area........ 34.1 | 12.8 | 6.8 | 15.6 | 10.8 Per cent of axis...... 45.4 | 46.8 | 41.1 | 48.0 | 38.0 Group 5: area........ 25.3 1208 6.4 Per cent of axis...... 47 .0 58.2 | 42.0 Group 6: area........ 17.8 Per cent of axis...... 47.1 Croup areaaceenr ee Percent of axis...... Operated Control nerve nerve Prox. Distal 57 .9 44 2 58.9 39.7 34.5 42.8 47 .4 44.5 45.7 38.7 28.3 39.5 32.5 44.3 39.5 39.0 Average weight of the three animals, 156 grams Average age of the three animals, 189 days Average of control nerve in group 1, 65.7 square micra Average of proximal measurements of group 1, 55.8 square micra Average of distal measurements of group 1, 29.9 square micra 502 M. J. GREENMAN SECTIONAL AREAS OF FIBERS FROM THE OPERATED NERVE We note in table 10, that the average sectional areas of the fibers of both the proximal and the distal ends of operated nerves are much less than the average sectional area of the fibers of the control nerves. Considering only the data given in group 1, we note that the average for the fibers from the proximal ends of the operated nerves is 55.8 square micra or 15 per cent less than the average for the fibers of the control nerves. Sections from the controls were taken midway in the course of the nerve and hence should be expected to show smaller fibers than those at the proximal end of the operated nerve. Also, the average of the fibers of the distal ends of the oper- ated nerves is 29.9 square micra or 54 per cent less than the average of the fibers of the control nerves. These last, however, are newly formed fibers and hence form a class different from either the control fibers or the proximal operated. From table 10, we note that no. 106 was an adult animal (276 days of age) which was killed 26 days after the operation. Its control fibers average 65.5 square micra, its proximal fibers 57 square micra and its distal fibers 20.7 square-micra. TABLE 11 AGE AT TIME ae yrean eSvAgiate Nees) oho AVERAGE OF AVERAGE OF Nk OF KILLING OPERATION CONTROL FIBERS | PROXIMAL FIBERS DISTAL FIBERS np xttting | IN SQUARE MICRA | IN SQUARE MICRA | IN SQUARE MICRA 154 | 129 98 57.9 58.9 34.5 106 276 26 65.5 57.0 20.7 No. 154 was a much younger animal (129 days of age) and was killed 98 days after operation. Its control fibers average 57.9 square micra, its proximal fibers 58.9 square micra and its distal fibers 34.5 square micra. Placing these data in tabular form for convenience of comparison, we have table 11. It is evident that no. 154, the younger animal, after a greater lapse of time (98 days) has been able to repair more completely the fibers in the proximal segment of the operated nerve and to regenerate in the distal segment fibers more nearly equal in REGENERATION OF PERIPHERAL NERVES 503 sectional area to its control fibers than no. 106, the older animal, surviving only 26 days after the operation. The objection may be made that the greater lapse of time in the case of no. 154 accounts for its greater development in size of fibers. This objection does not hold good if we compare nos. 114 and 154 in which the lapse of time is greater in the case of the older animal yet it fails to develop fibers as nearly equal to its control as no. 154, the younger animal. AXIS-SHEATH AREA RELATION: NORMAL ANIMALS For determining the area relations of axis and sheath, series of measurements were made on fibers from normal animals, on fibers from the control nerve of operated animals and on fibers from the proximal and the distal ends of operated nerves. A drawing of the magnified image of the fiber section was made outlining the entire fiber and the contained axis cylinder. The area of the entire fiber was first measured by the planimeter, the area of the axis cylinder was then measured and the area of the sheath computed. Referring to table 9, we see that the average sectional area of axis at the proximal end of the nerve in three normal animals is 71.2 square micra; the average sectional area of sheath is 61.3 square micra. The axis therefore constitutes 53.7 per cent of the fiber. Fibers of the distal end show a slightly different rela- tion. The average of the distal axes is 47.2 square micra, the average of the distal sheaths is 47.3 square micra. The axis here constitutes 49.9 per cent of the fiber. These data show that while both the axis and the sheath taper from the proximal to the distal end of the nerve, yet the axis constitutes a slightly less percentage of the fiber at the distal end, that is, the pro- portion of sheath has increased at the distal end. Combining the percentage values of both proximal and distal ends, we obtain the following average percentage for the area relation of axis to sheath: Axis 51.8 per cent Sheath 48.2 per cent for normal unoperated animals of 135 grams average weight. 504 M. J. GREENMAN Donaldson and Hoke (’05) in an examination of the nerve fibers from a large series of vertebrates which included the albino rat, found the area relation of axis to sheath to be approximately as one to one. The results here given are in substantial agree- ment with their observations. Dunn (712) found in the albino rat that medullated nerve ' fibers and their axis cylinders increase continuously in size until 270 days of age and that in old rats, about 640 days of age, there is a noticeable decrease in size of both the nerve fiber and its axis cylinder. Dr. Dunn also followed the changes in the axis sheath relations with age. AXIS-SHEATH AREA RELATION: OPERATED ANIMALS| Passing to the consideration of the axis-sheath area relation in operated animals, we observe from table 10 that the percent- age of axis in operated animals is less than in normal animals. | The average percentage value of the axis in the largest fibers of the controls shown in group 1 (nos. 106, 114 and 154) is 44.1 per cent. The sheath, therefore, constitutes 55.9 per cent, but it should be recalled that these fibers in the control nerve have suffered a diminution in total area of 42 per cent as the result of the operation. If we take from table 10 the average percentage value of the axis, and compute the sheath value, (1) of the controls and (2) of the proximal, and (3) distal ends of the operated nerves in the first four groups, and then arrange these values under their proper ‘headings, we get table 12. TABLE 12 OPERATED NERVE CONTROL TIME NERVE NO. AGE Sasa Proximal Distal _ |Per cent Fereent Percent per cent|Pereent fo) F ° . fo) of axis sheath of axis sheath of axis sheath 106 276 26 46.4 | 53.6 49.1 | 50.9 | 47.9 | 52.1 114 164 105 42.5 | 57.5 | 32.8 | 67.2 | 48.5 | 51.5 39.2 | 60.8 | 41.2 | 58.8 154 129 | 98 44.1 | 55.9 ANWeEragey. 25-1. 44.3 | 55.7 | 40.3 | 59.7 | 45.8 | 54.2 REGENERATION OF PERIPHERAL NERVES 505 From this summary of axis values, we observe that the axes in all controls of operated animals have been reduced below that of a normal fiber, the average for the three animals being 44.3 per cent, the percentage of sheath being correspondingly increased. In examining the percentage relations of the operated nerves as shown in table 12, it is evident that in the loss in size of fibers of operated nerves the reduction in the percentage of axis is more marked in the animals which are younger and which have lived longer periods since the operation as nos. 114 and 154. This would accord with Dunn’s observations (’12). hormal Canitrol Operated wey © O Lesion Mniddle ~ © © Qverage areas of largest nerve filers Figure 3 The control nerve of no. 106 varies less in its axis-sheath rela- tion from the normal than the control nerve of no. 114, a younger animal. It will be noted also that the deviation from normal re- lations in the distal ends of operated nerves is less marked than in the proximal ends, but the distal ends are newly formed while the proximal ends are simply showing modifications. The greater reduction in percentage relation of the axis in the proximal seg- ments of operated nerves as compared with the relation in the 506 = M. J. GREENMAN control, may be due to the branching which takes place in the proximal zone when new fibers are produced at the expense of the parent stem. In order to bring together the general results of the observa- tions on the relations of the area of the axis and of the sheath, figure 3 has been constructed. Jn this figure the total areas and the axis sheath relations of the averages from the ten largest fibers taken respectively from the normal, control and operated nerves are given. In each instance the area of the fibers at the proximal end, the middle zone and the distal end are drawn. Where the determination depends on direct observation, the medullary sheath is shown as a black ring, where the area has been computed the outlines of both sheath and axis are indicated by lines. The size of the fibers thus represented serves to show more clearly, than the simple measurements can, the striking modifications of the fibers which occur in the operated animals. LITERATURE The literature on the subject of regeneration has been care- fully reviewed by Ranson (Jour. Comp. Neur., vol. 22, no. 6, 1912). We will, therefore, call attention only to those papers dealing directly with the number of fibers (including branching), their size and the axis-sheath relation in the peripheral nervous system of vertebrates. Rudolph Wagner (’47) observed that in the distribution of primitive fibers in the electric organ of fishes there is true branch- ing and that one primitive fiber may have as many as twenty- five branches. He demonstrated that the branching of nerve fibers was a true branching and not the formation of a network. Schwalbe (’82) studied the size of fibers and the relation of length to diameter. He attempted to show that the larger fibers were distributed to the most distant parts. This was disproven by Dunn (’02) in her study of the nerve fibers of the frog’s leg. Dunn here found a conical diminution in the nerve fiber in its course. REGENERATION OF PERIPHERAL NERVES 507 Voischvillo (83) studied the numerical relations of sensory fibers to the skin of the extremities and of motor fibers to mus- cles which move rapidly and those which move less rapidly but with great force. He found that the skin of the upper extremi- ties was more abundantly supplied with sensory fibers than the skin of the lower extremities, also that the eye muscle received a greater proportion of nerve fibers to muscle fibers than the muscles of the extremities. Vashkevitch (’87) determined the number of fibers in the n. ischiadicus and n. medianus of bats, mice, rats, marmots, rabbits, cats and dogs and found that the absolute number of nerve fibers depended upon the weight of the central nervous system and the weight of the body, but that the increase in number of fibers does not progress at the same ratio with the increase in body weight. Fritsch (89) determined, in the torpedo the numerical relations of the elements of the electric organ to the nerve cells and nerve fibers. Dunn (’00) observed that practical equality exists in the num- ber of fibers in the legs of the two sides of the frog. Dunn also (02) showed by numerical determinations on the nerve distribution in the frog’s leg that the number of fibers normally increases by branching as one passes distally. Hatai (’02) in a study of the ganglion cells and the dorsal root fibers determined the ratio of nerve fibers to cells in the white rat at 10.3 grams body weight and at maturity. Hatai (’03) also found that the rate of increase in the number of fibers of the ventral roots of the spinal nerves of the albino rat was most rapid between the body weights of 10.8 and 25.4 grams and that the number found at maturity is 2.7 times that found in the 10.3 gram rat. Donaldson (’03) in considering the number of fibers distributed to the skin and muscles of the frog’s leg found that this dis- tribution followed a fixed law expressed as follows: The nerve fibers entering the leg of the frog (Rana virescens) by the sciatic and crural nerves, are distributed to the thigh, shank and foot 508 M. J. GREENMAN ‘ in numbers which, for each of these segments are equal to the sum of the efferent fibers—taken in proportion to the weight of the museles—and of the efferent fibers—taken in proportion to the area of the skin. Ingbert (’04) studied the areas of cross section in the ventral and dorsal roots of the spinal nerves and computed the number of sensory and motor fibers and showed that during the increase in the nerve supply the gain has been more in the sensory than the motor fibers. ; Donaldson and Hoke (’05) in a series of observations on the spinal nerves of a number of vertebrates found that the relation of axis cylinder to sheath was approximately as one to one. Boughton’s (’06) results on the albino rat and on the cat cor- respond with ours in finding an increase in the number of fibers and an increase in size of fibers as the animal increases in weight. He also points out that fibers which develop after the period of rapid growth never attain large size. Perroncito (06) showed that an axon begins to regenerate by sprouting from the proximal stump within three hours after section, and that in many cases a single fiber gives rise to many sprouts. Osborne and Kilvington (’08) proved that bifurcation of motor axons took place when a plurality of path was offered and that motor axons would bifurcate and follow a sensory path as well as a motor path.. In a later paper (’09) they supplement their results by further observations and state that bifurcation occurs to some extent at the point of section as well as at the point where plurality of paths is offered. Dunn (’09) in a paper on the albino rat observes the splitting of nerve fibers and notes the deviation from the one to one relation in the area of sheath and axis according to age. In a later paper (’11) she observes the loss of fibers in old animals. Ranson (712) observed the branching of medullated nerve fibers 5 mm. above the section. REGENERATION OF PERIPHERAL NERVES 509 SUMMARY OF RESULTS 1. The peroneal nerve of the normal albino rat of 135 grams body weight was found to contain 2288 medullated fibers in its proximal end and 2323 medullated fibers in its distal end. The middle zone is estimated to contain 2306 fibers. The portion of the nerve utilized for these experiments was 10 mm. long. 2. There is an increase in the number of fibers as we pass from the proximal to the distal end of this 10 mm. of nerve amounting to 1.5 per cent of the proximal number. 3. The number of fibers is approximately the same for each side. 4. The number of medullated fibers increases with body weight (= age) during the first 276 days of life. The increase between the 128-129-day group and the 276-day group (table 4) is about 5 per cent. 5. Crushing the nerve by the method used causes complete degeneration beyond the point of the lesion. 6. Four days after the-operation no normal fibers are to be found on the distal side of the lesion. The degeneration is assumed to have involved the entire distal portions of the fibers. 7. Complete degeneration also extends from 2 to 3.2 mm. on the proximal side of the lesion. 8. Characteristic loss of motor control always follows the oper- ation. In many cases this has seemingly disappeared at the end of ten days, probably as the result of compensatory adjustment. 9. The general effects of the operation are more pronounced on older animals. : 10. The control nerve of an operated animal contains fewer medullated fibers than the same nerve from a normal animal of the same age. This loss in number is one of the effects of the oper- ation, and in the cases here examined amounted to 16 per cent. 11. Following the degeneration in the operated nerve, regen- eration, accompanied by branching of axons, takes place and there is an increase of from 64 to 249 per cent in the number of medullated fibers on the proximal side of the lesion. 510 M. J. GREENMAN 12. The increase in number of fibers on the distal side of the lesion is less than on the proximal side but the number always exceeds that found on the control side. 13. The number of regenerated fibers rapidly increases as the region of the lesion is approached from the proximal side; the number decreases as we pass from the lesion distally. Over 13 per cent of the regenerated fibers arise from a point more than 7 mm. above the lesion. 14. The average sectional area of the 10 largest fibers in the middle zone of the peroneal nerve of a normal albino rat of 135 grams body weight was found to be 113.6 square micra. 15. The nerve. fiber tapers from its proximal to its distal end (see table 9). The sectional area of fibers increases with the age of the animal during the first 276 days of life. 16. The average sectional area of the 16 largest fibers from the control nerve of an operated albino rat of 156 grams body weight is 65.7 square micra. 17. One of the results of operation is, theréfore, a loss in sec- tional area of nerve fibers ef the control nerve. In this instance the loss amounts to 42 per cent. It is possible that this effect is general throughout the peripheral nervous system. 18. The sectional area of the 10 largest nerve fibers on the proximal side of the lesion is 55.8 square micra, or 15 per cent less than the area of the fibers of the control nerve. 19. The sectional area of the 10 largest regenerated nerve fibers on the distal side of the lesion is 29.9 square micra or 54 per cent less than the area of the fibers in the control nerve. 20. In the normal albino rat of 185 grams body weight, the axis-sheath relations of the fibers of the peroneal nerve are as follows: Area of axis 51.8 per cent. Area of sheath 48.2 per cent. 21. In a series of three operated animals of an average weight of 156 grams, the average axis-sheath area relation is as follows: Controls), 1 a eee Axis 44.3 per cent Sheath 55.7 per cent Proximal end of operated nerve...... .......Axis 40.3 per cent Sheath 59.7 per cent Distal end of operated NETVE Aes. eee ee eee Axis 45.8 per cent Sheath 54.2 per cent REGENERATION OF PERIPHERAL NERVES 511 22. Thus in the operated animal in which the fibers of both the control and operated nerves are all diminished in total area, the axis-sheath relation is such that in all three localities the area of the axis is relatively less than in the fibers from the normal animal. 23. This deviation from the normal in the case of the control nerves and the proximal end of the operated nerves represents an alteration in existing structures while in the fibers distal to the lesion, it appears in structures which have been newly formed. CONCLUSIONS The relation of the more important results here given to pre- vious information is as follows: The observations that medullated nerve fibers branch in their course and in a given nerve increase in number for a time, with age, 1s in accord with the findings of all the authors who have studied these matters. Also, in agreement with others is the fact that the number of fibers on the two sides of the same ani- mal is similar. In agreement with Schwalbe (’82) and Dunn (02) it is found that the largest fibers in the peroneal nerve undergo a conical diminution. The fact that in the operated animals the number of fibers is diminished on the control side has been reported by Dunn (’09) for the frog. The observations that the fibers of the control side are greatly diminished in diameter and that the area rela- tion of the axis and sheath is modified—also that the same is true for the fibers in the proximal portion of the operated nerve, are all new. The results on the area relation of the axis-sheath in the fibers of the normal nerve agree in general with the observations of Donaldson and Hoke (’05), but the determinations of this rela- tion in the newly formed fibers on the distal side of the lesion has not been previously made. The observations of Perroncito (06), Osborne and Kilvington (08-09) and the study of neuromata all indicate a tendency to branching in the regenerating fiber. Our observations give pre- cise information as to the amount of this branching, the general THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 23, NO. 5 AW M. J. GREENMAN distribution of the fibers in the nerve near the lesion and also show that the increase in number begins high up in the course of the proximal portion of the nerve. The fate of these excess fibers has not yet been determined. The most important outcome of this investigation is the evi- dence it furnishes of an alteration in the number and size of the fibers in the control nerve and in the proximal end of the operated as well as the accompanying changes in the diameter of the fibers and the area relation of the axis-sheath. These alterations, as they stand, apply to symmetrical periph- eral nerves, but it seems possible that they are more widely distributed. The sensitiveness of the area relation of the axis- sheath to disturbance of the normal conditions in the animal points to a high degree of nutritive response in the medullated fiber. , LITERATURE CITED Bout, Franz 1876 Studi sulle immagini microscopiche della fibra nervosa midollare. Nota del Prof. Franz Boll presentata dal Socio Tommasi- Crudeli nella seduta del 4 giugno. Boueuton, THomas H. 1906 The increase in the number and size of the medul- lated fibers in the oculomotor nerve of the white rat and of the cat at different ages. Jour. Comp. Neur., vol. 16, no. 2. Casat, 8S. Ram6n 1913 Estudios sobre la degeneracion y regeneracion del sis- tema nervioso. Imprenta de Hijos de Nicolas Moya. Madrid. Downaupson, H. H. 1903 Ona law determining the number of medullated nerve fibers innervating the thigh, shank and foot of the frog, Rana vires- cens. Jour. Comp. Neur., vol. 13, no. 3. Donatpson, H. H., anp Hoke, G. W. 1905 On the areas of the axis cylinder and medullary sheath as seen in cross sections of the spinal nerves of vertebrates. Jour. Comp. Neur., vol. 15, no. 1. Dunn, EvizaAsetH Hopkins 1900 The number and size of the nerve fibers in- nervating the skin and muscles of the thigh in the frog (Rana virescens brachycephala, Cope). Jour. Comp. Neur., vol. 10, no. 2. ’ 1902 On the number and on the relation between diameter and distri- bution of the nerve fibers innervating the leg of the frog (Rana virescens brachycephala, Cope). Jour. Comp. Neur., vol. 12, no. 4. 1909 A statistical study of the medullated nerve fibers innervating the legs of the leopard frog (Rana pipiens) after unilateral section of the ventral roots. Jour. Comp. Neur., vol. 19, no. 6. 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 albinorat. Jour. Comp. Neur., vol. 22, no. 2. REGENERATION GF PERIPHERAL NERVES Hills Fritscu, Gustav 1889 Das numerische Verhidltniss der Elemente des elek- trische Organs der Torpedineen zu den Elementen des Nervensystems. Sitz. d. K. Akad. der Wissen. Harpesty, Irvinc 1899 The number and arrangement of the fibers forming the spinal nerves of the frog (Rana virescens). Jour. Comp. Neur., vol. 9, no. 2. j 1900 Further observations on the conditions determining the number and arrangement of the fibers forming the spinal nerves of the frog (Rana virescens). Jour. Comp. Neur., vol. 10, no. 3. Harar, S. 1902 Number and size of the spinal ganglion cells and dorsal root fibers in the white rat at different ages. Jour. Comp. Neur., vol. 12, no. 2. : 1903 On the increase in the number of medullated nerve fibers in the ventral roots of the spinal nerves of the growing white rat. Jour. Comp. Neur., vol. 13, no. 3. INGBERT, C. 1903 On the density of the cutaneous innervation in man. Jour. Comp. Neur., vol. 13, no. 3. 1903 An enumeration of the medullated nerve fibers in the dorsal roots of the spinal nerves of man. Jour. Comp. Neur., vol. 13, no. 2. 1904 An enumeration of the medullated nerve fibers in the ventral roots of the spinal nerves of man. Jour. Comp. Neur., vol. 14, no. 3. Ossporne, W. A., AND Kitvineton, Basin 1908 Axon bifurcation in regener- ated nerves. Jour. Physiol., vol. 37. 1909 Axon bifurcation in regenerated nerves. Jour. Physiol., vol. 38. PrerRonciITO, ALpo 1906 La rigenerazione delle fibrenervose. Bollettino della Societa Medico-Chirurgica di Pavia. Ranson, 8S. WALTER 1912 Degeneration and regeneration of nerve fiber. Jour. Comp. Neur., vol. 22, no. 6. VoisHvi1LLo, EK. 1883 Materials for the study of the relation of the caliber of nerves to the skin and muscles of man. Investigation made upon the eye and the lower and upper extremity of man. St. Petersburg Print- ing office of M. Stasulewitch. VASHKEVITCH, F. U. 1889 Materiali k voprosu o chislie nervnikh volokon peri- fericheskoi nervnoi sistemi po otnoschenivu k viesu tiela mlekopi- tayushtshikh zhivotnikh. (Data on the number of nerve-fibers of the peripheral nervous system as related to the weight of mammals). Sborn. trud. Kharhovsk. Vet. Inst. (1887) 1889 i, 97-188. WaGneR, Rupo~pH 1847 Neue Untersuchungen iiber den Bau und die En- digung der Nerven und de Struktur der Ganglien. Supplement zu den Icones Physiologicae von Rudolph Wagner. Oey ne iar ane sighed) RE . Ml Pie 7 a eih a) ial Rar. "} i wid, tC ; 4 id rata tt) Vy Persie Nha 7 Ee Pa we eres al pir’ ey aL ae « f Cea . A COMPARATIVE STUDY OF THE BRAINS OF THREE GENERA OF ANTS, WITH SPECIAL REFERENCE TO THE MUSHROOM BODIES CAROLINE BURLING THOMPSON Department of Zoology, Wellesley College FORTY-TWO FIGURES CONTENTS Introduction Meterial and: methods 1.05.44 a carr tee esd wits s 8 0) os de ee ee ee ee 516 Ailvep ai tian ss a2). Loree £ Sir adheres oasierere, Sanu wik dw cota ag eee eee 518 iP AENISCOPIGAly AGCCOUNG cateqc atts es sss oh ses aia) ee wah are ee 518 ae General) anatomy 234.2. dt elie s occas Voc ee dene ce Jeane 520 Comparison of the brains of the castes 4 L,,., Vherqueen- rainy hice sede ck oni k evista as vlads alto a eee §21 2s, PN OLW OPO E RAM 5.5/1 cg eos bs ctathos essen tues ade otop adc A 523 on. Me THalenonaiiberie saa c0 sche eee eit c=]. Sous. sate. . 36 ea 524 The finer structure of the brain 15. Aer bTain: Seri Met neers cote cess cate eae ad one pig « ndar 50. 672 Cc. JUDSON HERRICK AND JEANNETTE B. OBENCHAIN Figs.5to12 Comprise series of cross sections through the brain of Ichthyomy- zon at the levels indicated on figure 3. X 36. Fig. 5 Section passing through the interventricular foramen and decussation of the optic tracts. Fig. 6 Section passing through the primordium hippocampi and lobus subhip- pocampalis a short distance behind the interventricular foramen. The lamina of cells representing the primordial corpus striatum (c.s.) is extensively developed, though for the most part it is withdrawn from the ventricular surface. Fig. 7 Section passing through the caudal end of the chiasma ridge. On the right side it passes through the habenula and eminentia fimbriae; on the left side through the caudal end of the primordium hippocampi. The cellular elements of the eminentia thalami (em. th.) are well developed between the sulcus medius and the suleus intermedius. Between this region and the eminentia fimbriae is an area containing fibers of the stria medullaris and poor in cells, which should probably be associated with the lobus subhabenularis. Fig. 8 Section through the middle of the right habenula and the rostral part of the thalamus. I Th.--- f relrof- 673 674 Cc. JUDSON HERRICK AND JEANNETTE B. OBENCHAIN Fig. 9 Section through the thalamus three sections (45 ») farther caudad than figure 8 and passing through the common vertical ridge formed by the medial and ventral lobes of the thalamus. Fig. 10 Section through the rostral end of the tuberculum posterius. The letters s./. mark the position of the sulcus limitans, which appears clearly defined a few sections farther caudad. The elements of the cell plate below this level are somewhat larger and more loosely arranged than those above it. Fig. 11 Section through the posterior commissure and the caudal end of the recessus mammillaris. Fig. 12 Section through the midbrain immediately in front of the commissura posterior tecti, illustrating the characteristic internal structure at the site of the sulcus limitans, through the sulcus itself is not evident on the ventricular sur- face (ef. fig. 3). ANATOMY OF A CYCLOSTOME BRAIN 675 com. post. c.gen. lal-y AiBAGredaum atl ] SUBJECT AND AUTHOR INDEX MBLYSTOMA. The primary ventral roots and somatic motor column of...... 121 Ants, with special reference to the mushroom bodies. A comparative study of the brains Giathreermeneratoln.. .. kemck cou sooner 515 Axis to sheath. Studies on the regeneration of the peroneal nerve of the albino rat: num- ber and sectional areas of fibers: area rela- [NYO GN (011 oe aR en om An ed che An atc ste ec 479 LACK, D. Davinson. The study of an atypical cerebral cortex................. 351 ———The central nervous system in a case of (HUG Gorey thay le s9 4 eAaadocsneuooonan antec 193 Brain: Ichthyomyzon concolor. Notes on the anatomy of a cyclostome.................. 635 of the albino rat. The effects of formal- Gehyd elomither wre casa ials seeecaz once rer ke 283 Brains of three genera of ants, with special ref- erence to the mushroom bodies. A com- Dardblve study: OL they, sce ssekieeee ae cen 515 AT. The course within the spinal cord of the non-medullated fibers of the dorsal roots: A study of Lissauer’s tract in the. 259 The nervus terminalis in the adult dog ENTS Sy Ager eee LCRA PREECE na heme 145 Cerebral cortex. The study of an atypical... 351 Coauitt, G. E. The primary ventral roots and somatic motor column of Amblystoma 121 Commissures in reptiles and mammals. The morphology of the septum, hippocampus, hots yor illu: |loenee Sey Ratto rereIO ne cero ADDS 371 Concer, A. C., Lanpacre, F. L. and. The origin of the lateral line primordia in Bepi GOStEUSTOSSCUSEe a -nece ceca coho 575 Cortex. The study of an atypical cerebral... 351 Cyclopia in homo. The central nervous sy: s CODA AICASE OLY came eei re oh ae teraee aleaeas 193 Cyclostome brain: Ichthyomyzon concolor. Notes on the anatomy of a................ 635 IGESTIVE tube. On the innervation of Ot Se Seite oe EEE OAT oo SN OAC ar 173 Dog and cat. The nervus terminalis in the Se ee Re! ea 145 Dorsal roots: A study of Lissauer’s tract in the cat. The course within the spinal cord of the non medullated fibers of the........... 259 | Dee MUSCLE nervesin Necturus. The. 153 IBERS of the dorsal roots: A study of Lis- sauer’s tract in the cat. The course with- in the spinal cord of the non-medullated 259 Formaldehyde on the brain of the albino rat. Theieflects Of ee ese occ see oe 283 ANGBIA in the pig. The development of the cranial sympathetic.............. 71 GREENMAN, M. J. Studies on the regenera- tion of the peroneal nerve of the albino rat: number and sectional areas of fibers: area relation of axis to sheath............ . 479 Growth of the human spinal cord. Prenatal 39 AIR of the white rat. The tactile........ 1 Hevpt, THomas J. Mollgaard’s reticu- Une Rnee es San Gab nine 56.5600 AACR Eee 315 Herrick, C. Jupson and OBENCHAIN, JEAN- NETTE B. Notesonthe anatomy ot a cy- clostome brain: Ichthyomyzon concolor... 635 Hippocampus, and pallial commissures in rep- tiles and mammals. The morphology of Thesep titty. pon ee ee en 371 CHTHYOMYZON concolor. Notes on the anatomy of a cyclostome brain:........... 635 Innervation of the digestive tube. Onthe.... OHNSTON, J. B. Nervus terminalis in reptiles and mamimalsin. es aee eee eee 97 The morphology of the septum, hippo- campus, and pallial commissures in rep- iilesfandemsamim Alston eee ee eee eee ING, Heten Dean. The effects of for- maldehyde on the brain of the albino rat. Kuntz, ALBERT. On the innervation of the digestive tubes .6=.ic ace hee Meee The development of the cranial sy mpa- thetic ganglia in the pig ANDACRE, F. L. and Concer, A. C. The origin of the lateral Jine primordia in hepidosteus/Osseusee--e eerie Lateral line primordia in Lepidosteus osseus. The origin of the Lepidosteus osseus. Line spEIMOrdiasineese aie eee eee eee Lissauer’s tract in the cat. The course within the spinal cord of the non-medullated fib- ers of the dorsal roots: A study of......... 259 Moa Routito E. The nervus ter- 575 ‘The origin of the lateral 575 minalis in the adult dog and cat........ 145 McKisBEN, Paut 8. The eye-muscle nerves In IN GCtUGUS ) o..c nists: coe sto ie eee ae nee 153 Mammals. Nervusterminalisin reptilesand 97 The morphology of the septum, hippo- campus, and pallial commissures in rep- tilesian dx en hk ho dere os ears Mriuuter, Max Mayo. Prenatal growth of the laqobamirhay jouer (orl aanencacaccasacceaee ss 39 Mollgaard!sireticulumy.: 43-4 cee ee eeine Motor column of Amblystoma. The primary ventral roots and somatic...........-.....- 121 Mushroom bodies. A comparative study of the brains of three genera of ants, with special reference to the................---- 515 Neca The eye muscle nerves in.... 153 Nerves in Necturus. The eye-muscle..... .-... 153 Nerve of the albino rat: number and sectional areas of fibers: area relation of axisto sheath. Studies on the regeneration of thesperoneal: : o/sicos 2B oose scien e jaca 479 Nervous system in a case of cyclopia in homo. Mhercentrals tee ee ee ee ee 193 Nervus terminalis in reptiles and mammals 97 in the adult dogand cat. The... 145 677 678 BENCHAIN, JEANETTE B., HERRICK, C. Jupson and. Notes on the anatomy of a cyclostome brain: Iehthyomyzon con- coler... IG. The development of the cranial sym- pathetic ganglia im the.................. ANSON, S. Water. The course with- in the spinal cord of the non-medullated fibers of the dorsal roots: A study of Lis- Sauers tract in the cat. s..-seene ee eee % Rat: number and sectional areas of fibers: area relation of axis to sheath. Studies on the regeneration of the peroneal nerve of ilavei fll tale REPRE nr ta seroma eaten diosa ———The effects of formaldehyde on the brain Ofte al bin ot .ss5e a e e or eee ere : = Whetactile harmon tMenwMibeseeeene seen oe Regeneration of the peroneal nerve of the al- bino rat: number and sectional areas of fibers: area relation of axis to sheath. Studiesion:thee seers et riettcrat rns Reptiles and mammals. Nervus terminalis The morphology of the septum, hippocampus, and pallial commissures i Reticulum. Molledardisqesceeree- eae ee Roots and somatic motor column of Amblysto- ma. The primary ventral... . ; INDEX Roots: A study of Lissauer’s tract in the cat. The course within the spinal cord of the non-medullated fibers of the dorsal........ EPTUM, hippocampus, and _ pallial com- missures in reptiles and mammals. The morphology ofithevat: |... 0.428. eee Sheath. 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