Progress in Brain Research Volume 25 The Cerebellum

PROGRESS I N B R A I N RE SE ARCH VOLUME 25 T H E CEREBELLUM PROGRESS I N B R A I N RESEARCH ADVISORY BOARD W. Barg...

Author: C.A. Fox | Ray S. Snider

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PROGRESS I N B R A I N RE SE ARCH VOLUME 25 T H E CEREBELLUM

PROGRESS I N B R A I N RESEARCH

ADVISORY BOARD

W. Bargmann

H. T. Chang E. De Robertis J. C. Eccles J. D. French

H. Hydtn J. Ariens Kappers S. A. Sarkisov

J. P. Schadt

F. 0. Schmitt

Kiel Shanghai Buenos Aires Chicago Los Angeles

Goteborg Amsterdam Moscow Amsterdam Brookline (Mass.)

T. Tokizane

Tokyo

J. 2. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 25

THE CEREBELLUM EDITED BY

C L E M E N T A. FOX, PH. D. Professor and Chairman, Department of Anatomy, Wayne State University School of Medicine, Detroit, Michigan AND

R A Y S. S N I D E R , PH. D. Director, Center for Brain Research, University of Rochester, Rochester, New York

ELSEVIER P U B L I S H I N G C O M P A N Y AMSTERDAM / L O N D O N / NEW YORK

1967

ELSEVIER P U B L I S H I N G COMPANY 335 J A N VAN GALENSTRAAT, P.O. BOX 21 1, AMSTERDAM

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LIBRARY O F C O N G R E S S CATALOG C A R D N U M B E R 67-12774

WITH 280 ILLUSTRATIONS

A L L R I G H T S RESERVED T H I S BOOK O R A N Y P A R T T H E R E O F MAY N O T BE R E P R O D U C E D I N ANY F O R M , I N C L U D I N G PHOTOSTATIC O R M I C R O F I L M FORM, W I T H O U T W R I T T E N P E R M I S S I O N F R O M T H E PUBLISHERS

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List of Contributors

V. BRAITENBERG, Laboratorio di Cibernetica del C.N.R., Istituto di Fisica Teorica, Universiti di Napoli, Naples (Italy). A. BRODAL, Anatomical Institute, University of Oslo, Oslo (Norway). C. R. DUTTA,Department of Anatomy, Marquette University School of Medicine, Milwaukee, Wis. (U .S.A.). C. A. FOX,Department of Anatomy, Wayne State University School of Medicine, Detroit, Mich. (U.S.A.). D. E. HILLMAN, Department of Anatomy, Wayne State University School of Medicine, Detroit, Mich. (U.S.A.). R. NIEUWENHUYS, Netherlands Central Institute for Brain Research, Amsterdam (The Netherlands). 0. POMPEIANO, Institute of Physiology, University of Pisa, Pisa (Italy). K. A. SIEGESMUND, Department of Anatomy, Marquette University School of Medicine, Milwaukee, Wis. (U.S.A.). R. S. SNIDER, Center for Brain Research, University of Rochester, Rochester, N.Y. (U.S.A.). Cajal Institute, Madrid (Spain). C. SOTELO, J. VOOGD,Laboratory of Neuroanatomy, Neurological Institute, University of Leiden, Leiden (The Netherlands). P. E. VOORHOEVE, Department of Neurophysiology, Jan Swammerdam Institute, University of Amsterdam, Amsterdam (The Netherlands)

Other volumes in this series:

Volume 1 : Brain Mechanisms Specific and Unspecific Mechanisms of Sensory Motor Integration Edited by G. Moruzzi, A. Fessard and H. H. Jasper

Volume 2: Nerve, Brain and Memory Models Edited by Norbert Wiener? and J. P. SchadC

Volume 3 : The Rhinencephalon and Related Structures Edited by W. Bargmann and J. P. Schadk

Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. SchadC

Volume 5 : Lectures on the Diencephalon Edited by W. Bargmann and J. P. SchadC

Volume 6: Topics in Basic Neurology Edited by W . Bargmann and J. P. SchadC

Volume 7: Slow Electrical Processes in the Brain by N. A. Aladjalova

Volume 8 : Biogenic Arnines Edited by Harold E. Himwich and Williamina A. Himwich

Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich

Volume 10: The Structure and Function of the Epiphysis Cerebri Edited b y J . Ariens Kappers and 3. P. SchadC

Volume 11 Organization of the Spinal Cord Edited by J. C. Eccles and J. P. SchadC

Volume 12: Physiology of Spinal Neurons Edited by J. C. Eccles and J. P. SchadC

Volume 13 : Mechanisms of Neural Regeneration Edited by M . Singer and J. P. SchadC

Volume 14: Degeneration Patterns in the Nervous System Edited by M . Singer and J. P. Schade Volume 15 : Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrea

Volume 16 : Horizons in Neuropsychopharmacology Edited by Williamina A. Himwich and J. P. Schadk Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener? and J. P.Schadk Volume 18: Sleep Mechanisms Edited by K. Akert, Ch. Sally and J. P. SchadC

Volume 19: Experimental Epilepsy by A. Kreindler Volume 20: Pharmacology and Physiology of the Reticular Formation Edited by A. V. Valdman Volume 21A: Correlative Neurosciences. Part A: Fundamental Mechanisms Edited by T. Tokizane and J. P. Schade Volume 21B : Correlative Neurosciences. Part B: Clinical Studies Edited by T. Tokizane and J. P. Schade

Volume 22: Brain Reflexes Edited by E. A. Asratyan

Volume 23 : Sensory Mechanisms Edited by Y.Zotterman Volume 24: Carbon Monoxide Poisoning Edited by H. Bour and I. McA. Ledingham Volume 26 : Developmental Neurology Edited by C. G. Bernhard Volume 27: Structure and Function of the Limbic System Edited by W.Ross Adey and T. Tokizane Volume 28 : Anticholinergic Drugs Edited by P. B. Bradley and M. Fink

Preface

The resurgence of interest in the cerebellum by various biological disciplines plus the expansion of research techniques which gives a new order of precision to the data collected, made it desirable to bring together a group of ‘specialists on the cerebellum’ who were active in these various fields. Dr. SchadC and his associates, with generous help from Dr. Winkler and his staff, were unusually successful at the skillful planning and execution of the hundreds of minute demands which such an international group can make. The organization of the working sessions, the informal discussions, the intermingling of the young and the mature scientists, and the friendly overtones of exchange of information were such a prominent part of the symposium that the formal reports included in this volume, while indicating many highlights of the meeting, do not give an adequate measure of its total success. The unwritten future contributions which will result from participants who became intellectually motivated by these sessions, and by the genuine enthusiasm of the workers in the field, are unmeasured but not unrecognized rewards of the symposium. Special attention was given to both the anatomy and physiology of the cerebellum. Under the former heading the topics ranged from general concepts resulting from phylogenetic studies involving extensive light microscopic observations on the cortex as well as afferent and efferent pathways, to the precise evaluation of ultrastructural details of the primate cerebellum. The physiological reports were more limited in scope, and were concerned with the detailed evaluation of data collected with the most modern electrophysiological methods. These studies ranged from minute analysis of the activity of single units in the cerebellar cortex to the ascending influences which the cerebellum exerts on centers in the diencephalon and telencephalon. Although the data were presented in a manner that allows each paper in this volume to be read as an entity, all participants felt that the bringing together of the various papers in a single volume would facilitate interdisciplinary interest and make a convenient reference for related disciplines. February 1967

C. A. Fox and R. S. SNIDER

Contents

................................. .....................................

List of contributors Preface

Comparative anatomy of the cerebellum R. Nieuwenhuys (Amsterdam, The Netherlands)

V .VIII

...................

Comparative aspects of the structure and fibre connexions of the mammalian cerebellum J. Voogd (Leiden, The Netherlands). . . . . . . . . . . . . . . . . . . . . . . .

.

1

94

Anatomical studies of cerebellar fibre connections with special reference to problems of functional localization 135 A. Brodal (Oslo, Norway) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The primate cerebellar cortex: A Golgi and electron microscopic study C. A. Fox, D. E. Hillman, K. A. Siegesmund and C. R. Dutta (Detroit, Mich., Milwaukee, Wis.,U.S.A.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Cerebellar neuroglia: Morphological and histochemical aspects C. Sotelo (Paris, France) . . . . . . . . . . . . . . . . .

.............

226

Intracerebellar inhibitory systems P. E. Voorhoeve (Amsterdam, The Netherlands)

. . . . . . . . . . . . . . . . . . . 251

Climbing fibre responses in cerebellar cortex P. E. Voorhoeve (Amsterdam, The Netherlands)

...................

Functional organization of the cerebellar projections to the spinal cord 0. Pompeiano (Pisa, Italy) . . . . . . . . . . . . . . . . . . . .

268

.........

282

............

322

........................... ................................... ...................................

334

Functional alterations of cerebral sensory areas by the cerebellum R. S. Snider (Rochester, N. Y.,U.S.A.) . . . . . . . . . . .

Is the cerebellar cortex a biological clock in the millisecond range? V. Braitenberg (Naples, Italy) Author Index Subject Index

347 352

This Page Intentionally Left Blank

1

Comparative Anatomy of the Cerebellum R U D O L F NIEUWENHUYS Netherlands Central Institute for Brain Research, Amsterdam (The Netherlands)

It is intended to present in this report a survey of the comparative anatomy of the cerebellum, by way of introduction to this meeting which will be devoted almost entirely to the structure and function of the mammalian cerebellum. With regard to proportionate size and shape the cerebellum is doubtless the most variable part of the central nervous system, and its histological structure is also subject to considerable differences among the various groups of vertebrates. It will be clear, therefore, that within the limits set for this report only a schematic picture of the evolutionary development of the cerebellum can be drawn. In the first part of the following account some general information on the development and structure of the cerebellum will be provided. Special attention will be paid here to J. B. Johnston’s classical conception of the origin of the cerebellum, and to the fundamental pattern of this brain part, as described by Larsell. Following these introductory notes the structure and connexions of the cerebellum in the various groups of submammalian vertebrates will be briefly considered. In the final section of this paper the main results will be surveyed, and some general comments will be presented. GENERAL I N T R O D U C T I O N

In all vertebrates the cerebellum develops from two bilaterally symmetrical anlagen, dorsally situated in the rostral end of the rhombencephalon (Fig. la). Initially these anlagen are only connected by the membranous roof of the fourth ventricle, but later their rostral parts fuse in the middle plane. During further development the rhomboid fossa widens and the angle between the cerebellar anlagen of the two sides increases, until a stage is reached in which the fused halves of the cerebellar primordium form a more or less transversely oriented, plate-like structure (Fig. lb). This cerebellar plate lies immediately behind the tectum mesencephali and, arching somewhat caudally, roofs the rostral part of the rhomboid fossa. It is connected with the tectum by a thin lamella, the velum medullare anterius, which constitutes the bottom of a transverse groove. The fibres of the trochlear nerve decussate in the velum medullare anterius and this crossing forms a landmark which may be of significance in establishing the rostral limit of the cerebellum. Rostrolaterally and laterally the cerebellar plate is directly continuous with the brain stem and it is here that the various afferent and efferent connexions of the cerebellum pass. Caudally the cerebellar primordium References p . 88-93

2

R. N I E U W E N H U Y S

ouric.

corn. cb

nl :ere

A

B

C

Fig. 1. Drawings to illustrate the morphogenesis of the cerebellum. A, Dorsal aspect of the rostral part of the rhombencephalonin a 10 mm human embryo (after Jakob, 1928). B, Outline of a horizontal section through the cerebellar region of a 13-day-old opossum embryo (redrawn from Larsell, 1935). C, Dorsal view of the cerebellar region of a salamander. auric. = auricle; cereb. = cerebellum; cereb. anl. = cerebellar adage; com. cb. = commissura cerebelli; mes. = mesencephalon; n N.= nervus trochlearis; r. lat. = recessus lateralis.

thins out into a seam or taenia, to which the ependymal roof of the fourth ventricle is attached. In the petromyzonts the cerebellum maintains a simple plate-like configuration (Figs. 5a and 8a), but in most groups of gnathostomes the medial parts of the cerebellar plate become transformed into a median cerebellar mass or corpus cerebelli. In most aquatic forms the lateral parts of the cerebellar plate evaginate rostrolaterally, a process in which generally not only nervous tissue, but also the ependymal roof of the fourth ventricle becomes involved. The so called auriculae cerebelli which result from this evagination surround blind pouches or diverticula of the rhomboid fossa (Fig. lc). The morphogenetic events leading to the formation of the corpus cerebelli show a remarkable variety among different groups of vertebrates. We shall see that local or generalized thickening, evagination, invagination and eversion may all play a rBle in the development of this structure. According to the classical conception of Johnston (1901, 1902a,b), the cerebellum can be defined as a correlation centre which has gradually evolved from the dorsal column of the medulla oblongata. It may be recalled that the rhombencephalic part of this column gives rise to two sensory areas, viz., the end station of the trigeminal nerve (area trigemini), and the nuclear complex of the 8th cranial and lateral line nerves (area octavo-lateralis). Johnston believed that the cerebellum must be considered a derivative of the rostral end of the area octavo-lateralis. In order to elucidate this view it seems desirable to comment briefly on the structural relations in the dorsal part of the rhombencephalon of lower forms. As its name implies, the octavo-lateral area receives impulses from two sources, namely, from the lateral line organs and from the labyrinth, by way of the 8th cranial nerve. In this context, I should like to mention that the lateral line organs and the labyrinth show a marked structural and functional similarity. Both contain groups of sense cells, the neuromasts and maculae respectively, whose constituent elements are pro-

A N A T O M Y OF T H E C E R E B E L L U M

3

vided with fine hairs that extend into a fluid, and both are concerned with the appreciation of fluid movements (Fig. 2). An important difference is that the system of lateral line organs is in open communication with the water that surrounds the animal, whereas the labyrinth consists of a closed system of cavities which contains a special fluid, the endolymph. The receptor cells in the neuromasts respond to current-like water disturbances, and serve mainly to detect and locate moving animals (Dijkgraaf, 1963). The sense cells in the maculae of the labyrinth furnish data for the control of balance and position. The area which receives the lateral line and octavus fibres contains in lower vertebrates a zone of periventricular gray, which consists mainly of small, granular cells. These granular elements are each provided with a few short dendrites and with a laterally directed axon that bifurcates longitudinally in the submeningeal area of the alar plate (Fig. 2: 1). Larger neurons, with long, peripherally extending dendrites, and with axons that curve ventromedially, lie scattered among the granular cells (Fig. 2 : 2), or may form local condensations. The long dendrites of the large cells and the unmyelinated axons of the small ones together form a zone characteristically known as tela choc

...............'.. i'

7-1

*.

...........-..

............

.........

.......... vent r i c Ie

.I.*

# i

y\ s.I im. H.

basal plate

Fig. 2. Diagrammatic transverse section through the medulla oblongata of a lamprey. crista cereb. = crista cerebellaris;n. lat. = nervus lateralis; tela chor. = tela chorioidea; s. lim. H. = sulcus limitans of His. Further explanation in the text.

the crista cerebellaris or cerebellar crest. Johnston's (1902a) investigations have revealed that in the lamprey, i.e. the lowest form with a clearly distinguishable cerebellum: (1) the cell layer of the lateral area continues directly into the cerebellar gray; (2) the cells which make up the cerebellar gray are almost identical to those of the octavo-lateral area; (3) the cerebellar crest is continuous with the fibre layer of the cerebellum; (4) the lateral line and octavus fibres terminate not only in the dorsal region of the medulla oblongata, but also in the cerebellum (Fig. 3); References p . 88-93

4

R. NIEUWENHUYS

(5) the fibres just mentioned form a large commissure, situated in the dorsal part of the cerebellar plate (Fig. 5a); ( 6 ) the cerebellum receives, in addition to octavus and lateral line fibres, trigeminal fibres and a small bundle originating from the lobus inferior hypothalami (tr. lobocerebellaris; Fig. 3). On the basis of these observations, and of his earlier studies on the brain of the sturgeon, Johnston (1898, 1901, 1902a,b) concluded that the cerebellum in its most simple form is nothing but a dorsal fusion or bridge of tissue between the two areae octavo-laterales (the ‘acustica’ in his terminology). Even in the lampreys this primitive condition is not wholly preserved; the presence of a lobo-cerebellar tract shows, according to Johnston, that in this group the cerebellum has already made the first step in the direction of the differentiation of a correlation centre. This differentiation is not appreciable as yet in the histological structure of the lamprey’s cerebellum; its large cells, for instance, are still identical to those in the area octavo-lateralis. However, it is these large elements which during further evolutionary differentiation develop

Fig. 3. Diagram of the afferent cerebellar tracts of the lamprey. com. cb. = commissura cerebelli; com. vest. lat. = commissura vestibulo-lateralis;N.L.A. = nervus lateralis anterior;N.L.P. = nervus lateralis posterior; tr. loboxb. = tractus lobo-cerebellaris; tr. sp.cb. = tractus spino-cerebellaris; tr. tect.cb. = tractus tecto-cerebellaris.


5

into the highly characteristic elements, known as Purkinje cells (Fig. 7 : Pc; Johnston, 1902a,b). Later investigations have shown that the sensory input of the lamprey's cerebellum is more diverse than Johnston believed. Tecto-cerebellar and spino-cerebellar tracts have been described by several authors, and in connexion with Larsell's conception of the fundamental plan of the cerebellum which will be discussed next, it is of particular interest that the spino-cerebellar fibres form a small commissure in the basal part of the cerebellum (Figs. 3 and 5a). For a consideration of the views of Larsell we can best proceed from the simple cerebellum of urodele amphibians, to which that author devoted some of his earlier studies (Larsell, 1920; 1931; 1932a). As in the lamprey the medial part of the urodelan cerebellum is represented by a plate-like structure; its lateral parts show, however, a further differentiation and have bulged out rostrolaterally, to form large auriculae (Figs. 4 and 45). The auriculae, which caudally are directly continuous with the areae octavo-laterales rhombencephali, receive numerous vestibular and lateral line nerve fibres. A number of these fibres decussate toward the contralateral auricula by way of a commissure, situated in the morphologically most dorsal part of the cerebellum (Fig. 4). This commissure, the commissura lateralis of Herrick (1914) or the commissura vestibulo-lateralisof Larsell(l957) also contains, apart from the components already mentioned, a number of true commissural fibres which connect the auriculae of

I

rlobocb.

'

\ \

I

MES

I

"

p.med. Iabvest lot.

Fig. 4. Diagram of the cerebellum and its chief afferent connexions in a newt (modified from Larsell, 1934). AURIC. = auricula; p. lat. lob. vest. lat. = pars lateralis lobi vestibulo-lateralis;p. med. lob. vest. lat. = pars medialis lobi vestibulo-lateralis;tr. tect. et tegm. cb. = tractus tecto-et tegmentocerebellaris. For other abbreviations see Fig. 3. References p . 88-93

R. NIEUWENHUYS

6

the two sides (Herrick, 1914; Larsell, 1931,1932a). From the wide cell layer of the auriculae bilateral strands of nerve cells extend medially along the vestibulo-lateral commissure. These cell strands and the auriculae were denoted by Larsell as the partes mediales and lateralis, respectively, of the lobus vestibulo-lateralis cerebelli (Fig. 4). The ventral and medial parts of the urodelan cerebellum were designated by Herrick (1914, 1924) and Larsell(l920, 1931) as the corpus cerebelli. This region, although not free from vestibular fibres, is dominated by systems from other sources: fibres originating from the spinal cord (tr. spino-cerebellaris), from the tectum and the tegmentum mesencephali (tr. tecto- et tegmento-cerebellaris), from the lobus inferior hypothalami (tr. lobo-cerebellaris), and from the chief sensory nucleus of the trigeminal nerve (tr. trigemino-cerebellaris) have all been traced toward the corpus cerebelli. The component last mentioned forms, together with spino-cerebellar fibres and with some direct trigeminal root fibres, a commissural system, known as the commissura cerebelli (Fig. 4). Extensive comparative studies have led Larsell to the conclusion that the two parts which can be recognized in the urodelan cerebellum, namely, the lobus vestibulolateralis or lobus auricularis, and the corpus cerebelli, can be recognized in most

corn vest I a t

tect. rnes.

A. PETROMYZON

tegrn.

c trigcer:

c IV

B, EMBRYO HOMO

spcec

17mm C.R.

C,BDELLOSTOMA

Fig. 5. Sagittal sections through the cerebellum of various vertebrates. A, Midsagittalsection through the cerebellum of Pefromyzon marinus (modified from Larsell, 1947a). B, Sagittal section through the cerebellum of a 17 rnm C.R. length human embryo (redrawn from Larsell, 1947~).C, Outline of a midsagittal section through the region of the midbrain of Bdellostoma, showing the position of the small vestibulo-lateral commissure (based on Larsell, 1947a). com.vest. = commissura vestibularis; com.vest.lat. = commissura vestibulo-lateralis; r.IV = root of the nervus trochlearis; tectmes. = tectum mesencephali; tegm. = tegmentum; tr.sp.cer. = tractus spino-cerebellaris; tr. trig.cer. = tractus trigemino-cerebellaris.

ANATOMY OF THE CEREBELLUM

7

groups of vertebrates, and must be looked upon as the fundamental divisions of the cerebellum. He observed that these divisions are often demarcated from each other by an external groove, the fissura postero-lateralis, and he emphasized that in those groups of vertebrates in which the cerebellum shows more than one transverse groove (crocodiles, birds, mammals), the posterolateral fissure is always the first to appear during development (Larsell, 1932; 1935; 1947c; Larsell and Dow, 1935). The two commissural systems, i.e. the commissura vestibulo-lateralis and the commissura cerebelli, present in the urodelan cerebellum, have been identified in several other groups of vertebrates including petromyzonts (Larsell, 1947a; Fig. 5a), anurans (Larsell, 1923), reptiles (1926, 1934, 1937), birds (Larsell, 1945, 1948; Whitlock, 1952), mammals (Larsell, 1935) and man (Larsell, 1947c; Fig. 5b). These commissures appear early in development and Larsell (1932a: Ambystoma, 1935: Didelpliys) observed that after their fibres have crossed the ventricular roof they are followed by nerve cells which proliferate and migrate medially from either side. Thus the vestibulo-lateral and the cerebellar commissures constitute the bridges across which the medial parts of the auricular lobe and the corpus cerebelli have been raised. Concerning the histogenesis of the cerebellum, the views of Larsell differ from those of Johnston. It has already been mentioned that the somatic sensory region of the rhombencephalon can be divided into two zones, namely, a dorsal one, from which the octavo-lateral area develops, and a more ventrally situated one, which gives rise to the area trigemini. We have seen that Johnston believed that the cerebellum develops exclusively from the dorsal zone, which is also known as the special somatic sensory area. However, according to Larsell (1957) the area just mentioned gives origin only to the lobus auricularis. The corpus cerebelli, on the contrary, is in Larsell’s opinion a derivative of the trigeminal or general somatic sensory area. The lobus auricularis, i.e. that part of the cerebellum dominated by vestibular and lateral line nerve fibres, is a prominent feature of the brain in most groups of fish and in the urodele amphibians. In the anuran tadpole similar relations prevail, but at metamorphosis the lateral line system disappears, which leads to a reduction of the auricular lobe (Larsell, 1925, 1929).In amniotes a lateral line system is entirely lacking and the auricular lobe receives only vestibular fibres. In most reptiles the auricular lobe is quite small, but in crocodiles, birds, and mammals it is larger. In these three groups the lateral parts of the ‘vestibulo-cerebellum’ (i.e. the homologues of the auriculae) are usually designated as the flocculi, whereas its median portion is termed the nodulus. These structures form together the flocculo-nodular lobe which, as stated already, is demarcated from the corpus cerebelli by the posterolateral fissure. The corpus cerebelli receives its sensory input from various sources. Spino-cerebellar and trigemino-cerebellar tracts, conveying proprioceptive and exteroceptive information toward this part of the cerebellum, have been described for all groups of vertebrates. In mammals the ‘direct’ spino-cerebellar tracts are augmented by several ‘indirect’ systems, which have a relay nucleus in the medulla oblongata (n. cuneatus externus, n. funiculi lateralis, n. reticularis paramedianus). It seems probable that at least some of these spino-bulbo-cerebellar pathways also exist in submammalian forms, but experimental evidence on their presence is wanting. References p . 88-93

8


In addition to the spino-cerebellar and trigemino-cerebellar tracts, which may be termed the ascending afferents, the corpus cerebelli receives numerous fibres which descend from more rostrally situated centres. A tecto-cerebellar system is probably present in all veitebrates, and for most groups of anamniotes hypothalamo-cerebellar (lobo-cerebellar) and tegmento-cerebellar tracts have been described. Direct tegmentocerebellar fibres have not been observed in higher vertebrates, but it is known that in mammals an indirect tegmento-cerebellar pathway, synaptically interrupted in the inferior olive, exists. In addition to the fibres from the midbrain tegmentum the mammalian lower olivary complex receives afferents from the cerebral cortex (tr. corticoolivaris), from the corpus striatum (tr. strio-olivaris), as well as from the spinal cord (tr. spino-olivaris). The descending and ascending impulses brought in by these fibre systems are all relayed toward the cerebellum by the olivo-cerebellar tract. Not only in mammals but also in birds and in some groups of fish an inferior olive and an olivo-cerebellar tract have been identified. However, little is known of the olivary afferents in these forms. The final afferent cerebellar connexion to be mentioned here is the ponto-cerebellar tract, a system which has so far been demonstrated only in birds and mammals. In the former group this tract originates from two small cell masses situated in the rostra1 part of the medulla oblongata, immediately beneath its ventral surface. These nuclei, which do not effect an externally visible pons, receive descending afferents from the tectum mesencephali and ascending ones from the spinal cord (Zecha, 1966). Their efferent fibres terminate predominantly in the lateral parts of the corpus cerebelli (Brodal et al. 1950;Whitlock, 1952).In mammals the pontine nuclei are better developed and, at least in higher mammals, larger in number than in birds. Tecto-pontine and spino-pontine fibres (cf. Brodal, 1954) have also been observed in mammals, but in this group quantitatively the most important afferent system of the pons originates from the cerebral cortex. The ponto-cerebellar tract terminates largely in the greatly expanded lateral parts of the mammalian cerebellum, known as the cerebellar hemispheres, but also sends a considerable amount of fibres to its unpaired central portion, i.e. the vermis (Brodal and Jansen, 1946; Voogd, 1964). In the older comparative neuroanatomical literature it is commonly stated that the pontine nuclei first appear in mammals, and exclusively mediate impulses from the cerebral cortex to the cerebellar hemispheres. The latter structures are accordingly considered as neocortical dependencies, and thus as new acquisitions to the cerebellum, which do not occur below the mammalian level. Edinger (1910) and Comolli (1910) have expressed this view by subdividing the mammalian cerebellum into a neocerebellar part, comprising the hemispheres, and a paleocerebellar part, comprising the vermis and the flocculi. The authors mentioned considered the cerebellum of birds in toto as a paleocerebellum and homologized the avian corpus cerebelli with the mammalian vermis. However, it will be clear that, in view of the recent work on the avian and mammalian cerebellum mentioned earlier, the conception of Edinger and Comolli cannot be maintained in its original form. The studies of Brodal and Jansen (1946) and Voogd (1964) have shown that in mammals the ponto-cerebellar tract does not project only to the hemispheres, but also to the vermis. Consequently a distinction


9

between a paleocerebellum and neocerebellum cannot be based on the distribution of this fibre system. Moreover, it has been demonstrated that pontine nuclei are not confined to mammals, but also occur in birds, and finally, the fact that the avian pontine nuclei send their fibres chiefly to the lateral parts of the cerebellum (Brodal et al., 1950), supports the view of Larsell(l937) that the mammalian cerebellar hemispheres represent lateral outgrowths of the submammalian corpus cerebelli rather than entirely new structures. The efferent fibres of the cerebellum can be divided into an ascending and a descending system. The ascending system or brachium conjunctivum passes forward and ventralward from the cerebellum into the caudal part of the midbrain. Here its fibres decussate below the ventricle and then pass forward to terminate in various centres of the mesencephalic tegmentum, in the oculomotor nucleus and in the thalamus. The descending system comprises a cerebello-vestibular and a cerebello-motor component. The fibres of the former end in the vestibular nuclear complex; those of the latter arch around the fourth ventricle and connect the cerebellum with the rhombencephalic reticular formation and with the motor nuclei of the IVth, Vth, VIth and VIIth cranial nerves. It is assumed that in lower vertebrates the cerebellofugal pathways chiefly consist of Purkinje cell axons, but also contain a number of fibres which take origin from a separate cell mass. This cell mass, which forms the most ventral part of the cerebellum, is situated laterally to the fourth ventricle in a small protrusion, known as the eminentia ventralis. It receives collaterals from several cerebellar afferent systems, but its main input is provided by Purkinje cells whose axons, unlike the ones mentioned above, do not leave the cerebellum. The cell mass under discussion shows a marked progressive development as the vertebrate series is ascended. In fish and amphibians its cells are either diffusely arranged or form a single nucleus, the nucleus (lateralis) cerebelli (Edinger, 1901: sharks; Larsell, 1923: frog). Reptiles show a differentiation into two nuclei, a medial and a lateral one (Van Hoevell, 1916; Larsell, 1926, 1932b; Weston, 1936), but in most birds and mammals three or more cerebellar nuclei can be distinguished (Cajal, 1908; Sanders, 1929; Weidenreich, 1899; Brunner, 1919). In the two classes last mentioned the complex attains a considerable size, and its right and left groups approach each other above the fourth ventricle. Thus the cerebellar nuclei, still laterally situated in the reptiles, form the central core of the avian and mammalian cerebellum. Herrick (1924) supposed that, parallel with the progressive development of the cerebellar nuclei, the proportion of Purkinje cells whose axons terminate in this complex becomes larger, whereas the number of axons contributing directly to the cerebellofugal fibre systems gradually decreases. It should be emphasized that this hypothesis, though probably correct, deserves confirmation by degeneration studies in so far as the submammalian groups are concerned. However, it has been established experimentally that in mammals the vast majority of the Purkinje cell axons terminate in the deep cerebellar nuclei and that the brachium conjunctivum and the cerebellomotor system (represented here by the direct and crossed fastigio-bulbar fibres) issue exclusively from these nuclei. In this group only the vestibular nuclei receive a certain References p . 88-93

10


.

.

.

- . .

. ..

'

.

str: rnol.

. . .. . . . . . ..

stc rnol.

. * '

,

n.cereb.

-

' '.

. '. _. -. . . b

Fig. 6 . The laminar organization of the cerebellar wall in a lamprey (a) and a mammal (b).

number of Purkinje fibres (for details cf. Jansen, 1954; Jansen and Brodal, 1958). We will conclude this introduction with a few remarks on the histological differentiation of the cerebellum. It has already been mentioned that in the lampreys very simple relations are found. In this group the cerebellum contains a narrow, periventricular cell layer, and a wider outer zone of neuropil, known as the molecular layer (Fig. 6a). In the cell layer small and larger cells are intermingled; the former are obviously equivalent to the granular cells of the gnathostome cerebellum (Fig. 7 : gr), the latter, which extend their long dendrites into the molecular layer, are according to Johnston (1902a) and Larsell (1947a) the precursors of the Purkinje cells. A periventricular cell layer, consisting of small granular, and of larger elements, is also found in most groups of fish, amphibians and reptiles. However, in these classes the large elements are usually not scattered among the small ones, but tend to form a separate layer in between the molecular zone and the granular cells. The Purkinje cells of higher vertebrates occupy a similar position and, hence, the large elements in the cerebellum of fish, amphibia and reptiles are usually designated with the same name. However, 'true' Purkinje cells are not only characterized by the size and the position of their perikarya, but also by the fact that their dendrites branch in a single plane and it should be noted that in several groups of lower vertebrates (primitive actinopterygians, dipnoans, urodeles) the large cerebellar elements do not show such an orientation of their dendritic trees. The avian and mammalian cerebellum distinguishes itself in three respects from that of the lower vertebrates (cf. Fig. 6b): (1) During ontogenesis most of its cells migrate outward and thus the three layers

11


str. alb.

str. gran. stcP

str. mol.

Fig. 7. A somewhat diagrammatic sagittal section through a folium of the mammalian cerebellum, showing typical neurons. Redrawn from Jansen and Brodal(1958). bc. = basket cell; clf. = climbing fibre; Gc = large Golgi cell; G I1 = Golgi type 2 cell of the granular layer; gr = granular cells; hc = horizontal cell; ic = interstitial cell; mf = mossy fibre; Pc = Purkinje cell; pf = parallel fibres; sc 1, sc 2 = stellate cells; str.alb. = stratum album; str.gran. = stratum granulare; str. mol. = stratum moleculare; str.P. = stratum Purkinje.

References p. 88-93

12


(molecular, Purkinje, granular) which in most lower vertebrates occupy the entire width of the cerebellar wall, constitute here a superficially situated cortex. (2) The afferent and efferent fibres form a separate fibre layer. (In lower vertebrates these fibres are generally embedded in the granular layer.) (3) The complex of cerebellar nuclei has considerably enlarged, and forms a deep, central layer of gray matter. Fig. 7 represents a schematic sagittal section through a lobule or folium of the mammalian cerebellum, and gives an impression of the histological organization of the cortex (for details I may refer to Dr. Fox’s contribution to this volume). The Purkinje cells (Pc) are arranged in a very regular layer. Their richly ramifying dendritic trees, which extend into the molecular layer, spread out at right angles to the long axis of the folium. The axons of the Purkinje cells form the efferent systems of the cerebellar cortex, and it has already been mentioned that these fibres terminate chiefly in the deep cerebellar nuclei. Together the Purkinje cell axons form the so-called corticonuclear projection. The afferent fibres of the cerebellar cortex are of two types, the climbing (clf) and the mossy fibres (mf). It is generally assumed that these two types represent different systems of extracerebellar tracts ; this matter will be further considered in the article by Dr. Voogd. The climbing fibres traverse the granular layer, reach the base of the dendritic tree of a Purkinje cell, and dividing and redividing, climb up along its branches. The other type of afferent fibre, the mossy fibre, ramifies and terminates in the granular layer, where it synapses with the dendrites of a great number of granular cells (gr). The latter elements send their axons into the molecular layer, where they form T-shaped divisions. The branches of these divisions run parallel to the surface and at right angles to the plane of branching of the Purkinje cell dendrites. These so-called parallel fibres enter into synaptic relation with numerous Purkinje cells. Apart from the granular and Purkinje cells, various other types of neurons are found in the cerebellar cortex (cf. Cajal, 1911). The molecular layer contains scattered elements, known as stellate cells. The more superficially situated cells of this type are provided with short, branching axons that presumably synapse with the dendrites of Purkinje cells. The deeper lying stellate cells are the so-called basket cells (bc). The axons of these elements run parallel to the surface just above the Purkinje cell layer and give off numerous short descending collaterals. The latter participate in the formation of complex entanglements (‘baskets’) around the perikarya of the Purkinje cells. The granular layer contains, in addition to the small neurons after which it is named, fusiform, horizontal cells (hc) and large Golgi cells (Gc). The dendrites of the latter extend into the molecular layer, but are less numerous than those of the Purkinje cells. Their axons enter the granular layer and break up almost immediately into a number of small branches, which form a dense plexus. These branches are in synaptic relation with the short claw-like dendrites of the granular cells (gr).


13

COMPARATIVE SURVEY

Cyclostomes Myxinoidea The question whether or not the myxinoids possess a cerebellum has received widely different answers in the literature. Holm (1901) and Edinger (1906) were of the opinion that a cerebellum is entirely lacking in this group, but Holmgren (1919) held that ‘die Hinterteile des Mesencephalons von Edinger und Holm vertreten ein grosses Cerebellum’. This cerebellum has, according to Holmgren, a small ventricle of its own. It receives afferents from the medulla oblongata (exact site of origin not mentioned), from the area octavo-lateralis (the ‘acusticum’ in Holmgren’s terminology) and from the tectum mesencephali. Holmgren traced, in addition, root fibres from the vagus and the trigeminal nerve toward the cerebellum. According to Holmgren, the fibres last mentioned, together with bulbo-cerebellar and with true commissural fibres, constitute a commissura cerebelli. Holmgren traced efferent cerebellar fibres to the hypothalamus, to the tectum, and to the medulla oblongata. Jansen (1930), who like Holmgren (1919) studied Myxine in extensive Golgi and silver impregnated material, arrived at a widely different interpretation s3 far as the cerebellum is concerned. This author pointed out that in myxinoids the lateral line organs are poorly developed, and that the internal ear of this group is the simplest found in the whole vertebrate range. Taking into consideration that the lampreys, in which the octavo-lateral system is much better developed than in the myxinoids, still have a small cerebellum, Jansen conjectured that it would be ‘astounding’ to find in myxinoids a large cerebellum. His observations led him to the conclusion that the region denoted as cerebellum by Holmgren represents the roof of the midbrain. Thus Jansen supported the interpretation given earlier by Holm (1901) and Edinger (1906). However, Jansen left open the possiblity that the commissura posterior tecti, i.e. the commissura cerebellaris of Holmgren, contains a few fibres of a rudimentary cerebellar commissure. Larsell (1947a), who studied the brain of Bdellostoma, concluded that in this form an externally visible cerebellum is lacking, but he found in the posterior part of the tectum a small octavo-lateral commissure, separate from the comissura posterior tecti (Fig. 52.). From the areae octavo-laterales, strands of cells extend medially along the fibres of this commissure. These cell strands approach the median plane, but do not reach it. Larsell concluded that the commissure under consideration marks the beginning of cerebellar structure and is comparable to the fibrous connections between the auricles of urodeles. The medial extensions of cells from the octavo-lateral areas are, according to Larsell, the forerunners of the gray substance of the cerebellum. Bone (1963) has recently examined the question as to whether or not cerebellar structures, similar to those described for Bdellostoma, also occur in Myxine, but he remained unable to discern a cerebellar rudiment in this form. Petromyzontida Several aspects of the morphology of the lamprey’s cerebellum have already been discussed in the introductory part of this paper, and therefore I will confine myself References p . 88-93

14


cereb

iob.oct. lat.

Fig. 8. Dorsal view (a) and midsagittal section (b) of the brain of a lamprey. Figure a is redrawn from Larsell(1947a). b.ol. = bulbus olfactorius; cereb. = cerebellum; dien. = diencephalon; lob. oct.lat. = lobus octavo-lateralis; n.lat.ant. = nervus lateralis anterior; n.lat.post. = nervus lateralis posterior; tectmes. = tectum mesencephali; tel. = telencephalon.

here to some additional notes and illustrations. We have seen that in the lamprey the cerebellum consists of a thin lamina, bridging over the medulla oblongata and uniting laterally with the areae octavo-laterales (Fig. 8). These latter areas contain at the level of entrance of the octavus and anterior lateral line nerves three cell masses: the nucleus dorsalis, the nucleus intermedius (in the literature generally infelicitously denoted as the n. medialis), and the nucleus ventralis. The dorsal and intermediate nuclei receive predominantly fibres from the lateral line nerves (Fig. 9). The ventral nucleus is primarily the region of termination of the octavus, i.e. the vestibular nerve. If we trace these nuclei rostralward (Figs. 9-11) it appears that the dorsal and ventral nuclei disappear before the level of the cerebellum is reached. The intermediate

A N A T O M Y OF THE CEREBELLUM

15

Fig. 9. A cross section through the brain stem of Lampetrafluviatilis. Bodian method. cr. cb.= crista cerebellaris; f.a.i. = fibrae arcuatae internae; M.c. = Miiller cells; N.1at.ant.d. = nervus lateralis anterior, pars dorsalis; N.1at.ant.v. = nervus lateralis anterior, pars ventralis; n.dors., nhterm., n.ventr. = nucleus dorsalis, nucleus intermedius and nucleus ventralis of the area octavo-lateralis; n.Vm. = nucleus motorius nervi trigemini; pl.ch. = plexus chorioideus; r.Vd. = ramus descendens nervi trigemini; VII s = radix sensibilis nervi facialis.

Fig. 10. A cross section through the brain stem ofLampetrafluviatilis,rostra1 to the plane o f Fig. 9. tr.lin.cb. = tractus lineo-cerebellaris; Vm. = radix motorius nervi trigemini. For other abbreviations see Fig. 9.

nucleus, however, extends further rostrally and continues into the cerebellar gray (Pearson, 1936; Larsell, 1947a). It has already been mentioned that according to Johnston (1902a) the cerebellar References p . 88-93

16


Fig. 11. A cross section through the cerebellar region OfLampetraPuviutilis. com.vest.lat. = commissura vestibulo-lateralis; n.cb. = nucleus cerebelli; n.oct.rn.ant. = nucleus octavo-motorius anterior; r.Vd. = rarnus descendens nervi trigemini; str.gris.cb. = stratum griseumcerebelli; str.mo1. = stratum moleculare; tr.lin.cb. = tractus lineo-cerebellaris; Vm. = radix motorius nervi trigemini; Vs. = radix sensibilis nervi trigemini.

Fig. 12. A cross section through the cerebellar region ofLampetrafluviatiZis, a few sections rostra1 to the plane of Fig. 11. d. IV = decussatio nervi trochlearis; N.IV = nucleus nervi trochlearis. For other abbreviations see Fig. 11.


17

gray of the lamprey contains cells of two types: small, granular elements, and large cells which he considered the forerunners of the Purkinje cells. These two cell types have also been observed by Larsell (1947a), but it should be noted that Tretjakoff (1909) and Heier (1948) have denied the occurrence of Purkinje-like cells in the lamprey. It is also worthy of note that the large Purkinje cells described by Jeleneff (1879, L. Jluviatilis), Schaper (1 899, L.Jluviatilis) and Saito (1 930, Entosphenus japonicus) presumably represent the elements of the trochlear nucleus. As early as 1883 Ahlborn showed that this somatic motor nucleus lies far dorsally in the lamprey (Fig. 12). He regarded its position as being intracerebellar, as did Addens (1933). Larsell(1947b), however, was of the opinion that the trochlear nucleus for the most part is not located in the cerebellum proper, but rather in the anterior medullary velum. Apart from a zone of periventricular gray the cerebellar region of the lamprey contains a group of more superficially situated small cells (Fig. 11: n.cb.). This group was first described by Van Hoevell (1916) under the name ‘cellulae cerebellares’. Later investigators, among them Larsell (1947a) and Riideberg (1961), termed it the nucleus cerebelli. Larsell has shown that the axons of this nucleus collect into a fairly compact cerebello-tegmental bundle, which in his opinion represents the beginning of the brachium conjunctivum of higher vertebrates. Little is known about the afferent connexions of the nucleus cerebelli, but Larsell(1947a) regarded it probable that some axons of primitive Purkinje cells terminate among its constituent elements. Ventrolaterally to the nucleus cerebelli a group of large cells is located, which has been called the nucleus octavo-motorius anterior (Van Hoevell, 1916 ;Ariens Kappers, 1921; Fig. 11). This nucleus receives vestibular impulses through ascending octavus fibres. Its coarse efferent fibres form the tractus octavo-motorius anterior (Fig. 12) which, like the cerebellar efferent system mentioned above, terminates in the tegmentum of the midbrain. The nucleus under discussion lies outside the cerebellar territory, but Larsell (1947a) observed that numerous dendritic processes of its cells extend dorsomedially into the cerebellum. With regard to the interpretation of the two nuclei just discussed there is some disagreement in the literature. Van Hoevell(l916) and Ariens Kappers (1921) believed that the deep cerebellar nuclei of higher vertebrates have their origin in relation to the vestibular nuclear complex. They advanced the theory that in the course of phylogeny the cells of the cerebellar nuclei have become more distinctly segregated from this complex and have shifted from a subcerebellar to an intracerebellar position. This migration was thought to have occurred under the neurobiotactic influence of the Purkinje cell axons. On the basis of this hypothesis Ariens Kappers considered the nucleus octavo-motorius anterior or its immediate surroundings to be a forerunner of the deep cerebellar nuclei. Van Hoevell thought it probable that the large-celled nucleus octavo-motorius and the small-celled nucleus cerebelli (his ‘cellulae cerebellares’) together represent a primordium of the cerebellar nuclei of higher vertebrates. Larsell(1947a) and Riideberg (1961), on the other hand, were of the opinion that only the small-celled nucleus is homologous to the deep cerebellar nuclei. The last mentioned author has recently made an extensive comparative study of the development of the cerebellar nucIei. He did not observe any migration of cells from the vestibular region References p . 88-93

18


into the cerebellar anlage, and arrived at the conclusion that the cerebellar nuclei, 'must be considered as taking their embryologic origin, at least to their greatest extent, from the cell-masses of the cerebellum itself' (Riideberg, 1961, p. 121). Finally, it should be mentioned that Heier (1948) regarded the cell mass called here the nucleus cerebelli as a primordial lobus auricularis. However, in agreement with Larsell (1947a), I think it more probable that not this group of migrated cells, but rather the cerebellar periventricular gray of the lamprey corresponds to the auricles of fish and urodeles. The present day knowledge of the connexions of the petromyzontian cerebellum is primarily based on the studies of Johnston (1902a), Clark (1906), Tretjakoff (1909), Pearson (1936), Larsell(1947a) and Heier (1948). The following afferent systems have been described (cf. Figs. 3 and 13). (1) Primary fibres from the anterior and posterior lateral line nerves (= tr. lineocerebellaris of Heier, 1948; Fig. 10). (2) Primary octavus fibres. (3) Secondary fibres arising in the area octavo-lateralis. Pearson (1936) named this system the tractus bulbo-cerebellaris. It receives, according to his observations, fibres from all three of the nuclei of the area in question. Larsell (1947a), however, stated that only the dorsal and intermediate ('medial') nuclei distribute fibres to the cerebellum. The fibre systems mentioned under 1-3 terminate both homolaterally and contra-

\ n m TECT. MES

Fig. 13. A diagram presenting the afferent connexions ofthe petromyzontian cerebellum, projected on a sagittal plane. The labels do not require explanation.

19


laterally in the cerebellum. The decussating fibres form a conspicuous commissure, situated in the most dorsal part of the cerebellar plate (Fig. 5a). A large proportion of the primary and secondary octavus and lateral line fibres run superficially through the dorsal part of the alar plate, and participate in the formation of the crista cerebellaris (Figs. 2,9 and 10). In the introductory part of this paper it has already been mentioned that this fibre and neuropil zone is rostrally continuous with the molecular layer of the cerebellum. (4) Primary and secondary trigeminus fibres. The latter arise, according to Pearson (1936), from the nucleus of the descending root of the trigeminus. (5) Tractus spino-cerebellaris. This tract was first described by Pearson (1936), who observed that its fibres originate from the lateral funiculus of the cord and, after having traversed the medulla oblongata, arch around the trigeminus nerve. Its fibres reach the cerebellar gray of the same and the opposite side. Larsell (1947a) has confirmed the description of Pearson, and has pointed out that the decussating spinocerebellar fibres constitute a separate commissura cerebelli, situated rostroventrally to the large commissura vestibulo-lateralis (cf. Fig. 5a). (6) Tractus tecto-cerebellaris. This is a small bundle which passes through the velum medullare anterius. (7) Tractus lobo-cerebellaris. This tract connects the lobi inferiores hypothalami with the basal part of the cerebellum. It has been observed by most students of the lamprey’s brain, but Heier (1948) reported that he was unable to demonstrate its presence.

tegm.

Fig. 14. A diagram showing the efferent connexions of the petromyzontian cerebellum, projected on a sagittal plane. br.conj. = brachium conjunctivum; f.1.m. = fasciculus longitudinalis medialis; n.ret.tegm. = nucleus reticularistegmenti mesencephali;n.111, n.V, n.VII = motor nuclei; tr.cb.mot. = tractuscerebello-motorius;tr.cb.tect. = tractus cerebello-tectalis;tr.cb.tegm. = tractus cerebellotegmentalis. References p . 88-93

20


The efferent fibres of the cerebellum (Fig. 14) leave the cerebellar region as a single system, which later splits up into a dorsal, an intermediate and a caudal component. According to Johnston (1902a) these efferent fibres arise from the large primordial Purkinje cells. Larsell (1947a) arrived at a similar conclusion, with the reservation that some fibres of the intermediate component are made up of fibres from the nucleus cerebelli. The dorsal component of the cerebellar efferent system consists, according to Pearson (1936), of cerebello-tectal fibres, which enter the superficial layers of the tectum. Clark (1906) and Tretjakoff (1909) also noted a cerebello-tectal connection, but Larsell (1947a) denied its presence. Heier (1948) regarded it probable that fibres of cerebellar origin pass to the tectum as well as to the primordial torus semicircularis (Fig. 14: tr.cb.tegm.). According to Larsell(1947a), the intermediate component represents the beginning of the brachium conjunctivum of higher vertebrates. Its fibres pass rostroventrally and fan out in the mesencephalic reticular nucleus. The caudal component of the cerebellar efferent system constitutes the so-called tractus cerebello-motorius. The more rostra1 bundles of this system distribute, according to Pearson (1936), to the oculomotor nuclei. The more caudal bundles form both crossed and uncrossed connexions with the motor nuclei of the medulla oblongata, whereas other fibres join the fasciculus Iongitudinalis medialis (Pearson, 1936; Heier, 1948). Chondrichthyes

The cerebellum of cartilaginous fishes (sharks and rays) is considerably larger and much further differentiated than that of cyclostomes. In early developmental stages it consists, just as that of the adult lamprey, of a simple plate-like structure, but during further development two processes take place which give the chondrichthyan cerebellum its characteristic appearance. These are :(1) bilaterally a rostrolaterally-directed lengthening and outpouching of the caudolateral parts of the cerebellar anlage, a process in which not only the nervous, but also the adjacent chorioid tissue is involved, and (2) a dorsalward evagination of the rostromedial parts of the cerebellar plate. The former event leads to the formation of the auriculae, the latter gives rise to the corpus cerebelli (Fig. 15). The auricles, which can be divided into a rostromedial upper leaf and a caudolateral lower leaf (Fig. 15a), surround extensive recesses of the fourth ventricle. The lower leaf is continuous with the lobus lineae lateralis anterior (Fig. 18); the upper leaves of both auricles are connected over the fourth ventricle by a band of nervous tissue, known as the ‘lower lip’ (cf. here Figs. 15b and 18). Dorsally the auricles are entirely covered by lateral extensions of the tela chorioidea of the fourth ventricle. These membranous structures, which are attached to the external margins of the auricles, are not represented in Figs. 15 and 16, but in Fig. 18 they are clearly visible. The upper leaves of both auricles and the lower lip are separated from the corpus ceiebelli by a deep groove. Voorhoeve (1917) termed the lateral parts of this groove the sulci


21

-I V

0 Fig. 15. The brain of the shark Squalus acanfhias. a and b, Dorsal view of the brain; in figure b the caudal half of the corpus cerebelli (corp. cer.) has been removed in order to show the ‘lower lip’ which is laterally continuous with the upper leaf of the auriculae (auric.). Modified from Haller v. Hallerstein (1934). c, Median section through the brain. For abbreviations see Fig. 8.

para-auriculares and the intermediate part (i.e. the part which marks the boundary between the lower lip and the corpus cerebelli) the sulcus postremus. The auriculae are dominated by the vestibular and lateral line systems. Together with the lower lip they represent according to Larsell (1957) the lobus vestibulo-lateralis cerebelli. The sulci para-auriculares and the sulcus postremus are in Larsell’s opinion equivalent to the fissura postero-lateralis of urodeles and amniotes. The central, unpaired corpus cerebelli encloses a large ventricular cavity. Its dilated dorsal part extends rostrally over the roof of the midbrain and caudally over the lower lip (Figs. I5 and 16). On the basal part of the corpus cerebelli four walls may References p . 88-93

22

R. NIEUWENHUYS

a

b

C

Fig. 16. Dorsal view of the brain of the shark Carcharias (spec?). Modified from Haller v. Hallerstein (1934). For abbreviationssee Fig. 8. Fig. 17. Median section through cerebella of sharks. a, Scylliorhinus eaniculus; b, Galeus canis; c, Lamnacornubica. After Voorhoeve (1917).

be distinguished: a iostral one, a caudal one, and two lateral ones. The rostra1 wall is continuous with the tectum via a very short velum medullare anterius.The caudal wall is connected with the lower lip and with the upper leaves of the auricles. The lateral walls, finally, pass over into the medulla oblongata. At their medial side they show a marked intraventricular protrusion, known as the eminentia ventralis cerebelli (Fig. 19). This eminence contains a mass of diffusely arranged large cells, which was first described by Edinger (1901), under the name nucleus lateralis cerebelli (Fig. 19). It should be noted that Edinger and most other studentsof thechondrichthyan cerebellum (Van Hoevell, 1916; Voorhoeve, 1917 ; Ariens Kappers, 1921; Larsell, 1957) considered this nucleus to be a subcerebellar structure. However, Riideberg (1961), who studied the ontogenetic development of the nucleus lateralis cerebelli, concluded that this nucleus originates from the cerebellar anlage, and lies within the cerebellum. The extensive comparative studies of Voorhoeve (1917) have shown that the chondrichthyan corpus cerebelli, with regard to its external configuration, is a remarkably variable structure. In primitive sharks like Hexanchus and Heptranchius the cerebellum is smooth or shows only a slight median groove. Similar relations are


23

found in small species of the more advanced sharks (e.g. Scylliorhinus canicnlus, Fig. 17a). In larger sharks and in all rays the corpus cerebelli displays transverse grooves, the number of which increases with the size of the body. The most common transverse groove is the sulcus transversus primus which divides the corpus cerebelli into an anterior and a posterior lobe of about equal size (Fig. 15a). In small species this groove is shallow and single, but in larger animals it is deep and two other sulci appear, one before and the other behind the sulcus transversus primus (Fig. 15b). These grooves divide the lobus anterior and the lobus posterior each into two lobuli. In the largest sharks and rays accessory sulci divide the four cerebellar lobuli into numerous sublobuli (Fig. 1%). A further complication which occurs in some large chondrichthyans is an asymmetry of the corpus cerebelli (Fig. 16). It should be emphasized that the convolution of the cerebellum as seen in chondrichthyans differs from that in higher vertebrates. In the former the entire wall is involved in the folding, whereas in the latter only the external surface is convoluted (cf. Fig. 15 with Fig. 57). Before leaving the gross anatomy of the chondrichthyan cerebellum it should be mentioned that not only the corpus cerebelli, but also the auriculae may show considerable differences in their development. However, whereas the size of the corpus cerebelli increases with an increase in the size of the body, the degree of development of the auriculae is independent of body size. Voorhoeve (1917) and Ariens Kappers (1921) have pointed out that this difference is correlated with the difference in sensory input of the two structures named. According to these authors the corpus cerebelli receives chiefly general somatic impulses, brought in by spinocerebellar and olivocerebellar tracts, whereas the auricles are supplied by primary and secondary vestibular and lateral line fibies. In Fig. 16 a brain with strongly developed, convoluted auriculae is represented. Histologically the chondrichthyan cerebellum is much better developed than that of the cyclostomes. Its wall shows a differentiation into four layers: the granular layer, the fibre zone, the layer of Purkinje cells, and the molecular layer. The auriculae contain a wide granular layer throughout their extent but in the corpus cerebelli the granule cells are concentrated in two longitudinal ridges (Figs. 18 and 19). These ridges, the prominentiae granulares, are situated on either side of the median plane. They begin immediately behind the trochlear decussation, curve along the inside of the wall of the corpus cerebelli, and terminate as two slight ventral protrusions of the lower lip (Fig. 18). A fibre layer is distinguishable only in the lateral parts of the corpus cerebelli (Fig. 19). Its constituent elements form a fan which diverges from the site where the corpus cerebelli is connected with the wall of the medulla oblongata. In the medial parts of the corpus cerebelli and in the auriculae the fibres are at most places diffusely embedded in the granular layer. The median zone of the cerebellum, which is rather narrow due to the fact that external and ventricular sulci approach each other here (cf. Figs. 18 and 19), is passed by numerous fibre bundles. These bundles form a continuous series which extends from the rostra1 part of the corpus cerebelli into the lower lip. In the corpus cerebelli many decussating fibres can be traced into the (lateral) fibre layer. The most caudal bundles of decussating fibres represent the commissura References p. 88-93

24


Fig. 18. Transverse section through the caudal part of the cerebellum of Scylliorhinus caniculus. auric. = auricula; cr.cb. = crista cerebellaris; 1ob.lin.lat.ant. = lobus lineae lateralis anterior; N.lat. ant.d. = nervus lateralis anterior, pars dorsalis; N.1at.ant.v. = nervus lateralis anterior, pars ventralis; pl.ch. = plexus chorioideus; r e d a t . = recessus lateralis ventriculi quarti; str.gran. = stratum granulare; strmol. = stratum moleculare; str.Purk. = stratum Purkinje; ventr.cb. = ventriculus cerebelli.

Fig. 19. Transverse section through the middle of the cerebellum of Scylliorhinus caniculus. emin. ventr. = eminentia ventralis; n.cb. = nucleus lateralis cerebelli. For other abbreviations see Fig. 18.

vestibulo-lateralis, described by Larsell (1957). The Purkinje cells are arranged in a regular layer one to two cells thick. This layer extends through the entile wall of the corpus cerebelli with the exception of the paramedian regions (Figs. 18 and 19). In the auriculae the Purkinje cells are less regularly

25

ANATOMY OF THE C E R E B E L L U M

arranged and less numerous. I am under the impression that in some parts of the upper leaves these elements are even entirely lacking. The submeningeal zone of the cerebellum is occupied by the molecular layer. This layer is thick in the corpus cerebelli, but very thin in certain parts of the upper leaf of the auricles (Fig. 18). The molecular layer of the lower leaf is continuous with the crista cerebellaris which, as in cyclostomes, covers the area octavo-lateralis. Golgi studies (Schaper, 1898; Houser, 1901) have revealed that the chondrichthyan Purkinje cells spread their dendrites in a single plane, and that these dendrites are covered with numerous fine spines (Fig. 20: P). Their branching is, however, much simpler than in higher vertebrates. The axons of the Purkinje cells pass through the

Y

sl

.................

.............

Fig. 20. Cell types in the cerebellum of sharks. Combined from figures of Schaper (1898) and Houser (1901). G = large granule cell; gr = small granule cells; P = purkinje cell; s 1, s 2 : stellate cells in the molecular layer.

subependymal fibre layer toward the cerebellar peduncle. Some authors (Van Hoevell, 1916; Voorhoeve, 1917) thought it probable that a certain proportion of Purkinje cell axons terminate in the nucleus lateralis cerebelli. It should, however, be stressed that the problem : if and to what extent the chondrichthyan cerebellar efferent systems are synaptically interrupted in the lateral cerebellar nucleus, is still awaiting its solution. The granular layer contains two kinds of cells: small elements which iesemble in every respect the granule cells of higher vertebrates (Fig. 20: gr), and larger Golgi type 2 cells (Fig. 20: G). Mossy fibres have not been demonstrated as yet in the granular layer of chondrichthyans. The molecular layer contains: (1) Purkinje cell dendrites, (2) axons of granule cells and their branches (i.e. parallel fibres), (3) a primitive type of climbing fibres (Ariens Kappers et ul., 1936), and (4) scattered stellate cells (Fig. 20: sl,s2). The axons of some of the last mentioned elements run horizontally but it has not been observed that they form pericellular baskets. References p . 88-93

26


The cerebellar fibre systems of cartilaginous fishes are larger and more compact than those of cyclostomes. The following afferent systems have been described (Fig. 21) :

CORP CEREB

7 Fig. 21. Diagram illustrating the afferent connexions of the selachian cerebellum. N.L.A. = nervus lateralis anterior; N.L.P. = nervus lateralis posterior;n.cb. = nucleus lateralis cerebelli; tr. lobo-cb. = tractus lobo-cerebellaris;tr.mes.cb. = tractus mesencephalo-cerebellaris; tr.ol.cb. = tractus olivocerebellaris; t.spino.cb. = tractus spino-cerebellaris.

(1) Root fibres from the anterior and posterior lateral line nerves. These fibres reach, according to Voorhoeve (1917) and Ariens Kappers (1921), the auricle (particularly the lower leaf) and the nucleus lateralis cerebelli. (2) Root fibres from the vestibular nerve. Voorhoeve (1917) and Ariens Kappers (1921) traced these fibres exclusively to the auricle, but Jansen and Brodal (1958), probably quoting Larsell (1957), mention that a considerable number of vestibular fibres reach the caudal part of the corpus cerebelli. (3) A secondary system, connecting the octavo-lateral area with the cerebellum. This has been reported by Jansen and Brodal (1958). (4) Trigemino-cerebellar fibres. These have been described by Haller (1 898) and Edinger (1901). Ariens Kappers et al. (1936, p. 722) stated, however, ‘It is uncertain as to whether or not direct root fibres of the trigeminal reach the body of the cerebellum or the auricles in selachians’. (5) A spino-cerebellar system, comprising a small rostral portion, and a large caudal portion. This terminates in the corpus cerebelli (Ariens Kappers, 1921). The rostral portion reaches the cerebellum by way of the velum medullare anterius, and is believed to be homologous with the ventral spino-cerebellar tract of higher vertebrates. The caudal portion, which would represent the dorsal spino-cerebellar tract, ascends toward the corpus cerebelli behind the eminentia ventralis cerebelli, through the basal part of the auricles. The greater part of the spino-cerebellar system terminates ipsi-

A N A T O M Y OF THE C E R E B E L L U M

27

laterally, but some of its fibres decussate through the commissural plate of the corpus cerebelli to the other side (Ariens Kappers et al., 1936). (6) An olivo-cerebellar system. This accompanies the caudal portion of the spinocerebellar tract toward the corpus cerebelli. The fibres of this system originate from the contralateral inferior olive, and terminate in all parts of the body of the cerebellum (Ariens Kappers, 1921). (7) A tractus mesencephalo-cerebellaris superior which has been described by Voorhoeve (1917) and Ariens Kappers (1921). This bundle runs caudally through the midbrain tegmentum and the isthmus region. In the rostral part of the medulla oblongata its fibres arch dorsally and pass rostrally to the eminentia ventralis toward the corpus cerebelli. Ariens Kappers (1921) considered it probable that at least a part of this system originates from the rostral part of the tectum mesencephali, but thus far the exact origin of this bundle has not been determined (Ariens Kappers et al., 1936). (8) A lobo-cerebellar tract, originating from the lobus inferior hypothalami, which has been reported by Ariens Kappers (1906) and Voorhoeve (1917). Ariens Kappers et al. (1936), however, were not convinced of the presence of such a system. Before considering efferent systems it should be mentioned that Edinger (1901), on the basis of some Marchi experiments, has concluded that root fibres of the VIIIth, IXth and Xth cranial nerves enter the corpus cerebelli, in which they partly decussate toward the other side. Voorhoeve (1917) and Ariens Kappers (1921) have denied the existence of these connexions, but it should be noted that since the attempts of Edinger no other experimental studies on the chondrichthyan cerebellar afferent systems have been reported. CORP CEREB

AURIC. C

\

n4!

I! Y'\yn.cb. "

W

2 HYPOTH. \ Fig. 22. Diagram to show the efferent connexions of the selachian cerebellum. brxonj. = brachium conjunctivum;n.cb. = nucleus lateralis cerebelli;n.ret.med. = nucleus reticularis medius; n.ret.mes. = nucleus reticularis mesencephali;n.ret.sup. = nucleus reticularis superior; n III, n V, n VII = motor nuclei. References p . 88-93

28


Turning now to the efferent connexions, the fibres which have been described include a tractus cerebello-vestibularis et cerebello-bulbaris rectus, a tractus cerebellomotorius, and a brachium conjunctivum (Fig. 22). The tractus cerebello-vestibularis et cerebello-bulbaris rectus consists of fibres which leave the cerebellum posterior to the eminentia ventralis and terminate in the area octavo-lateralis. The tractus cerebello-motorius (Ariens Kappers, 1906; Sterzi, 1909) arises from the corpus cerebelli and the auricles. Its fibres arch around the fourth ventricle and reach the motor and reticular nuclei of the lower brain stem largely by way of the ipsilateral and contralateral fasciculus longitudinalis medialis. Some of the fibres of this system pass rostrally and are believed to reach the eye muscle nuclei. The brachium conjunctivum leaves the cerebellum in the region of the nucleus cerebelli, but it is unknown whether or not this bundle actually originates from this nucleus. Wallenberg (1907) observed that in Scyliorhinus the brachium conjunctivum decussates completely and distributes its fibres to the nucleus reticularis mesencephali (designated as nucleus ruber by Wallenberg), to the oculomotor nucleus, and to the hypothalamus. A descending branch of the brachium conjuntivum reaches, according to Wallenberg, the nucleus raphes medullae oblongatae. Osteichthyes Introductory notes The class of Osteichthyes (higher bony fishes) includes two subclasses, namely the Actinopterygii (ray-finned fishes) and the Sarcopterygii (fish with fleshy-lobed fins). The subclass Actinopterygii, which encompasses more than 30,000 species, can be divided into four superorders : the Palaeoniscoidei, the Chondrostei, the Holostei, and the Teleostei. The vast majority of the recent bony fishes belongs in the Teleostei; the other groups mentioned are generally considered to be primitive groups of which only a small number of species have survived. The Sarcopterygii comprise the Dipnoi or lung fishes and the Crossopterygii. At one time it was believed that the Dipnoi gave rise to the land vertebrates, but it is now known that this honour is due to the crossopterygians. In the ensuing survey the cerebellum of the Polypteriformes (living representatives of the ancient Palaeoniscoidei), the Chondrostei, the Holostei, the Teleostei, the Dipnoi, and that of the sole surviving crossopterygian Latimeria will be dealt with briefly. It is, however, important to note that of the various groups of bony fishes it was the teleosts whose cerebellum was first studied microscopically. As a result, the cerebella of the other osteichthyan groups have usually been interpreted in the light of the knowledge of the teleosts. Before dealing with the primitive actinopterygian groups it is therefore necessary to comment briefly on two structures which were first described in teleosts, namely the eminentiae granulares and the valvula cerebelli. The eminentiae granulares are bilateral masses of superficially situated granular cells, which constitute the lateral parts of the teleostean cerebellum (Figs. 31 and 32).


29

.Ariens Kappers (1921) believed that the eminentiae granulares arise from a fusion of the lobus lineae lateralis anterior with the auricles. Herrick (1924) stated that the eminentiae granulares receive vestibular and lateral line nerve fibres, just as is the case for the chondrichthyan auricles. He also pointed out that the two granular eminences are connected across the caudoventral border of the corpus cerebelli by a band of granular cells which resembles the lower lip (cf. also Van der Horst, 1926). On the basis of these similarities Herrick regarded the eminentiae granulares as equivalent to the auricles. According to Herrick, in the teleosts the lateral recesses of the fourth ventricle are during development completely filled with granule cells. This would explain the fact that the eminentiae granulares are solid structures in adult teleosts. As regards the relationship between the granular eminences and the auricles, the results of Hocke Hoogenboom (1929) and of Pearson (1936) differ from those of Herrick. The first mentioned author held that in the chondrostean Polyodon large auricles are present, and that the granular eminences have been partly incorporated in these auricles, to form part of their medial walls. Pearson (1936) also concluded that the auricles and the granular eminences are different structures. In the holostean Amia and in the teleosts the auricles are, according to this author, represented by masses of granular cells, which surround small recesses of the fourth ventricle. These structures (and not the eminentiae granulares) are, according to Pearson, connected by a band of granular cells, which forms the morphologically most caudal part of the cerebellar gray. Pearson observed that the eminentiae granulares are situated rostrolaterally to the auricles, and that they are separated from the latter by fibre bundles. Finally it should be mentioned that the embryological investigations of Rudeberg (1961) have not confirmed the statement of Herrick that in teleosts the lateral recesses are gradually filled up with granule cells. The valvula cerebelli is a pouch-like structure which projects forward into the ventricle of the midbrain (Fig. 31 b). Ventrally this structure is continuous with the rostrobasal part of the corpus cerebelli and dorsally it is connected with the caudal part of the tectum. Its rostrolateral surface is often fused with the tegmentum of the midbrain. The structure as a whole encloses an extension of the cranial cavity (Fig. 31b). Histologically the valvula shows a typical cerebellar structure. Its three layers correspond to, and are caudally directly continuous with, the molecular, the Purkinje, and the granular layers of the corpus cerebelli. The fibre connexions indicate that the valvula cerebelli serves as a centre of the lateral line system. It receives some direct root fibres from the anterior lateral line nerve (Addison, 1923; Pearson, 1936), but its main input is formed by a tertiary system, the tractus mesencephalo-cerebellaris posterior, which arises from the nucleus lateralis valvulae (Fig. 36). The latter nucleus is situated in the tegmentum of the midbrain. It is the end station of a secondary pathway, which originates from the lateral line nerve centres in the medulla oblongata. Ariens Kappers (1907) and Franz (19 11 a) believed that the teleostean valvula cerebelli is homologous to the rostra1 part of the chondrichthyan corpus cerebelli. The only difference would be that the former structure extends dorsal to the tectum, whereas the latter occupies a subtectal position (cf. Fig. 1% with Fig. 31b). Herrick (1924), on the other hand, regarded the valvula as a new feature, ‘grown up in correReferences p . 88-93

30


lation with the differentiation within the midbrain of special correlation centres (poorly developed in sharks) related with the lateral line nerves’ (Herrick, 1924, p. 19). This view was accepted by Larsell (1929) and others. Ariens Kappers (1929) interpreted the valvula as ‘a frontal extension of the basiauricular (vestibulo-lateral) region of the cerebellum’ whose outgrowth ‘is caused by impulses, which reach it from the tegmentum of the midbrain’ (Ariens Kappers, 1929, pp. 118-119). The formation of the valvula is, according to Ariens Kappers, a typical example of neurobiotaxis. It seems to me that if the teleostean valvula represents an outgrowth of a pre-existing part of the cerebellum, it must have taken its origin from the corpus cerebelli, rather than from the vestibulo-lateral cerebellar region. The fact that in many teleosts the valvula lies morphologically in front of the trochlear decussation, whereas in all other vertebrates the corpus cerebelli begins behind this decussation, seems to favour the theory that the valvula is a new structure, so to speak a ‘cerebellisation’ of the velum medullare anterius. There is, however, evidence that among the teleosts the trochlear decussation has shifted from a prevalvular toward a retrovalvular position (for details see: Franz, 1911c, and Van der Horst, 1916). Jt appears that a reliable landmark allowing a sharp demarcation of the teleostean valvula from the corpus cerebelli is lacking, and it must be concluded that the structure under discussion is only characterised by its fibre connexions. The valvula is mostly defined as a subtectal structure. However, it should be noted that such purely topographical descriptions are of little significance for the identification of structures in the central nervous system (cf. Nieuwenhuys, 1966, 1967). This may be illustrated by the fact that in the Mormyridae the enormously hypertrophied valvula has become a superficial structure, which covers all other parts of the brain, inclusive of the tectum mesencephali (Franz, 1911b, 1920; Stendell, 1914; Suzuki, 1932; Figs. 33 and 34). Polyp ter iformes

The cerebellum of the Polypteriformes shows a very unusual configuration. Only its lateral and morphologically most caudal parts are externally visible. The former surround lateral recesses of the fourth ventricle and are homologous to the auricles of the Chondrichthyes*; the latter connects the upper leaves of the auricles and represents, notwithstanding its superficial position, the chondrichthyan ‘lower lip’ (cf. Fig. 23 with Fig. 15). The corpus cerebelli has invaginated into the ventricular system (Fig. 23b). Its caudal and central parts protrude into the fourth ventricle. Its rostra1 part thrusts forward under the tectum. The polypteriform cerebellum as a whole encloses an extension of the extracerebral cavity, and thus shows a most remarkable mirrorlimage of the evaginated cerebellum of the Chondrichthyes (cf. Fig. 23b with Fig. 1% and Fig. 24 with Fig. 18). In most actinopterygians the two halves of the cerebellum show a more or less extensive fusion in the midplane, but the cerebellum of the Polypteriformes maintains

* A more exact determination of the extension of the polypteriform auricles will be attempted after the relations as found in the Chondrostean and HoIostean cerebellum have been dealt with (see p.41).


31

cereb.

I cereb. \

Fig. 23. Dorsal view (a) and median section (b) of the brain of the primitive bony fish Polypterus. The cerebellum has invaginated into the fourth ventricle. Figure a shows a brain from which the choroid roof of the fourth ventricle has been removed, so that the caudal pole of the corpus cerebelli can be seen. auric. = auricula; b.01. = bulbus olfactorius; cereb. = cerebellum; tect.mes. = tectum mesencephali; tel. = telencephalon.

its paired character in the full grown animal. In this group the two halves of the cerebellum are only connected by ependyma and a narrow layer of fibres (Figs. 24 and 25). Microscopically, the polypteriform cerebellum appears to contain a molecular layer, a zone of Purkinje cells and a zone of granule cells, but these structures are not arranged in the usual laminated pattern. The granule cells are concentrated in the lateral parts of the corpus cerebelli ; they surround slit-like, lateral extensions of the extracerebral cavity and bound laterally, dorsally and ventrally the ventricular surface of the corpus cerebelli (Figs. 24 and 25). Medially and dorsally the masses of granule References p. 88-93

32


Fig. 24. A cross section through the caudal part of the cerebellum of Polypterus ornatipinnis. Bodian method. auric. = auricula; cr.cb. = crista cerebellaris; extracerebr.cav. = extracerebral cavity; str.gran. = stratum granulare; str.mo1. = stratum moleculare; str.Purk. = stratum Purkinje.

Fig. 25. A cross section through the middle of the cerebellum of Polypterus ornntipinnis. corp.cb. corpus cerebelli; em.gran. = eminentia granularis; rec.lat. = recessus lateralis ventriculi quarti; IV nervus trochlearis. For other abbreviations see Fig. 24.

= =

cells are covered by a molecular layer. The perikarya of the Purkinje cells form a triangular band which, partly separating the molecular and granule zones, extends through the entire cerebellum (Figs. 24 and 25). Up to the present time the literature on the central nervous system of the Polypteriformes is scanty. The only author who, to my knowledge, has dealt with the cerebellum of this group is Van der Horst (1919, 1925). His views, which in some respects differ from those presented above, may be summarized as follows: (1) In the Polypteriformes the dorsal roots of the anterior lateral line nerves are small. In relation herewith the


33

lobi lineae laterales dorsales are slightly developed and the cerebellar auricles are almost (Pulypterus), or entirely (Culurnoichthys) lacking. (2) The masses of granule cells, lying dorsally to the lateral extensions of the extracerebral cavity (cf. Figs. 24 and 25) represent the eminentiae granulares. (3) The large intraventricular part of the polypteriform cerebellum is homologous to the valvula of the teleosts; only its dorsocaudal portion represents the corpus cerebelli. With regard to this last point it should be noted that in my opinion the question as to whether the polypteriform cerebellum is divisible into a corpus cerebelli and a valvula can only be decided by a study of its afferent connections. So far such a study has not been made.

Chondrostei The cerebellum of the Chondrostei (Figs. 26-28) presents in its general structure a peculiar combination of features reminiscent of the Chondrichthyes, the Polypteriformes and the Teleostei. Its lateral parts are formed by very large auriculae which rostrally terminate in blind sacs (Fig. 28). Medially the auricles fuse with a massive central body which protrudes into the ventricle. As in the Polypteriformes the central body of the chondrostean cerebellum surrounds an extension of the extracerebral cavity, but in the latter this extension is very slight and only represented by a rostroventrally directed transverse groove (Fig. 26c). According to Hocke Hoogenboom (1929) this groove indicates the boundary between the subtectally situated valvula and the corpus cerebelli, hence she termed it the plica valvulae. The caudal part of the auriculae contains a wide molecular layer, which is continuous with the crista cerebellaris (Fig. 27). The granular layer is confined here to a narrow periventricular zone which at places consists of sparsely distributed cells. In the rostral part of the auricles, on the contrary, the layer of granule cells is very wide and only partially covered by a thin molecular zone (Fig. 28). Hocke Hoogenboom (1929) identified an equivalent of the eminentia granularis of the teleosts in the cerebellum of Polyodon. The principal part of this bilateral granular mass lies, according to her observations, in the corpus cerebelli, but its lateral part has become incorporated in the medial wall of the auricle. It has already been mentioned that the central body of the chondrostean cerebellum has invaginated into the rhombencephalic and mesencephalic ventricle. The lateral parts of this body are formed by masses of granule cells, which in the most rostral, and notably in the more caudal parts of the cerebellum protrude into the ventricularcavity (Figs. 26b and 27: prominentiae granulares). In between these masses of granule cells, but extending further rostrally, ventrally and caudally, there is an unpaired zone of molecular substance, which dorsally passes over into the molecular layer of the auricles (Figs. 27 and 28). The auricles as well as the central body contain typical Purkinje cells. A number of these elements lie in rows, close to the granular layer, but others are scattered through the molecular zone~(Johnston,~l901; Hocke Hoogenboom, 1929). Studies on the morphogenesis of the cerebellum of the Chondrostei have not been reported. It seems, however, probable that during development the walls of the two References p . 88-93


34

cereb.

Fig. 26. The brain o he sturgeon. a, Dorsalview of the brain of Acipenser rubicunc...s. _,The roof of the midbrain and the cerebellum of the brain represented in figure a have been removed and inverted to show the prorninentia granularis (proragran). Figures a and b have been drawn from photographs

of Johnston (1901). c, Median section through the brain of Acigenserfulvescens. For abbreviations see Fig. 8.

halves of the cerebellar anlage fold inward, and that the medial surfaces of these halves, thus brought into close apposition, fuse in the midplane (Johnston, 1901). The afferent connexions to the chondrostean cerebellum include : (1) Direct fibres from the vestibular and lateral line nerves. According to Johnston most of these fibres terminate in the auricles (his ‘lateral lobes’), but some enter the corpus cerebelli. (2) Trigemino-cerebellar fibres, reaching the corpus cerebelli (Johnston). (3) A largely uncrossed spino-cerebellar system, which corresponds to the ventral spinocerebellar

35


cell. Purk.

A

st f: mol.

.prom.

CT: cb.

Fig. 27. A cross section through the cerebellar region of Acipenser fulvescens. cell.Purk. = cells of Purkinje; cr.cb. = crista cerebellaris; prom.gr. = prominentia granularis; strmol. = stratum moleculare.

Fig. 28. A cross section through the cerebellar region of Acipenser fulvescens, rostra1 to the plane of Fig. 27. auric. = auricula; corp.cb. = corpus cerebelli; rec.lat. = recessus lateralis ventriculi quarti.

tract of the teleosts (Hocke Hoogenboom, 1929). (4) A tractus mesencephalo-cerebellaris posterior (= bundle y of Johnston). This bundle originates, according to Hocke Hoogenboom, partly from the nucleus lateralis valvulae and partly from other tegmental centres. It passes through a bridge of tissue which connects the tegmentum mesencephali with the valvula, and terminates, largely decussating, in the latter structure. (5) A tecto-cerebellar, and (6) a lobo-( = hypothalamo-) cerebellar system. The former passes (unlike its teleostean equivalent) directly from the lateral margin of the tectum into the valvula; the latter reaches the cerebellum by way of the bridge between the tegmentum and the valvula. Both systems terminate, according to JohnReferences p . 88-93

36


ston, in the valvula, the corpus, and the auricles. The author just mentioned, who investigated the brain of Acipenser with the Golgi method, also reported that all axons entering the cerebellum, save for some tecto-cerebellar fibres, end in the granular layer. The cerebellar efferent fibres constitute two bundles: the tractus cerebello-motorius and the brachium conjunctivum anterius (Hocke Hoogenboom, 1929). The cerebellomotor tract originates probably from the Purkinje cells of the auricles. Its fibres decussate in the raphe and join the fasciculus longitudinalis medialis. The fibres of the brachium conjunctivum pass from the base of the cerebellum ventrally and decussate in the tegmentum of the isthmus region. Their site of termination is not known. Hocke Hoogenboom (1929) found in Polyadon a homologue of the nucleus lateralis cerebelli of the Chondrichthyes, and she thought it probable that the brachium conjunctivum partly arises from this cell mass. Holostei and Teleostei The embryological studies of Schaper (1894a,b: Salmo) have shown that the cerebellum of the teleosts, just as that of other vertebrates, at the beginning of its development consists of a simple transversely oriented plate which, together with the caudal wall of the tectum, bounds a deep fissura rhombo-mesencephalica. Initially the teleostean cerebellar plate shows a slight rostral bend, but soon its dorsal and lateral sides curve caudally, roofing in the rostral part of the fourth ventricle. Ventrally the area surrounding the deepest part of the fissura rhombo-mesencephalica starts to grow rostrally into the ventricle of the midbrain, thus forming the pouch-like anlage of the valvula. Somewhat later the caudal parts of the now curved cerebellar plate grow downward, so that the cerebellar anlage gets a ventricular cavity of its own : the cavum cerebelli primitivum of Schaper. During the morphogenetic processes just sketched the lateral walls of the dorsal part of the cerebellar anlage (i.e. the future corpus cerebelli) thicken and later bulge into the cerebellar ventricle. Finally these bilateral ‘Seitenwiilste’ (Schaper) fuse in the median plane. Through this fusion the teleostean corpus cerebelli becomes a very compact structure (Fig. 32), in which the ventricular cavity is reduced to a small basal recess (Fig. 3I b) and a narrow median canal situated rostrally, dorsally (Fig. 32: ventr.cb.), and caudally to the coalesced ‘Seitenwiilste’. More ventrally the lateral walls of the cerebellar anlage show another thickening. Contrary to the ‘Seitenwulste’, these ventral thickenings project laterally, so that ventrally to the corpus cerebelli a pair of swellings, the eminentiae granulares, appear (Fig. 31a and 32). Caudal to the eminentiae granulares are found the lateral recesses of the fourth ventricle. These recesses are relatively small and do not extend into lateral diverticula of the cerebellum. In other words, externally visible auriculae are not present in the teleosts. The studies of Palmgren (1921) and Pearson (1936) have revealed, however, that small masses of granular cells, most probably representing the auricular gray, surround the teleostean lateral recesses. Moreover, Palmgren and Pearson have shown that, at least in some teleosts, the cell masses related to the lateral recesses are connected


37

Fig. 29. Dorsal view (a) and median section (b) of the brain of Amiu culvu. b.ol. = bulbus olfactorius; cereb. = cerebellum; corp.cer. = corpus cerebelli; emgran. = eminentia granularis; tect.mes. = tectum mesencephali; tel. = telencephalon; valv.cer. = valvula cerebelli.

across the caudoventral border of the cerebellum by a band of granular cells. This band of cells (the pars medialis auriculi of Palmgren; the stratum granulare, pars ventralis of Pearson: Fig. 32), which forms the morphologically most caudal part of the central cerebellar mass, may well be equivalent to the ‘lower lip’ of the chondrichthyan cerebellum. The valvula cerebelli varies considerably in size and shape. In species like Gobius and Lophius it is merely represented by a small subtectal fold, but in most teleosts the valvula occupies a considerable part of the optic ventricle. Its wall is often thrown into folds (Fig. 31b). In the Cyprinidae and the Siluridae the valvula is so strongly developed that it effects a lateral displacement of the caudal parts of the tectum mesencephali. In these forms the valvula can be differentiated into an intermediate portion and two lobi laterales (Franz, 191la). It is these lobi laterales valvulae which in the Mormyridae attain amazing dimensions (Franz, 191lb). In this group the valRefQrencQsp . 88-93

38


I _ _

--

str. gran.

st. r m o

em.gran.

V

a

str. mol. str. Purk. s t r gran.

auric . gr.

b

Fig. 30 a, Transverse section through the brain of Amiu culvu at the level of the cerebellum. WeigertPal. b, Semidiagrammatic drawing of a transverse section through the cerebellar region of Lepisosteus osseus. corp.cb. = corpus cerebelli; em.gran. = eminentia granularis; rec.x.,y. = recesses of the fourth ventricle discussed in the text; str.gran. = stratum granulare; str.mo1. = stratum moleculare; str.Purk. = stratum Purkinje; ventr.cb. = ventriculus cerebelli.

vula grows out of the ventricle of the midbrain and becomes a superficially situated structure which, as has already been mentioned, may cover all other parts of the brain. Reference to the Figs. 33 and 34 shows that the valvula of the mormyrids is a much folded structure, whose outer surface is considerably enlarged by the formation of fine transverse ridges. These ridges are only visible in that part of the valvula that

ANATOMY OF T H E CEREBELLUM

39

covers the caudal part of the brain; its rostra1 portion has developed in such a fashion that its originally internal surface has become a part of the outer, dorsal surface of the brain (Fig. 34). Reference to Fig. 35b shows the remarkable fact that, microscopically, the ridges of the mormyrid valvula only comprise the molecular layer and the Purkinje cells with some surrounding elements. The granular layer does not participate in these superimposed folds. A description of the earlier phases of the morphogenesis of the brain of the mormyrid Gymnarchus has been given by Assheton (1907). There is evidence that the development of the valvula is proportionate to the differentiation of the lateral line system (cf. Berkelbach van der Sprenkel, 1915; Lissman, 1958).

cor p. cer:

Fig. 31. Dorsal view (a) and median section (b) of the brain of Sulmofurio. For abbreviations see Fig. 29.

The size of the corpus cerebelli, which also shows considerable variations, is said to be related to the complexity of locomotory performance. In most teleosts the corpus cerebelli has a bend in the caudal direction (Fig. 31b), but in the Siluridae it extends Refirences p. 88-93

40


forward over the tectum (Fig. 36). Aronson (1963) found in Acunthurus and in some other species a particularly strongly developed corpus cerebelli which, projecting rostrally, does not only cover the tectum, but also a part of the forebrain. The development of the corpus cerebelli attains, however, its greatest height in the mormyrids. In this group the corpus cerebelli is convoluted and differentiated into several lobes (Fig. 34). The relations in the holostean cerebellum (Ariens Kappers, 1907; Pearson, 1936; Fig. 29) correspond essentially to those described for the teleosts. The following differences, some of which clearly reflect a less specialized condition, should, however, be noted. (1) The concrescence of the Seitenwulste in the corpus cerebelli is more restricted and the valvula is smaller than in most teleosts (cf. Fig. 29b with Fig. 31b). (2) Whereas in the teleosts the cell mass in the eminentia granularis is generally adjacent to the lateral surface of the brain (Fig. 32), its holostean homologue is largely covered by a molecular layer (Fig. 30). (3) In the holostean cerebellum there are lateral recesses of the fourth ventricle which in every respect correspond to those found in the teleosts (Fig. 30b: rec.x.), but there are, in addition, two more dorsally situated ventricular recesses (Fig. 30b:

Fig. 32. A transverse section through the cerebellum and medulla oblongata of Sulmofurio. em.gran. = eminentia granularis; str.gran.p.princ. = stratum granulare, pars principalis; str.gran.p.ventr. = stratum granulare, pars ventralis; str.mol.p.princ. = stratum moleculare, pars principalis; str.mo1.p. ventr. = stratum moleculare, pars ventralis; str.Purk. = stratum Purkinje; ventr.cb. = ventriculus cerebelli.


41

rec.y.). The latter extend into the eminentiae granulares and give these structures the appearance of rostrolaterally directed evaginations (Fig. 30b). In Lepisosteus they are more pronounced than in Amia. Space does not permit here a detailed comparative consideration of the interesting and still not entirely clarified relations in the lateral parts of the actinopterygian cerebellum. Suffice it to say that my observations, and especially the facts mentioned above under 2 and 3, have led me to the following tentative conclusions : (1) The cerebellar tissue covering and partly surrounding recess x (Fig. 30b) of the holostean fourth ventricle corresponds to the caudal part of the auricula of the Chondrostei. (2) The holostean eminentia granularis represents the entire rostra1 portion of the chondrostean auricula, and not, as Hocke Hoogenboom (1929) believed, only its rostromedial wall (cf. Fig. 28 with Fig. 30b). (3) In the teleosts both the small mass of granular cells surrounding the lateral recess and the non-diverticular eminentia granularis together constitute the homologue of the large auricles of the Chondrostei and the Chondrichthyes. This interpretation is at variance with that given by Pearson (1936), but tallies so far as the eminentia granularis is concerned with that of Herrick (1924). It has, however, already been mentioned that the supposition of the latter that the teleostean auricles initially contain lateral recesses, which are later filled with granular cells, has not received embryological support. (4) In view of the relations found in the Chondrostei and the Holostei it seems to me that in the Polypteriformes, auricles are not only present, but are even extremely large. I believe that in this group the auricles contact each other in front of the ‘lower lip’, above the ventrally displaced corpus cerebelli (cf. here Fig. 25 with Figs. 28 and 30). This interpretation implies that the mass of granular cells situated dorsally to the intracerebellar extension of the cranial cavity probably represents the eminentia granularis, as has been supposed by Van der Horst (1925). With regard to the histology of the holostean and teleostean cerebellum it may be stated that granular, molecular and Purkinje cell layers can be distinguished in the corpus as well as in the valvula cerebelli. The same layers can also be recognized in the holostean eminentia granularis, but the teleostean homologue of this structure adheres more strictly to its name and consists only of granular elements. The granular layer occupies a central position in th‘e corpus cerebelli. In the holostean the cerebellar ventricle separates it into bilateral halves (Fig. 30); but due to the elaborate fusion of the ‘Seitenwiilste’ already discussed, it appears in most teleosts as a single mass of cells (Fig. 31). Laterally the granular layer of the corpus cerebelli is continuous with the eminentia granularis (Fig. 32), and rostrally it passes over into the granular zone of the valvula. Pearson (1936) divided the granular layer of the central part of the holostean and teleostean cerebellum into a pars principalis and a pars ventralis. The former constitutes, as its name implies, the main portion of the cerebellar gray; the latter consists of a relatively narrow band of periventricular cells, situated directly in front of the attachment of the tela chorioidea. In the holosteans the most caudal part of the cerebellar ventricular surface is recurved dorsally (Fig. 29b), References p. 88-93

42


Fig. 33. Dorsal view of the brain of Mormyrus cushive. The only visible structure is the enormously hypertrophied valvula cerebelli, which covers all other parts of the brain. Magn. 5 x .

A N A T O M Y OF THE CEREBELLUM

43

Fig. 34. A sagittal section through the brain of Petrocephalus bovei. Bodian method. corp.cb. = corpus cerebelli; dienc. = diencephalon; 1ob.lin.lat. = lobus lineae lateralis; str.gran. = stratum granulare; strmol. = stratum moleculare; str.Purk. = stratum Purkinje; valv.cb. = valvula cerebelli.

and hence the pars ventralis of the stratum granulare forms the most caudal part of the cerebellar gray. In most teleosts, however, due to the caudal bent of the corpus cerebelli, the pars ventralis of the granular layer is situated ventrally to the main mass of granular cells (cf. Fig. 31b with Fig. 32). It has already been mentioned that the pars ventralis of the stratum granulare is rostrolaterally continuous with small masses of gray adjoining the lateral recesses of the fourth ventricle, and probably corresponds to the ‘lower lip’ of the chondrichthyan cerebellum. The afferent and efferent fibres of the cerebellum traverse the granular layer, in which they constitute compact bundles of considerable size (Fig. 32). Some of these bundles may pass close to the ventricular surface, but a continuous deep cerebellar fibre layer, as present in the higher vertebrates, is not found in the holostean or teleostean cerebellum. Golgi studies (Schaper, 1893; Franz, 191la) have shown that the granule cells of teleosts closely resemble those of other vertebrate groups. They are provided with a few short, claw-like dendrites, and their axons enter the molecular layer, where they dichotomize and form the parallel fibres. In addition to small granule cells the superficial zone of the granular layer contains a number of large irregularly shaped cells (Schaper, 1893). These elements, which probably correspond to the large Golgi cells of higher vertebrates, extend their dendrites into the molecular layer. Their axons ramify and become lost among the granular cells. The Purkinje cells are at many places arranged in several layers. In the holosteans these elements are smaller and placed less regularly between the granular and molecular layers than in the teleosts (Figs. 30b and 35a). As in the Chondrichthyes the Purkinje cell dendrites of the teleosts are oriented in a sagittal plane. The branching of these dendrites is, however, in the latter group generally much more complex than in the former (Fig. 63b,c). References p . 88-93

44


It is interesting to note that in the cerebellum of mormyrids the Purkinje cell dendrites show a remarkably regular arrangement, being oriented rigorously perpendicular to the external cerebellar surface (Figs. 35a,b). The axons of the Purkinje cells usually pass over some distance parallel to the cerebellar surface, before joining the fibre bundles which traverse the granular layer. Franz (191 la) observed that some Purkinje cell axons terminate in the most superficial part of the granular layer, and he believed that these fibres synapse with other Purkinje cells. Catois (1901) traced neuraxes of Purkinje cells directly into the brachium conjunctivum. The molecular layer is divisible into a pars principalis and a pars caudalis, which parts cover externally the portions of the granular layer of the same name (Fig. 32). In the teleostean molecular layer two types of cells have been found (Schaper, 1893): small stellate elements, whose axonal connexions have not been established as yet, 1

Fig. 35a.


45

Fig. 35b.

Fig. 35. The cerebellum of the rnorrnyrid Petrocephalus bovei. Details from the section represented in Fig. 34; (a) corpus cerebelli; (b) valvula cerebelli. gr. = granular layer; m. = molecular layer; P = Purkinje cell layer. References p. 88-93

46


and larger fusiform cells, whose axons and dendrites are oriented in a sagittal plane. These fusiform elements are provided with two extremely long main dendrites which extend forward and backward, parallel to the cerebellar surface. The axons of these cells send out a number of collaterals toward the Purkinje cell layer. It is believed that the cells under discussion represent forerunners of the basket cells of higher forms, but it has not actually been observed that their axon terminals do form pericellular entanglements around the perikarya of the Purkinje cells (Schaper, 1893, 1898). The afferent fibre systems of the teleostean cerebellum form three different types of endings : (1) mossy fibres in the granular layer, (2) climbing fibres around the proximal parts of the Purkinje cell dendrites, and (3) terminals which branch freely in the molecular layer. The endings mentioned under 2 and 3 were first described by Schaper (1893). Several authors have identified a primordium of the deep cerebellar nuclei of higher vertebrates in the teleostean brain. Catois (1901) described a nucleus, situated at the base of the cerebellum, which contributes fibres to the brachium conjunctivum.

v\ t r . m.cb.ant.

valvsb.

em

Fig. 36. A diagrammatic representation of the cerebellar afferent systems in a teleost (Ameiurus). Redrawn from Herrick (1924). a.ac. = areaacustico-lateralis(=area octavo-lateralis);aq. = aqueductus mesencephali; corp.cb. = corpus cerebelli; cr.cb. = crista cerebellaris; em.gr. = eminentia granularis; n.lat. = nervus lateralis; tect. = tecturn mesencephali; torus = torus semicircularis; tr.mes.cb.ant. = tractus rnesencephalo-cerebellarisanterior; tr.rnes.cb.p. = tractus rnesencephalocerebellaris posterior; tr.sp.cb. = tractus spino-cerebellaris; valv.cb. = valvula cerebelli.


47

Likewise, Franz (191la) observed that the cerebello-tegmental fibre system partly originates from a group of diffusely arranged fusiform cells, lying at the site of junction of the cerebellum and the medulla oblongata. Pearson (1936) finally found in the holostean Amiu as well as in teleosts laterally to the ventricle a distinct nucleus cerebelli, giving rise to cerebello-tegmental and cerebello-motor fibres. With regard to this cell mass Pearson (p. 262) stated : ‘Its position, partly at the base of and partly within the peduncle, marks the beginning phylogenetically of the inclusion of the cerebellar nuclei within the cerebellum proper’. Data on the fibre connexions of the holostean cerebellum are found in the studies of Ariens Kappers (1907) and Pearson (1936). The cerebellar fibre systems of the teleosts have been considered by a great number of authors, among whom Franz (191la,b), Stendell (1914, mormyrids), Ariens Kappers (1921), Addison (1923), Tuge (1934, 1935), and Ariens Kappers et ul. (1936) may be especially mentioned. The following brief survey of the pathways related to the holostean and teleostean cerebellum is primarily based on the literature quoted. The afferent connexions (Fig. 36) include: (1) Primary fibres of the anterior and posterior lateral line nerves, which reach the eminentia granularis and the valvula cerebelli. A certain number of these fibres decussate in the rostra1 part of the corpus cerebelli to be distributed to the contralateral side of the valvula. Other root fibres of the lateral line nerves constitute together with bulbo-cerebellar fibres (see 4) a commissure in the pars ventralis of the stratum moleculare, i.e. in the morphologically most caudal part of the cerebellum. This commissure corresponds probably to the commissura octavo-lateralis, which has been described by Larsell (1931, 1932a, 1947a) for cyclostomes and urodeles. (2) Primary octavus fibres, which terminate in the eminentia granularis and possibly in the valvula. (3) A tractus bulbo-cerebellaris (Pearson, 1936) or tractus nucleo-cerebellaris (Ariens Kappers, 1907). This system connects the area octavo-lateralis with the eminentia granularis. According to Pearson (1936) its fibres reach, in addition, the corpus and the valvula cerebelli. (4) A tractus trigemino-cerebellaris. The fibres of this system originate from the nucleus descendens nervi trigemini and accompany the spino-cerebellar and bulbocerebellar tracts to the cerebellum. Woodburne (1936) observed that a number of secondary trigemino-cerebellar fibres decussate in the raphe and join the contralateral spino-cerebellar tract. Pearson (1936) traced some root fibres of the trigeminal nerve to the commissura cerebelli (see below), but he remained unable to establish their site of termination. (5) A tractus spino-cerebellaris. This tract constitutes a large bundle of fibres which passes rostrally along the lateral periphery of the spinal cord and the medulla oblongata. At the level of the trigeminal nerve its fibres arch dorsally in a broad curve and reach the cerebellar peduncle. The fibres of this system end in the ipsilateral and contralateral halves of the corpus cerebelli and, according to Pearson (1936), also in the eminentia granularis and in the valvula. The contralateral spino-cerebellar fibres decussate in the rostrobasal part of the corpus cerebelli and form part of a complex commissural system, known as the commissura cerebelli. Apart from spino-cerebellar References p . 88-93

48


fibres this commissure contains bulbo-cerebellar fibres and true commissural fibres interconnecting the eminentiae granulares, the nuclei cerebelli and the two halves of the corpus cerebelli (Pearson, 1936). (6) A tractus olivo-cerebellaris. Ariens Kappers (1921) and Addison (1923) observed in teleosts a small olivo-cerebellar tract, which after total decussation in the medulla oblongata joins the spino-cerebellar system. (7) A tractus mesencephalo-cerebellaris anterior or tractus tecto-cerebellaris. This tract originates, according to Addison (1923), from the region where the anterior border of the tectum opticum, the torus longitudinalis and the tegmentum come together. Burr (1928) believed that in M d a this system arises principally from the pretectal nucleus and hence he called it tractus pretecto-cerebellaris. Pearson (1936) stated that the majority of the fibres of the tractus mesencephalo-cerebellaris anterior can be traced from the efferent layer of the tectum. The system under discussion passes caudally through the tegmentum of the midbrain and enters the cerebellum through the valvula cerebelli (cf. Fig. 36). Its fibres distribute to the structure last named and to the body of the cerebellum. In Amiu the tecto-cerebellar tract is made up of a lateral and a medial portion. The lateral portion runs through the tegmentum of the midbrain and corresponds to the system present in teleosts. The medial portion, however, passes directly from the tectum to the cerebellum by way of the velum medullare anterius (Pearson, 1936). The tractus mesencephalo-cerebellaris anterior is large in species with a highly developed optic system, and is believed to mediate visual impulses toward the cerebellum (Franz, 191l a ; Addisson, 1923; Burr, 1928; Ariens Kappers et aZ., 1936). It is, however, worthy of note that Charlton (1933) found a distinct anterior mesencephalo-cerebellar tract in blind cave fishes. (8) A tractus mesencephalo-cerebellaris posterior or tractus tegmento-cerebellaris. This tract, which originates largely from the nucleus lateralis valvulae in the tegmentum of the midbrain, joins the tractus mesencephalo-cerebellaris anterior, and passes with this system to the valvula and the corpus cerebelli. It has been pointed out already that there is a positive correlation between the size of the tractus mesencephalo-cerebellaris posterior and the development and differentiation of the lateral line system. (9) A tractus lobo-cerebellaris, which passes from the posterior part of the lobus inferior hypothalami toward the corpus cerebelli. The efferent system of the holostean and teleostean cerebellum comprises four components, namely the tractus cerebello-tectalis, the brachium conjunctivum, the tractus cerebello-motorius, and the tractus cerebello-octavo-lateralis (cf. Fig. 37). The cerebello-tectal tract passes frontalward through the tegmentum and enters the rostra1 part of the tectum mesencephali. The brachium conjunctivum (= tractus cerebello-tegmentalis mesencephalicus of Tuge, 1935) forms a conspicuous decussation in the raphe and terminates in the nucleus reticularis mesencephali (which may contain a primordium of the nucleus ruber), in the oculomotor nucleus, and probably in the nucleus of the fasciculus longitudinalis medialis. Goldstein (1905) and some others traced fibres from the brachium conjunctivum toward various diencephalic centres, but Tuge (1935) concluded from his Marchi experiments that no fibres of

49


Fig. 37. Diagram showing the cerebellar efferent pathways in the teleost Curussius uurufus.area acust. = area acustico-lateralis (= area octavo-lateralis); f.1.m. = faeciculus longitudinalis rnedialis; lob. fac. = lobus facialis; 1ob.vag. = lobus vagi; nuc.f.1.m. = nucleus of the fasciculus longitudinalis medialis; nuc.rnot.teg.ant. = nucleus motorius tegmenti anterior; nuc.rnot.teg.post. = nucleus rnotorius tegrnenti posterior; nuc.ret.rnes. = nucleus reticularis rnesencephali; nuc.rub. = nucleus ruber; s m . = somatic musculature; torus sern. = torus semi-circularis; valv.cereb. = valvulacerebelli; 1 = tractus cerebello-tectalis; 2 = tractus cerebello-tegrnentalis mesencephalicus; 3 = tractus cerebellotractus tegrnentalis bulbaris anterior; 4 = tractus cerebello-tegmentalis bulbaris posterior; 5 cerebello-acustico-lateralis.(H.TUGE,(1935); J . csmp. Neurol., 61,360, Fig. 5.) :

this system reach farther rostrally than the midbrain. The tractus cerebello-motorius (= tractus cerebello-tegmentalis bulbaris pars rostralis and pars caudalis of Tuge, 1935; cf. Fig. 37) originates from the valvula as well as from the corpus cerebelli and distributes to various bulbar centres of the same and opposite side. After leaving the cerebellum the fibres of this tract pass medially close to the ventricular floor and reach the motor zone of the tegmentum. Here many of its fibres turn caudally and run in or near the fasciculus longitudinalis medialis toward their destination. According to Tuge (1935) the fibres of the cerebello-motor tract terminate in the nucleus motorius tegmenti, in the nucleus raphes, in the trochlear nucleus, in the motor facial nucleus, and possibly also in the abducens nucleus and in the motor trigeminal nucleus. The tractus cerebello-octavo-lateralis, finally, passes through the cerebellar crest and distributes, as its name implies, to the area octavo-lateralis (Tuge, 1935). Another cerebello-octavo-lateral connexion has been described by Pearson (1936) who traced a number of cerebello-motor fibres toward the ventral nucleus of the octavo-lateral complex. References p . 88-93

50


rec. la t.

++

cereb

Fig. 38. Dorsal view (a) and median section (b) of the brain of the lungfish Protopterus annectens. Modified from Burckhardt (1892). auric. = auricula; cereb. = cerebellum;rec.lat. = recessus lateralis ventriculi quarti; tect.mes. = tectum mesencephali;tel. = telencephalon.

With regard to the exact origin of the cerebellofugal fibres it has already been mentioned that Purkinje cell axons as well as fibres arising from the nucleus cerebelli have been traced into the efferent cerebellar pathways. However, no data are available concerning the relative number of fibres in these two categories. Tuge (1935) and Ariens Kappers et al. (1936) regarded it probable that most of the cerebellofugal fibres originate directly from Purkinje cells.

Dipnoi The dipnoan cerebellum is relatively simply built as compared to that of the other groups of fish. In the African lungfish, Protopterus, it consists of two large auriculae, whose upper leaves pass over into a plate-like corpus cerebelli. A median ventricular furrow betrays the bilateral origin of the latter (Figs. 39 and 40). The auriculae are prominent and overhang the lateral sides of the midbrain, but the corpus cerebelli is


lob. lin. fat

51

an

Fig. 39. A cross section through the medulla oblongata, the cerebellum and the tectum ofthelungfish Protopterus dolloi. Bodian method. cr.cb. = crista cerebellaris; corp.cb. = corpus cerebelli; 1ob.lin. 1at.ant. = lobus lineae lateralis anterior; N.1at.ant.d. = nervus lateralis anterior dorsalis; N.1at.ant.v. = nervus lateralis anterior ventralis; rec.lat. = recessus lateralis ventriculi quarti; tectmes. = tectum mesencephali.

n.mes.V

3t.

Fig. 40. A cross section through the brain ofthe lungfish Protopterus dolloi, slightly rostral to Fig. 39. auric. = aricula; n.mes.V. = nucleus mesencephalicus nervi trigemini; r.Vd. = ramus descendens nervi trigemini; strmol. = stratum moleculare; str.Purk. = stratum Purkinje. For other abbreviations see Fig. 39.

largely covered by the tectum (Figs. 38-40). Save for a stronger development of the corpus, the cerebellum of Protopterus shows in its overall structure a striking resemblance to that of the urodeles (cf. Fig. 38 with Fig. 45). In the Australian lungfish, Neoceratodus (Holmgren and Van der Horst, 1925) the auricles are smaller, but the corpus cerebelli is much larger than in Protoperus. The structure last named is here not covered by the tectum, and its curved wall caps the rostral part of the fourth ventricle. In this respect the cerebellum of Neoceratodus reminds us somewhat of that of the turtle (cf. Fig. 50A). The middle portion of the corpus is thickened in the Australian lungfish and forms a ‘median cerebellar ridge’ References p . 88-93

52


Fig.41.Sagittalsectionthroughthecerebellumofthelungfish Protopterusdo1loi.Bodian method.gr= granular celllayer; m = molecular layer; P = Purkinje celllayer; t = tectum mesencephali.

(Holmgren and Van der Horst), which protrudes into the ventricle. Microscopically, the three characteristic layers - the molecular, the Purkinje and the granular -can be identified in the dipnoan cerebellum (Figs. 40 and 41). It should,

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53

however, be noted that the Purkinje cells are rather irregularly distributed. In some parts of the cerebellum they are quite numerous, but in others (the paramedian zones, the most caudal region) they are entirely lacking. Where present the Purkinje elements tend to form a zone separating the granular and the molecular layers. Many of them lie, however, scattered through the two layers last mentioned. The auricles of Protopterus consist largely of crowded granular cells, but in Neocerutodus these structures contain distinct granular and molecular layers. The presence of Purkinje cells in the auricles has not been reported. The fibre connections of the cerebellum of Neocerutodus have been studied by Hormgren and Van der Horst (1925). These authors described the following afferent systems: (1) ‘A large bundle that may consist of direct or indirect lateralis and perhaps octavus fibres’ (Z.C., p. 125). Some of the fibres of this bundle reach the auricle, but most of them spread in the cerebellum in front of the auricle. From this observation Holmgren and Van der Horst (1925, p. 125) concluded : ‘So the small auricles are not of so great importance for the lateralis connections in Cerutodus as they are in Selachians according to Voorhoeve and also in the frog according to Larsell’. (2) Trigemino-cerebellar fibres, which enter the corpus cerebelli together with (3) spinocerebellar fibres. A certain proportion of these fibres decussate toward the contralateral side. (4) Diffusely arranged tecto-cerebellar fibres. As regards the efferent cerebellar connections, Holmgren and Van der Horst mention only the presence of a brachium conjunctivum. This bundle originates, according to their observations, from a laterally situated nucleus, which projects into the ventricle. They thought it probable that this nucleus is homologous to the nucleus lateralis cerebelli of the Chondrichthyes and to the nucleus cerebelli of the frog. The brachium consists of small fascicles which pass rostrally and ventrally close to the ventricular wall. Holmgren and Van der Horst noted that this system distributes to the tegmentum of the midbrain. A decussation of the brachia was not found and the site of termination of their fibres could not be established.

Fig. 42. The brain stem and cerebellum o f the crossopterygianLatimeria chalumnae midsagittallycut and seen from the ventricular side. Based on a photograph in Millot and Anthony (1956). corp.cb. = corpus cerebelli;tect.mes. = tectum mesencephali. References p . 88-93

54


Crossopterygii The work of Millot and Anthony (1956, 1965) has revealed that the cerebellum of the sole surviving crossopterygian Latimeria is well developed, and can be clearly differentiated into a pair of auriculae and a central corpus cerebelli (Figs. 42-44). The corpus cerebelli has evaginated dorsally and encloses a rather narrow ventricular cavity. Its caudal wall passes over into a horizontally oriented lamella, which is most probably equivalent to the ‘lower lip’ of the chondrichthyan cerebellum (Fig. 42). The auriculae are extremely large in Latimeria, a fact which is doubtless related to

auric.

Fig. 43. A diagrammatic cross section through the medulla oblongata and the cerebellum of the crossopterygian Latimeria chalumnae. Based on a photograph in Millot and Anthony (1956). a u k . = auricula; corp.cb. = corpus cerebelli; cr.cb. = crista cerebellaris; str.gran. = stratum granulare; strmol. = stratum moleculare; str.Purk. = stratum Purkinje.

rec.

auric.

Fig. 44.A schematic cross section through the medulla oblongata and the cerebellum of the crossopterygianLatimeriachalumnae,rostralto theplane of Fig. 43. Based on a photograph in Millot and Anthony (1956). auric. = auricula; corp.cb. = corpus cerebelli; rec.lat. = recessus lateralis ventriculi quarti; ventr.cb. = ventriculus cerebelli.


55

the strong development and high degree of differentiation of the lateral line system. The upper leaves of the auriculae are mediocaudally continuous with the corpus cerebelli. Their lower leaves pass over into the lobi lineae laterales. The auriculae contain extensive lateral recesses which, just as in the Chondrichthyes, are laterally bounded and closed by an extension of the choroid roof of the fourth ventricle (Figs. 43 and 44). Microscopically the corpus as well as the auriculae of the latimerian cerebellum appear to contain distinct granular and molecular layers. In the corpus cerebelli these two layers are separated by a remarkably regular layer of Purkinje cells. In the auriculae the presence of Purkinje cells has also been demonstrated, but here these elements are smaller and less regularly arranged than in the corpus cerebelli (Millot and Anthony, 1965). Amphibia Urodela The cerebellum of the urodeles consists of a medial band- or ridge-like corpus cerebelli and two lateral auricular lobes (Figs. l c and 45). Numerous authors, among them Kingsbury (1895), Herrick (1914, 1924), Larsell(l931, 1932a), have commented upon the simple structural relations of the urodelan cerebellum, and the statement of Herrick (1924) that the cerebellum of lower urodeles resembles that of the lamprey more nearly than that of fish has been repeated many times in the literature. However, in fact it is the cerebellum of the dipneumonian lungfishes (i.e. Protopterus and Lepidosiren) which approaches the urodelan condition most closely (cf. Figs. 8a, 38a and 45:). The gross morphology of the parts of the urodelan cerebellum can best be described in their relation to the recessus lateralis rhombencephali. Reference to Fig. 45a shows that this recess is very extensive in Necturus, and this feature prevails in all urodeles. In many species the lateral recess extends rostrally, lateral to the midbrain, forming a blind pouch, the diverticulum anterius. This elongation of the lateral recess is marked in Siren (Rothig, 1927), Proteus (Kreht, 1931) and in Triturus (Larsell, 1931), but only slight in Salamandra (Kreht, 1930). The nervous tissue which constitutes the rostrolateral wall and the rostral part of the bottom of the lateral recess represents the auricula. Caudally the structure last named passes insensibly over into the area octavolateralis, which also forms part of the wall of the lateral recess (Fig. 45a). In most urodeles the roof of the lateral recess is entirely membranous (Fig. 46). However, in Necturus (Herrick, 1914) the rostral end of the diverticulum anterius is completely surrounded by nervous tissue. The lamina of nervous tissue which forms the anteromedial wall of the lateral recess represents the corpus cerebelli. This structure arches around the tectum mesencephali, to which it is attached by a short velum medullare anterius. Laterally the corpus cerebelli is fused with the auricular lobes, and ventrally it passes over into the bulbar tegmentum (Fig. 46). In species considered as ‘lower’ urodeles (e.g. Amphiuma, Necturus) the corpus cerebelli is clearly a paired structure; that is to say, no cerebellar tissue is present in the mid-dorsal plane, except a band of References p. 88-93

56


tec t

Fig. 45. Dorsal view (a) and median section (b) of the brain of the urodele Necturus. Modified from Kingsbury (1895). ar.oct.lat. = area octavo-lateralis; dien. = diencephalon. For other abbreviations see Fig. 38.

commissural fibres. In ‘higher’ urodeles, like Ambystoma, however, the bilateral halves of the corpus cerebelli have fused. The neurons of the urodelan cerebellum, like those of the other parts of the brain, constitute a layer of periventricular gray. A stratum album or molecular layer overlies the entire external surface of the cerebellum, and continues caudolaterally into the superficial neuropil zone of the area octavo-lateralis (i.e. the equivalent of the cerebellar crest of fishes). In the auricular lobe as well as in the corpus cerebelli the central gray contains cells of two types, namely small, granule cells, and larger elements which are usually considered as primitive Purkinje cells. As in all vertebrates the


/

57

Com.vest. l a t

carp. cb

tr

Fig. 46. A cross section through the medulla oblongata and the cerebellumof the urodele Ambystoma tigriizum. Redrawn from Herrick (1948). com.cb. = commissuracerebelli; com.vest.lat. = commissu-

ra vestibulo-lateralis; corp.cb. = corpus cerebelli; n.cb. = nucleus cerebelli; n.lat.ant. = nervus lateralis anterior; rec.lat. = recessus lateralis ventriculi quarti; tr.sp.cb. = tractus spino-cerebellaris.

granule cells posses a few tortuous dendrites. Their axons ascend toward the molecular layer, where they give rise to typical parallel fibres (Larsell, 1932a). The primitive Purkinje cells form in Ambystoma (Larsell, 1920) and in Salamundra (Kreht, 1930) a fairly regular layer, but in most urodeles they are scattered in the outer zone of the central gray. The simple dendritic trees of the primitive Purkinje cells, which are not clearly arranged in a single plane, extend into the molecular layer. It is believed that many of the Purkinje cell axons contribute directly to the efferent cerebellar pathways. The molecular layer contains, apart from parallel fibres and Purkinje dendrites, some small stellate cells. Elements comparable to the basket cells of higher vertebrates have not been reported. The fibre connexions of the urodelan cerebellum show a great resemblance to those of fish. The auricles receive (cf. Figs. 4 and 47) : (1) Root fibres of the octavus nerve. Larsell (1931) has pointed out that in Triturus this is the largest and most important fibre bundle related to the cerebellum. (2) Root fibres of the lateral line nerves (Herrick, 1914; Larsell, 1920, 1931; Rothig, 1927). (3) Bulbo-cerebellar fibres, originating from the gray of the area octavo-lateralis (Larsell, 1920; Rothig, 1927). A number of fibres of this system pass with the connexion known as the tractum B of Kingsbury (1895) toward the auricle (Larsell, 193I). It should be noted that, in addition to the lateral line and vestibular stimuli relayed over the systems just mentioned, the auricle probably also receives muscle sense and References p. 88-93

58

R. NIEUWENHUYS

Fig. 47. Diagram showing the afferent connexions of the cerebellum in a urodele. com.cb. = commissura cerebelli; com.vest.lat. = commissura vestibulo-lateralis;f.po.lat. = fissura postero-lateralis; L.AUR. = lobus auricularis; N.L. = nervus lateralis;n.cb. = nucleus cerebelli;tr.lobo.-cb.= tractus lobo-cerebellaris; tr.spino-cb. = tractus spino-cerebellaris; tr.tect.cb. = tractus tecto-cerebellaris, tr.tegm.cb. = tractus tegmento-cerebellaris.

related stimuli, by way of the spino-cerebellar and spinc-tectal tracts. Larsell (1932a) observed that in Ambystoma cells of the auricles send their dendrites into the region of these fibre systems. The auricles are interconnected by a commissure that passes through the dorsal part of the cerebellar plate. This is the commissura lateralis (Herrick, 1914) or the commissura vestibulo-lateralis (Larsell, 1931, 1957). Apart from the true commissural fibres already indicated this system contains some direct vestibular root fibres, and secondary fibres of the lateral line system (Herrick, 1914, 1948; Larsell, 1931, 1932a). In the introductory part of this paper it has already been mentioned that small bilateral strands of cells accompany the vestibulo-lateral commissure along the caudolateral border of the cerebellar plate, and it was pointed out that Larsell termed these strands of cells, together with the auriculae, the lobus vestibulo-lateralis or lobus auricularis. Larsell considered this lobe as the primordium of the lobus flocculonodularis of higher vertebrates. In Triturus and other urodeles a transverse external groove, believed to be the precursor of the fissura postero-lateralis, demarcates the medial parts of the auricular lobe from the corpus cerebelli (Larsell, 1937; Fig. 47). The urodelan corpus cerebelli receives four principal groups of fibres, namely : (1) direct and secondary trigeminal fibres (Herrick, 1948); (2) spino-cerebellar fibres; (3) tecto-cerebellar fibres, which arise from the nucleus posterior tecti, an undifferen-


59

tiated primordium of the inferior colliculus (Herrick, 1925; Rothig, 1927; Larsell, 1931, 1932a); and (4) secondary fibres from the area octavo-lateralis (Larsell, 1920, 1957). A hypothalamo-cerebellar or mammillo-cerebellar tract is probably also present (Herrick, 1914, 1948; Larsell, 1920, 1931). A compact fascicle of well-myelinated fibres connects the bilateral halves of the corpus cerebelli. This fascicle, the commissura cerebelli (Figs. 46 and 47), contains (1) spino-cerebellar fibres; (2) fibres originating from the superior sensory trigeminal nucleus; the majority of these fibres form an intertrigeminal commissure, but others terminate in the corpus cerebelli; (3) fibres connecting the two sides of the corpus cerebelli (Rothig, 1927; Herrick, 1930); and (4) some direct ascending trigeminal root fibres. The efferent system of the urodelan cerebellum consists of internal arcuate fibres which, arising from both the corpus cerebelli and the auriculae, pass forward, downward, and backward in the tegmentum (Herrick, 1914, 1930; Larsell, 1920, 1931). Most of these fibres are in dispersed arrangement; only those which are directed farthest forward form a compact bundle. This bundle, which decussates in the floor of the midbrain, represents the brachium conjunctivum. The remainder of the efferent fibres is believed to provide direct and crossed connections with the rhombencephalic reticular formation (tractus cerebello-tegmentalis) and with motor cranial nerve nuclei (tractus cerebello-motorius, Rothig, 1927; Kreht, 1930; Ariens Kappers et al., 1936). The efferent cerebellar path is in part directly provided by Purkinje cell axons, but Herrick (1914) observed that a number of fibres of the brachium conjunctivum arise from a small ventricular eminence, the eminentia ventralis cerebelli, situated in the borderland of the corpus cerebelli and the tegmentum. On account of the origin of these fibres Herrick considered the ventral cerebellar eminence as the primordium of the deep cerebellar nuclei of higher vertebrates. Later, Herrick (1948) designated the neurons in this eminence as the ‘nucleus cerebelli’ (Fig. 46: n.cb.), although these elements cannot be demarcated from the general ventricular gray in which they are embedded. The cells of the eminentia ventralis cerebelli are in synaptic relation with the tractus spino-cerebellaris, with ascending fibres of the area octavo-lateralis, and with secondary fibres of the sensory trigeminal nucleus (Larsell, 1929; Ariens Kappers et al., 1936). Larsell (1923) was under the impression that in anurans numerous Purkinje cell axons terminate in the ‘nucleus cerebelli’, but so far such relations have not been described for urodeles.

Anura The anuran cerebellum shows the same general features as that of the urodeles. The corpus cerebelli is, however, much more massive (Figs. 48 and 49), and its constituent tissue elements are more highly differentiated (Larsell, 1923). This applies in particular to the Purkinje cells, which constitute a distinct zone in between the granular and molecular layers. The dendrites of the Purkinje cells are clearly oriented in a sagittal plane and show a more complex ramification than those of the urodeles (cf. Cajal, 1911). Larsell(1923) observed that the secondary and tertiary dendritic branches of the Purkinje cells are studded with spines. References p. 88-93

60


cereb.

cereb. \

Fig. 48. Dorsal view (a) and median section (b) of the brain of the frog. Modified from Gaupp (1899). b.01. = bulbus olfactorius; cereb. = cerebellum: dien. = diencephalon;tect.mes. = tectum mesencephali; tel. = telencephalon; msp. = nervus spinalis.

The superficial zone of the granular layer contains a fairly compact zone of myelinated fibres which, according to Ariens Kappers et al. (1936), mainly belong to the spino-cerebellar tracts. Axons leaving this fibre zone ascend toward the Purkinje cell layer and terminate as typical climbing fibres (Larsell, 1923). Concerning the auriculae it should be noted that during larval life these structures are well marked, and receive both lateral line and vestibular connexions. However, during metamorphosis the lateral line organs and nerves degenerate and disappear, which leads to a reduction of the auriculae (Larsell, 1923, 1925, 1929). In the adult frog the structures last named are represented by small lateral projections, with only

A N A T O M Y O F THE C E R E B E L L U M

61

Fig. 49. A transverse section through the medulla oblongata and the cerebellum of the frog R u m cutesbiunu. rec.lat. = recessus lateralis ventriculi quarti; str. fibr. = stratum fibrosum; str.Purk. = stratum Purkinje; tr.sp.cb. = tractus spino-cerebellaris.

vestibular connexions. The anuran ‘nucleus cerebelli’ shows the same relations as the corresponding structure in the urodeles, but is, according to Larsell (1923), more definitely a part of the cerebellum. The fibre systems of the anuran cerebellum closely correspond to those of the urodeles and need no separate consideration here. It should, however, be noted that the spino-cerebellar system is strongly developed (‘the most prominent bundle connected with the cerebellum’, Larsell, 1923), and that according to Rothig (1927), the tectocerebellar system comprises both a superficial and a deep tract in this group. The superficial tract passes along the caudal margin of the tectum ; the deep system probably includes an isthmo-cerebellar as well as a postico-cerebellar component. The observations of Rothig have been confirmed by Kreht (1940), but this author considered both tracts as efferent with respect to the cerebellum. Finally, it should be mentioned that a mammillo-cerebellar tract has been observed in larval stages, but not in the adult frog (Larsell, 1923, 1925). Reptiliu The cerebella of the various groups of reptiles show considerable differences in both form and size. In the Chelonia (Larsell, 1932b)the cerebellum forms a caudally directed arch which roofs the rostra1 part of the fourth ventricle (Fig. 50A), but in the rhynchocephalian Sphenodon (Hindenach, 1931; Fig. 50B) and in many lizards the cereReferences p . 88-93

62


bellar plate is tilted forward or everted so as to bring the granular layer to the dorsal surface. Among the lizards the eversion is especially marked in Ymznus (De Lange, 1917) and in Chameleon (Shanklin, 1930). In these forms the cerebellum is large and extends over the caudal part of the midbrain (Fig. 50C). However, in many other lacertilians (e.g. Lucertu) the cerebellum is smaller and the eversion is less pronounced. The simplest cerebellum in the reptiles appears to be that found in the limbless lizard Anniellu (Larsell, 1926). In this species the organ consists merely of a commissure accompanied by a small mass of granular cells. The cerebellum of snakes, although larger than that of Anniella, also presents simple relations. It consists of a flat lamella which, projecting caudally, covers the rhomboid fossa (Fig. SOD).

A,Testudo

B,Sphenodon

D, Thornnophis

C , Varanus

E, Alligator

Fig. 50. Sagittal sections through the cerebellum of various reptiles. A, B, D, and E show the cell picture,C, themyelinatedfibres.Figure B is redrawn from Hindenach (1931). f.prima. = fissuraprima; f.sec, = fissura secunda.

63


mes.

auric corp.cec

Fig. 51. Dorsal view (a) and median section (b) of the brain of Alligator rnississipiensis. auric. = auricula (= flocculus + paraflocculus); corp.cer. = corpus cerebelli; tectmes. = tectum mesencephali; tel. = telencephalon; tr.01. = tractus olfactorius.

The most highly developed cerebellum among reptiles is found in the crocodilians. In these forms the cerebellar wall is strongly curved and surrounds a distinct ventriculus cerebelli (Figs. 50E and 51). Two transverse grooves, the sulcus anterior and the sulcus posterior (De Lange, 1917; Ingvar, 1918), divide the crocodilian cerebellum externally into three lobes. These lobes were denoted by De Lange and Ingvar as lobus anterior, lobus medius, and lobus posterior. Both of these authors, and Larsell as well (1932b), considered the sulcus anterior to be equivalent to the fissura prima of the mammalian cerebellum. The sulcus posterior was believed by Ingvar to correspond to the fissura prepyramidalis, but according to Larsell(l934) it represents the fissura secunda. If Larsell's interpretation of the sulci is correct the lobes of the crocodilian cerebellum can be homologized with the cerebellar folia of birds (Larsell, 1948; cf. Fig. 57b) and mammals (Larsell, 1952) in the following way: the lobus anterior References p.188-93

64


corresponds to folia I through V, the lobus medius to folia VI, VII and VIII, and the lobus posterior to folia IX and X. The reptilian cerebellum, like that of fish and amphibians, can be divided into a corpus cerebelli and an auricular lobe. The latter structure is, however, slightly developed in most reptiles. In lizards and snakes it is represented merely by the most lateral parts of the granular layer, which have been shown to receive primary and secondary vestibular fibres*. The 'vestibulo-cerebellum' is somewhat larger in the chelonians but of all reptiles this entity is best developed in the crocodilians. The auricular lobe consists in these forms of a band of nervous tissue which arches around the lateral recess of the fourth ventricle and extends medially along the caudal margin of the cerebellum. The lateral part of this band represents the auricle or flocculus**,

Fig. 52. Cerebellar and neighboring regions of the brain of an older Alligator embryo. Modified from Larsell(1932b).

whereas its medial part may be regarded an incipient nodulus (Larsell, 1932b). Both structures are demarcated from the corpus cerebelli by an external groove. This groove, the homologue of the postero-lateral fissure of higher forms, is shallow in the adult alligator but conspicuous in embryonic stages (Larsell, 1932b; Fig. 52). The basal portion of the crocodilian cerebellum shows on both sides a small but distinct lateral projection, which is delimited from the main cerebellar mass by an extension of the sulcus posterior (= fissura secunda of Larsell). De Lange (1917) and Ingvar (19 18) considered these projections to be homologous to the auricles of fish

* The lateral line connexions of cyclostomes, fish and amphibians are, of course, entirely lacking in the reptiles. ** Larsell (1932b) has suggested to reserve the term flocculus for those groups in which only the vestibular part of the static connexions are present (i.e. the reptiles, birds, and mammals).


65

and amphibians. Larsell (1932b, 1934, 1937) advocated a similar interpretation*, but this author later believed (cf. Goodman and Simpson, 1960) that the projections in question largely correspond to the mammalian paraflocculi and that only their caudal parts represent the flocculi (i.e. the homologues of the auricles of lower forms). Before turning to the microscopical structure of the reptilian cerebellum another division of this organ, proposed by Larsell (1926), should be briefly mentioned. Studying the garter snake and a variety of lacertilians, this author noticed that the cerebellum is narrow in legless forms and in species in which the limbs are feebly developed whereas in forms with strongly developed limbs the cerebellar plate is much wider. On the basis of these observations Larsell divided the reptilian cerebellum into two parts: (1) on each side a pars lateralis, supposed to be concerned with the movements of the paired appendages, and (2) a pars interposita in between, supposed to be related functionally with the musculature of the trunk and possibly the tail. Referring to the work of Bolk (1906), Larsell (1926) and Larsell and Dow (1939) regarded it likely that the pars lateralislforeshadows the lobulus ansiformis of the

s t r mol. stc Purk. s t r gran n.med.cb.

n. I a t . cb. n.vest. sup.

a

b

Fig. 53. Transverse sections through the cerebellar region of the turtle Chrysemys murginutu (a), and of .4/liguformissisxipiensis (b). Redrawn from Weston (1936). I.aur. = lobus auricularis (= flocculus); n.lat.cb. = nucleus lateralis cerebelli; n.med.cb. = nucleus medialis cerebelli; n.vest.sup. = nucleus vestibularis superior; str.gran. = stratum granulare; strmol. = stratum moleculare; str.Purk. = stratum Purkinje.

* In his 1932b and 1934 papers Larsell indicated the lateral part of the sulcus posterior as the sulcus parafloccularis. This, however, without the intention to suggest the presence of a paraflocculus (cf. Larsell, 1937, p. 584.) References p . 88-93

66

K. N I E U W E N H U Y S

mammalian cerebellum. In his 193213 paper Larsell applied the subdivision just indicated also to the cerebellum of the chelonians and the crocodilians. In so far as the latter forms are concerned, however, the ideas of Larsell have not been corroborated by the work of Goodman and Simpson (1960). These authors, who stimulated the cerebellum of Caiman scferops by means of implanted electrodes, concluded (p. 133): ‘Indeed the present study has demonstrated that this lateral region of the corpus cerebelli is concerned with the control of postural modifications of the limbs, but no more so than the medial region. Moreover, the postural effects on the trunk region are equivalent in both the lateral and medial parts of the corpus cerebelli’. The histological structure of the reptilian cerebellum has been described by numerous authors, among whom P. Cajal (cf. Cajal, 191I), Larsell (1926, 1932b), Hindenach (1931) and Weston (1936) may be mentioned. From these studies it appears that the three typical cerebellar layers are present in all reptilian groups. The disposition of the Purkinje cells shows a considerable variation, however. In the chelonians (Figs. 50A and 53a) and in the snakes (Fig. 50D) these elements form a rather diffuse zone between the granular and molecular layers. In Sphenodon (Fig. 50B)and in most lizards the Purkinje cell layer is more compact, although at many places up to five cells in thickness. Only in the crocodilians (Figs. 50E and 53b) and in certain lizards (e.g. chameleon; Shanklin, 1930) is the avian and mammalian condition of a very regular Purkinje layer of one cell deep approached. The Golgi studies of P. Cajal (see Cajal, 1911) and Larsell (1932b) have revealed that the dendritic branching of the Purkinje cells is richer than in amphibians and is most clearly oriented in a sagittal plane (Fig. 54). In the turtle all dendrites except the main trunk are densely studded with gemmules (Fig. 63D). It is believed that most of the Purkinje cell axons terminate in the cerebellar nuclei to be discussed below.

Fig. 54. Cross section through the cerebellum of a lizard. Golgi method. After Cajal(l911). A, molecular layer; B, Purkinjecelllayer; C, granular layer; D, ependymallayer; E, fibre bundle; a, neuron in the molecular layer; b, dendritic trees of Purkinje cells; d, Purkinje cell; e, granule cells; h, mossy fibres; i, stellate cell; j, ependymalcell.


67

The granular cells show in Golgi preparations the characteristic short dendrites with claw-like endings. Their slender axons enter the molecular layer, where they bifurcate into parallel fibres (Fig. 54). Numerous typical mossy fibres ramify and terminate in the granular layer, but it is noteworthy that in the turtle these fibres have less numerous terminal tufts than those of mammals (Larsell, 1932b). The molecular layer contains typical basket cells along with stellate elements having freely branching axons. In the chameIeon the axons of the basket cells are provided with numerous collaterals that break up in terminal arborizations around the bodies of the Purkinje cells (P. Cajal). In the turtle the axons of the basket cells show a similar relation but are less branched than in the chameleon and make contact with no more than two or three Purkinje cells (Larsell, 1932b). As regards the localization of the fibres in the reptilian cerebellum it should be remarked that in turtles and snakes the afferent and efferent axons are scattered among granular cells. A similar condition prevails in the lizards, but in these groups the fibres tend to concentrate in the external part of the granular layer (Fig. 50C). In Sphenodon a compact band of fibres separates the granular zone from the Purkinje cell layer. In the crocodilians, however, the fibres are concentrated in a stratum album profundum so that the granular layer is clearly detached from the ventricular surface (Fig. 50E). On these grounds it may be stated that the crocodilians possess, as do the birds and the mammals, a true cerebellar cortex. It has been observed that in lower vertebrates the efferent cerebellar pathways originate in part from a group of cells situated laterally to the ventricle, close to the cerebello-tegmental junction. Similar observations have been made for reptiles, but here the cell group in question is more distinct and is usually differentiated into a large-celled nucleus medialis cerebelli and a small-celled nucleus Iateralis cerebelli (Ariens Kappers, 1921; Larsell, 1926, 1932b; Weston, 1936). In the chelonians these nuclei occupy a position corresponding to that of the single diffuse nucleus cerebelli of sharks and amphibians (cfi Fig. 53a with Figs. 19 and 46): but in the crocodilians they show a considerable extension into a medio-dorsal direction (Fig. 53b). Several authors (Van Hoevell, 1916; Ingvar, 1918; Ariens Kappers, 1921; Herrick, 1924; Ariens Kappers et al., 1936) have expressed the opinion that in the course of phylogeny the cerebellar nuclei have shifted from a subcerebellar position into the cerebellum itself. Referring to the condition found in the crocodilians it was stated, then, that the definite incorporation of these nuclei into the cerebellar area has occurred at the reptilian level. Rudeberg (1961), however, concluded on the basis of extensive comparative embryological studies that in all vertebrates the nuclei cerebelli lie within cerebellar territory and develop from the cerebellar anlage. Concerning the homology of the reptilian cerebellar nuclei, it should be noted that Ariens Kappers (1921) and Larsell and Dow (1939) regarded the medial nucleus as equivalent to the nucleus fastigii of mammals. The lateral nucleus corresponds, according to Larsell (1957), with at least part of the nucleus interpositus. With regard to the fibre connexions of the reptilian cerebellum I will confine myself to a brief enumeration of the major pathways, relying chiefly on the studies of De Lange (1917), Ingvar (1918), Larsell (1926, 1932b), Shanklin (1930) and Weston Refirences p. 88-93

68


(1936). It is important to note that all of these studies are based exclusively on normal material. The afferent systems include : (1) The vestibulo-cerebellar tract, which terminates in the flocculus and in the nucleus medialis cerebelli. This tract is largely composed of fibres originating from the nucleus vestibularis ventrolateralis and the nucleus vestibularis dorsolateralis (Weston), but it also contains some direct vestibular root fibres. A small bundle of fibres belonging to this system passes along the caudal margin of the cerebellum to reach the contralateral flocculus (Larsell, 1932b, 1934). This decussating bundle corresponds, according to Larsell, to the commissura vestibulolateralis of amphibians and other lower vertebrates. (2) Cochleo-cerebellar fibres, which accompany the vestibulo-cerebellar tract to the cerebellum. Some of these fibres are direct root fibres of the cochlear nerve (Beccari, 1912; Weston, 1936) but most of them arise from the nucleus laminaris. (3) A well developed spino-cerebellar system, which is clearly divisible into a dorsal and a ventral tract. The latter sends many of its fibres into a large commissure situated in the rostrobasal part of the cerebellum. This commissure, which is known as the commissura (inferior) cerebelli, also contains tecto-cerebellar fibres and efferent cerebellar fibres, originating from the nucleus medialis cerebelli. As regards the site of termination of the spino-cerebellar fibres, there is no unanimity in the literature. Ingvar (1 918) held that they end in the rostral portion of the cerebellum but according to Larsell (1932b) they distribute, at least in the Alligator, to both rostral and caudal parts of the corpus cerebelli. Weston (1936) noted that the dorsal spino-cerebellar tract terminates laterally and caudally in the body of the cerebellum, and possibly to a slight extent in the homolateral flocculus. The ventral spino-cerebellar tract distributes, according to Weston, to the homolateral nucleus medialis cerebelli and, by way of the cerebellar commissure, to the rostral portion of the contralateral part of the corpus cerebelli. (4) Reticulo-cerebellar fibres (Weston). (5) An olivo-cerebellar tract, which accompanies the spino-cerebellar system, has been described by several authors (Ariens Kappers, 1921; Shanklin, 1930; Larsell, 1932b). The presence or at least the distinctness of this tract has been questioned by Weston however. (6) The trigemino-cerebellar system. This system comprises, again according to Weston, (1) a homolateral, dorsal trigemino-cerebellar tract arising from the chief sensory nucleus of the trigeminal nerve, and (2) a crossed, ventral trigemino-cerebellar tract which takes origin from the nucleus of the trigeminal. The homolateral tract is augmented, according to Weston (1 936) and Woodburne (1 936), by some direct trigeminal root fibres. (7) The tecto-cerebellar tract. This tract originates mainly from the deep fibre layer of the tectum opticum but also receives fibres from the inferior colliculus (Weston). Its fibres pass through the velum medullare anterius to the cerebellum, where many of them enter the cerebellar commissure. There is evidence that fibres of this system terminate as climbing fibres in the molecular layer (cf. Weston, 1936, p. 161). The efferent connexions of the reptilian cerebellum may be divided into three systems,


69

viz. the tractus cerebello-bestibularis et cerebello-spinalis, the tractus cerebellomotorius et cerebello-tegmentalis, and the brachium conjunctivum. The tractus cerebello-vestibularis et cerebello-spinalis arises chiefly from the medial cerebellar nuclei of both sides, but also receives Purkinje cell axons (Weston). Its fibres loop around the lateral recess of the fourth ventricle, course caudally close to the ventricular surface, and terminate in various nuclei of the vestibular complex and in the reticular gray adjacent to this complex. Some of its fibres are said to reach the spinal cord by way of the vestibulo-spinal tract. The bundle just described is probably the representative in reptiles of the crossed and the direct fastigio-bulbar fibres. The tractus cerebello-motorius et cerebello-tegmentalis. A system of fibres connecting the cerebellar with the motor and reticular nuclei of the rhombencephalon has been described by several authors. Ariens Kappers (1921) noted the presence of a pathway, comparable to the tractus cerebello-motorius of sharks, which probably originates from the nucleus medialis cerebelli (Dachkern). Huber and Crosby (1926) described a tractus cerebello-tegmentalis the fibres of which distribute to the motor nucleus of the trigeminal nerve, to the reticular nuclei of the rhombencephalon, and to ‘other bulbar nuclei’. A certain proportion of the fibres of this tract join the fasciculus longitudinalis medialis. Huber and Crosby make no specific statement about the origin of the cerebello-tegmental tract. They mention, however, that its fibres constitute a portion of the inferior cerebellar commissure. Weston described the cerebello-tegmental tract of Huber and Crosby under the name of tractus cerebello-motorius et tegmentalis bulbaris. According to him, this tract originates chiefly from the ipsilateral nucleus lateralis cerebelli but also comprises a considerable number of fibres which pass through the cerebellar commissure (origin unknown), and probably some Purkinje cell axons as well. The brachium conjunctivum, finally, takes its origin from the lateral cerebellar nucleus (Shanklin, 1930; Larsell, 1932b). It decussates under the fasciculus longitudinalis medialis, and terminates, according to most authors, in the nucleus ruber, and in the tegmental gray surrounding that nuclear mass. Weston, however, has pointed out that the reptilian brachium conjunctivum, like its mammalian equivalent, contains both a tegmental and a motor component. He traced the fibres of the latter to the oculomotor and abducens nuclei.

Aves The cerebellum of birds is distinguished from that of the reptiles by being more massive and more complexly fissured (cf. Fig. 55 with Fig. 51). The fissures, which as in crocodilians, are transversely oriented, divide the avian cerebellum into a rostrocaudal series of lobules or folia. Just as in sharks (Fig. 17) and mammals the number of lobules increases with the size of the body (Fig. 58). Embryological studies (Ingvar, 1918 ; Larsell, 1948) have shown that the cerebellum of birds, like that of all vertebrates, develops from two bilateral thickenings of the alar plates (Fig. 56A and C), which later fuse in the median plane to form a single cerebellar plate (Fig. 56B). Already before the onset of this fusion slight grooves References p . 88-93

70

K. N I E U W E N H U Y S

mes. Vlll

paraf locc ulus

cere b.

Fig. 55. Dorsal view (a) and median section (b) of the brain of the pigeon Columba liviu.

appear on each side which mark off the most caudal and lateral parts from the remainder of the cerebellar anlagen (Fig. 56C). These grooves deepen in successively later stages and approach each other medially, to form the fissura postero-lateralis (Larsell, 1948; Figs. 563 and D). According to Larsell this groove divides the avian cerebellum into the two fundamental parts, common to all classes of vertebrates: the lobus flocculo-nodularis and the corpus cerebelli. During further development the curvature of the cerebellar plate increases, so that a cerebellar ventricle is formed (Fig. 56C), and gradually fissures appear on the surface of the corpus cerebelli. Ingvar (1918) observed that in the chick three grooves, designated by him as the fissures x, y and z, develop particularly early. He regarded these sulci homologous with the fissura prima, the fissura prepyramidalis and the fissura secunda, respectively, of the mammalian cerebellum (Fig. 56D and E). Larsell (1948) confirmed Ingvar’s observations and

* (See p. 71) Larsell noted, however, that the fissures which Ingvar in his pictures of embryonic avian cerebella labelled as y and z, do not correspond to the fissures similarly designated in his (Ingvar’s) figures of the cerebella of adult birds.

71


concurred with his interpretation of the three sulci mentioned*. After the appearance of the first sulci the mass of cerebellar tissue increases rapidly. The ventricular cavity becomes reduced to a small slit, and gradually new fissures appear on the surface of the corpus cerebelli (Fig. 56F). On the basis of extensive comparative studies on

.

.. .

. . . . .. ... .. .. . A

corp. cb. fl.

D

f. ppa.

E F

Fig. 56. The development of the avian cerebellum. A, Transverse section of the cerebellar anlage of a chick embryo of 5 days’ incubation. Modified from Ingvar. B, Median section through the cerebellum of a 15 mm embryo of Hirundo rustica. After Seatersdal. C , Dorsal view of the cerebellum of a chick embryo, incubated 8 days. D, Dorsoposterior view of the cerebellum of a chick embryo, incubated 9+ days. E, Median section of the cerebellum of a cormorant embryo of 65 mm totallength. F, Midplane of the cerebellum of a duck embryo incubated 13 days. Figures G-F are redrawn from Larsell(1948). cereb.an1. = cerebellar anlage; corp.cb. = corpus cerebelli; f.ppd. = fissura prepyramidalis; f.pr. = fissura prima; f.sec. = fissura secunda; f.po.lat. = fissura postero-lateralis; fl. = flocculus; v.cb. = ventriculus cerebelli. I to X indicate the primary folia, according to Larsell. Va, VIIIb and similar combination of roman numerals and letters indicate secondary folia. References p . 88-93

72


embryonic and adult avian and mammalian material Larsell(l948, 1952) reached the following conclusions : (1) The fissures divide the developing avian cerebellum into 10 primary folia. (Larsell numbered these folia I to X, beginning anteriorly). (2) During later development a varying number of these primary folia is further subdivided into secondary or tertiary folia. (As illustrated by Fig. 58 in the small

Fig. 57a. A paramedian section through the brain stem and the cerebellum of the pigeon. Weigert-Pal paracarmine.


73

folium+ /- ,-fis.

tuber vermis p r epy r a mi d.

(Sacte r sda I)

lob. centralis

Fig. 57b. Outline of the section represented in Fig. 57a. The lobules and fissures are named according to the terminology of Larsell(1948).

humming bird only two folia (VI and IX) are subdivided, but in the eagle all primary folia, save for I and X, show subfoliation). (3) The avian folia can be homologized in detail with the lobules of the mammalian cerebellum, as follows (Fig. 57b): folium I corresponds to the lingula, folia I1 and LII to the lobulus centralis, folia IV and V to culmen, folium VI to declive, folium VII to

A

B

Fig. 58. Median section of the cerebellum of the humming bird (A), and of the bald eagle (B), X 4. Figure A is modified from Craigie (1928); figure B is redrawn from Larsell(l948). Both figures are labelled according t o Larsell. References p . 88-93

74


folium plus tuber vermis, folium VIII to pyramis, folium IX to uvula, and folium X to nodulus. After the appearance of Larsell’s (1948) study, Saetersdal (1959) published a thorough analysis of the fissuration of the cerebellum in 26 avian species,including 2 of the 3 species used by Larsell. This author confirmed that the avian cerebellar plate early in ontogenesis is divided into a corpus cerebelli and a lobus flocculo-nodularis by the fissura postero-lateralis. With regard to the identification of the fissura prima the results of Saetersdal are, however, at variance with those of Larsell. Saetersdal pointed out that the fissure labelled as such by Larsell generally develops very late in ontogenesis, as compared to the other sulci. Another sulcus in the anterior cerebellar surface, designated by Larsell as sulcus intraculminaris appears, according to Saetersdal, much earlier, i.e. about simultaneously with the fissura prepyramidalis. This sulcus, then, is one of the three primary fissures of the avian corpus cerebelli, and therefore represents in Saetersdal’s opinion the mammalian fissura prima (Fig. 57b). In connexion with this ‘ventral shift’ of the fissura prima Saetersdal suggested several changes in Larsell’s interpretation of the avian cerebellar folia. Reference to Figs. 55 and 56C,D shows that the avian cerebellum possesses on either side a small lateral projection. In early embryonic stages these projections are exclusively formed by the anlagen of the flocculi, but during later development lateral outgrowths of the caudal part of the uvula (subfolium IXc) appear, which blend with the floccular anlagen. These uvular extensions grow very rapidly and gradually surpass and partly surround the flocculi. In full-grown birds only the caudomedial parts of the auriculae consist of the flocculi, whereas their entire rostra1 and lateral portions are formed by the lateral continuations of the uvula. The latter were designated by Larsell (1948) as the paraflocculi. He considered these structures homologous to a part of the paraflocculi of the mammals. From the foregoing it appears that the lateral projections of the avian cerebellum are not equivalent to the auricles of amphibians and other lower forms. They comprise apart from the lateral portions of the ‘vestibulo-cerebellum’, i.e. the flocculi, a large uvular or parafloccular component (Larsell, 1957). Numerous previous workers, among them Brandis (1 894), Edinger (1910), Comolli (1910), Shimazono (1912) and Huber and Crosby (1929), considered the large central body of the avian cerebellum as the homologue of the vermis of the mammalian cerebellum. However, Larsell(1948) directed attention to the fact that the basis of the corpus cerebelli of birds on either side shows a small swelling, covered with nonfoliated cerebellar cortex (Fig. 59b: cortex lat.). He believed this swelling to ‘represent the region which, in mammals, becomes the lateral cerebellar hemisphere’ (Larsell, 1948, p. 172). This hypothesis has received considerable support from the work of Brodal et al. (1950). On the basis of analysis of the ponto-cerebellar projection in the chick these authors concluded that the unfoliated lateral cortex and the adjacent lateral parts of the folia V-VIII represent the avian homologue of the mammalian cerebellar hemispheres. Microscopically the avian cerebellum shows a pattern similar to that of mammals : externally a greatly expanded, very regularly arranged cortex, and centrally a compact

75


layer of fibres (Figs. 57-59). The latter sends out rays into the folia and subfolia, which together give the characteristic picture of the arbor vitae (Fig. 57a). The histological organization of the cerebellar cortex of birds is so similar to that of mammals that a separate discussion is felt unnecessary (cf. General Introduction). Mention may be made of the facts that typical basket cells, resembling in every respect those of mammals, have been identified (Dogiel, 1896), and that the Purkinje cell dendrites show a very complex ramification (Fig. 63E). A point of difference would be that the axons of the avian Purkinje cells have less numerous collaterals than those of mammals (Cajal, 1911). The cerebellar nuclear complex of birds is considerably larger than that of reptiles. Its right and left groups approach each other above the fourth ventricle, and are only separated by a narrow ventricular slit. Within this complex Brandis (1 894) distinguished on either side two nuclei, a nucleus medialis and a nucleus lateralis. A similar subdivision has been made by Shimazono (1912), Ariens Kappers (1947) and Zecha (1966). Cajal(1908) recognized four nuclear groups within the avian deep cerebellar gray, namely : a nucleus internus, which corresponds to the nucleus medialis of the authors mentioned above, a nucleus intercalatus, a nucleus intermedius, and a nucleus lateralis. This subdivision was adopted by Sanders (1929) and Doty (1946), but the latter emphasized that the different nuclei are connected by cell bridges, constituting (p. 23) : ‘a single gray band greatly folded and convoluted, but continuous throughout’.

IXC

-X

Fig. 59a. A cross section through the medulla oblongata and the caudal part of the cerebellum of the pigeon. The cerebellar folia are labelled according to Larsell. References p. 88-93

76

R. NIEUWENHUYS

Fig. 59b. A cross section through the medulla oblongata, the middle of the cerebellum and the caudal part of the tectum mesencephali of the pigeon. The cerebellar folia are labelled according to Larsell. cortex lat. = cortex lateralis. nn.cer. = nuclei cerebelli.

As regards the homology of the deep cerebellar nuclei it is commonly agreed that the medial or internal nucleus of birds represents the mammalian nucleus fastigii. Shimazono (1912) and Ariens Kappers (1947) considered the lateral nucleus of their descriptions homologous to the nucleus dentatus of mammals. Cajal(l908) compared the four nuclear masses which he distinguished in the avian cerebellum directly with those of Man. Thus he homologized the avian nucleus internus, intercalatus, intermedius and lateralis with the nucleus fastigii, globosus, emboliformis, and dentatus, respectively. Larsell(l957) believed that the nucleus intermedius and lateralis of birds have developed from the nucleus lateralis of reptiles. He compared the nucleus intermedius of birds with the mammalian nucleus interpositus. The avian lateral nucleus represents, according to Larsell, a primordium of the nucleus of the same name in mammals, as defined by Brunner (1919). The homologies indicated above are all based on study of the positional relations and fibre connexions of the nuclei in adult material. It is important to note that Riideberg (1961), who approached the problem of the homologization of these nuclei in an entirely different way, namely by analysing their ontogenetic development, concluded that a direct morphologic comparison between the individual cerebellar nuclei in birds and mammals cannot be made. While the cerebellar fibre systems in lower vertebrates have been investigated


77

almost exclusively in normal material, in birds these connexions have been the subject of a considerable number of experimental studies. Most important among these are the Marchi studies of Friedlander (1898), Frenkel (1909) and Shimazono (1 912), the work of Brodal et al. (1950) with the method of retrograde degeneration, and the study of Whitlock (1952), who used, apart from the Marchi and the Gudden techniques, also electrophysiological methods. The ensuing survey of the connexions of the avian cerebellum is chiefly based on the publications just mentioned. The following afferent connexions have been described (cf- Fig. 60): (1) A tractus vestibulo-cerebellaris which, according to Whitlock, contains both direct vestibular root fibres, and axons arising from the vestibular nuclear complex. Whitlock traced in Marchi preparations both fibre categories to the uvula (folium IX) and nodulus (folium X), and to the lateral extension of these folia, i.e. the flocculusparaflocculus complex. In normal embryonic material Whitlock observed that direct and secondary vestibular fibres enter the deep cerebellar nuclei, and that other fibres of these types constitute a small commissura vestibularis in the most caudal part of the cerebellum. The presence of vestibular root fibres passing to the cerebellum was earlier advocated by Wallenberg (1900), on the basis of a not entirely convincing Marchi experiment, and by Sanders (1929), who studied normal material. Trendelenburg (1907) and Frenkel (1909), however, denied their existence. The secondary vestibulo-cerebellar connexion, described by Whitlock, corresponds to the tractus octavo-floccularis of Shimazono (19 12). (2) A spino-cerebellar system. This system is strongly developed and receives contributions from all levels of the cord (Friedlander). Most authors distinguish a dorsal and a ventral spino-cerebellar tract. The fibres of the ventral tract pass the level of the velum medullare anterius and then curve dorsalward and backward into the cerebellum. Those of the dorsal tract follow a more direct course (Ariens Kappers et al., 1936). The cells of origin of the spino-cerebellar system have not been determined as yet. It is worthy of note, however, that a probable avian homologue of the column of Clarke of mammals (which is known to give rise to the dorsal spino-cerebellar tract) has been described by Streeter (1904) and by Huber (1936). Both the ventral and the dorsal tracts distribute to the ipsilateral as well as to the contralateral side of the cerebellum. Whitlock traced a bundle from the ventral spino-cerebellar tract into the commissura cerebelli, which is situated just caudal to the lobulus centralis. According to Whitlock, this commissure also contains primary and secondary trigeminal fibres. The Marchi studies of Shimazono (I 912), Ingvar (1 9 18) and Whitlock (1952) have shown that, as regards its termination, the avian spino-cerebellar tract shows a marked similarity to its mammalian homologue. The bulk of its fibres ends in the rostra1 portion of the cerebellum (folia I1 through V, and VIa,b). A smaller number appears to reach folia VIII and IX. The experiments of Shimazono and Ingvar indicate that there is no marked somatotopical arrangement within the termination of the spino-cerebellar system. Some authors (Frenkel, 1909; Sanders, 1929) have traced spino-cerebellar fibres References p . 88-93

78

A. N I E U W E N H U Y S

Fig. 60. A diagrammatic representation of the afferent systems of the avian cerebellum. Partly based on Shimazono (1912). CORP.CB. = corpus cerebelli;cortex lat. = cortex lateralis; FLOC. = flocculus; N.CB. = nuclei cerebelli; N.PONT. = nuclei pontini; N.VEST. = nuclei vestibulares; tr.01. tr.ret.cb. = tractus olivo-cerebellarisand tractus reticulo-cerebellaris; tr.pont.cb. = tractus pontocerebellaris; tr.tect.cb. = tractus tecto-cerebellaris;tr.sp.cb. = tractus spino-cerebellaris; tr.vest.cb. = tractus vestibulo-cerebellaris.

+

to the deep nuclei. On the other hand Shimazono (1912) has emphasized that although numerous spinocerebellar fibres skirt along the deep nuclei, none of them actually terminates in these cell masses. More recently Whitlock (1952) has reached a similar conclusion. (3) A trigemino-cerebellar system. This tract connects the chief sensory trigeminal nucleus with the corpus cerebelli (Shimazono, 1912; Craigie, 1928; Sanders, 1929; Woodburne, 1936). Its fibres, which partly decussate in the cerebellar commissure, according to Whitlock terminate in folia V, VI, and VII. The question of whether the avian trigemino-cerebellar system contains direct trigeminal root fibres cannot be definitely answered. Some authors (Woodburne, 1936; Whitlock, 1952) have observed


79

such fibres in normal material but their observations have not been experimentally verified. Frenkel(l909) did not find any degeneration in the cerebellum after tearing out the trigeminal nerve. (4) An olivo-cerebellar connexion. The existence of this system in birds was first demonstrated by Yoshimura (1910) who, following partial extirpation of the cerebellum, found massive retrograde cellular changes in the contralateral olive. His observations have been confirmed by Brodal et al. (1950), and by Whitlock (1952). Using the Marchi method, the latter demonstrated in addition that the olivo-cerebellar system terminates in all folia of the cerebellum. (5) A ponto-cerebellar system. Although several authors (Brouwer, 1913 ; Papez, 1929; Craigie, 1930; Ariens Kappers et al., 1936; Ariens Kappers, 1947) had suggested the presence of primordial pontine nuclei in the avian brain, it was Brodal et al. (1950) who first conclusively demonstrated the existence of such nuclei in birds. Following extensive lesions of the cerebellum of chickens, these authors found severe retrograde cellular changes and cell loss in two small well defined cell groups situated along the ventral surface of the upper medulla. It appeared that the more medial of these two nuclei projects chiefly to the opposite side, whereas the lateral nucleus projects chiefly to the same side of the cerebellum (Fig. 60). Comparison of the retrograde cellular changes resulting from a variety of more restricted lesions revealed that the majority of pontine fibres pass to the lateral parts of folia VI-VIII and to unfoliated cortex, which forms a lateral continuation of these folia. The paraflocculus also appeared to receive numerous pontine fibres. Little is known as yet of the afferent connexions of the avian pontine nuclei. It seems probable, however, that the tractus tecto-bulbaris superficialis non cruciatus, described by Miinzer and Wiener (1898) and by Boyce and Warrington (1899) and which was thought by these authors to terminate in the trapezoid body, in reality represents a tecto-pontine system. The existence of telencephalo-pontine and spino-pontine fibres in birds has been demonstrated recently by Zecha (1966). (6) A tecto-cerebellar tract. Fibres passing from the tectum mesencephali to the cerebellum have been traced by means of the Marchi technique by several authors. There appears to be no unanimity, however, about the origin and the course of this tract. Munzer and Wiener (1898) and Boyce and Warrington (1899) indicate that its fibres arise from the ventral and caudal parts of the tectum. Frenkel (1909) stated that the tecto-cerebellar fibres degenerate only after deep tectal lesions involving the area situated medial and ventral to the ventricle, but Shimazono (1912) observed degeneration in the tract in question following lesions of the dorsal part of the tectum. Whitlock (1952) indicated that the tecto-cerebellar tract originates from the ‘medial tectal gray’, thus partially confirming the observations of Frenkel. With regard to the course of the tecto-cerebellar tract, Whitlock reported that this system reaches the cerebellum via the lateral aspects of the velum medullare anterius but most previous workers believed that its fibres pass through the cerebellar peduncle. Shimazono stated that the tecto-cerebellar fibres enter the peduncle more caudally than do those of the spino-cerebellar system. The tecto-cerebellar tract terminates largely but not entirely in the ipsilateral Refermces p. 88-93

80


part of the cerebellum. According to Shimazono and Whitlock its fibres distribute principally to the caudal lobules (folia VI-VIII). (7) Tractus reticulo-cerebellaris. In mammals several nuclei of the reticular formation project onto the cerebellum (cf- Brodal, 1954) but very little is known about the existence of such connexions in birds. Brandis (1894) reported having observed reticulo-cerebellar fibres in normal Weigert stained avian material. Following a lesion in the region of the lower olive in a pigeon, Shimazono traced in Marchi preparations: ‘Fasern aus der Formazio Reticularis der Medulla Oblongata mit der unteren Olive zum gekreuzten Kleinhirn’ (cf- Fig. 60). He considered it likely, however, that all of the observed fibres originated in fact from the inferior olivary nucleus. (8) Tractus strio-cerebellaris. Fibres connecting the striatal region of the forebrain with the cerebellum have been described by some authors (Craigie, 1928; Huber and Crosby, 1929), but these findings have not yet been verified experimentally. (9) Tractus cochleo-cerebellaris or lamino-cerebellaris. In embryonic material Bok (1915) traced fibres from the cochlear group of nuclei to the cerebellum, and Sanders (1929) observed a similar connexion in normal adult material. The existence of this system has not been substantiated, however, by the experimental work of Whitlock (1952). Before turning to the efferent connexions it should be noted that Shimazono, on the basis of numerous Marchi experiments, concluded that the afferent fibres to the avian cerebellar cortex do not terminate in the granular layer but reach the layer of Purkinje cells, where they apparently participate in the formation of pericellular baskets. The efferent fibres of the avian cerebellar cortex, i.e. the axons of the Purkinje cells, fall into two categories: (1) long corticofugal fibres which pass directly toward extracerebellar structures, and (2) short corticofugal fibres which terminate in the central cerebellar nuclei, thus forming the corticonuclear projection. According to Frenkel (1909), the long corticofugal fibres distribute to the ‘area acustica’, and Shimazono (1912) traced them in his Marchi material as far as the vicinity of the nucleus of Deiters. In Fig. 61 I have indicated that these fibres originate from the flocculus. However, this should be merely regarded as an extrapolation from the mammalian condition since the exact origin of the long efferents of the avian cerebellar cortex is still unknown. The Marchi experiments of Friedlander (1898), Frenkel (1909) and Shimazono (1912) have shown that the short corticofugal fibres far outnumber the long direct efferents, but data on the anatomical organization of the former are entirely wanting. The recent neurophysiological studies of Goodman, Horel and Freeman (1964) indicate, however, that the avian cerebellar cortex can be divided into three longitudinally arranged functional zones. It seems reasonable to assume that, as in mammals, this functional organization is correlated with an orderly medio-lateral arrangement of the cortico-nuclear fibres*.

* On the basis of experimental anatomical analyses of the corticonuclear projection, Jansen and Brodal (1940, 1942) and Voogd (1964) concluded that in mammals the cerebellar cortex can be divided into a number of longitudinal zones, each one connected with a definite part of the central cerebellar nuclei. The physiological studies of Chambers and Sprague (1955a, b) have revealed that this zonal organization of the cortico-nuclear projection is paralleled by a distinct functional localization.


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We have already seen that the central cerebellar nuclei are strongly developed in birds and it is from these centres that the two chief efferent systems, i.e. the tractus cerebello-bulbo-spinalis and the brachium conjunctivum, arise. The tractus cerebello-bulbo-spinalis originates from the medial cerebellar nuclei of both sides (Cajal, 1908; Frenkel, 1909; Shimazono, 1912; CJ Fig. 61), descends through the peduncle to the medulla oblongata, and passes caudally in close relation to the spino-cerebellar system. It distributes fibres to various bulbar centres during its course, including the motor nuclei of the Vth (Frenkel) and the VIIth (Frenkel, Shimazono) cranial nerves, the reticular formation, and the inferior olive (Boyce and Warrington, 1899; Shimazono, 1912). Cerebello-vestibular fibres, which have been described by Shimazono, probably also belong to the cerebello-bulbar tract but it has not been conclusively shown that these fibres originate specifically from the medial

floc.

trcb. bulb

Fig. 61. A diagram showing the efferent systems of the avian cerebellum. Partly based on Shimazono (1912). br.c. = brachium conjunctivum; f.1.m. = fasciculus longitudinalis medialis; floc. = flocculus; n.cb. = nuclei cerebelli; n.rub. = nucleus ruber; n.vest. = nuclei vestibulares; N.111, nucleus nervi oculomotorii; N.VlI, nucleus nervi facialis; p = Purkinje cell axons (= cortico-nuclear projection); tr.cb.bulb.sp. = tractus cerebello-bulbo-spinalis. References p . 88-93

82


cerebellar nucleus. Several authors (Friedlander, 1898; Frenkel, 1909; Shimazono, 1912) have observed that some fibres of the system under consideration descend into the spinal cord, thus forming a cerebello-spinal tract. Following a lesion of the medial cerebellar nucleus, Shimazono traced these fibres in Marchi preparations as far as the lower thoracic region. Boyce and Warrington (1899), on the other hand, stated that lesions limited to the cerebellum do not give rise to any degeneration in the spinal cord, and Yoshimura (1910) reported that according to his observations the cerebellospinal tract of Friedlander has almost vanished at the level of the bulbo-spinal junction. The brachium conjunctivum originates, according to Wallenberg (1898), Shimazono (1912), Ariens Kappers et al. (1936) and Zecha (1966), from the lateral cerebellar nucleus but Ariens Kappers (1947) and Larsell (1957) believed that both the medial and the lateral nuclei give rise to this system. Its fibres enter the tegmentum, decussate in the raphe, and terminate in the red nucleus. According to Sanders (1929) the brachium conjunctivum distributes to the reticular gray of the mesencephalon in addition, and Wallenberg (1904) observed that fibres of this system reach the oculomotor nucleus by way of the fasciculus longitudinalis medialis. Efferent cerebellar fibres which join the fasciculus longitudinalis medialis have also been described by Frenkel (1909), Ariens Kappers (1934, 1947) and others, but Shimazono (1912) and Zecha (1966) did not find degenerating fibres in this bundle following lesions confined to the cerebellum.

SUMMARY AND C O N C L U D I N G REMARKS

1. Embryological investigations have shown that in all groups of vertebrates the cerebellum develops from bilateral anlagen, which form the most rostra1 parts of the rhombencephalic alar plates. These anlagen become interconnected early in development by decussating fibres. They eventually fuse in the median plane to form a single cerebellar plate. The cerebellum is small in adult petromyzonts and maintains a simple plate-like configuration, but in most other groups of vertebrates a further growth and elaboration of cerebellar tissue occurs. The modes in which the embryonic cerebellar plate is transformed into the adult organ differ widely among the various groups of gnathostomes. 2. There is some difference of opinion in the literature as to whether or not the Myxinoidea possess a cerebellum. Holmgren believed that Myxine has a large cerebellum, but others (Holm, Edinger, Jansen, Bone) have questioned or denied the presence of a cerebellum in this form. According to Larsell, the cerebellum in Bdellostoma is represented by a small commissure, consisting of octavus and lateral line nerve fibres, which is accompanied by bilateral strands of nerve cells. The latter extend medially from the octavo-lateral areas. 3. Tn petromyzonts the simple platelike cerebellum structurally closely resembles the adjacent area octavo-lateralis, but differs from the latter in having a much more diverse input. The petromyzontian cerebellum receives spino-cerebellar, trigeminocerebellar, lobo-( = hypothalamo-)cerebellar, as well as tecto-cerebellar fibres, in


83

addition to primary and secondary octavus and lateral line afferents. Two commissural systems are present: a large, dorsal commissura octavo-lateralis and a smaller, more basally situated commissura cerebelli. The latter is composed of trigeminal and some spino-cerebellar fibres. 4.The studies of Larsell have revealed that in most groups of fish, in urodeles and in larval anurans, the cerebellum consists of two fundamental divisions, viz., a caudobasal lobus vestibulo-lateralis which, as its name indicates, receives vestibular and lateral line fibres, and a more rostrally situated corpus cerebelli, which is dominated by spino-cerebellar, trigemino-cerebellar, tecto-cerebellar and other systems. A similar division can be made in adult anurans, reptiles, birds and mammals, but a lateral line system is absent in these groups and the caudo-basal part of the cerebellum only receives primary and secondary vestibular fibres. Apart from the difference in sensory input between the two main parts of the cerebellum, Larsell has pointed out the following particulars pertaining to this subdivision. (a) The lobus vestibulo-lateralis (c.q. vestibularis) and the corpus cerebelli are often demarcated from each other by an external groove, thefissura postero-luteralis. (b) The two commissural systems present in the petromyzontian cerebellum (viz., the com. vestibulo-lateralis, and the com. cerebelli), also occur in most gnathostomes. In these groups the commissures can be clearly related to the two fundamental parts of the cerebellum. (c) The cerebellar commissures appear early in ontogenesis and nerve cells proliferate medially along their fibres from either side. The commissures thus constitute the framework upon which the central parts of the cerebellum are raised. (d) The two fundamental divisions of the cerebellum are derived from different parts of the rhombencephalic alar plates. The lobus vestibulo-lateralis ( c q . vestibularis) develops from the special somatic sensory column, whereas the corpus cerebelli arises from the general somatic sensory column. There can be no doubt that the subdivision worked out by Larsell is a major contribution to the comparative anatomy of the cerebellum. It should be pointed out, however, that although the lobus vestibulo-lateralis (c.q. vestibularis) is certainly the most important cerebellar end station for vestibular fibres in all groups of vertebrates, a certain proportion of these fibres terminates in the corpus cerebelli in many groups (sharks, teleosts, amphibians, birds, mammals). It may also be noted that the question of whether Larsell’s conclusion that the two fundamental parts of the cerebellum are derived from different ‘functional’ columns or subcolumns of the oblongata is based upon conjecture or upon direct observation can only be answered after the appearance of his extensive monograph, which is expected shortly. So far only Jansen and Brodal’s (1958) brief summary of this work is available. 5. The lobus vestibulo-lateralis consists in most groups of fish and in urodeles of a small intermediate portion and an enlarged lateral portion on each side. These lateral portions, or auriculae cerebelli, are the result of a laterally or rostro-laterally directed evagination, a process in which not only cerebellar tissue but also the adjacent roof of the fourth ventricle has become involved. The auriculae are particularly large in References p . 88-93

84


groups with a strong development and high degree of differentiation of the lateral line system (e.g. Chondrichthyes, Polypteriformes and the crossopterygian Latimeria). 6 . Auriculae, in the sense of lateral projections of cerebellar tissue surrounding ventricular cavities, are lacking in teleosts. The basolateral portions of the cerebellum are formed in this group by solid masses of granule cells, the eminentiae granulares. There is some controversy in the literature concerning the morphological significance of these structures. The present author considers it likely that they are homologous to the rostral parts of the auriculae of primative actinopterygians and other fish. 7. The auriculae and the eminentiae granulares both represent primary and secondary vestibular and lateral line nerve centres. In actinopterygians the cerebellum contains, in addition, a tertiary centre of the lateral line nerve system. This centre, known as the valvula cerebelli, is not a derivative of the auriculae but is rather a rostral outgrowth of the corpus ceiebelli. This portion of the cerebellum usually occupies a subtectal position, but in one group of teleosts, the mormyrids, the valvula is greatly hypertrophied and has become a superficial structure which covers all other parts of the brain. This hypertrophy is related to the high degree of differentiation of the lateral line system in these fish. 8. In anurans and in most groups of reptiles the lobus vestibularis is only feebly developed, but it is larger in alligators, birds and mammals and can be divided into an intermediate part, the nodulus and aJEocculuson each side. Together, these structures are commonly designated as the lobus JEocculo-nodularis. 9. The corpus cerebelli is, with regard to its shape, doubtless the most variable structure of the central nervous system. The following conditions can be found among the gnathostomes; (a) In the dipnoans Protopterus and Lepidosiren, in urodeles, anurans, and in snakes the corpus cerebelli maintains a plate-like configuration. (b) In Chondrichthyes, Holostei, Teleostei, Crossopterygii (Latimeria), Crocodiles, birds and mammals the corpus cerebelli has evaginated. (c) A condition intermediate between (a) and (b) is found both in the dipnoan Neoceratodus and in Chelonia. The corpus cerebelli is represented by a curved plate in these forms. (d) In some primitive actinopterygian groups (Polypteriformes, Chondrostei) the cerebellum has inuuginated into the ventricular system. (e) A clearly everted cerebellum is found in the rhynchocephalian Sphenodon, and in many lizards. 10. In many Chondrichthyes, some teleosts, and in crocodilians, birds and mammals the corpus cerebelli shows transversely oriented external grooves. In the first mentioned group the entire wall is involved in the folding, but only the external surface is convoluted in the others. The crocodilian cerebellum has two grooves, which Larsell regarded as homologous to the fissura prima and the fissura secunda in mammals. The cerebellum of birds is more complexly fissurated than that of the crocodilians. Comparative embryological and anatomical studies have led Larsell to the conclusion that the fissures, and consequently also the lobules or folia of the avian cerebellum, can be homologized in detail with those of mammals. According to

85


Larsell, the corpus cerebelli is divisible both in birds and in mammals into nine primary folia, conventionally indicated with roman numerals (the lobus flocculonodularis is in both classes designated as the tenth primary folium). Saetersdal(l959) has also reached the conclusion that the avian cerebellum can be divided into ten primary folia. There is a discrepancy between the two authors, however, in respect to the localization of the fissura prima and, consequently, in the delineation of the lobules. 11. Most previous workers have considered the entire corpus cerebelli of birds to be a homologue of the mammalian vermis. Larsell (1948), however, has regarded it probable that a small swelling covered with non-foliated cortex, situated on either side of the basis cerebelli, represents a primordial hemisphere. The views of Larsell have received considerable support from the work of Brodal et al. (1950), which has revealed that in birds the laterally situated non-foliated cortex and the adjacent parts of the folia V-VIII receive a considerable ponto-cerebellar projection, just as do the mammalian cerebellar hemispheres. 12. The major Cerebellar afferent connexions which have been reported for the various groups of submammalian vertebrates are listed in Table I. The existence only of those systems labelled with an exclamation mark has been confirmed experimentally. It should be noted that, except for the birds, practically nothing is known about the exact termination of the various systems that enter the corpus cerebelli. 13. The eferentfibres ojthe cerebellum can be divided into a rostral and a caudal system. The rostral system, or brachium conjunctivum, decussates in the tegmentum and distributes its fibres to the mesencephalic reticular formation, and in amniotes especially to the red nucleus. In several groups (sharks, teleosts, reptiles, birds) fibres of the brachium conjunctivum have been traced to the oculomotor nucleus. The caudal system includes cerebello-vestibular, cerebello-reticular and probably direct cerebello-motor fibres. The latter are said to terminate in the nuclei of the trigeminal and facial nerves. For most groups of submammalians it has been reported that a certain proportion of the cerebello-reticular and cerebello-motor fibres reach their destination by way of the medial longitudinal fasciculus. TABLE I CEREBELLAR AFFERENTS

Petrom.

tr. lineo-cb. tr. vestib.-cb. tr. trigemino-cb. tr. spino-cb. tr. tegmento-cb. tr. tecto-cb. tr. hypothalamo-cb. tr. olivo-cb. tr. ponto-cb. References p . 88-93

+ + + +

+

+

Chondrichth. Actinopt.

+ + + +!

+ + +! +

Urod.

Anur

+ + + +

+ +

.

Aept.

Birds

+ + +

+ +

+

+

+ +

+!

+ !

i! +! +! +!

86


0

a

. .

b

--

.

C

,.. '. . . . *

d

.-

'.

..

.

e

Fig. 62. Sections showing the cell picture of the cerebellum of s o r e representative vertebrates. (a) lamprey; (b) lungfish; (c) turtle; (d) lizard; (e) pigeon.

14. In lower vertebrates the cerebellar efferent systems are probably mainly composed of axons of Purkinje cells. It is believed, however, that Purkinje axons terminate more and more in the cerebellar nuclei as one ascends the vertebrate scale, and that the latter have gradually become the chief site of origin of the cerebellar efferents. 15. The cerebellar nuclear complex shows a progressive development. In anamniotcs it is represented by a group of diffusely arranged cells or by a single nucleus on each side. Reptiles show a differentiation into two nuclei, and in most birds and mammals three or more cerebellar nuclei can be distinguished. 16. The classical conception concerning the origin and localization of the cerebellar nuclei (Van Hoevell, 1916; Ariens Kappers, 1921) can be summarized as follows. (a) In anamniotes the cerebellar nuclei develop outside the cerebellum proper, in close relation to the vestibular nuclei, and also in the adult stage occupy a subcerebellar position. (b) In the course of phylogeny the cerebellar nuclei have become more distinctly segregated from the vestibular complex, and have shifted from their original position into the cerebellum itself. (c) The definite incorporation of these nuclei within the cerebellum has occurred at the reptilian level; 'true' cerebellar nuclei are thus found in amniotes only. This conception was based almost exlusively upon studies of adult material. Rudeberg (1961), however, on the basis of a thorough embryological analysis, reached the conclusion that in all classes of vertebrates the cerebellar nuclei develop from the cerebellar anlage and lie within the cerebellum proper. 17. Histologically, the cerebellum of petromyzonts contains an outer fibrous layer and an inner cellular zone (Fig. 62a). The latter contains cells of two types, viz. small, granular cells and larger elements which are considered to be primordial Purkinje cells. In the cerebellum of most groups of gnathostomes the three characteristic layers can be distinguished: molecular, Purkinje and granular (Figs. 62 b-e). The arrangement of the Purkinje cells shows a considerable variation, however. In many

87


LAMPREY

8,SHARK

D, TURTLE

E, BIRD

A.

C,TELEOST

MAN

Fig. 63. Purkinjecellsofvariousvertebrates.Based on Johnston(1902a): A ; Schaper (1898): B; Schaper (1893): C; Larsell(1932b):D; Cajal(l911): E; and Kolliker (1896): F.

anamniotes they constitute a rather diffuse zone or tend to form small clusters. In teleosts and reptiles the Purkinje layer may be up to four or five cells in thickness. Only in birds and mammals are the Purkinje elements invariably arranged in a single row. 18. The three layers mentioned occupy the entire thickness of the cerebellar wall in most groups of vertebrates. In crocodiles, birds and mammals, however, the granular layer is clearly separated from the ventricular surface by a continuous fibre layer. It may therefore be stated that only these three latter groups possess a true cerebellar ‘cortex’. 19. The large cerebellar cells of petromyzonts, urodeles and dipnoans have a relatively simple dendritic tree which does not clearly show an orientation into a single plane. These elements are therefore designated ‘primordial’ rather than ‘typical’ Purkinje cells. Typical Purkinje elements have been found in most groups of gnathostomes, and there seems to be an increasing complexity in the branching of their dendrites as one ascends the phylogenetic scale (Fig. 63). An exception to this trend is found in the teleosts, however, where the Purkinje cell dendrites are more richly branched than in reptiles. 20. There is some evidence that not only the Purkinje but also the Golgi type 2 cells show an evolutionary increase in the complexity of their dendritic branching. The granule cells, on the other hand, show a remarkable constancy in appearance throughout the vertebrate series. 21. Basket cells have been observed in amniotes only. References p . 88-93

88


22. In conclusion it is emphasized that, whereas our knowledge of the mammalian cerebellum has recently been considerably extended and deepened, most of the fundamental problems concerning the structural organization of the cerebellum of lower forms are still awaiting solution.

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* The unpublished manuscript of Larsell to which these authors had access, had not yet appeared at the time that the present article was prepared.

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WOODBURNE, R. T., (1936); A phylogenetic consideration of the primary and secondary centers and connections of the trigeminal complex in a series of vertebrates. J . comp. Neurol., 65, 403-501. YOSHIMURA, K., (1910); Experimentelle und vergleichend-anatomische Untersuchungen uber die untere Olive der Vogel. Arb. neurol. Znst. W e n . Univ., 18,46-59. ZECHA,A., (1966); Personal communication.

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Comparative Aspects of the Structure and Fibre Connexions of the Mammalian Cerebellum J. V O O G D Laboratory of Neurounatomy, Neurological Institute, University of Leiden, Leiden (The Netherlands)

1. I N T R O D U C T I O N

Most subdivisions of the mammalian cerebellum were developed as a short recapitulation of a special anatomical investigation, but often they have taken on a life of their own, and served as a skeleton for later work. Portions of old nomenclatures, like Edinger’s division in a Palaeo- and a Neocerebellum, Bolk’s lobulus simplex and Hayashi’s pars intermedia of the anterior lobe, live on, suggesting a degree of morphological differentiation far beyond their original significance. It is the purpose of this review to reconsider the evidence for some of these subdivisions, in order to indicate their limitations and to point out their usefulness in future comparative anatomical investigations. A subdivision of the cerebellum is essentially a subdivision of its cortex. Owing to the uniform structure of the cortex it must be based on other criteria, such as regional differences in its histogenesis, the ontogeny and the adult condition of the folial pattern, or the-afferent and efferent connexions.

Fig. 1. Subdivision of the mammalian cerebellum according to Edinger (1910) and Comolli (1910). For abbreviationssee p. 131.

When we compare the diagram of the unfolded cerebellar cortex published by Edinger and Comolli some 50 years ago, with a diagram taken from the more recent review of the cerebellum of Jansen and Brodal (Figs. 1 and 2) we find some striking

STRUCTURE OF MAMMALIAN CEREBELLUM

95

PafYJflok. ac. Fig. 2. Subdivision of the mammalian cerebellum according to Jansen and Brodal(l958, Fig. LOO). For abbreviations see p. 13 1 .

similarities. In both, a number of distinct transverse fissures divide the cortex into a series of transverse lobules and both combine this division with a further division of the cortex into a number of rostro-caudally directed longitudinal zones. In the diagram of Edinger and Comolli this longitudinal division is based on postulated differences in phylogenetic age of the respective zones. The middle zone, the vermis, corresponds to almost the entire avian cerebellum and the lateral zones, the hemispheres, are new acquisitions in mammals, connected with the pons. Their transverse division is based on different considerations, namely on the first two fissures which appear during the ontogeny of the mammalian cerebellum. In the diagram of Jansen and Brodal, the inclusion of transverse fissures which appear later, leads to the distinction of more transverse lobules. Their more intricate longitudinal division, however, is no longer based on phylogenetic differences but on regional differences in histogenesis and on the fibre connexions of the cortex. Both diagrams therefore are intriguing combinations of transverse and longitudinal divisions, which are based on quite different kinds of morphological evidence. 2.

T H E E A R L Y D E V E L O P M E N T OF T H E M A M M A L I A N C E R E B E L L U M

At stages which show the first indications of a transverse division of the cerebellum, it is still difficult to delimit the cerebellar anlage from the rest of the rhombencephalon. Students of its early histology have therefore arrived at different conclusions. According to Van De Voort (1960, Fig. 3) the cerebellum of the rat develops from the rostra1 portion of his ‘rhombic list’. This list is formed by the dorsal part of the alar plate, which provides the attachment to the thin roofplate of the fourth ventricle. Like the rest of the alar plate, it is composed of a ventricular matrix and a mantle References p.1132-134

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-

Fig. 3. The development of the cerebellum of the rat. Reproduced from Van De Voort (1960, Fig. 1). ---_ - matrix of alar plate; 1 I 1 1 I 1 1 = matrix of rhombic list; = secondary (superficial) matrix. For abbreviations see p. 131.

S T R U C T U R E O F M A M M A L I A N CEREBELLUM

97

layer, but it lacks a cell-free marginal layer which is present in the rest of the rhom.bencephalic wall. The rostral portion of the list, rostral to the greatest width of the fourth ventricle, becomes the cerebellum and its caudal portion gives rise to other structures. Owing to the increase in prominence of the pontine flexure between the 12th and 16th intra-uterine day, the rostral alar plate with the rostral list which borders it becomes located dorsal to the caudal part of the rhombencephalon (Fig. 3). In addition to the ventricular matrix the rostral list now possesses a superficial matrix which, from the region of attachment of the roof of the fourth ventricle, migrates over the external surface of the list. Together, both matrices furnish the cells of the future cerebellar cortex and central nuclei. Externally the cerebellar anlage is bordered by the line formed by the joining of the superficial matrix and the cell-free marginal zone of the alar plate. At the ventricular side its border is indicated by a shallow furrow located between the ventricular matrices of the rhombic list and the rest of the alar plate. A part of the fused alar plates therefore separates the cerebellar anlage from the tectum mesencephali.

Mi9r.B Fig. 4A. Section passing through the cerebellar anlage of Bos taurus; 26.5 mm. Reproduced from Riideberg (1961, Fig. 61). Notethe presence of migrationA2B1 in Fig. 4A. Fig. 4B. Section passing through the cerebellar anlage of a rat embryo of 16 days. Reproduced from Van De Voort (1960, Plate It3. Compare Fig. 3. Forabbreviationsseep. 131.

According to Rudeberg (1961) it is difficult in small rodents to trace the migrations which give rise to the cerebellum. In Bos taurus and in man he was able to trace the development of the cerebellum from two migration layers, A and B, derived from the dorsolateral cell column, the most dorsal of the five longitudinal cell columns present in the rhombencephalic wall. The most superficial migration layer A gives rise to cells References p . 132-134

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of the superficial matrix and, together with part of the deep layer B, to the anlage of the lateral cerebellar nucleus (A2B1). A comparison of his illustrations with those of Van De Voort (Fig. 4A and B) shows that his delimitation of the cerebellar anlage includes a much larger part of the alar plate. The portion marked A2B1 moreover clearly possesses a marginal layer, which according to Van De Voort is lacking in the primordium of the cerebellum. Reference to the problem of the demarcation of the cerebellum is made because it has a bearing on Larsell’s fundamental division of the mammalian cerebellum into a vestibular flocculo-nodular lobe and a somesthetic corpus cerebelli. In the opossum

D

E

Fig. 5. Horizontal sections passing ventrally (A) to dorsally (E) through the cerebellar anlage of the Virginia opossum. Reproduced from Larsell(l935, Figs. 29-33). For abbreviations seep. 131.

embryo Larsell(1935) described the ingrowing of fibres of the vestibular and trigemin d roots into the dorsally fused alar plates of the rostral rhombencephalon. Here they cross and constitute a vestibular commissure (Fig. 5, colat), located closely rostral to the attachment of the roofplate of the fourth ventricle and a ‘cerebellar’ commissure (cocb) containing trigeminal and spinocerebellar fibres, located more rostrally. A fissure, the posterolateral fissure (fPL), subsequently develops along the vestibular commissure and soon separates a vestibular region, the future flocculo-nodular lobe, from the more rostrally located, somesthetic corpus cerebelli (CCB). In view of the evidence which Van De Voort and Riideberg derived from the early histology of the mammalian cerebellum it is necessary to reconsider Larsell’s description. Adopting Van De Voort’s definition of the cerebellar anlage as the rostra1 part of the rhombic list, the vestibular commissure is probably located within it. The rostral, cerebellar commissure and the somestheticcorpus cerebelli, however, develop rostrally to it in Van De Voort’s alar plate or near the region A2Bl of Rudeberg, which develops

99


into part of the central nuclei. Larsell's conception of a causal relationship between the two commissures and the development of the cortices of the vestibular flocculonodular lobe and the somesthetic corpus cerebelli therefore must be the subject of renewed investigation.

3.

GROWTH, TRANSFORMATION

AND

MATURE CHARACTERISTICS OF

THE

CEREBELLAR SURFACE

( A ) The subdivisions of Bolk and Lmsell The spatial and temporal patterns in the transformation of the external cerebellar surface, from its original smooth condition into its mature foliated one, are essentially similar in most mammals. In the adult cerebellum the interrelations of lobes and lobules fall into a similar, though slightly modified pattern, the modifications consisting of a loss of prominence of some early fissures, the disappearance of regional differences in the differentiation of the cortex and the addition of characteristic features late during development.

I1 IPS

fPPD

1SEC.

IPL

llNCUS TERMINAL!S

A : BOLK

FLO

8:development of t h e folial

C. LARSELL

p u t t e r n [Larsetl,l952)

Fig. 6 . The nomenclature of the cerebellum. (A) Nomenclature of Bollc. Areas usually devoid of cortex are shaded. (B) Three stages in the development of the folial pattern in a mammal, according to the description of Larsell(1947, 1952,1954). (C) Nomenclature of Larsell. Paramedian lobule shaded. For abbreviations see p. 131.

The surface characteristics of the adult mammalian cerebellum described by Bolk (1906) (Fig. 6A) include the absence of a clear demarcation between vermis and References p . 132-134

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hemispheres in the rostral, and the presence of such a paramedian sulcus in the caudal, part of the cerebellum. In its rostral portion, consisting of the anterior lobe and the simple lobule, the fissures present in the midsagittal plane can be followed laterally over a variable distance, some continuing to the lateral margin of the cerebellar surface. Caudally the medial (vermal) part of the simple lobule continues in a chain of folia, the vermis and its lateral (hemispheral) portion passes over into the folial chain of the hemisphere. In the paramedian SUICUS, between vermis and hemisphere, many of their fissures and part of their cortex may be interrupted. To use Bolk’s terms, the folial chains of vermis and hemisphere therefore represent mutually independent ‘growth centres’, Both may be divided into a number of segments, or lobuli; the folial chain of the hemisphere, moreover, is thrown into a number of loops. Irrespective of the changing direction of inter- and intralobular fissures of the chain, the Purkinje cells keep their orientation perpendicular to them. The lobules of the hemisphere can be defined by their position in the chain and by the degree of continuity of their fissures and their cortex with a particular segment of the vermis. In the rostral part of the chain Bolk distinguished a lobulus ansiformis with laterally diverging folia, followed by the paramedian lobule, situated alongside the vermis. The interlobular fissures of this region of the hemisphere and of portion C of the caudal vermis (comprising lobules VI, VII and VIII of Larsell) are (usually) continuous, while the intralobular fissures are not. Between the ansiform lobule and the vermis the cortex is always interrupted, but between the paramedian lobule and the vermis it is continuous in some species. The terminal portion of the folial chain of the hemisphere (Bolk’s formatio vermicularis) consists of a laterally directed loop, the paraflocculus, and a medially directed one, the flocculus. Here neither the inter- nor the intralobular fissures continue into those of the caudal portion of the vermis, lobules A and B (Larsell’s lobules IX and X respectively). Between the paraflocculus and lobule B the cortex is usually absent, and between the flocculus and lobule A a bridge of the cortex is present in some species. The intricate relationship between the lobules of vermis and hemisphere has been clarified by the ontogenetic studies of Larsell and the authors before him. His way of dividing the avian cerebellum, which lacks a paramedian sulcus, into 10 transverse lobules, led him to a similar distinction of the lobules I to X in the developing cerebellum of the rat which lacks a paramedian sulcus till late in its development (1952). In the cerebellum of the rat, and in other mammalian cerebella described so far, the posterolateral fissure, separating the flocculus and the nodule (lobule X of Larsell) from the rest of the cerebellum, is the first to appear (Fig. 6B). At first it is present only laterally, but at a later stage it runs along the whole caudal margin of the cerebellar anlage. In the medial part of the cerebellum the primary fissure is the next to appear, followed by the posterior superior, the prepyramidal and the secondary fissures caudal to it and a number of fissures in the anterior lobe rostral to it. They delimit Larsell’s 10 lobules in the medial region and start growing laterally. By now the intraparafloccular, the parafloccular, the ansoparamedian, the intercrural and the lateral part of the posterior superior fissures have become visible in the lateral part of the cerebellum.

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Growing medially, they either fuse with fissures in the medial region or, bypassing them, enter the future vermis of the cerebellum. Ultimately the fissures of the anterior lobe delimit the lobules I to V (Fig. 6C). Caudal to the primary fissure, the medial and lateral parts of the posterior superior fissure meet and delimit lobule VI caudally. The intercrural and ansoparamedian fissures either fuse with the posterior superior fissure or continue in the medial portion of lobule VII. The prepyramidal fissure, which borders lobule VII caudally, does not fuse with a laterally arising fissure, but continues laterally, often to the lateral margin of the cerebellum. The parafloccular fissure grows medially into the medial portion of lobule VIII, dividing it into two portions, VIII A and VIII B. The secondary fissure, which separates lobule VIII from lobule IX, fuses laterally with the interparafloccular fissure. The earliest developed fissure, the posterolateral, delimits lobule IX from lobule X, the flocculo-nodular lobe. (B) The morphology of the cerebellum in some mammalian species Bolk‘s and Larsell’s conceptions on the mammalian cerebellum can be verified in a number of mammalian species, which have been available for experimental investigations (Figs. 7 and 8). The anterior lobe shows a uniform pattern. Larsell’s lobules I to V which he described in the rat (1952) and the cat and the monkey (1953) can be recognized from the arbor vitae. The interlobular fissures all extend to the lateral margin of the cerebellum. In Myoris, the anterior lobe is divided by one fissure only, the two lobules probably correspond to lobules I to 111and IV to V respectively. In midsagittal section, lobule V shows the greatest variation, and its rostrocaudal extent is largest in Macaca and man. Corresponding variations are observed in the width of the anterior lobe. Lobules I and I1 do not reach very far laterally. Conspicuous differences are found between the short medio-lateral extent of the lobules IV and V in Hyrax, goat and cat, and the much greater length of their folia in ferret, Macaca and man. Although a distinct paramedian sulcus was found to be lacking in all species, slight changes in the surface contour of the anterior lobe in some of them suggest its longitudinal subdivision. The region just caudal to the primary fissure, Bolk‘s lobulus simplex or Larsell’s lobule VI, can hardly be distinguished from the anterior lobe. A faint constriction of its folia divides it into a medial and a lateral part or, as in Macaca, into medial, intermediate and lateral ones. The fissures present in its medial portion continue in its lateral part. The last of these continuous fissures is the posterior superior fissure, separating the lobulus simplex from lobule VII, which shows a distinct division into vermis and hemispheres. In a midsagittal section of the cerebellum the posterior superior fissure can be recognized by its depth in all mammals. Laterally in the larger cerebella of goat, ferret, cat, monkey and man, it usually follows an asymmetrical course. No distinct border is therefore present between the lateral part of the simple lobule and the ansiform lobule, the first segment of the folial chain of the hemisphere. The fissures delimiting the lobules of the caudal vermis can usually be recognized in References p. 132-134

102

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Figs. 7 and 8. Caudolateral aspect and midsagittal section of some mammalian cerebella. In the diagrams to the right the shaded areas indicate the extent of the areas devoid of cortex. The distribution of spino-cerebellar mossy fibre degeneration, as present in Nauta-stained series of cases of high hemichordotomy, is indicated by dots. The distribution in the cat was copied from Grant's diagrams (1962b). In the human cerebellum the homologues of the pyramis and its lateral extensions according to Larsell (1959) are shaded. For abbreviations see p. 131. References p . 132-134

I04

J. V O O G D

a midsagittal section of the cerebellum, and can be further identified by tracing them laterally. Of the fissures separating the lobules of the caudal vermis, the prepyramidal fissure, between the lobules VII and VIII, can usually be traced into the hemisphere. It stops near (ferret, man) or at the lateral border of the cerebellar surface (opossum, Hyrax, rat, hedgehog, rabbit, goat, Mucacu). The posterolateral fissure, between the lobules IX and X, in the vermis is very conspicuous in all mammals, but it can be traced into the hemisphere only in Hyrax and opossum. Neither the secondary fissure, between the lobules VIII and IX, nor any of the intralobular fissures of the lobules V11 to X can be traced beyond the paramedian sulcus. The identification of the secondary fissure therefore mainly depends on its depth. On the basis of the continuity of the fissures, the folial chain of the hemisphere can be divided into three segments. The first lies between the posterior superior and the prepyramidal fissure. It corresponds to the hemispheral portion of lobule VII, or Bolk’s ansiform and the rostral part of his paramedian lobule. In its most simple form as seen in Hyrax, it consists of three folia. In most other mammals it consists of two or more rosettes of folia, radiating from a common centre formed by an extension of the paramedian sulcus. In all mammals investigated, with the exception of Hyrax, this centre is devoid of cortex, and the isolation of the hemisphere in this region therefore is complete. The changes in direction of the folial chain in this region, caused by the succession of rosettes of diverging folia, have led to the further subdivision of the region into Crus I, Crus 11, ansula and paramedian lobule. The great variations in the morphology of this complicated region between the species reduce the comparative anatomical value of these subdivisions. Bolk’s statement that the ansoparamedian lobule should be studied as a whole in comparative anatomy is worthy of repetition. The second and third segments of the folial chain of the hemisphere are divided by the lateral part of the posterolateral fissure. They correspond to the caudal part of Bolk’s paramedian lobule with the paraflocculus and the flocculus respectively. In their morphology they show only minor variations from one species to another. The caudal part of the paramedian lobule is represented by one or two folia (Hyrax, opossum, Myotis, rat, hedgehog, rabbit) or by a rosette of folia (goat, ferret, cat, monkey, man). Laterally the last folium of the paramedian lobule continues into the first of the paraflocculus. The latter consists of a chain of rather small folia, which initially is directed rostraIly and subsequently is bent upon itself. The paraflocculus thus encloses a deep fossa, the interparafloccular sulcus, which at the bottom is devoid of cortex. Rostra1 to the interparafloccular sulcus the cortices of the dorsal and the ventral limb of the paraflocculus are continuous, caudally and medially this sulcus opens into the paramedian sulcus. At its end the medially directed folial chain of the hemisphere, represented by the ventral paraflocculus, abruptly turns laterally and terminates in one folium or a rosette of folia, the flocculus (the lateral part of Larsell’s lobule X or the ‘uncus terminalis’ of Bolk’s terminology). Both of the latter names are appropriate because in Hyrax the cortex of lobule IX and X continues in the cortex of the last folium of the ventral paraflocculus and the flocculus respectively, the posterolateral fissure intervening between them. In the


105

opossum the area devoid of cortex in the interparafloccular sulcus completely separates the cortex of lobule IX from the ventral paraflocculus, and the medial portion of the posterolateral fissure does not continue in a fissure between the flocculus and the ventral paraflocculus, although a small rim of cortex interconnects the vermal logule X and the flocculus. In the other species investigated, even this cortical connexion is absent. Here, the area devoid of cortex in the interparafloccular fissure extends along the bottom of the paramedian sulcus and completely separates the vermal lobules X, IX (rat, hedgehog, rabbit), part of lobule VIII (goat, ferret, monkey) and even lobule VII (cat) from the hemisphere. The cortex of the ventral paraflocculus and that of the flocculus, however, are continuous. Subdivisions of this part of the folial chain of the hemisphere, such as the caudal part of the paramedian lobule, the dorsal and ventral paraflocculus and the flocculus, can only be distinguished by the more or less sudden changes in direction of its folia. The comparison of these subdivisions in different species is therefore hindered by the difficultyin identifying the particular fissures separating them. In the human cerebellum the portion of the folial chain of the hemisphere caudal:to the prepyramidal fissure is represented by the caudal part of the gracile lobule, the biventral lobule, the tonsilla and the flocculus. Like other mammals this part of the chain encloses a parafloccular sulcus which at the bottom is devoid of cortex. Medially this bare area continues on the lateral surface of the lobules VIII and IX of the caudal vermis. The cortex of lobule IX continues laterally in that of the last folium of the tonsilla, a configuration reminiscent of Hyrax; the cortices of vermal lobule X and the flocculus are discontinuous, however. The cortex of the flocculus, moreover, is completely separated from the rest of the folial chain of the hemisphere, a situtation which is never encountered in other mammals. The different topographical relations cf this part of the hemisphere in man may be explained by the great width of the folia of the gracile and biventral lobules as compared with the caudal parts of the paramedian lobule and the dorsal paraflocculus in other mammals. In conclusion, we can state that Bolk and Larsell each accentuated different characteristics of the mammalian cerebellum. Bolk’s description of its composition as an undivided rostra1 portion and a caudal ‘lobus complicatus’ consisting of vermis and hemispheres, adequately summarized its structure. The apparent independence of the folial chains of vermis and hemispheres is accentuated by the discontinuity of most of their fissures and part of their cortex in the paramedian sulcus. Larsell’s distinction of 10 lobules is a suitable basis for the description of the cerebellum. His description of the development and the mature relations of the prepyramidal and posterolateral fissures, moreover, facilitates the understanding of the interrelations of the lobules of vermis and hemispheres. His conception of the cerebellum as a series of transverse lobules, all divided into a vermal and two hemispheral parts, however, is questionable. In most mammals the folial pattern of the anterior lobe and the simple lobule does not justify such a distinction. Also, the detailed relationship between the lobules and sublobules of vermis and hemisphere in the region between the posterior-superior and prepyramidal fissures and between the latter and the posterolateral fissure, as described by him, could not be substantiated in my material. References p . 132-134

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( C ) The interrelation of vermis and hemispheres and the focal differentiation of the cerebellar cortex Although the development of the folial pattern has been studied in great detail by macroscopicinspection, the underlying histogenetic events have been largely neglected. In the literature only few studies are reported which biidge the gap in our knowledge between the early development of the cerebellar anlage, reported in the first section of this paper, and its adult appearance. All of them have been carried out in man.

ROSTRAL

CAUDAL Fig. 9. Rostra1 and caudal views of a cerebellum of a human embryo of 4 months. From Langelaan (1919). For abbreviations seep. 131.


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As pointed out by Langelaan (1919) the development of transverse fissures is usually preceded by a proliferation of cells perpendicular to the direction of such a fissure. Other fissures, the paramedian sulcus in particular, ‘seem to be formed by differences in growth between the regions adjoining the groove and those forming the bottom of the groove’ (p. 134). The paramedian sulcus, however, is not the only longitudinal groove present in the cerebellum. In embryos of 3 and 4 months Langelaan depicts (Fig. 9) the presence of one midsagittal groove and two, and in the caudal half of the cerebellum even four, parasagittal grooves. According to him, the second of these grooves corresponds to the paramedian sulcus. Together with the transverse fissures the longitudinal grooves form a kind of lattice. The elevations found in its interstices were called ‘foci’ by Langelaan. If his description of the origin of the paramedian sulcus also holds for the other longitudinal grooves, a focus is the result of the different mode of origin of the transverse and the longitudinal division of the cerebellum. This assumption receives support from the work of Hayashi (1924) and Jakob (1928) who similarly observed the first signs of the longitudinal division of the cerebellum in a stage prior to the appearance of the first transverse fissures in the corpus cerebelli. They described a midsagittal and two parasagittal grooves delimiting a medial, an intermediate and a lateral zone (Fig. lo), but Langelaan’s distinction of additional grooves in the caudal part of the cerebellum was not confirmed by them. Moreover, Hayashi differs from the others because he recognized grooves in the region of the future anterior lobe only.

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Fig. 10. Caudal view of a cerebellum of a human embryo of 9.2 cm. CF. Redrawn from Jakob (1928). a = medial zone (vermis) of the anterior lobe; b = intermediate zone of the anterior lobe. For abbreviations see p. 131.

Even before the appearance of the external granular layer, the three zones which are delimited by the longitudinal grooves, show differences in cortex differentiation. In Jakob’s low power photographs the cortical plate gives the impression of being beaded References p . 132-134

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and the cellular material destined for a special zone appears to be sharply demarcated, but the external granulai layer does not show such beading. This can be observed in several of Hochstetter’s pictures (1929), one of which is reproduced in Fig. 1 1 and in some figures from Rudeberg’s thesis (1961).

Fig. 11. Human embryo of 104 mm. Reproduced from Hochstetter (1929, Fig. 191) showing the subdivision of the cortical plate.

In later stages these differences in histogenesis and the longitudinal grooves disappear completely, with the exception of the paramedian sulcus separating the verniis from the hemisphere. According to Jakob and Hayashi the vermis arises from the fusion of the medial zones only. In their diagram of the cerebellum (Jakob, 1928, p. 743) they therefore distinguish an ‘intermediate zone’ located between the vermis and the lateral part of the anterior lobe, but they are not sure about the localization of an intermediate zone in the caudal part of the cerebellum. Langelaan, however, states that the veimis is formed by the fusion of the foci belonging to both the medial and intermediate zones, the intermediate zone of Hayashi and Jakob therefore is incorporated in the vermis and present in both the rostra1 and the caudal portion of the cerebellum. The old controversies about the role of the ventricular matrix and the external granular layer in the formation of the cerebellar cortex have been largely settled by the autoradiographic studies of Miale and Sidman (1961) in the mouse. According to them, the future Purkinje cells are formed in the ventricular matrix and subsequently migrate outwards to become located in the cortical plate, a layer which remains separated from the pial surface by the future molecular layer. At a stage in which the activity of the ventricular matrix has stopped, the external granular layer arises from the region of attachment of the roof of the fourth ventricle. It constitutes a superficial matrix


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which remains active until well after birth. It gives rise to the granular cells which reach their ultimate position by moving inward. It is possible that the beaded appearance of the cortical plate and the early longitudinal fissuration of the cerebellum described by Hayashi and Jakob are produced by the future Purkinje cells. Their subsequent rearrangement in a layer of single cells may be the cause of the disappearance of this longitudinal pattern. Similarly it is probable that the proliferation which, according to Langelaan, precedes the appearance of the transverse fissures, takes place in the external granular layer, the only cortical layer able to proliferate. Further investigations of the complicated histogenesis of the cerebellar cortex are necessary, however, to settle these points. In the light of the different modes of origin of the transverse and longitudinal fissures of the cerebellum, the question of the interrelation of vermis and hemispheres is much clarified. It must be kept in mind, however, that the histogenetical evidence points to a more complicated longitudinal division of the cerebellum than that in vermis and hemisphere alone. 4.

A F F E R E N T C O N N E X I O N S OF T H E CEREBELLUM

The afferent connexions of the cerebellum can be divided into those terminating as mossy fibres and those which terminate in a different manner. The arguments for such a distinction can be found in the papers of Carrea et al. (1947) and Szenthgothai and Rajkovits (1959). ( A ) Fibre systems terminatimg as mossy fibres (1) The vestibulo-cerebellar fibres. The termination of the vestibular root fibres was studied recently by Carpenter (1960) and Brodal and Herivik (1 964) ;the mode of termination of the secondary vestibulo-cerebellar fibres is unknown. (2) The spino-cerebellar tracts (Grant, 1962b). (3) The reticulo-cerebellar fibres arising from the reticular nuclei of the medulla oblongata were reviewed by Brodal (1957), Busch (1961) and Voogd (1964). (4) The cuneo-cerebellar fibres, arising from the external cuneate nucleus, were recently studied by Grant (1962a). (5) The brachium pontis, which arises from ipsi- and heterolateral basal pontine nuclei, the nucleus reticularis tegmenti pontis and the nucleus raphe pontis located in the pontine tegmentum (for references see Jansen and Brodal, 1958, and Voogd, 1964). It can be divided into : (a) the ‘spinal system’ (spinal in the sense of caudal), which consists of crossed and uncrossed, coarse and medium-sized fibres, arising from the tegmentum pontis and the caudal, dorsal and lateral parts of the basal pontine nuclei. (b) the ‘cerebral’ system (cerebral in the sense of rostral) which consists of mainly crossed, fine fibres arising from the rostral, ventral, and medial parts of the basal pontine nuclei. This list is not complete; fibre systems of uncertain mode of termination such as the References p.1132-134

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fibres of the perihypoglossal nuclei and those from the raphe nuclei have not been included. Moreover, the termination of these fibre systems is not necessarily restricted to the cortex, and occasionally terminations in the central nuclei have been described, but a systematic study of the extracerebellar afferents of the central nuclei is still lacking.

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Fig. 12. Diagram of the extra- and intracerebellar course of the main fibre systems terminating as mossy fibres in the cerebellum of the cat. Thin lines: ponto-cerebellarfibres. Heavy lines: bulbo- and spinocerebellar systems. For abbreviations see p. 131.

In Fig. 12, based on my investigations in the cat, the course of some of the fibre systems terminating as mossy fibres is indicated. The fibres of the ‘cerebral’ system, which arise from the rostra1 part of the pes pontis, are located in the superficial part of the brachium. They are directed caudally and cross over the fibres of the ‘spinal’ system, which arise from the tegmentum and the caudal part of the pes pontis and occupy the deep layers of the brachium. Part of the fibres of the ‘cerebral’ system fan out laterally to terminate in the hemisphere, others reach the vermis in broad curves, sweeping through the caudal and dorsal regions of the cerebellum. Some of these fibres run medially in the bottom of the parafloccular sulcus to terminate in lobule IX of the vermis. In normal material these fibres may give the impression of a connecting stalk between lobule IX and the paraflocculus. Others, running medially in the dorsocaudal apex of the cerebellum, form the floor of the region devoid of cortex in the centre of the ansoparamedian lobule. These fibres either enter the dorsocaudal part of the cerebellar commissure or terminate in the cortex of this part of the vermis. The fibres of the ‘spinal’ system enter the cerebellum medially and rostrally to


111

those of the 'cerebral' system. Some leave the main body, immediately after it emerges from under the cover of the 'cerebral' system, to terminate in the flocculus. Others, running medially, describe similar but smaller curves than the fibres of the latter system and reach the cerebellar commissure ventrally and rostrally to those of the 'cerebral' system. Some fibres pursue a still more rostral course, enter the lateral part of the anterior lobe, and proceed medially in the top of its lobules. The bulbar components of the restiform body take a similar course in a still more rostral and ventral plane, and in the cerebellar commissure the decussating fibres of the restiform body occupy a position ventral and rostral to those of the 'spinal'

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Fig. 13. Diagrams of the unfolded cerebellar surface of the cat, showing thelocalization of the main afferent fibre systems in the cortex of the cat. The diagram of the vestibulo-cerebellar projection is based on the investigations of Brodal and H ~ i v i k(1964). The diagrams of the spino- and bulbocerebellar projections are based on the studies of Grant (1962a, b) and Voogd (1964). The diagrams of the ponto-cerebellar projections are based on my earlier investigations (Voogd, 1964) and on unpublished materialillustrated in Fig. 14. For abbreviations see p. 131. References p . 132-I34

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Fig. 14. Sections selected from a transverse and a parasagittal series illustrating the distribution of degenerated ponto-cerebellar mossy fibres 10 days after a total, midsagittal transection of the pes pontis. Cat. Nauta stain. For abbreviations see p. 131.

system. The dorsal and ventral spino-cerebellar fibres, which enter the cerebellum nearest the midsagittal plane, cross in the most ventral and rostral part of the cerebellar commissure. Together the medially directed, so-called ‘semicircular’ fibres of these afferent systems constitute a continuous layer, located rostrally, dorsally and laterally to the central cerebellar nuclei. It extends from the ventral spino-cerebellar tract in the anterior medullary velum into the white matter of lobule VII in the caudo-dorsal part of the cerebellum. Medially this layer continues in the cerebellar commissure. From all over the layer, fibres and/or collaterals branch off both in a rostral and a caudal direction to terminate in the cortex. In this way, the concentrically arranged afferent cerebellar systems within the layer of semicircular fibres are projected both on the rostral and the caudal cerebellar surface, where their sites of termination show the same concentric disposition (Fig. 13). Pontocerebellar fibres of the ‘cerebral’ system terminate in lobule VII of the caudal vermis, located at the junction of the rostral and the caudal cerebellar surface and in the adjoining part of the ansoparamedian lobule. The fibres of the ‘spinal’ system

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terminate both rostrally and caudally to the ‘cerebral’ system, but they do not spread as far laterally as those of the latter. In the rostral cerebellar surface they project as far as the dorsal and lateral parts of lobule VI and the adjoining part of the ansoparamedian lobule. More rostrally, they terminate in the cortex on the top and the extreme lateral parts of the lobules V to I1 of the anterior lobe (Fig. 14). In the caudal cerebellar surface the terminations of the two pontocerebellar systems cannot be differentiated, but here also ponto-cerebellar fibres terminate in the apex of lobule VIII and in the lateral part of the paramedian lobule. Bulbo- and spino-cerebellar fibres terminate still more rostrally and caudally. In lobule VI and lobule V of the anterior lobe, bulbo-cerebellar fibres terminate ventrally and medially to those of the ‘spinal’ system. Some spino-cerebellar fibres terminate in patches of cortex at the base of these lobules, but most of them terminate in still more rostral parts of the anterior lobe. The projection of the bulbo-cerebellar systems to the caudal cerebellar surface includes the rostral parts of lobule VIII and the adjoining paramedian lobule, but spino-cerebellar fibres terminate in their caudal parts. Vestibular root fibres (Carpenter, 1960; Brodal and Hsivik, 1964) terminate in the most rostral and caudal cerebellar lobules, lobule I and lobule X with the adjoining part of lobule IX and the flocculus. A second zone of termination of the ‘cerebral’ system, composed of the rostral half of lobule IX and the paraflocculus, lies intercalated between the spinal and the vestibular projections on the caudal cerebellar surface. Caudally it is bordered by the projection of the spinal system of the brachium pontis to the flocculus. A simple division of the cerebellar surface in vestibular, spinal, bulbar and pontine regions (Ingvar, 1919; Marburg, 1924; Larsell, 1958) obviously can no longer be held. In some places the borders of these regions, which give the impression of contour lines, are located at the bottom of a fissure, but mostly they run along its sides between the base and the apex of the lobules, dividing them in proximal and distal portions. Each lobule or sublobule therefore displays its own characteristic distribution of afferent fibres. With respect to their afferent connexions, they represent regional specializations rather than mere expansions of the cerebellar surface. A similar relationship between the folial pattern and the projections of the afferent fibre systems can be observed in a number of mammals in which a high hemicordotomy is performed (Figs. 7 and 8). In all of them mossy fibre degeneration is present in the anterior lobe, in the bottom of the primary fissure and in the sides of the secondary fissure in lobules VIII and IX and the adjoining part of the paramedian lobule. The simplest configuration is seen in the anterior lobe of Myotis, which is completely occupied by spino-cerebellar fibres. In the hedgehog, the rat and the opossum the distal, dorsal part of the lobules IV and V and the caudal part of lobule I are devoid of spino-cerebellar afferents. In the rabbit, the cat, the ferret, Hyrax and the goat, the latter area is more extensive and includes the proximal parts of lobules 11,111and IV. In Macaca the distal portions of lobules 11,111, IV and V lack spino-cerebellar afferents, which in this animal prove to terminate between the apex and the base of the lobules I1 to IV and in a small area around the base of lobule V. The origin of fibres terminating in lobule I and in the proximal parts of the lobules I1 to IV is unknown, References p . 132-134

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but probably they are vestibulo-cerebellar fibres. In accordance with the findings in the cat, the distal parts of the lobules IV and V probably receive bulbo-cerebellar fibres. The latter area, which apparently is small in insectivores and somewhat larger in rodents, carnivores, and ungulates, apparently reaches its peak of development in Macuca, with the incorporation of the distal parts of lobules 111and 11. The concentric disposition of the projections of the different afferent systems, which is responsible for the differences in afferent connexions between the apex and the base of the lobules in the cat, can therefore also be recognized in mammals of other orders. In the hedgehog, the ferret, the goat and Mucucu, spino-cerebellar mossy fibre degeneration is present in lobule VIII, whereas in the other species it is also present in the rostral part of lobule IX on the caudal side of the secondary fissure. In all species investigated, with the exception of Myotis and the rat, the rostral part of lobule VIII lacks spinocerebellar afferents, whereas in the cat bulbo-cerebellar fibres terminate in this region. In the paramedian lobule, the spino-cerebellar fibres reach further rostrally than in vermal lobule VIII and in most species they even surpass the prepyramidal fissure. This part of the hemisphere and lobule VIII reach their highest development in Mucacu. The projection of spino-cerebellar fibres to this region therefore forms the mirror image of their distribution in the anterior lobe. The terminations are concentrated in the caudal parts of lobule VIII and the paramedian lobule, and more rostrally they are present in the bottom of the fissures only, diminishing in the lateral part of the paramedian lobule. Judging from Smith’s description (1961), the same distribution can be observed in the human cerebellum. According to Smith, the fibres terminate in the pyramid and the adjoining part of the biventral lobule, the homologues of lobule VIII and the caudal part of the paramedian lobule respectively (Larsell, 1959). The explanation of the differences between the folial pattern and the distribution of afferent systems may be found in the regional variations in the histogenesis of the cortex. According to Van Valkenburg (1913), Winkler (1927), Jakob (1928) and Langelaan (1919) the histogenesis starts in the rostral part of the anterior lobe, the bottom of the incipient primary, prepyramidal and secondary fissures, a region corresponding to the distribution of the vestibular and spino-cerebellar fibres in many mammals. From these early loci the differentiation of the cortex proceeds in a concentric manner, finally to reach the apex of the lobules, the portion of the vermis intercalated between the primary and prepyramidal fissures (lobule VII) and the lateral parts of the hemisphere, i.e. the region known to receive ponto-cerebellar fibres. Keeping this in mind, we can state that the transverse folial pattern is an expression of the distribution of the afferent fibre systems. The increase in surface area of the lobes, lobules and individual folia, both during phylogeny and ontogeny, therefore probably occurs by ‘intussusception’(Winkler) of bulbo- and ponto-cerebellar projection areas. It may be questioned why the longitudinal division of the cortex during its differentiation does not leave its traces in the distribution of the afferent systems; in fact however such traces probably are left. Spino-cerebellar mossy fibres in the rabbit (Van Rossum, unpublished, Fig. 15) show a similar division into clusters. In the lobules 11, 111 and IV maxima of spino-cerebellar degeneration are present, one in the midsagittal plane, and four at each side. The second of these maxima, located at an inclination of

S T R U C T U R E OF MAMMALIAN CEREBELLUM

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Fig. 15. The distribution of mossy fibre degeneration in the cerebellum of the rabbit 7 days after a rightsided hemicordotomy at Th. 10/11. Nauta stain. Unpublished observations of Van Rossum. For abbreviations see p. 131.

the folia, is also present in lobule V. In the bottom of the primary fissure, one midsagittal and two parasagittal maxima are again present. In lobule IX one mid- and one parasagittal maximum are seen and in lobule VIII an additional one extends into the caudal part of the paramedian lobule. A similar division of the spino-cerebellar afferents is observed in transversely sectioned series of other species, including man (Nikitin, 1925).

(B) Fibre systems not terminating as mossy fibres Of the two kinds of terminals in the cerebellar cortex, the mossy and the climbing fibres, the origin of the latter is still a matter of conjecture. With respect to Carrea, Reissig and Mettler’s experimental demonstration of their origin within the central cerebellar nuclei (1947), Szenthgothai and Rajkovits (1 959) considered their degeneration criteria to be inconclusive. According to them, the characteristic terminal portion of the climbing fibre along the dendrites of the Purkinje cell fails to become impregnated with any of the known silver methods for degenerating axons, and degenerating climbing fibres can consequently only be recognized by their unbranchedcoursethrough the granular layer and their collaterals running through the layer of Purkinje cells (Scheibel and Scheibel, 1954). The visible portion of the degenerating climbing fibre therefoie follows the same course as the recurrent collaterals of Purkinje cell axons, which constitute the system of short and long association fibres described by Eager (1963a). References p . 132-134

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In this way Szenthgothai and Rajkovits established that the olivo-cerebellar system terminates as climbing fibres. According to them degenerating climbing fibres are moreover present after lesions of the brachium pontis and the ventral spino-cerebellar tract, findings which could not be corroborated in my material. The climbing fibre degeneration in the flocculus observed by Szenthgothai and Rajkovits after lesions, including the brachium conjunctivum observed by CupCdo (1963) in the rat, was identified with the degeneration of the tecto-cerebellar tract which CupCdo traced to the flocculo-nodular lobe, lobules IX and VIII, the paraflocculus and the rostra1 part of the anterior lobe. The olivo-cerebellar system stands apart from the fibre systems terminating as mossy fibres, not only by its mode of termination but also by the uniform, small size of its fibres, and by their intracerebellar course between the layer containing the mossy fibre systems and the central cerebellar nuclei with their efferent tracts. Their position in close proximity to the central cerebellar nuclei offers an additional argument against Carrea, Reissig and Mettler's supposition that climbing fibres arise in the central nuclei, since lesions of the latter usually include the olivo-cerebellar system. The main interest in this system lies in the remarkably sharp and detailed correlation between parts of the inferior olive and special lobules of the cerebellum and in the evidence it provides for the longitudinal division of the anterior lobe (Brodal, 1940; Jansen and Brodal, 1958).

CAT

(BRODAL'LO)

MAN ( J A N S E N

AND BRODAL'SB)

OLIVO-CEREBELLAR PROJECTION Fig. 16. The projection of the dorsal accessory olive and the principle olive on the anterior lobe in cat and man.For abbreviations see p. 131.

The latter aspect is illustrated in Fig. 16. In the anterior lobe of the cat, the vermis (small dots) is arbitrarily defined as a zone of the same width as the caudal vermis. It receives fibres from the lateral part of the heterolateral dorsal accessory olive. The


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medial part of the latter projects to the lateral part of the anterior lobe of the cat. An additional projection of the rostra1 pole of the principal olive to the lateral part of the anterior lobe can be observed in rabbit and man, but not in the cat. In the anterior lobes of rabbit and man, therefore, three longitudinal zones can be distinguished, a vermis and a paravermal region, receiving fibres from the dorsal accessory olive, and a lateral zone, which receives fibres from the principal olive, whereas in the cat this lateral zone is absent or very small. Brodal compared this paravermal zone with the pars intermedia of the anterior lobe of Hayashi and Jakob, which can be distinguished during certain stages of the development of the human cerebellum. It should be remembered, however, that according to Langelaan the pars intermedia later on becomes incorporated in the vermis. Contrary to my observations on the systems terminating as mossy fibres, which show a mainly transverse orientation and which expand by intussusception, the olivocerebellar system shows evidence of a longitudinal organisation and the apposition of a lateral zone receiving fibres from the principle olive. 5.

T H E S T U D Y OF T H E E F F E R E N T C O N N E X I O N S O F T H E C E R E B E L L U M

In order to describe the efferent connexions of the cerebellar cortex and the central cerebellar nuclei, there must be an agreement on their respective subdivisions. Because most authors divided the central nuclei into medial, intermediate and lateral parts, the corresponding division of the cerebellar cortex may be expected to be a mediolateral one, i.e. a division into longitudinal zones, rather than a division into transverse lobules. The evidence available on the longitudinal division of the cortex and the possibilities of dividing the central nuclei will therefore be considered first, and subsequently the cortico-nuclear interrelations will be considered. ( A ) Substantiation of the longitudinal division of the cerebellum

Part of the following substantiation of the longitudinal division of the cortex is provided by the study of its development and afferent connexions already considered. Further evidence can be derived from the structure of the white matter of the cerebellum as well as from its histochemical differentiation. (1) The paramedian sulcus divides the caudal part of the mammalian cerebellum into the folial chains of vermis and hemispheres. In lobule VI and in the anterior lobe a paramedian sulcus cannot usually be identified. According to some authors the paramedian sulcus arises as a border between regions differing in their degree of cortex differentiation. (2) In the human cerebellum, Jakob and Hayashi described three such zones on each side of the midsagittal plane. The fused medial regions become the vermis and the middle, paravermal region is known as their ‘pars intermedia’. In the anterior lobe their borders disappear without a trace, but in the caudal part of the cerebellum the lateral border of the vermal region remains as the paramedian sulcus. According to Langelaan both the medial and intermediate regions fuse to become the vermis, and References p . 132-134

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the paramedian sulcus arises from the border between the middle and the lateral regions. The region of the future biventral lobule and the tonsilla, moreover, shows evidence of a further longitudinal division (Fig. 9). (3) Another indication of a longitudinal division of the cerebellar cortex is found in the distribution of the spino-cerebellar mossy fibres. In the anterior lobe of the rabbit (Fig. 15) a midsagittal and four parasagittal maxima of spino-cerebellar mossy fibre degeneration are present, and the same is seen in the posterior part of the cerebellum. Taken as the borders of longitudinal cortical zones, these maxima delimit five cortical zones on each side of the anterior lobe. (4) On the basis of the olivo-cerebellar projection to the anterior lobe, Brodal(l940) distinguished an arbitrarily delimited vermis, a paravermal intermediate zone and a lateral zone.

Fig. 17. The distribution of the enzyme 5’-nucleotidase in the molecular layer of the cerebellar cortex of the mouse. Photograph of a horizontal section from Scott (1964, Fig. 3). Compare Figs. 15, 18 and 19.

( 5 ) Scott (1964) found the enzyme 5‘-nucleotidase in the molecular layer of the cerebellum of the mouse to be distributed in a series of rostrocaudally directed bands (Fig. 17). One band is seen in the midline, the remainder are symmetrically disposed on either side of it. At most, five such bands are present, delimiting six longitudinal cortical zones. In the cortex of the flocculus and the paraflocculus three such bands are present. (6) In Haggqvist-stained material of the ferret, sectioned perpendicular to the direction of the fibres in the white matter of the folia, the latter appears to be divided into a number of compartments. In the midsagittal plane the white matter is constricted and shows an accumulation of small fibres. More laterally these are replaced by larger fibres (3-4 p), presumably Purkinje cell axons. Still more laterally, the number of small fibres with darkly staining myelin sheaths increases again and suddenly makes room for another bundle of Purkinje cell axons. In the anterior lobe of the ferret, five of these more or less abrupt changes in fibre pattern are present on each side of the mid-


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A

Fig. 18. The longitudinal subdivision of the white matter of the cerebellum of the ferret. The borders of the compartments (the ‘raphes’) are indicated by heavy lines. For description see text, Compare the localization of the spino-cerebellar terminations in the granular layer (Fig. 15) and the localization of the enzyme 5’-nucleotidase in the molecular layer (Fig. 17). Central nuclei shaded. For abbreviations see p. 131.

Fig. 19. The longitudinal subdivision of the white matter in the anterior lobe of the ferret, level of Fig. 18a. 18 x . Haggqvist stain. References p. 132-134

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Fig. 20. The border between the medial and the next paramedial compartment in the white matter of lobule 11. Same preparation as in Fig. 19. 180 x . Haggqvist stain.

Fig. 21. The longitudinal subdivision of the white matter in the paraflocculus of the ferret. 42 x . Haggqvist stain.

Fig. 22. Border between the dorsal and middle compartments of a folium of the dorsal paraflocculus. Same preparation as in Fig. 21. 180 x. Haggqvist stain.


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sagittal plane (Figs. 18-22). The second of these is less abrupt, the third is an abrupt change from a medial large fibre region into a region of smaller fibres. In the white matter of the lobules VII, VIII and IX two or three of these compartments are present on both sides, and in the adjoining hemisphere three or four other compartments can be distinguished. The white matter of the paraflocculus shows a similar tripartition. The borders between the compartments of adjoining folia show a tendency to deviate laterally in the dorsal part of the cerebellum. The tracing of these borders from the anterior lobe into the more caudal parts of the cerebellum is hindered by bundles of afferent, semicircular fibres and by the change in direction of the folia of the hemisphere. These sudden changes in fibre pattern were formerly identified in the cerebellum of the cat and called ‘raphes’. In the anterior lobe of the cat only the midsagittal ‘raphe’, the first (the ‘parasagittal raphe’) and one of the more lateral (the ‘lateral raphe’) were identified, in addition to one of the parasagittal borders in lobules VIII and IX and the tripartition of the paraflocculus. When projected on the surface of the folia and lobules the ‘raphes’ delimit longitudinal cortical zones. On this basis, a longitudinal division of the cerebellum of the cat was proposed (Voogd, 1964), which needs to be reconsidered, however, with respect to my observations on the ferret. Studies at present under way indicate that a similar longitudinal division of the white matter of the lobules is also present in other mammals. A comparison of the division of the anterior lobe by the distribution of the spinocerebellar fibres in the rabbit (Fig. 15), the bands of 5’-nucleotidase activity in the mouse (Fig. 17) and the compartments in the white matter of the folia in the ferret (Fig. 18) strongly suggests their correlation, although this has not been definitely proved. Although nothing is known as to the development of these longitudinal patterns in the adult, it seems reasonable to keep in mind the mediolateral differences in cortical differentiation described by Hayashi, Jakob and Langelaan. Brodal‘s tripartition of the anterior lobe as yet stands apart and cannot be correlated with the other modes of longitudinal division. (B) The subdivision and the eflerent projection of the central cerebellar nuclei in mammals

Most subdivisions of the central cerebellar nuclei are based on their fibre architecture and that of the white matter surrounding them, and cytoarchitectonic criteria are usually considered to be less important. Because of the differences in fibre architecture, application of the same criteria in different mammals does not lead to subdivisions which are functionally equivalent. This only can be decided upon after an analysis of both the afferent and efferent connexions, which is far from completed as yet. Generally, a medial or fastigial nucleus is distinguished, which is bordered laterally by a system of long corticofugal fibres. Ventrally and caudally the fastigial nucleus is continuous with the interposed nucleus, the border between them being arbitrarily determined at the narrowest part of their connexion. The subdivision of the interposed and lateral nuclei is subject to much variation. According to Brunner (1919) the border between both nuclei is indicated by an indentation of their dorsal and rostral surface only (Fig. 23) and it cannot be determined with accuracy. References p. 132-134

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BRUNNER:

MED

INT

LAT

WEIDENREICH OGAWA Fig. 23. Horizontal section of the cerebellum of the cat, illustrating the mediolateral subdivision of Brunner and the rostrocaudal subdivision of the central cerebellar nuclei of Weidenreich-Ogawa. Hatched: fastigial nucleus (F). Small dots: posterior interposed nucleus (IP). Filled circles: anterior interposed nucleus (IA) pars convexa of the lateral nucleus (Lc) and pars rotunda of the lateral nucleus (Lr). For abbreviations see p. 131.

Brunner’s division of the central nuclei into a medial, an interposed and a lateral nucleus can be carried out in most mammals, but not in chiropters and insectivores, in which no division is feasible. In Ogawa’s modification (1935) of Weidenreich’s subdivision (1899), a fibre lamella divides the nuclei into a caudomedial and a rostrolateral group. The former consists of the fused fastigial and caudal part of the interposed nucleus (the nucleus interpositus posterior), the latter of the rostral part of the interposed nucleus (the nucleus interpositus anterior) and the lateral nucleus. In the cat (Voogd, 1964) the lateral nucleus was further subdivided into a pars convexa (Fig. 24), located dorsally and caudally to its hilus, fusing medially with the nucleus interpositus anterior, and a pars rotunda, located ventrally and rostrally to the hilus, and dorsally to the peduncle of the flocculus. The rostrocaudal subdivision of Weidenreich-Ogawa can be recognized in most mammals. In Cetacea (Ogawa, 1935) this leads to the distinction of an enormous posterior interposed nucleus, whereas the anterior interposed-lateral nuclear complex is small. Otherwise, the posterior interposed and fastigial nuclei show little variation in most species. In several so-called lower mammals, such as the phalanger (Trichosuris vulpecula, Fig. 24) the anterior interposed nucleus and the pars convexa of the lateral nucleus cannot be separated and a large, reticular region intervenes between them and the fastigial nucleus. In Macaca ira (Fig. 24), the separation of the anterior interposed nucleus and the pars convexa is much more distinct. The latter already shows a differentiation into a rostral magnocellular and a caudal parvocellular limb, probably corresponding to the palaeo- and neo-dentatum distinguished in the human dentate nucleus (Gans, 1924; Demole, 1927). The pars rotunda of the lateral nucleus can be recognized in most mammals, and in Macaca ira it is probably represented by a cell column located ventrally to the rostral limb of the pars convexa. In Cetacea, the subdivision of Weidenreich-Ogawa has been confirmed by the study of their development (Korneliussen and Jansen, 1965). Unfortunately this is not the case in the earlier investigations of Rudeberg (1961) with regard to Bos taurus and man.

123


CA T

rostrcile

PHALANGER

MONKEY

---*

caudal

Fig. 24. The subdivision of the central nuclei of the cat, the phalanger (Trichosuris vulpecula) and a monkey' (Maeaca ira) according to Weidenreich-Ogawa and Voogd. Symbols as in Fig. 23. Open circles: pars rotunda. For abbreviations see p. 131.

Using Brunner's nomenclature in the description of the mature nuclei in Bos taurus, he made no attempt to distinguish between the development of the anterior and posterior interposed nuclei, although they can be fairly well distinguished in this species. References p.Il32-134

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Moreover, his description of the development of both the anterior and posterior interposed nuclei of man from one single migration (his migration Bz) differs from the observation of Korneliussen and Jansen in Cetacea, that this migration gives rise to the posterior interposed nucleus only. The posterior and anterior nuceli of different species are therefore not necessarily homologous in the sense of Riideberg. In the cat the subdivision of the central nuclei of Weidenreich-Ogawa is substantiated by the study of their efferent connexions (Voogd, 1964). The efferent connexions of

Fig. 25. The initial course of the fastigio-fugal and long corticofugal fibres in the cat, depicted in diagrams of transverse sections passing through the cerebello-bulbar junction at the stereotactic planes P6 (upper diagram), P8 (middle diagram) and PI0 (lower diagram). After Voogd, (1964). (A) The uncinate tract (black arrows) and the direct fastigio-bulbar tract (white arrows). (B) Long corticofugal fibres arising from the anterior lobe, passing to the lateral vestibular nucleus (fine lines) and from the Rocculo-nodular lobe, passing to the superior, the descending and the medial vestibular nuclei (heavy lines). For abbreviations see p. 131.

the fastigial nucleus are generally admitted to be different from the other nuclei (for references see Walberg, Pompeiano, Brodal and Jansen (1962) and Voogd, 1964). They consist of the uncinate tract and the direct fastigio-bulbar tract (Fig. 25A). The former decussates within the cerebellum and the anterior medullary velum, and subsequently


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passes over the brachium conjunctivum to the vestibular nuclei. Within the lateral and descending vestibular nuclei its fibres lie laterally, whereas the direct fastigio-bulbar tract fibres are located medially. Both terminate in the vestibular nuclei, with the exception of the lateral and the superior nucleus, and in the reticular formation. Long corticofugal fibres, which can be recognized by their smaller size, occupy an intermediate position in the lateral and descending nuclei and terminate in all vestibular nuclei (Fig. 25B). Both the uncinate and the direct fastigio-bulbar tracts give rise to ascending fibres. The former ascend without crossing again to the mes- and diencephalon (accessorisch Bindearmbundel of Probst, 1901;uncrossed ascending limb of the brachium conjunctivum of Carrea and Mettler, 1954). Coarse fibres arising from the homolateral fastigial nucleus are located ventromedially in the medial third of the brachium conjunctivum, together with medium and small fibres from the posterior interposed nucleus (Fig. 26A). In the cat this medial third of the brachium conjunctivum is sharply demarcated from the lateral two thirds, which contains large fibres from the anterior interposed and lateral nuclei. Here the fibres of the anterior interposed nucleus and the pars convexa are located medially to those of the pars rotunda of the lateral nucleus (Fig. 26). All fibres of the brachium conjunctivum decussate in the mesencephalon. The components of the brachium differ further with respect to their localization in the decussation and the ascending and descending limbs of the brachium, as well as with respect to their termination in the reticular formation, the red nucleus, the central gray, the nucleus of Darkschewitsch and the subthalamus. The confusion in the literature about the localization within the brachium conjunctivum and the termination of its components is probably due to the general use of Brunner’s nomenclature, in which no distinction is made between the anterior and posterior interposed nuclei. Moreover, many authors generalize the results of small lesions in the central nuclei, using the terms ‘interposed’ and ‘lateral’ nuclei where a more detailed description would have been more appropriate. After reviewing the literature in more detail, I reached the conclusion (Voogd, 1964) that the relationship of the subdivisions of the central nuclei of Weidenreich-Ogawa to the components of the brachium conjunctivum is similar in the cat and the other species studied. This conclusion is supported by the observation (Verhaart, unpublished) that in most mammals the fibre pattern of the medial third of the brachium conjunctivum, containing the fibres of the fastigial and posterior interposed nuclei, differs markedly from that of the lateral two thirds, containing the fibres of the anterior interposed and lateral nuclei. ( C ) The corticonuclear projection

The results of experimental investigations of the corticonuclear projection with the Marchi and the Nauta method (for references see Jansen and Brodal, 1958; Eager, 1963b; Voogd, 1964) are generally in good agreement. In order to determine the degree of convergence and divergence in the projection of certain parts of the cerebellar cortex on the subdivisions of the central nuclei, however, a larger number of References p. 132-134

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Fig. 26. Localization within the brachium conjunctivum of the cat. (A) Diagrammatic representation of the localization of the fibres arising from the subdivisions of the central nuclei in the brachium conjunctivum prior to its decussation. From Voogd (1964). Symbols as in Figs. 23 and 24. (B) The brachium conjunctivum of the cat prior to its decussation. Note the sharp border between its medial third (left) and its lateral two thirds. 55 x .Haggqvist stain. For abbreviations see p. 131.


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small, representative lesions are necessary. Moreover, the interpretation of the corticonuclear projection is hindered by the lack of uniformity in the subdivision of the cortex and the central nuclei. Clarke and Horsley concluded from their classical investigation of the corticonuclear projection (1905) that all parts of the cortex are connected with the nearest part of the central cerebellar nuclei. This projection is therefore diffuse and there is no important divergence in the projections of adjacent cortical areas. In their conception, there is no specific relationship between the corticonuclear projection and the subdivision of the central nuclei, the latter being decided by their morphology and their extracerebellar connexions. [n a rostrocaudal direction, most experiments indeed show a convergent projection of adjacent lobules to the same subdivisions of the central nuclei. An exception to this rule is formed by the projection of the flocculo-nodular lobe on the superior vestibular nucleus, a connexion which is not observed after lesions of the adjacent parts of vermis and hemisphere (Voogd, 1964) (Fig. 25B). Mediolateral differences in corticonuclear projection are postulated in the concept of its organization in longitudinal zones, the efferent fibers of one zone converging on one nucleus or group of nuclei. Unlike the rostrocaudal and mediolateral differences in efferent connexions, the possibility of a divergent projection of the base and the apex of the lobules, as suggested by the differences in afferent connexions, has not been considered in the literature. The concept of the longitudinal organization of the corticonuclear projection was actually introduced by Clarke and Horsley who showed that the projection of the vermis to the fastigial nucleus differs from the projection of the hemisphere to the more laterally located nuclei. Some years later Hohman (1929) delimited an additional zone in the anterior lobe of the cat projecting to the lateral cerebellar nucleus, located between a medial (vermal) zone projecting to the fastigial nucleus and the hemisphere. In several mammals Jansen and Brodal (1940) described symmetrically disposed medial, intermediate and lateral zones in the whole of the cerebellum, projecting to the fastigial and vestibular nuclei, to the interposed nucleus and to the lateral nucleus of Brunner respectively. As pointed out before, the border between Brunner’s interposed and lateral nuclei is an arbitrary one, and the border between the intermediate and lateral zones consequently depends on other localization criteria, provided by the distribution of the olivo-cerebellar fibres described by Brodal (1940). The arbitrary border between the interposed and lateral nuclei, once established on the basis of the afferent connexions of the anterior lobe, was used by these authors to group the lobules of the rest of the cerebellar hemisphere in intermediate and lateral zones, according to their projection to the interposed and lateral nuclei respectively. When the cerebellum is divided in this way, however, the relationship between the zones and their afferent connexions, established for the anterior lobe, is completely disregarded, for the rest of the cerebellum and the zones are characterized by their efferent projections to hypothetical subdivisions of the central nuclei only. The definition and the efferent connexions of these zones were recently reconsidered by Walberg and Jansen (1964), adopting the less equivocal subdivision of WeidenreichOgawa instead of Brunner’s in the description of the projections to the central nuclei. References p . 132-134

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J. VOOGD

Fig. 27. Diagram of the corticonuclear projection in the cat, from Jansen and Brodal(l940, reproduced from Jansen and Brodal, 1958, Fig. 181).

Concerning this subdivision they conclude, however, that there is no specific relationship between the medial, intermediate and lateral zones and the rostrocaudal division of Weidenreich-Ogawa. Although the existence, in principle, of longitudinal zones projecting exclusively to Brunner's three subdivisions of the central nuclei is thus maintained by them, they state (p. 351) '...the projection of each small area of cortex spreads out in a fan-like manner in the anter-posterior as well as medio-lateral direction. Hence there can be no sharp boundaries between the longitudinal zones, indeed one may rather say that there are no boundaries at all. This is of course just what one would expect considering the uniformity and continuity of the cerebellar cortex in the medio-lateral direction. On the other hand it should be emphasized that narrow longitudinal areas with a projection to only one of the nuclei certainly do exist.' This apparent lack of a mediolateral divergence in the efferent projection of the cortex de-


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prives the concept of the longitudinal projection zone of its original significance. In its present form therefore Jansen and Brodal‘s concept of the corticonuclear projection practically amounts to Clarke and Horsley’s statement that all parts of the cortex are connected with the nearest part of the central nuclei. Their intermediate and lateral zones are ill-defined regions which are distinguished only as a consequence of the choice of a mediolateral subdivision of the central cerebellar nuclei. Another interpretation of experiments on the corticonuclear projection is illustrated in Fig. 28. It is based on the observation that the efferent fibres of the longitudinal

Fig. 28. Diagram of the corticonuclear projection in the cat. From Voogd (1964). Symbols as in Figs. 23,24 and 26. For a description see the text. For abbreviations see p. 131.

zones in the cerebellar cortex in the white matter of the cerebellum lie collected in sharply circumscribed bundles. The borders between these bundles, called ‘raphes’ in the cat, can often be traced into the borders of the subdivisions of the central nuclei distinguished by Weidenreich and Ogawa. In the anterior lobe, four such zones were distinguished, projecting to the fastigial nucleus (zone A), the lateral vestibular (zone B), the anterior interposed (zone C ) and the pars rotunda of the lateral nucleus (zone References p. 132-134

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J. V O O G D

D) respectively. The zones C and D also were distinguished in the more caudal parts of the hemisphere, zone C projecting to the pars convexa of the lateral nucleus, which in the cat is indistinguishable from the anterior interposed nucleus. In the hemisphere an additional zone B’ was discerned projecting to the posterior interposed nucleus. After Walberg and Brodal’s analysis of the corticonuclear projection and my observation of the presence of more than four zones in each half of the cerebellum of the ferret and other mammals, my diagram should be modified. Its essential feature formed by the mediolateral divergence in the corticonuclear projection and the convergence of the efferents from a zone on a specific part of the central nuclei, however, can be maintained. In this respect it may be mentioned that the anterior lobe probably also projects to the posterior interposed nucleus and that a distinctionmust bemadebetween its projection to the anterior interposed nucleus and different parts of the lateral nucleus. The zones A and B could also be distinguished in the anterior lobe of the ferret. In this animal, zone C divides into a medial zone probably projecting to the anterior interposed, and a more lateral, fourth parasagittal zone projecting to the posterior interposed nucleus. The latter appears to be absent in lobules I and I1 and present in the rest of the anterior lobe. In the lateral part of the anterior lobe, a fifth and a sixth zone are present projecting to different parts of the lateral nucleus. The most lateral zone probably corresponds to zone D of the cat. In the more caudal parts of the hemisphere, the relations are not immediately clear; in the paraflocculus the three zones B’, C’ and D’ which were described in the cat are clearly present. The subdivisions of the central nuclei of Weidenreich-Ogawa and the corresponding longitudinal division of the cerebellar cortex therefore offer a reliable basis for further experimentation and for the comparative anatomical evaluation of the corticonuclear projection.

6.

CONCLUSIONS

(1) A subdivision of the mammalian cerebellum is usually considered to be a combination of a transverse and a longitudinal division of its surface, based on macroscopic inspection. Microscopic investigations into the histogenesis of the cerebellar cortex indicate a different mode of origin of these transverse and longitudinal patterns. In the last 30 years, however, the study of the histogenesis of the mammalian cerebellum has been neglected in favour of investigations of the cerebellum in the adult. (2) The cerebellar cortex and the central nuclei are considered to be essential links in pathways involving the cerebellum, short circuiting by extracerebellar fibres terminating in the central nuclei has never been systematically analysed. Our knowledge of afferent fibre systems terminating as mossy fibres by far exceeds that of the systems terminating in a different way. A reinvestigation of the olivo-cerebellar projection is necessary. (3) Transverse and longitudinal localizations in the cerebellar cortex were described. With respect to their afferent connexions, the lobules and folia of the cerebellum were found to be specialized structures. Vestibulo- and spino-cerebellar fibres terminate at their base, bulbo- and ponto-cerebellar fibres at their apex and in their lateral parts.


131

On a larger scale this concentric arrangement of the terminations of afferent systems is also present in their distribution to the cerebellum as a whole. This distribution can be correlated with temporal differences in cortex differentiation. In this way the folial pattern, which suggests a sharp, transverse localization, is an expression of the much less distinct, transverse localization of the afferent cerebellar systems. Contrary to what one may expect longitudinal localizations both during ontogeny and in the mature cerebellum were found to be distinct. Sharply delimited longitudinal zones can therefore be distinguished which were found to diverge in their efferent corticonuclear projection. ACKNOWLEDGEMENTS

I gratefully acknowledge the financial assistance of the Dutch association for pure scientific research (Z.W.O.). Several of the species used in the investigation of the spino-cerebellar projection were made available by the anatomical department (Head : Professor H. J. Lammers) of the university of Nijmegen and by Dr. A. M. Husson (Museum of Natural History, Leiden, Holland). The Hyrax were a gift from Professor Dr. J. C . E. Kaufmann of the South African Institute for Medical Research, Johannesburg. Abbreviations used in a1lJigure.s A

a A1 AzBi

ang. lat. ANS ANSU ANT AP B b bc BaP C1,CZ

CCB cocb

colat cr

cu DaO De

sublobule A of Bolk's nomenclature = medial zone of the anterior lobe of Jakob and Hayashi = migration A1 of Rudeberg = migration AZBI of Riideberg = angulus lateralis = ansiform lobule == ansula (Bolk) = anterior lobe = alar plate = sublobule B of Bolk's nomenclature = intermediate zone of the anterior lobe of Jakob and Hayashi = brachium conjunctivum = basal plate = sublobule CI, CZof Bolk's nomenclature = corpus cerebelli = somestheticcerebellar commissure of LarseIl = vestibular lateral commissure of Larsell = restiform body = Culmen = dorsal accessory olive = Deciive

References p. 132-134

dsc

dorsal spino-cerebellar tract = descending (spinal) vestiDV bular nucleus = fastigial nucleus F = anso-paramedian fissure fAPM f Hor = horizontal (intercrural) fissure fI = primary fissure fIC = intraculminate fissure f ICE = intracentral fissure = intercrural fissure f ICRUR = flocculus FLO FORM VERM = formatio vermicularis (Bolk) = preculminate fissure f PC f PCE = precentral fissure f PL = posterolateral fissure f PPD = prepyramidal fissure = posterior superior fissure f PS f.ret. = reticular formation f.SEC = secondary fissure F.v. = Folium vermis GNV = trigeminal ganglion IA = nucleus interpositus anterior IP = nucleus interpositus posterior = lateral cerebellar nucleus L = lobulus centralis L.C. =

132 Lc

J. V O O G D =

= Li LOB ANS (IF) = = LOB ANT Lob. flocc. nod. = LOB PMD = = Lr

LV Ma0 MES MIGR A ; B

=

MV N NuA NOD NupB

=

0s

= =

=

= =

= = =

P PFL D or V

=

PMD PO Porn

=

=

=

Pars convexa of the lateral cerebellar nucleus Linguia lobulus ansiformis anterior lobe flocculo-nodular lobe paramedian lobule Pars rotunda of the lateral cerebellar nucleus lateral vestibular nucleus medial accessory olive mesencephalon migration A or B of Riideberg medial vestibular nucleus Nodulus arcuate nucleus Nodulus nucleus of the pontobulbar body superior olive Pyramis (ventral or dorsal) paraflocculus paramedian lobule principle olive pontine migration

:

Pr.M.

=

RLc rostr.1.

=

sc

= =

SIM sIPFL SM

=

sPFL sPM

=

Sulc.b, d, taen. chor. T.v. tV tvq U v. 4 vma vsc

=

sv

= =

= = =

= = = =

= = =

primary (ventricular) matrix rhombic list, caudal part rhombic list, rostra1 part spino-cerebellar fibres lobulus simplex (Bolk) intraparafloccular sulcus secundary (superficial) matrix parafloccular sulcus paramedian sulcus superior vestibular nucleus sulcus b, d, of Riideberg taenia chorioidea Tuber vermis root of the trigeminalnerve taenia chorioidea Uvula fourth ventricle velum medullare anterius ventral spinocerebellar tract

The numbers I to X refer to Larsell's nomenclature.

REFERENCES BOLK,L., (1906); Das Cerebellum der Saugetiere. Jena, Gustav Fischer. BRODAL,A., (1940); Experimentelle Untersuchungen iiber die Olivo-Cerebellare Lokalisation. Z. Neurol., 169,1-153. A., (1957); The Reticular Formation of the Brain Stem. Anatomical Aspects and Functional BRODAL, Correlations. Edinburgh, Oliver and Boyd. BRODAL, A., AND HerrvI~,B., (1964); Site and mode of termination of primary vestibulocerebellar fibres in the cat. An experimental study with silver impregnation methods. Arch. ital. Biol., 102, 1-21. BRUNNER, H., (1919); Die zentralen Kleinhirnkerne bei den Saugetieren. Arb. Neurol. Znst. Wiener Univ., 22, 20C272. BUSCH,H. F. M., (1961); An Anatomical Analysis of the White Matter in the Brain Stem of the Cat. Assen, Van Gorcum. CARPENTER, M. B., (1960); Experimental anatomical-physiological studies of the vestibular nerve and cerebellar connections. G. L. Rasmussen and W. F. Windle, Editors, Neural Mechanisms of the Auditory and Vestibular Systems, Springfield, Thomas, pp. 297-323. CARREA, R. M. E., AND METTLER, F. A., (1954); The anatomy of the primate brachium conjunctivum and associated structures. J. comp. Neurol., 103, 565-690. R. M. E., REISSIG,M., AND METTLER,F. A., (1947); The climbing fibers of the simian and CARREA, feline cerebellum. Experimental inquiry into their origin by lesions of the inferior olives and deep cerebellar nuclei. J. comp. Neurol., 87, 321-365. H., AND HORSLEY, V., (1905); On the intrinsic fibers of the cerebellum, its nuclei and its CLARKE, efferent tracts. Brain, 28, 13-29. A., (1910); Per una nuova divizione delcervelletto delmammaferi. Arch. ital. Anat. Embriol., COMOLLI, 9, 247-273. CUPBDO, R. N. J., (1963); Het tecto-cerebellaire system van de albino rat. Thesis, Groningen. With a summary in English. DEMOLE, V., (1927); Structure et connexion des noyeaux denteles du cervelet. 11. Schweiz. Arch. Neurol. Psychiat., 21, 73-110.

S T R U C T U R E O F M A M MA LI A N CEREBELLUM

I33

EAGER,R. P., (1963a); Cortical association pathways in the cerebellum of the cat. J . comp. Neurol., 121, 381-394. EAGER, R. P., (1963b); Efferent cortico-nuclear pathwaysin thecerebellum of thecat. J. comp. Neurol., 120, 81-104. EDINGER, L., (1910); Ueber die Eintheilung des Zerebellums. Anat. Anz., 35, 319-323. GANS,A., (1924); Beitrag zur Kenntnis des Aufbaus des Nucleus Dentatus aus zwei Teilen, namentlich auf Grund von Untersuchungen mit der Eisenreaktion. Z. ges. Neurol. Psychiat., 93,750-755. GRANT,G., (1962a); Projection of the external cuneate nucleus onto the cerebellum in the cat: An experimental study using silver methods. Exp. Neurol., 5, 179-195. GRANT,G., (1962b); Spinal course and somatotopically localised termination of the spinocerebellar tracts. Acta physiol. scand., 56, Suppl. 193. HAYASHI, M., (1924); Einige wichtige Tatsachen aus der ontogenetischen Entwicklung des menschlichen Kleinhirns. Dtsch. 2.Nervenheilk., 81, 74-82. HOCHSTETTER, F., (1929); Beitrage zur Entwicklungsgeschichte des menschlichen Gehirns. Wien und Leipzig, Deuticke. HOHMAN, L. B., (1929); The efferent connexions of the cerebellar cortex. Investigations based on experimental extirpation in the cat. Ass. Res. nerv. Dis. Proc., 6,445460. S., (1919); Zur Phylo- und Ontogenese des Kleinhirns. Folia neuro-biol. ( L p z ) . 11, 205495. INGVAR, JAKOB,A., (1928); Das Kleinhirn. Von MollendorfF, Herausg., Handbuch der mikroskopischen Anatomie des Menschen. IV/l, Berlin, Springer, pp. 674-916. JANSEN, J., AND BRODAL, A., (1940); Experimentalstudies on the intrinsic fibres of the cerebellum. 11. The cortico-nuclear projection. J. comp. Neurol., 73, 267-321. JANSEN, J., AND BRODAL,A., (1958); Das Kleinhirn. Von Mollendofi, Herausg., Handbuch der mikroskopischen Anatomie des Menschen. IV/8. Berlin, Springer. KORNELIUSSEN, H. K., AND JANSEN, J., (1965); On the early development and homology of the central cerebellar nuclei in Cetacea. J. Hirnforsch., 8, 47-56. J. W., (1919); On the development of the external form of the human cerebellum. Brain, LANGELAAN, 42, 130-170. LARSELL, O., (1935); The development and morphology of the cerebellum in the opossum. Part I. Early development. J . comp. Neurol., 63, 65-94. O., (1947); The development of the cerebellum in man in relation to its comparative LARSELL, anatomy. J. comp. Neurol., 87, 85-129. LARSELL, O., (1952); The morphogenesis and adult pattern of the lobules and fissures of the cerebellum of the white rat. J. comp. Neurol., 97, 281-356. LARSELL, O., (1953); Cerebellum of cat and monkey. J. comp. Neurol., 99, 135-200. LARSELL, O., (1954); The development of the cerebellum of the pig. Anat. Rec., 118, 73-102. LARSELL, O., (1958); Lobules of the mammalian and human cerebellum. Anat. Rec., 130, 329. O., (1959); Development of tonsilla, accessory paraflocculus and biventral lobule in man. LARSELL, Anat. Rec., 133, 302. O., (1924); Die Anatomie des Kleinhirns. Dtsch. Z. Nervenheilk., 81, 8-35, MARBURG, MIALE,I. L., AND SIDMAN, R. L., (1961); An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp. Neurol., 4, 271-296. NIKITIN,M., (1925); Sclerosis cerebello-pyramido-intercorticalis,als eine besondere Form der systematischen Erkrankung des Grosshirns und Riickenmarks. Arch. Psychiat. Nervenkr., 75, 472489. OGAWA,T., (1935); Beitrage zur vergleichenden Anatomie des Zentralnervensystems der Wassersaugetiere. Ueber die Kleinhirnkerne der Pinnipedien und Cetaceen. Arb. anat. Znst. Sendai, 17, 63-136. PROBST, M., (1901); Zur Kenntnis des Bindearms, der Haubenstrahlung und der Regio Subthalamica. Mschr. Psychiat. Neurol., 10, 288-309. S. I., (1961); Morphogenetic studies on the cerebellar nuclei and their homologization in RUDEBERG, different vertebrates including man. Thesis, Lund. SCHEIBEL, M. E., AND SCHEIBEL, A. B., (1954); Observations on the intracortical relations of the climbing fibers of the cerebellum. A Golgi Study. J. comp. Neurol., 101, 733-763. SCOTT, TH. G., (1964); A unique pattern of localization within the cerebellum of the mouse. J. comp. Neurol., 122, 1-8. SMITH,M. C., (1961); The anatomy of the spino-cerebellar fibers in man. 11. The distribution of the fibers in the cerebellum. J. comp. Neurol., 117, 329-354. J., AND IL\lKOMTS, K., (1959); The origin of the climbing fibres of the cerebellum. 2. SZENT~GOTHAI, Anat. Entwick1.-Gesch., 121, 130-141.

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J. V O O G D

VALKENBURG, C. T. VAN,(1913); Bijdrage tot de kennis eener localisatie in de menschelijke kleine hersenen. Ned. T. Geneesk., 6-24. VOOGD,J., (1964); The Cerebellum of the Cat. Structure and Fibre Connexions.Assen, Van Gorcurn. VOORT,M. R. J. VANDE,(1960); De ontwikkeling van de ruitlijst bij de witte rat. Thesis, Nijmegen. With a summary in English. F., AND JANSEN, J., (1964); Cerebellar corticonuclear projection studied experimentally WALBERG, with silver impregnation methods. J. Hirnforsch., 6, 338-354. WALBERG, F., POMPEIANO, O., BRODAL, A., AND JANSEN, J., (1962); The fastigiovestibular projection in the cat. An experimental study with silver impregnation methods. J. comp. Neurol., 118, 49-75. WEIDENREICH, F., (1899); Zur Anatomie der zentralen Kleinhirnkerne der Sauger. Z . Movphol. Anthropol., 1, 259-312. WINKLER, C., (1927); Manuel de Neurologie. L'anatomie du Systeme Nerveux. Tome I, 3erne partie, Le cervelet. Haarlem, De Erven F. Bohn.

135

Anatomical Studies of Cerebellar Fibre Connections with Special Reference to Problems of Functional Localization ALF BRODAL Anatomical Institute, University of Oslo, Oslo (Norway)

INTRODUCTION

In spite of the fact that there are still many and large gaps in our knowledge of the organization of the fibre connections of the cerebellum, it is not possible to give more than a sketchy review of the subject in this lecture. I have chosen, therefore, to select some data and to consider them with special reference to certain problems of general interest. I will deal successively with the following: The information given by studies of cerebellar fibre connections on the significance of cerebellar fissures as a basis for subdivisions of the cerebellum; evidence bearing on the existence of a functional localization within the cerebellum ; evidence bearing on the problem of somatotopical localization within the cerebellum; and information on the modes of ending of the cerebellar afferent and efferent fibres. Since these items overlap to some extent, some repetition cannot be avoided. Only a limited number of references to the literature will be included. My presentation will be based largely on studies performed in the Anatomical Institute in the University of Oslo. Experimental anatomical studies of cerebellar fibre connections have been pursued since the 1890's, as have parallel studies on human pathological material. Most of this work has been done by means of the Marchi method which - as is well known demonstrates degenerating myelinated fibres, and which permits the tracing of a fibre tract to its site of termination. Important information has further been provided by the study of the retrograde cellular changes which occur in a perikaryon when its axon is transected. In this way the origin of a transected fibre bundle can be determined. With the introduction of silver impregnation methods for the study of degenerating nerve fibres possibilities arose for more detailed studies of the course and termination of transected fibres. Not only do these methods, as for example the methods of Glees (1946), Nauta and Gygax (1954) and Nauta (1957) allow the tracing of unmyelinated fibres as well as myelinated ones, but it is possible to determine precisely the cells on which the degenerating fibres end. Furthermore, it can usually be decided whether the fibres of the system under study contact perikarya or dendrites or both. In the study of retrograde cellular changes the use of very young animals in the References p. 169-173

136

A. B R O D A L

so-called modified Gudden method (Brodal, 1940a) has turned out to be advantageous in many instances. Sometimes the neuroanatomist is asked the question : Is it not possible to.do without these traditional methods when we have now got the electron microscope and with the great advances which are made in neurophysiology? I don’t believe so. In fact, I think we need these methods perhaps even more than ever before. I will return to this question at the end of my presentation, T H E A L L E G E D U N I F O R M I T Y OF T H E C E R E B E L L A R C O R T E X

Cerebellar folia and fissures It is generally said that the cerebellar cortex, in contrast to the cerebral, is uniformly structured throughout. While this seems to be true as far as the principal organization in layers and their main cellular elements are concerned, it is certainly not valid as concerns details. Riese (1925) and Jakob (1928) drew attention to the existence of regional differences in the pattern of myelinated fibres in the folia of the human cerebellum, and noted that especially the flocculus is characterized by a greater density of radial fibres in the granular layer than most other parts of the cortex. The intra- and supra-granular plexuses are particularly well developed and characterized by the presence of relatively thick myelin sheaths. The same picture is found in the nodulus and uvula, although somewhat less marked*. There are, however, other regional differences. We have recently found that there are two types of mossy fibres within the cerebellar cortex of the cat and rat (Brodal and Drablss, 1963). In addition to the classical type, depicted in all textbooks (Fig. 1, above), there is another type (Fig. 1, below). These fibres differ from the classical ones by being provided with great numbers, and closely packed groups, of terminal boutons and by the frequent occurrence of short, fairly straight, parts of fibres. These differences are seen in Golgi as well as in silver impregnated sections. It is to be noted, however, that these fibres do not occur all over the cerebellar cortex. In the nodulus and flocculus the majority of mossy fibres are of this type, which, therefore, was called the ‘nodular’ type. Furthermore, a fair number occur in the ventral folia of the uvula and in the ventral paraflocculus; in the dorsal paraflocculus there are scattered ones. The distribution and relative density of fibres of the ‘nodular’ type are shown in Fig. 2. The distribution covers the areas mentioned by Riese and Jacob as having a particular pattern of myelinated fibres. According to Jakob (1928) the number of Golgi cells is greater in the flocculus and the inferior vermis than in other parts of the cerebellum. We have been able to confirm this, and to observe that the relative proportion of Golgi cells, especially of the largest ones, appears to be particularly great not only in the flocculus and nodulus, but in the ventral paraflocculus and ventral part of the uvula as well (Brodal and Drablss, 1963). The distribution thus appears to be much the same as of the ‘nodular’ type of

*

Nothing is said about the pattern in the tonsilla, the human homologue of theventralparaflocculus.

137

CEREBELLAR F I B R E C O N N E C T I O N S

Glees

Reumont-Lhermitte

Golgi

Fig. 1. Drawings showing the different morphology of the mossy fibres and their terminals in the vermis of the anterior lobe (above) and in the nodulus (below), as seen when impregnated with three different methods. The sections used for the Golgi preparations (Golgi-rapid method) are from 2- to 6-day-old rats, the others from adult cats. Note more profuse branching and greater number and density of terminal globules in the ‘nodular’ type of mossy fibres (below) than in the ‘classical’type (above). From Brodal and Drablm (1963).

mossy fibres. These regions of the cerebellar cortex thus show definite differences from the cortex of the anterior lobe, which was studied as a control. These findings lend support to the suspicion that there may be other regional differences within the cerebellar cortex as well. There are some other observations which support this view. Thus, Grant (1962a) found that most of the (dorsal and ventral) spinocerebellar fibres ending in the paramedian lobule and in the dorsal paraflocculus are of a fine calibre, contrasting with the majority of such fibres to the anterior lobe. The Cajal-Smirnow fibres, according to Shntha (1931), occur particularly in the cortex of the flocculus, lingula and lobulus centralis. Landau has noted regional variations in the granular layer (for references, see Jansen and Brodal, 1958, p. 92), but no systematic study of this subject appears to have been made. Differences References p. 169-173

138

A. B R O D A L

pfl d

’

(uv.)

Fig. 2. Diagrams showing the distribution (dotted areas) of mossy fibre terminals of the ‘nodular’ type (see Fig. 1, below) in the cerebellum of the cat. The spacing of the dots indicates the approximate relative density of terminals of the ‘nodular’ type. To the left a midsagittal section of the cerebellum (lobule markings according to Larsell, 1953), to the right part of a transverse section through the cerebellum. Note distribution of ‘nodular’ type endings beyond the region of the flocculonodular lobe. From Brodal and Drables (1963).

in calibre between the corticonuclear fibres from various parts of the hemisphere were noted by Voogd (1964, p. 110). When more regional differences have so far not been described this is probably because they have not been looked for since their presence has not been suspected. Histologists apparently have chosen their samples for study of the cerebellar cortex from the most easily accessible parts. The morphological differences between the two types of mossy fibres probably reflect functional differences. It appears likely that fibres of the nodular type belong to one or more particular afferent fibre systems. In fact, there is indirect evidence that the primary vestibular fibres end as mossy fibres of this kind. In an experimental study in the cat (Brodal and Herivik, 1964) these fibres were found to end as mossy fibres. Furthermore, the distribution of degenerating mossy fibres following interruption of the vestibular nerve corresponds very closely to the regions harbouring mossy fibres of the nodular type (Fig. 3). The picture of the degenerating mossy terminals in these cases differs somewhat from that of degenerating mossy fibres in the anterior lobe following lesions of the spinocerebellar tracts (Brodal and Grant, 1962) but, as is well known, differences among degenerating fibres are difficult to evaluate*. The findings reviewed above make it clear that the cerebellar cortex is not uniformly structured throughout. However, they have other implications as well. The cortex where mossy fibres of the nodular type occur (Fig. 2) and which receives primary vestibular fibres (Fig. 3) extends considerably beyond the fissura posterolateralis (and uvulonodularis) and thus exceeds the borders of the floceulonodular lobe. These features are of interest for comparative-anatomical considerations, since they tend to minimize the significance which has often been attributed to the fissures of the cere-

* No primary vestibular fibres were found to end in the fastigial nucleus while a certain number end in the parvicellular part (Flood and Jansen, 1961) of the dentate or lateral nucleus (p in Fig. 3).


139

bellum as indications of borders between functionally dissimilar subdivisions. Data on the sites of termination of other afferent cerebellar fibres, to be considered below, lend support to this notion, as do also studies of the embryological development of the cerebellar cortex (see Korneliussen, 1966). It may be surmised that future studies of the minute histology of the cerebellar cortex, when carefully undertaken, will

Fig. 3. Diagram of the cerebellar surface of the cat (imagined unfolded) and of the intracerebellar nuclei showing the sites and relative density of termination (dots) of degenerating primary vestibular fibres following transection of the left vestibular nerve. Note distribution beyond the borders of the flocculonodular lobe. From Brodal and H ~ i v i k(1964).

bring out further regional differences, and that the borders between such regions will not necessarily coincide with the fissures. If so, this would be comparable to the situation in the cerebral cortex, which has been studied in far more detail, and in which the regional differences are more marked than in the cerebellar cortex. FUNCTIONAL LOCALIZATION W I T H I N THE CEREBELLUM

When we speak of a functional localization within the cerebellum we generally have in mind the problem: to what extent can different regions or parts of the cerebellum be considered as being related to particular functions? The answer to this question will, of course, depend on how the relevant functions are defined. However, for our purpose it will be sufficient to use the conventional designations and concepts. Information on functions has to be derived from physiological and clinical studies. However, anatomical investigations, not least of fibre connections, may give useful clues. In addition, they are necessary for the correct interpretation of physiological References p. 169-173

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A. B R O D A L

data. In the following I will consider some anatomical data bearing on the problems of functional localization in the cerebellum. The comparative anatomical studies and studies of fibre connections of the cerebellum (for reviews, see Dow, 1942; Larsell, 1945;Jansen and Brodal, 1958)performed until about 1940 had led to a subdivision of the mammalian cerebellum into three parts : (1) the archicerebellum, also called the ‘vestibulocerebellum’,comprising the flocculus and nodulus or flocculonodular lobe, (2) the palaeocerebellum or ‘spinocerebellum’, consisting of the vermis of the anterior lobe and part of the posterior vermis, and (3) the neocerebellum, often called the ‘pontocerebellum’, made up of the middle part of the vermis and the cerebellar hemisphere. The three major subdivisions were dominated by afferents indicated by their names, vestibular, spinal and pontine, respectively. Further research has made necessary modifications and elaborations of this simple scheme. In the first place the existence of a longitudinal zonal subdivision in addition to the transverse has been firmly established. The suggestion set forth by Hayashi (1924) and Jakob (1928) received considerable support when it was shown by Marchi studies in the rabbit, cat and monkey (Jansen and Brodal,

dent

I

A! vestib.

Fig. 4. Diagram of the main principles in the corticonuclearprojection in the cerebellum of the cat as established in Marchi studies. The mutually interconnecteddivisions of the cerebellar cortex and the intracerebellar nuclei are marked with identical symbols. The drawings of the transversely sectioned nuclei are arranged from rostra1 (above) to caudal (below). Note longitudinal zonal pattern. Cf.text. From Jansen and Brodal(1940).

CEREBELLAR FIBRE CONNECTIONS

141

1940, 1942) that there is an orderly arrangement in the projection from the cerebellar cortex onto the intracerebellar nuclei as seen from Fig. 4, showing our findings in the cat. The vermis proper projects to the fastigial nucleus, what we called an intermediate zone, sends its fibres to the nucleus interpositus, and the hemisphere projects onto the dentate 01 lateral nucleus. Since the efferent projections from the three intracerebellar nuclei are not identical, it seems a likely conclusion that the longitudinal zones differ functionally. Following the first confirmation by Chambers and Sprague (1955a, b) this has been amply demonstrated in physiological studies. However, further studies, anatomical as well as physiological, have made it clear that the borders between the longitudinal as well as the transverse zones or subdivisions are far from being sharp, and that a clear functional subdivision is scarcely permissible neither with reference to the transversely oriented lobules nor as concerns the longitudinal zones. Only some of many data will be mentioned to illustrate this, and emphasis will be put on evidence provided by studies of cerebellar fibre connections.

( A ) Afferent connections The most clearcut information is obtained from an analysis of the uflerent connections. As already mentioned the flocculonodular lobe cannot alone be considered to represent a ‘vestibulocerebellum’ since the terminal area of primary vestibular fibres extends considerably beyond the territory of the flocculonodular lobe (Brodal and H ~ i v i k 1964) , and includes the major part of the uvula, the ventral paraflocculus and to a lesser degree the dorsal paraflocculus (Fig. 3). The secondary vestibular fibres, which are derived from certain regions of the ipsilateral medial and descending vestibular nucleus (Brodal and Torvik, 1957) and from the group x of Brodal and Pompeiano (1957), were traced by Dow (1936) to the nodulus, flocculus and uvula. However, Dow did not find such fibres to the paraflocculus in his Marchi preparation. To decide whether this is supplied by secondary as well as primary vestibular fibres it will probably be necessary to use silver impregnation methods*. However, it is clear from the available anatomical data, that the ‘vestibulocerebellum’, if defined as that part of the cerebellum which receives vestibular impulses, extends considerably beyond the confines of the flocculonodular lobe as discussed above (see Fig. 3). Evoked potentials have been recorded in the uvula as well as in the flocculus and nodulus (Dow, 1939) following vestibular stimulation* *. The term ‘spinacerebellum’ has been used to cover those areas of the cerebellum which are dominated by impulses from the spinal cord. Originally it was thought to

* Authors working with the Marchi method have described some primary and secondary vestibular fibres to the fastigial nucleus. With silver impregnation methods we (Brodal and Hsivik, 1964) were, however, not able to find evidence of terminations of primary vestibular fibres here, although such fibres traverse the nucleus. * * It appears likely on the basis of certain data of fibre connections as well as from physiological studies that the two parts of the flocculonodular lobe, the flocculus and nodulus, differ in some respects (see Brodal and Jansen, 1954, p. 286 ff, for a discussion). This, as well as some of the data mentioned above indicates that there exists within the vestibulocerebellum (in the sense used here) a functional subdivision. References p.-169-I 73

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A. B R O D A L

consist of the vermis proper of the anterior Iobe and the caudal part of the vermis of the posterior lobe, particularly the pyramis and to a lesser extent the uvula (Larsell’s folia VIII and IX). Subsequent studies have shown that this delimitation requires modification. When, on the basis of studies of cerebellar fibre connections, we (Jansen and Brodal, 1940, 1942) became aware of the justification for distinguishing an intermediate part of the anterior lobe, we had the opportunity to study the distribution of the spinocerebellar fibres in a human case of chordotomy (Brodal and Jansen, 1941). Within the anterior lobe the area of termination extends beyond the vermis proper (Fig. 5) and includes our intermediate zone. This lateral distribution has been confirmed in an extensive study of human material by Marion Smith

P‘.imp

Fig. 5. Diagram showing the distribution of degenerating fibres (dots) in the human cerebellar cortex as studied in Marchi sections following bilateral chordotomy at the level of Th4-5. Below diagrams showing the lesions (hatchings) and (to the right) the distribution of degenerating ascending fibres (dots) at a level a little above the lesion. Note distribution within the anterior lobe beyond the vermis proper. From Brodal and Jansen (1941).

(1961). In animals the situation is the same. Although some previous workers had mentioned the distribution of some spinocerebellar fibres to the lateral parts of the anterior lobe, it remained for Grant (1962a), by using silver-impregnation methods, to demonstrate this conclusively. Fig. 6 shows Grant’s findings in a case with a lesion of the dorsal spinocerebellar tract. This distribution is in agreement with physiological determinations of the spinal regions (Grundfest and Campbell, 1942; Adrian, 1943; Snider and Stowell, 1942, 1944, and others). The posterior region of termination of spinocerebellar fibres has likewise been found to extend beyond the vermis propel and to include part of the paramedian lobule (and a small part of the dorsal paraflocculus) as shown by Grant (1962a; see Fig. 6). This again is in agreement with the physiological observations of several authors (see Dow and Moruzzi, 1958 and Oscarsson, 1965a, for references).

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143

While the ‘spinocerebellum’ thus has a greater lateral extension than originally assumed, its longitudinal representation in the posterior vermis has been narrowed. According to Grant (l962a) spinocerebellar fibres to the posterior vermis end almost exclusively in lobulus VIII of Larsell, the pyramis (Fig. 6). Only few fibres reach the

Fig. 6. Diagram of the cerebellum of the cat, imagined unfolded, showing the sites of termination of dorsal spinocerebellarfibres (dots) as seen in sections impregnated according to the Nauta method following a lesion of the dorsal spinocerebellar tract at C6 (below). The distribution covers the ‘hindlimb regions’. Cerebellar lobules labelled according to Larsell (1953). From Grant (1962a).

adjoining part of the uvula (not shown in Fig. 6). In addition to being in agreement with electrophysiological data, this restriction is in accord with the observations, mentioned above, that most of the uvula is to be considered as belonging to the ‘vestibulocerebellum’. The diagram of Fig. 6 shows the distribution of the dorsal spinocerebellar fibres. The fibres of the ventral spinocerebellar tract terminate in the same lobules and sublobules. However, apart from containing different proportions of crossed and uncrossed fibres the two tracts differ somewhat with regard to the mediolateral distribution of their terminal regions. These differences (Grant, 1962a) appear to be more clearly evident in physiological studies of the two tracts (see Oscarsson, 1965a), References p . 169-173

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and indicate a further elaboration of the longitudinal pattern in the vek-mis and intermediate zone*. The dorsal spinocerebellar tract is concerned in the transmission of impulses from the hindlimb and lower trunk only. (The same appears to be true for the ventral spinocerebellartract; see Grant, 1962a;Oscarsson, 1965a).The cervical homologue to the nucleus of Clarke (giving rise to the fibres of the dorsal spinocerebellar tract) is the external cuneate nucleus. Using the method of retrograde cellular changes (modified Gudden method, Brodal, 1940a)it could be shown (Brodal, 1941) that the fibres from this nucleus reach not only the vermis proper of the anterior lobe but its intermediate part as well. This was confirmed recently by Grant (1962b), who, tracing degenerating fibres (Nauta method) following lesions of the nucleus, in addition could define furtheridetails, among other things a termination of a certain number of such fibres in the paramedian lobule (Fig. 7)**.

Fig. 7. Diagram of the cerebellum of the cat, imagined unfolded, showing the sites of termination (dots) of cerebellar afferents from the external cuneate nucleus, as studied experimentally with the Nauta method. The distribution covers the ‘forelimb regions’. From Grant (196213).

* As will be discussed in the following section and as seen in Fig. 6, when studied with silver impregnation methods the terminal areas of the dorsal and ventral spinocerebellar fibres in the anterior lobe are restricted to its anterior part, leaving most of the culmen free. Z.e. the spinocerebellar fibres supply the ‘hindlimb area’ but not the ‘forelimb area’ of the anterior lobe. In Marchi studies of animal and human material this restriction is not clear (see Fig. S), although some indications of its existence have been found in a few studies (Chang and Ruch, 1949; Vachananda, 1959). It is difficult to explain this discrepancy. It appears likely that it may be due to a better possibility to identify truly degenerating fibres with the Nauta method than with the Marchi method. Correspondingly the terminations in the posterior vermis have been identitied with greater precision with the former than with the latter method (cp. Figs. 5 and 6). ** Recent physiological studies have brought forward evidence that there is a forelimb equivalent also to the ventral spinocerebellar tract. This has been called the rostra1 spinocerebellar tract (Oscarsson and Uddenberg, 1964; Oscarsson, 1965a, b) but has so far not been studied specifically with anatomical methods.

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In addition to the spino- and cuneocerebellar pathways there are other, more indirect routes, for impulses from the cord to the cerebellum. The lateral reticular nucleus of the medulla (nucleus of the lateral funiculus) receives fibres from the cord (see Fig. 13) which end chiefly in its parvicellular part (Brodal, 1949, cat; Mehler, Feferrnan and Nauta, 1960, monkey; and others). The receiving area projects onto the ipsilateral cerebellar vermis (Brodal, 1943) and probably to the paramedian lobule as well (see Jansen and Brodal, 1958, pp. 224-226), but details concerning the sites of termination of the fibres within the cerebellum could not be worked out with

R flocc.

Fig. 8. A summarizing diagram of the localization within the olivocerebellar projection in the cat as determined experimentally. Above a diagram of the cerebellar surface, imagined unfolded. Below a diagram of the inferior olivary complex, reconstructed and imagined unfolded. (The medial accessory olive is represented twice.) The Roman numerals refer to levels of transverse sections, on which the reconstruction is based. Three of these sections are shown in the middle row. Regions of the cerebellum are indicated with the same symbols as the parts of the olive which send fibres to them. From Brodal (1940b). References p . 169473

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the method used (retrograde cell changes following cerebellar lesions). It appears from the physiological studies of Bohm (1953) and Combs (1954, 1956) that this pathway, which transmits cutaneous impulses, ends in the vermis and intermediate part of the anterior lobe and in the paramedian lobule. More precise anatomical data are available as concerns the spino-olivo-cerebellar pathway. It has been possible to determine the topography of the olivocerebellar projection in the cat and rabbit in great detail (Fig. 8) by studying the localized distribution of retrograde cellular changes in the olive which follow restricted lesions of the cerebellum (Brodal, 1940b). As seen from the diagram the various lobuli of the vermis receive their fibres from particular regions of the medial and dorsal accessory olives. In silver-impregnation studies the terminal areas of the spinal afferents within the olive were determined (Brodal, Walberg and Blackstad, 1950). Fig. 9 shows in

pJ .::: .:.:_:

._..._.. ...._.. .

a

3 1 9 I,"

Fig. 9. Diagram showing some details in the spino-olivo-cerebellarpathway in the cat. The three differently labelledregions of the cerebellar vermis indicated in the sagittal section of the cerebellum in C receive their olivary afferents from the correspondingly labelled parts of the medial accessory olive (seen in reconstructionin B). In A the parts of the medial accessory olive which receive spinal afferents are indicated by solid vertical lines. Stippled lines indicate transitional regions receiving a few spinal afferents. Most of the terminal area in A projects onto the vermis of the anterior lobe (cf. Fig. 8), part of it relays spinal impulses to the pyramis, as seen from a comparison with B. From Brodal, Walberg and Blackstad (1950).

A the terminal area in the medial accessory olive. This can be correlated with the areas sending fibres to parts of the vermis. Spinal afferents end in that part of the medial accessory olive which projects onto the vermis of the anterior lobe and onto the pyramis (cf. Fig. B and C). Other spinoolivary fibres end in the part of the dorsal accessory olive which supplies the posterior part of the vermis proper of the anterior lobe (cp. Fig. 8). The regions of the cerebellum receiving spinal impulses via the olive are mapped in Fig. 10. The restricted termination of this pathway to the vermis proper and the pyramis with the adjoining part of the uvula is particularly interesting when considered in connection with the lateral extension of the zones of termination of the dorsal and ventral spinocerebellarfibres in the anterior lobe, and the termination of some fibres of both these tracts in the paramedian lobule, which does not appear to receive spinal impulses via the olive. These observations represent further evidence in support of the existence of a longitudinal subdivision within the 'spinal area' of the cerebellum, as far as its types of afferents are concerned. This tallies well with anatomical and physiological results concerning the efferent projections of the 'spinal area' to be considered briefly below. In addition to the spinocerebellar routes mentioned above there are others as well, although they appear to be quantitatively less important. The paramedian reticular


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nucleus (Brodal, 1953) projects onto the cerebellar spinal area (Brodal and Torvik, 1954) as seen from Fig. 11*. This nucleus receives some afferents from the spinal cord (Brodal, 1953; Brodal and Gogstad, 1957), but their number is modest. The

N.fast. Hind- limb Fig. 10. Diagram of the cerebellum of the cat, imagined unfolded, showing the areas to which spinal impulses from the left side of the body may be conveyed via the inferior olive (cp. Figs. 8 and 9). Density of markings indicates approximately relative number of spinal fibres to parts of olive projecting onto cerebellar areas. No clearcut somatotopical pattern, but inflow from the hindlimb is more abundant than from the forelimb. Note restriction to vermis proper in the anterior lobe. From Brodal, Walberg and Blackstad (1950). 0

Fore - limb

0

perihypoglossal nuclei, like the paramedian reticular nucleus, send their efferents to the cerebellum (Brodal, 1952), apparently to the same areas as the latter (Torvik and Brodal, 1954), and receive a modest number of fibres from the spinal cord (Brodal, 1952)**. Finally, spinopontinefibres, to be considered below, should be listed among links in possible pathways from the cord to the cerebellum. As will appear from the above, the ‘spinocerebellum’, when defined as those regions which receive impulses from the cord, extends beyond the vermis proper and includes parts of the original ‘pontocerebellum’. This does not mean, however, that the latter is reduced in size. On the contrary, if defined as those regions of the cerebellum which receive fibres from the pons the ‘pontocerebellum’ overlaps with the ‘spinocerebellum’ and includes even parts of the vermis proper since a considerable number of fibres

* As seen from the diagram, evidence for a projection of the nucleus onto the paramedian lobule was not obtained in this study which was made by studying the distribution of retrograde cellular changes following lesions of the cerebellum. This negative finding is, however, not decisive, since if only a small number of cells are changed their identification may be impossible. Furthermore, some reservations have to be made as to the inclusion of the intermediate part of the anterior lobe in the terminal region, as discussed in the original publication. ** Spinal afferents to the paramedian reticular nucleus and the perihypoglossal nuclei have been found in the monkey by Mehler, Feferman and Nauta (1960). References p. 169-173

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from the pontine nuclei end in the spinal areas. This was demonstrated by early workers in neuroanatomy and is seen from the map of the entire pontocerebellar

N.

L.

R.

Fig. 11. Diagram showing the distribution within the erebellurn of the cat of fibres from the paramedian reticular nucleus of the medulla. Below a section showing the three subdivisions of the paramedian reticular nucleus (a = accessory group; d = dorsal group; v = ventral group). See also footnote on p. 147. From Brodal and Torvik (1954).

projection in the cat (Fig. 12) as determined (Brodal and Jansen, 1946) on the basis of a study of the retrograde cellular changes in the pontine nuclei following lesions of various parts of the cerebellum by means of the modified Gudden method (Brodal, 1940a). As is seen from Fig. 12 fibres to the vermis are derived from the lateral and medial parts of the pontine nuclei, and in addition from the nucleus reticularis


149

,

Fig. 12. Diagram of the pontocerebellar projection in the cat as determined by a study of retrograde cellular changes in the pontine nuclei following lesions of the cerebellum. Below transverse sections through the pontine nuclei arranged from rostra1 (X)to caudal (I). The main subdivisions of the cerebellum and the areas of the pontine grey predominantly connected with them are labelled with identical symbols. Theprojection is more diffuse than appears from the diagram. The question marks indicate that the projection of these parts could not be definitely settled. From Brodal and Jansen (1946). References p. 169-173

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tegmenti pontis*. These regions of the pontine nuclei, like the others, receive fibres from the cerebral cortex (Nyby and Jansen, 1951 and others). The electrophysiological studies of Dow (1939) and Jansen, Jr. (1957) are in agreement with these anatomical findings on the pontocerebellar projection. Voogd (1964) when studying the pontocerebellar fibres following lesions of the pons made largely corresponding findings as Brodal and Jansen (1946) but could provide more detailed information on some points. It is particularly interesting that pontine fibres reach the rostra1 part of the uvula while they appear to be absent in the regions near the posterolateral fissure, dominated by vestibular afferents (see Fig. 3). The ‘pontocerebellum’ thus overlaps with the ‘spinocerebellum’. A further point emphasizing this is the following: The pontine nuclei receive spinal afferents (Walberg and Brodal, 1953), and the sites of termination of these fibres are not restricted to those pontine areas which project onto the cerebellar vermis**. In our study (Brodal and Jansen, 1946) we were not able to decide whether the pontine nuclei give of€fibres to the flocculus and nodulus. However, Voogd (1964) recently described pontine fibres to the flocculus. Although he did not find fibres to the nodulus, a projection to the flocculus indicates that the ‘pontocerebellum’ overlaps with the ‘vestibulocerebellum’ as well, as follows also from our demonstration (Brodal and Harivik, 1964) that (particularly the ventral) paraflocculus receives primary vestibular fibres, since the paraflocculus receives a considerable projection from the pontine nuclei (Fig. 12)***. When considering the problem of a functional localization in the cerebellum from the point of view of its afferents, an additional feature should be mentioned, which was not known when the terms ‘vestibulocerebellum’, ‘spinocerebellum’ and ‘pontocerebellum’ were coined. Following the demonstration by Snider and Stowell (1942, 1944) that acoustic and visual stimuli give rise to potentials in the middle part of the vermis, this subject has been extensively studied by neurophysiologists (see Fadiga and Pupilli, 1964, for a recent review). The main acoustic and visual cerebellar areas overlap and are found in the middle part of the vermis, chiefly in Larsell’s (1953) lobules VI and VII. The pathways for visual and acoustic impulses to the cerebellum are, however, insufficientlyknown. It appears from physiological experiments that they involve the colliculi, but the evidence in favour of tectocerebellar fibres in mammals is meagre (see Jansen and Brodal, 1958; Altman and Carpenter, 1961, for reviews). The assumption that the impulses reach the cerebellum via tectopontine and pontocerebellar fibres (Brodal and Jansen, 1954, p. 344) receives some support from the observations that under certain experimental conditions visual and acoustic responses

* In the diagram of Fig. 12 only the ventral part of this (N.r.t.) is shown as projecting onto the cerebellum. In subsequent studies it has been found that the entire nucleus takes part in the projection (see Taber, Brodal and Walberg, 1960). * * It is of interest in this connection that birds possess a pontine homologue, which has been shown experimentally to project onto the cerebellum (Brodal, Kristiansen and Jansen, 1950). *** The different relations of the nodulus and flocculus to the pons may be taken as evidence that the two parts of the flocculonodular lobe are not functionally entirely similar (see also footnote on p. 141).

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can be recorded from fairly extensive regions of the dorsal aspect of the cerebellum in the cat (see Fadiga and Pupilli, 1964). Responses in the visual and auditory areas of the cerebellum have also been recorded following stimulation of the visual and auditory regions of the cerebral cortex (Hampson, 1949; Snider and Eldred, 1951, 1952; Jansen, Jr. and Fangel, 1961). The pathways used by these impulses are not known. The cortico-ponto-cerebellar route may be involved, but another possibility would be a pathway via the colliculi and the pons (cortico-colliculo-ponto-cerebellar). As mentioned by Fadiga and Pupilli (1964) it seems less likely that the reticular formation is a station in these pathways. The tactile .face region in the cerebellum discovered by Adrian (1943) and Snider and Stowell (1942, 1944), appears to be more or less coextensive with the visual and acoustic areas. Also here information of possible pathways is insufficient (see Jansen and Brodal, 1958, for a review). There is some evidence that fibres from the mesencephalic trigeminal nucleus pass to the cerebellum (see Brodal and Fegersten Saugstad, I965), and Carpenter and Hanna (1961) experimentally traced secondary trigeminocerebellar fibres from the spinal trigeminal nucleus (the subnuclei oralis and interpolaris) which appear to terminate largely in Larsell’s lobules V and VI. I n spite of the incomplete knowledge of the pathways transmitting optic, acoustic and facial impulses to the cerebellum, the existence of a ‘face’ area in the middle part of the vermis is established. Judging from the physiological data its borders are not sharp and it extends into neighbouring regions of the cerebellum belonging to the ‘spinocerebellum’ as well as the ‘pontocerebellum’. ( B ) Efferent connections

It appears from the above that a functional subdivision of the cerebellum on the basis of its afferent connections can only be done with certain qualifications. The afferents from various sources overlap to a considerable extent. If we turn to the efferent connections of the ‘vestibulo-’, ‘spino-’ and ‘pontocerebellum’ it becomes clear that the situation is similar. Since some relevant points will be considered in the following section of this review, it will suffice to mention a few data only. In the first place the efferent connections bear witness of the existence of an extremely intimate collaboration between ‘vestibular’ and ‘spinal’ parts of the cerebellum. Among other things, in addition to fibres from the ‘vestibular’ parts of the cerebellum the vestibular nuclei receive a fairly massive projection from the ‘spinocerebellum’, particularly from the vermis proper of the anterior lobe, as recently studied by Walberg and Jansen (1961). These fibres end in the lateral and descending vestibular nuclei. Furthermore, the fastigial nucleus, dominated by afferents from the vermis proper, gives off fibres to the vestibular nuclei (for a review, see Brodal, Pompeiano and Walberg, 1962). The intermediate zone of the cerebellum, belonging to the classical ‘pontocerebellum’ and receiving pontine as well as spinal afferents, may act on the spinal cord by way of the pathway from the nucleus interpositus to the red nucleus and the rubrospinal tract. These and other connections, some of them to be considered below, provide anatomical evidence that although certain major subdivisions of the cerebellum may be References p . 169-173

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particularly concerned in ‘controlling’ the vestibular mechanisms, the spinal mechanisms and the function of the cerebral cortex, respectively, there are a number of possibilities for an intimate collaboration between these regions. It is, therefore, artificial to consider these subdivisions more or less as functional units. On the other hand, there is anatomical and physiological evidence that the major subdivisions may be further subdivided into minor parts which differ mutually with regard to connections and also functionally. Mention was made above (p. 144) of anatomical and physiological observations which indicate that in the anterior lobe the vermis proper as well as the intermediate zone may be subdivided into longitudinal subzones as far as the influxes from the spinal cord are concerned. The same appears to be true for the paramedian lobule (see Szabo and Albe-Fessard, 1954; Grant, 1962a). Studies of the cerebellar ontogenesis (Korneliussen and Jansen, 1965) further support this view. AS to the efferent projections, the studies of Moruzzi and Pompeiano and their collaborators have demonstrated a corresponding zonal subdivision. Stimulation of the paravermal strip of the intermediate zone results in flexion of the ipsilateral limbs, while stimulation of the lateral part of this zone results in an inhibition of flexor motoneurones and increased extensor tonus in the ipsilateral limbs (Pompeiano, 1958). These effects are mediated via the rostromedial and rostrolateral parts, respectively, of the nucleus interpositus (Pompeiano, 1960a). Opposing effects are also obtained on stimulation of the caudomedial and caudolateral parts, respectively, of the nucleus interpositus (Maffei and Pompeiano, 1962a) which receives fibres from the paramedian lobule. In much the same way the rostrolateral part of the fastigial nucleus has an inhibitory, the rostromedial part an augmentatory, effect on postural tonus (Moruzzi and Pompeiano, 1957; Batini and Pompeiano, 1958). The available anatomical data on the connections from the cerebellar cortex to the intracerebellar nuclei are as yet not sufficiently detailed to permit a satisfactory correlation with these recent careful physiological studies. However, it may be stated that the original view, based on anatomical studies (Jansen and Brodal, 1940, 1942, see Fig. 4) is still generally valid: The vermis proper sends at least the overwhelming majority of its fibres to the fastigial nucleus, the intermediate zone including the paramedian lobule projects predominantly onto the nucleus interpositus, even if it appears from later studies with silver-impregnation methods (Eager, 1963; Walberg and Jansen, 1964) that there is a greater spread of the corticonuclear fibres in the lateromedial direction within the nuclei than evident in Marchi studies. However, it is worthy of notice that ‘certain narrow longitudinal cortical lesions produce degeneration within the boundaries of one cerebellar nucleus’ (Walberg and Jansen, 1964, p. 351), and Voogd (1964) advocates the existence of fairly separate sagittal bands of projection fibres from various of his longitudinal zones. It appears that so far the longitudinal zonal subdivisions can indeed be more clearly demonstrated with physiological than with anatomical methods. The demonstration within the vermis and intermediate part of the cerebellum of longitudinal subzones bears witness of the existence of a rather elaborate functional and structural differentiation within the ‘spinocerebellum’. It does not appear un-

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likely that future research may reveal other examples of functional parcellation within the other main divisions of the cerebellum. Some evidence in favour of this as concerns the ‘vestibulocerebellum’ has been mentioned (p. 141), and concerning the cerebellar hemispheres and the paraflocculus there are likewise data, e.g. with regard to fibre connections, which are highly suggestive. However, these will not be discussed here. It is deemed more appropriate to devote some attention to another aspect of functional localization, namely the question whether a cerebellar region may be further subdivided according to somatotopical principles. S OMAT OT OP ICAL L O C A L I Z A T I O N W I T H I N THE C ER EBELLU M

The idea that there is within the cerebellum a somatotopical localization, i.e. that certain parts of the organ are related to different bodily regions, appears to have been formulated first by Bolk in his monograph from 1906. Although his conclusions concerning the pattern have not stood the test of time, the principle of a somatotopical localization has been shown to be valid, so far only for the ‘spinocerebellum’ in the extended sense. The well known pattern of a ‘representation’ of various bodily regions in the anterior lobe and the paramedian lobules was established first by Snider and Stowell (1942, 1944) and Adrian (1943) in electrophysiological studies. These discoveries and their further extension presented challenging problems to the anatomist. It is difficult to conceive of a somatotopically organized transmission of impulses along pathways which are anatomically entirely diffusely organized. However, about 1940 little or nothing was known of the existence of somatotopical anatomical patterns in cerebellar connections, afferent or efferent. During recent years this situation has been radically changed. In the following I will make an attempt to review some of the new observations. ( A ) Aflerent connections In spite of numerous studies with the Marchi method, in various animals and man, until recently no convincing evidence for the existence of a somatotopical organization within the spinocerebellar tracts had been found, although the findings of Chang and Ruch (1 949) and Vachananda (1959) were highly suggestive. Using silver-impregnation methods Grant (1962a), however, could define the termination of the dorsal and ventral spinocerebellar tracts in the cat with great precision. As seen from Fig. 6, the termination of the dorsal spinocerebellar tract in the vermis and the intermediate zone of the anterior lobe is restricted to lobules 11-IV and the adjoining folia of lobulus V and, furthermore, covers only the posterior part of lobulus VIII (pyramis) and the caudal folia of the paramedian lobule, i.e. those regions which by physiological methods have been shown to receive impulses from the hindlimb. The same distribution, approximately, was found for the ventral spinocerebellar tract*. As referred to above, the ex-

* Apart from some difference with regard to the lateromedial distribution of the two tracts, as mentioned in the preceding section, they appear to differ in so far that there are only few ventral tract fibres to the paramedian lobule and the pyramis. It appears that the ventral spinocerebellartract does not carry impulses from the forelimb (see Grant, 1962a; Oscarsson, 1965a). The ‘rostra1 spinocerebellar tract’ which appears to be a forelimb equivalent to the ventral spinocerebellar tract was referred to above (footnote on p. 144). References p . 169-1 73

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ternal cuneate nucleus may be considered as the cervical cord equivalent to the column of Clarke. This is finally confirmed by the study of Grant (1962b) which showed that the fibres from the external cuneate nucleus terminate in the forelimb regions of the cerebellum, i.e. in the posterior parts of the anterior lobe, the anterior parts of the paramedian lobule and pyramis (Fig. 7). Physiologically there are, however, some minor differences between the cuneocerebellar tract and the dorsal spinocerebellar tract (Holmqvist, Oscarsson and RosCn, 1963 ; Oscarsson, 1965a). So far the spinocerebellar tracts and the cuneocerebellar tract are the only routes from the spinal cord to the cerebellum in which a somatotopical pattern has been convincingly demonstrated anatomically. Originally these tracts were believed to be concerned in the transmission of proprioceptive impulses only, while physiologically the pattern of a somatotopic propagation of spinal impulses to the cerebellum was most clearly seen for tactile impulses. However, recent studies have shown that the dorsal spinocerebellar and the cuneocerebellar tract convey impulses not only from muscle spindles and tendon organs but from cutaneous receptors as well, while the ventral and rostra1 spinocerebellar tracts appear to be devoted to transmission of proprioceptive impulses (for a recent review, see Oscarsson, 1965a). Another pathway for exteroceptive cutaneous impulses to the cerebellum is the route via the lateral reticular nucleus or nucleus of the lateral funiculus (Bohm, 1953;

8808 forelimb ..+.'.hjnd/imb Fig. 13. Diagram of the experimentally determined sites of termination of fibres from the spinal cord within the lateral reticular nucleus (nucleus funiculus lateralis) in the cat. The nucleus is shown in a series of horizontal sections. The fibres terminate with some overlapping in a segmental pattern within the parvicellular part (P.c.) and the adjoining region of the magnocellular part (m.c.). For the sake of simplicity distinction is made only between fibres from the cervical and lumbosacral cord, labelled forelimb and hindlimb fibres, respectively. From Brodal (1 949).

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Combs, 1954, 1956). The spinal afferents to this nucleus end in a somatotopical pattern within it (Fig. 13) as shown experimentally (Brodal, 1949). From a renewed analysis (see Jansen and Brodal, 1958, p. 224-226) of the material on which the study of the projection of this nucleus onto the cerebellum was based (Brodal, 1943) it appears likely that the second link in the pathway is likewise somatotopically organized. The physiological studies of Combs (1954, 1956) lend support to this assumption, but more detailed anatomical studies are needed. Lamarche and Morin (1957) who following stimulation of cutaneous nerves studied somatotopically localized responses in the paramedian lobule and the pyramis, do not commit themselves as to the pathway followed by the cutaneous impulses. As to the other indirect spinocerebellar routes, mentioned in the preceding section, there is no anatomical evidence available as to whether these betray any somatotopical organization. However, they are all quantitatively less important than the others, and it is not known from which type of receptors they convey impulses. It may be suggested that they perhaps are concerned in maintaining a diffuse background activity in the ‘spinal areas’ of the cerebellum, upon which the localized impulses play. The somatotopical pattern in the ‘spinal part’ of the cerebellum has been demonstrated to be valid for the impulses entering the cerebellum from the somatosensory and somatomotor areas of the cerebral cortex as well (Adrian, 1943; Hampson, 1949; Snider and Eldred, 1951, 1952). So far, however, there appears to be no anatomical demonstration of a somatotopical arrangement within the cerebrocerebellar pathways. The most likely candidates for such routes would be the cortico-ponto-cerebellar pathway via certain of the pontine nuclei and the cortico-olivo-cerebellar pathway. As to the latter, there is a discrete topical projection onto the anterior lobe from the accessory olives (Brodal, 1940b). As can be seen from Fig. 8 the regions of the olive projecting onto the various lobules of the anterior lobe can be indicated. Fibres from the sensorimotor region of the cerebral cortex end in that part of this area which projects onto the intermediate zone (but not onto the vermis proper) in the anterior lobe, as well as in certain other regions, among these the area projecting onto the paramedian lobule (Walberg, 1956). From the findings in two cases Walberg did not venture conclusions as to the existence of a somatotopical arrangement within this cortico-olivary projection. A detailed study of the cortico-olivary projection from the central region might, therefore, be of interest. As to the cortico-ponto-cerebellar projection this appears on the whole to be rather diffusely organized (Fig. 12). However, the regions of the pontine nuclei which project onto the vermis of the anterior lobe (Brodal and Jansen, 1946) appear to be among those in which descending fibres from the sensorimotor cortex terminate (Nyby and Jansen, 1951). A more detailed study of the cortico-ponto-cerebellar pathways with special reference to the possible existence of a somatotopical organization within its two links might be rewarding and of interest to physiological interpretations. It appears from the electrophysiological studies of Jansen, Jr. (1957) that the pontine nuclei are more important than the inferior olive as mediators of cortical impulses to the spinocerebellum. (For a discussion of pertinent neurophysiological problems, see Dow and Moruzzi, 1958 and Fadiga and Pupilli, 1964.) References p . 169-I 73

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( B ) Eflerent connections Just as the somatotopical localization in the spinocerebellum (in the extended sense) with regard to its afferent impulses was first demonstrated by physiologists, the existence of a somatotopical pattern in the effects exerted by the cerebellum on the effector mechanisms was established in physiological studies. The pioneer observations of Hampson, Harrison and Woolsey (1 952) have subsequently been confirmed and extended by several students, not least by Moruzzi and Pompeiano and his collaborators (see, for example, Moruzzi and Pompeiano, 1957; Maffei and Pompeiano, 1962a; Pompeiano, 1962). The pattern agrees with that derived at on the basis of studies of the afferent responses. In view of the unequivocal somatotopically localized effects which can be obtained on stimulation or ablation of appropriate parts of the ‘spinocerebellum’, one would expect a correspondingly clear pattern to be evident in the anatomy of the pathways transmitting impulses from the cerebellum to the spinal cord. As will be shown below, a pattern of this type has indeed been found in certain links of these pathways. However, in the first of them, the corticonuclear projection, localization does not appear to be very sharp (Jansen and Brodal, 1940; 1942, see Fig. 4). Even a relatively smaIl lesion in the cerebellar cortex gives rise to degenerating fibres which spread in a fan-like manner in the sagittal plane (Eager, 1963; Walberg and Jansen, 1964). There appears thus to be a considerable degree of overlapping between fibres from neighbouring somatotopical regions. There is, however, one point which may indicate that the localization within the corticonuclear projection is in fact sharper than appears from the anatomical studies published. Usually the lesions have had a fairly long extension in the sagittal plane, affecting a number of folia more or less superficially. Lesions of one or two folia only, but extending to their bases, would be expected to give a more selective distribution of the afferent fibres. Such lesions are, however, obviously very difficult to achieve. On account of the difficulties involved in producing lesions of restricted parts of the intracerebellar nuclei without damaging fibres passing through the place of the lesion but coming from other parts of the injured nucleus, from other nuclei or from the cerebellar cortex and passing in the vicinity of the nuclei, studies of the possible existence of a localization within the terminations of the efferent fibres from the intracerebellar nuclei are beset with great technical difficulties. For this reason and because of the usage of differing nomenclature and different subdivisions of the intracerebellar nuclei, the reports in the literature on the efferent projections from the intracerebellar nuclei are far from unanimous. Only by employing a large material with restricted lesions and comparing the distribution of degenerating fibres following differently placed lesions in the nuclei can one hope to achieve results. On a material of this kind we have so far analysed the projections of the fastigial nucleus onto the vestibular nuclei and the reticular formation. These projections must be links in the pathways which mediate the effects from the vermis proper, while those appearing on stimulation of the intermediate zone of the lobus anterior and the paramedian lobule must be mediated via efferent projections from the nucleus interpositus. It will be practical


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to treat the two routes for cerebellospinal impulses separately, and to consider first the pathways from the vermis proper. There is evidence that the components of these which involve the lateral vestibular nucleus are somatotopically organized throughout. The results of our studies of its final link, the projection of the nucleus of Deiters onto the cord, will be dealt with first. By using the modified Gudden method (Brodal, 1940a) it has been possible to demonstrate a somatotopical arrangement in the vestibulospinal projection (Pompeiano and Brodal, 1957a). Within the lateral vestibular nucleus of Deiters* a ‘fore-

I

rostra1

3 ~

,‘dorsal! vent r a I medial

lateral

4 caudal

I

@

,., +

--

-17-

thoracic

-n-

lumbosocral--

Fig. 14. Diagram of the somatotopical projection of the nucleus of Deiters onto the spin:.- cord as determined experimentally. To the left a series of drawings of transverse sections through the nucleus showing the sites of origin of the fibres to the cervical, thoracic and lumbosacral cord. To the right a drawing of a reconstruction of the nucleus in the sagittal plane where the somatotopical pattern is more clearly seen. From Pompeiano and Brodal (1957a).

* To avoid misunderstanding it should be mentioned that the term ‘nucleus of Deiters’ is taken to denote that part of the vestibular nuclear complex where the giant cells of Deiters form a characteristic cytoarchitectonic element (Brodal and Pompeiano, 1957). However, there are numerous small cells among the large ones. This use of the term is in agreement with the usage of authors such as Cajal (1909) and Kappers, Huber and Crosby (1936) and its correctness is borne out by studies of the fibre connections of the vestibular nuclear complex (see Brodal, Pompeiano and Walberg, 1962). Voogd (1964) uses the term ‘nucleus of Deiters’ differently. This should be recalled when his results are compared with ours. References p. 169-173

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limb and neck region’, a ‘trunk region’ and a ‘hindlimb region’ can be distinguished (Fig. 14). These regions give off fibres to the cervical, thoracic and lumbosacral segments of the cord, respectively. This has been confirmed by Nyberg-Hansen and Mascitti (1964) who traced the fibres degenerating after lesions of the different regions of the nucleus with silver impregnation methods. The functional validity of the anatomical findings is established in Pompeiano’s (1960b) physiological studies with liminal stimulation of various parts of the nucleus, and Ito et al. (1964) confirmed the pattern of localization by single unit recordings from the nucleus of Deiters following antidromic stimulation of the vestibulospinal tract*. Having found the somatotopical pattern in the vestibulospinal projection, it was deemed to be of particular interest to investigate whether there is a corresponding arrangement in the projection of the cerebellum onto the nucleus of Deiters. In the first place Walberg and Jansen (1961), therefore, studied the fibres passing directly from the cerebellar vermis to the vestibular nuclei. It turned out that there are fibres from the ‘hindlimb’ and ‘forelimb regions’ of the vermis of the anterior lobe to the corresponding regions of the lateral vestibular nucleus of Deiters, as shown in the left hand diagram in Fig. 15. There are also fibres from the posterior vermis, but whether there is any topical arrangement within this has so far not been decided. It is to be noted that the direct cortical cerebellovestibular fibres terminate in the dorsal half of the nucleus of Deiters only (see Fig. 15). N o fibres could be traced to the superior and medial vestibular nucleus, while there are fibres to the descending nucleus (not shown in Fig. 15), chiefly dorsally, but this nucleus does not give off fibres to the cord (Nyberg-Hansen, 1964). Fig. I5 shows to the right in a diagrammatical way the pattern of the other component of the pathways under consideration, the cerebellovestibular projection via the fastigial nucleus onto the nucleus of Deiters. The diagram is based on experimental studies of the fastigiovestibular projection (Walberg et al., 1962a) combined with the data on the corticofastigial projection. (The fastigial nucleus in addition sends fibres to restricted parts of the other vestibular nuclei, but these do not concern us here.) Even if it is far from being a point to point projection the principle is clear as illustrated diagrammatically in Fig. 15 (to the right). There is a somatotopical pattern throughout in the pathways from the vermis of the anterior lobe via the fastigial nucleus to the cord, and the findings are highly suggestive that a similar pattern is present within the pathways from the posterior vermis. It is interesting to note that the cerebellar impulses to the nucleus of Deiters which pass via the rostra1 part of the fastigial nucleus and come from the anterior lobe vermis reach the dorsal part of the ipsilateral nucleus only, as do the direct corticovestibular fibres. The pathway from the caudal vermis, however, which passes through the caudal part of the fastigial nucleus and leaves this by way of fibres of the hook bundle reaches the contralateral nucleus of Deiters, and its fibres are distributed to its ventral half only.

* It should be noted that axons of small as well as large cells in the nucleus of Deiters take part in the projection onto the cord (Pompeiano and Brodal, 1957a). This fits in with the wide range of conduction velocities of fibres in the vestibulospinal tract, 24-140 m/sec (Ito et al., 1964).

159


w

forelimb + hindlimb 9

Fig. 15. Diagrammatic representations, showing the principles of somatotopical organization within the two pathways from the cerebellar vermis onto the lateral vestibular nucleus as determined experimentally in the cat. Above sagittal sections of the cerebellum (lobules indicated according to Larsell, 1953), below diagrams of sagittal sections through the nucleus of Deiters. The oblique lines in these diagrams indicate the approximate border between a rostroventral forelimb and a dorsocaudal hindlimb region (cp. Fig. 14). To the left is shown the direct projection from the cerebellar vermis to the nucleus of Deiters. Note somatotopical pattern within the projection from the anterior lobe vermis and restriction of terminal area to the dorsal half of the ipsilateral nucleus. To the right is shown the projection via the fastigial nucleus. Note distribution of fibres from the rostra1 part of the fastigial nucleus to the dorsal half of the ipsilateral nucleus of Deiters and from the caudal part of the fastigial nucleus via the hook bundle to the ventral half of the contralateral nucleus of Deiters. The caudal spinal area in the cerebellum covers too much of the uvula in these diagrams, while the pyramis on account of its curving appears too small. From Brodal, Pompeiano and Walberg (1 962).

It is extremely likely from these anatomical observations that the somatotopically localized effects on the motor mechanisms of the cord following stimulation or ablation of specific regions of the vermis proper are mediated via the lateral vestibular nucleus of Deiters. Since the fastigioreticular (Walberg et al., 1962b) and the reticulospinal projection (Torvik and Brodal, 1957) are both diffusely organized, these pathways can scarcely be responsible for the localized effects, even if they undoubtedly exert an action on the spinal mechanisms. Strong support for the view expressed above comes from physiological studies with recordings from single units in the nucleus of Deiters following liminal D C stimulation of the vermis of the anterior lobe. Pompeiano and Cotti (1959) in this way confirmed the existence of a distinct correspondence between points in the ‘forelimb region’ of the cerebellum with points in the ‘forelimb region’ of the nucleus of Deiters. Indeed, a large proportion of the cells in Deiters’ nucleus could be influenced by stimulation of one cerebellar folium only, while they were unresponsive t o stimulation of immeReferences p. 169-173

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diately neighbouring folia. This study indicates that the localization is more precise than can be revealed by anatomical methods, where a larger number of fibres must always be studied simultaneously. However, it was not possible for Pompeiano and Cotti (1959) to decide whether the responses they recorded are mediated via the direct corticovestibular route or utilize the pathways through the rostral part of the fastigial nucleus. On the whole, it appears that the available anatomical evidence permits the conclusion that the routes for impulses from the vermis proper via the lateral vestibular nucleus to the cord present a sufficient degree of localization to make possible a somatotopically organized cerebellar influence on the spinal mechanisms. However, the routes for impulses from the vermis proper of the anterior lobe pass via another part of the nucleus of Deiters than do those from the posterior vermis via the fastigial nucleus (Fig. 15), indicating that the rostral and caudal ‘spinal areas’ of the vermis are functionally not entirely similar. The functional differences between the rostrolateral and rostromedial and between the caudolateral and caudomedial parts of the fastigial nucleus were referred to above (p. 152). Little is known of anatomical differences in the efferent projections of these subdivisions which may be correlated with these functional observations. It is clear, however, that physiologically as well as anatomically the apparatus mediating the cerebellar influences on the cord is organized in an extremely complex manner. This holds true not only for the relation of the vermis proper, but for the intermediate zone of the cerebellum as well. AS to the pathways to the cord mediating the somatotopical responses following stimulation of the intermediate zone of the anterior lobe and the paramedian lobule, anatomical data are far from complete. However, the somatotopical pattern in the vestibulospinal projection (Pompeiano and Brodal, 1957a) is paralleled by a corresponding anatomical organization in the rubrospinal tract, the last link in the pathway for impulses from the intermediate zone of the vermis and the paramedian lobule to the cord. By studying the retrograde cellular changes in the red nucleus following lesions at different levels of the cord, the pattern shown in Fig. 16 could be determined (Pompeiano and Brodal, 1957b). There is a ‘forelimb and neck region’, a ‘trunk region’ and a ‘hindlimb’ region. The somatotopical pattern was further confirmed in silverimpregnation studies of the rubrospinal tract following discrete lesions of the red nucleus (Nyberg-Hansen and Brodal, 1964), and agrees with that determined physiologically by Pompeiano (1957) and Maffei and Pompeiano (1962b). (See also Massion and Albe-Fessard, 1963.) It has been clearly demonstrated physiologically that somatotopical responses can be elicited on stimulation of the intermediate zone of the anterior lobe (Pompeiano, 1958) and from the paramedian lobule (Maffei and Pompeiano, 1962a) as well as from rostral and caudal parts of the interpositus complex (Pompeiano, 1959, 1960a; Maffei and Pompeiano, 1962a), which receive fibres from these parts of the cerebellar cortex. In view of the somatotopical pattern in the rubrospinal projection (Fig. 16) and the regular distribution of the fibres from the cerebellar cortex to the nucleus interpositus (Fig. 4), it appears a likely assumption that the pathway from the latter onto the red nucleus anatomically is organized in a somatotopical pattern. Although


161

the efferent projections from the nucleus interpositus have been studied by numerous authors (for a review, see Jansen and Brodal, 1958; see also Cohen, Chambers and Sprague, 1958; Voogd, 1964) none of them appears to have taken up this problem for serious consideration. Voogd (1964) describes a differential lateromedial distribution in the red nucleus of cerebellar fibres from different subdivisions of the intracerebellar nuclei, and suggests that there may be a somatotopical arrangement in the

,4

ventrolateral

medial caudal

A

B

W ' . .

caudal F

fibres t o cervicol

cord

0

-31-

thoracic

+

-v-

lurnbosocrol-1)-

-71-

Fig. 16. Diagram of the somatotopical pattern in the rubrospinal projection as studied experimentally in the cat. In A a series of transverse sections through the red nucleus, indicating the areas which give off rubrospinal fibres to the various levels of the cord. In B a reconstruction of the nucleus (in the plane indicated by broken lines in the series of drawings in A). From Pompeiano and Brodal (1957b).

projection of his cerebellar zones B' and C-C', which appear to correspond to our intermediate zone of the anterior lobe. It is, however, difficult to evaluate the findings on which his conclusions on the somatotopical projection are based. It is obvious from the recent physiological studies of Pompeiano (1959, 1960a) and Maffei and Pompeiano (1962a, b) that there are within the rostra1 as well as the caudal part of the nucleus interpositus regions which exert an opposite action on the motor References p . 169-173

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activities of the spinal cord (the rostrolateral and caudomedial parts inhibit the activity of flexor motoneurones). It appears, furthermore, that destruction of the red nucleus abolishes only the flexor responses, but not the facilitatory effect on extensor motoneurones (Maffei and Pompeiano, 1962a). This effect, therefore, appears to be mediated via other routes than interpositorubral fibres (see Maffei and Pompeiano, 1962b). We are far from possessing the necessary anatomical knowledge on the efferent fibres from the nucleus interpositus to make possible correlations with the recent physiological findings. There are several reasons for this. In most studies the lesions made have not been restricted to the nucleus interpositus; the part of the complex labelled thus differs among authors, and as touched upon above, efferent fibres from noninjured parts of the nuclear complex may easily be damaged. Flood and Jansen (1961) in a recent study of the cytoarchitecture of the intracerebellar nuclei in the cat have shown the importance of differentiating between a nucleus interpositus anterior and posterior. Except for a minor discrepancy concerning the border between the lateral nucleus and the nucleus interpositus anterior the subdivisions distinguished coincide with those made on the basis of silver impregnated sections (Courville and Brodal, 1966). It is hoped that studies in progress (Courville), undertaken with due reference to the nuclear topography, will bring out the true efferent connections specific to the nucleus, without contamination of fibres from the dentate nucleus. Furthermore, it is hoped that it will be possible to decide whether there is within the projection onto the red nucleus a somatotopical pattern. That this is indeed present might be surmised, not only on account of the physiological data, but also because there is within the pathway passing in the opposite direction, the far less heavy rubrocerebellar projection (Brodal and Gogstad, 1954), an indication of a pattern of this type (Courville and Brodal, 1966). The fact that the majority of the rubrocerebellar fibres end in the nucleus interpositus anterior, some in the interpositus posterior, while no fibres could be traced to the fastigial and lateral nuclei points to a particularly close relationship between the red nucleus and the interpositi, which appears to be reflected in the cerebellorubral projections as well. However, some fibres from the nuclei interpositi pass rostrally beyond the red nucleus, as appears from the study of Jansen and Jansen (1955). The available literature does not permit a final conclusion as to the sites of termination of these fibres, nor is it possible to judge whether they are concerned in the transmission of somatotopically localized impulses from the ‘spinal areas’ of the cerebellum to the sensorimotor cerebral cortex (Henneman, Cooke and Snider, 1952). Since these projections will be considered by Dr. Snider, they will not be discussed here. It appears from the above that in several of the afferent as well as efferent connections of the ‘spinocerebellum’ there is an anatomical pattern of somatotopical organization, which fits entirely with the results of physiological studies. Even if there is for all fibre systems studied so far no point to point relationship in the anatomical sense but a considerable degree of overlapping, the pattern is sufficiently clear to explain the marked degree of localization brought out with physiological methods. Whether there is a somatotopical pattern within the hemispheres as well has been


163

much debated. Unequivocal evidence in favour of this view does not appear to have been produced, although there are some suggestive findings. Thus Jansen, Jr. (1957) found an indication of a pattern of this kind in the crus I1 following stimulation of cortical somatic area 11. In order to get information of this question further studies, anatomical as well as physiological, are needed. MODES OF E N D I N G OF CEREBELLAR FIBRE CONNECTIONS

This subject deserves some comments, since it is of interest for electron microscopical as well as neurophysiological studies. Only a few examples will be mentioned. As to the afferent connections it may be considered as established that the spinocerebellar (Miskolczy, 1931; Brodal and Grant, 1962 and others) and pontocerebellar fibres (Snider, 1936) as well as those from the external cuneate nucleus (Grant, 1962b) and the primary vestibular fibres (Brodal and Hnrivik, 1964) end as mossy fibres. It appears likely, but is so far not decided, that the cerebellar afferents from the lateral reticular nucleus, the paramedian reticular nucleus and the perihypoglossal nuclei likewise end as mossy fibres, and the findings of Carrea, Reissig and Mettler (1947) indicate that secondary vestibular fibres belong to the mossy fibres. The sources of the climbing fibres have been more difficult to discover, and conflicting opinions have been expressed (see Jansen and Brodal, 1958,p. 137, for a review). The most convincing finding must be attributed to Szentagothai and Rajkovits (1959), who from experimental silver-impregnation studies in the cat conclude that the great majority of climbing fibres come from the inferior olive*. In view of the far more selective terminal distribution of the climbing fibres than of the mossy fibres, it is interesting to recall the remarkable precise localization within the olivocerebellar projection (Fig. 8). If the olive is the main source of climbing fibres to the cerebellum, it must presumably play a particular role in the function of the cerebellum, an assumption which receives some support from the fact that of all regions sending fibres to the cerebellum, the inferior olive appears to be the only one which supplies the entire cortex. The evoked potentials in the cerebellar cortex following stimulation of the pons and the inferior olive differ conspicuously (Jansen, Jr., 1957). When considered in conjunction with the anatomical data (mossy fibres from the pons, climbing fibres from the olive), this may indicate important functional differences between the two types of fibres. However, there appear to be differences between mossy fibres as well. This is shown by our findings of fibres of a particular type in the vestibular part of the cerebellum. Physiological observations indicate that the fibres of the dorsal spinocerebellar tract influence a small group of cortical cerebellar neurones, while the ventral and rostra1 spinocerebellar fibres influence cortical cells scattered in a wide area (see Oscarsson, 1965a). These features probably reflect differences between the mossy fibres of these tracts with regard to the profuseness of their branching.

* In addition these authors advocate the existence of a smaller number of climbing fibres from other sources, such as fibres to the vermis from the dorsomedial pontine nuclei or ventromedial regions of the reticular formation. Refei-rncrs p . 169-173

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Studies of endings of the afferent cerebellar fibres with silver-impregnation methods meet with considerable difficulties. It appears likely that additional information on details may be obtained in electron microscopical work. Degenerating terminals can be identified following experimentally produced lesions, as seen from Fig. 17, which shows an example of a degenerating spinocerebellar mossy fibre studied by Mugnaini and Walberg.

Fig. 17. Electronmicrograph showing a degenerating terminal of a mossy fibre in the granular layer of the right lobulus IV 4 days following transection of the ipsilateral dorsal and ventral spinocerebellar tracts in the cat. Several dendrites (d) and a process of an astrocyte (9) are apposed to the degenerating terminal (b). The arrows point to synaptic complexes with dendrites, the solid triangle to a dendritic contact. Scale line 1 p. Courtesy of E. Mugnaini and F. Walberg.


165

The efferent cerebellar fibres likewise do not behave uniformly as concerns their endings. This is of interest from a functional point of view. As an example may be mentioned some data from the nucleus of Deiters. Silver impregnation studies show that the direct corticovestibular fibres establish contact chiefly with the giant cells of the nucleus (Walberg and Jansen, 1961), while fibres from the fastigial nucleus end largely on small cells (Walberg et al., 1962a). Small as well as large cells project onto the spinal cord (Pompeiano and Brodal, 1957a). It is tempting to hypothesize that the different synaptic relationships of the two cerebellovestibular pathways may in some way be related to the role played by the cerebellum in linking the influences on a- and y-neurones in the cord (Granit, Holmgren and Merton, 1955). In any case the preferential terminations of the two groups of cerebellovestibular fibres on small or giant cells, respectively, indicate that these cells in the nucleus of Deiters are functionally not identical. This assumption is further supported by the observations that primary vestibular fibres end chiefly, perhaps exclusively, on small cells in the nucleus (Walberg, Bowsher and Brodal, 1958), while the spinovestibular fibres contact chiefly giant cells (Pompeiano and Brodal, 1957~). In experimental studies both types of cerebellovestibular fibres are found in contact with somata as well as proximal dendrites of the cells (Fig. 18). The fibres from the

Fig. 18. Photomicrograph of a Nauta impregnated section from the lateral vestibular nucleus of the cat following a lesion of the ipsilateralfastigial nucleus. Two giant cells are contacted along soma and dendrites by degenerating fine fibres with swellings, presumably terminal boutons. (Most fastigiovestibular fibres end on small cells, cp. text.) x 270. From Walberg er al. (1962a).

cerebellar cortex ending in the intracerebellar nuclei establish contact in the same way. In Golgi sections it can be seen that virtually a single fibre passes along a proximal dendrite and the soma of a single cell. References p . 169-173

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In silver impregnated sections it is difficult, and usually impossible, to decide whether there are contacts between degenerating fibres and the distal, thin parts of dendrites. However, this problem can now be tackled in electron microscopical studies of degenerating nerve terminals following cerebellar lesions. Walberg recently studied the nucleus of Deiters and the intracerebellar nuclei. As seen in Fig. 19, degenerating terminal boutons establish contact with very thin dendrites. Obviously there

Fig. 19. Electronmicrograph from the dorsal part of the lateral vestibular nucleus in the cat following a lesion of the vermis of the anterior lobe of the cerebellum. A degenerating bouton ( b ~lies ) close to a dendrite (d) about I p in diameter. Arrow points to synaptic complex. bz is a normal terminal bouton. Scale line 1 p. Courtesy of F. Walberg.

are thus axodendritic as well as axosomatic synapses of cerebellar fibres on these cells. A detailed knowledge of the types and sites of synapses is essential for correlations with electrophysiological studies of inhibitory and facilitatory synapses. According to Ito and collaborators stimulation of appropriate parts of the cerebellar cortex produces predominantly inhibitory postsynaptic potentials (monosynaptic) in the cells of the nucleus of Deiters (It0 and Yoshida, 1964) and in the intracerebellar nuclei (Ito, Yoshida and Obata, 1964).

CEREB E L L AR F IBR E C O N N E C T I O N S

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Only some aspects of the fibre connections of the cerebellum have been considered in this account. Many details have been left out, and the subject has been considerably simplified. Even so, however, I think it will appear that there is still very much to do before we know the morphology of the cerebellar connections sufficiently well. In concluding I would like to return to the question which was brought up in the beginning of this presentation: Do we need further studies of fibre connections, when we have got the electron microscope and can record from single units? We are all aware of the vast number of data on the ultrastructure of nervous tissue which have been made available by the use of the electron microscope. Their importance for our understanding not only of the structure but also of the function of the nerve cells and fibres is apparent. For example, the finding in silver impregnation studies of argyrophilic particles attached to a cell surface is highly suggestive that the particles represent true synaptic contacts, but they are not decisive. Thin sheets of glia, interposed between an apparent terminal and the cell can not be recognized in such preparations. The final proof that true synapses are present is to be obtained in electron microscopical studies, performed on experimental material. Furthermore, the many morphological varieties among synapses which have been disclosed in electron microscopic studies must be assumed to reflect functional differences. In a single nucleus or cell group there are often synapses of different types. In order to evaluate the functional implications of this it is essential to know to which afferent fibre systems the various types of synapses belong. This can only be determined experimentally by studying with the electron microscope the degenerative changes which occur in terminal boutons when the axons to which they belong have been transected. On account of the minute size of the pieces of tissue which can be selected for electron microscopy, it is a necessary prerequisite for such studies that one knows precisely where to search for changes in the boutons. This requires a preliminary study by means of classical methods, preferably one of the silver-impregnation methods. It should be noted that such studies have to be very detailed, since there are within many nuclei variations with regard to the sites of termination of the different afferent influxes, as exemplified by the nucleus of Deiters. The situation is much the same with regard to the evaluation of many results of the recent refined studies in neurophysiology. Intracellular recordings from single units following natural or artificial stimulation of receptors or other parts of the nervous system can only be properly evaluated when one knows the pathways which mediate the afferent impulses to the nucleus in question. Again it is necessary to know the sites of termination as well as the pattern of synaptic contacts in great detail. Otherwise misinterpretations are apt to occur. A further field where studies of the fibre connections are of use is in the evaluation of data of comparative anatomical research. A thorough knowledge of the mutual connections between the many parts of the central nervous system is essential, if we want to understand the brain and its function properly. Recent neurophysiological research brings to light more and more data References p . 169-1 73

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which demonstrate an extensive and intimate degree of collaboration between various parts of the brain. The time when one could speak of ‘systems’ is finished, or ought at least to be finished. To anatomists engaged in studies of fibre connections this does not come as a surprise. In fact, it becomes more and more obvious the more we get to know of the extremely complex patterns of interconnections which are by and by revealed by systematic studies. Yet, even if we get to know these patterns in very great detail, we are far from our goal. There still remain the vast number of ultrathin fibres, demonstrated with the electron microscope, but not visible at all in the light microscope. One may indeed ask: Shall we ever be able to find out how these fibres are arranged and how they function, inaccessible as they apparently are even to present day refined electrophysiological recording methods? This, however, should not discourage us in using our present methods as far as they will go. If better methods will in time be found, we may at least be assured that future studies will have to build on evidence obtained by the procedures which are at our disposal at present and certainly will serve as useful tools for many years to come. SUMMARY

Certain problems within the subject covered in the title have been selected for consideration. (1) Studies of cytoarchitectonics and fibre connections demonstrate that regional variations exist within the cerebellar cortex. For example, in the flocculus, nodulus, the caudal part of the uvula and the dorsal paraflocculus there are mossy fibres which differ morphologically from the classical type. The distribution of these mossy fibres corresponds to the sites of termination of primary vestibular fibres. The latter fact shows that the ‘vestibulocerebellum’ extends outside the flocculonodular lobe, and casts doubt on the correctness of considering cerebellar fissures as borders between functionally dissimilar parts of the cerebellum. (2) As concerns the functional subdivision of the cerebellum, studies of its fibre connections demonstrate a considerable overlapping between the sites of termination of fibres transmitting afferent impulses from the cerebral cortex (via various intercalated stations), and from the spinal cord. On the efferent side there is evidence for a particularly clearcut collaboration between the ‘spinal parts’ and the ‘vestibulocerebellum’ since there are well developed connections from the anterior and posterior vermis to the vestibular nuclei. It appears from anatomical as well as physiological studies that the longitudinal subdivision of the cerebellum into vermis proper, intermediate zone and lateral or hemispheral part may be pursued further. (3) Anatomical evidence has recently been produced which provides the morphological basis for the physiological observations on a somatotopical pattern within the vermis and intermediate zone. This applies on the afferent side particularly to the spinocerebellar tracts, on the efferent side particularly to the vestibulo-cerebello-spinal pathways. The cerebello-rubro-spinal pathway appears to be organized in the same way.


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(4) Studies of the modes of termination of afferent and efferent cerebellar fibres have given valuable information of details concerning synaptic relations. These are of particular interest in the interpretation of electrophysiological results. ( 5 ) In the future the electron microscope will undoubtedly be an important tool in the study of the fine details in synaptic relationships. Its fruitful use on problems of this type, however, requires that the region under study has previously been closely mapped with reference to its connections with classical neuroanatomical methods.

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ADDENDUM

Since this paper was submitted for publication there has appeared a report on the anatomy of the climbing fibres which is of relevance to the problem discussed on p. 163. HAMORI,J., AND SZENTAGOTHAI, J., (1966); Identification under the electron microscope of climbing fibers and their synaptic contacts. Exp. Brain Res., 1, 65-81.

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The Primate Cerebellar Cortex : A Golgi and Electron Microscopic Study CLEM ENT A. FO X, D E A N E. H ILLMAN, K E N N E T H A. S I E G E S M U N D A N D CHITTA R. D U T T A Anatomy Departments, Wayne State University School of Medicine, Detroit, Mich., and Marquette University School of Medicine, Milwaukee, Wis. (U.S.A.j

INTRODUCTION

Ramon y Cajal’s first success with the Golgi technique came with his study of the cerebellar cortex and it is no accident that its structure is as well known as it is. The capricious method of Golgi, which now and then reveals the external morphology of a nerve cell and all its processes more completely than any other technique, has been most successful when applied to the cerebellar cortex. It is the easiest central nervous system center to impregnate. Consequently its fine structure has been known longer and better than that of any other center. In his last communication on the cerebellar cortex Ramon y Cajal (1926) noted that nearly all that is known of this structure - the detailed morphology of its nerve cells, their synaptic relationships and the direction of their axons -was due to studies made with the protoplasmic procedures, i.e. the methods of Golgi and Ehrlich. He pointed out that, although there is perhaps no nervous center in which the fine structure is better known, our knowledge of its circuitry still had many gaps. To resolve some of the interesting problems of the cerebellar cortex, he proposed that studies, scrupulously made with protoplasmic procedures, be undertaken since only these methods are capable of showing sharply in toto the extent of neuronal protoplasm. Today neuroanatomists repeat his counsel. Commenting on the recent complementary developments in electron microscopy and electrophysiology Palay (1965) says : ‘The refinement of detail now obtainable in morphology and physiology makes it imperative that the investigation of the architecture of the nervous system by means of optical microscopy should be intensified, especially by using Golgi techniques’. Investigators have studied aspects of the cerebellar cortex by electron microscopy: Fernandez-Moran. (1957), Palay (1958, 1964), Hager (1959), Gray (1961), Hager and Hirschberger (1960), Dahl et al. (1962), Palay et al. (1962), Fox (1962), Smith (1963), Herndon (1963, 1964), Fox et al. (1964), Szentigothai (1965a,b). No doubt the superficial position of the cerebellar cortex and its well known structure make it ideal for such studies. Certainly, the rigid orientation of its elements in the molecular layer - the granule cell axons parallelingeach other; the transverse spread

CEREBELLAR CORTEX

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of the Purkinje and stellate cell dendrites; the presence of medullated fibers in the most inferior portion of the molecular layer; the palisade arrangement of the Bergmann fibers; and the basket formations about the Purkinje cell bodies - all supply valuable clues for the correlation of electron microscopic observations with those of light microscopy. A quotation from a previous account of the cerebellar cortex (Fox, 1962) is now timely: ‘Rambn y Cajal(l91 l), impressed by the vast divergence and convergence and the resultant multiplications of synapses from stage to stage in the cerebellar cortex, concluded there is ‘avalanche conduction’ in the cerebellar cortex. It would be wrong, however, to assume that all these connections result in excitation. Nervous activity has another aspect, namely, inhibition. If more were known about the nature of inhibition, or better still, if an anatomical basis for inhibition could be established, our detailed anatomical knowledge of the circuits in the cerebellar cortex could be more readily translated into precise physiological terms. Meanwhile, we must be content to use the term ‘influences’instead of ‘excites’ or ‘inhibits’ when describing the activity of individual elements in the cerebellar cortex’. This is now changed. Thanks to the excellent contributions of Eccles and his collaborators and Ito and his collaborators it is possible to use the terms ‘excites’ or ‘inhibits’ when describing the activity of individual elements in the cerebellar cortex. In the present study features of the cerebellar cortex are illustrated with Golgi impregnations of the macaque monkey (Macaca mulatta) and with electron micrographs of man, the macaque monkey (Macaca mulatta) and the squirrel monkey (Suimiri sciureus). It will be seen that the Golgi preparations provide an invaluable guide; without them interpretations of the electron micrographs would have been difficult, if not impossible. The discussion is interspersed in the presentation in order to explain immediately the interpretations given the electron micrographs. T o save space the figures are described in the text. THE G R A N U L A R LAYER

The granular layer, sandwiched between the medullary and molecular layers, has cells intermingling with the peripheral fascicles of the medullary layer, inferiorly, and cells invading the intervals between the Purkinje cells, superiorly. Considerably thicker at the summits than at the depths of the folia, in Nissl sections (Fig. 2, Macaca mulaatta) it appears as an enormous quantity of intensely stained, small, naked nuclei pressed one against the other. These nuclei, granule cell nuclei, characterize the layer and are so prodigious in number that residual space seems insufficient to accommodate their processes, the fibers of passage, and other intrinsic cells. The latter, infinitely less numerous, are the large stellate, or Golgi cells, and the glial cells. Distinguishing these cells from each other and from the granule cells is no problem; one needs only to take into account the structure and the volume of their respective nuclei (Rambn y Cajal, 1911). The granule cells, round or oval in shape and ranging in diameter from 5 ,u to 8,u, resemble lymphocyte nuclei and have chromatin granules aggregated against their References p. 222-225

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c. A,

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nuclear membranes and centrally clumped. Herndon (1964) attributes this chromatin arrangement - seen also in Gray’s electron micrographs (1961) and apparent in the electron micrographs (Fig. 1, human; Fig. 4, Macaca mulatta) - to poor fixation. In material fixed by the perfusion method of Palay et al. (1962), Herndon finds the chromatin evenly distributed in the nucleus with no clear areas, though in instances the pattern of granularity may be a little coarser towards the center of the nucleus. One of the larger intensely stained chromatin clumps may be the nucleolus (Rambn y Cajal, 1911). Nucleoli are identifiable in electron micrographs of the monkey granule cells (Dutta et al., 1963). The nakedness of granule cell nuclei is due : (1) to the complete absence of discrete Nissl granules ; correspondingly, membrane profiles of the endoplasmic reticulum are scant and RNP particles are dispersed in the cytoplasm (Fig. 4, Macaca mulatta); (2) to the thinness of the rimming cytoplasm which varies from several hundred A to a little more than a micron. Nonetheless, these karyochrome neurons have all the cytoplasmic organelles found in cytochrome neurons : a few mitochondria; an occasional dense body (DB, Fig. I , human); centrioles (Palay et al., 1962); multi-vesicular bodies (Herndon, 1964); a Golgi apparatus. This latter organelle (GH, Fig. 5 , Macaca mulatta), composed of an agranular reticulum and varying sized vesicles, is considerably less extensive than that in most large neurons. Interestingly, Rambn y Cajal (1911) in reduced silver material found a very thin, juxtanuclear dark line in the thickest part of the cytoplasm and considered it an extremely simplified GolgiHolmgren apparatus. Elements of the cerebellar islands. The cerebellar islands, known also as plasma islands (Rambn y Cajal, 1911) and cerebellar glomeruli (Held, 1897), are the cell free areas bounded by the clustering granule cells. Here granule cell dendrites, mossy fibers and Golgi cell axons form complex synapses and, as is known from Schroeder (1929), Boeke (1942) and others, astroglial processes are also present. All these elements are refractory to Nissl staining and in such preparations the islands (I, Fig. 2 ) appear as clear spaces. But in electron micrographs (I, Fig. I , human) the islands are solidly packed with the profiles of these various elements. In this figure note the partial profiles of 9 granule cells surrounding the island; also note, in the lower part of the figure, the two dendrites emerging from two apposed granule cells and entering the island side by side. The tight packing of the granule cells with their plasma membranes in immediate apposition, apparent here (Fig. l), has been observed by Hager and Hirschberger (1960), Gray (1961) and Palay et al. (1962). Proximally the granule cell dendrites have conspicuous dendritic tubules. They are clearly displayed in the fortunate section (D, Fig. 4, Macaca mulatta) which shows two dendrites emerging from the same cell. The intrinsic granular layer astrocytes display luxurious processes bristling with fine appendages (As, Fig. 3, Macaca mulatta);the adjacent impregnated granule cell (GrC) affords a good-sized comparison. Profiles of astrocytic processes in the islands are seen in Figs. 1 and 4. The literature on the cerebellar glomeruli, reporting results obtained by a variety of methods, is both extensive and confusing. Fortunately, this lengthy literature has been well reviewed by Jansen and Brodal (1958) and, better still, recent electron

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Figs. 1-5. Fig. 1. Low power electron micrograph of a human cerebellarisland. Fig. 2. Oil immersion photomicrograph of granule cells. Nissl preparation; Mucucu muluttu. Fig. 3. An astrocyte and a granule cell. Golgi prepration; Mucucu muluttu. Fig. 4 . Electron micrograph of a granule cell with two emerging dendrites. Mucuca muluttu. Fig. 5 . Electron micrograph of a small portion of the nucleus and cytoplasm of a granulecell showing Golgi apparatus. Mucucu muluttu. For abbreviations see p. 221. References p . 222-225

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microscopic studies (Gray, 1961; Palay et ul., 1962; and Szentagothai, 1965a,b) are doing much to eliminate this confusion. But these studies, excellent as they are, would not have been possible were it not for the revelations obtained by the Golgi method, particularly those of Ram6n y Cajal(l911). This will become apparent after elements of the cerebellar glomeruli are presented in a series of Golgi impregnations and electron micrographs. MossyJibers.A mossy fiber (MF) ramifying into branches that bear the excrescences Ram6n y Cajal (1911) designated ‘rosettes’ (R) is shown in the Golgi impregnation (Fig. 6, Mucucu rnuluttu). The fine beaded fibers in the same field are endings of Golgi axons. Ram6n y Cajal(1937) discovered the mossy fibers in 1888 and it is now known from experimental evidence based on the axonal degeneration that they are the terminals of the spinal cerebellar tracts (Miskolczy, 1931; Rosiello, 1937), the ponto-cerebellar fibers (Snider, 1936; Mettler and Lubin, 1942) and the vestibulo-cerebellar fibers (Snider, 1936). In a remarkable impregnation Ramdn y Cajal (Fig. 41, 1911) demonstrated single mossy fibers distributing to two cerebellar folia. Considering their branching by bifurcations in the medullary layer and by secondary and tertiary branches in the granular layer, he estimated that a single mossy fiber may have as many as 20 or 30,or more, secondary and tertiary branches. It was his opinion that all branches of the mossy fibers are denuded of myelin as they enter the granular layer. But Dogie1 (1896), in Ehrlich material of the dove, revealed nodes of Ranvier and myelin sheaths on branches of the mossy fibers in the granular layer. Dogiel’s observations are now substantiated by electron microscopy (Gray, 1961). A medullated mossy fiber (MF) of the granular layer losing its myelin sheath and in continuity with a rosette (R) is shown in the electron micrograph (Fig. 7, Mucacu muluttu). Note the astrocyte and its process (As) surrounding this segment of the fiber just beyond the medullary sheath region. Palay (personal communication) has seen myelinated fibers on apposing sides of a cerebellar island in continuity with a single rosette. Is it possible that the segments of the mossy fibers between the rosettes are myelinated and that the rosettes are modifications of the nodes of Ranvier? The rosettes are synaptic sites clasped by the claw-like dendrites of the granule cells. The evidence for this is shown in the rare Golgi impregnations (Figs. 8-11, Mucuca rnuluttu). Two of the granule cells (GrC, Fig. 8) related to a rosette (R) are impregnated. Three of the granule cells (GrC, Fig. 9) related to a rosette (R) are impregnated but one of them is out of the plane of focus. On this same mossy fiber, but out of the field, are two more impregnated granule cells related to a rosette. Seven of the granule cells (Fig. 10) related to a rosette (R) are impregnated. Two groups of granular cells (1 and 2, Fig. 11) cluster about two successive rosettes on a single mossy fiber. By carefully focusing on group 2 in the latter figure, 14 granule cells related to a single rosette can be counted. Apparently Ram6n y Cajal (191 1) observed the connections between the granule cell dendrites and the mossy fiber rosettes and, advancing various arguments to prove this connection, he adds ‘In some cases, very few in number it is true, in preparations made by the methods of Golgi and Ehrlich the meeting and the intimate engagement

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Figs. 6-11. Fig. 6. A branching mossy fiber showing rosettes. Golgi preparation; Mucucu muluttu. Fig. 7 . Electron micrograph of a mossy fiber losing its myelin sheath and entering a cerebellar island. Mucucu muluttu. Fig. 8 . Two granule cells contacting a mossy fiber rosette. Golgi preparation; Macacu mulutru. Fig. 9. Two granule cells contacting a mossy fiber rosette. Golgi preparation; Mucucu muluttu. Fig. 10. Seven granule cells contacting a mossy fiber rosette. Golgi preparation; Mucucu muluttu. Fig. 11. Two clusters of granule cells related to successive rosettes on a mossy fiber. Golgi preparation; Mucucu muluttu. For abbreviations see p. 221

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of mossy fiber appendages and the digitations of granule cells can be clearly seen in the interior of the cerebellar islands.’ Even as early as his Croonian lecture (1894) he mentions this observation made in Ehrlich preparations but nowhere does he illustrate it. Why he resorts to arguments and does not offer convincing illustrations may seem strange. But then, in our opinion, illustrating these complex synapses in the era of the bitter ‘contactxontinuity’ battle would have been disadvantageous and awkward for this champion of ‘contact’ versus ‘contiguity’. Simultaneous impregnation of the rosettes and their specifically related granule cells (Figs. 8-1 1) obliterates the details of the rosettes and the granule cell dendrites in a common mass of impregnation. Only in electron micrographs are the intimate relationships of these elements, the rosettes and the dendritic digits, resolvable (Fig. 13, Macaca mulatta; Fig. 15, human). The mossy fiber granule cell synapse allows for considerable divergence. On one of the mossy fibers distributing to two folia in Ram6n y Cajal’s Fig. 41 (1911) we counted 44 rosettes. If on an average a rosette is contacted by 15 granule cells, then some extra cerebellar neuron sending its axon into the granular layer diverges to at least 660granule cells. Undoubtedly this is a minimal estimate for most likely the impregnations seen in our Fig. 11 and in the figure of Ram6n y Cajal, referred to above, are incomplete. Also this estimate does not consider the possibility of other branchings of a mossy fiber in its course through the medullary layer. Structure of the rosettes. Under low power, the rosettes (R, Fig. 6) appear to be solid structures but when observed under oil immersion (Figs. 12, 14 and 19, Mucaca rnulatta) sharp impregnations of the rosettes appear to be very coiled, convoluted fibers. Electron microscopic observations in no way contradict this latter view of the rosette configuration. Compare the impregnations (Figs. 12 and 14) with the rosette profiles in the electron micrographs (Fig. 13, Mucuca mulatta; Fig. 15, human) taken from relatively thick sections - which show the rosette profiles (R) darkened with synaptic vesicles and a concentration of mitochondria standing out in sharp contrast to the lighter profiles of the dendritic digits. The profile (R, Fig. 13) could well be a thin section through one of the looped fibers in the rosette (Fig. 12); similarly the profile (R, Fig. 15) could well be a thin section through a portion of the rosette (Fig. 14). If the rosettes were solid structures, solid rosette profiles as large as 15 p x 20 p should sometimes be encountered in electron micrographs. We have never found solid rosette profiles of this dimension. Also, if the rosettes were solid structures, it would be impossible for the dendritic digits to enter a rosette and lose their identity as they do in Figs. 8, 9 and 10. Dogiel’s (1896) Ehrlich preparations of the mossy fibers show the rosettes as rounded, oval or irregular formations of convoluted fibers which he called ‘Endknauel’ formations. Their resemblance to our preparations is striking. Further, Craigie’s (1926) comparative study of mossy fibers in birds and mammals,. done by neurofibrillar techniques, is reconcilable with our interpretation of the rosettes, He finds, for example, in man that the fibers of the rosettes are relatively slender, unbranched and frequently coiled. Granule cell dendrites. Though granule cells are essentially alike, each has its own individuality. Variations are mainly in the dendrites : their lengths ; their branchings ; the pattern of their claw-like endings. Obviously the short digit-like branchlets

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Figs. 12-15. Fig. 12. An oil immersion photomicrograph of a mossy fiber rosette. Golgi preparation; Macaca mulatta. Fig. 13. An electron micrograph of a mossy fiber rosette. Macaca mulatta. Fig. 14. An oil immersion photomicrograph of a mossy fiber rosette. Golgi preparation; Macaca mulatta. Fig. 15. An electron micrograph of a mossy fiber rosette; human. For abbreviations see p. 221. Refrrences p . 222-225

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sprouting still shorter protoplasmic processes, are dendritic devices designed for articulation with the rosettes, while the dendritic lengths are a function of the cell body’s distance from the related rosette. The usual extremes of dendritic lengths are illustrated here (Figs. 16, 18). They are relatively long in the impregnation (Fig. 16). Here the upper dendrite branches and each resulting branch terminates in dendritic digits. In addition, on the right branch another set of dendritic digits arises immediately beyond the bifurcation. Occasionally we have observed dendrites 3 and 4 times as long as the upper dendrite in Fig. 16. In the impregnation (Fig. 18) the two inferior dendrites are extremely short and the terminal dendritic digits arise 1 p and 3 ,u respectively, from the cell body. The upper dendrite on the same cell is of moderate length, Note particularly its dendritic digits and their short, stubby, spine-like, lateral extensions. For comparison, view the longitudinally sectioned dendritic digit in the electron micrograph (DD, Fig. 20) with its serrated edge dovetailing the mossy fiber rosette (R). The ‘gearing’ in this fortunate section would have delighted Ram6n y Cajal (1954) who, in his now classical classification of synaptic patterns, placed the granule cell-mossy fiber articulation in the category ‘axo-dendritic connections by gearing’. Many interesting comparisons can be made between the Golgi and the electron microscopic preparations. Compare, for example, the dendritic digits (DD) in Figs. 17 and 21. Microspines. In certain regions of the dendritic digit-rosette apposition small protrusions of the dendritic surface, 400 to 1200 8,in length and 400 to 600 8,in width, invaginate the rosettes. These structures, here called microspines (MS, Figs. 23 and 24), bear ‘spiked halos’ on their presynaptic surfaces. Vesicles 800 8, in diameter frequently occur within the rosettes not far from these invaginations. They have short, radiating linear structures on their surfaces (SpV, Fig. 25). These spiny vesicles, undoubtedly, are the complex vesicles of Gray (1961) and the ‘spiked’ vesicles of Andres (1964). Andres regards the microspines as micropinocytotic invaginations and, theorizing, relates the microspines and the ‘spiked’ vesicles to a process of pinocytosis in which degradation products of the synapse are recirculated to the Golgi apparatus for resynthesis. In the developing cerebellar cortex (Larramendi, personal communication) synaptic vesicles are present before the microspines develop. As previously mentioned, mitochondria are numerous in the rosettes and they are also present in the dendritic digits. In all probability the mitochondria are the neurosomes, i.e. the granules Held (1897) described in the cerebellar glomeruli in material stained by the Altman-Arnold method. Occasional granular, dark-core synaptic vesicles are found in the rosettes (Fig. 22). Their significance is unknown. Dendritic digit aggregates and the attachment plaques. Considering the number of granule cells related to a single rosette (Group 2, Fig. 11) and the number of digits and their processes on a single dendrite (DD, Fig. 17) it is not surprising to findlarge aggregates of these closely packed profiles in electron micrographs of the cerebellar islands (DD, Figs. 1, 4, 13, 15, 20 and 21). In these aggregates there are many symmetrical attachment plaques (AP, Figs. 22 and 24) on apposing dendritic membranes. Gray (1961), who first described them, was undecided whether they occur between the dendritic branches of the same or different neurons. Never having found dendritic digits

Figs. 16-21. Fig. 16. An oil immersion photomicrograph of a granule cell. Golgi preparation; Macaca mulatta. Fig. 17. An oil immersion photomicrograph of a granule cell dendrite. Golgi preparation; Macaca mulatta. Fig. 18. An oil immersion photomicrograph of a granule cell. Golgi preparation; Macaca mularta. Fig. 19. An oil immersion photomicrograph of a mossy fiber rosette. Golgi preparation; Macaca mulatta. Fig. 20. An electron micrograph of a granule cell dendritic digit synapsing with a mossy fiber rosette; human. Fig. 21. An electron micrograph showing branching of a granule cell dendritic digit; Macuca mulutta. For abbreviations see p. 221. References p. 222-225

Figs. 22-29. Fig. 22. An electron micrograph showing attachment plaques bctween granule cell dendritic digits. Note also the dark core vesicles in the rosette; human. Fig. 23. An electron micrograph showing microspines on a granule cell dendritic digit; Squirrel monkey. Fig. 24. An electron micrograph showing microspines on dendritic digits of granule cells. Note also the attachment plaque; Mucucu muluttu. Fig. 25. An electron micrograph showing ‘spiked vesicle’ in a rosette; Mucucu rnuluttu. Fig. 26. Photomicrograph of a granule cell axon arising from a dendritic digit. Golgi preparation; Mucuce rnulutru. Fig. 27. Photomicrograph showing granule cell axon arising from a dendrite. Golgi preparation; Mucucu mulatiu. Fig. 28. Photomicrograph showing a granule cell axon arising from cell body and its T-shaped bifurcation in the molecular layer. Golgi preparation; Macacu rnulutiu. Fig. 29. An electron micrograph showing a crescent bundle of ascending granule cell axons in the granular layer; squirrel monkey. For abbreviations see p. 221.


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this close together on the same dendrite we are forced to conclude that they must exist between different neurons. The possibility has been considered (Palay, 1956; Gray, 1959; Van der Loos, 1963) that the material producing the thickenings on certain sites of the pre- and postsynaptic membranes (Figs. 20 and 24) may be attachment plaques. It is well known that synaptic contacts are not easily broken in the central nervous system and must, therefore, have an adhesive quality. Undoubtedly the so-called ‘second system of axons’ on Deiters’ (1865) teased ventral horn neurons are adhering synaptic endings with their preterminal fibers. Carpenter (1911) described preterminals remaining attached to the cells of the ciliary ganglion of the chick in teased preparations, and Hyden (1960) shows neurons isolated from Deiters’ nucleus with adhering synaptic knobs on their surfaces. Conceivably, then, the entire assemblage of dendritic digits related to a single rosette (Group 2, Fig. 11) adhere to each other and to the rosette by attachment plaques which stabilize this complex synapse into a single formation. Granule cell axons. These axons emerge either from a cell body (A, Fig. 28) or from a dendrite (A, Figs. 26 and 27). If the latter gives rise to the axon, it is usually an ascending and not a horizontal or descending dendrite. This illustrates one of Ramon y Cajal’s (1909) generalities on the nervous system, ‘the law of economy of matter’. The axon ascends to the molecular layer where it bifurcates T-shaped (Fig. 28) and then parallels the long axis of the folium; hence, the designation (Ramon y Cajal, 1911) ‘parallel fibers’. Ascending through the granular layer these axons are bundled together. Note in the electron micrograph (block arrows, Fig. 29) the crescent shaped aggregate of crosscut ascending granule cell axons in the granular layer. In general, granule cells low in the granular layer give rise to parallel fibers low in the molecular layer, and granule cells high in the granular layer give rise to parallel fibers high in the molecular layer (Ram6n y Cajal, 1911). This can easily be confirmed in the present material. A granule cell in the inferior part of the granular layer with its axon bifurcating in the inferior part of the molecular layer is shown in Fig. 28. Details of the parallel fibers will be considered when the molecular layer is described. Dense staining cells. An occasional densely stained cell of the type Gray (1961) suggests it might be a microglial cell shown in the electron micrograph (DkC, Fig. 30). Interestingly, Schroeder (1929) reports that microglial nuclei stain more intensely than macroglial nuclei. Palay et al. (1962) find no dense cells in their osmium tetroxide perfused material. Large stellate or Golgi cells. The large stellate cells, called Golgi cells by Retzius (1892), are easily recognized in Nissl preparations. Usually 2 or 3 can be seen in a low power field but fields with more, 7 or 8, are also encountered (Jakob, 1928). It is the latter author’s impression that they are more numerous in the inferior vermis and in the flocculus. The Golgi cell bodies, stellate, polygonal, and even triangular in shape, are located most frequently in the vicinity of the Purkinje cell layer (Fig. 31); occasionally deep in the granular layer (Fig. 33); in rare instances in the molecular layer (Fig. 32). These cells are considerably larger than the granule cells (Fig. 30) and have large clear nuclei with a prominent nucleolus. Their cytoplasm contains mitochondria, a Golgi apparaReferences p . 222-225

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Figs. 30-33. Fig. 30. An electron micrograph showing a Golgicell; Macucamulatta. Fig. 31. Photomicrograph of a Golgi cell near the Purkinje cell layer. Golgi preparation; Mucuca mulatta. Fig. 32. Photomicrograph of a Golgi cell displaced in the molecular layer. Golgi preparation; Macacu mulatta. Fig. 33. Photomicrograph of a Golgi cell deep in the granular layer. Golgi preparation; Macaca mulatta. For abbreviations see p. 221.

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tus (GH, Fig. 87) and the arrangement of endoplasmic reticulum (NB) seen in somatochrome neurons. Cytoplasmic protrusions extend into the nucleus (arrows, Fig. 87). According to Ram6n y Cajal (1911) and others the Golgi cell dendrites, which ascend vertically in the molecular layer to the pial surface (Figs. 3 1 and 32), spread in a loose arrangement and are not flattened in a single plane. Even deep Golgi cells send a few dendrites into the molecular layer (Ram6n y Cajal, 1911). The Golgi cell dendrites are contacted by the parallel fibers but to our knowledge their contacts in the granular layer have not been definitely established. Szentiigothai (1965a) says: ‘The dendrites in the granular layer are with their ends engaged in the cerebellar glomeruli. Here they obviously synapse with the mossy fibers’. This may be true but in our material we have not found evidence to support it. Unfortunately, in our electron micrographs we have never succeeded in identifying Golgi cell dendrites, either in the granular or the molecular layer. Golgi cell axons. The density of Golgi cell axonic plexuses (GA, Figs. 31-33) is well known. Simultaneous impregnation of a number of these cells can blacken the granular layer. Both Van Gehuchten (1890) and Retzius (1892) have noted these heavy impregnations and we, too, have observed them. Usually the axonic arborizations from a single cell distribute to the region of the granular layer immediately beneath the spread of its dendrites in the molecular layer. The possibility that Golgi cells have supplementary axons should be considered. Apparently this concerned Ram6n y Cajal for he is critical of Retzius’ (Fig. 3. 1892) representation of a Golgi cell showing the axonic plexus not only arising from two points on the cell body but also from horizontal dendrites. Ram6n y Cajal (1911) refers to these processes arising from dendrites as axoniform dendrites. We have studied a large number of Golgi cell axonic plexuses and have never succeeded in tracing all the fine beaded fibers back to a single point of origin on the cell body. The axonic plexus (GA, Fig. 31) comes from the inferior portion of the cell body and the horizontally running dendrite (arrow) also contributes to this plexus. This is clear, of course, when traced under the microscope. Similarly the two descending processes from the displaced Golgi cell (Fig. 32) participate in the formation of the axonic plexus (GA). The descending process on the left shortly after its emergence gives rise to an ascending dendrite (arrow). Are the supplementary processes from dendrites which seem to contribute to the axonic plexus, axoniform dendrites or true axons? Structurally they are similar. The displaced Golgi cells are rare. Actually the only one we have seen is that in Fig. 32 and to our knowledge is the only one thus far reported in the monkey cerebellar cortex. Ram6n y Cajal (191 1) found them only in the rabbit where he identified them in Golgi and Nissl preparations. In the latter (see his Fig. 33, 1911) he observed an unusual collection of small cells about their inferior ends and suggested that they are glial cells insulating the descending axon from molecular layer elements. There is considerable convergence in the axonic plexuses generated by neighboring Golgi cells. When two nearby Golgi cells are simultaneously impregnated, their axonic plexuses overlap and the delicate nest formations (N, Fig. 33) are prominent. These nests, made up of fine beaded fibers, are obviously the result of convergence References p. 222-225

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from more than one Golgi cell, and as Ramon y Cajal(l911) indicated, they are located in the cerebellar islands. A portion of one of these nests is shown in the oil immersion photomicrograph (N, Fig. 35). The shapes of these terminals with their characteristic constrictions and dilations supply the clue for their identification in electron micrographs (Fig. 36). This section cuts through a nest of Golgi axon endings (G). Compare the dumb-bell shaped profile (inked outline, arrow, Fig. 36) with the similarly shaped endings in the impregnation (arrow, Fig. 35). The Golgi axons are distinguishable from the rosettes by their size and shape and as a rule they contain fewer synaptic vesicles. They tend to be at the periphery of the cerebellar islands where they are in apposition with the rosettes and the dendritic digits. But we have found thickening of the synaptic membrane only where they contact the dendritic digits. Since such thickenings are presumed to indicate synaptic relationship, it is our conclusion that they are functionally related to the granule cell dendrites. To be more certain of our identification of the Golgi endings several experiments were undertaken. Cat cerebellar folia were undercut by fusing a series of stereotaxically placed electrolytic lesions made by 10 parasagittal electrode tracts 2 mm apart in the vermis. To minimize damage of the surface vessels, electrolysis was commenced 2 mm after the electrode pierced the cortex and each millimeter thereafter until the electrode penetrated to a depth of 12 mm. Survival times were 4 and 33 days, respectively. The degenerating rosette (DR, Fig. 37, four days survival) stands out from the dendritic digits because of its light appearance. Its elongated irregularly curved profile is consistent with the concept that the rosettes are convoluted fibers. A clearer picture of a rosette degenerated for 4 days (DR), showing depletion of synaptic vesicles and the clumping of the remaining vesicles, is seen in the electron micrograph (Fig. 38). Note the normal Golgi axon ending (G). The degenerating rosettes in our preparations show no neurofibrillar hypertrophy such as reported by Gray and Hamlyn (1962), Colonnier and Guillery (1964) and Guillery (1965) in degenerating terminals. This may be due to swelling resulting from undercutting the cerebellar folia (Mugnaini, personal communication). Degenerating mossy fiber rosettes, following section of the spinal cerebellar tracts (Mugnaini and Walberg, 1966) do not have this appearance. After 33 days’ degeneration the mossy fibers are completely eliminated and gliosis is extensive. Scattered granule cells and patches of normal appearing areas remain (arrows, low power electron micrograph, Fig. 39). Two of these normal appearing patches are shown in the electron micrograph (arrows, Fig. 40). Note the hypertrophied astrocytic processes (As) loaded with glial filaments. Another normal patch is shown in higher magnification in the electron micrograph (Fig. 42). It contains Golgi axon endings (G) synapsing on the dendritic processes (DD) of granule cells. Compare the shape of the profile (G) with synaptic vesicles in its bulbous extremities (arrows) with the shape of the Golgi axon ending in the impregnation- (Fig. 41). Clearly, here we are dealing with the endings of Golgi axons since all of the mossy fibers have been eliminated. SzentPgothai’s (1965b) experimental results obtained in isolated cerebellar folia are similar. It is his opinion as well as ours that the Golgi cell

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Figs. 34-36. Fig. 34. Photomicrograph showing two Golgi cells and nest formations generated by their axonic plexuses. Golgi preparation ; Mucuca muluttu. Fig. 35. An oil immersion photomicrograph of a nest formation in a Golgi cell axonic plexus. Fig. 36. An electron micrograph of Golgi axon endings in a cerebellar island; Mucuca mulutru. For abbreviations see p. 221. References p . 222-225

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Figs. 37-38. Fig. 37. An electron micrograph showing a mossy fiber rosette degenerated four days; cat. Fig. 38. An electron micrograph showing a mossy fiber rosette degenerated 4 days; cat. For abbreviations see p. 221.

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Figs. 3 9 4 2 . Fig. 39. Low power electron micrograph showing narrow patches (white arrows) in granular layer after 33 days’ degeneration; cat. Fig. 40. Electron micrograph showing two normal patches (arrows) in granular layer after 33 days’ degeneration. Fig. 41. Oil immersion photomicrograph of Golgi axon endings. Golgi preparation; Macaca muluttu. Fig. 42. Electron micrograph of normal Golgi axon endings and granule cell dendritic digits after 33 days’ degeneration; cat. For abbreviations see p. 221. Referiwccs p . 222-225

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Figs. 4 3 4 5 . Fig. 43. Photomicrograph of a Purkinje cell. Golgi preparation; Macaca mulatra. Fig. 44. Oil immersion photomicrograph of Purkinje cell dendrites. Golgi preparation; Macaca mulatta. Fig. 45. Electron micrograph of Purkinje cell dendrite; human. For abbreviationsseep. 221.

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axons are found peripherally in the normal cerebellar glomeruli. In a few instances we have observed small synaptic endings, presumably endings of Golgi cell axons, on the more distal portions of the main trunks of granule cell dendrites. Szentagothai also reports some transneuronal degeneration in granule cell dendrites. These findings confirm the closed looped circuit schematized by Ram& y Cajal (Fig. 103; 1911), e.g. granule cell axons contacting Golgi cells and Golgi cell axons contacting granule cells. With the disclosure that the Golgi cells are inhibitory (Eccles et al., 1964b, 1966c) it is now demonstrated that this loop provides a negative feedback. MOLECULAR L A Y E R

Fiber background. A glance at the numerous crosscut axon profiles of varying caliber in the low power electron micrograph (PFC and PFD; Fig. 45, human) shows why Ram6n y Cajal(l911) preferred the term plexiform layer rather than molecular layer. With few exceptions they are the fibers resulting from the T-shape bifurcations of the granule cell axons which run longitudinally in the folium and parallel each other. The parallel fiber density is frequently revealed in Golgi preparations (Fox and Bar1964). The magnitude of this unmedullated plexus is a corollary nard, 1957; Fox et d., of: (a) the granule cell density in the granular layer and (b) the parallel fiber lengths. Estimates are that there are more than 2 million granule cells per cubic millimeter of granular layer in the monkey (Fox and Barnard, 1957) and more recent estimates in human material place this figure between 3 and 7 million granule cells per cubic millimeter of granular layer (Braitenberg and Atwood, 1958). Calculations based on fragmentary Golgi observations estimate that the parallel fiber lengths range from 3 mm for the longest to 1 mm, or less, for the shortest fibers. These measurements refer to the total lengths of individual parallel fibers and include both segments of the fibers issuing from the point of bifurcation. Braitenberg (personal communication) now finds parallel fibers 5 mm in length in man, a figure which accords well with the electrophysiological data of Dow (1949) who, while recording potentials along the axis of the parallel fibers, was unable to obtain records when the stimulating and pickup electrodes were separated by distances greater than 5 mm. Eccles (personal communication) and his collaborators now verify Dow’s results. According to Ram6n y Cajal (191 1) the parallel fibers vary in diameter between 0.5 p and 0.2 p. He observed (Ram6n y Cajal, 1903) that only parallel fibers in the lower third of the molecular layer stain in reduced silver preparations. The superior parallel fibers’ refractoriness to reduced silver suggested to Ramdn y Cajal ( 1 926) that they are thinner than the inferior parallel fibers. In Golgi preparations of the monkey Fox et al. (1950) and Fox and Barnard (1957) demonstrated that the parallel fiber caliber gradually decreases in the molecular layer from below upwards. Electron microscopic preparations reveal (Fox et al., 1964) that the diameters of the inferior, heavy caliber, parallel fibers are slightly more than 1 ,LA and the thinnest superior parallel fibers approximately 0.1 p. In the present study heavy caliber, inferior parallel fibers have not been found in electron micrographs of the human cerebellar cortex. Is their R&rences p . 222-225

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absence in the human cerebellar cortex related, in part at least, to the different granule cell counts obtained in monkey and in man? Parallel fibers maintain the plane adopted at their bifurcations and their general direction is not altered by the short flexuosities imposed on them by the numerous dendritic processes dispersed in their course. These flexuosities are more pronounced in Golgi sections parallel to the molecular surface than they are in sections perpendicular to this surface (Fox and Barnard, 1957). Golgi preparations facilitate the interpretation of the parallel fiber profiles seen in electron micrographs (Fox et al., 1964). It is apparent that the dilated portions of the parallel fibers (PFD, Figs. 46, 47, human) are the synaptic regions described by Estable (1923) as boutons and warty rosettes, and the hook-like endings observed by Fox and Barnard (1957). They are filled with synaptic vesicles and are in synaptic relationship with the dendritic spines. The constricted portion of the parallel fibers (PFC, Figs. 46-48, human) are essentially preterminals preceding successive synaptic regions. In them synaptic vesicles are absent; they contain several or more longitudinally running axonic tubules about 200 A in diameter. Glial background. The Bergmann fibers (BF) are the ascending extensions of the Golgi epithelial cells (GE, Figs. 31, 92). These neuroglial elements resemble retinal Muller cells (Ram6n y Cajal, 1911) and there is general agreement that they are a special form of astrocyte (Penfield, 1932) indigenous to the molecular layer. The cell bodies giving rise to these upward extensions are situated in the vicinity of the Purkinje cell layer. Descriptively called ‘candelabra1 cells’ by the French, they have 2, 3, 4, or even more ascending processes which emit short lateral extensions. The upward prolongations run perpendicularly in the molecular layer forming straight palisades and terminating at the surface of the cortex in conical expansions, the bases of which are directed toward the external folial surface. The abutment of these conical expansions, the subpial astrocytic endfeet, form the ‘basal membrane’ of the older anatomists (Ram6n y Cajal, 1911). The palisade-like elongated profiles of the Bergmann fibers and their peripheral conical expansions making up the basal membrane have been demonstrated electron microscopically (Fox et ~ l .1964). , In longitudinal sections of the folia Ram6n y Cajal(l911) has shown that the Bergmann fibers are sandwiched between the dendritic arborizations of consecutive Purkinje cells. Mugnaini and Forstronen (1965), studying the Golgi epithelial cells in chick embryos, find that their plasmalemma is directly opposed to that of the Purkinje cells for long distances so that Purkinje cells lie in a bath of astrocytic protoplasm. These authors consider it likely that the Golgi epithelial cells are nutritional satellites of Purkinje cells. Herndon (1964) has given a n account of the ultrastructure of the Golgi epithelial cell bodies. One of these cells (GE) is shown in the electron micrograph (Fig. 74). Oligodendroglia are sparse in the molecular layer. According to Schroeder’s (1929) exhaustive study they are found: never in the outer, rarely in the middle, and only occasionally in the lower third of the layer. They are most numerous in the granular-molecular layer transition where they occur between the Purkinje cells in groups of 2 or 3 and even 5 and 7. Their presence here may be related to the medullated supra- and infra-ganglionic plexuses. An oligodendroglial cell in the transition region

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seemingly related to a myelinated fiber is shown in the electron micrograph (0, Fig. 76). There is an oligodendroglia cell in the Golgi impregnation (Fig. 78). Nearby granule cells (GrC) in each figure afford a size comparison. The Purkinje cell. A Purkinje cell with its rich dendritic arborization arising from the summit of its pear-shaped cell body and spreading out in the molecular layer is shown in the Golgi impregnation (Fig. 43, Mucuca rnuluttu). As is well known the dendritic system of each Purkinje cell is, without exception, flattened in the transverse plane of the folium. This dendritic orientation was revealed in carmine material by the older anatomists: Stieda (1864) who first noted the flatness of the dendrites; Obersteiner (1869) who likened the Purkinje cells to trees, not free standing trees but espalier trees; and Henle (1879) who disclosed that the dendrites spread in the transverse plane of the folia. Golgi (1894) who first saw the richness of the Purkinje cell dendritic ramifications and commented that it is easier to draw than to describe, indicated that there is a first, a second, a third, and a fourth order of dendritic branching. Ramdn y Cajal’s (1891) discovery of the peridendritic spines refined Golgi’s account by distinguishing primary, secondary and tertiary smooth branches (ramure) and spine-laden terminal branchlets (ramuscles terminaux) which emerge from all the smooth branches in great numbers. This led to the important observation that specific parts of these neurons are contacted by specific afferents: the cell bodies by the nest formations of the basket cell axons; the smooth branches by climbing fibers; and the spiny branchlets by parallel fibers (Ram6n y Cajal, 1895, 1911). Smooth branches (SmB) and spiny branchlets (SB) are displayed in the Golgi preparation (Fig. 44, Macaca rnulutta) and in the electron micrograph (Fig. 45, human). In the latter a secondary smooth branch arching across the field gives rise to a tertiary smooth branch sprouting a spiny branchlet (SB). Note the climbing fiber (CF) ending on the smooth branch and the spines (S) on the branchlet. The spiny branchlets. The branchlets moderately branched and roughly the same in length are enormous in number. Estimates are that a monkey Purkinje cell has more than40 mm of spiny branchlets(Fox and Barnard, 1957). They arisefrom all the smooth branches : primary, secondary, and tertiary. Some ascend, some run transversely and obliquely, some descend. They occupy a zone coextensive with the distribution of the parallel fibers, i.e. from the pial surface to the summits of the Purkinje cell bodies. The branchlets from a single cell are staggered in planes 2 or 3 deep and some veer off at slight angles to these planes - an arrangement assuring contacts with as many parallel fibers as possible (Fox and Barnard, 1957). Electron microscopic studies of the branchlets in the rat (Palay, 1964), cat and monkey (Fox et al., 1964) reveal that they are densely packed with mitochondria, contain an occasional multivesicular body and have few dendritic tubules. Human spiny branchlets are displayed in the electron micrographs (Figs. 46 and 47). Ram6n y Cajal (1909) regarded the spines, known also as thorns or gemmules, as dendritic devices for increasing synaptic surfaces and rendering synapses more intimate. This interpretation was controversial (see literature in Fox and Barnard, 1957) until Gray’s (1959) electron microscopic studies revealed dendritic spines on rat pyramidal cells. Similar disclosures in different centers now amply verify Rambn y Cajal’s Rcfrrenrrs p . 222-225

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Figs. 4 6 4 8 . Fig. 46. Electron micrograph of an ideally sectioned spiny branchlet with emerging spines; human. Fig. 47. Electron micrograph of a spiny branchlet and emerging spine synapsing with a parallel fiber; human. Fig. 48. Electron micrograph of a Purkinje cell smooth dendrite; human. For abbreviations see p. 221.

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conjecture. Purkinje cell dendritic spines have been demonstrated by Gray (1961), Palay (1964), and Fox et al. (1964). On an average the Purkinje cell dendritic spines in the monkey are approximately 1 ,u; in man they are slightly longer (Figs. 46 and 47). With their enlarged tips the spines (S) engage the parallel fibers by indenting or invaginating the dilated, synaptic vesicle-filled portion of these fibers (PFD, Fig. 47, human). Earlier estimates of 15 spines per 10 ,u length of spiny branchlet, hence a total of 60 thousand spines on a monkey Purkinje cell (Fox and Barnard, 1957), were based on figures cautiously derived from counts on thick Golgi sections under oil immersion. Emergence of the spines from all sides of the branchlets complicated the procedure and a most conservative estimate was given. After studying the spiny branchlets in cat, in monkey (Fox et al., 1964) and now in man, the inadequacy of this estimate is apparent. On the ideally sectioned branchlet (Fig. 46, human) 14 spines emerge in a distance of 8 p. Sectioning this branchlet in a mid-longitudinal plane at right angles to the present plane would most likely show a similar number of spines, and it is possible that even these two planes would not reveal all because the spines are staggered. Perhaps the estimate of 60 thousand spines on a Purkinje cell should be doubled. Ram6n y Cajal (1954) used the parallel fiber Purkinje cell synapse as a prototype to illustrate the class of synapses he called ‘cruciform axo-dendritic connections of great length’. This type of synapse allows a single neuron t o contact many fibers and a single fiber to contact a number of neurons. Considering the serial arrangement of the Purkinje cells, the dense spread of their dendrites, the lengths of the parallel fibers, and the density of the parallel fiber, it is obvious that this architecture permits maximal convergence and maximal divergence in minimal space. Furthermore, considering the overlapping of the Purkinje cell dendrites in the transverse plane and the continuous arrival and termination of parallel fibers, it is also obvious that this is the ideal architecture for fractionation of synaptic pool. Smooth branches. The smooth branches are distinguishable from the branchlets by their size and internal structure. These branches contain mitochondria elongated in their longitudinal direction; however, the mitochondria are less numerous per cubic volume than they are in the branchlets. The smooth branches are occupied throughout by fine longitudinal canaliculi closely spaced and parallel to each other. The canaliculi, barely visible at the magnification shown in the longitudinally sectioned smooth branch (Fig. 48, human), are distinct in the large transversely sectioned smooth branch (DT, Fig. 49, human). In the higher power electron micrograph (DT, Fig. 50, human) they appear as small, thick-walled tubules approximately 200 A in diameter. Palay (1956, 1958) regards them as extensions of the endoplasmic reticulum and estimates (Palay, 1964) their walls are approximately 60 A thick. Gray (1959) named them dendritic tubules. Membranes of the longitudinally disposed cisternae of the endoplasmic reticulum, usually adjacent to the dendritic surface, are prominent especially in the large dendrites (ER, Figs. 48,49 and 5 1, human). The smooth branches also contain occasional dense bodies (DB, Fig. 49) and occasional rosette-like clusters of RNP particles (Palay, 1964; Fox et al., 1964). Portions of Purkinje cell bodies are visible in the electron micrographs (Figs. 72, 73, References p . 222-225

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Figs. 49-51. Fig. 49. Electron micrograph of a transversely sectioned Purkinje cell smooth dendrite; human. Fig. 50. Electron micrograph showing dendritic tubules in a transversely sectioned smooth Purkinje cell dendrite; human. Fig. 51. Electron micrograph of a Purkinje cell smooth dendrite showing endoplasmic reticulum; human. For abbreviations see p. 221.

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74, 76, 80, 82). Their fine structure is not considered here since excellent accounts are available in the literature (Niklowitz, 1962; Herndon, 1963; Palay, 1964). Bergmann fibers and the Purkinje cells. Processes of Bergmann fibers (BF, Fig. 48) insulate the smooth branches (SmB) from the passing parallel fibers. This insulation is absent only where climbing fibers (CF, Fig. 49) come into synaptic relationship with the smooth branches. These astrocytic processes contact spiny branchlets and form a cuff around every spine (Figs. 46 and 47). They also are in apposition with fascicles of parallel fibers but do not separate the individual parallel fibers from each other. Climbingjibers.According to classical descriptions (Ram6n y Cajal, 1911) the climbing fibers follow an unbranched course through the granular layer. On reaching the Purkinje cell layer they either arch around or contact one side of the Purkinje cell body. Then, climbing upward, they make ‘longitudinal axo-dendritic connections’ with its smooth dendritic branches - a type of synapse (Ram6n y Cajal, 1954) that allows a single fiber to make multiple contacts with the dendrites of the single cell. In addition, collaterals of the climbing fibers have synaptic relationships with upper stellate and lower stellate (basket) cells in the molecular layer (Scheibel and Scheibel, 1954). Some of the above features are illustrated in our Golgi and electron microscopic preparations. Thus, the straight radial course of a climbing fiber (CF) in the granular layer, its branching in the molecular layer and its collateral branches contacting a stellate cell’s (SC) dendrites, are seen in the Golgi impregnation (Fig. 56, Macaca mulutta). Aspects of the climbing fiber (inset, Fig. 52) are displayed in greater detail in the oil immersion montage (Fig. 53). It will be seen that this axon has a relatively thick smooth portion zig-zagging in the molecular layer and giving off finer beaded terminal branches. The smooth branches of climbing fibers (CF, electron micrograph, Fig. 54, human) contain fine filaments and are filled with synaptic vesicles in regions where the axon synapses with Purkinje cell dendrites. Shortly after they emerge some of the beaded branches (Fig. 53, closed block arrow) closely follow the course of the parent fiber and most likely contact the Purkinje cell dendrite. A beaded collateral of the climbing fiber in synaptic relationship with a Purkinje cell smooth dendrite is seen in the electron micrograph (BCF, Fig. 55, human). Turning again to the Golgi impregnation (Fig. 53) it will be noted that some of the beaded collaterals diverge from the course of the parent fiber. Some of them may be the collaterals that Scheibel and Scheibel (1954) showed synapsing with stellate cells. We have never observed them in electron micrographs. More peripherally (Fig. 53, open block arrow) the smooth axon divides into two thinner branches that, since they run close together, must contact the same dendritic branch. At the height of its course (Fig. 53, arrow) this climbing fiber forms a network of beaded terminals. Scheibel and Scheibel (1954) described retrograde collaterals of climbing fibers that return to the granular layer. According to the experimental findings of Szenthgothai and Rajkovits (1959) the retrograde collaterals contact Golgi cells. The origin of the climbing fibers has been a perennial problem. Older experimental work (Carrea et al., 1949) suggests climbing fibers are collaterals of the deep cerebellar nuclei but Ule’s (1957) observations on three patients with almost complete degeneraReferences p . 222-225

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Figs. 52-54. Fig. 52. Photomicrograph of a climbing fiber. Golgi preparation; Macaca mulatta. Fig. 53. Oil immersion montage of a portion of a climbing fiber in Fig. 52. Golgi preparation; Macucu rnuluttu. Fig. 54. Electron micrograph of a climbing fiber on a smooth dendritic branch; human. For abbreviations see p. 221.

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tion of the dentate nuclei cast doubt on this interpretation. A later investigation attempting to verify the cerebellar nuclei as a source of the climbing fibers failed but brought forth good evidence indicating that some climbing fibers arise from the inferior olivary nucleus (Szenthgothai and Rajkovits, 1959). These investigators also found extensive climbing fiber degeneration in the contralateral flocculus following lesions in the superior cerebellar peduncle and some degenerating climbing fibers in parts of the vermis after destruction of the superior medial tegmental nucleus of Bechterew in the pons. Utilizing this information, Eccles et al. (1964a, 1966a, 1966d) recorded a ‘climbing fiber response’ intra- and extracellularly from Purkinje cells in the contralateral vermis, fallowing stimulation of the medial accessory olive. They concluded ‘... the climbing fiber is the most powerful and specific excitatory synapse yet discovered in the central nervous system’ (Eccles et al., 1966a). According to Rambn y Cajal(l911) a single climbing fiber innervates one Purkinje cell. But Scheibel and Scheibel(l954) report that a climbing fiber distributes to several adjacent Purkinje cells. Eccles et al. (1966a, see their discussion) comment on this point. Their electrophysiological evidence favors the view that the relationship between the Purkinje cells and climbing fibers is ‘one to one’ although they have no positive evidence excluding the alternative possibility. In our preparations (Figs. 52, 53 and 56) climbing fibers are impregnated on a clear background and the related dendrites are invisible. Deep and superjicial stellate cells of the molecular layer. The stellate cells are the intrinsic neurons of the molecular layer. Little is known concerning the upper stellate cells. Their cell bodies are found in the outer half and particularly in the outer third of the molecular layer. The deep stellate cells are the well known ‘basket cells’. Scheibel and Scheibel(l954) divide stellate cells into two categories: (1) stellate cells with short axons which break up a short distance from their cell bodies and within their own dendritic arborizations ; (2) cells with long transversely running axons. Respective samples of these cell types (SC1, SC2) along with a basket cell (BC) are shown in the Golgi impregnation (Fig. 57, Macaca nmlatta). The Scheibels consider it probable that all cells of the second type, i.e. those with transversely running axons, have descending collaterals which enter into basket formations about Purkinje cell bodies. However, we have not found descending collaterals of the upper stellate cells participating in basket formations. In general stellate cells high in the molecular layer have longer descending dendrites and stellate cells low in the molecular layer have longer ascending dendrites (Rambn y Cajal, 191 1). These dendrites, like the dendrites of the Purkinje cells, radiate in the transverse direction of the folium. Compare the disposition of the basket cell dendrites from a longitudinal section of a folium (Fig. 58) with the disposition of the basket cell dendrites from a transverse section of a folium (Fig. 59). In Ehrlich preparations (Rambn y Cajal, Fig. 18, 1911) the stellate cell dendrites are beaded. Ram& y Cajal (1909) regarded this beading as an artifact and attributed it to uneven retention of the methylene blue at different points along the dendrite. However, beading of basket cell and stellate cell dendrites can be observed in Golgi and electron microscopic preparations. Note the beading on the basket cell dendrites in the Golgi preparation References p . 222-225

Figs. 55-59. Fig. 55. Electron micrograph showing beaded fiber synapsindwith Purkinje cell smooth dendritic branch; human. Fig. 56. Photomicrograph showing climbing fiber crossing the granular layer and contacting a stellate cell in the upper part of the molecular layer. Golgi preparation; Mrscaca mulatta. Fig. 57. Photomicrograph showing upper stellate cells and basket cells in the molecular layer. Golgi preparation; Mneaea mulaffa. Fig. 58. Photomicrograph showing orientation of basket cell in a longitudinal section of the folium. Golgi preparation; Macaca mulatta. Fig. 59. Photomicrograph showing orientation of basket cell in a transverse section of the folium. Golgi preparation; Macaca mulatta. For abbreviations see p. 221.

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Figs. 60-62. Fig. 60. Electron micrograph showing a stellate cell with an emerging bifurcating dendrite and swellings (arrows) on dendrites; human. Fig. 61. Photomicrograph of a basket cell. Golgi preparation; Mucucu muluttu. Fig. 62. Electron micrograph of a basket cell dendrite showing a swelling (arrow) and synapsing with parallel fibers; squirrel monkey. For abbreviations see p. 221. Re/ercrrces p. 222-225

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(arrow, Fig. 61) and on the stellate and basket cell dendrites in the electron micrographs (arrows, Figs. 60, 62). Dendritic tubules are prominent in these dendrites but in the regions of the swellings they either disappear or are disarrayed (arrow, Fig. 62). The stellate and basket cell dendrites synapse with the dilated portions of parallel fibers (PFD). At the open block arrow (Fig. 60) there is a parallel fiber synapsing both with a stellate cell dendrite (D) and with a Purkinje cell dendritic spine (S). The stellate and basket cells have dendritic spines (Ram6n y Cajal, 1911). One is seen in the electron micrograph (SS, Fig. 60). The lone myelinated fiber in this electron micrograph (Fig. 62) indicates (vide infru) this section is low in the molecular layer; therefore, the dendrite in this electron micrograph must be a basket cell dendrite. The stellate cells are characterized by their axons. This is particularly true of basket cells. The main stem of the basket cell axon is the transversal fiber (T, Figs. 59 and 61) which courses just above the Purkinje cell layer and gives off ascending (AC, Fig. 59), descending and longitudinal collaterals. Most of the descending collaterals form complicated nests (BN, Figs. 57,58 and 71) about Purkinje cell bodies. The longitudinal collaterals (L, Figs. 67 and 70) come off regularly from both sides of the transversal fibers and are visible only in tangential sections cutting through the molecular layer in a plane parallel to and just above the Purkinje cell layer. These right angle collaterals in turn have beaded right angle collaterals (block arrow, Fig. 67). Longitudinal collaterals have been observed by Lugaro (1895), Estable (1923), Fox et ul. (1950), Szenthgothai (1965a). Estable (1923) called them longitudinal fibers because they have the same direction as the parallel fibers and run longitudinally in the folia. The basket cell axon emerges usually from a triangular or conical prolongation of the cell body and then for some distance, 40 u , or more, it is reduced to a thin delicate strand. These features are illustrated in the Golgi preparation (Fig. 61, Mucacu muluttu) and in the electron micrograph (Fig. 63, human). The axon then thickens and pursues its course as the transversal fiber. These fibers (T) are easily identified in Golgi impregnations (Figs. 59, 61, 67, 70 and 71) and in electron micrographs (Figs. 64, 65, 66, 68 and 69) by their orientation and their structure. In Golgi preparations they have a relatively smooth contour and in electron micrographs they display a large number of wavy filaments. The thickest transversal fibers are 4 p or more in diameter and they are located most inferiorly in the molecular layer. A surprising finding in the present investigation is that, although the smooth contoured transversal fibers give no indications of having synaptic relationships in Golgi preparations, it is evident in electron micrographs that they synapse with spiny branchlets (SB, Fig. 68, squirrel monkey) and dendritic spines (S, Fig. 69, squirrel monkey) of Purkinje cells. The transversal fibers have short, beaded ascending collaterals (AC, Fig. 59). Ram6n y Cajal (Fig. 21, 1911) found nest-like formations on them which he thought might be pericellular formations about other basket and stellate cells. The ascending collaterals are involved in the reciprocal connections - depicted by Scheibel and Scheibel(l954) - of the basket and other stellate cells with each other. These authors have also observed that the ascending collaterals have axo-axonic connections with the climbing fibers. We have never been able to find these connections in our Golgi or electron microscopic preparations. The synaptic vesicle-filled bulbous tip of the

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Figs. 63-64. Fig. 63. Electron micrograph of a basket cell showing an emerging axon; human. Fig. 64. Electron micrograph of a basket cell axon (transversal fiber) with an ascending collateral; squirrel monkey. For abbreviations see p. 221.

References p . 222-225

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Figs. 65-67. Fig. 65. Electron micrograph of basket cell axon (transversal fiber) with an emerging longitudinal branch; squirrel monkey. Fig. 66. Electron micrograph of basket cell axon (transversal fiber) of a descending collateral; squirrel monkey. Fig. 67. Photomicrograph of a horizontal section in the inferior part of the molecular layer showing basket cell axons (transversal fibers) with longitudinal collaterals. Golgi preparation; Macacu mulatta. For abbreviations see p. 221.

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Figs. 68-70. Fig. 68. Electron micrograph of basket cell axon (transversal fiber) synapsing with spiny branchlet; squirrel monkey. Fig. 69. Electron micrograph of a basket cell axon (transversal fiber) synapsing with Purkinje cell dendritic spines; squirrel monkey. Fig. 70. Photomicrograph of a horizontal section in the inferior part of the molecular layer showing basket cell axon (transversal fiber) with longitudinal branches. Golgi preparation; Mucucu muluttu. For abbreviations see p. 221. References p . 222-225

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ascending collateral in the electron micrograph (AC, Fig. 64, squirrel monkey) is in apposition with a small segment of a spiny branchlet and our suspicion is that the basket cell ascending collaterals contact the spiny branchlets of Purkinje cells. The transversal fibers also have beaded descending collaterals (DC, Fig. 59) which stay within the confines of the molecular layer and descend to about the summits of the Purkinje cells. One of them with its tips filled with synaptic vesicles is shown in the electron micrograph (DC, Fig. 66, squirrel monkey). Our guess is that these beaded descending collaterals also contact the spiny branchlets of Purkinje cells. The transversal fibers vary in length. The shorter ones from smaller basket cells, extend for distances of 8 to 10 Purkinje cells (Ramon y Cajal, 1911). We have followed longer ones for distances of 18 to 20 Purkinje cells and have followed the right angle collaterals, the longitudinal fibers, which emerge from both sides of the transversal fibers, for distances of 6 Purkinje cells. The longitudinal fiber (L) emerging from the transversal fiber (T) in the electron micrograph (Fig. 65, squirrel monkey) is in apposition with a spiny branchlet (SB). Synaptic relationship is not apparent here but we have observed it in other electron micrographs. This confirms Estable’s (1923) observation that the longitudinal fibers contact the spiny branchlets of Purkinje cells. Thus, the axons of the larger basket cells are capable of influencing Purkinje cells in a rectangular patch, 20 Purkinje cells long and 12 Purkinje cells wide, i.e. 240 Purkinje cells. The best known collaterals of the transversal fibers are the descending collaterals that embrace Purkinje cell bodies with pericellular nests (BN, Figs. 57, 58, 59 and 71) and terminate in the so-called ‘paint brush tips’ (PT, Figs. 71, 77 and 79). Their discoverer, Ramon y Cajal (191 I), found them in 1888 and studied them by a variety of techniques including the method of Boveri. In the latter procedure material is processed in an osmic acid-silver nitrate mixture and the ‘paint brush tips’ appear as a cone of fibrous and granular substance immediately below the Purkinje cell bodies, while the naked Purkinje cell axon is seen traversing the axis of the ‘paint brush‘ and acquiring its myelin sheath after it passes the ‘paint brush tip’. The emerging Purkinje cell axon (PA) in the electron micrograph (Fig. 80, squirrel monkey) traversing the conical assemblage of basket cell axon profiles (BA) cannot be followed to the point where it receives its myelin sheath, but we have seen one of Professor Palay’s preparations in which this is possible. It is exactly as Ramon y Cajal(l911) described it. The basket cell axon’s attraction for Purkinje cell axons is emphasized by Estable’s rare observation (see his Fig. 14, 1923) that when a Purkinje cell axon emerges from a large dendrite instead of the cell body the basket formation surrounds the axon and not the cell body. In the Golgi preparation (Fig. 77, Mucucu mulutta) the basket cell axon (BA) completely avoids the Purkinje cell body and its ‘paint brush tip’ (PT) comes into relationship with the Purkinje cell axon (PA). Our impression from studying electron micrographs is that collaterals like these may be quite numerous and have gone undetected because Golgi impregnations of the pericellular nests, in most instances, are incomplete. Observe, for example, the horizontally sectioned basket cell axon profiles (BA) not adjacent to the Purkinje cell body in the electron micrograph (Fig. 76, squirrel monkey) and the similar peripheral profiles (BA) in the electron


209

Figs. 71-72. Fig. 7I.Photomicrograph of a tangential section through the Purkinje cell layer showing a basket cell axon (transversal fiber) and basket cell axon pericellular nest of Purkinje cell bodies. Golgi preparation; Macaca rnulatta. Fig. 72. Electron micrograph showing basket cell axon synapsing with Purkinje cell body; human. For abbreviations see p. 221. References p. 222-225

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Figs. 73-75. Fig. 73. Low power electron micrograph showing basket cell axon on Purkinje cell body; Macacu mulatta. Fig. 74. Electron micrograph showing profiles of basket cell axons of a pericellular nest on one side of the Purkinje cell body; Macaca mulatta. Fig. 75. Photomicrograph of a basket formation about a Purkinje cell body. Golgi preparation; Mucaca mulatta. For abbreviations see

p. 221,

Figs. 76-80. Fig. 76. Electron micrograph of a horizontal section showing profiles of basket cell axons on one side of a Purkinje cell body; squirrel monkey. Fig. 77. Photomicrograph of a basket cell axon (BA) not in contact with a Purkinje cell body. Its ‘paint brush tip’ (PT) is closely associated with the Purkinje cell axon (PA). Golgi preparation; Mucucu muluttu. Fig. 78. Photomicrograph showing granule cells and an oligodendroglial cell. Golgi preparation; Mucucu muluftu. Fig. 79. Photomicrograph showing basket formation about a Purkinje cell body and its ‘paint brush tips’. Golgi preparation; Mucucu mululta. Fig. 80. Electron micrograph showing profiles of basket cell axons (BA) about an emerging Purkinje cell axon (PA); squirrel monkey. For abbreviations see p. 221. References p . 222-225

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micrograph (Fig. 74, Mucucu mulutta). It is very probable that they never contact the Purkinje cell bodies and are profiles of collaterals similar to the collateral (BA) in the Golgi impregnation (Fig. 77). Most collaterals contact Purkinje cell bodies and many like the collateral (BA) in the low power electron micrograph (Fig. 73, Macaca mulutta) and the collaterals (BA) in the Golgi impregnation (Fig. 75) follow the contour of the Purkinje cell body. In regions where no astrocyte processes (As) intervene they come into synaptic relationships with the Purkinje body (Fig. 72, human; Fig. 74, Mucaca mulatta). It should be mentioned that the basket formation about any Purkinje cell body is formed by contributions from several or more basket cells. For a discussion of convergence and divergence in the basket cell system see Szentagothai (1965a). A portion of the Purkinje cell axon (Fig. 80) is enlarged in the electron micrograph (Fig. 83). Arrows point to profiles of astrocytic processes intervening between the Purkinje cell axon (PA) and the surrounding basket cell axon profiles (BA). Actual axo-axonic contacts are difficult to find and where they exist few, if any, synaptic vesicles are present. Palay, who illustrates this relationship in a transverse section through the Purkinje cell axon and identifies the very fine terminals of the ‘paint brush tips’ (see his Fig. 23, 1964), regards this as a highly unusual synapse similar to the Mauthner cell axon cap described by Robertson et al. (1963). He also suggests its electrical characteristics may resemble those Furshpan and Furukawa (1962) and Furukawa and Furshpan (1963) disclosed in goldfish Mauthner cells. The transversal fibers, the longitudinal fibers and the collaterals in the basket formations are easily recognized in electron micrographs. Characteristically they display numerous wavy whorled filaments and irregular little lines of an electron dense material (open arrows) arrayed in a direction transverse to the filaments (Figs. 64,65, 72, 74, 80 and 82). The basket and upper stellate cells are interspersed between the dendritic arborizations of consecutive Purkinje cells (see Ram6n y Cajal’s Fig. 51, 1911). Thus a beam of parallel fibers coextensive with the dendritic spread of a Purkinje cell excites a longitudinal series of Purkinje cells and a series of similarly disposed basket and stellate cells. The latter by means of their transversely oriented axons influence Purkinje cell spiny branchlets, cell bodies and axons in the areas immediately flanking and outside of the excitatory beam of parallel fibers. Exploiting this anatomical arrangement, Andersen et al. (1 964), and Eccles et al. (1966b, 1966c) demonstrated that basket and stellate cells inhibit Purkinje cells. We have not identified upper stellate cell axons in electron micrographs, but since basket cell transversal fibers contact Purkinje cell spiny branchlets we suspect upper stellate cell transversal fibers have similar connections. Szentagothai (1965a) thinks it improbable that stellate cells with short axons distributing essentially within their own dendritic arborizations are inhibitory and he suggests that they may be excitatory. Judging from the design of the experiments of Eccles and his collaborators, the influence of these cells ( X I , Fig. 57) would be difficult to ascertain; most likely they were not studied by Eccles and his collaborators. Stellate cells and the glial processes. The stellate (Fig. 60) and basket (Fig. 63) cell

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Figs. 81-82. Fig. 81. Photomicrograph of an intermediate cell of Lugaro with its cell body and dendrite contacted by basket cell axons. Golgi preparation; Mucaca mulutru. Fig. 82. Electron micrograph showing the profiles of basket cell axons contacting a Purkinje cell (PC) and an intermediate cell of Lugaro; squirrel monkey. For abbreviations see p. 221. Refercnces p . 222-225

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bodies together with their dendrites appear to be in direct apposition with the passing parallel fibers, and their surfaces unlike those of the Purkinje cells are not insulated by Bergmann fibers. Intermediate cells of Lugaro. These neurons (ICL, Fig. Sl), the fusiform horizontal cells, have cell bodies and dendrites elongated in the transverse direction of the folia. They are located immediately below the Purkinje cell layer where basket cell axons (BA) contact their dendrites and cell bodies (Fox, 1959). In the electron micrograph (Fig. 82, squirrel monkey) a Purkinje cell body (PC) is contacted by a basket cell axon. The nerve cell (ICL) on the left of the figure (note its nucleus, Nu) is not a Purkinje cell. Since it is in synaptic relationship with a basket cell axon, it is interpreted as an intermediate cell of Lugaro. Observe the large accumulation of synaptic vesicles adjacent to the presynaptic membrane in the basket cell axon. The axons of the intermediate cells of Lugaro enter the molecular layer obliquely and give rise to branches that course longitudinally in the inferior part of this layer where they emit short beaded collaterals that engage the basket cell bodies in axosomatic synapses (Fox, 1959). Purkinje cell axons. The Purkinje cell axon arises from the inferior pole of the cell by a conical prolongation of the cell body (Fig. 80) and then descends (PA, Figs. 84 and 85) to the medullary layer. At times the cone of origin may be on one side of the cell body; when this occurs the axon (PA, Fig. 92) descends obliquely towards the medullary layer. Rambn y Cajal (see his Fig. 1, 1911) calls attention to fine Nissl granules in the axonic cone and notes that in Purkinje cells it is difficult to draw a sharp linelof demarcation between the chromatic portion of the soma and the achromatic portion of the axon. Palay (1964), who has made a careful electron microscopic study of the Purkinje cell axon’s unmedullated segment, observes ‘. .. it has a characteristic internal structure more like that of the large dendrite than that of an axon’. The initial naked segment of the Purkinje cell axon (PA, Fig. 83) contains longitudinally arrayed fine filaments (block arrow), clusters of RNP particles (open block arrows) such as found in large dendrites, and tubules (Tu) of the endoplasmic reticulum associated with vesicles. Do the latter, i.e. the tubules associated with vesicles, represent some modification of the Golgi apparatus extending into the axon? Recurrent collaterals of the Purkinje cell axons. Purkinje cell axons give off recurrent collaterals as they cross the granular layer (RC, Fig. 84) and as they course in the medullary layer (Figs. 85 and 92). With Ehrlich’s procedure Rambn y Cajal (see his Fig. 12,1911) proved that these collaterals, after some branching, have secondary branches forming a myelinated plexus beneath the Purkinje cells and tertiary branches forming a myelinated plexus above the Purkinje cells. These plexuses are the infra- and supraganglionic plexuses of Jakob (1928). Without knowing their origin, Henle (1879) illustrated the myelinated fibers above the Purkinje cells and showed that their orientation is mainly in the longitudinal direction of the folia. The transversely sectioned myelinated fibers in the electron micrographs (Figs. 62, 63, 64, 66 and 95) are Purkinje cell axon recurrent collaterals in the supraganglionic plexus and their presence indicates these sections are deep in the molecular layer.

SIZ

X 3 1 X O 3 XV17383X33

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Figs. 86-88. F&. 86. Photomicrograph of a portion of the field seen in Fig. 85 showing a Purkinje cell axon recurrent collateral endings (E) in the granular layer, near the Purkinje cell layer. Golgi preparation; Macaca mulatta. Fig. 87. Electron micrograph showing synaptic ending (E) interpreted here as a Purkinje cell axon collateral ending on a Golgi cell; Macaca mulatta. Fig. 88. Electron micrograph showing an enlargement of the ending (E) in Fig. 87; Macaca mulatta. For abbreviations see p. 221.

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Figs. 89-91. Fig. 89. Photomicrograph of a Purkinje cell and its axon emitting nearby recurrent collaterals. Golgi preparation; Macacu rnuluttu. Fig. 90. Oil immersion photomicrograph of a portion of the field in Fig. 89 showing synaptic ending (E), i.e. a recurrent collateral terminating on a Purkinje cell body (PC). Fig. 91. Oil immersion photomicrograph of a portion of the field in Fig. 89 showing rzcurrent collateral ending (E) terminating on a Golgi cell body (GC). Golgi preparation; Mucacu rnuluttu. For abbreviations see p. 221.

Figs. 92-95. Fig. 92. Photomicrograph of a Purkinje cell axon (PA) giving off recurrent collaterals (RC). One of these collaterals enters the infraganglionic piexus (IP). Golgi preparation; Macaca mulatta. Fig. 93. Photomicrograph of a portion of the field in Fig. 92 showing collateral branch from a fiber of infraganglionic plexus UP) contacting a Purkinje cell body (PC). Golgi preparation; Maeaca mulatta. Fig. 94. Electron micrograph showing a portion of the field in Fig. 95. The dendrite (D) is contacted by two synaptic endings (E, E?); human. Fig. 95. Electron micrograph of a horizontal section in the inferior portion of the molecular layer. The myelinated fiber (RC) is a recurrent collateral and its ending (E) contacts the dendrite @). The ending (E?) may also be the ending of a Purkinje cell axon recurrent collateral. For abbreviations see p. 221.

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Lugaro (1895) reports that some recurrent collaterals terminate in the granular layer. This can be confirmed in our Golgi preparations. Usually these endings (E, Figs. 85, 86 and 91) are found near the Purkinje cell layer. Because of this location and other evidence to be presented it is our opinion they end on Golgi cell bodies. The shape of these endings in the Golgi impregnation (E, Fig. 86) is similar to the shape of the ending on the Golgi cell in the electron micrograph (E, Fig. 87); an enlargement of this ending is seen in the inset (E, Fig. 88). Obviously it is not a mossy fiber or a Golgi cell axon ending and therefore must be the ending of a Purkinje cell axon recurrent collateral. The Golgi preparation (Fig. 89) shows a Purkinje cell axon and some of its nearby recurrent collaterals. The photomicrographs (Figs. 90 and 91) were made by focusing on this same field under oil immersion and searching hopefully for the shadowy outlines of faintly chromated unstained cell bodies in the background. In Fig. 90 one of these collaterals ends (E) on a Purkinje cell body (PC). In Fig. 91 a Y-shaped branch of a recurrent collateral embraces the outline of the cell, interpreted here as a Golgi cell body (GC) and another collateral with a club-shaped ending (E) appears to be in apposition with the same cell body. It is possible in our preparations to follow recurrent collaterals into the infraganglionic plexus but we have never succeeded in following them into the supraganglionic plexus. The latter either are not impregnated or, when they are, impregnation of other elements such as glia obscure their course. This may be the reason why Ram6n y Cajal (1911) succeeded in showing them only in Ehrlich preparations. The branch (IP, Fig. 92) resulting from the T-shaped bifurcation of the recurrent collateral (RC) on the right is in the infraganglionic plexus. A portion of this field shown at higher magnification in the inset (Fig. 93) reveals that the horizontally running fiber in the infraganglionic plexus sends an ascending beaded branch onto the side of the Purkinje cell body (PC). There can be no doubt that some of these fibers have synaptic relationships with Purkinje cells. Knowing that the molecular layer medullated fibers (supraganglionic plexus) are Purkinje cell collaterals, we have searched for one of them synapsing on some identifiable structure. A number of such fibers losing their myelin sheaths have been found but in most instances they pass out of the plane of section before they terminate. The electron micrograph (Fig. 95, human) is a horizontal section of the molecular layer; this is obvious from the orientation of the parallel fibers (PFC and PFD). In this section the myelinated recurrent collateral (RC) loses its sheath and ends (E) on the dendrite (D). On the opposite side of this dendrite is a similar type of ending (E?) but it is not in contiguity with the myelinated portion of its axon. The dendrite and these endings containing synaptic vesicles are shown in the enlargement (Fig. 94). A transversely sectioned dendrite here in the inferior part of the molecular layer, which is neither a Purkinje cell smooth branch nor a spiny branchlet, can only be a basket or Golgi cell dendrite. The above findings may be of some interest in view of recent electrophysiological disclosures. Purkinje cell axons inhibit neurons of Deiters nucleus (It0 and Yoshida, 1964) and neurons of the cerebellar nuclei (It0 et al., 1964). Since it is postulated that all synapses of a neuron’s axonal branches have a similar function, the endings of Purkinje cell collaterals on other Purkinje cells should be inhibitory. Eccles et al. (I966b) References p. 222-225

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find in recordings made in chronically deafferented (mossy fibers degenerated) folia that the Purkinje cell collaterals inhibit other Purkinje cells, basket cells and Golgi cells. Purkinje cell axon collaterals inhibiting basket cells which in turn inhibit Purkinje cells, and Purkinje cell axon collaterals inhibiting Golgi cells which in turn inhibit granule cells are circuits similar to the disinhibitory circuit Wilson and Burgess (1962) revealed in the spinal cord. If the Purkinje cells are strictly inhibitory, the cerebellar cortex functions in a manner hitherto unexpected. There must then exist excitatory pathways to the deep cerebellar nuclei, bypassing the cerebellar cortex. Goldman and Snider’s(1955) electroanatomical investigations suggest this possibility. In this regard Brodal’s (1940) discovery that, in addition to projecting on the cerebellar cortex, the olivocerebellar fibers project in an orderly manner on the intracerebellar nuclei and Eccles et al. (1966a) disclosure that the olivocerebellar fibers are powerfully excitatory, take on added significance. The long and short associational fibers of the cerebellar cortex must be Purkinje cell axon collaterals. They are medullated fibers (Jansen, 1933). Eager (1963) finds in Nauta material that they distribute in the region of the supraganglionic plexus. More recently Eager (1965), studying the mode of termination and the temporal course of the degenerating cortico-cortico association pathways in the cat cerebellum, cautiously suggests that it is ‘... necessary to continue to consider associational pathways as possible sources of at least part of the climbing fiber population of the cerebellum’. In the past, associational pathways have been implicated as a possible source of the climbing fibers (Popoff, 1896; Held, 1897; Lorente de N6, 1924). Our criticism is: if Purkinje cell axon recurrent collaterals have the same pattern in other folia as they do in the folia of their cells of origin (see again Ram6n y Cajal’s Fig. 12, 191l), they cannot be climbing fibers. Recurrent collaterals branch and re-branch in the granular layer and the climbing fibers cross the granular layer without branching. SUMMARY

The following over-simplified summary of the circuitry of the cerebellar cortex is based largely on older anatomical and recent electrophysiological information available in the literature and on a few contributions in the present communication. Olivocerebellar fibers, conceivably, drive the intracerebellar nuclei and continuing on as climbing fibers stimulate the Purkinje cells. The climbing fiber-Purkinje cell ratio may be one to one. Purkinje cells actively inhibit the cerebellar nuclei and Deiters nucleus. This inhibition is continuously modified by circuits activated by mossy fibers, arriving from spinal, reticular, vestibular, pontine and tegmental sources. A single mossy fiber has a wide distribution. Some distribute to at least two adjacent folia and their ramifying branches in the granular layer have many nodal points (rosettes) each contacted by a group of granule cells. In turn each granule cell, having 3 to 6 dendrites, is contacted by 3 to 6 different mossy fibers. Therefore, at the input stage in the cerebellar islands there is much divergenceand some convergence. The dense beam of parallel fibers generated by granule cells activates longitudinal series of Purkinje, upper stellate, basket and Golgi cells in the molecular layer. The

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activity induced by the parallel fibers on Purkinje cell spiny dendritic branchlets must differ qualitatively from that induced by climbing fibers on Purkinje cell smooth dendritic branches : the spiny branchlets are more distant from the Purkinje cell axon hillock; furthermore, the numerous and varying calibered parallel fibers allow for a greater temporal dispersion of arriving impulses. The basket and stellate cells, activated by a beam of parallel fibers, inhibit by means of their transversely running axons rows of Purkinje cells flanking the excitatory beam of parallel fibers. Golgi cells with their nest-like endings in the cerebellar islands have a negative feedback to the granule cells. The recurrent collaterals of Purkinje cells inhibit other Purkinje cells and inhibit basket and Golgi cells. The output stage of the cerebellar cortex feeds back to the input stage of the cerebellar cortex by means of Purkinje cell axon collaterals contacting Golgi cells, which in turn contact granule cells in the cerebellar islands. The significance of the intermediate cells of Lugaro is unknown. ACKNOWLEDGEMENTS

This investigation was supported by Public Health Service Research Grant NO. NB 0531 5-02 from the National Institute of Neurological Diseases and Blindness. We are indebted to Dr. Sanford J. Larson of Marquette University for specimens of human cerebellar cortex. Abbreviations used in the figures

A AC AP As BA BC BCF BF BN CF D DB DC DD DGC DkC DR DT E

ER G GA

axon ascending collateral of transversal fiber attachment plaque astrocyte basket cell axon basket cell beaded portion of climbing fiber Bergmann fiber basket axon nest formation climbing fiber dendrite dense body descending collateral of transversal fiber dendritic digits of granule cell displaced Golgi cell dark cell degenerating rosette dendritic tubule synaptic ending of Purkinje cell recurrent collateral cisternae of endoplasmic reticulum Golgi cell axonic ending Golgi cell axonic plexus

References p. 222-225

GC GE GH GrC GrL I ICL IP L M MdL MF ML MS N NB Nu Nuc 0 PA PC PF PFC

Golgi cell Golgi epithelial cell Golgi apparatus granule cell granular layer plasma islands intermediate cell of Lugaro infraganglionic plexus longitudinal collateral of transversal fiber mitochondria medullary layer mossy fiber molecular layer microspine nest formation of Golgi cell axons Nissl body nucleus nucleolus oligodendroglial cell Purkinje cell axon Purkinje cell parallel fiber parallel fiber constricted portion

222 PFD PT R RC

S SS

c. A. FOX et al. parallel fiber dilated portion ‘paint brush tip’ formed by basket cell axons mossy fiber rosette recurrent collateral of Purkinje cell axon Purkinje cell dendritic spine stellate cell dendritic spine

SB SbC SC SmB SpV T Tu

Purkinje cell spiny dendritic branchlet subsurface cisternae stellate cell smooth dendritic branch spiked vesicle transversal fiber of basket or stellate axon tubule of endoplasmic reticulum

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SCHROEDER, A. H., (1929); Die Gliaarchitektonik des menschlichen Kleinhirns. J. Psychol. Neurol. (Lpz.), 38,234-257. SMITH,K. R., JR., (1963); The cerebellar cortex of the rabbit. An electron microscopic study. J . comp. Neurol., 121, 459483. SNIDER, R. S., (1936); Alterations which occur in mossy terminals of cerebellum following transection of brachium pontis. J . comp. Neurol., 64,417435. STIEDA,L., (1864); Zur vergleichenden Anatomie und Histologie des Cerebellum. Arch. Anal. Physiol., 407-433. SZENT~GOTHAI, J., (1965a); The use of degeneration methods in the investigation of short neuronal connexions. Progress in Brain Research, Vol. 14, Degeneration Patterns in the Nervous System, M. Singer and J. P. SchadB, Editors, Amsterdam, Elsevier, pp. 1-30. SZENThGOTHAI, J., (196513); Complex synapses. Aus der Werkstatt der Anatomen, W. Bargmann, Editor. Stuttgart, Georg Thieme Verlag, pp. 147-167. SZENT~GOTHAI, J., AND RAJKOVITS, K., (1959); uber den Ursprung der Kletterfasern des Kleinhirns. Z. Anat. Entwick1.-Gesch., 121, 130-141. ULE,E., (1957); uber die zentralen Kleinhirnkerne als moglichen Ursprungsort der Kletterfasern. Z. Zellforsch., 46, 286-316, VANDER Loos, H., (1963); Fine structure of synapses in the cerebral cortex. Z. Zellforsch., 60,815825. VANGEHUCHTEN, A., (1890); La moelle epinikre et le cervelet. Cellule, Bd. 6, 81-122. WILSON,V. .IAND ., BURGESS,P. R., (1962); Disinhibition in the cat spinal cord. J. Neurophysiol., 25, 392404.

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Cerebellar Neuroglia : Morphological and Histochemical Aspects c. SOTELO’ Department of Histochemistry, Paris University School of Medicine, Paris VI (France)

INTRODUCTION

During the year 1909 Cajal published his, now classic, book ‘Histologie du Systtme Nerveux de 1’Homme et des Vert6br6s’. In this book Cajal pointed out that, up to that time, the function of the neuroglia in the central nervous system was unknown and, moreover, no methods were available for solving this problem. Nevertheless, from time to time, hypotheses have been advanced to give an explanation of the glial function. The first of these hypotheses -the principal one during the 19th century - considered the glial cells to be tissues of support in the nerve centers. Even today this hypothesis has some adherents. But soon some opposing concepts began to appear. In 1887, Nansen attributed to the neuroglia an important functional role, the site of intelligence, his evidence being the increase in their number with age. In the year 1910, Nageotte and Mawas independently advanced the hypothesis of the glandular function of the neuroglia. They based this concept on the presence of some granulations - the gliosomes - in the cytoplasm of protoplasmic astrocytes. Achucarro (19 13,1915) defended Nageotte’s concept and added new arguments: Ci) the attachments of the astrocytes to blood vessels (vascular feet); and (ii) the presence of cytoplasmic granulations. in the protoplasmic neuroglia of the pineal body, this body having an endocrine function. Achucarro reached the conclusion that the neuroglial tissue can be considered as an endocrine interstitial gland of the nervous system. During the last few years, the idea of a trophic role of glial cells has become the most widely-accepted hypothesis of the functional significance of neuroglia. On the other hand, some objective observations were made: those of Kuntz and Sulkin (1947) who, after electrical stimulation of preganglionic fibers of sympathetic ganglia, found an increase in the number of perisomatic gliocytes. Kulenkampff (1952) and Kulenkampff and Wiistenfeld (1954) subjected white mice to a physiological stimulation (3 to 4 h swimming in warm water at 37”)and found hypertrophy and a quantitative increase in the number of glial cells in the anterior horn of the spinal cord. All these observations suggest a direct involvement of neuroglia in the maintenance of nerve cell activity.

*

Of the Cajal Institute, Madrid (Spain)

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One of the ways to arrive at the knowledge of the mechanism that can explain the glial involvement in neural activity is to study neuroglial metabolism and its correlations with neuronal metabolism. This is the purpose of our work. The classical biochemical methods - analysis of the nervous tissue and its components - do not permit the study of neuroglial metabolism, for the great diversity of cell types and the structural complexity of the central nervous system make impossible the dissociation of the metabolic activity of neuroglia from the whole of the nervous tissue metabolism. To this problem we can only apply the methods which show a correlation between biochemical and histological aspects of the neuroglia. These methods are numerous, but they can all be included under the generic term 'histochemical methods'. In this group of methods, enzyme histochemistry, when it is used critically and with care to avoid erroneous interpretations, principally due to the diffusion of the enzyme - a frequent artifact in enzyme histochemistry - is particularly useful in the study of certain problems, because it can locate enzymic activities in the different tissues and even cellular structures. MATERIAL A N D METHODS

The observations reported in this work were made on the cerebella of 120 rats and 30 rabbits obtained from the Medical Schools of Paris and Cologne. The rats (males and females) from 150 to 300 g weight, and the rabbits (males and females) from 1500 to 3000 g, were kept for at least 1 week on a standard laboratory diet. The rats were killed by direct decapitation, without narcosis; and the rabbits by injection of 10 ml of air into the marginal vein of a n ear. Animals were quickly exsanguinated, and the cerebellum was dissected out, mounted with appropriate orientation on a cryostat tissue holder, and quickly frozen by immersion in dry ice or preferably in liquid nitrogen. This procedure permits the preservation of morphology and reduces enzymic inhibition to a minimum. The whole of these operations (killing, dissection, freezing) took 3 to 4 min for the rats, and 8 to 10 min for the rabbits. The freezing acts as an instantaneous stabilizer which keeps enzymes and morphology in good condition. The tissue blocks were placed in the cryostat, where they were warmed to its temperature. Optimal temperature for section cutting was found to be between -15" and -17". Two series of frozen sections were prepared, the first 8 to 10 p thick for cytochemical study, and the second 15 to 20 ,u thick for the study of neuroglial relationships with the neurons, blood vessels and nerve fibers. The sections were affixed to slides by melting, after which they were air dried for 2 to 4 min. The slides were then treated for the localization of enzyme activities. Novikoff et al. (1958 and 1963) recommended the use of fixatives (cold formolcalcium, cold acetone) for the preservation of morphology and especially to avoid diffusion into the incubation medium, which occurs in the unfixed cryostat-cut sections. On the other hand, they admitted that the fixation has some important disadvantages, such as the strong inhibition of some enzyme activities, especially the hexose monophosphate shunt enzymes. We have preferred to work with unfixed cryostatcut sections, because, for our material, the possible diffusion of dehydrogenases or References p. 248-250

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reduced coenzyme into the incubation medium does not disturb the staining results at the cellular level; and in some fixation tests we have not noted any improvement in the morphology or in the formazan deposit at the cellular level. We have studied the histochemically demonstrable enzymic activities which give a general idea on the metabolic pathways and derivatives of carbohydrate metabolism. The following techniques for studying enzyme activities have been used. ( a ) Phosphorylase. Two methods were employed: (1) Guha and Wegmann’s technique (1959) on unfixed cryostat-cut sections; (2) Friede’s method (1959) on small unfixed blocks of cerebellum measuring about 1 mm3. In each method the newlyformed glycogen was demonstrated with Lugol solution and by the method of McManus (PAS) after lipid extraction. (b) Oxidative enzymes. For most of this work, we used Nitro BT (Grade I11 of Sigma) as the electron acceptor. The incubation time was never longer than 45 min: after this time the control slides were always negative. The substrate concentrations were the same as previously described (Wegmann and Sotelo, 1962; Sotelo and Rudolph, 1964). The oxidative enzymes we have studied in the present work are: (a) succinate tetrazolium reductase system; (b) NADP enzyme system-nicotinamide adenine dinucleotide phosphate tetrazolium reductase (NADPH reductase), glucose-6-phosphate and 6-phosphogluconate dehydrogenases (G-6-PDH and 6-P-GlDH) ; (c) NAD enzyme system-nicotinamide adenine dinucleotide tetrazolium reductase (NADH reductase) and lactic dehydrogenase (LDH). For the morphological study of the cerebellar neuroglia, we used the metallic impregnations recommended by the Spanish school : (a) the gold sublimate method of Cajal; (b) the Golgi-Rio Hortega method; and (c) the silver carbonate methods of Rio Hortega for the visualization of macroglia and oligodendroglia. M O R P H O L O G I C A L ASPECTS OF CEREBELLAR N E U R O G L I A

It is well known that in the cerebellar cortex the two types of glial cells, astrocytes and oligodendrocytes, are found (Fig. 1). (A) Astroglia. In the molecular layer, the astroglia are represented by a morphological variety different from the classical type: the Bergmann cells or Golgi’s epithelial cells are located between the perikarya of Purkinje cells, surrounding them completely, and they can be considered as the perineuronal satellite neuroglia of the Purkinje cells. These cells extend two to three long processes from the Purkinje cell layer towards the surface of the cerebellar convolution (Fig. 7), and there they terminate by a conical swelling, the base of which is located under the pia mater, constituting the glial limiting membrane. With the help of the gold sublimate of Cajal, Faiiands (1 916) has described another variety of astroglia (FaiianBs cells) located in the inferior part of the molecular layer and whose processes do not reach the glial limiting membrane. This kind of neuroglia has the same functional signification as Bergmann cells. Both glial cells are clustered closely against the neuronal elements, mainly with the Purkinje cell dendrons.

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Fig. 1. Diagram showing the distribution of the cerebellar neuronal elements and neuroglia. P = Purkinje cell; cc = basket cell; ce = stellate cell; cg = granule cell; G = glomeruli cerebellaria; Z = cell of Golgi's type 11; fg = climbing fibers; fm = mossy fibers; o = oligodendroglia; op = perivascular oligodendroglia; AP = protoplasmic astrocyte; B = Bergmann cell; AF = fibrous astrocyte.

In the granular layer the astrocytes are principally of the protoplasmic type, and in the white matter they are of the fibrous type (Fig. 13). They extend some fine processes from the white matter, through the granular and molecular layers, towards the glial limiting membrane. ( B ) oligodendroglia. The presence of oligodendroglia in the gray matter of the cerebellar cortex has been discussed, mainly for the molecular layer. In the granular layer, all the authors (Rio Hortega, 1928; Schroeder, 1929; De Castro, 1946; etc.) agree on the presence of a large number of oligodendrocytes, mainly of type I. SomeReferences p. 248-250

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Figs. 2 and 3. Legend p. 231.


23 1

Fig. 4. Rat cerebellum. NADH tetrazolium reductase. A high activity in the Purkinje cell perikarya. A weaker one in the Purkinje cell dendrons. Some perikarya of Bergmann cells (B) show a rather high activity.

times it is difficult to differentiate them from the granule cells. Some authors, for example De Castro (1946), think that, in the cerebellar molecular layer, constituted by diffuse synaptic systems, there are almost no oligodendrocytes, in contrast with other regions, such as the spinal cord with its circumscript synapses, where the oligodendrocytes are numerous. Other authors, for example Schroeder (1929), have expressed the idea that in the cerebellar molecular layer there is a relatively large number of Fig. 2. Total phosphorylase activity (Guha and Wegmann’s method). The white matter (sb) shows a high activity. A rather high activity too, was in the molecular layer (cm). The granular layer (cg) is practically devoid of phosphorylase activity. Fig. 3. Phosphorylase activity (Friede’s method). The arrows show the negative shape of Purkinje cells. The newly-formed glycogen round the Purkinje cells belongs t o the perikarya of the Bergmann cells. In the molecular layer it is possible to observe some Purkinje cell dendrons, but mainly the Bergmann fibers predominate. References p . 248-250

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oligodendrocytes, placed only in the inferior part of this layer. The electron microscope (Palay et al., 1962) shows the presence of oligodendrocytes at this level. After studying the histochemical slides we think that the oligodendrocytes are relatively numerous and scattered all along the thickness of the molecular layer, and not only in its deeper part. We have found some cells, which have a spherical form and an eccentric nucleus (negative shape in our slides) and very few cytoplasmic processes. These cells can be considered as oligodendrocytes. They can be seen near the terminal feet of the Bergmann cells, in the superficial zone of the molecular layer, under the glial limiting membrane. We have tried metallic impregnation of these cells in young cat’s cerebellum (30-day-old) with the Rio Hortega silver carbonate and the Golgi-Rio Hortega techniques. The differentiation between oligodendrocytes (type I) and the superficial stellate cells is difficult but we have observed oligodendrocytes in all parts of the molecular layer (Fig. 8).

Fig. 5. Rat cerebellum. NADH tetrazolium reductase. The reaction is weak in the Purkinje cells (P). The Bergmann cell perikarya (B) and the Bergmann fibers are more reactive than the neuronal elements.


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In the white matter, the oligodendrocytes are very numerous; they occur in rows between the nerve fibers (interfascicular oligodendroglia), or sometimes they surround the blood vessels (Fig. 10) (perivascular oligodendroglia). The oligodendrocytes of type I1 are numerous in the axial region of the cerebellar convolution, and they have some long processes attached to nerve fibers (Figs. 15 and 17). Oligodendrocytes of types 111 and IV are rare.

Fig. 6. Rat cerebellum. Glucosed-phosphate dehydrogenase. The Purkinje cells (P) are feebly reactive. On the contrary, the Bergmann cell perikarya (B) and the Bergmann fibers show a high activity. The arrows show this enzyme activity localized in the oligodendroglia (type I) of the molecular layer. References p . 248-250

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C. SOTELO HISTOCHEMICAL R E S U L T S

Rat cerebellum

In the rat cerebellum sections which we studied, it was possible to see the cerebellar cortex and some central gray nuclei. From these last, we studied the dentate nucleus, which consists of 6 to 10 irregular rows of neurons very near to each other. These neurons are separated by a large number of glial cells, mainly protoplasmic astrocytes. ( A ) Gray matter of the rat cerebellar cortex ( 1 ) Bergmann cells (Golgi epithelial cells) (a) Phosphorylase activity. A study of this enzyme activity has shown that the

Fig. 7. Golgi method. Bergmann fibers.


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Fig. 8. Cat cerebellum. Silver carbonate according to Rio Hortega. Molecular layer. P = Purkinje cell; o = oligodendrocytes (type I); m = microglia; e = stellate cell.

Bergmann cells are the most reactive of all cerebellar cortex elements. Most Purkinje cells are negative for this reaction (Wegmann and Sotelo, 1962). In Fig. 3 it is possible to see the negative shape of the Purkinje cell perikarya, outlined by newly-formed glycogen grains in the Bergmann cell perikarya. Glycogen grains are also seen in its processes (Bergmann fibers) up to the glial limiting membrane. (b) Succinate tetrazolium reductase system. Although this system has always been studied on unfixed cryostat-cut sections, we observed no activity in Bergmann cells, even with the use of phenazine methosulfate (PMS) or menadione as electron transport agents. All the molecular layer gave a homogeneous and diffuse reaction, where it was impossible to recognize cellular shapes, which we have interpreted (Rudolph and Sotelo, 1964) as the formazan deposit on the mitochondria of the different synaptic types localized in this layer. When we studied mitochondria with the Polak technique (Wegmann and Sotelo, 1962; Sotelo and Wegmann, 1962) we obtained a References p . 248-250

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Fig. 9. Rabbit cerebellum. G-6-PDH. Molecular layer. Note the similarity of this histochemical picture with the silver impregnation (Fig. 8). P = Purkinje cell; o = oligodendrocyte; e = stellate cell. This picture is different from that obtained, for the same enzyme, in the rat cerebellum (Fig. 6).

picture similar to that of reaction for SDH activity. With the method praised by Potanos et al. (1959), the small block tissue incubation, the results are different. In the molecular layer, it was possible to distinguish the Purkinje cell dendrons, their primary branches, and sometimes even the secondary ones, as well as the stellate cell perikarya. It was even possible to distinguish the Bergmann fibers with a low activity, and certain cellular elements, with spherical perikarya, distributed throughout the molecular layer, and which we have identified as oligodendrocytes of type I. We think that these results, in accord with those of Potanos’, are not specific for the succinate tetrazolium reductase system, because if small blocks of cerebellum are incubated in a medium containing a tetrazolium salt, even without substrate, they are stained by a tetrazolium reduction to a depth of 0.5 mm. This fact can be interpreted as attributable to the existence, in the block tissue, of some endogenous substrates (lactic, pyruvic and succinic acids, and so on)


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and coenzymes (NAD, NADP) capable of tetrazolium reduction by the endogenous dehydrogenases and tetrazolium reductase action. This method probably allows the study of the tissue oxidase-reductase activity, but is not useful for the study of specific dehydrogenase activities. The Purkinje cells have shown a high reaction for this enzymic system (Wegmann and Sotelo, 1962). But not all the Purkinje cells are positive; almost 90% of these cellular perikarya showed a high succinate tetrazolium reductase activity. In the remaining 10% it was possible to see different degrees of activity, and some cells were completely devoid of this enzyme activity, which we have interpreted as a different functional activity in the Purkinje cells. With the autoradiographic method, Oehlert et al. (1958) reached the same conclusion : there is no regular protein turnover in all the Purkinje cell perikarya. Most ofthem have a high protein turnover, but some of them show few silver grains or are even completely devoid of them.

Fig. 10. Silver carbonate according to Rio Hortega. Cerebellar white matter showing a perivascular oligodendrocyte. References p. 248-250

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Fig. 11. Rat cerebellum. G-6-PDH. The enzyme activity is localized in the cytoplasm of perivascular oligodendroglia (arrows)and in some interfascicular oligodendrocytes.

(c) NADP enzyme system. These enzymes showed a higher activity in Bergmann cells than in Purkinje cells. Of these three enzymes, the glucose-6-phosphate dehydrogenase gave the sharpest picture of Bergmann cells, superimposable on the Cajal gold-sublimate pictures. The Bergmann cell perikarya are full of formazan grains coupled to each other, which give the aspect of a uniformly stained cytoplasm. The Bergmann fibers are also highly positive, and it is possible to follow them to the glial limiting membrane (Fig. 6). The NADPH tetrazolium reductase showed the same localization as the Gd-PDH (Fig. 5). The 6-P-GlDH was very slightly positive, and it was sometimes necessary to study these slides with the phase contrast microscope to be certain of the localization of this enzyme activity. The 6-P-GlDH activity showed the same localization as the other dehydrogenases of the NADP system, and it was always higher in the Bergmann cells than in the Purkinje cells. (d) NAD enzyme system. This group of enzymes was, on the other hand,


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more active in the Purkinje cells than in the Bergmann cells. The NADH tetrazolium reductase (Fig. 4) and the lactic dehydrogenase reactions showed the same localization. The formazan was found in some rare Bergmann cell perikarya, and mainly in the stellate neurons of the molecular layer. (2) Granular layer. Due to the complex structure of this cerebellar layer, the visualization of neuroglia at this level was difficult. We have not been able to recognize oligodendrocytes with certainty with any of the studied enzymes. Granule cells, positive for most of these enzymic reactions, have shown, in the enzymic histochemical sections, a morphology so similar that it was impossible to differentiate them. The protoplasmic astrocytes, numerous in this layer, have not shown any activity of the oxidative enzymes revealed by histochemical methods, even with the use of thick sections (I 5 to 20 p).

Fig. 12. Rat cerebellum. NADPH tetrazolium reductase. The formazan grains are in the cytoplasm of interfascicular oligodendroglia. The capillaries and some axoplasms are positive. References p . 248-250

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Fig. 3. Golgi-Rio Hortega method. Cerebellar white matter. A = fibrous astrocyte with vascular feet (arrow). 0 1 = oligodendrocyte type I.

( B l White matter of the rat cerebellar cortex (1 Phosphorylase activity. The white matter shows high phosphorylase activity, which it is possible to reveal by Guha and Wegmann’s method (Fig. 2) as well as by Friede’s method. The shapes of the figurative elements in the white matter are clearer with the use of Guha and Wegmann method. The more active elements are the fibrous astrocytes. Their perikarya and their processes are full of newly-formed glycogen grains. The vascular feet of these astrocytes show, as a rule, a high phosphorylase activity allowing the location of the capillaries by their glial membrane coloration. We have not, with certainty, found oligodendrocyte pictures in our slides. It is possible that the oligodendrocytes have a phosphorylase activity although always weaker than that of the astrocytes. (2) Succinate tetrazolium reductase system. The cerebellar white matter was completely negative for this enzymic system, even when the incubation time was prolonged


24 1

to 60 min (Sotelo and Wegmann, 1964). With the use of PMS, the reaction was always negative for the white matter; but for the gray matter it was quicker and higher. In a 10-min incubation, the formazan deposit was stronger than in a 1-h incubation without this electron transport agent. The best histochemical results for the succinate tetrazolium reductase system were obtained with the use of menadione as electron transport agent. The technical conditions were similar to that of PMS. It was possible to observe a weak, but clear, reaction in the cerebellar white matter localized in the axoplasm and in the glial sheath of the axons. Some blue formazan grains were mainly visible in the interfascicular oligodendrocyte cytoplasm. The myelin was always completely negative. (3) NADP enzyme system. The three enzymes belonging to this system have shown a rather high activity ifi the white matter. The NADPH tetrazolium reductase(Fig. 12), the G-6-PDH and the 6-P-GlDH had the same activity localization. As we have said, the last of these three enzymes showed a weak coloration, and sometimes it was necessary to use the phase contrast microscope to allow its fine localization. Among all the

Fig. 14. Rabbit cerebellum. G-6-PDH activity in the interfascicular oligodendroglia of the white matter. References p . 248-250

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Fig.15. Golgi-Rio Hortega method. A = fibrous astrocyte; 0 1 = oligodendrocyte type I; 011 oligodendrocyte type 11.

=

enzyme activities which we have studied, the NADP enzyme system was the most characteristic of the white matter. In the histochemical sections it was easy to distinguish the blood vessels because their walls were stained by the formazan grains. The glial cells showed a high activity. Their nuclei were negative, and each one was surrounded by a cytoplasm rich in formazan grains. The formazan deposit was extended through the proximal part of their processes.From the shapes of the cytoplasm and its processes and from its localization in relation with the nerve fibers and with the blood vessels, we can assert that the G-6-PDH is present in the interfascicular and perivascular oligodendroglia (Fig. 1 1). In a relatively small percentage of the rats examined, we have found the hexose monophosphate shunt enzyme activities in some cells resembling the normal fibrous astrocytes. This observation has induced us (Sotelo and Wegmann, 1964) to suggest that the fibrous astrocytes of the rat cerebellar cortex are rich in pentose cycle enzymes, although a s we have noted, the oligodendrocytes presented the highest oxidative activity among all the cerebellar glial cells.


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(4) N A D enzyme system. The two enzyme activities studied in this system have not shown a high intensity at the white matter level. Both enzymes had a strong activity in neuronal structures in comparison with glial structures. The interfascicular oligodendrocytes were the cells which presented the highest reaction in the white matter. The fibrous astrocytes were negative for both enzymes. The axoplasm was positive, but with a rather weak activity. The myelin was always negative. The blood vessels showed a strong coloration - the sign of a high enzyme activity - in their walls. ( C ) Dentate nucleus of the rat cerebellum It was possible to obtain positive results in the neuroglia only with the NADP linked enzymes. When the thick cryostat-cut sections were studied, mainly for the

Fig. 16. Rabbit cerebellum. NADPH tetrazolium reductase. Reaction is present in the interfascicular oligodendrocyte cytoplasm . References p. 248-250

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Fig. 17. Golgi-Rio Hortega method. Oligodendrocyte type I1 of the axial region of the cerebellar circumvolution.

NADPH tetrazolium reductase and G-6-PDH, it was possible to locate, by its rather high reaction, the protoplasmic astrocytes which surround the neurons of the cerebellar olive. The perikarya of these astrocytes were full of formazan grains. Sorr.etirnes (Fig. IS) it was possible, in the G-6-PDH preparations, to see some interneuronal astrocytes making the neuron-surrounding medium, and some other processes, equally stained, directed towards the blood vesseIs constituting the vascular feet. This picture can be interpreted as the requirement of hexose monophosphate shunt enzymes to transport the nutritional materials coming from the blood vessels to the nerve cells through the protoplasmic astrocytes. For the NADP enzyme system, the glial cells of the dentate nucleus were always more active than the nerve cells. For the other oxidative enzymes it was not possible to recognize the protoplasmic astrocytes, whereas the nerve cells were very reactive.


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Rabbit cerebellum The results with the rabbit were different, from a qualitative point of view, from those obtained with the rat cerebellum. It was impossible to observe any oxidative enzymes in Bergmann cells. The molecular layer showed, for the NADP linked dehydrogenases (Fig. 9), a picture quite different from that of the rat cerebellum. It was possible to distinguish some cellular elements distributed throughout the molecular layer. Some of these cells were star-shaped (stellate cells) and most of them were similar to those described as oligodendrocytes. The results obtained for the glial cells in the granular layer and white matter (Figs. 14and 16)were similar to those described for the rat cerebellum. The only exception was that, whereas in the rabbit cerebellar white matter, we have never found any hexose monophosphate shunt enzyme activity in the protoplasm of fibrous astrocytes, for certain rats (Sotelo and Wegmann, 1964) we have found some fibrous astrocytes positive for the G-6-PDH reaction. There is then a metabolism specificity for the astrocytes in relation to the animal species. Otherwise it has been possible to show a metabolic uniformity for the oxidative enzymes of the Purkinje cells of different mammals; On the other hand, from the observations recorded in the present work, we are led to think that there are metabolic differences between the astroglia cells of the various species of mammals. The same observations were made at the dentate nucleus level. In the rabbit dentate nucleus, in contrast with that of the rat, the protoplasmic astrocytes have

Fig. 18. Nucleus dentatus of the rat cerebellum. G-6-PDH. Interneuronal protoplasmic astrocytes (A) making the neuron surrounding medium. c = capillaries. The arrows show the vascular feet of these interneuronal astrocytes. References p. 248-250

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not shown any oxidative enzymic activity; on the other hand the oligodendrocytes of type I showed an enzymic pattern typical of all oligodendrocytes. DISCUSSION

We think it necessary to avoid the limitations and possible artifacts of enzyme histochemistry, by comparing these results with those obtained by biochemical methods. The macrochemical works that have studied the respiration of the gray matter of the cerebral cortex (oxygen consumption), making a comparison between the respiratory rates of neuronal and non-neuronal elements - formed mainly by glial cells - are numerous (Elliott and Heller, 1957; Korey and Orchen, 1959; Tower, 1960). The most important fact is that although the glial cells are much more numerous, the nerve cells consume 80% of the total oxygen, and only 20% is consumed by the non-neuronal elements. From a carbohydrate metabolism point of view we can sum up these results by saying that the aerobic glycolysis is higher in the nerve cells than in the glial cells, which is in accord with our results. The cerebellum has been studied by Buell, Lowry et al. (1958) and by Robins et al. (1953 and 1957) by microchemical methods of quantitative histochemistry. Our results are in agreement with Lowry’s. The G-6-PDH activity is: (a) very high in the white matter, being localized primarily in the oligodendrocytes and secondarily in the axoplasm, and (b) a IittIe less active, but with an important degree of reaction, in the molecular layer, the enzyme activity being localized in Bergmann fibers and in oligodendrocytes, type I, for the rat cerebellum, and only in oligodendrocytes for the rabbit cerebellum; and (c) least positive in the granular layer. For the phosphorylase activity our results are also in agreement with Lowry’s results, with the exception that we have not been able to observe a high reaction in the granular layer. On the other hand, and only in a comparative way, because the Hydtn group has not, till now, studied the cerebellum, there is a discrepancy between our results and those of Hydtn (1959) and Hamberger (1963). HydCn has mainly studied Deiters’ nucleus and some enzymic activities (cytochrome oxidase, succinate oxidase, a-ketoglutarate oxidation and glutamate oxidation). He found that the cytochrome oxidase and the succinate oxidase activities were higher in the neuronal glia than in the nerve cells. Although we are convinced of an important oxidative metabolism in the neuronal neuroglia in a general way, except for the hexose monophosphate shunt enzymes, the oxidative activities we have studied in this work were always higher in the nerve cells. Tolani and Talwar (1963), using preparative cytochemical methods, studied the mitochondrial fractions of the cerebellar and cerebral cortices, in comparison with the mitochondria1 fraction of the corpus callosum. They found that the cytochrome oxidase activity was nearly 20 times higher in the mitochondria of neuronal origin than in the mitochondria of neuroglial origin. Although the number of mitochondria is smaller in the neuroglia than in the nerve cells (Palay, 1958) there is a chemical heterogeneity between neuronal and neuroglial mitochondria. This fact seems to support our results. With the same enzyme histochemical methods, Friede (1965) studied the glial cells

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in the central nervous system. He expressed the opinion that the Bergmann cells of the cerebellum and the Miiller cells of the retina (Kuwabara and Cogan, 1960) must be considered enzymically as astrocytes and not as oligodendrocytes. He studied 12 species of animals and never found any increase in the enzymic activity in Bergmann cells of normal animals. Our experience is quite different. We also support the astroglial metabolism nature of Bergmann cells, but in this work we found a difference, for oxidative enzymes, between the Bergmann cells of rat and rabbit cerebellum. CONCLUSIONS

The neuroglia has its own metabolism, qualitatively different from neuronal metabolism. With histoenzymic methods, it is not possible to find any difference between the enzyme pattern of neuronal neuroglia and interfascicular neuroglia in the cerebellar cortex. In both situations, glial anaerobic glycolysis is high, whereas the aerobic glycolysis is secondary and much weaker than in the nerve cells. The hexose monophosphate shunt is important in the neuroglia and slightly active in nerve cells. Both glial cell types, oligodendroglia and astroglia, did not show the same enzyme pattern. There were metabolic differences between the astroglial cells of the various species of mammals, as follows: (i) Rat cerebellum. The Bergmann cells and the interneuronal protoplasmic astrocytes of the dentate nucleus showed an oxidative reaction closely similar to that of the oligodendroglia. (ii) Rabbit cerebellum. The astroglia were devoid of oxidative enzymes. The oligodendroglia showed a rather high oxidative reaction similar to the reaction exhibited by the oligodendroglia of the rat. There is another difference between oligodendroglial and astroglial metabolism. The metabolism of glycogen, its storage and its degradation by phosphorylase action, is almost limited to astrocytes. Hypothesis on glial function ( A ) Neuronal neuroglia. The hypothesis advanced by Cajal(l897) on the existence of neurono-neuroglial symbiosis may have a biochemical basis. The neurons always showed a high activity for the enzymes of the Krebs cycle (liberation of the energy needed mainly for the synthesis of acetylcholine and for ion transport in the nerve cells) and a weak activity for the enzymes of the hexose monophosphate shunt. The neuroglia, on the contrary, shows an opposite enzymic pattern, relatively feeble for the enzymes of the Krebs cycle and intense for the oxidative enzymes of the hexose monophosphate shunt (reduction of NADP). The neuronal neuroglia may present the nerve cells with NADPH, the coenzyme necessary for the principal reactions of cellular biosynthesis. The neuronal neuroglia can be considered as an auxiliary metabolic part of the neurono-neuroglial axis. ( B ) Interfascicular neuroglia. Del Rio Hortega (1928) offered the hypothesis of a myelination function of oligodendroglia. Based on electron microscope studies (De Robertis et al., 1958; etc.) the Del Rio Hortega hypothesis has been corroborated. Referenres p . 248-250

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The oligodendroglia plays a leading part in myelination of central nerve fibers. The histochemical results give biochemical arguments to this role of the interfascicular neuroglia in the chemical maintenance and renovation of myelin. The myelin, from its lipoprotein constitution, and mainly its sphingomyelin fraction, requires NADPH for its biosynthesis. This reduced coenzyme is provided, we may suppose, by the hexose monophosphate shunt, which is very active in the oligodendrocyte cytoplasm. SUMMARY

The cerebellar neuroglia of rat and rabbits has been studied with conventional metallic impregnations and enzyme-histochemical methods. The presence has been proved of some oligodendrocytes of type I scattered all along the thickness of the molecular layer. These oligodendrocytes can vary in number in different species of mammals. In the rabbit cerebellum they are more numerous than in the rat cerebellum. The metabolism of glycogen, its storage and its degradation by phosphorylase, was chiefly dependent on astroglia cells. In a general way, the oligodendrocytes were the most active glial cells for the oxidative enzymes. The Bergmann cells had a different enzyme pattern in the rat than in the rabbit cerebellum. In the first mammal, these cells showed a strong activity for the NADP linked dehydrogenases; whereas in the second, the Bergmann cells were free of any oxidative activity. This means that there is not a chemical unity - for oxidative enzymes - in the astroglial cells of the various species of mammals. The neuronal neuroglia showed an enzyme pattern complementary to that of the neurons : weak activity for the succinic dehydrogenase and relatively strong activity for the NADP linked dehydrogenases. These findings permit conclusions regarding the functional role of the cerebellar neuroglia.

REFERENCES ACHUCARRO, N., (1913); La estructura secretora de la glandula pineal humana. Bol. Soc. ~ s p . Biol., 2, 83-88. ACHUCARRO, N., (1915); De I'Cvolution de la nCvroglie et spicialement de ses relations avec l'appareil vasculaire. Trab. Lab.Invest, Biol. (Madrid), 13, 169-212. BUELL,M. V., LOWRY,0.H., ROBERTS, N. R., CHANG, M. W., AND KAPPHAHN, J. I., (1958); The quantitative histochemistry of the brain. V. Enzymes of glucose metabolism. J. b i d . Chem., 232, 979-993. CAJAL, S. R A M ~YN , (1909); Histologie du Syst2me Nerveux de I'Homme et des Vertibris. Vol. 1, Paris, Maloine. S . R A M ~Y,NAND OLORIZ, F., (1897); Los ganglios sensitivoscranesles de 10s mamiferos. Rev. CAIAL, trim. microgr., Vol. 2. DECASTRO, F., (1946); Sobre el comportamientoy significacidn de la oligodendrogliaen la substancia gris central y de 10s gliocitos en 10s glfinglios nerviosos perifCricos. Arch. Histol. ( B . Aires), 3, 317-343. DE ROBERTS,E., GERSCHENFELD, H. M., AND WALD,F., (1958); Cellular mechanism of myelination in the central nervous system. J. biophys. biochem. Cyfol.,4, 651458.

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ELLIOm, K. A. c.9AND HELLER, I. H., (1957); Metabolism of neurons and glia. D. Richter, Editor. Metabolism of the Nervous System, London, New York, Paris, Los Angeles, Pergamon Press, pp. 286-290. J. RAMON Y, (1916); Contribucion al estudio de la neuroglia del cerebelo. Trab. Lab. Invest. FARANAS, Biol. (Madrid), 14, 163-179. FRIEDE, R. L., (1959); Histochemical demonstration of phosphorylase in brain tissue: association of postmortal neuron changes with the phosphorylase activity. J . Histochem. Cytochem., 7 , 34-38. FRIEDE, R. L., (1965); Enzyme histochemistry of neuroglia. E.D.P. de Robertis and R. Carrea, Editors, BioIogy of NeurogIia, Vol. 15, Progress in Brain Research, Amsterdam, Elsevier, pp. 3 5 4 7 . GUHA,S., AND WEGMANN, R., (1959); Une nouvelle methode demise en evidence de la phosphorylase. Ann. Histochim., 4, 103-115. HAMBERGER, A., (1963); Difference between isolated neuronal and vascular glia with respect to respiratory activity. Acta physiol. scand., 58, Suppl. 203. HYD~N H.,, (1959); Biochemical changes in glial cells and nerve cells at varying activity. Biochemistry of the Central Nervous System. 4th. Int. Congr. Biochem., Vol. 111, F. Brucke, Editor, London, New York, Paris, Los Angeles, Pergamon Press, pp. 64-90. KOREY,S. R., AND ORCHEN, M., (1959); Relative respiration of neuronal and glial cells. J . Neurochem., 3,277-285. KULENKAMPFF, H., (1952); Das Verhalten der Neuroglia in den Vorderhornern des Ruckenmarks der weissen Maus unter dem Reiz physiologischer Tatigkeit. Z. Anat. EntwickLCesch., 116, 304-312. KULENKAMPFF, H., AND WUSTENFELD, E., (1954); Funktionsbedingte Veranderungen der Kerngrosse von Gliazellen im Grau des Ruckenmarkes der weissen Maus. Z. Anat. Entwick1.-Gesch., 118, 97-101. KUNTZ,A., AND SULKIN,N. M., (1947); The neuroglia in the autonomic ganglia: cytologic structure and reactions to stimulation. J. comp. Neurol., 86,467477. KUWABARA, T., AND COGAN,D. G., (1960); Tetrazolium studies on the retina. 111. Activity of metabolic intermediates and miscellanous substrates. J. Hisrochem. Cytochem., 8 , 2 1 4 2 2 4 . MAWAS,J., (1910); Note sur la structure et la signification glandulaire probable des cellules n6vrogliques du systeme nerveux central des vertebrks. C. R . Soc. Biol. (Paris), 69,45-46. NAGEOTTE, J., (1910); Phenomenes de skretion dans le protoplasme des cellules nevrogliques de la substance grise. C. R. Soc. Biol. (Paris), 68, 1068-1069. NANSEN, F., (1887); The structure and combination of the histological elements of the central nervous system. Bergens Mus. Aarsber, 1886, Bergen, 1887, pp. 29-214. NOVIKOFF, A. B., (1963); Electron transport enzymes: Biochemical and tetrazolium staining studies. First Znt. Congr. Histochem. Cytochem., R. Wegmann, Editor. Oxford, London, New York, Paris, Pergamon Press, pp. 465-481. NOVIKOFF, A. B., AND MASEK,B., (1958); Survival of lactic dehydrogenase and DPN-diaphorase activities after forrnol-calcium fixation. J. Histochem. Cytochem., 6, 217. OEHLERT, W., SCHULTZE, B., AND MAURER,W., (1958); Autoradiographische Untersuchung der Grosse des Eiweissstoffwechsels der verschiedenen Zellen des Zentralnewensystem. Beitr. path. Anat., 119, 343-376. PALAY, S. L., (1958); An electron microscopical study of neuroglia. Biology of Neuroglia, W. F. Windle, Editor. Sprinfield, Thomas, pp. 24-38. PALAY, S. L., Mc. GEE-RUSSELL, S. M., GORDON, S., AND GRILLO,M. A., (1962); Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide. J . Cell Biol., 12, 385410. POTANOS, J. N., WOLF,A,, AND CQWEN,D., (1959); Cytochemical localization of oxidative enzymes in human nerve cells and neuroglia. J. Neuropath. exp. Neurol., 18, 627-635. RIO-HORTEGA, P. DEL,(1928); Tercera aportacibn a1 conocimiento morfdogico e interpretacih funcional de la oligodendroglia. Mem. Real SOC.ESP. Hist. Nut., 14, 5-122. ROBINS, E., AND SMITH,D. E., (1953); A quantitative histochemical study of eight enzymes of the cerebellar cortex and subjacent white matter in the monkey. Ass. Res. nerv. Dis. Proc., 32, 3 05-327. ROBINS,E., SMITH,D. E., AND JEN,M. K., (1957); The quantitative distribution of eight enzymes of glucose metabolism and two citric acid cycle enzymes in the cerebellar cortex and its subjacent white matter. Progr. Neurobiol., 2,205-214. RUDOLPH,G., AND S O ~ L OC., , (1964); Enzymhistochemischer Kachweis spezifischer Dehydrogenasen in der Kleinhrnrinde von Ratten. In press.

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SCNROEDER, A. H., (1929); Die Gliaarchitektonik des menschlichen Kleinhirns. J . Psychol. Neurol. (LPz.), 38,234-257. SOTELO, C . AND RUDOLPH, G., (1964); Etude histoenzymologique du metabolisme des glucides au niveau des gliocytes des ganglions rachidiens. In press. SOTELQ, C., AND WEGMANN, R., (1962); Aspects enzymohistochimiques des glomerules ckrebelleux: Correlation entre leur structure, leur fonction et la production d’energie. Ann. Histochim., 7 , 105-110. SOTELO, C., AND WEGMANN,R., (1964); DBc5rences du metabolisme des glucides de la substance blanche et de la substance grise du cervelet. Acra hisrochem., 18, 125-136. TOLANI, A. J., AND TALWAR, G. P., (1963); Differential metabolism of various brain regions. Biochemical heterogeneity of mitochondria. Biochem. J., 88, 357-362. TOWER,D. B., (1960); Summary statement neurochemistry symposium : Some neurochemical aspects of cortical neurobiology. D. B. Tower and J. P. Schad6, Editors. Strucrure and Function of the Cerebral Correx. Amsterdam. Elsevier (pp. 41 1424). WEGMANN, R., AND SOTEU), C., (1962); Aspects cytoenzymatiques du metabolisme des glucides de la cellule de Purkinje. Ann. Histochim., 7 , 65-81.

25 1

Intracerebellar Inhibitory Systems P. E. VOORHOEVE Department of Neurophysiology, Jan Swammerdam Institute, University of Amsterdam, Amsterdam (The Netherlands)

INTRODUCTION

The cerebellum is considered to be an integrating centre for movement and posture. For the neurophysiologist this means that one would expect excitatory and inhibitory mechanisms to co-operate in such an organ in the same way as excitatory and inhibitory influences converge on the motoneurone which is the final common path for the execution of a movement. A considerable amount of work has been done in the past on the function of the cerebellum as a whole, on the localization of certain cerebellar functions and on the topology and modalities of different afferent systems. Much less is known, however, from the physiological point of view about the intracerebellar organization ; and the existence of intracerebellar inhibitory mechanisms, especially, was highly questionable. ‘We must concede that we know practically nothing about the events that take place between the arrival of the afferent signals and the response of the Purkinje neurons or the modulation of their discharge’, as Dow and Moruzzi (1958) put it succinctly in their monograph. One of the pioneer investigations in this field, though performed for another purpose, was done by Dow (1949) who studied the field potentials on local stimulation of the parallel fibres in a folium. Buser and Rougeul (1954) working on a pigeon were, to my knowledge, the first investigators who succeeded in recording intracellularly from Purkinje cells. They found an initial high-frequency discharge which stopped when the cell under study was sufficiently depolarized. We would now call this type of discharge an injury discharge. Japanese workers (Suda and Takahira, 1954) had, in the same year, apparently obtained similar results. Brookhart et al. (1950) made an analysis of single unit discharges. One of their conclusions was that ‘activity in cerebellarneuronesisinherently asynchronous’. They also noted, in the decerebrate preparation, activity in erratic bursts separated by silent intervals, but they did not determine the factors controlling the resting frequency and the timing of the periods of rest and activity. Granit and Phillips’s papers in 1956 and 1957 are a landmark in the field of cerebellar organization. These authors were the first to define ‘excitatory and inhibitory processes References p . 266-267

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acting upon individual Purkinje cells’, which was the title of their first paper. They gave criteria for identifying Purkinje cells, and distinguished two ways of stopping Purkinje cell firing. The most commonly observed type of inhibition was through excessive depolarization of the cell, causing inactivation of the spike generating mechanism. Granit and Phillips suggest that this may be tentatively ascribed to activity in basket cells, having axons around the Purkinje cell and the bottle-neck ofitsexit point which can, so to speak, strangulate the cell. The second form of inhibition observed was by hyperpolarization of the Purkinje cell. This seems to be the appropriate moment to define what we understand by ‘inhibition’. One can say that a nerve cell is inhibited when the generation of impulses by that cell is depressed as a consequence of impulses from other cells impinging on it. This general definition includes the phenomena of electrical and presynaptic inhibition (Eccles, 1964). We will use the term here in the more limited sense of so-called postsynaptic inhibition which is due to an increased permeability of the subsynaptic membrane to K+ and CI- ions, caused by a specific chemical transmitter that is released by an inhibitory interneuron. This transmitter, or these transmitters by the way, are as yet unidentified in the mammalian CNS. Inhibition thus defined is usually accompanied by hyper-

In

LOCAL STIMULATION

whtte matter

STlMULATlO N

Fig. 1 . Diagram of the cerebellar cortex and the experimentalarrangement.A, the surface stimulating electrodes, the concentric electrode for antidromic stimulation and the micro-electrodetrack. P is a Purkinje cell with its axon Pa, €3 a basket cell with its axon Ba. The mossy fibres mf make contact with a granule cell G that sends its axon as a parallel fibre pf along the superficial molecular layer. B shows the increase in the excited beam of parallel fibres with increasing stimulus strength and in the dotted line the tangential approach to impale Purkinje cells (Andersenet a/., 1964~).

polarization of the cell membrane. It is important to realize that inhibition is only functionally effective when it stops a cell firing or diminishes its firing frequency but, on the other hand, cessation of cell firing does not necessarily mean that the cell under observation is inhibited. In this respect I would like to refer to an observation by Arduini and Pompeiano (1957) that rostro-median fastigial units could be depressed by polarizing the vermis. These authors suggested that this could be due to a cessation of cortico-fastigial drive if Purkinje cells were somehow suppressed by surface polariza-

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tion. This suggestion is refuted by the evidence of Ito et al. (1964), to be presented today, that Purkinje cells are purely inhibitory. In 1957, Purpura and Grundfest published a paper in which it is stated that ‘the cerebellar cortex is but poorly endowed with inhibitory synapses’. This conclusion was reached on several assumptions, such as that surface positivity stands for a hyperpolarizing postsynaptic potential and surface negativity for a depolarizing PSP, which need not necessarily be so. Pharmacological arguments were that d-tubocurarine and strychnine block inhibitory synapses and that heparine protects excitatory synapses against the blocking action of dtubocurarine. Our own approach to the cerebellum in 1963 with Eccles and Andersen was primarily not for the cerebellum’s sake, but to test a hypothesis concerning the inhibitory function of the basket cells. By a combination of intracellular recording and a detailed analysis of the extracellular field potentials, Andersen et al. (1964a, b) had shown that in the fields CAI and CA3 in the hippocampus a powerful inhibition is exerted on the cell bodies of the pyramidal cells. By localizing the site of the inhibitory action they identified the basket cells as being the inhibitory cells.

RESULTS

1. Basket cell and stellate cell inhibition of Purkinje cells

In the initial experiments use was made of the special anatomical features offered by the cerebellar cortex. By putting a bipolar electrode gently on the cerebellar surface one can activate a narrow ‘beam’ of parallel fibres which in its turn activates synaptically all cells having their dendritic tree in this beam, i.e. Purkinje cells, basket cells, stellate and Golgi cells. The histological arrangement is such that the basket cell bodies are located eccentrically to their own field of action, for they send their axon transversely across the folium making synaptic contact with the cell bodies of about 10 Purkinje cells on either side (Cajal, 1911 ; Szenthgothai, 1965). In order to investigate the effects of basket cell activity on Purkinje cells uncontaminated by the direct excitatory action of the parallel fibres on the Purkinje cells themselves, one simply has to penetrate with the exploring micro-electrode just ‘out of line’ with the beam of excited parallel fibres. It is well known that Purkinje cells are not easy to impale with a micro-electrode. This may be due partly to a tearing to pieces of the dendritic tree before penetration, and to a dense glial covering around the cell body. In order to avoid damage to the dendritic tree we have tried oblique and tangential approaches without much success.With some practice, however, it is often possible to impale cells and keep them for several minutes. Fig. 1 illustrates the experimental set-up. Purkinje cells were encountered at a depth of about 0.35 mm on perpendicular insertion in the middle of a folium, and could often be antidromically activated from a juxta-fastigial electrode. In cells penetrated slightly out of the line of the excited parallel fibres, slow hyperpolarizing potentials were recorded on local stimulation. This hyperpolarization occurred without initial firing of the cell, and it diminished and even reversed on hyperpolarization of the cell membrane or on injection of C1- ions. It is References p . 266-267

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considered to be an inhibitory postsynaptic potential (IPSP) (Fig. 2). On recording more in line with the excited parallel fibres the IPSP was preceded by an initial depolarization, an excitatory postsynaptic potential (EPSP).

1 m sec

-LLLL lOmsec

~ l ~ l l ~ l \ l l l l l l l : 10mrec

Fig. 2. Intracellularly recorded Purkinje cell IPSP at different sweep speeds from A to C and increasing stimulus strength from above downward. Stimulus strength expressed as multiples of threshold value 1.O T. The upper beam in each record represents the intracellular, the lower beam the just extracellular recording. In the lower row the just extracellular potentials have been subtracted from the intracellular record for 4 different stimulus strengths in order to disclose the actual potential across the membrane. Observe that the initial depolarization in most records disappears after subtraction, indicating that it represents a focal potential andnot an EPSP generated across the membrane of this particular cell. Increase in stimulus strength causes a progressive decrease in the latency of the IPSP, a shortening of its rising phase and a gradual increase in size. The initial downward deflection in B and C is a negative going monitor pulse (Andersen et al., 19%).

Fig. 3 shows that this method of parallel fibre stimulation has indeed an inhibitory effect on two cells in the Purkinje cell layer that were spontaneously firing with a high

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Fig. 3. Inhibition of the discharges of two spontaneously firing Purkinje cells by surface stimulation of the parallel fibres. Repetitive stimulation at the indicated frequencies is shown. At a frequency of lO/sec the cell with the large spike is totally inhibited and the one with the small spikes resumes firing at the end of the inhibitory period. At a rate of 16/sec and more, both cells are completely suppressed (Andersen ei al., 1964~).

frequency. The pause in firing for the more remote cell with the smaller spike lasted for 100 msec, that for the cell with the large spike some 120 msec. Repetitive stimulation at lO/sec completely stopped the first cell firing, whereas a repetition rate of 16/ sec was needed to suppress the second cell. After a period of stimulation a t 100/sec causing complete suppression of firing it took but slightly longer for both cells to resume their activity, indicating their strong excitatory drive and, thus, the potency of the inhibitory influence. To identify the site of the inhibitory action, i.e. the place where the IPSP is generated one can, in a uniformly polarized structure such as the cerebellar cortex, best perform an analysis of laminar field potentials. As already mentioned, Dow (1949) has shown that a stimulus applied to the surface of the cerebellar cortex evoked a brief surface negative potential in a narrow strip in line with the course of the parallel fibres up to a distance of about 5 mm. This negative potential was preceded by a small positive potential, and was followed by a potential of much longer duration that was usually negative a t the surface. Depth tracking showed that the whole complex had a dual composition. The initial positive-negative sequence behaved differently from the later slow potential. The former was, in accordance with Dow, interpreted as being the approaching volley, recorded in volume, in the References p. 266-267

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excited beam of parallel fibres, as has been more fully corroborated by later experiments (Eccles et al., 1966b). When a micro-electrode was inserted perpendicularly to the surface of the folium, a large increase in the initial negative potential was recorded with a maximum, depending on the stimulus strength, between 0. I5 and 0.30 mm. At greater depths this negative potential disappeared, and a later large slow positive potential was dominant. In the Purkinje cell and granule cell layer, from 0.30-0.50 mm depth, unitary activity was recorded in the early phase of the potential. The slower positive wave, having its maximal amplitude around a depth of 0.35 mm, declined rapidly towards the surface. It often reversed superficially and turned into. the slow negative wave that has been mentioned earlier. Deeper than 0.40 mm this positivity gradually decreased and did not reverse. When the electrode penetrated slightly out of line with the excited beam of parallel fibres the initial brief negative wave was lost and the whole picture was dominated by the positive wave thus recorded without contamination by the direct excitatory effects of the parallel fibre volley. In Fig. 4 these potentials are presented in three different tracks and in C the amplitude of the,positivity is plotted against depth of recording. The whole behaviour of . . 01

.

02

t

0.3 O.'t

Fig. 4. Focal potentials at different distances from the line of parallel fibre stimulation. A, responses obtained at the indicated depths, recorded with negativity upward.*At distance 0, in the line of stimulation the electrode is optimally located for recording the excitatory responses. The positive wave is developing beyond 0.2 mm depth, and unitary activity is recorded between 0.25 and 0.5 mm. More laterally the initial negative wave disappears and the later positive becomes more prominent: in C the amplitudes of the positive waves, measured at a latency of 6.6 msec at the dotted lines, are plotted against the depth of recording. The points for the curves at 0.4 and 0.5 mm were obtained with slightly stronger stimulation. In B a Purkinje cell is schematically drawn on the same scale and the arrows indicate the direction and the density of the ionic currents flowing from the soma into the dendrites (Andersen et al., 1964c).

this positive wave corresponds closely to that of the positivity as recorded in the hippocampus (Andersen et al., 1964a). In the cerebellum it attains its maximum at the layer of the Purkinje cell bodies (0.35 mm) and decreases steeply towards the surface.


257

The steepness of this decline can be regarded as a measure of the intensity of current flowing outward from the soma into the dendritic tree and indicates that the dendrites act as an efficient sink for this current. That dendritic depolarization is not the primary cause for this potential profile is shown by the fact that it corresponds with a simultaneous hyperpolarization of the soma membrane as revealed by intracellular recording (Gloor, 1963; Andersen et al., 1963a). There is, however, some evidence that dendritic depolarization is the principal cause of the positivity at depths of 0.10-0.25 mm in line with the parallel fibre beam. These findings are in accord with the postulate that the IPSPs recorded from Purkinje cells are generated by synapses on or close to their somata, and thus by impulses in basket cells which alone have their terminals concentrated on the Purkinje soma and adjacent regions of the axon. The projection of basket cell axons across the folium by as much as 1.O mm explains

A

h---

Concentric

dep:h 20

420 520

-

I

'V !i,y -w500-

I

Fig. 5. Laminar field potentials obtained by different means of parallel fibre stimulation, and recorded at the indicated depths. In A the stimulus was applied through a concentric surface electrode, activating only a superficial beam of parallel fibres. The profile illustrated in B was evoked by unipolar stimulation. In C the positivities, measured at the latency indicated by the dotted lines, are plotted against the depth of recording. Note the superficial peak in the curve for concentric stimulation indicating hyperpolarization in the dendrites evoked by stellate cell activation and the more deeply located maximum for unipolar stimulation, indicating somatic hyperpolarization evoked by the basket cells. D shows schematically the position of recording and stimulating electrodes. SC stands for stellate cells, the other symbols as in Fig. 1 (Eccles, 1965). References p . 266-267

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why this positive wave can be recorded for almost the same distance out of line of the beam of excited parallel fibres as shown in Fig. 4 C. Our initial experiments were mainly concerned with the slow wave, but later work (Eccles et al., 1966b) has revealed that all of the components of the potential waves evoked by parallel fibre stimulation had not been fully appreciated. Part of the earlier negativity at depths of about 0.30 mm appeared to be due to direct stimulation of mossy fibres and subsequent activation of granule cells. This work revealed that there was a difference accordiiig to whether the parallel fibres were stimulated by a coiicentric electrode, activating a rather superficial beam of fibres, or by a unipolar electrode activating the more deeply located fibres also. The difference in depth profiles thus obtained is shown in Fig. 3. The profile with its maximum positivity at a depth of 0.10-0.20 mm, much of which is due to passive sources to superficial active sinks, is attributed to an inhibitory action of stellate cells that are situated at this level and make synapses in the superficial layer on the dendrites of the Purkinje cells.

2. Inhibitory interneurones In view of the long duration of the rising phase of the IPSP it can be postulated that the responsible interneurones will have a long-lasting repetitive discharge, as is the case with basket cells in the hippocampus (Andersen et al., 1964b), and Renshaw cells (Eccles et al., 1954). Occasionally cells have been encountered in our series at the depth of the bodies of the Purkinje cells that behaved differently, as can be seen in Fig. 6. In contrast to the Purkinje cells, these cells fired with an increasing number of spikes when the stimulus strength was increased, the initial frequency being as high as 300/sec. With two stimuli of equal strength the number of spikes in the second burst was even higher than in the first. These findings have now been extended considerably (Eccles et al., 1966a). It turned out that such cells could never be fired antidromically from a juxta-fastigial electrode. Responses with longer latency have been evoked, however, by juxta-fastigial stimulation. This excitation presumably involved the mossy fibre-granule cell pathway or the climbing fibre collaterals that have been ascribed to basket cells in the cerebellar cortex (Scheibel and Scheibel, 1954; Szenthgothai, 1965). So far the initial hypothesis that basket cells are inhibitory had been verified. There appeared, however, one marked difference from the situation in the hippocampus. In the cerebellum this form of inhibition is not recurrent, whereas the hippocampal basket cells are activated by collaterals from the pyramidal cell axons. 3. Golgi cell inhibition of mossy Jibre input

The Golgi cells, or large stellate cells of the granular layer, have their dendrites oriented in the molecular layer in a similar fashion as the Purkinje cells and basket cells. They send a short axon to the granular layer where it branches extensively and forms a thick plexus that makes synaptic contacts in the so-called glomeruli. In these

259


glomeruli are the expanded terminal endings of the mossy fibres, which are numerically the most important afferent fibres to the cerebellar cortex, and here they make axodendritic synapses with the clawlike endings of the granule cell dendrites. Degeneration studies (Hamori, 1964; Szentagothai, 1965) have shown that the Golgi cell axon terminals in the glomerulus also make synaptic contact with the granule cell dendrites, and not with the mossy fibre terminal as had been thought (and which would provide the possibility for presynaptic inhibition). Golgi cells can be activated by parallel fibre stimuIation, and their actions have been studied by micro-electrode recordings

D

C

B

A

-/--I-

E

Antid.

~oc.

-rrrr F ~oc.+~oc.

1Omsec lOmrec

Fig. 6. Extracellular recording of a repetitively discharging cell in the Purkinje cell layer. In A and B the number of discharges, marked by dots, is seen to increase with increasing strength of parallel fibre stimulation. Stimulus strength expressed in volts. In C another cell is seen to follow repetitive stimulation with constant strength at different frequencies. D illustrates how another, comparable cell could not be activated by antidromic stimulation of the Purkinje cell axons. The same cell responded with a burst of discharges to surface stimulation in E, and a second stimulus applied in F increased the duration of the discharge. The monitor pulse at the start of each sweep indicates negativity (Andersen et a].,1964~).

throughout the cerebellar cortex with the emphasis on the effect on granular cell activity (Eccles et al., 1966~). A micro-electrode inserted into the granular layer records a negative field potential on activation of the mossy fibres that presumably consists mainly of the envelope of individual granule cell spikes that have been synaptically generated. This mossy fibre References p . 266-267

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P. E. VOORHOEVE

Fig. 7. Field potentials evoked at a depth of 0.65 mm by transfolial (T.F.) and juxta-fastigial (J.F.) stimulation at 4 different strengths, and their inhibition by local parallel fibre stimulation. In the upper records A-D, downward arrows, nl, indicate the presynaptic spike in mossy fibres, and upward arrows, n2, the postsynaptic response of granule cells. In the lower records of A-D this response is conditioned by local stimulation which suppresses almost completely the postsynaptic response n2. In the upper records, E-H, the same response is evoked by juxta-fastigial stimulation. Note the large presynaptic spike NI, partly composed of antidromic Purkinje axon and climbing fibre spikes, and the huge postsynaptic response Nz.Preceding local stimulation greatly diminishes the postsynaptic response. The initial spike N1 is also partly suppressed, presumably owing t o depression of the antidromic spike in the Purkinje cell axons by parallel fibrebasket cell activity (Eccles et al., 19664.

activation can be achieved in several ways, e.g. by stimulating an adjacent folium through a sort of axon reflex in fibres that branch sufficiently to embrace two folia, by electrical stimulation of a peripheral nerve, or by stimulation through a juxta-fastigial electrode. Fig. 7 shows this negative potential, recorded at a depth of 0.65 mm as evoked by such transfolial stimulation a t different strengths. Preceding Golgi cell activation by parallel fibre stimulation suppressed the granule cell spikes leaving only a trace of a slow negative potential, which most likely represents the mossy fibre-granule cell EPSP. The optimal interval for this suppression was about 20 msec, the whole effect lasting some 100 msec. A similar result was obtained when a Golgi cell-discharge conditioned granule cell activation by juxta-fastigial stimulation. Apparently the synaptic drive was much stronger here, and not all cells could be completely suppressed. The presynaptic spike visible with both types of granule cell activation represented the incoming volley in the mossy fibres, and in E-H (Fig. 7) also represented impulses in passing climbing fibres and antidromically activated Purkinje cell axons. Extracellular-recorded activity in individual granule cells, either occurring spontaneously or evoked by peripheral nerve stimulation, was also very effecLively depressed by parallel fibre stimulation (Fig. 8). Fig. 9 gives another example of Golgi cell inhibition. It represents an intracellular recording from a Purkinje cell that was synaptically activated by transfolial stimula-

26 I


tion via the mossy fibre - granule cell - parallel fibre route. Fig. 9A shows the already familiar EPSP-IPSP sequence. Conditioning by local stimulation (Fig. 9B) very effectively suppressed both the transfolial evoked synaptic responses. When, on the other hand, in Fig. 9C the testing stimulus was applied directly to the parallel

A SR

I

t LOC

SR

tLOC

C I

.o.’\

TJ.F.

I

SR

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Fig. 8. A-D. ExtracelIular recording of inhibition of a spontaneously k i n g granule cell, recorded at 0.6 mm depth, by local, inferior olive and juxta-fastigial stimulation. All these modes of stimulation eventually excite Golgi cells. E illustrates another granule cell that was excited through its mossy fibre input by stimulation of the superficial radial nerve. In F-H this stimulus is preceded by local stimulation of the parallel fibres at different intervals. There is complete suppression of discharge at an interval of about 50 msec; at an interval of 75 msec the first spike reappeared (Eccles et al., 1966~).

fibres one synapse later in the chain, a preceding parallel fibre stimulation did not suppress the test response at this interval. The EPSP was even enhanced, as described in the preceding section. This result indicates that the inhibitory effect was exerted earlier in the pathway, i.e. at the mossy fibre-granule cell synapse in the glomerulus. This conclusion is in agreement with the results obtained by analysis of the field potentials recorded in the granular layer. 4 . Miscellaneous observations

Recent results by Ito and his group (It0 and Yoshida, 1964; Ito et al., 1964a, b) have disclosed that Purkinje cell activation evokes monosynaptically an IPSP in Deiters’ neurones and in cells of the intracerebellar nuclei. Reports have been published so far on 92 cells in which this result has been obtained. I t is hard to evaluate, with the data available at this moment, the full meaning of this highly important finding. After it had been established that all identifiable cells in the cerebellar cortex, with the exReferences p . 266-267

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ception of the granule cells of course, are inhibitory it now appears that the only efferent cell in the cerebellum, the Purkinje cell, is inhibitory too! This is easy to accept for the cerebellar influence on Deiters' neurones, which has been known to be inhibitory in nature for a long time. It is more difficult to understand, however, for the action on the cerebellar nuclei, for which the Purkinje axons are, quantitatively at least, the main afferent line. It would be interesting to know from where these nuclei receive their excitatory input, for that must be very powerful. In a few weeks, at the XXIII International Physiological Congress in Tokyo, there

t t

L

T.F.

Fig. 9. Intracellular recording from a Purkinje cell. A, EPSP and beginning of IPSP evoked by mossy fibre activation via transfolial stimulation (T.F.). Local stimulation in C also evokes an EPSP and IPSP. When, in B, local stimulation precedes transfolial stimulation, the response to the second stimulus is almost completely suppressed. When, in D, local stimulation conditions local stimulation the second response is even enhanced by virtue of the hyperpolarization of the cell membrane. The suppressing influence on the second response in B is thus exerted a t the earlier synapse in the chain, i.e. in the glomerulus. (Eccles et al., 1964).

will be a symposium devoted to recent developments in cerebellar physiology, and we must wait till that information is available. I will here only briefly mention a suggestion by Hamori and Szentigothai (1965) about the possibility of electrical inhibition in the Purkinje cell baskets. Based on Gesell's (1940) original remarks on the strategic localization of inhibitory synapses on the axon hillock and the similarity with the axon cap synapse of the Mauthner cell, it was suggested that part of the basket cell inhibitory action might be by electrical hyperpolarization of the axon-hillock region. It is known that the axon hillock of the Mauthner cell is hyperpolarized by electrical transmission in the axon cap (Furukawa and Furshpan, 1963), but so far no signs have been found that could support this suggestion for the cerebellar cortex. 5. Some morphological aspects of cerebellar inhibition

Electronmicroscopists distinguish, on morphological grounds, axo-dendritic type 1


263

from axo-somatic type 2 synapses (Gray, 1959, 1961; Hamlyn, 1962). The axosomatic synapses on the following cells belong to type 2, and are shown by physiologists to be inhibitory in nature: hippocampal pyramidal cells (Andersen et al. 1964b; Blackstad and Flood, 1963), cerebellar Purkinje cells (Andersen et al., 1964c; Palay, personal communication) and dentate granule cells (Andersen et al., 1966; Blackstad and Dahl, 1962). The hypothesis of type 2 synapses being inhibitory seemed to be refuted when Hiimori and Szentiigothai (1966) described type 2 synapses on the secondary branches of Purkinje cell dendrites. The main input to the stem of the Purkinje cell dendrites are the climbing fibres, and they have a very powerful excitatory action (cf. next section). These type 2 synapses might, however, belong to superficial stellate and basket cells, or to Purkinje cell collaterals that also make contact with the dendritic tree, and in view of the findings outlined above, might well be inhibitory. The whole question remains open and it seems worthwhile to pay attention to it. 6. Some pharmacological aspects of cerebellar inhibition

Until recently strychnine has been considered to be an effective competitor for inhibitory transmitters, but a number of exceptions has now been found (Crawford et al., 1963; Andersen et al., 1963b, 1966). In the older literature one finds that strychnine causes high frequency bursts of individual cells in the cerebellar cortex (cf. Dow and Moruzzi, 1958) and enhances the surface negativity of evoked potentials (Bremer, 1958). As already mentioned, Purpura and Grundfest (1957) found but little effect that could be ascribed to a blocking effect of strychnine on inhibitory synapses. After having established at least one definite inhibitory system in the cerebellar A

.

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Fig. 10. A, Duration of the inhibitory pause evoked by parallel fibre stimulation in a spontaneously discharging Purkinje cell is plotted against the frequency of this discharge. The control values, dots, do not markedly differ from those obtained after intravenous injection of 0.33 mg/kg of strychnine hydrochloride, crosses. (Crawford et al., 1963.) C-E, Focal potentials recorded in the layer of the Purkinje cells evoked by parallel fibre stimulation at different stimulus strengths, expressed as multiples of the threshold value, 1.0 T. C is the control response, and D and E were obtained after intravenous injection of 0.1 mg/kg and 0.2 mg/kg strychnine hydrochloride respectively. Note the increase in excitatory negative potential and unitary discharges, whereas the positive inhibitory potential remains unaltered (Andersen et af., 1963b). References p . 266-267

264


Fig. 11. Diagram of the inhibitory influence that mossy fibre activation can exert on both sides of a simultaneously activated row of Purkinje cells. The cluster of activated granule cells is outlined by a dotted line. The projection of the parallel fibres that originate in this activated spot to Purkinje cells and basket cells (black) is viewed in longitudinal and transverse section through the folium and from above. The shading of the area on either side of the beam of activated parallel fibres, and Purkinje cells, indicates the degree of inhibition. In reality the basket cell axon embraces some 10 Purkinje cells having the densest contact with the 3rd-5th cell. This degree of inhibition is also indicated in the transverse section by different hatching of Purkinje cells (Szenthgothai, 1965).


265

cortex it seemed worthwhile to look at the effect of strychnine on this system (Crawford et al., 1963). Fig. 1OA shows the duration of the pause induced in a spontaneously firing Purkinje cell by parallel fibre stimulation. There was a considerable variation in the length of this pause, but even a highly convulsive dose of 0.3 mg/kg strychnine did not significantly alter it, and the same was found for the rather high firing rates of Purkinje cells induced by close electrophoretic application of DL-homocysteic acid. Neither was there any decrease in the size of the extracellularly-recorded IPSP’s as revealed by the slow potentials in Fig. 1OC-E, nor any change in the inhibitory effect on the second of two potentials evoked by local parallel fibre stimulation. Picrotoxin has also been reported to increase the surface-negativity of evoked potentials (Curtis, 1940), but proved ineffective on the inhibitory pause of firing Purkinje cells. Similarly, pentamethylenetetrazol and dihydro-p-erythroidine did not have any influence on this inhibition. A special feature of the cerebellar cortex is the rigid orientation of the Purkinje cell and stellate and basket cell dendrites in respect to their input through the parallel fibres. We have seen that a narrow beam of activated parallel fibres excites Purkinje cells and basket cells in its way, and that the synaptically activated basket cells exert their action on Purkinje cells on either side of this beam. Szentagothai (1965) has postulated that this arrangement would provide an ideal situation for lateral inhibition if basket cells were inhibitory, and the physiological results have fully confirmed this hypothesis. In the normally functioning organism such an activated beam would originate in a small cluster of granule cells, that in its turn is synaptically excited by impulses in a small bundle of mossy fibres, as could well be caused by some local activation in the periphery. The fields of action of the basket cells thus border that of the primarily activated Purkinje cells, but apparently they evoke inhibition instead of avalanche conduction as was once thought. The same argument holds true for the stellate cells that operate on a more superficial level, but in an essentially similar fashion. A certain mossy fibre input thus exerts a type of feed-forward inhibition on Purkinje cells that are neighbours of those in the core of the excited parallel fibre beam (Fig. 11). The very strategic location of the basket cell inhibitory synapses on the base of the soma and the axon hillock of the Purkinje cell, the site for impulse generation in these cells (Eccles et al., 1966d), should be emphasized. At this site the most effective control on cell discharge is exerted, but we shall see in the next section that even this powerful inhibitory mechanism is not able completely to suppress the very potent excitatory influence from the climbing fibres. The stellate cells, with their inhibitory synapses on the dendrites, are less effective in preventing cell discharge, but they may help to set the general level of excitability, and probably some integrative action already operates way out in the periphery of the Purkinje cell. SUMMARY

Several inhibitory systems in the cerebellar cortex are described : basket cells and stellate cells exert a postsynaptic inhibitory action on Purkinje cells, the former on the References p . 266-267

266


soma and axon hillock, the latter on the dendrites. Golgi or large stellate cells, also activated by the parallel fibres, participate in the mossy fibre-granule cell synapse in the glomerulus and inhibit impulse transmission. Apparently, Purkinje cells themselves are also inhibitory to cells in the roof nuclei and in the nucleus of Deiters. Some morphological and pharmacological aspects of these inhibitory synapses are discussed. ACKNOWLEDGEMENT

The author is deeply indebted to Drs. Eccles, Llinhs and Sasaki for permission to quote extensively from work unpublished a t the time this Summer School was held. REFERENCES ANDERSEN, P., ECCLES, J. C., AND UYNING, Y., (1963a); Identification of inhibitory neurones in the hippocampus. Nature, 199,699-700. ANDERSEN, P., ECCLES,J. C., UYNING, Y., AND VOORHOEVE, P. E., (1963d); Strychnine-resistant central inhibition. Nature, 200, 843-845. ANDERSEN, P., ECCLES, J. C., AND U m c , Y., (1964a); Location of postsynaptic inhibitory synapses on hippocampal pyramids. J. Neurophysiol., 27, 592-607. ANDERSEN, P., ECCLES, J. C., AND UYNING, Y., (1964b); Pathway of postsynaptic inhibition in the hippocampus. J. Neurophysiol., 27, 608-619. ANDERSEN, P., ECCLES, J. C., AND VOORHOEVE, P. E., (1964~);Postsynaptic inhibition of cerebellar Purkinje cells. J. Neurophysiol., 27,1138-1 153. ANDERSEN, P., HOLMQVIST, B., AND VOORHOEVE, P. E., (1966); Entorhinal activation of dentate granule cells. Acta physiol. scand., 66,448-460. ARDUINI, A., AND POMPEIANO, O., (1957); Microelectrode analysis of units of the rostra1 portion of the nucleus fastigii. Arch. ital. Biol.,95, 56-70. BLACKSTAD, TH.W., AND DAHL, H. A., (1962); Quantitative evaluation of structures in contact with neuronal somata. An electron-microscopic study on the fascia dentata of the rat. Aeta morph. need-scand., 4, 3 29-343. BLACKSTAD, T. W., AND FLOOD, P. R., (1963); Ultrastructure of hippocampal axo-somatic synapses. Nature, 198, 542-543. BREMER, F., (1958); Cerebral and cerebellar potentials. Physiol. Rev., 38, 357-388. BROOKHART, J. M., MORUZZI, G., AND SNIDER, R. S., (1950); Spike discharges of single units in the cerebellar cortex. J. Neurophysiol., 13, 465-486. BUSER,P., AND ROUGEUL, A., (1954); La reponse electrique du cervelet du pigeon a la stimulation de la voie optique et son analyse par microelectrodes. J. Physiol. (Paris), 46, 287-291. CAJAL, S. RAMONY, (1911); Histologie du Systgme Nerveux de I'Homme et des Vertibre's. 11. Paris, Maloine, 993 pp. CRAWFORD, J. M., Cmns, D. R., VOORHOEVE, P. E., AND WILSON,V. J., (1963); Strychnine and cortical inhibition. Nature, 200, 845-846. CURTIS, H. J., (1940); Cerebellar action potentials in response to stimulation of cerebral cortex. Proc. SOC.exp. Biol. ( N . Y.),44, 664-668. Dow, R. S., (1949); Action potentials of cerebellar cortex in response to localelectrical stimulation. J . Neurophysiol., 12,245-256. Dow, R. S., AND MORUZZI,G., (1958); ThePhysiology andPathology ofthe Cerebellum, Minneapolis, Univ. Minnesota Press, p. 373. ECCLES, J. C., (1964); The Physiology of Synapses, Berlin, Gottingen, Heidelberg, Springer Verlag, 316 pp. ECCLES, J. C., (1965); Functional meaning of the patterns of synaptic connections in the cerebellum. Perspect. Biol. Med., WII (3), 289-310. ECCLES, J. C., FAIT, P., AND KOKETSU, K., (1954); Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J . Physiol., 216, 524-562. ECCLES,J. C., LLINLS,R., AND SASAKI,K., (1964); Golgi cell inhibition in the cerebellar cortex. Nature, 204, 1265-1266.


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ECCLES, J. C., LLINAS,R., AND SASAKI, K., (1966a); The inhibitory interneurones within the cerebellar cortex. Exp. Brain Res., 1, 1-16. ECCLES,J. C., LLINAS,R., AND SASAKI,K., (1966b); Parallel fibre stimulation and the responses induced thereby in the Purkinje cells of the cerebellum. Exp. Brain Res., 1, 17-39. K., (1966~);The mossy fibre-granule cell relay of the cereECCLES, J. C., LLINAS,R., AND SASAKI, bellum and its inhibitory control by Golgi cells. Exp. Brain Res., 1, 82-101. ECCLES, J. C., LLINAS,R., AND SASAKI, K., (1966d); The action of antidromic impulses on the cerebellar Purkinje cells. J. Physiol., 182, 316345. FURUKAWA, T., AND FURSHPAN, E. J., (1963); Two inhibitory mechanisms in the Mauthner neurons of goldfish. J. Neurophysiol., 26, 140-176. GESELL,R., (1940); A neurophysiological interpretation of the respiratory act. Ergebn. Physiol., 43, 477-639. GLOOR,P., (1963); Identification of inhibitory neurones in the hippocampus. Nature, 199, 699-700. C. G., (1956); Excitatory and inhibitory processes acting upon individual GRANIT,R., AND PHILLIPS, Purkinje cells of the cerebellum in cats. J. Physiol., 133,520-547. R., AND PHILLIPS,C. G., (1957); Effects on Purkinje cells of surface stimulation of the cereGRANIT, bellum. J. Physiol., 135, 73-93. GRAY,E. G., (1959); Axosomatic and axodendritic synapses of the cerebral cortex: an electron microscopy study. J. Anut., 93, 420-433. GRAY,E. G., (1961); Ultra-structure of synapses of the cerebral cortex and of certain specialisations of neuroglial membranes. Electron Microscopy in Anatomy, Ed. Boyd et al. London, Edward Arnold, pp. 54-73. HAMLYN, L. H., (1962); The fine structure of the mossy fibre endings in the hippocampus of the rabbit. J. Anat. (Lond.), 96, 112-120. HAMORI, J., (1964); Identification in the cerebellar isles of Golgi I1 axon endings by aid of experimental degeneration. 3rd European Regional Conference on Electron Microscopy. Prague, Publishing House of the Czechoslovak Academy of Sciences. H ~ O R J., I , AND SZENTAGOTHAI, J., (1965); The Purkinje cell baskets: Ultrastructure of an inhibitory synapse. Acta biol. Acad. Sci. hung., 15, 465-479. J., (1966); Identification under the electron microscope of climbing HAMORI,J., AND SZENTAGOTHAI, fibers and their synaptic contacts. Exp. Brain Res., 1, 65-81. ITO, M., OBATA,K., AND OCHI,R., (1964a); Initiation of IPSP in Deiters’ and fastigeal neurones associated with the activity of cerebellar Purkinje cells. Proc. Japan Acad., 40, 766-768. M., (1964); The cerebellar-evoked monosynaptic inhibition of Deiters’ ITO, M., AND YOSHIDA, neurones. Experientia, 20, 515. ITO,M., YOSHIDA,M., AND OBATA, K., (1964b); Monosynaptic inhibition of the intracerebellar nuclei induced from the cerebellar cortex. Experientiu, 20, 575. H., (1957); Physiological and pharmacological consequences of PURPURA,D., AND GRUNDFEST, different synaptic organizations on cerebral and cerebellar cortex. J . Neurophysiol., 20, 494-522. M. E., AND SCHEIBEL, A. B., (1954); Observations on the intracortical relations of the climSCHEIBEL, bing fibers of the cerebellum. J. comp. Neurol., 101, 733-760. E., (1954); Studies on the laminar structure of the central nervous system SUDA,I., AND TAKAHIRA, by means of microelectrodes. I, II. Cerebellar cortex. (In Japanese.) J. Physiol. SOC. Japan, 16, 303 and 17, 144. SZENTAGOTHAI, S., (1965); The use of degeneration methods in the investigation of short neuronal connexions. Progress in Brain Research, Vol. 14, Degeneration Patterns in the Nervous System. M. Singer and J. P. Schade, Editors. Amsterdam, Elsevier, pp. 1-30.

268

Climbing Fibre Responses in Cerebellar Cortex P. E. VOORHOEVE Department of Neurophysiology, Jan Swammerdam Institute, University of Amsterdam, Amsterdam (The Netherlands)

INTRODUCTION

It has been known since their discovery by Ram6n y Cajal that climbing fibres in the cerebellum make an extensive synaptic contact with the primary and secondary dendrites of the Purkinje cells in a 1 : 1 ratio, i.e. each fibre being related to one cell only. The origin of the climbing fibres had been long in doubt until a very careful degeneration study by Szenthgothai and Rajkovits (1959) disclosed that the great majority of climbing fibres originated in the inferior olivary complex. This anatomical arrangement provides a possibility for selective stimulation of climbing fibres by inserting a stimulating electrode in the inferior olive, and thus to study the physiological properties of this synapse. That climbing fibres make synaptic contact with other cells too (Hhmori and Szenthgothai, 1966; Scheibel and Scheibel, 1954) does not invalidate this argument, because the synapses on the Purkinje cells are the most abundant, and Purkinje cells can be identified electrophysiologically. As the recordings were made in the anterior lobe of the vermis, the stimulating electrode had to be placed in the accessory olive. An electrode inserted in close proximity to the fastigial nucleus will, as has been described in the preceding section, stimulate Purkinje axons antidromically simultaneously with mossy and climbing fibres. This admixture can to a large extent obscure the antidromically evoked potentials (Eccles et al., 1966b). For more precise location of the juxta-fastigial stimulus the electrode actually consisted of an array of three independent concentric electrodes cemented together. R E S U LTS

1. Potentials$elds

Fig. 1 gives a schematic representation of the set-up as used in these experiments. When an electrode was inserted in the usual way perpendicular to the surface of a folium it recorded a typical set of potentials at different depths on stimulation of the inferior olive as shown in Fig. 2A. This potential profile is dominated by an initial negative wave having its maximum amplitude around a depth of 0.15 mm. This negativity has a rather steep rise and a more gradual decline to a positive wave that increases in size at deeper levels depending on the strength of stimulation. Small spike-like potentials were often detectable on the falling phase of this potential. It can

269

CEREBELLAR C O R T E X R E S P O N S E S

-0 IJ * 100

- 200 ML

600

Fig. 1. Schematic representation of the cerebellar cortex, and the experimental arrangement. The outline of one Purkinje cell is drawn, and the extent of the molecular (ML), Purkinje (PL) and granular layer (GL) are indicated on the right-hand scale. The local stimulating electrode (L.S.). the surface recording electrode (S.R.)and the microelectrode are shown on the surface of the folium. The other stimulating electrodes are placed in the inferior olive (1.0) and close to the fastigial nucleus (J.F.); the latter consists of an array of three concentric electrodes. The other cellular elements are granule cells (GC), mossy fibres (MF) and Purkinje cells (P) with their axons (PA). The olivo-cerebellar pathway (OCP) enters through the inferior peduncle (Eccles et al.,1966a).

be seen that the focal potentials obtained are essentially similar at the superficial level, but the deeper positivity is more dominant with the stronger stimuli. This positivity is probably produced, in part, by the stimulation of other structures, such as reticulocerebellar fibres, that surround the olivary complex. References p. 280-281

270


A depth

kJ

30

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1.0.

c n

80

B

~0~41.0.

c

LOC+l.O

CON

t

t

130

180

230

280

330

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430

530

Fig. 2. Field potentials evoked in the cerebellar cortex by climbing fibre activation through stimulation of the inferior olive. In A this response is shown at different depths, as indicated on the scale. In B this test response is preceded at an interval of 19 msec by a local stimulus to the parallel fibres, and falls in the declining phase of the hyperpolarization synaptically evoked thereby. Note the increase in amplitude of the negative wave. In C this potential is recorded at a fixed depth of 0.15 mm at different intervals after the local stimulus. CON gives the control response, the arrows indicating the stimulus artifact of the 1.0.stimulus (Eccles et al., (1966~).

These large extracellular negative potentials are in accord with the postulated strong excitatory action exerted by the climbing fibres on the Purkinje cells. The later and deeper positivity represents the phase when the somata and axons of the Purkinje cells act as passive sources for the active sinks on the main dendrites. In view of the presence of powerful inhibitory mechanisms in the cerebellar cortex, as outlined in the preceding section, it seemed interesting to test the strong excitatory climbing fibre drive on this background of inhibition. Fig. 2B and C illustrate the focal potentials, evoked by inferior olivary stimulation, during the period of basket cell and stellate cell inhibition. Fig. 2B shows the effect at a fixed interval of parallel fibre and inferior

271

CEREBELLAR CORTEX RESPONSES

olive stimulation at different depths, and Fig. 2C at a constant depth of 0.15 mm with variable stimulus intervals. Although the period of inhibition has a duration of some 100 msec the climbing-fibre-evoked negativity never diminished and usually increased beyond a stimulus interval of about 10 msec!

-10mil A

D

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I .o.

+

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1 \1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I Fig. 3. Inferior olive activation of Purkinje cells. A, B; extracellular recording at two different stimulus strengths. Note the difference in latency and the all-or-none character of the response with the weaker stimulus in B. The same cell responds to juxta-fastigial stimulation in C with an initial antidromic invasion (up-going arrow), an early response (down-going arrow) due to direct activation of the climbing fibre and a late response (down-going arrow) that is ascribed to reflex activation of the inferior olive. D and E show the intracellularly-recorded, all-or-nothing, response in D on inferior olive stimulation, and the simultaneously recorded surface potential in E. The all-or-none character of the response is also illustrated by another Purkinje cell in F and G on the extracellular recording (Eccles et al., 1966a). References p. 280-281

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2. Responses of individual Purkinje cells

When the exploring microelectrode is fairly close to a Purkinje cell it can record its activity almost in isolation from activity in neighbouring cells. As can be seen in Fig. 3, especially in 3B and F, a single stimulus to the inferior olive evoked a compound response of large positive-negative ‘giant’ spikes in this cell in an all-or-none fashion. Either the cell responded with a series of discharges of decreasing amplitude, or it did not respond at all, and only a low background potential remained. This slow wave is the focal potential generated by neighbouring Purkinje cells; it is negative when the recording electrode is rather superficial,and positive at greater depths. The decrease in amplitude for the later spikes in the burst can be attributed to the remaining depolarization of the cell and to the relative refractoriness at this high discharge rate. Juxtafastigial stimulation gave an initial antidromic response, then a secondary complex discharge evoked by direct excitation of the passing climbing fibres, and a late complex response which is presumably due to reflex activation of the inferior olive and hence to a second discharge in the climbing fibres, as will be explained later. When a conditioning local stimulus to the parallel fibres preceded the climbing fibre excitation, the Purkinje cell response was, as expected, depressed. This depression involved, however, the later components of the compound response exclusively. The initial spike response remained the same at all intervals. In Fig. 4 the subsequent grouped discharge was suppressed in number and amplitude at intervals from 4.3 msec to 33 msec. At shorter intervals (Fig. 4C) a facilitatory inLOC+I.O.*

Fig. 4. Extracellular recording of the Purkinje cell response to inferior olive stimulation at different intervals after a local conditioning stimulus. A and B are the control responses simultaneously recorded with the surface potential in A. C shows the enhancement of the compound response at the shortest interval. In D-H there is a depression of the later components of this response, but the initial spike response is unaffected at all intervals. Note the different time scales for A, B F and G-H (Eccles et al., 1966~).

fluence could be detected that increased the amplitude ofthe later spikes especially. The conditioning stimulus in itself also evoked, through parallel fibre excitation, a complex response consisting of a large initial spike with a subsequent variable complex of smaller discharges.

213


A similar result was obtained when local stimulation preceded the direct climbing fibre response evoked by juxta-fastigial stimulation. It was noteworthy, however, that this way of conditioning was strong enough to inhibit the concomitant antidromic invasion of the Purkinje cell over a wide range of intervals but that the initial climbingfibre-evoked spike came through undiminished. This illustrates the extremely powerful excitatory synaptic action exerted by the climbing fibres on the Purkinje cell. The increased spike amplitude a t short intervals is explained by the remaining hyperpolarization in the later phase of the IPSP that partly restores the membrane potential. Further analysis by intracellular recording from Purkinje cells revealed a similar picture, as can be seen in Fig. 5. Inferior olive stimulation evoked an initial spike

A

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Fig. 5. Intracellular records from two different Purkinje cells activated by inferior olivary and juxtafastigial stimulation. A-C is one cell. In B the juxta-fastigial stimulus evoked an initial antidromic spike; in C the stimulus strength was diminished and subthreshold for this particular Purkmje axon. Note the difference in latency for 1.0. and J. F. stimulation. D shows the compound response in another cell on 1.0.stimulation, and E the response in the same cell at faster sweep on J.F. stimulation. The second spike in record E is evoked by mossy fibre activation through the granule cell to parallel fibre pathway (Eccles et at., 1966a).

discharge followed by a prolonged depolarization on which small spike-like discharges were superimposed. Juxta-fastigial stimulation evoked an early antidromic discharge in this cell, identifying it as a Purkinje cell, with a subsequent complex discharge as in Fig. 5A by direct climbing fibre excitation. When Purkinje cells were sufficiently depolarized by the impalement with a microelectrode the spike generating mechanism became more and more depressed, and inferior olive stimulation evoked a complex depolarizing potential, an EPSP. It is generally assumed that excitatory synapses act by temporarily creating a high conductance in the subsynaptic membrane for all available species of ions which then References p. 280-281

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P. E. VOORHOEVE

move along their respective concentration gradients. It is therefore possible to alter the size and the polarity of the EPSP by hyperpolarizing or depolarizing the membrane potential. Complete reversals of the EPSP have been obtained in relatively few types of cell because it is difficultto depolarize the cell sufficiently. Fig. 6 illustrates the effect of hyperpolarizing and depolarizing currents on two Purkinje cells. With strong depolarizing currents the climbing fibre EPSP is reversed. With strong hyperpolarizing currents the membrane potential is sufficiently restored in these deteriorated cells to reactivate the spike generating mechanism (Fig. 6E). The remarkable fact about this A

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Fig. 6. The effect of hyperpolarizing and depolarizing currents on the intracellulary recorded EPSP in a Purkinje cell. A and B illustrate the EPSP evoked in this cell by climbing fibre activation through a stimulus in the inferior olive, together with the simultaneously recorded surface potential in B. In C it is seen that the amplitude of this EPSP increased during the passing of a hyperpolarizing current, while it decreased during depolarization and reversed with stronger depolarizing currents. The control response is shown between the arrows. D illustrates a similar effect in another cell that was activated by juxta-fastigial stimulation. Note that the later part of the compound response, as marked by arrows, behaved in the same way as the directly-evoked EPSP. E represents the same cell as D, but the recordings were made a t lower gain and slower sweep speed. A current pulse of 3.6 x A is given for comparison. Note that with the larger hyperpolarizing currents the spike generating mechanism is partly restored (Eccles et al., 1966a).

climbing fibre EPSP is that it appeared in an all-or-nothing fashion, strongly suggesting that it is evoked by an impulse in only one fibre, which agrees well with the histologicapicture. Only in two out of more than a hundred cells was there any evidence of c o d

275


vergence of two fibres to the same cell. The ripple on the declining phase of this EPSP, however, could indicate delayed synaptic bombardment or prolonged residual transmitter action, as occurs for example with Renshaw cell activity. These complexities disappeared as the cell deteriorated and the EPSP became almost smooth, which indicates that they did not arise from delayed synaptic actions but from local responses that failed to initiate a full-sized spike, as illustrated in Fig. 7.

1.0.

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Fig. 7. Intracellular records from a Purkinje cell of potentials evoked by inferior olive stimulation. The sequenceillustratesthe different responseswhich a single climbingfibre evokedin a slowly deteriorating cell. In A the synaptic activation produced a series of small synchronized responses on the declining phase of the EPSP. In B-E these local responses gradually disappear and in F there is an almost smooth decay. Note the all-or-none character of the response in C and F (Eccles ef al., 1966a).

Delayed synaptic bombardments, however, do occur on stimulation of the inferior olive, as is indicated by several lines of evidence. In the first place stimulation of the inferior olive with increasing stimulus strength, evoked a sequence of all-or-nothing EPSP responses in a deteriorated Purkinje cell a t about 2 msec intervals. In these circumstances the latency of the first response can be shortened stepwise with increasing stimulus strength. It seems as if the weaker stimuli only excite presynaptic pathways to the cell of origin of the climbing fibre to the Purkinje cell under observation, and the olivarycell itself is onlyexcited directly with the stronger stimuli. Thestep-likevariations in latency can be explained either by several relays in this presynaptic pathway or by postulating excitatory recurrent axon collaterals from inferior olive cells to other cells in the nucleus. This hypothesis is supported by the results obtained with juxtafastigial stimulation. As we have seen in Fig. 3C this evokes, in Purkinje cells in good condition, after the antidromic invasion, a complex discharge consisting of two groups separated by an interval of some 6 msec apparently evoked by the impulse in a single climbing fibre. When deterioration of the Purkinje cell by the impaling microelectrode impairs the spike generating mechanism, the antidromic invasion disappears, and the first complex turns into a smooth EPSP. Thus, when the local responses are References p . 280-281

P. E. VOORHOEVE

276

eliminated, this direct climbing fibre response corresponds to a single impulse in the fibre. The second, delayed response, however, retains its repetitive character (Fig. 8H).

J.F.

Fig. 8. Extracellular and intracellular recording of potentials evoked in a Purkinje cell by reflex activation of a climbing fibre by juxta-fastigial stimulation. A and B show the initial antidromic invasion, then the direct climbing fibre response marked by an arrow and in B the second response indicated by the upward arrow. In C and D, after impalement of the cell, there is the same sequence of events. E-G illustrate these responses at slower sweep speed. In F a spontaneous spike prevented antidromic invasion in one of the two superimposed sweeps. H and I were taken after deterioration of the cell when the spike generating mechanism was depressed. It is clearly seen that the direct response now has a smooth decline whereas the reflexly evoked one retains its repetitive character. J illustrates the spontaneous climbing fibre response that was occasionally observed in some cells (F!ccIes et al., 1966a).

It is therefore concluded that this delayed response is evoked by a repetitive discharge from reflexly activated inferior olive cells. It is not yet clear whether this repetitive discharge is due to activation of excitatory recurrent collaterals from the inferior olive

277


cells themselves, or to excitation from mossy fibre collaterals to the inferior olivary complex. It is known that many incoming fibres, which might well be mossy fibres, give collaterals to the inferior olive. Additional evidence for the reflex excitation of climbing fibres by juxta-fastigial stimulation is obtained from observations with combined juxta-fastigial and inferior olive stimulation. When the juxta-fastigial stimulus followed that to the inferior olive by a few milliseconds it often failed to evoke a direct response in the Purkinje cell, presumably owing to refractoriness of the climbing fibre, but it caused a delayed EPSP with a latency of some 7 msec. This means that the juxta-fastigial stimulus can evoke a reflex discharge in the inferior olive independently of whether or not it directly excited the climbing fibre that made synaptic contact with the Purkinje cell under observation. Latency differences for these two modes of stimulation of the same cell disclosed a conduction velocity of some 5-20 msec for the climbing fibres. Interaction experiments with local inhibitory stimulation on to the climbing fibre response conform with the general picture that has been developed so far. At short intervals between the conditioning and testing stimulus there was a diminution of the excitatory response, whereas slightly later in the declining phase of the conditioning IPSP the climbing fibre EPSP increased in amplitude. This is illustrated in Fig. 9 for the response evoked by direct stimulation of the climbing fibre. These results conform with, and explain the changes in, focal potentials when the climbing fibre response is displayed on a background of basket cell and stellate cell inhibition. LOC+J.F.

LOC

-

1.F

Fig. 9. Intracellular records of a Purkhje cell showing the time course of the interaction between an IPSP evoked by parallel fibre stimulation (LOC) and the climbing fibre EPSP evoked by juxta-fastigial stirnulation. A shows the control EPSP. B-F and G-L give two series from the same cell at different sweep speeds when this EPSP is superimposed after various intervals on the IPSP. The upper drawing in M represents the time course of the synaptic potentials in this cell by the local stimulation as such. In the lower graph the amplitude of the EPSP in mV is plotted against the stimulus interval in msec. Note the correspondence between the depression of the EPSP and the rising phase of the IPSP and the enhancement of the EPSP in the declining phase of the IPSP (Eccles ef a/.,1966~). References p . 280-281

278

P. E. VOORHOEVE

It is now generally accepted that the hyperpolarization of postsynaptic inhibition is caused by ionic currents that flow through subsynaptic areas of high conductance for K+ and C1- ions. Inhibition is thus effected by the shunting effect of these patches of high conductance on the opposite ionic currents generated in the excitatory synapses. This mechanism holds true during the rising phase of the IPSP. On the other hand, hyperpolarization per se increases the amplitude of the EPSP, as occurs especially during the decline of the IPSP when the shunt is closed and the membrane potential returns to its ‘resting’level with a time course determined by the electrical properties of the membrane. As we have seen, extracellularly as well as intracellularly, the climbing fibre response in Purkinje cells is diminished during the early phase of the postsynaptic inhibition evoked by a parallel fibre volley, and increased in the later period of the inhibition. The increased negative field potentials are in part due to these increased EPSP’s and partly to depression of the later spike complex. The superficial dendritic regions act as passive sources to sinks produced by impulses that invade only the basal part of the dendritic tree, and this superficial positivity is, in recording, subtracted from the superficial negative focal potential due to climbing fibre EPSP. When these later spikes are suppressed the superficial dendritic region does not act as a source and does not obscure the negative wave.

3. Repetitive stimulation of climbing fibres In Purkinje cells having a low membrane potential a climbing fibre impulse evokes an EPSP uncomplicated by spike potentials, hence these cells are suitable for the study of the effect of a second impulse along the climbing fibre. The response to a second impulse was found to be depressed for several hundred milliseconds after the first. If, in these circumstances, the number of impulses was increased, a progressive depression was seen for the first 6-8 impulses in the train, with a steady level of about 30-40 % of the amplitude of the initial response. On cessation of the tetanus there was usually no sign of a remaining depolarization that is characteristic for residual transmitter action. A remaining depolarization, on the other hand, is obvious in the series illustrated in Fig. 1OK-0, evoked by juxtafastigial stimulation. The first stimulus in the series directly evoked a climbing fibre response, followed by a reflex discharge from the inferior olive; subsequent stimuli only evoked the direct response. Extracellular recording from uninjured cells showed that repetitive activation of the climbing fibre synapses had a remarkable ability to discharge Purkinje cells, in spite of the depression of the EPSP. This is well illustrated in Fig. 11. With juxta-fastigial stimulation at the high repetition rates of 108/secthe antidromic invasion soon failed whereas the complex climbing fibre response remained unaltered (Fig. 11E). Still higher stimulation frequencies (180/sec in Fig. 10F) failed to fire the cell under observation, but on cessation of the tetanus there was a prolonged after-discharge at an initially high frequency of 350/sec. Comparable results obtained by Granit and Phillips (1956) on juxta-fastigial stimu-

279

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1.0.

1.0.

J.F.

--lOmsec

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Fig. 10. Intracellular records of EPSP’s evoked in Purkinje cells by repetitive inferior olive (1.0.) and juxta-fastigial (J.F.) stimulation. A-E and F-J represent two different cells; K - 0 is the same cell as in F-J .The stimulation frequency in pulses per second is indicated at the left side of each record. Note that the complex response in F-J and K-O occurs only on the first stimulus of the train (Eccles et al., 1966~).

lation can now, in the light of these results, be attributed to climbingfibrestimulation. The initial depression of the EPSP a t repetitive stimulation is attributed to depletion of the transmitter. In this respect, as in reaching a steady level after a few impulses, the climbing fibre synapse conforms with the known properties of other synapses (Eccles, 1964). The transmittability in this synapse, however, is so powerful that even with the considerable depression of the EPSP a t frequencies up to 100/sec, each impulse continues to evoke a multiple discharge. The failure of the Purkinje cell to fire a t even higher stimulation rates is due to the intense depolarization produced by this bombardment. This is evidently the inactivation response of Granit and Phillips because the spike generating mechanism is suppressed. In our initial experiments we had suggested that recurrent facilitation from Purkinje axon collaterals explained the high frequency discharges on juxta-fastigial stimulation. We soon found, however, that these responses were never obtained on pure antidromic activation of the Purkinje cell, but only occurred when truly afferent fibres were stimulated too. SUMMARY

By stimulating the inferior olive, it is shown that climbing fibres have an extremely powerful excitatory synaptic action on Purkinje cells. Some of the basic properties of References p. 280-281

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9

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J .F.

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a

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Fig. 11. Intracellular and extracellular recorded spike activity evoked in Purkinje cells by tetanic juxta-fastigial stimulation. A shows at fast sweep speed the antidromic spike (ax followed by the directly evoked climbing fibre response (cf.) In B-D the stimulation frequency is augmented to 33/sec, 5O/sec and 70/sec respectively, and each time the response consists of the compound a and cf spikes. In E, at a stimulation frequency of 108/sec, the antidromic invasion fails after the first stimulus but the cf response remains unaltered. In F,at 18O/sec there is a failure of the large spike potentials after 6 stimuli and a continuation of irregular small spikes. Shortly after cessation of the tetanus there is an after-discharge of large spikes commencing at the high frequency of 350/sec and declining to 180/sec before failing. G-H show similar responses in another Purkinje cell. I is the intracellular response in still another cell. It illustrates the prolonged depolarization during the tetanus and the spike inactivation (Eccles et al., 1966~).

this synapse are displayed on a background of inhibition or repetitive synaptic excitation. The notion that each climbing fibre activates only one Purkinje cell is confirmed by the physiological evidence. ACKNOWLEDGEMENT

The author wishes to express his indebtedness to Drs. Eccles, Llinhs and Sasaki for putting at his disposal many data that were otherwise not available at the time when this paper was presented. REFERENCES ECCLES,J. C., (1964); The Physiology of Synapses. Berlin, Gottingen, Hejdelberg, Springer Verlag, Ch. VI.

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28 1

ECCLES, J. C., LLINAS,R., AND SASAKI,K., (1966s); The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum. J. Physiol., 182, 268-296. ECCLES, J. C., LLINAS,R., AND SASAKI, K., (1966b); The action of antidromic impulses on the cerebellar Purkinje cells. J.Physiol., 182,316-345. ECCLES, J. C., LLINAS,R., SASAKI,K., AND VODRHOEVE, P. E., (1966~);Interaction experiments on the responses evoked in Purkinje cells by climbing fibres. J. Physiol., 182, 297-315. GRANT,R., AND PHILLIPS, C. G., (1956); Excitatory and inhibitory processes acting upon individual Purkinje cells of the cerebellum in cats. J. Physiol., 133, 520-547. HAMORI,J., AND SZENT~GOTHAI, J., (1966); Identification under the electron microscope of climbing fibres and their synaptic contacts. Exp. Brain Res., 1, 65-81. SCHEIBEL, M. E., AND SCHEIBEL, A. B., (1954); Observations on the intracortical relation of the climbing fibers of the cerebellum. J. cornp. Neurol., 101, 733-760. SZENTAGOTHAI, J., AND RAJKOVITS, K., (1959); Uber den Ursprung der Kletterfasern des Kleinhirns. Z . Anat. EntwickL-Gesch., 121, 130-141.

282

Functional Organization of the Cerebellar Projections to the Spinal Cord 0.POMPEIANO Institute of Physiology, University of Pisa, Pisa (Italy)

INTRODUCTION

In the last few years, a considerable amount of information has been published on the anatomical and functional organization of the efferent projections from the cerebellum to the spinal cord. The present report represents an attempt to integrate the most recent findings on this subject. Attention will be devoted to the problem of cerebellar localization, particularly to the selective control that the different corticonuclear regions of the paleo-cerebellum exert on the spinal motoneurones and to the somatotopic organization of these cerebellofugal influences on the spinal cord.

Fig. 1 . Diagram illustrating the pattern of somatotopical localization in the cat’s cerebellum as determined by observation of overt movements produced by stimulation of the cerebellar cortex in the decerebrate animal (Hampson et al., 1952).

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The pattern of localization of the postural responses to stimulation of the anterior and posterior lobe of the cerebellum has been clearly demonstrated by Hampson et al. (1945, 1946, 1952), who found that the forelimbs were represented in the culmen, the hindlimbs in the lobulus centralis, and the tail in the lingula. Just the opposite pattern of localization was found within the posterior lobe of the cerebellum, particularly within the paramedian lobule, because the upper folia influenced the facial musculature, the middle folia influenced the arm, while the lowermost folia were concerned with leg and tail musculature (Fig. 1). It will be shown here that the localization of these responses is due to somatotopical organization of the efferent cerebellar projections to the lateral vestibular (Deiters’) nucleus and to the red nucleus, and that these cerebellar systems control respectively the activity of the extensor and flexor motoneurones. S O M A T O T O P I C O R G A N I Z A T I O N O F T H E CEREBELLAR P R O J E C T I O N S T O

THE

SPINAL CORD

Jansen and Brodal(1940, 1942) have shown that in the rabbit, cat and monkey there is a well-localized projection from the cerebellar cortex to the cerebellar nuclei. In particular the vermis projects to the fastigial nucleus, the medial part of the hemisphere or pars intermedia to the interposite nucleus, while the lateral part of the hemisphere projects to the dentate nucleus. Apparently the vermis is independent of the rest of the cerebellar cortex since no associative fibres connect this medial area with the most lateral cortical regions (Eager, 1963b, 1965). This subdivision of the cerebellum into longitudinal zones is also associated with a localization of the corticonuclear projection in a rostro-caudal direction. The latest anatomical studies (Vachananda, 1959; Eager, 1963a; Goodman et ~ l .1963 , ;Walberg and Jansen, 1964; Voogd, 1964) have basically confirmed this pattern although some variants of this picture have been underlined. In the discussion of the efferent cerebellar projections to the spinal cord only those intermediate stations of the brain stem will be considered which receive afferent projections from the vermal and the paravermal (or intermediate) parts of the cerebellum and send efferent projections to the spinal cord in a somatotopically organized way. It is known that the brain stem reticular formation represents an intermediate station (cf. Jansen and Brodal, 1954, 1958) which has been considered as responsible for some important effects exerted by the cerebellum on the spinal cord (cf. Dow and Moruzzi, 1958). Reticulospinal fibres originate from the pons and the medulla which reach not only the cervical and thoracic (cf. Torvik and Brodal, 1957; Brodal, 1957), but also the lumbosacral segments of the spinal cord (Verhaart, 1953; Kuru et al., 1959, 1960; Staal, 1961; Petras, 1962; Magni and Willis, 1963; Wolstencroft, 1964; Nyberg-Hansen, 1965) where they terminate in layers VII and VIII of Rexed but not in layer IX where the motoneurones are located (cf. Nyberg-Hansen, 1965). The localization of the responses induced by stimulation or ablation of the cerebellum (cf. Dow and Moruzzi, 1958) would seem not to be mediated by the reticular formation, since it has been shown that both the fastigio-reticular (Walberg et al., References p. 316-321

284

0.POMPEIANO

1962b) and the reticulospinal projections (Torvik and Brodal, 1957; Brodal, 1957)are diffusely organized. Attention has therefore been directed to the lateral vestibular (Deiters') nucleus and to the red nucleus; these also receive numerous efferent fibres from the paleocerebellum and project even to the caudalmost segments of the spinal cord. Cerebellar projections to the lateral vestibular (Deiters') nucleus

Observations performed on cats using the method of retrograde degeneration, after lesions at different levels of the spinal cord, have shown that the vestibulospinal projection originating from Deitei s' nucleus is clearly organized somatotopically (Pompeiano and Brodal, 1957a). Specifically the rostro-ventral part of the nucleus sends fibres to the cervical segments of the spinal cord; the dorso-caudal region projects to the lumbosacral segments; while the thoracic segments receive fibres from the

91

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Fig. 10. Monosynaptic excitatory connections from the ipsilateral Deiters’ nucleus to extensor motoneurones. A-D are from a tibia1 (Tib), E-H from a posterior biceps-semitendinosus (PBSt), motoneurone. Upper traces in each record are intracellular recordings with microelectrode. For voltage calibration see inset. Lower traces are records from L7 dorsal root entry zone. A and E show the maximal monosynaptic Ia EPSPs, B and F antidromic spikes obtained by slightly higher stimulus intensities applied to the homonymous nerves. C and G show effects of stimulation of Deiters’ nucleus with low, and D and H with high stimulus intensities. See text for explanation of the drawings &und and Pompeiano, 1965).

(1954, 1956c, 1957a) found that high-frequency stimulation of the ipsilateral vermis of the anterior lobe yielded a clear-cut increase in the extensor rigidity of the ipsilateral limbs with the same parameters of stimulation (300/sec, 1 msec, threshold voltage) which usually produced the typical inhibition of the decerebrate rigidity. This is interpreted as being due t o interruption of most or all of the inhibitory pathways. Under these experimental conditions a facilitatory effect, otherwise concealed by an overwhelming inhibition, is brought to light. This extensor response, on the other hand, is abolished by destruction of the rostro-medial part of the fastigial nucleus. Ablation experiments (Batini and Pompeiano, 1956, 1958) indicate that both the inhibitory and the excitatory influences on the extensor motoneurones, exerted by or via the rostro-

CEREBELLAR PR O J E C T IO N S TO S P I N A L C O R D

A

Slack

4 mm

295

10 mm

Fig. 11. Responses of afferent from muscle spindle in the soleus muscle to stimulation of Deiters’ nucleus in the anaesthetized cat (chloralose-nembutal). A = resting (above) and driven (below) responses (30 superimposed sweeps each) at three different degrees of muscle extension: slack, 4 and 10 mm, respectively. Stimulus strength 5 V, duration 2.1 msec. Time 10 msec. B = single sweep record of driven response with myogram (calibration 300 g) at slower speed. Muscle at 4 mm, stimulus as in A, time 100 c/sec. C = site of stimulation, marked 2, in the hindlimb region of the ipsilateral Deiters’ nucleus. D = from left to right, responses obtained by stimulating at sites marked 1, 2 and 3, respectively, in C; muscle at 4 mm, stimulus 3.2 V, 2.1 msec, time as in A (Granit et nl., 1959).

lateral (Figs. 13A, B) and the rostro-medial (Figs. 13C, D) parts of the fastigial nucleus, respectively, are tonic in nature*. -

*

In order to avoid the interruption of the crossed fastigio-bulbar fibres, the rostra1 lesions were made after bilateral and symmetrical destruction of the caudal parts of the fastigial nuclei, from which crossed fibres originate. The decrease in the decerebrate rigidity contralateral to the side of the fastigial lesion in Fig. 13B is due to crossed inhibitory influences originating from the overstretched proprioceptors of the ipsilateral limbs made spastic by the lesion. On the other hand the increase in the decerebrate rigidity contralateral to the side of the fastigial lesion in Fig. 13D is due to release of the decerebrate rigidity from crossed inhibitory influences, which are greatly reduced when the ipsilateral limbs are made atonic by the lesion. This mechanism of crossed reflex inhibition which exaggerates the postural asymmetry following a fastigial lesion (cf. Moruzzi and Pompeiano, 1955, 1957b) is illustrated in Figs. 18C and D. References p . 316-321

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Moruzzi and Pompeiano (1957a) put forward the hypothesis that the abolition of the vermal inhibition of the anterior lobe following electrolytic lesion of the rostrolateral part of the fastigial nucleus is due to destruction of fastigial neurones or to interruption of long cerebellar corticofugal fibres passing through the rostro-lateral part of the fastigial nucleus. Recent anatomical studies (Walberg and Jansen, 1961 ;cf. also Eager, 1963b) have shown that long corticofugal fibres to the vestibular nuclei from the cortex of the vermis of the anterior lobe pass through the rostro-lateral part of the fastigial nucleus (Fig. 12 B).

Fig. 12. A = microphotograph showing a dorso-lateral lesion of the rostra1 part of one fastigial nucleus yielding reversal of the inhibitory response of the vermal part of the anterior lobe. The lesion occupies the rostro-lateral and dorsal, parvicellular part of the right fastigial nucleus, while the rostro-medial, magnocellular part is intact. Stimulation of the right herniverrnis of the anterior lobe increased ipsilateral extensor rigidity, while the inhibitory response was obtained from the left hemivermis (Moruzzi and Pompeiano, 1957a). B = diagrammatic representation of the cerebellar cortico-vestibular fibres mainly directed to Deiters’ nucleus, on their way through the cerebellum (Walberg and Jansen, 1961). These corticofugal fibres mainly penetrate the rostro-lateral part of the fastigial nucleus, i.e. that region whose destruction abolishes the inhibitory effects induced by vermal stimulation of the anterior lobe.

It has been suggested, therefore (Brodal et al., 1962),that thelong cerebellar corticovestibular fibres are responsible for the vermal inhibition of the decerebrate rigidity. It is of interest that numerous units recorded from Deiters’ nucleus can be inhibited by stimulation of the vermal cortex of the anterior lobe (De Vito e l al., 1956; Pompeiano and Cotti, 1959a-d) (Fig. 14). According to Brodal et al. (1962) ‘the pathway for the inhibitory action on these units is related to the rostro-lateral part of the fastigial nucleus through which the long cerebellar cortico-vestibular fibres pass, making it likely that the stimulation of these fibres is involved in the effect’ (pp. 145-1 55). Recent studies confirm this hypothesis. With intracellular microelectrodes, Ito and Yoshida (1964), Ito and Kawai (1964) and Ito, Obata and Ochi (1964), recorded the activity of neurones in Deiters’ nucleus identified by antidromic stimulation of the vestibulospinal tract, and found that stimulation of the vermis of the anterior lobe gives monosynaptic inhibitory postsynaptic potentials (IPSPs) on the Deiters’ cells.

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The collapse of the extensor rigidity induced by high-frequency stimulation of the vermis of the anterior lobe is due to inhibition of the tonogenic centres (including Deiters’ nucleus) which are responsible for decerebrate rigidity. Recent studies using intracellular recording seem to support this hypothesis. It has been observed that stimulation of the vermal cortex of the cerebellum produces hyperpolarization of the membrane of the motoneurones (Terzuolo, 1959; Llinas, 1964), and that this increase in membrane potential is not reversed by passing a hyperpolarizing current through the membrane (Fig. 15), nor is it associated with the conductance changes typical of the IPSPs. The conclusion from these observations was that the cerebellar inhibition is due to suppression of tonic excitatory influences acting upon the motoneurones. It is of interest that Deiters’ nucleus exerts a monosynaptic excitatory influence on the extensor motoneurones (Lund and Pompeiano, 1965). One may conclude, therefore, that the inhibition of the decerebrate rigidity induced by cerebellar stimulation is due to an abrupt suppression of the vestibulospinal excitatory input.

Fig. 13. Effects of lesions of the rostro-lateral and rostro-medial parts of the fastigial nucleus in the decerebrate cat after chronic bilateral destruction of the caudal part of the same nucleus. A = symmetrical distribution of extensor rigidity after chronic electrolytic destruction of the caudal halves of both fastigial nuclei followed, 16 days later, by intercollicular decerebration. B = ipsilateral increase and contralateral decrease of extensor rigidity after electrolytic lesion of the rostro-lateral part of the left fastigial nucleus made in the same decerebrate preparation. C = new experiment. Symmetrical distribution of extensor rigidity after chronic electrolytic destruction of the caudal halves of both fastigial nuclei followed, 14 days later, by intercollicular decerebration. D = the same cat after electrolytic lesion of the rostro-medial part of the left fastigial nucleus. Note marked decrease in extensor rigidity on the left side and increase of extensor rigidity on the right side (Batini and Pompeiano, 1958). References p . 316-321

298


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Fig. 14. Inhibition of a single unit in the right Deiters' nucleus to anodal polarization of a single folium in the cortex of the vermis of the anterior lobe. Decerebrate and curarized cat. A = spontaneous discharge. B = no response to polarization of the right side of folium Vb with 0.4 mA. C = control after stimulation. D = arrest of discharge on polarization with 0.3 mA of the right side of the most ventral folium of sublobulus V A. E = control after stimulation. F = spontaneous activity after local application on the active lamella of 6% Nembutal for 1 min. G = noeffect on polarization with 0.3 mA of the right side of the most ventral folium of sublobulus V A (cf. record D). H = control after stimulation. I = spontaneous activity after washing the cortex with Ringer solution for 2 min. L = on polarization with 0.3 mA of the point stimulated in D and G the arrest of discharge reappears. M = control after stimulation (Pompeiano and Cotti, 1959d).

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This new interpretation of cerebellar inhibition differs from the old, i.e. that the inhibition of decerebrate rigidity elicited by stimulation of the vermis of the anterior lobe or of the fastigial nucleus is due to excitation (through fastigio-reticular pathways) of the inhibitory region of the reticular formation (cf. Dow and Moruzzi,

A

8

C

0

Fig. 15. Absence of reversal of the increase in membrane potential due to cerebellar inhibition during the flow of inward current. A = inhibitory postsynaptic potential recorded from a bicepssemitendinosus motoneurone following stimulation of the quadriceps nerve. Cerebellar inhibition increases the membrane potential (€3). In C and D a steady inward current is applied so that a reversal of the inhibitory postsynaptic potential into a depolarizing one is obtained. Cerebellar inhibitory stimulation applied in this condition still increases the membrane potential. The top line is only a reference, and the major displacement in d.c. potential between the upper and the lower pairs of records is not a measure of the hyperpolarization of the membrane due to the flow of inward current. Time, 1 msec. Calibration, 5 mV (Terzuolo, 1959).

1958). If this were so the hyperpolarization produced by cerebellar stimulation should present the same characteristics as the hyperpolarization produced by stimulation of the medial reticular formation. It is known, however, that the last effect is not due to a suppression of tonic excitatory influences acting on the motoneurones but is due to true IPSPs, because the IPSP can be reversed by passing hyperpolarizing current through the membrane, and is associated with conductance changes typical of the IPSP induced by peripheral afferent volleys (Fig. 16) (Llinas et al., 1962; Llinas and Thomas, 1962; Janskowska et al., 1964a, b; Llinas and Terzuolo, 1964, 1965). Regarding the facilitatory responses obtainable by stimulation of the vermis of the anterior lobe, the experimental evidence (Moruzzi and Pompeiano, 1954, 1956c, 1957a) indicates that they depend upon the integrity of the rostro-medial part of the ipsilateral fastigial nucleus. The possibility that these responses are partially, at least, due to excitation of Deiters’ neurones is supported by the fact that the rate of discharge of a certain number of units in Deiters’ nucleus is increased following stimulation of the vermis of the anterior lobe (Fig. 17) (De Vito, et al. 1956; Pompeiano and Cotti, 1959a-d; Ito and Yoshida, 1964; Ito and Kawai, 1964). Recent studies have shown that Deiter’s neurones sometimes receive monosynaptic (and polysynaptic) excitatory postsynaptic potentials (EPSPs) from the vermal cortex of the anterior lobe (Ito and Yoshida, 1964; Ito and Kawai, 1964). This effect has been explained as References p. 316-321

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being due to antidromic excitation of some cerebellar afferent fibres and to monosynaptic excitation through collaterals of neurones of Deiters’ nucleus. This interpretation does not take into account the possibility that stimulation of the vermal cortex of the anterior lobe also influences the fastigial projection to Deiters’ nucleus originating from the rostra1 part of the fastigial nucleus, which is left intact when the inhibitory cerebellar cortico-vestibular projection is interrupted by the lesion. It is interesting that Deiters’ units respond to localized stimulation of single folia of the vermis of the anterior lobe (Pompeiano and Cotti, 1959a-d). In particular it appears that neurones in the rostralmost part of Deiters’ nucleus, which are known to project to the cervical segments of the spinal cord (Pompeiano and Brodal, 1957a), are influenced by the folia of the culmen closer to the fissura prima; while A

Brain stem

FDL

M. D. C.r. rr.sp.

c

r.sp.v

8~10-~A

nBrain stem 8 x 1 0 - 9 ~

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Fig. 16. Postsynaptic inhibition in motoneurones evoked from the lower reticular formation. Precollicular decerebrate cat under Flaxedil. A-D = gastrocnemius-soleus motoneurone. The upper traces show the potentials recorded intracellularly with a K-citrate microelectrode (resistance 3.5 Ma). Intracellular potential change in a positive direction is shown as an upward deflection. The lower traces were recorded from the dorsal root entry zone in lower L6 against an indifferent electrode in the muscle. Upward deflection denotes negativity of the central electrode. Record A shows a steady increase in membrane potential elicited by repetitive stimulation (150/sec) of the medial reticular formation of the medulla. Record B shows the IPSP produced in the same motoneurone by strong stimulation of the nerve t o the flexor digitorum longus. Records C and D show the reversal A) of both these responses when the membrane was hyperpolarized by an inward current (17 x applied through the recording microelectrode. E-F = posterior biceps-semitendinosusmotoneurone. Change in membrane potential induced by rectangular pulse of depolarizing current (8 x A) (E) and its depression (F)by repetitive stimulation (280/sec) of the medullary reticular formation. On the right hand side are drawings of transverse and parasagittal section of the brain stem showing the region of the medial reticular formation whose repetitive stimulation produced IPSPs in both extensor and flexor motoneurones of the ipsilateral hindlimb. The inhibitory region (hatched) mainly covers the nucleus reticularis pontis caudalis, gigantocellularis, ventralis and lateralis of Meessen and Olszewski as well as the region of the medial longitudinal fasciculus (Jankowska et ul., 1964b).


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those situated in the caudalmost part of the nucleus, which is known to project to the lumbosacrai segments of the spinal cord (Pompeiano and Brodal, 1957a), are under the influence of the ventralmost folia of the anterior vermis. As already stated (see above) anatomical studies have fully confirmed the existence of such a pattern of somatotopical organization of the cerebellar projections to Deiters' nucleus (Walberg and Jansen, 1961; Walberg et al., 1962a).

A

B C

D

E

F

G

Fig. 17. Regular increase in the rate of discharge of a unit in Deiters' nucleus during stimulation of a single folium of the vermal cortex of the anterior lobe. Decerebrate cat. A = spontaneous discharge of a unit localized in the left Deiters' nucleus. B = increase in the frequency of discharge evoked by anodal polarization of the left side of folium IV b with 0.3 mA. C = control after the stimulation. D = the unit activity is unaffected by polarization of the left side of folium V c with 0.7 mA. E = control after the stimulation. F = the polarization of the left side of folium IV b with 0.3 rnA reproduces the effects illustrated in B. G = control after stimulation (Pompeiano and Cotti, 1959d).

The results of physiological observations (Pompeiano and Cotti, 1959a-d), correlated with the anatomical studies of the vestibulospinal projection (Pompeiano and Brodal, 1957a) and of the efferent cerebellar projections to the vestibular nuclei (Walberg and Jansen, 1961;Walberg et ul., 1962a) explain the somatotopical organiReferences p . 316-321

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zation of the postural responses to stimulation (Hampson e l al., 1945, 1946, 1952; Moruzzi and Pompeiano, 1957a) or ablation (Soriano and Fulton, 1947; Chambers and Sprague, 1955a,b) of the vermal cortex of the anterior lobe, the forelimb being represented in the culmen, the hindlimb in the lobulus centralis (cf. Dow and Moruzzi, 1958). The function of the posterior part of the cerebellar vermis which projects to the caudal part of the fastigial nucleus must now be considered. Acute (Moruzzi and Pompeiano, 1956a,b) or chronic (Batini and Pompeiano, 1955, 1957; Cohen ef al., 1958) electrolytic lesions limited to the caudal part of the fastigial nucleus increase the extensor tonus in the ipsilateral limbs but reduce it strikingly in the contralateral limbs (Fig. 18). This syndrome, the so-called ‘crossed fastigial atonia’, is also observed following unilateral ablation of the pyramis and uvula which are known to project

Fig. 18. Disappearance of extensor rigidity following contralateral caudo-fastigial lesion and its reappearance after contralateral deafferentation. A and B = bilateral extensor rigidity in fore- and hindquarters following precollicular decerebration. C = rigidity disappears in right legs after lesion of the caudal pole of the left fastigial nucleus. D = after deafferentation of left forelimb (C5 to T2) extensor rigidity returns in the right foreleg, while Sherringtonian flaccidity is observed in the deafferent4 limb. Previous fastigial asymmetry persists in the hindquarters (Moruzzi and Pompeiano,

195%).

on to the caudal part of the fastigial nucleus (Jansen and Brodal, 1940, 1942). The opposite effect, i.e. ipsilateral inhibition and crossed facilitation of extensor rigidity, is observed when the caudal part of the fastigial nucleus is stimulated electrically (Moruzzi and Pompeiano, 1956a, b) or during stimulation of the pyramis and uvula (Bremer, 1922; Hampson et al., 1945, 1946, 1952; Sprague and Chambers, 1954;


303

Chambers and Sprague, 1955a; Moruzzi and Pompeiano, 1956a,b). It was therefore concluded that the caudal part of the fastigial nucleus exerts a tonic excitatory influence on the extensor motoneurones of the contralateral side. This effect is supposed to be mediated via the hook bundle originating to a large extent from the caudal part of the fastigial nucleus (Jansen and Jansen, 1955; Batini and Pompeiano, 1955, 1957). Recent experiments support the view that Deiters’ nucleus is involved in the effects under consideration (Pompeiano, 1961,1962). It was found that when a lesion involving the rostral part of the caudal region of the fastigial nucleus was made, the ensuing atonia was limited to the contralateral forelimb, while the hindlimbs did not show any postural asymmetry (Figs. 19A,B). On the other hand, destruction of the extreme caudal pole of the fastigial nucleus abolished the extensor rigidity in the contralateral hindlimb only (Figs. 19C,D). Discrete effects, but opposite in character, were elicited after localized stimulation of the same nuclear regions. The somatotopical

Fig. 19. Effects of localized destruction of the caudal part of the fastigial nucleus. A = symmetrical distribution of extensor rigidity after precollicular decerebration. B = disappearance of extensor rigidity of the right foreleg after a lesion of the rostral part of the caudal third of the left fastigial nucleus. C = another experiment showing symmetrical rigidity after precollicular decerebration. D: disappearance of extensor rigidity of the right hindleg following lesion of the caudal pole of the left fastigial nucleus. (Pompeiano, 1962.)

pattern in the postural responses to local stimulation, or destruction, of the caudal part of the fastigial nucleus is in complete accord with the pattern of localization of the anatomical projection of the caudal part of the fastigial nucleus in to the contralateral Deiters’ nucleus (Walberg et ~ l . 1962a). , These findings, therefore, strongly support the conclusion that the excitatory influence exerted from the caudal part of References p . 316-321

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the fastigial nucleus on the extensor motoneurones of the contralateral side is mediated via Deiters’ nucleus. Strictly related to the problem of the somatotopical organization of the crossed fastigio-vestibular tract is the problem of the localization of the postural responses elicited by stimulation or destruction of the different folia of the posterior vermis. This problem has not been considered so far by those authors whose experiments involved stimulation or ablation of this cortical area. Hampson et al. (1952), however, illustrate a localization of the postural responses elicited by stimulating the pyramis and uvula (Fig. I), but they do not mention this point in their results. An electrophysiological study of the responses of single units in Deiters’ nucleus to local stimulation of separate folia of the posterior vermis and of the caudal third of the fastigial nucleus might shed light on the problem of the somatotopical organization of the projections from the posterior vermis. Furthermore a study of this kind might provide information as to whether the ipsilateral hemivermis of the anterior lobe and the contralateral one of the posterior lobe are able to influence the activity of the same unit in Deiters’ nucleus even if the pathways from the two regions of the vermis have different terminal regions in this nucleus (cf. Walberg et al., 1962). CEREBELLAR I N F L U E N C E S O N T H E R E D N U C L E U S

In the past great emphasis has been placed on the problem of the cerebellar control of postural extensor tonus (Dow and Moruzzi, 1958). It has been shown in the previous paragraph that Deiters’ nucleus is, at least in part, involved in the cerebellar control of the extensor motoneurones. Recent observations indicate that the cerebellar control of flexor motoneurones stems from the intermediate cortex of the cerebellum and the interposite nucleus, and that this influence is transmitted to the spinal cord through the red nucleus. Previous experiments have shown that threshold stimulation of the red nucleus produces, in the decerebrate animal, an active flexion of the contralateral limbs, which on suprathreshold stimulation is associated with an increase in the extensor tonus of the ipsilateral limbs (Pompeiano, 1956a, 1957). These responses, which are opposite in sign to those of the typical tegmental reaction (Ingram et al., 1932), are somatotopically organized. Stimulation of the dorsal part of the red nucleus produces an active flexion of the contralateral forelimb, while stimulation of the ventral part of the red nucleus produces an active flexion of the contralateral hindlimb only (Pompeiano, 1957) (Fig. 20). Recent anatomical observations have shown that the rubrospinal path is organized in a somatotopical manner (Figs. 2A,B) agreeing with the pattern described from physiological experiments (Pompeianoland Brodal, 1957b). The excitatory influence exerted by the red nucleus on the contralateral flexor motoneurones has been confirmed not only in the decerebrate preparation (Maffei and Pompeiano, 1961c, 1962b) but also in the intact animal (Gassel et al., 1965). It is of interest that the crossed flexor responses to stimulation of the red nucleus have been observed even on a background of a-rigidity, such as occurs in the decerebrate, cerebellectomized animal (Pompeiano, 1956a, 1957). Later experiments have shown that


305

A

B

C

D Fig. 20. A series of drawings showing the localization of the postural responses to stimulation of the red nucleus. The drawings represent transverse sections of the cat’s midbrain, taken at equal intervals, and labelled successively in a rostro-caudal sequence. The dots indicate the points of the red nucleus which on liminal stimulation yielded a localized flexor response in the contralateral forelimb, the triangles the points which gave a localized flexor response in the contralateral hindlimb, while the sauares indicate points from which stimulation with the same voltage elicited flexor responses in both the contralateral fore- and hindlimb (Pompeiano, 1957; Maffei and Pompeiano, 1962b).

stimulation of the red nucleus produces a discharge which can be recorded from contralateral flexor nerves only, and not from contralateral extensor nerves or from ipsilateral nerves (Sasaki et al., 1960; Thulin, 1963). Even the monosynaptic flexor reflex on the contralateral side is facilitated, while the monosynaptic extensor reflex is unmodified or only slightly depressed (Sasaki et al., 1960; Thulin, 1963). Stimulation of the red nucleus also produces EPSPs and spike discharges in the contralateral flexor motoneurones, while IPSPs occur in the contralateral extensor motoneurones. On the other hand no change takes place in the extensor or flexor motoneurones of the ipsilateral side (Sasaki et al., 1960). The spinal mechanisms of activation of the flexor motoneurones by the rubrospinal tract have not yet been analysed. Recent anatomical observations (Nyberg-Hansen and Brodal, 1964) show that the rubrospinal fibres in the cat do not terminate on the motor cells of the anterior horn, but only on the interneurones of layers V-VII of Rexed. Interneurones can actually be influenced by stimulation of the red nucleus References p. 3163.21

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(Sasaki et al., 1960; Hongo et al., 1965). Stimulation of the red nucleus also gives an excitatory influence on fusimotor flexor neurones (Appelberg and Kosary, 1963) (Fig. 21A)*. Therefore both skeletomotor and fusimotor flexor neurones are under the control of the (red) nucleus. This effect is abolished by nembutal anaesthesia (Fig. 21B).

Fig. 21. Excitation of flexor fusimotor neurones by electrical stimulation of the red nucleus. A = simultaneous recording from extensor (middle beam) and flexor (lower beam) spindle afferent. Note inhibition of extensor spindle and facilitation of flexor spindle during stimulation in the red nucleus (broad line above). B = same as A but recording made approximately 15 min after the injection of an additional dose (25 mg) of nembutal. Note lack of facilitation of flexor spindle. Time calibration, 1 sec (Appelberg and Kosary, 1963).

It is generally assumed that electrical stimulation of the intermediate cortex of the anterior lobe increases the extensor tonus of the ipsilateral limbs (Stella, 1944; Hampson et al., 1945, 1946, 1952; Moruzzi, 1947; Sprague and Chambers, 1954). On the other hand, stimulation of the same cortical area has been reported to produce sporadically an active flexion of the ipsilateral limbs (Sprague and Chambers, 1954; Chambers and Sprague, 1955a). In all instances the responses of the intermediate cortex are not abolished by acute destruction of the vermal cortex (Stella, 1944) nor by a bilateral destruction of the fastigial nucleus (Sprague and Chambers, 1953; Moruzzi and Pompeiano, 1957a), but they are abolished by destruction of the interposite nucleus (Chambers and Sprague, 1955a). Because suprathreshold stimulation of a limited region of the cerebellar cortex may activate cerebellar regions nearby, the hypothesis has been advanced that in the decerebrate preparation the responses are often the result of activation of different cerebellar mechanisms (Pompeiano, 1956b, 1958). In order to avoid these complications, experiments were performed in decerebrate animals submitted to bilateral complete lesions of the fastigial nuclei (Pompeiano 1956b, 1958). In these preparations, with a completely inexcitable vermis, an increase in the extensor tonus of the ipsilateral limbs, associated with de-

* The inhibitory effect on the y flexor motoneurones, which has been stated to occur on stimulation of the red nucleus (Appelberg, 1962a,b) can be explained by spread of current to the mesencephalic reticular formation.


307

Fig. 22. Localization of the postural responses to electrical stimulation of the rostra1 part of the interposite nucleus. The drawings correspond to serial histological sections of the cerebellum taken at equal intervals. They are numbered in a rostro-caudal direction from 1 to 10. The following abbreviations have been used: B.c. = brachium conjunctivum; C.r. = corpus restiforme; D = dentate nucleus; F = fastigial nucleus; I = interposite nucleus; L = lateral vestibular nucleus of Deiters; M = medial vestibular nucleus; S = superior vestibular nucleus. On the left side of the drawings the points are indicated whose threshold stimulation produced an extensor response of the ipsilateral forelimb (dots), of the ipsilateral hindlimb (filled triangles), or of both ipsilateral limbs (filled squares) (Pompeiano, 1960~). With other symbols the points are indicated whose threshold stimulation produced a flexor response of the ipsilateral forelimb (circles), of the ipsilateral hindlimb (triangles), or of both ipsilateral limbs (squares) (Pompeiano, 1959).

pression of flexor motoneurones, occurs on stimulating the lateral strip of the intermediate part of the anterior lobe. These effects are followed by a flexor rebound*. On the contrary, stimulation of the paravermal strip of the intermediate cortex of the anterior lobe produces an active flexion of the ipsilateral limbs. This effect has

* It is of interest that primary afferent depolarization in Ib and cutaneous afferents can be evoked from the most lateral strip of the intermediate region, but not from the anterior vermis (Carpenter et al., 1966). References p . 316-321

308


been confused by previous authors (cf. Hampson et al., 1945, 1946, 1952; Sprague and Chambers, 1954; Chambers and Sprague, 1955a) with the response elicited by the contiguous vermal part of the anterior lobe. Experimental stimulations of the rostral part of the interposite nucleus have shown that this nuclear region mainly exerts an excitatory influence on the ipsilateral flexor motoneurones (Figs. 22 and 23). There are, however, points particularly localizedin the rostrolateral part of the interposite nucleus which produce a predominantly excitatory effect on extensor motoneurones (Fig. 22).

Fig. 23. Localization of the flexor responses to electrical stimulation of the rostro-medial part of the interposite nucleus. A = symmetrical rigidity after precollicular decerebration. B = stimulation of the rostro-medial part of the left interposite nucleus at level 2 of Fig. 22 produces a flexion of the ipsilateral hindlimb and a slight increase in the extensor rigidity of the contralateral hindlimb. Note the absence of any response of the forelimbs. C = stimulation of the rostro-medial part of the left interposite nucleus at level 5 of Fig. 22 yields a flexion of the ipsilateral forelimb and a slight increase in the extensor rigidity of the contralateral forelimb. Note the absence of any postural changes in the hindlimbs. D = taken a few seconds after C, during the same stimulation (Pompeiano, 19591.

Electrolytic destruction of the contralateral red nucleus, or retrocollicular decerebration (Pompeiano, 1956b, 1958; Pompeiano et al., 1953, 1954) does not modify the extensor responses from the intermediate cortex. On the other side it abolishes: (i) the flexor responses induced by stimulation of the paravermal strip of the intermediate cortex of the anterior lobe and of the rostral part of the interposite nucleus; and (ii) the depression of flexor motoneurones and the ensuing flexor rebound which occur during and after stimulation of the lateral part of the intermediate cortex of the anterior lobe (Pompeiano, 1956b, 1958). The inability of Calma and Kidd (1959) to observe an increase in the activity of flexor motoneurones by stimulating the intermediate cortex of the anterior lobe

CEREBELLAR PROJECTIONS TO S P I N A L C O R D

309

could be due to anatomical and functionalchangesin the red nucleusproduced duringthe decerebration. On the other hand EPSPs have been recorded from flexor motoneurones after stimulating the ipsilateral interposite nucleus (Sasaki and Tanaka, 1963, 1964). Because the red nucleus excites the flexor motoneurones of the contralateral limbs (Pompeiano, 1956a, 1957) one may conclude that an interposito-rubrospinal path originates from the rostral part of the interposite nucleus and exerts an excitatory influence on the flexor motoneurones. The depression of the activity of flexor muscles, which occurs during stimulation of the lateral areas of the intermediate part of the anterior lobe, followed by a flexor rebound at the end of stimulation (Pompeiano, 1958), has been supposed to be due to a suppression, by the cerebellar cortex, of excitatory interposito-rubral influences (cf- Maffei and Pompeiano, 1962a). Recent experiments have shown that the interposite nucleus monosynaptically excites the neurones of the contralateral red nucleus (Tsukahara et al., 1964; cJ: also Appelberg, 1961; Massion and Albe-Fessard, 1960, 1963; Massion, 1961; Davis, 1964). On the other hand, weak stimulation of the intermediate part of the anterior lobe does not produce postsynaptic potentials in the cells of the red nucleus but simply reduces the EPSPs induced by stimulation of the interposite nucleus (Tsukahara et al., 1964). This reduced excitability of the neurones ofthe interposite nucleus should be referred to monosynaptic IPSPs produced in interposite neurones after stimulation of the intermediate cortex of the anterior lobe (Ito, Yoshida and Obata, 1964; cf- also Tsukahara et al., 1965). If this is so, one should expect that the hyperpolarization of flexor motoneurones induced by stimulating the intermediate cortex of the anterior lobe is not associated with the conductance changes which are typical of the IPSPs, but is due to lack of supraspinal excitatory impingement. Future experiments should test this hypothesis. The problem of localization of the responses will now be considered. Similarly to the postural responses induced by stimulation of the intermediate cortex of the anterior lobe (Hampson et a t , 1945, 1946, 1952) even the flexor responses produced by stimulation of the rostral part of the interposite nucleus show a corresponding pattern of somatotopical organization (Pompeiano, 1959, 1960a). For example, the caudal part of the rostral half of the interposite nucleus produces responses localized to the ipsilateral forelimb; whereas the most rostral part influences, selectively, the ipsilateral hindlimb (Figs. 22 and 23). Since both the cerebellar corticonuclear projection (Jansen and Brodal, 1940, 1942) and the rubrospinal tract (Pompeiano, 1957: Pompeiano and Brodal, 1957b) show a clear pattern of localization, we conclude that the entire pathway mediating the flexor components of the posturalFresponses (intermediate cortex of the anterior lobe rostral part of the interposite nucleus --f red nucleus spinal cord) is somatotopically organized (Figs. 4 and 5). The observation that the red nucleus receives cerebellofugal fibres not only from the rostral, but also from the caudal part of the interposite nucleus (Jansen and Jansen, 1955) has led to the study of the effects exerted on flexor motoneurones from the caudal part of the interposite nucleus and the paramedian lobule, which projects upon this nuclear region (Maffei and Pompeiano, 1961a,b; 1962a).

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References p . 316-321

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The experiments of stimulation of the paramedian lobule reported in the literature showed contradictory results (for refs. see Maffei and Pompeiano, 1962a). In our experiments (Maffei and Pompeiano, 1961a,b, 1962a) the most regular response induced by stimulation of the paramedian lobule in the precollicular decerebrate cat was a slight increase in the ipsilateral extensor tonus. This was accompanied by inhibition of the flexor musculature and followed at the end of the stimulation by a flexor rebound. Experiments to stimulate the caudal part of the interposite nucleus have shown that this nuclear region exerts an excitatory influence on the ipsilateral flexor motoneurones (Fig. 24). There are, however, points mainly localized in the caudo-

Fig. 24. Localization of the postural responses t o electrical stimulation of the caudal part of the interposite nucleus. The drawings correspond to serial histological sections of the cerebellum taken at equal intervals. They are numered in a rostro-caudal direction from 1 to 10. The following abbreviations have been used: B.c. = brachiurn conjunctivum; Dt. = dentate nucleus; F = fastigial nucleus; 1 = interposite nucleus; L = lateral vestibular nucleus of Deiters; S = superior vestibular nucleus. On the left side of the drawings the points are indicated whose threshold stimulation produced an extensor response of the ipsilateral forelimb (dots), of the ipsilateral hindlimb (filled triangles), or of both ipsilateral limbs (filled squares). With other symbols the points are indicated whose threshold stimulation produced a flexor response. of the ipsilateral forelimb (circles), of the ipsilateral hindlimb (triangles), or of both ipsilateral limbs (squares). The symbol x indicates points whose stimulation had no effect on the postural tonus (Maf€ei and Pompeiano, 1962a).


31 1

medial part of the interposite nucleus which cause a predominantly excitatory influence on the extensor motoneurones (Fig. 24). The ipsilateral increase in the extensor tonus elicited by stimulating the cortex of the paramedian lobule is transmitted through the caudo-medial part of the interposite nucleus to ipsilateral structures of the brain stem placed caudally to the red nucleus. On the contrary, electrolytic destruction of the contralateral red nucleus abolishes : (i) the flexor responses produced by stimulation of the caudal part of the interposite nucleus; and (ii)the depression of flexor motoneurones and the followingflexor rebound which appear during and after stimulation of the cortex of the paramedian lobule. The influence of the paramedian lobule on the contralateral red nucleus has been recently confirmed by Massion (1961; cf. also Whiteside and Snider, 1953) with the evoked potential technique. No detailed analysis of this projection, however, was made by these authors. Because the red nucleus excites the flexor motoneurones of the contralateral side (Pompeiano, 1956a, 1957), one may conclude that an interpositorubrospinal path originates from the caudal part of the interposite nucleus, which is excitatory to flexor motoneurones. The depression of the activity of flexor muscles, which in the precollicular decerebrate cat represents an important component of the response of the paramedian lobule, is likely to be due to inhibition from the cerebellar cortex of the paramedian lobule of those neurones of the interposite nucleus which have an excitatory influence on the red nucleus. In agreement with the present hypothesis is the fact that stimulation of the interposite nucleus produces monosynaptic EPSPs in neurones of the contralateral red nucleus. IPSPs, on the contrary, are absent (Tsukahara et al., 1964, 1965). Next to be considered is the problem of localization of the responses. Our experiments show that the flexor responses to stimulation of the caudal parts of the interposite nucleus are organized somatotopically. The rostral part of the caudal half of the interposite nucleus produces responses localized to the ipsilateral forelimb; while the caudal part selectively influences the ipsilateral hindlimb (Fig. 24). A similar pattern of localization was found for the postural responses elicited by stimulation (Hampson et al., 1946, 1952; Snider and Magoun, 1949; Snider et al., 1949) or ablation of the paramedian lobule (Chambers and Sprague, 1955b). Because not only the cerebellar corticonuclear projection (Jansen and Brodal, 1940, 1942), but also the rubrospinal tract (Pompeiano, 1957; Pompeiano and Brodal, 1957b), show a clear pattern of localization, we conclude that the entire pathway mediating the flexor components of the postural responses (lobulus paramedianus caudal part of the interposite nucleus red nucleus -+ spinal cord) is somatotopically organized (Figs. 4 and 5). It should be mentioned that the responses induced by stimulating both the paramedian lobule and the caudal part of the interposite nucleus were less marked than the corresponding responses elicited (for the same voltages and in the same preparation) by stimulating the intermediate cortex of the anterior lobe and the rostral part of the interposite nucleus. This observation is in agreement with those of Appelberg (1961) and Massion (1961) who found that rubral responses were more easily obtained by stimulating the rostral part of the interposite nucleus.

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It is of interest that the same cortical areas of the intermediate part of the anterior lobe and of thebaramedian lobule which decrease the activity of flexor muscles also exert an increase in the extensor rigidity of the ipsilateral limbs (Pompeiano, 1956b, 1958; Maffei and Pompeiano, 1961a, 1962a). These extensor responses are somatotopically organized in a manner similar to that of the flexor responses induced by stimulating these cortical areas. And a similar pattern of somatotopical organization has been found for the extensor responses elicited by stimulation of the rostro-lateral (Pompeiano, 1960b,c) and the caudo-medial (Maffei and Pompeiano, 1961a,b, 1962a) parts of the interposite nucleus. The problem of the identification of the pathways and of the brain-stem structures which transmit these extensor responses has been discussed elsewhere (cf. Maffei and Pompeiano, 1962a). The extensoi responses are still observed after intercollicular decerebration (Sprague and Chambers, 1953, 1954; Chambers and Sprague, 1955a; Cohen et al., 1958) or midsagittal section of the midbrain affecting the decussation of the superior cerebellar peduncles (Pompeiano et al., 1953, 1954; Pompeiano, 1958, 1960~). We may conclude that the functional organization of the cauda1,part of the interposite nucleus, which is under the influence of the paramedian lobule, is a mirrorimage of that of the rostral part of the interposite nucleus, which is under the influence of the intermediate cortex of the anterior lobe. The similarity concerns both the localization of function (Pompeiano, 1956b, 1958) and the localization of the limbs (Pompeiano, 1959, 1960~).While both the rostral and the caudal parts of the interposite nucleus exert an excitatory influence on the flexor motoneurones, extensor responses may be elicited by stimulating the rostro-lateral and the caudo-medial parts of the interposite nucleus. Besides this the most rostral points of the interposite nucleus influence the hindlimb muscles. The most caudal points of the rostral part of the interposite nucleus influence the forelimb muscles. The opposite picture is seen in the caudal part of the interposite nucleus (Fig. 5). DISCUSSION

The results of recent work have shed some light on the anatomical and functional organization of the cerebellar projections to the spinal cord although several aspects of the problem still remain to be clarified. It is known that the areas of the cerebellar cortex influencing the spinal cord, namely the vermal and the intermediate cortex, project to the fastigial nucleus and the interposite nucleus respectively and have a we11 established pattern of localization. Anatomical (Walberg and Jansen, 1961; Walberg et al., 1962a) and physiological studies (Pompeiano and Cotti, 1959d; Pompeiano, 1959, 1960c, 1962; Maffei and Pompeiano, 1962a)have shown that the efferent projections from the cerebellar cortex and the fastigial nucleus to Deiters’ nucleus (Walberg and Jansen, 1961;Walberg et al., 1962a; Pompeiano and Cotti, 1959d; Pompeiano, 1962)and from the interposite nucleus to the red nucleus (Pompeiano, 1959,1960~;Maffei and Pompeiano, 1962a) are somatotopically organized. It has also been found that both the vestibulospinal (Pbmpeiano and Brodal, 1957a) and the rubrospinal projections (Pompeiano and Brodal,


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1957b) are somatotopically organized. Physiological observations have shown that Deiters’ nucleus excites the a-extensor motoneurones (Pompeiano, 1960d,e), while the red nucleus excites the flexor motoneurones (Pompeiano, 1956a, 1957). Parallel influences are exerted by Deiters’ nucleus (Granit et al., 1959) and the red nucleus (Appelberg and Kosary, 1963) on the fusimotor extensor and flexor neurones respectively. It has also been found that these influences appear to be somatotopically organized in full agreement with the anatomical pattern. On the basis of the functions attributed to Deiters’ nucleus and the red nucleus, and of the organization of the efferent cerebellar projections to these nuclei, one may tentatively divide the part of the cerebellum influencing the spinal cord into two large areas : (i) the first region, which includes the vermal cortex and the fastigial nucleus, is capable of influencing the extensor motoneurones, an effect that is at least in part mediated by Deiters’ nucleus; and (ii) the second region, which includes the paravermal or intermediate cortex of the cerebellum and the interposite nucleus, is capable of influencing the flexor motoneurones. These effects are mediated by the red nucleus. This simplification of the sphere of influence of the different cerebellar cortical areas on the spinal cord is complicated by the fact that the vermal and the intermediate cortices of the cerebellum also project through the fastigial and the interposite nuclei on to the brain-stem reticular formation (cf. Jansen and Brodal, 1954, 1958), but unfortunately the final effect on the motoneurones of the influences exerted by these cerebello-reticularprojections is almost unknown. What is certain is that the cerebello-reticular projections cannot be responsible for the localization of the limb responses, because the efferent projections from the cerebellar nuclei to the brain-stem reticular formation are diffuse (Walberg et al., 1962b). The evidence presented in the present report supports the hypothesis (Sprague and Chambers, 1953) that the cerebellar nuclei represent tonogenic centres which exert their excitatory influences on the brain-stem structures. In particular the rostral and the caudal parts of the fastigial nucleus exert an excitatory influence on the ipsilateral and contralateral extensor motoneurones. These effects are likely to be mediated through the direct and the crossed fastigio-vestibular pathways to Deiters’ nucleus. On the other hand, both the rostral and the caudal parts of the interposite nucleus exert an excitatory influence on the flexor motoneurones. This effect is mediated by two parallel interposito-rubral pathways. In the interposite nucleus, however, there are regions which produce an increase in the activity of the extensor motoneurones. The brain-stem mechanisms responsible for these responses are still little known. It has been suggested that the activity of the cerebellar nuclei, which is likely to be sustained by extracerebellar afferents, is inhibited by the cerebellar cortex (Sprague and Chambers, 1953; Moruzzi and Pompeiano, 1954, 1957a; Stella et al., 1955; Batini and Pompeiano, 1957, 1958). This hypothesis is supported by recent findings showing that the cerebellar Purkinje cells are inhibitory and so produce monosynaptically an IPSP in their target neurones, which are mainly located among the cerebellar nuclei and Deiters’ nucleus (It0 and Yoshida, 1964; Ito, Yoshida and Obata, 1964; Ito and Kawai, 1964; Ito, Obata and Ochi, 1964). References p. 316-321

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The inhibition of the ipsilateral extensor motoneurones, elicited by stimulating the vermis of the anterior lobe, is likely to be due to direct inhibitory influences exerted by the cerebellar cortex on Deiters’ neurones. This inhibitory iduence, which is produced monosynaptically by the Purkinje neurones on the Deiters’ cells (It0 and Yoshida, 1964; Ito and Kawai, 1964; Ito, Obata and Ochi, 1964), is probably due to activation of the direct cerebellar cortico-vestibular projection, because it is abolished after electrolytic destruction of the rostro-lateral part of the fastigial nucleus (Moruzzi and Pompeiano, 1954, 1957a)’ where the long cerebellar cortico-vestibular fibres run (Walberg and Jansen, 1961). In agreement with this hypothesis is the fact that the hyperpolarization of extensor motoneurones, induced by stimulation of the vermis of the anterior lobe, is likely to be due to lack of excitation since it is not associated with the conductance changes typical of the IPSP (Terzuolo, 1959). The monosynaptic inhibition of Deiters’ neurones induced by the activity of Purkinje cells would actually produce a striking defacilitation of the extensor motoneurones, because these motoneurones receive in turn a monosynaptic excitatory impingement from Deiters’ nucleus (Lund and Pompeiano, 1965). In a similar manner the abolition of the flexor muscle activity which occurs by stimulating the intermediate cortex of the anterior lobe and the paramedian lobule (Pompeiano, 1958; Maffei and Pompeiano, 1962a) is likely to be due to inhibition of neurones of the interposite nucleus, which exert a monosynaptic excitatory influence on the red nucleus (Tsukahara et al., 1964, 1965). There are, however, the following phenomena which cannot be easily explained with this inhibitory hypothesis: (i) the increase in the activity of the extensor motoneurones by low rate stimulation of the ipsilateral vermis of the anterior lobe (Moruzzi, 1950a,b) or by high rate stimulation of the vermis of the anterior lobe after electrolytic destruction of the inhibitory cortico-vestibularpathway (Moruzzi and Pompdano, 1957a);(ii) the increase in the activity of the extensor motoneurones by stimulation of the contralateral posterior hemivermis and the abolition of the rigidity of the contralateral limbs after ablation of this cortical area, these effects being similar to those induced respectively by stimulation or ablation of the corresponding caudal part of the fastigial nucleus (cf. Moruzzi and Pompeiano, 1956b); (iii) the increase in the activity of the flexor motoneurones induced by stimulation of the paravermal region of the intermediate cortex of the anterior lobe (Pompeiano, 1958); and finally (iv) the increase in the activity of extensor motoneurones resulting from stimulation of the lateral region of the intermediate cortex of the anterior lobe (cf. Pompeiano, 1958) and the paramedian lobule (Maffei and Pompeiano, 1962a). Future experiments should be devoted to an analysis of the mechanisms which are responsible for these facilitatory effects. One should take into account that electrical stimulation of the cerebellar cortex might excite not only the cerebellar Purkinje cells but also all the other nervous elements of the cerebellar cortex, such as the climbing fibres, the mossy fibres and the parallel fibres. Excitatory effects might tentatively be attributed to: (i) antidromic stimulation of axon collaterals which, according to Carrea et al. (1947), extend up to the cerebellar cortex from the intracerebellar nuclei as climbing fibres ; (ii) antidromic stimulation of the cerebellopetal fibres, including


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the mossy fibres, and orthodromic excitation, through their collaterals, of neurones localized in the cerebellar nuclei or in other brain-stem structures; and (iii) stimulation of parallel fibres, which would influence the Purkinje neurones located in contiguous regions of the cerebellar cortex either directly or indirectly, through the basket cells and the Golgi cells. It has been shown that, while the parallel fibres excite the Purkinje cells, the basket cells exert inhibitory postsynaptic potentials on the Purkinje neurones (Andersen et al., 1964) while the Golgi cells inhibit the granular cells (Eccles et al., 1964). Under certain conditions the result of excitation of parallel fibres could be that of blocking the activity of the inhibitory pathways originating from neighbouring Purkinje neurones. Very little too, is known about the anatomical and functional significance of the interconnections between different cerebellar cortical areas (cf. Jansen, 1933; Barnard and Woolsey, 1950; Eager, 1963b, 1965), between the different cerebellar nuclear regions, and finally between the cerebellar nuclei and the brain-stem structures which in their turn may project back to the cerebellar nuclei and/or to the cerebellar cortex. Because of these gaps in our knowledge, it would be advisable that future physiological work be performed in unanaesthetized preparations because it is likely that any kind of anaesthesia may alter the normal picture. SUMMARY

(1) The selective control that the different corticonuclear regions of the paleocerebellum exert on spinal motoneurones, and the somatotopical organization of these cerebellofugal influences are discussed. (2) Deiters’ nucleus exerts an excitatory influence on extensor motoneurones, while the red nucleus exerts an excitatory influence on flexor motoneurones. Both these supraspinal influences are somatotopically organized. (3) The fastigial and the interposite nuclei can be considered as tonogenic centres exerting an excitatory influence on extensor and flexor motoneurones. These effects are mediated respectively by Deiters’ nucleus and the red nucleug. (4) There are two fastigio-vestibular projections, originating from the rostral and the caudal parts of the fastigial nucleus, which exert an excitatory influence on the ipsilateral and the contralateral Deiters’ nucleus respectively. Similarly there are two interposito-rubral projections, originating from the rostral and the caudal part of the interposite nucleus, which run parallel and exert an excitatory influence on the contralateral red nucleus. All these pathways are somatotopically organized. (5) This picture of the functional organization of the cerebellar projections to the spinal cord is complicated by the influence that the fastigial and the interposite nuclei exert on the brain-stem reticular formation. (6) The hypothesis that the cerebellar cortex exerts an inhibitory influence on the relay nuclei is supported by the fact that stimulation of the vermis of the anterior lobe greatly reduces the activity in the extensor motoneurones. This effect is due to the inhibitory influence exerted by the long cerebellar cortico-vestibular projection on to Deiters’ nucleus. On the other hand stimulation of the intermediate cortex of the References p . 316-321

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anterior lobe and the paramedian lobule reduces the activity in the flexor motoneurones, and this effect has been referred to the direct inhibitory influence exerted by the cerebellar cortex on the interposite nucleus. (7) There are, however, other effects of stimulation of different cerebellar cortical areas which cannot be explained by a direct application of this hypothesis. ACKNOWLEDGEMENT

The investigations of Gassel et al. (1965) reported in this review article were carried out with the support of the PHS research grant NB 02990 from the National Institute of Neurological Diseases and Blindness, N.I.H., Public Health Service, U.S.A.

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Functional Alterations of Cerebral Sensory Areas by the Cerebellum * R A Y S. S N I D E R Center for Brain Research, University of Rochester, Rochester, N . Y. (U.S.A.)

Recognition of cerebral influences on the cerebellum came early in physiological research. Sherrington (1906), for example, repeatedly referred to the large corticopontocerebellar system in higher mammals and speculated on its function. However, recognition by physiologists of cerebellar influences on the cerebrum has been slow. Rossi (1912) was one of the earliest contributors to the field. He showed that electrical stimulation of the cerebellar hemispheres resulted in an increased excitability of the cerebral motor area. Surprisingly, there was little interest in this aspect of neurophysiology until Bremer (1935) repeated Rossi's observations, and Walker (1938) showed that electrical excitation of Crus I and I1 led to marked alteration of spontaneous activity of the cerebral motor area. Walker's work was an extension of his anatomical studies (1938) in which he showed unequivocal projections from the nucleus ventralis lateralis thalami (nucleus to which the brachium conjunctivum sends many of its thalamic fibers) to the pre-central gyrus. For an extensive review of the early anatomical literature the reader is referred to Gerebtzoffs (1936) article. The major purpose of the present article is to review in concise form material which has recently been published relevant to cerebellar influences on cerebral sensory systems. Influences on motor systems cannot be covered nor can cerebro-cerebellar relationships. EZecfroanatomicaZ studies. Detailed studies of cerebello-cerebral relationships were carried out by Snider and associates following the original descriptions of the sensory areas of the cerebellum by Snider and Stowell (1944). The earliest report in the series was the one by Henneman et aZ. (1950) which showed a more extensive cerebellar projection to the cerebrum than was indicated by earlier work. For example, if depressant anesthetics such as barbiturates were not used except in conjunction with chloralosane, or if animals were immobilized under curare-like drugs and low levels of ether anesthesia, it could be shown that the tuber vermis portion of the auditory area of the cerebellum projected to the auditory area of the cerebrum and the so-called

* Supported in part by research grants NB 04592 and NB 04596 from the National Institute of Neurological Diseases and Blindness.

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tactile areas of the cerebellum projected to the tactile areas of the cerebrum. Although interrelationships between the cerebellar and cerebral visual areas were observed the responses were so capricious that they could not be studied in detail. Projections to the motor areas were easiest to demonstrate in that thresholds were lower and the responses were more resistant to the depressant action of anesthetics. Electrical stimuli applied directly to the central cerebellar nuclei induced widespread responses in the cerebrum which showed lowest thresholds when projecting to the motor areas with latencies as short as 3 msec (measured to beginning of the rising phase of the responses). This study proved to be an important one since it established for the first time a cerebellar influence on a sensory system at a cerebral level. It also showed that in the case of the tactile system when careful attention was given to anesthetic levels and electrical thresholds an ‘area to area’ relationship could be obtained for a cerebellocerebral projection. Then followed the detailed studies of Whiteside and Snider (1953), which paid particular notice to pathways involved. By means of the evoked response technique and multiple stereotaxic electrodes placed in the upper brain stem, a very extensive cerebellar projection to the thalamus and diencephalic tegmentum was found. These workers found in addition to the well-accepted projection to the nucleus ventralis lateralis, definite though less prominent projections to ventralis posterior, medial geniculate nucleus, Forel’s fields, centralis medialis, centralis lateralis, centrum medianum and parafascicularis nuclei. In addition, there were widespread influences exerted on the mesencephalic tegmentum including the ventral part of the periaqueductal gray substance. In essence, these data showed cerebellar influences exerted on the midbrain reticular formation as well as the diencephalic reticular formation including parts of the so-called reticular activating system and, in addition, showed for the first time a sizeable projection to the so-called sensory relay nuclei of the thalamus. Such projections imply that the cerebellum may influence sensory systems ascending to the cerebrum. Some functional implications of these hitherto unrecognized projections of the cerebellum are considered below. The above mentioned electroanatomical studies have been supported in part by recent anatomical studies. Hassler (1950), studying the human, found projections in addition to ventralis lateralis, to centrum medianum and mesencephalic gray substance. Cohen et al. (1958), studying the cat, traced fibers through mesencephalic tegmentum to Forel’s field H, subthalamus ventralis lateralis, ventralis posterior, lateralis posterior, centrum medianum, centralis lateralis, reticularis, ventralis anterior, ventralis medialis and dorso-lateral hypothalamus. Earlier, Jansen and Jansen (1955) found that one-third of fibers of the brachium conjunctivum going cephalad of the nucleus ruber pass to zona incerta, parafascicular nucleus, subthalamic nucleus, and the medial nucleus of thalamus. Niimi et al. (1962), using the Nauta anatomical method, have described the most extensive projection of the brachium conjunctivum and come closest to confirming the electrical studies of Whiteside and Snider (1953). They found cerebellar efferent fibers passing to mesencephalic reticular formation, Forel’s field, zona incerta, and to thalamic nuclei - parafascicularis, laminaris, ventralis (pars posterior and anterior), medialis dorsalis, centre medianum, medial geniculate nucleus, and pulvinaris. Thus, ample recent anatomical evidence substanReferences p . 332-333

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tiates our thesis that the cerebellum sends sizeable projections to the ascending reticular formation and to some of the so-called sensory relay nuclei. Functional studies. Earlier functional studies stressed cerebellar influences on motor systems. Walker (1938), working in Bremer’s laboratory, concluded that ‘excitation of the cerebellar hemispheres produces a marked increase in the amplitude and frequency of the cortical action potentials from the motor areas of the cat’, and this mechanism ‘normally maintains a coordinating influence upon volitional movement’. Curiously, later workers failed to recognize that Walker also recorded cortical changes of a ‘lesser extent from the parietal and temporal regions’ but tended to disregard these observations since he states that in the cat ‘it is possible that the response may be entirely confined to the motor cortex in animals in which the cerebral cytoarchitecture is better differentiated’. Moruzzi (1 950) reviewed his earlier studies on cerebellar effects on motor areas of the cerebrum and added additional data to Rossi’s eailier findings, Henneman et al. (1950) could do nothing more than speculate on the function of the cerebella-cerebral projections to sensory areas. Cooke and Snider (1953) saw extensive low voltage fast EEG activation-like activity in both sensory and motor cortices of the cat which continued for as long as 2 min after withdrawal of the electrical stimuli to the cerebellum. Stimulation of the so-called audio-visual area was usually followed by generalized cortical activation although focal changes in the ectosylvian area were observed which were more prolonged and of greater amplitude than elsewhere. Stimulation of the paramedian lobule (caudal tactile area) produced generalized cortical activation which was especially pronounced in the sensorimotor area of the cerebrum. Although possible spread of current to the nearby brain stem (when higher voltages of stimulation were used) was an ever present hazard, this could not have occurred when lower voltages (as low as 0.5 V) were employed, and in studies on the tuber vermis and lobulus simplex there was even greater distance to the brain stem, hence less hazard. This was the first of a series of studies which implicated the cerebellum as part of a generalized system going to the cerebrum to involve sensory as well as motor cortices, a hitherto unrecognized function. However, it was an outgrowth of an earlier observation of Snider and Magoun (1949) on facilitation produced by cerebellar stimulation. Cooke and Snider (1955) extended the above mentioned work into mechanisms involved in cerebral seizure activity. They showed in curarized animals that not only could cerebellar stimulation alter the electrical patterns of the seizuring cerebrum but that there could be generalized stoppage of the seizure with stimulation voltages as low as 7 V in a cat which required 60 V (applied to the cerebrum) to induce the seizure. Further, it was shown that stimulation of the afferent systems to the cerebellum could be effective, especially the brachium restiformis and inferior olive. Stimulation of the brachium pontis was less effective, but stimulation of the central cerebellar nuclei was more effective in inducing seizure stoppage than was excitation of the cortex. Since an activation-like pattern often appeared in the postictal period, it was tempting to believe that cerebellar excitation of the reticular activating system may have been responsible for the breakdown of the synchronized discharges observed during the seizure intervals.

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Dondey and Snider (1955) attempted to work out some of the physiological mechanisms involved in the above mentioned cerebellar investigations. They showed that a prominent slow potential shift (as much as 5 mV) could be induced in the sensory and motor areas of the cerebrum by faradization of the cerebellar cortex. This effect was also obtained in animals in which electrically induced seizure patterns were present in the cerebrum. Not infrequently the slow shift accompanied stoppage of electrical signs of seizure activity and could be recorded in nucleus ventralis lateralis thalami (VL) as well as in the cerebrum. Although it was established that VL was an intermediate center, it could not be determined that it was the only center involved, nor could the cellular mechanisms be studied. However, Cohen et ul. (1962) have done an excellent study on some of these mechanisms. They have shown that intrathalamic regulation of the cerebellocerebral projection exists. Stimuli pass from the brachium conjunctivum to VL to motor cortex and the pyramidal tract within 4 msec and can be driven as fast as 140 pulses per second. Slow frequency (7/sec) stimulation of the medial thalamic nucleus produced early facilitation and long latency prolonged inhibition of the cerebellar effects. The inhibitory effects were eliminated by 25 to 50/sec stimulation of the non-specific thalamic system. Low frequency stimulation in VL elicited augmenting responses in the motor cortex which were associated with short latency pyramidal tract discharge, but augmenting responses could not be obtained by brachium conjunctivum stimulation alone. These studies followed those of Casey and Towe (1 961) which showed that pyramidal tract discharges were augmented by electrical stimulation of the anterior vermis for about 10 msec after the testing shock. Then they were depressed for about 400 msec and were followed by a period of augmentation lasting for 1000 msec. Curiously enough, the positive component of the evoked primary response of the pericruciate gyrus was depressed while the negative component remained unaltered and the excitability of the pyramidal tract neurons did not correlate with the amplitude of the positive component. It is unfortunate that such detailed electrophysiological studies have not been extended to sensory cortices. Ablution studies. Ablation studies have uniformly shown prominent motor deficits with few subtle sensory disturbances. Indeed, it is difficult to evaluate sensory phenomena when there are such obvious signs of motor incoordination. The task is made more difficult when one realizes how few studies have been done with cerebellar lesions limited to the so-called sensory areas of the cerebellum. The most detailed observations have been made by Chambers and Sprague (1955) who removed the tuber vermis and folium vermis and noted that the cats had difficulty ‘in gauging distances properly when jumping’ as well as a diminution in ‘sensory attention’ characterized by the reduction in startle response to loud noises and a ‘marked tendency for visual attention to become fixed’. The auditory losses were more stable than the visual ones. Sprague and Chambers, in their 1959 paper, report a ‘loss’ in tactile placing reactions following lesions of the nucleus interpositus and the nucleus dentatus while lesions in the nucleus fastigii and the vermal cortex of the anterior lobe induced hyperactive tactile placing reactions. Without giving details as to how the animals were tested they also report ‘response to acoustic and visual stimuli were hypoactive’ following lesions of the tuber and folium while the same responses were References p?332-333

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‘hyperactive’ following lesions of the nucleus fastigii. Further, there was a ‘marked increase in threshold to nociceptive stimuli of ipsilateral limbs and trunk following dentate destruction’. Although the behavioral evaluation of the deficiencies reported needs much refinement, these experiments have the advantage over previous ones in that the extent of the lesions was accurately controlled. Certainly the authors do not believe there was a so-called ‘conscious loss of sensory function’. The experiments bring to mind the earlier ones of Sjoqvist and Weinstein (1942) on chimpanzees trained to discriminate weight differences. Lesions limited to either the medial lemniscus or the superior cerebellar peduncle failed to produce an enduring loss of the proprioceptive skill. However, there was a marked permanent loss when the two lesions were combined. Clearly this implies that cerebellum is directly involved in this learned sensory task. Unfortunately, no recent studies on possible sensory loss in man following cerebellar lesions have been reported and the earlier studies of Holmes (1939) are negative. Goldstein (1927), on the other hand, has reported abnormal responses in man to tactile, auditory and visual stimuli which he believes are not related to motor deficits. Electrical action with subcortical centers. Iwata and Snider (1959) studied interaction of the cerebellum and the hippocampus. They showed that cerebellar stimulation could induce an activation-like pattern in the cerebral cortex and simultaneous synchronized slow waves in the hippocampus. In a well-responding (cat) preparation, it was usually not possible to obtain one effect independent of the other and both effects were readily blocked by barbiturate anesthesia. If stronger pulses were applied to the hippocampus with resultant seizure dischargeswhich may or may not visibly invadethe cerebral cortex, stimulation of the tuber vermis would quickly stop the discharges and a fast activation-like pattern in the cortex would result. Seizure patterns could appear in the hippocampus and/or the cerebrum without invading the cerebellum although this was not always the case. Probably the most significant contribution resulting from this study was the observation that by alternately stimulating the cerebellum and hippocampus, cortical activity could be driven repeatedly toward either a low voltage activation pattern or toward a slow wave pattern, and recalls the earlier work of Cooke and Snider (1955) on stoppage of cerebral seizure activity by cerebellar stimulation. Clearly, this work opens new possibilities on concepts of cerebellar function and brings to mind the review paper of Snider (1950) in which some speculation was given to possible functional significance of the cerebro-cerebello-cerebral circuits. Why do these feedback sensory loops exist? The plea was made to broaden the older concepts which limited cerebellar functioncto that of an ‘organ of muscular synergy’. The suggestion was made that this organ may act to alter thresholds of excitability (either motor or sensory) depending on physiologic need. The research which followed in the 1950’s added only mild support to this concept. However, there has been an increased enthusiasm for electrophysiological studies on the cerebellum in the 1960’s accompanied by a new depth of understanding for the complexity of its function. Fanardjian and Donhoffer (1964) not only confirmed Iwata and Snider’s observations (1959), but pointed out that, in the unrestrained cat, hippocampal cerebellar

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evoked responses (but not cerebello-hippocampal responses) could be obtained. The intermediate centers which were involved were not studied. Dow et al. (1962) and Fernandez-Guardiola et al. (1962) extended the studies on cerebellar effects on seizure activity to include unitary analyses of cerebellar and cerebral neurons after injection of convulsant drugs and noted that cerebellar units exhibited, first, acceleration of discharge when the rest of brain was normal, then cerebral activity increased, and during the clonic period of convulsive activity cerebellar units were depressed. They confirmed Cooke and Snider’s (1955) observation that the cerebellum can inhibit cerebral seizure activity. During seizures induced by cobalt powder, total ablation of the cerebellum induced a ‘hypersynchrony of the electrocorticogram and enhanced clinical and electrocortical manifestation of the experimental epilepsy’. Intermediate centers which were involved were not studied. However, temporary cooling of the cerebellar surface produced a reversible increase in spontaneous and sensory activated epileptic manifestations which would indicate that the experiments were not subject to the usual claim that there was spread of stimulating current to the brain stem. Grigoryan (I 962) reported that neocerebellar stimulation increased the spontaneous electrical activity of contralateral sensorimotor cortex while paleocerebellar stimuI ation produced a diffuse influence on the electrocorticogram which desynchronized a synchronized cortex and vice versa. Steriade and Stoupel (1960) not only confirmed the cerebellar projection to the auditory area of cerebrum first reported by Henneman et al. (1950), but studied differences in responses in primary area versus EPI (posterior ectosylvian). They reported occlusion of effects of test and conditioning stimuli a t intervals of 10 to 70 msec similar to that reported by Snider and Sat0 (1958). They reported a dual cerebello-cerebral projection, one which goes through the reticular formation and the other through the thalamus but not the medial geniculate nucleus. Yamaguchi et al. (1963) studied the functional relationships of the non-specific thalamic nuclei and cerebellum and noted upon electrical stimulation that the centrum medianum showed the longest latency (more than 30 msec) and the reticularis thalami the shortest latency to the cerebellum (about 12 msec). The projections were widespread in the cerebellum and fast stimulation failed to alter the electrocerebellogram, whereas slow (7.5/sec) stimulation appeared to facilitate subliminal stimulation of the sciatic nerve. These experiments are difficult to interpret since thalamo-corticocerebellar circuits might have been involved. The experiments of Sawyer et a]. (1 961), while primarily concerned with autonomic changes, showed EEG changes of special interest. Stimulation of the anterior lobe induced an arterial pressor effect and an ‘aroused’ EEG in the rabbit, while similar stimuli applied to the posterior lobe occasionally produced an ‘aroused’ EEG accompanied by a depressed arterial response with a pressor rebound upon withdrawal of the stimulus. Occasional stimuli applied to the pyramis and uvula evoked a sleep-like EEG with sleep spindles accompanied by parasympathetic effects which changed to sympathetic phenomena and an aroused EEG upon withdrawal of the stimulus. These experiments recall the earlier ones of Simkina (1948) which were not well References p . 332-333

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controlled but which with chronically implanted electrodes in unrestrained animals showed drowsiness and sleep during 4 min of stimulation and arousal upon withdrawal of the stimuli. The so-called ‘rebound effects’ following stimulation of the cerebellum reported above reminds the author of the earlier work of Clark (1939), who stimulated the cerebellum in free moving animals and noted that at times there was ‘evidence of hypersensitivity to touch in the involved extremity and it will withdraw suddenly on being stroked‘. Sprague and Chambers (1959) noted that lesions in the nuclei of the vermal and paravermal zones produce symptoms which are the reciprocal of those characteristic of their respective cortices. Clark and Ward (1952) speak of a ‘march’ of motor symptoms during ‘cerebellar seizures’ which either appear during or upon withdrawal of stimulation. The work of Whiteside and Snider (1953) established unequivocally a dual projection system going cephalad to motor and sensory areas. Whether or not the ‘rebound’ effects are primarily intracerebellar cannot be decided on the basis of the present data. However, the ‘aroused EEG’ (Cooke and Snider, 1953) plus the ‘threshold to nociceptive stimuli of ipsilateral trunk and limb increases after dentate destruction’ (Chambers and Sprague, 1955) in addition to the work of Sawyer et ul. (1961) can be interpreted to mean that the cerebellum may function to help control ‘levels of excitability’ in the ascending reticular formation. However, this is not the only ascending influence of the cerebellum because, as shown by Steriade and Stoupel (1960), a cerebellar projection was demonstrable to cerebral auditory area I following bilateral lesions of the midbrain reticular formation. This second ascending system would have to pass along or near the classical sensory pathways to the thalamus. Snider and Sat0 (1958) and Snider et ul. (1964) plotted curves of excitability of the cerebello-cerebral (auditory) pathway and found with conditioning electrical shocks followed by click test shocks there was depressed excitability up to 80 msec which decreased progressively as the interval augmented. Following this diminution there was usually a period of waxing and waning of excitability for another 80 to 100 msec. These observations were made during the influences of Flaxedil medication, and when chloralosane was used there was depression of excitability for almost 400 msec. Similar studies were also made on the medial geniculate nuclei and, although some waxing and waning of excitability was seen, the electrical conditioning shock to the cerebellum usually produced depression (for 300-400 msec) to the click response, thus implicating this structure in the ascending auditory pathway. Similar studies on the visual and tactile systems have not been reported although Cohen et al. (1958) have shown anatomically that the cerebellum projected to the superior colliculus and to the ventralis posterior thalami. Parenthetically, the visual system has been the most difficult system to work with, and the reader is referred to the excellent review of Fadiga and Pupilli (1964), not only for a comprehensive review of the literature on cerebellar teleceptive responses, but also for problems concerning the visual area of the cerebellum. They point out that Meulders and Colle’s work (1963) showed that cerebellar responses evoked by electrical stimulation of the optic tract were clearly enhanced if elicited during continuous illumination of the retina. Bilateral destruction of the cerebral visual area blocked the effect. These


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studies also argue strongly for feedback circuits between the cerebellum and cerebrum which Snider stressed so firmly in the 1950 review. More recently, Munson and Snider (1965) have implicated the midbrain colliculi in feedback circuits to the cerebellum. By far the most detailed electrical investigations on the human cerebellum have been done by Wetzel and Snider (1957) and Snider and Wetzel (1965). These workers showed unequivocal changes in the human EEG following cerebellar stimulation during operative procedures under local anesthesia. The usual response when posterior cerebellar structures were stimulated was a change from low voltage a-like frequency or fast frequency patterns to high voltage slow ones. In an occasional patient a low voltage fast record resulted, but this was seen more frequently when the anterior (including anterior lobe) rather than the posterior cerebellum was stimulated. Usually trains of stimuli of 1 sec or more ranging in frequency from 10 to 300/sec were effective. However, in one patient (there were 26 tested) there were EEG changes induced by single electrical shocks. Neither disturbances of consciousness nor muscular movements were observed although it must be noted that the welfare of the subject made it necessary to keep stimulating voltages low. Thus EEG observations which have been made on lower mammals have been extended to the human. This appears to be particularly important in view of the extensive enlargement of the human cerebellum since it not only indicates that electrical excitability is possible but it also lends credence to a projection to the ‘reticular activating system’, and reemphasizes the need for answering the questions posed by Snider (1950) who postulated a ‘modulator’ function of the cerebellum on related centers depending on physiologic need of these centers to maintain certain ‘levels’ of activity. The cerebellar influence. This paper brings together recent data and older relevant data in an attempt to analyze the role which the cerebellum plays on higher sensory centers, particularly the cerebral ones. By necessity the analysis is strongly influenced by the author’s own studies. There can be little doubt that the cerebellum has widespread ascending projections to the mesencephalic and diencephalic reticular formation and when one considers both the ascending and descending connections of the cerebellum to the reticular formation then, with the exception of the major ascending sensory systems, it is difficult to find structures which have more extensive ‘input’ to the reticular formation. These observations point to the question of whether or not the cerebellum may be one of the suprasegmental regulators of reticular activity. The answer to this question, when limited to descending (motor) influences is an unqualified ‘yes’. The early work of Snider et al. (1949) showed that so-called cerebellar inhibition acted by way of the bulbar reticular pathway, and Scheibel et al. (1955) showed unequivocally that cerebellar polarization yielded three types of unitary responses in the bulbar reticular formation which could also be modified by sensory stimuli. Von Baumgarten et al. (1954) showed that the same bulbar reticular unit which was inhibited by cerebellar stimulation was also modified by stimulation of the cerebral motor area. Thus unitary studies added considerable strength to Snider et al.’s (1949) observations that strong cerebellar influences could be exerted via the reticular formation. The intriguing experiments of Granit et al. (1955) on a- and y-motor neurons, after showing that the y-motor system could act as an ‘ignition Rqferences p. 332-333

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mechanism’ to initiate movement and maintain tonus, showed that the anterior lobe of the cerebellum was a major center in regulating muscle spindle discharge rates which resulted from y-motor neuron activity and that so-called ‘y-paralysis’ could result from anterior lobe destruction. Thus the cerebellum could ‘gate’ a- or y-motor neuron firing and influence postural tonus. Furthermore, these experiments showed that both a tonic and a phasic influence could be exerted on the so-called ‘proprioceptive input’ from the extremities. Unfortunately, it has not been possible to carry out such precise studies on ascending cerebellar systems. However, if an analogy can be made from functional influences on lower spinal centers to the higher centers, it would be possible to assign a dual ascending influence, i.e., a tonic one via the ascending reticular formation and a phasic one via the so-called sensory relay nuclei of the thalamus. In the control of the interaction between these two ascending systems the low voltage fast (300/sec) basic frequency of the cerebellum could play a modulating role to establish threshold levels of sensory responsiveness which, when acting through lower centers, would find expression in a motor act. Since behavioral data are not yet available, one cannot at this time relate these observations to the mechanisms of perception although this was clearly in the authors’ mind during the stimulation studies on the human cerebellum (Snider and Wetzel, 1965) despite the fact that they were not able to collect reliable data on this phenomenon from these ‘conscious’ individuals. In unpublished studies, the author has mapped the ‘typical’ fast cerebellar-like activity through widespread cephalic regions of the reticular activating nuclei only to lose it in the thalamo-cortical projection pathways. It is tempting to believe that this fast activity exerts some as yet undefined tonic influence on the reticular activating system and this explains, in part, the unexpected findings of Iwata and Snider (1959) and is related to the variation in intrathalamic activity observed by Cohen et al. (1962) when different frequencies of stimulation were used. It is more difficult to explain the effects which these workers observed with low frequencies (below l2/sec) of stimulation. However, a phasic output from the cerebellum cannot be considered an unexpected finding in view of the numerous microelectrode studies which have been made on the cerebellar cortex. The early studies of Brookhart et al. (1951) noted clusters of single unit firing in some of their studies and Granit and Phillips (1957) speak of a sudden ‘arrest of Purkinje firing’ of several milliseconds following surface stimulation. More recently, Andersen et al. (1963) have assigned the waxing and waning of Purkinje cell activity and the silent period between to be due to inhibitory functions of the basket cells. On the other hand, Suda and Amano (1964) postulated that it might be due to nuclear feedback to the Purkinje cells. Regardless of the mechanism it is well known that single unit firing patterns in efferent tracts of the cerebellum appear in clusters (with silent interperiods) when relatively strong stimuli are applied to the cerebellar cortex or to afferent systems. Indeed this may be the mechanism whereby strong ‘inputs’ to the cerebellum are ‘broken down’ and transmitted to efferent centers. As the strong tonic firing to the cerebellar cortex weakens there may be less and less periodicity to the cerebellar output and the interaction between tonic and phasic systems becomes reestablished. In some as yet to be determined manner similar


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to this, cerebellar ‘switching’ could regulate threshold levels of sensory input to the cerebrum. Speculative as this concept has to be a t this time, it is not entirely unbelievable since it carries information now known about cerebellar influences on lower centers to the higher ones, but it must await behavioral studies in order to determine what the cerebellum is doing to so-called ‘meaningful’ information to the organism. It would be desirable, for example, to ask what is the role of the cerebellum in an animal learning to execute a motor task in response to threshold levels of sensory stimulation. The author has been impressed by the well known but seldom cited observations that many afferents to the cerebellar cortex also project to the central cerebellar nuclei. This suggests that a mechanism, when properly activated, may be available to by-pass the cortex, and that the signals of the cerebellum can be grouped into those (a) relayed directly to the nuclei from the afferent fibers, (b) relayed through the nuclei via the cortex, and (c) relayed through the cortex without synapse in the central cerebellar nuclei. If the cerebellar cortex inhibits nuclear activity, then some as yet unknown intrinsic nuclear activity needs to be studied as well as possible nuclear influences on the cerebellar cortex. Data storage and processing in this cortex needs to be worked out since speculations about a possible cerebellar role in the mechanisms of memory are becoming numerous. It is not too early to ask about a cerebellar role in the mechanisms of perception. The answers are not yet available. However, neurophysiological concepts of cerebellar function are broadening and behavioral studies are overdue. In closing, the author wishes to quote from Torgersen’s (1954) paper on the evolution of the occiput, posterior cranial fossa and the cerebellum. ‘Man is by far the youngest among the higher species and the human brain is in a stage of reorganization which tends to eliminate the cortical representation of ancient structures which are about to lose their functional significance. The net result will be an increase in relative cerebellar weight. Most probably the cerebellum is a ‘coming’ organ in human evolution. The frequent disturbances of coordination in higher functions such as speech indicate that man is in a stage of what may be called cerebro-cerebellar schizophrenia, the cortex being precociously evolved compared with the cerebellum.’ SUMMARY

(1) Well documented physiological studies indicate that the cerebellum can influence electrical activity of the sensory areas of the cerebrum. (2) There appear to be two major ascending systems from the cerebellum to the cerebrum via (a) the diffuse projection system of the thalamus, and (b) the specific relay nuclei of the thalamus. (3) The thesis is developed that the cerebellum may help to regulate levels of responsiveness in sensory systems by acting as a temporal modulator to ‘switch or gate’ in either phasic or tonic ascending activity (via the two systems noted above) depending on the ‘neurologic need’ of the organism. An analogy is drawn from what is known of descending cerebellar influences in addition to what is known of corticonuclear interaction. References p . 332-333

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REFERENCES

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MORUZLI,G., (1950); Problems in Cerebellar Physiology. Springfield, 111. Charles C. Thomas, MUNSON,J. B., AND SNLDER, R. S., (1965); Cerebral modulation of auditory and visual input to the cerebellum. Fed. Proc., 24, 206. NIIMI, K., FUJIWARA,N., TAKIMOTO, T., AND MATSUGI, S., (1962); The course and termination of the ascending fibers of the brachium conjunctivum in the cat as studied by the Nauta method. Tokushima J. exp. Med., 8,269-284. Ross~,G., (1912); Suglieffetti consequenti alla stimolazione contemporanea della corteccia cerebrale e di quella cerebellare. Arch. Fisiol., 10, 389-399. SAWYER, C. H., HLLIARD, J., AND BAN, T., (1961); Autonomic and EEG responses to cerebellar stimulation in rabbits. Amer. J. Physiol., 200, 405412. SCHEIBEL, M., SCHEIBEL, A., MOLLICA,A., AND MORUZZI,G., (1955); Convergence and interaction of afferent impulses on single units of reticular formation. J. Neurophysiol., 18, 309-331. SHERRINGTON, C. S., (1906); The Integrative Action of the Nervous System. New York, Charles Scribner’s Sons. SIMKINA,A. M., (1948); [Present views on the cerebellar influence on the autonomic and sensory functions.] Usp. sovrem. Biol., 25, 345-370 (in Russian). SJ~QVIST, O., AND WEINSTEIN, E. A., (1942); The effect of section of the medial lemniscus on proprioceptive functions in chimpanzees and monkeys. J. Neurophysiol., 5, 69-74. SNIDER,R. S., (1950); Recent contributions to the anatomy and physiology of the cerebellum. Arch. Neurol. Psychiat. (Chic.), 64, 196-219. SNIDER,R. S., AND MAGOUN,H. W., (1949); Facilitation produced by cerebellar stimulation. J. Neurophysiol., 12, 335-345. SNIDER,R. S., MCCULLOCH, W. S., AND MAGOUN,H. W., (1949); A cerebello-bulbo-reticularpathway for suppression. J. Neurophysiol., 12, 325-334. SNIDER,R. S., AND SATO,K., (1958); Some cerebellar influences on evoked cerebral auditory response. Fed. Proc., 17, 152. SNIDER, R. S., SATO,K., AND MIZUNO,S., (1964); Cerebellar influences on evoked cerebral responses. J. neurol. Sci., 1, 325-339. SNIDER,R. S., AND STOWELL, A., (1944); Receiving areas of the tactile, auditory and visual systems in the cerebellum. J. Neurophysiol., 7, 331-357. SNIDER, R. S., AND WETZEL, N., (1965); Electroencephalographic changes induced by stimulation of the cerebellum of man. Electroenceph. elin. Neurophysiol., 18, 176-1 83. SPRAGUE, J. M., AND CHAMBERS, W. W., (1959); An analysis of cerebellar function in the cat as revealed by its partial and complete destruction and its interaction with the cerebral cortex. Arch. ital. Biol., 97, 68-88. STERIADE, M., AND STOUPEL, N., (1960); Contribution a l’ktude des relations entre l’aire auditive du cervelet et l’korce ckrebrale chez le chat. Electroenceph. clin. Neurophysiol., 12, 119-136. SUDA,I., AND AMANO,T., (1964); An analysis of evoked cerebellar activity. Arch. ital. Biol., 102, 156-1 82. TORGERSEN, J., (1954); The occiput, the posterior cranial fossa, and the cerebellum. Aspecrs of Cerebellar Anatomy, J. Jansen and A. Brodal (Eds.?. Oslo, J. Grundt Tanurn Forlag, pp. 396418. VON BAUMGARTEN, R., MOLLICA,A., AND MORUZZI,G., (1954); Modulierung der Entladungsfrequenz einzelner Zellen der Substantia reticularis durch Corticofugale und Cerebellare Impulse. Arch. ges. Physiol., 259, 56-78. WALKER,A. E., (1938); An oscillographic study of the cerebro-cerebellar relationships. J. Neurophysiol., 1, 16-23. WETZEL, N., AND SNIDER,R. S., (1957); Electrical stimulation of the human cerebellum. Surg. Forum, 7,533-535. WHITESIDE, J. A., AND SNIDER,R. S., (1953); Relation of cerebellum to upper brain stem. J. Neurophysiol., 16, 397413. YAMAGUCHI, N., MASAHASHI, K., ANDO,K., YAGO,S., KUMADA, T., KAzom, K., AND SHIMAZONO, Y., (1 963); Electrophysiologicalstudies on the functional relationship between the nonspecific thalamic nuclei and the cerebellum in dogs. Electroenceph. elin. Neurophysiol., 15, 1006-1016.

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Is the Cerebellar Cortex a Biological Clock in the Millisecond Range? V. BRAITENBERG Laboratorio di Cibernetica del C.N.R., Istituto di Fisica Teorica, Universita di Napoli, Naples (Italy)

I shall summarize here an argument which I have put forward in various places (Braitenberg and Atwood, 1958; Braitenberg, 1961; Braitenberg and Onesto, 1962). It seemed to me that many morphological features of the cerebellar cortex become understandable if this structure is interpreted as a mechanism specialized for accurate measurements of time intervals. (1) The cerebellar cortex shares a cortex-like organization with the cerebral cortex, with the optic tectum, with the retina, and with some ganglia of invertebrate brains. By cortex we mean a piece of gray matter (essentially, an aggregate of synapses) in which we can easily define everywhere a vertical direction (histologically, not topographically, speaking!) with a certain synaptic organization, typical for each cortex, and a plane perpendicular to it, in which this organization is repeated, as it were, by apposition. Thus the cerebellar cortex, like the others, can be considered as a sheet of uniform thickness, containing neurons whose synaptic relations depend on their position in the thickness (in the layers) of the cortex and on their distance in the plane. The latter statement has to be refined. (2) The synaptic relations between different parts of the cortex are simply a function of metrical distance if the structure is (at least statistically) isotropic. This is not so for the cerebellar cortex (nor, for that matter, for some invertebrate ganglia), which has a lattice type structure, i.e. two distinct sets of connexions (axons and dendrites) appear in histological preparations cut in planes perpendicular to two well-defined axes of the plane, the anteroposterior and the laterolateral axis (Fig. l a and b). Hence, the functional relations between two portions of the cortex, if we may judge from the histology, besides being a function of their distance in the plane, should be a function of the angle which the line connecting the two forms with the two axes. Again, judging from the histology, their relation should be symmetrical, i.e. reciprocally identical, since statistically, to every fiber running in one direction corresponds a fiber running in the opposite direction. Thus, in summary, the synaptic relations between the elements of the cerebellar only on the distance, not on the position, and (c) along the anteroposterior axis symmetrical relations of another kind, again depending only on the distance. ( 3 ) Comparative invariance. What has just been said is valid for all animals in which

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a cerebellum can be recognized (by its relative position with respect to other parts of the brain), i.e. for all vertebrates. Moreover, we can assemble a more detailed generalized cerebellum as the set of properties which have been found to be valid for all cerebella examined. (a) There are two main layers of the cerebellar cortex, the molecular layer and the granular layer. (b) All fibers leaving the cerebellar cortex are axons of Purkinje cells. These are characterized by their dendritic trees which occupy a region of the molecular layer flattened in the laterolateral direction (i.e. the branching is mainly in a sagittal plane) and not overlapping with each other (Fig. 2). (c) There are two sets of incoming fibers, each distributed throughout the cerebellar cortex, the mossy fibers and the climbing fibers of Cajal. (d) Each climbing fiber makes synaptic contacts with the dendrites of Purkinje cells in a very narrow region of the molecular layer, perhaps with one Purkinje cell only. (e) Each mossy fiber makes synaptic contacts in a delimited region of the granular layer. (f) The synaptic pathway from the mossy fibers to the output Purkinje cells is indirect via the granular cell and parallel fiber system (Fig. 3). These fibers run in the laterolateral direction and relay the same mossy fiber signal from the granular layer to a row of at least 100 Purkinje cells (to the right and to the left of the point of entrance of the mossy fiber) in the molecular layer. These facts (a) to (f) are summarized in the diagram of Fig. 4 which represents a fragment of a laterolateral row of Purkinje cells with the associated input, output and relay fiber systems. (8) Where there is folding of the cerebellar cortex, the folds run almost exclusively in the laterolateral direction. Thus the flat dendritic trees of Purkinje cells are kept parallel as much as possible. (4) Size and shape. I may refer to Nieuwenhuys (this volume) for a review of the astonishing variation in the gross shape of cerebella in various vertebrates. The human cerebellar cortex, ideally stretched and flattened in such a fashion as cortex must follow the pattern of symmetry which we discover in the histology: (a) along the vertical axis (from layer to layer) we expect asymmetrical relations, (b) along the laterolateral axis we expect symmetrical relations of one kind, depending to make the maximal anteroposterior extension as well as the maximal laterolateral width correspond to actual measurements, has the shape outlined in Fig. 5 . It is about 1.20 m long, a sheet about two thirds of the length of a human figure, folded and compressed into the small tentorial cavity. Mammalian cerebella, reviewed by Riley (1928) all seem to fit into the scheme established by myself for cat, dog, monkey and human cerebella (Fig. 6). Neglecting the folding, the cerebellar cortical sheet forms two lateral baglike extensions, the hemispheres, and a median posterior extension, the vermis. While a large part of the cerebellar cortex is continuous over the midline between the right and left half, without any interruption of the cortical structure, the posterior vermis is truly separated from the lateral portions. References p . 346

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a

n

Fig. 1. Low-power (about 70 x ) micrographs of the molecular layer of the monkey cerebellum as it appears in transversal cuts (a) and sagittal cuts (b). Golgi preparations. Dendritic trees of Purkinje cells are marked Pje. They appear as cypress trees in Fig. la and as widely fanning trees in Fig. lb. Some parallel fibers have also been stained by the (unsystematic) Golgi staining procedure. They appear as horizontal streaks in Fig. l a and are invisible in the sagittal cut of Fig. lb, where they run perpendicular to the plane of the picture.

CEREBELLAR CORTEX, A B I O L O G I C A L C L O C K ?

337

338

V. BRAITENBERG

Fig. 2. Tangential section through the molecular layer of the cerebellar cortex of man. Golgi preparation. The micrographshows, so to say, a top view of the dendritic trees ofseveralPurkinje cells to show their flattening in the transversal direction (which corresponds to the horizontal co-ordinate of this picture).

If the cerebellar cortex is projected on to a system of Cartesian coordinates in such a fashion as to orient all folia (parallel fibers) parallel to the horizontal axis, we obtain Fig. 7. Here the horizontal lines represent the length of the folia as measured on a human cerebellum. The anteroposterior distances are not isometric, because of the curvature of the sheet, but are represented by the number of horizontal lines. Each line represents 10 folia and corresponds to about 3 cm of cortex in the anteroposterior direction .


339

Fig. 3. Transversal cut through the molecular layer of the human cerebellum. Golgi preparation. Some parallel fibers are stained and can be seen originating as horizontal branches of fibers ascending from the granular layer (lower third of the picture). Magnification is the same as in Fig. 2. The two photographs should be mentally superimposed since they represent elements that co-exist in the molecular layer.

The curvature evident in Figs. 6 and 7 imposes exceptions on the regularity of the histological lattice previously described. In fact, evidently not all folia (hence, not all parallel fibers) can be followed through from the left to the right margin of the cerebellar sheet, since there is convergence as well as divergence of folia, implying that not all parallel fibers are truly parallel. It is interesting to observe the following regions of the cerebellar cortex (Fig. 8): A, an anterior region, in which the number of folia is

Fig. 4. Scheme of the connections in the generalized cerebellum as described in the text. Ka = climbing fibers; Kt = terminations of climbing fibers; Pd = dendritic fields of Purkinje cells; Pa = axons of Purkinje cells; Gd = dendritic fields of granular cells; Ga = axons of granular cells; Gt = terrninations of granular cells (parallel fibers); Ma = mossy fibers; Mt = terminations of mossy fibers. From Braitenberg, Naturwissenschaften, 48 (1961) 489. References p . 346

340

V. B R A I T E N B E R G

h

3

Fig. 5. Representation of the sheath of the human cerebellar cortex in which the maximum longitudinal and transversal extensions are respected. From Braitenberg and Atwood, 1958.

highest on the midline and decreases laterally; B, a region in which on each side there is a maximum of folia which decreases laterally as well as toward the midline; C, two posterior extensions of B, in which all folia terminate medially; D, an unpaired poste-

Fig. 6. Simplifiedtopographic diagram of the right half of the mammaliancerebellar cortex, neglecting the folding. From Braitenberg and Atwood, 1958.

CEREBELLAR CORTEX, A BIOLOGICAL C L O C K ?

34 1

Fig. 7. Planar diagram of the right half of the human cerebellar cortex derived from measurements of the length of the folia, and counts of folia in sagittal cuts. Three horizontal lines represent 10 folia. From Braitenberg and Atwood, 1958.

rior extension characterized by short folia crossing the midline and interrupted laterally. The key for the identification of these regions with the established anatomical nomenclature is given in Braitenberg and Atwood, 1958. This pattern implies that in some regions the cerebellar cortex can operate con-

Fig. 8. Characteristic zones of the cerebellar cortex based on the pattern of foliation. From Braitenberg and Atwood, 1958. References p. 346

342


tinuously between right and left, while in other regions its operation must be limited to one side only. (5) Some histological invariants. The following statements are based on measurements obtained from human cerebella, but were found to be valid for monkeys and dogs as well. What I intend to collect here, however, is not evidence of invariant structure in the zoological variety, but rather some quantitative relations between different parts of the cerebellar cortex which are invariant under the transformations imposed by the folding and other local variations of the cerebellum. (a) The molecular layer has a constant thickness everywhere (Fig. 9). This implies

Fig. 9. Low-power micrograph of a sagittal cut through the vermis. Human cerebellum, Nissl preparation.

that the topographical relations of the elements within the molecular layer have functional importance and are kept fairly constant. This fact has no counterpart in the cerebral cortex, where no layer is unaffected by the variations in thickness between the summit and the depth of the convolutions (Von Economo and Koskinas, 1925; Bok, 1959). (b) On the contrary, the thickness of the granular layer varies greatly, from a minimum in the valleys to a maximum on the summits. However, the ratio of the volume of the molecular layer to that of the granular layer is found to be approximately constant, if the cerebellar cortex is divided into portions defined by sections which follow the histological vertical direction, i.e. the direction of fanning of the incoming and outgoing fibers (Figs. 9 and lo). Since the density of granular cells in the granular layer is not subject to noticeable variations, this fact implies that there is a constant number of granular cells per volume of molecular layer, and therefore also a constant number of parallel fibers per volume of molecular layer. As a corollary we may derive


343

that the length of the parallel fibers must be everywhere the same, unless we invoke a variation in thickness of the parallel fibers to compensate possible variation of length, so that the volume of the molecular layer remains constant per volume of granular layer. But from electron microscopy (Palay, this meeting) we learn that, at least in rats, the thickness of parallel fibers is remarkably constant. (c) When the cerebellar cortex is cut into portions defined as in Fig. 10, also the

Fig. 10. Diagram of a convolution of the mammalian cerebellum to show constant ratios between volume of molecular layer, number of Purkinje cells and volume of granular layer. From Braitenberg and Atwood, 1958.

number of Purkinje cells per volume of molecular layer, hence per volume of granular layer and per number of granula, is found to be constant. In fact on the summit of the convolutions the Purkinje cell bodies are packed much more densely than in the valleys, as can easily be seen on tangential cuts (Fig. 11). The ratio, number of granular cells : number of Purkinje cells is between about 3000 : 1 and 7000 : 1, theuncertainty being due to systematic errors in the procedure of counting granula. There are about 300 Purkinje cells in 1 mm2 of cortical surface, and 15,000,000 Purkinje cells in the whole cerebellar cortex. The number of granula is 900,0W2,100,000 per mm2 of cortical surface, and between 1010 and 1011 in the whole cortical sheet of the human cerebellum. (d) In any particular region of the cerebellar cortex, the ratio, volume of the molecular layer : number of afferent and efferent fibers is approximatelyconstant. This can be deduced from measurements of the surface of the molecular layer in a certain region and the cross section of the corresponding white core. Since this is true both for small portions of a convolution and for large complex convolutions, it implies that there is no population of ‘U-fibers’, as there is in the cerebral cortex, i.e. the white core contains only afferent and efferent fibers. A population of associational U-fibers of equal density everywhere would make the ratio cortex :white core smaller for small convolutions than for larger ones. Taken together, these histological invariants (a)-(d) strengthen our impression that the molecular layer of the cerebellum performs some simple operation on the incoming signals, in which the afferent fiber systems, the granular cell-parallel fiber neurons and the Purkinje cells are involved as a unitary mechanism. This is particuReferences p. 346

344


Fig. 11. Some tangential cuts through the layer of Purkinje cells (indicated by small circles). Human cerebellum. No regular arrangement of Purkinje cell bodies is apparent. They are more crowded on the surface of convolutions (a,b) than in the depth of the furrows (c,d).

larly plausible on the basis of the constant numerical relations between these elements. Also, the constant thickness of the molecular layer and the absence of folding in the direction across that of the folia make it likely that distance within the molecular layer plays a much more radical functional role than for instance in the cerebral cortex, where the folding seems to preserve neighborhood relations, but not distances in any direction. (6) Local variation. The ratio cortex :white core, which we have described as constant within any narrow region of the cerebellar cortex, is the only histological feature which we have observed to vary from region to region (see however Brodal, this volume). In fact, the ratio, surface of cortex : cross section of the corresponding white core was found to vary between 66 : 1 and 420 : 1. These ratios seem to be correlated with the pattern of convergence and divergence of folia, shown in Fig. 7. There is more white matter per unit surface of the cortex in the center of the hemispheric expansion than on the margin, and more near the midline in the anterior cerebellum than in the lateral portions, etc. (7) Teleological interpretation. If the structure of the cerebellar cortex is viewed in the same spirit in which we would analyze an unknown machine, the following inference can readily be made. Since it seems that the same signal in the mossy fiber system will reach hundreds of Purkinje cells in one laterolateral row through the parallel fiber system, there is a multiplicity of pathways between the input and the output, which appears to be wasted unless one invokes the different delays which are due to the different distances of Purkinje cells from the locus of the arriving signals. The same argument applies, of course, if one considers on the contrary the convergence of many afferent mossy fiber pathways on to one and the same Purkinje cell. The importance of delays is quite plausible, since parallel fibers are very likely the longest very thin fibers discovered in brains. Thin fibers conduct slowly, according to the well known law established by Gasser, but are generally shorter than thick fibers, so that in most situations their importance as delay elements may be neglected or may just be subsumed together with other delays in the concept of ‘synaptic delay’. Here the situation is different, since conduction in the laterolateral direction

CEREBELLAR

CORTEX, A

BIOLOGICAL

CLOCK?

345

is practically only due to the system of parallel fibers in which conduction velocity, extrapolated from data obtained from peripheral nerves, should be about 0.5 mm/ msec. Thus the delay furnished by one parallel fiber, for which a length of the order of 10 mm may be assumed (Braitenberg and Atwood, 1958), is of the order of 20 msec, while a chain of parallel fibers reaching from one side of the (human) cerebellum to the other (about 100 111111) will give a total maximum delay of 200 msec. A number of structural traits of the cerebellar cortex become understandable if its importance as a system of delay lines is recognized. Flattening of the dendritic trees of Purkinje cells provides for a better temporal definition of the arrival of impulses in the parallel fiber system. Absence of foldfng keeps the flat dendritic trees of Purkinje cells parallel to each other and therefore parallel to an approaching wave front in the parallel fiber system. Multiplication of lines in the parallel fiber system (one afferent mossy fiber relays excitation to many parallel fibers) may be a compensation for the small amount of excitation carried by individual parallel fibers. The small caliber of the parallelfibers with consequent slow conduction is a necessity when relatively long delays have to be accommodated without undue enlargement of the geometrical dimensions. Climbingfibers may be understood as the mechanism through which individual Purkinje cells are set to fire at the arrival of a signal in the parallel fiber system, and may therefore represent the necessary implementation for a delay line which can be tapped at various places for different delays. According to this interpretation, we may write the functional dependence of the excitation of a Purkinje cell P(t) at time t from the state of climbing fibers C(t) and of mossy fibers M(t) in the same laterolateral row y of the cerebellar cortex as

indicating with x and y the positions of the elements in the laterolateral and anteroposterior coordinate respectively, with v the conduction velocity in parallel fibers and with d the distance between the mossy fibers and the Purkinje cell in question. The form of the functional dependence f is left unspecified. (8) Possible importance of a chronometric device in the brain. It is not difficult to invent possible uses for such a mechanism in the neurological machinery, but direct neurophysiological support is lacking at present. (a) Delays may be interposed between the various components of rapid voluntary movements. Critical timing with a precision approaching milliseconds must play an important role, as one can easily convince himself by examining rapid movements in handwriting, sports, music, etc. The cerebellum, according to our model, provides a temporal definition better than milliseconds. (b) Delays are essential components of devices which perform auto- and crosscorrelation of functions of time, i.e. operations of fundamental importance in the detection of signals in noise, in the detection of movement, etc. (c) Delays may be used to measure other delays, e.g. between successive spikes in References p . 346

346


spike sequences emitted by a neuron, for which a principle of pulse interval coding has sometimes been postulated (MacKay, 1962; see also my review in this volume). ACKNOWLEDGEMENT

The research reported in this document has been sponsored in part by the 6570th Medical Research Laboratories under Grant AF EOAR 65-44 through the European Office of Aerospace Research (OAR) United States Air Force. REFERENCES

BOK,S. T., (1959); Histonomy of the Cerebral Cortex. Amsterdam, Elsevier. BRAITENBERG, V., (1961); Functional interpretation of cerebellar histology. Nature, 190, 539-540. BRAITENBERG, V., AND ATWOOD, R. P., (1958); Morphological observations on the cerebellar cortex. J . comp. Neurol., 109, 1-34. N., (1962); The cerebellar cortex as a timing organ. Discussion of an BRAITENBERG, V., AND ONESTO, hypothesis. Proc. 1st. Znt. Conf. Med. Cybernet., Naples, Giannini, pp. 1-19. MACKAY,D., (1962); Self-organization in the time domain. Sef’Orgunizing Systems. M. C . Yovits, G. T. Jacobi and G. D. Goldstein, Editors, Washington, Spartan Books, pp. 3748. RILEY,H. A., (1928); The mammalian cerebellum. Arch. Neurol. Psychiat., 20,l-34. VONECONOMO, C., AND KOSKINAS, G. N., (1925); Die Cytoarchitektonik der Hirnrinde des erwachsenen Menschen. Vienna and Berlin, Springer.

347

Author Index * Achucarro, N., 226 Addens, J. L., 17 Addison, W. H. F., 29,47,48 Adrian, E. D., 142, 151, 153, 155 Ahlborn, F., 17 Albe-Fessard, D., 152, 160, 309 Altman, J., 150 Amano, T., 330 Andersen, P., 212,252-259, 263, 315, 330 Ando, K., 327 Angaut, R., 287 Anthony, J., 53-55 Appelberg, B., 306, 309, 311, 313 Arduini, A., 285,296,299 Ariens Kappers, C. U., 17,22, 23,25-30,40,47, 48, 50, 59, 60, 67, 69, 75, 76, 79, 82, 86, 157 Aronson, L. R., 40 Assheton, J. G., 39 Atwood, R. P., 193, 334, 340, 341, 343, 345 Ban, T., 327, 328 Bard, D. S., 285 Barnard, J. W., 193-195, 197, 315 Batini, C., 152,285,290,293, 294, 302, 303, 313 Beccari, N., 68 Berkelbach van der Sprenkel, H., 39 Birch-Andersen, A., 174 Blackstad, T., 146, 147, 263 Bodenheimer, T. S., 212 Boeke, J., 176 Bohm, E., 146, 154 Bok, S. T., 80, 342 Bolk, L., 65, 94, 99-101, 104, 105, 153 Bone, Q., 82 Bowsher, D., 165,287 Boyce, R., 79, 81, 82 Braitenberg, V., 193, 334-346 Brandis, F., 74, 75, 80 Bremer, F., 263, 302, 322 Brittin, G. M., 285 Brodal, A., 8-11, 26, 74, 77, 79, 80, 83, 85, 94, 95, 109, 111, 113, 116118, 124, 125, 127-130, 135-173, 220, 283-290, 292, 296, 3W305, 309, 311-313 Brookhart, J. M., 251, 330 Brouwer, B., 79 Brunner, H., 9,76, 121-123, 127, 128 Brusa, A., 296,299

*

Buell, M. V., 246 Burckhardt, R., 50 Burgess, P. R., 220 Burr, H. S., 48 Busch, H. F. M., 109 Buser, P., 251 Cajal, S. Ramon y, 9, 12, 59, 66, 67, 75, 76, 81, 87, 156, 174-176, 178,180, 182,185, 187, 188, 193-195, 197, 199, 201, 204, 208, 212, 214, 219, 220, 226, 247, 253, 268 Calma, I., 308 Campbell, B., 142 Carpenter, M. B., 109, 113, 150, 151, 185, 285, 287, 307 Carrea, R. M. E., 109, 115, 116, 125, 199, 314 Casey, K. L., 325 Catois, E. H., 44, 46 Chambers, W. W., 80, 141, 161, 285, 287, 293, 302, 303, 306, 308, 311-313, 323, 325, 328 Chang, H. T., 144,153 Chang, M. W., 246 Charlton, H. H., 48 Clark, M. B., 18, 20 Clark, S. L., 328 Clarke, H., 127, 129, 144, 154 Cogan, D. G., 247 Cohen, D., 161,285,287,302,312, 325,328, 330 Colle, J., 328 Colonnier, M., 188 Combs, C. M., 146, 155 Comolli, A., 8, 74, 94, 95 Cooke, P. M., 162, 322,324,326-328 Cotti, E., 159, 160, 285, 296, 298-301, 312 Courville, J., 162 Cowen, D., 236 Craigie, E. H., 78-80, 180 Crawford, J. M., 263, 265 Crosby, E. C., 25-27, 48, 50, 59, 60, 67, 69, 74, 77, 79, 80, 82, 157 Cuptdo, R. N. J., 116 Curtis, D. R., 263, 265 Dahl, H. A., 263 Dahl, V., 174 Davis, R., 309 De Castro, F., 229, 231 Deiters, O., 185

Italics indicate the pages on which the paper of the author in these proceedings is printed.

348

AUTHOR INDEX

De Lange, S . J., 62-64, 67 Demole, V., 122 De Robertis, E., 247 De Vito, R. V., 296, 299 Dijkgraaf, S., 3 Dogie], A. S., 75, 178, 180 Dondey, M., 325 Donhoffer, H., 326 Doty, E. J., 75 DOW,R. S., 7, 65, 67, 140-142, 150, 155, 193, 251,255,263,283,285,292,293,299,302,327 Drablnrs, P. A., 136138 Dutta, C. R., 174-225 Eager, R. P., 115, 125, 152, 220, 283, 285, 296, 315 Eccles, J. C., 175, 193, 201, 212, 219, 220, 252-263,265,268-277,279,280,315,330 Edinger, L., 8, 9, 13, 22, 26, 27, 74,82, 94, 95 Eldred, E., 151, 155 Elliott, K. A. C., 246 Engberg, I., 307 Estable, C., 204, 208 Fadiga, E., 150, 151,155,328 Faihnhs, J., Ram6n y, 228 Fanardjian, V. V., 326 Fangel, C., 151 Feferman, M. E., 145, 147 Fernandez-Guardiola, A., 327 Fernandez-Moran, H., 174 Flood, P. R., 263 Flood, S., 138, 162 Forstronen, P. F., 194 FOX,C. A., 174-225 Franz, V., 29, 30, 37, 43, 44,47 Freeman, F. R., 80 Frenkel, B., 77, 7%82 Friede, R. L., 228, 231, 246 Friedlander, A., 77, 80, 82 Fujiwara, N., 323 Fulton, J. F., 302 Furshpan, E. J., 212,262 Furukawa, T., 212,262 Gans, A., 122 Gassel, M. M., 304 Gerebtzoff, M. A., 322 Gerschenfeld, H. M., 247 Gesell, R., 262 Glees, P., 135 Gloor, P., 257 Gogstad, A. C., 147, 162 Goldman, M. A., 220 Goldstein, K., 48, 326 Golgi, C., 180, 195 Goodman, D. C., 65, 66,80,283 Gordon, S.,Jr., 174, 176, 178, 185,232 Granit, R., 251,252, 278, 292, 295, 313, 329

Grant, G., 103, 109, 111, 137, 138; 142-144* 152-154, 163 Gray, E. G., 174, 176, 178, 182, 185, 188, 195, 197,263 Grigoryan, R. A., 327 Grillo, M. A., 174, 176, 178, 185, 232 Grundfest, H., 142,253,263 Guha, S., 228,231,240 Guillery, R. W., 188 Gygax, P. A., 135 Ha, H., 292 Hager, H., 174, 176 Haller, B., 26 Haller v. Hallerstein, V., 21, 22 Hallett, R. E., 283 Harnberger, A., 246 Hamlyn, L. H., 188,263 Hhmori, J., 259, 262, 263, 268 Hampson, J. L., 151, 155, 156, 282, 283, 302, 304,306,308,309 Hanna, G. R., 151 Hannet, F. I., 304 Hamson, C. R., 156, 282, 283, 302, 304, 306, 308,309 Hashiramoto, S., 305, 306 Hassler, R., 323 Hauglie-Hanssen, E., 283, 304, 313 Hayashi, M., 94, 107-109, 117, 121, 140 Heier, P., 17-20 Held, H., 176, 182,220 Heller, I. H., 246 Henle, J., 195 Henneman, E., 162, 322, 324, 327 Herndon, R. M., 174,176,194, 199 Hemck, C. J., 5,6,9,29,30,41,46,55,57-59,67 Hilliard, J., 327, 328 Hillman, D. E., 174-225 Hindenach, J. C. R., 61, 66 Hinman, A., 287 Hirschberger, W., 174, 176 Hochstetter, F., 108 Hocke Hoogenboom, K. J., 29, 33, 35, 36,41 Hohman, L. B., 127 H&ivik, B., 109, 111, 113, 138, 139, 141, 150 Holm, I.,13,82 Holmes G., 326 Holmgren, B., 329 Holmgren, N., 13, 51-53 Holmqvist, B., 154, 263 Hongo, T., 158,290,306 Horel, J. A., 80 Horsley, V., 127, 129 Housepian, E. M., 325, 330 Houser, G. L., 25 Huber, G. C., 25-27, 48, 50, 59,60, 67, 69, 74, 77, 79, 80, 82, 157 Hydkn, H., 185, 246

AUTHOR I N D E X

Ingram, W. R., 304 Ingvar, S., 63, 64, 67-70,77, 113 Tto, M., 158, 166, 219, 253, 261, 290, 296, 299, 309, 313, 314 Iwata, K., 326, 330 Jakob, A., 2, 107-109, 114, 117, 121, 136, 140, 185,214 Jansen, J., 8-11,13,26,74,77,79,80,82,83,85, 94, 95, 116, 122, 124, 125, 127-129, 137, 138, 14&142, 145, 148-152, 155, 156, 158, 159, 161-163, 165, 176, 220, 283, 285-288, 292, 296, 301-304, 309, 311-315, 323 Jansen, Jr., J., 150, 151, 155, 162, 163, 303, 323 Janskowska, E., 299, 300, 306 Jeleneff, A., 17 Sen, M. K., 246 Johnston, J. B., 2-5, 10, 15, 18, 19, 33-35, 87 Kapphahn, J. I., 246 Kato, M., 287, 290 Kaufrnan, R. P., 285,287 Kawai, N., 296, 299, 313, 314 Kazorna, K., 327 Kidd, G. L., 308 Kingsbury, B. F., 55-57 Kolliker, A., 87 Korey, S. R., 246 Korneliussen, H. K., 122, 124, 139 Kosaka, K., 309, 311, 314 Kosary, I. Z., 306, 313 Koskinas, G. N., 342 Koyama, Y.,283 Kreht, H., 55, 57, 59, 61 Kristiansen, K., 8, 9, 74, 77, 79, 85, 150, 152 Kulenkarnpff, H., 226 Kumada, T., 327 Kuntz, A., 226 Kurati, T., 283 Kuru, M., 283 Kuwabara, T., 247 Lamarche, G., 155 Langelaan, 5. W., 106, 107, 114, 117, 121 Larsell, O., 2, 5-7, 9, 10, 13-15, 17-22, 24, 26, 47, 53, 55, 57-76, 82-85, 87,98-101, 103, 105, 113,138,140,143,150,151 Lissman, H. W., 39 Llinas, R., 193,201,212, 219, 220,256,258-262, 265, 268-277, 279, 280, 297, 299, 315 Lorente de N6, R., 220 Lowry, 0.H., 246 L~yning,Y., 252-258,263 Lubin, A. J., 178 Lugaro, E., 204,219 Lund, S., 290,293, 294,297, 300,314 Lundberg, A., 300, 306, 307

349

McCulloch, W. S., 329 McGee-Russell, S. M., 174, 176, 178, 185, 232 MacKay, D., 346 Maffei, L., 152,156,16&162,287-289,305,309 310,312, 314 Magni, F., 283 Magoun, H. W., 311,324, 329 Manni, E., 327 Marburg, O., 113 Marchiafava, P. L., 304 Masahashi, K., 327 Mascitti, T. A., 158, 292 Masek, B., 227 Massion, J., 160, 309, 311 Massopust, L. C., 193,204 Matsugi, S., 323 Maurer, W., 237 Mawas, J., 226 Mehler, W. R., 145, 147 Mettler, F. A., 109, 115, 116, 125, 178, 199, 314 Meulders, M., 328 Miale, I. L., 108 Millot, J., 53-55 Miskolczy, D., 163, 178 Mizuno, S., 328 MoIlica, A., 329 Mori, K., 290 Morin, F., 155 Morton, P. A., 329 Moruzzi, G., 142, 152, 155, 156, 251, 263, 283, 285, 290, 292, 293, 295, 296, 299, 302, 303, 306, 313, 314, 324, 329, 330 Mugnaini, E., 164, 188, 194 Munson, J. B., 329 Nageotte, J., 226 Namikawa, A., 305, 306 Nansen, F., 226 Nauta, W. J. H., 135, 144, 145, 147 Nieuwenhuys, R., 1-93 Niimi, K., 323 Nikitin, M. ,115 Niklowitz, W., 199 Novikoff, A. B., 227 Nyberg-Hansen, R., 158, 160,283,287,291, 305 Nyby, O., 150, 155 Obata, K., 158, 166,219,253,261,290,296,309, 313,314 Obersteiner, H., 195 Ochi, R., 253,261,296, 313, 314 Oehlert, W., 237 Ogawa, T.,122-125, 127-130, Okada, Y.,158,290 Olson, S., 174 Onesto, N., 334 Orchen, M., 246 Oscarsson, O., 142-144, 153, 154, 163

350

A U T H O R IN D E X

Palay, S. L., 174, 176, 178, 185, 195, 197, 199, 214, 232,246,263, 343 Palmgren, A., 36, 37 Papez, 5. W., 79 Pearson, A. A., 15, 18-20, 29, 36,40,41, 47-49 Petras, J. M., 283 Phillips, C. G., 251, 252, 278 Pompeiano,O., 124,141,151,152,155,157-162, 165,252,282-321 Popoff, s., 220 Potanos, J. N., 236 Probst, M., 125 Pupilli, G. C., 150, 151, 155, 328 Purpura, D., 253,263, 325, 330 Rajkovits, K., 109, 115, 116, 199, 201, 268 Ranson, S. W., 304 Reissig, M.,109, 115, 116, 199, 314 Retzius, G., 185, 187 Ricci, G. F., 308, 312 Riese, W., 136 Riley, H. A., 335 Rio-Hortega, P. del, 229, 237, 247 Roberts, N. R., 226 Robertson, J. D., 212 Robins, E., 246 Rothig, P., 55, 57, 59, 61 Rosen, I., 154 Rosiollo, L., 178 Rossi, G., 322 Rougeul, A., 251 Ruch, T. C., 144, 153 Rudolph, G., 228 Rudeberg, S., 17, 18, 22, 29, 67, 76, 86, 97, 98, 108, 122, 124 Saetersdal. T. A. S., 74, 85 Saito, T., 17 Sanders, E. B., 9, 75, 77, 78, 80 SBntha, K. v., 137 Sasaki, K., 193,201,212,219,220,256,258-262, 265,268-277,279,280,290,305,306,309,315 Sato, K., 327, 328 Saugstad, L. F., 151 Sawyer, C. H., 327, 328 Schaper, A., 17, 25, 36,43, 44,46, 87 Scheibel, A. B., 115, 199, 201, 258, 268, 329 Scheibel, M. E., 115, 199, 201, 258, 268, 329 Schroeder, A. H., 176, 185, 194,229,231 Schultze, B., 237 Scott. Th. G., 118 Shanklin, W. M., 62, 66, 67, 69 Sherrington, C. S., 322 Shimazono, J., 74-82, 327 Siegesmund, K. A., 174-225 Sidman, R. L., 108 Simkina, A. M., 327 Simpson, J. T., 65, 66 Sjoqvist, O., 326

Smith, D. E., 246 Smith, K. R., Jr., 174 Smith, M. C., 114, 142 Snider, R. S., 142, 150, 151, 153, 155, 162, 178, 220,25 1, 311, 322-333 Soriano, V., 302 Sotelo, C., 226-250 Sperti, L., 293, 313 Sprague, J. M., 80, 141, 161, 285, 287, 292, 293, 302,303,306,308,311-313,323,325,328 Staal, A., 283 Stage, D. E., 212 Stella, G., 293, 306, 313 Stendell, W., 30, 47 Steriade, M., 327 Sterzi, G., 28 Stevens, G. H., 287 Stieda, L., 195 Stoupel, N., 327, 328 Stowell, A., 142, 150, 151, 153, 322 Streeter, G. L., 77 Suda, I., 251, 330 Sulkin, N. M., 226 Suzuki, N., 30 Szabo, T., 152 SzentBgothai, J., 109, 115, 116, 174, 178, 187, 188, 201,204, 212,253,258,259,262-265,268 Taber, E., 150 Takahira, E., 251 Takimoto, T., 323 Talwar, G. P., 246 Tanaka, T., 290,309 Terwitliger, E. H., 304 Terzuolo, C. A., 297, 299, 314 Thomas, D. M., 285,287 Thomas, K., 299 Thomas, R. C., 287,290 Thulin, C.-A., 305 Tolani, A. J., 246 Torgersen, J., 331 Torvik, A., 141, 147, 148, 158. 283, 284 Towe, A. L., 325 Tower, D. B., 246 Toyama, K., 309, 311, 314 Trendelenburg, W., 77 Tretjakoff, D., 17, 18, 20 Tsukahara, N., 309, 311, 314 Tuge, H., 47-50 Ubeda-Purkiss, M., 193, 204 Uddenberg, N., 144 Udo, M., 311, 314 Ule, E., 199 Vachananda, B., 144, 153,283 Van der Horst, C. J., 29,30,32,41,51-53 Van der Loos, H., 185 Van de Voort, M. R. J., 95-98

AUTHOR I N D E X

Van Gehuchten, A., 187 Van Hoevell, J. J. L. D., 9, 17, 22, 25, 67, 86 Van Rossum, 114, 115 Verhaart, W. J. C., 283 Von Baumgarten, R., 329 Von Economo, C., 342 Voogd, J., 8, 80, 94-134, 157, 161, 285, 288,290 Voorhoeve, J. J., 20, 22, 25-27, 53, 201, 212, 251-267,248-281,3 15,330 Walberg, F., 124, 127, 130, 146, 147, 150-152, 155-159,164-166,188 283,285-288,296,301, 303, 304, 312-314 Wald, F., 247 Walker, A. E., 322, 324 Wallenberg, A., 28, 77, 82 Waltman, B., 292, 295, 313 Ward, J. W., 328 Warrington, W. B., 79, 81, 82 Wegmann, R.,228,231,235,237,240342,245 Weidenreich, F., 9, 122-125, 127-130 Weinstein, E. A,, 326 Welch, R. B., 283 Weston, J. K., 9, 65-69 Westrum, L. E., 283, 304, 313

351

Wetzel, N., 329, 330 Whiteside, J. A., 311, 323, 328 Whitlock, D. G., 8, 77-80 Willis, W. D., 283 Wilson, J. H., 327 Wilson, V. J., 220,263, 265, 281, 290 Winkler, C., 114 Wolf, A., 236 Wolstencroft, 3. H., 283 Woodbume, R. T., 42, 68,78 Woolsey, C. N., 156, 282, 283, 302, 304, 306, 308, 309, 3 15 Wiistenfeld, E., 226 Yago, S., 321 Yamaguchi, N.. 327 Yoshida M., 158, 166, 219, 253, 261, 290, 296, 299, 309, 313, 314 Yoshimura, K., 79, 82 Zanchetti, A., 308, 312 Zatti, P., 293, 313 Zecha, A., 8,75, 79, 82 Zeiss, F. R., 304

352

Subject Index Amphibians, cerebellum comparative survey, 55-61 histogenesis, 5-7 Anura, cerebellum, development, 59-61 Archicerebellum, mammals, 140 Astroglia, and cerebellum, molecular Iayer, 228, 229 Aves, cerebellum, comparative survey, 69-82 embryology, 69-74 fibre connections, 76-82 histology, 74-76 Basket cells, and cerebellum, molecular layer, 201-214 and Purkinje cell, inhibition, 253-258 Bergmann cells, and enzyme activity, 234-239 Bergmann fibers, and Purkinje cell, 198, 199, 235 Cerebellum, acoustic areas, 150, 151 afferent connections, 109-1 17 afferent systems, 85 anatomy, comparative, 1-93 and cerebral sensory areas, 322-331 ablation studies, 325, 326 ascending systems, 322-331 electro-anatomicalstudies, 322-324 functional studies, 324, 325 comparative anatomy, 1-93 cortex, Bergmann cells, 234-239 and biological clock, 334-346 and cerebrum, comparative invariance, 334, 335 synaptic relations, 334 time intervals, 334 and chronometric device, 345,346 electron microscopy, 174-221 fibres, structure, 136-1 39 flocculus, structure, 136-139 glial cells, 228-233

Golgi study, 174-221 granular layer, 10-12, 174-193, 239 histological invariants, 341-344 and inhibitions, experiments, 252, 253 molecular layer, 10-12, 193-220 primates, 174-221 responses, and climbing fibres, 268-280 potential fields, 268-271 structure, 136-139 teleologicalinterpretation, 244,245 white core, local variation, 344, 345 white matter, enzyme activity, 240-243 and Deiters’ nucleus, 288-304 inhibition, 299, 300 development, 1-12 efferent connections, 117-130 face region, 151 fibre connections, anatomical studies, 135-168 comparative aspects, 94-1 31 fibre systems, 109-117 function, definition, 2 functional localization, 139-153 functional organization, 135-168 granular layer, attachment plaques, 182-185 axons, 184, 185 dendrites, 180, 182, 183 Golgi cells, 185 inhibition, 258-261 island elements, 176-178 microspines, 182, 183 mossy fibres, 178-180 rosettes, 179-181 and hippocampus, interaction, 326-329 histogenesis, 7 inhibition, morphological aspects, 262, 263 pharmacological aspects, 263-265 mammals, morphology, 101-1 05 subdivision, 140, 141 molecular layer, basket cells, 201-214 fibre background, 192-195

SUBJECT INDEX

Cerebellum, molecular layer, Purkinje cells, 192, 195-220 stellate cells, 201-214 morphogenesis, 1-12 mossy fibres, and Golgi cell, inhibition, 258-261 neuroglia, 226-248 enzyme activity, 228 nuclei, phylogenesis, 17 pathways, cerebellofugal, and Purkinje cells, 9, 10 postural responses, localization, 309- 312 projections, and Deiters’ nucleus, 284-286 functional organization, 282-316 and red nucleus, 287,288 somatotopic organization, 283-288 and spinal cord, 282-316 and Purkinje cells, neurobiotaxis, 17 rat, dentate nucleus, 243-246 gray matter, 234-239 white matter, 240-243 and red nucleus, 304-312 somatotopical localization, 153-163 stimulation, and rebound effect, 328 structure, comparative aspects, 94-131 surface, characteristics, 99-109 growth, 99-109 nomenclature, of Bolk, 99-101 of Larsell, 99-101 transformation, 99-109 subdivisions, 99-101 vertebrates, shape, 335-342 size, 335-342 visual areas, 150, 151 Chondrichthyes, cerebellum, afferent systems, 26, 27 development, 23 efferent systems, 27, 28 granular layer, 25 histology, 23-25 molecular layer, 25 Chondrostei, cerebellum, afferent systems, 34-36 efferent systems, 36 granular layer, 33 molecular layer, 33 morphogenesis, 33, 34 Purkinje cells, 33 structure, 33

Cyclostomes, cerebellum, comparative survey, 13-20 morphology, 13-20 Deiters’ nucleus, and cerebellar control, 288-304 cerebellar inhibition, and reticular formation, 299, 300 and cerebellar projections, 284-286 somatotopical projection, 157-1 60 stimulation, and moloneurones, 290-293 Dentate nucleus, enzyme activity, 243, 244 Dipnoi, cerebellum, fibre connections, 53 morphogenesis, 50, 51 Enzymes, activity, and cerebellum, gray matter, 234-239 white matter, 240-243 and dentate nucleus, 243, 244 Granular layer, and cerebellar cortex, 335, 339, 343 Golgi cell, inhibition, and granular layer, 258-261 and mossy fibres, 258-261 Hippocampus, and cerebellum, interaction, 326-329 Holostei, cerebellum, fibre connections, 47-50 morphogenesis, 40,41 Inhibitory systems, intracerebellar, 25 1-266 morphological aspects, 262,263 pharmacological aspects, 263-265 Lamprey, cerebellum, afferent systems, 18, 19 efferent systems, 19, 20 histological differentiation, 10 histology, 3-5, 15-20 Mammals, cerebellum, afferent connections, 109-1 17 corticonuclear projection, 125-1 30 development, autoradiographic studies, 108 early -, 95-99 efferent connections, 117-130 efferent projection, 121-125

353

3 54

SUBJECT I N D E X

Mammals, cerebellum, fibre connections, comparative aspects, 94-131 fibre degeneration, 101-103, 112-1 14 fibre systems, 109-117 folial patterns, 113-1 15 focal differentiation, 106-109 histogenesis, 11. 12 longitudinal division, 117-121 morphology, 101-105 olivo-cerebellar projectiop, 116, 117 ontogenesis, 10-12 palaeocerebellum, 140 structure, comparative aspects, 94-131 subdivision, 94,95 Molecular layer, and cerebellar cortex, 335--339, 343 Neocerebellum, mammals, 140 Neuroglia, histochemical aspects, 228, 234246 interfascicular, function, studies, 247, 248 metabolism, enzyme pattern, 247 morphological aspects, 226-233 morphology, techniques, 228 neuronal, enzyme activity, 247 Oligodendroglia, cerebellum molecular layer, 229-233 Osteichthyes, cerebellum, comparative survey, 28-55 Palaeocerebellum, mammals, 140 Polypteriformes, cerebellum, histology, 3 1-33 Pontocerebellum, afferent connections, 155-163 modes of ending, 136-166 efferent connections, 150, 151 fibres, connections, 147-151 mammals, 140, 147, 150 Purkinje cells, axons, 214220 and Bergmann fibres, 198,199 and cerebellar cortex, 335-339,343 and cerebellum, efferent systems, 9-12 histogenesis, 108, 109 histology, 86, 87 neurobiotaxis, 17

climbing fibres, 199-202 stimulation, 278, 279 and Deiters’ neurones, IPSP, 261,262 Purkinje cells, distribution, chondrostei, 33 inhibition, and basket cells, 253-258 morphological aspects, 262, 263 and stellate cells, 253-258 and inhibitory interneurones, 258, 259 and intracerebellar nuclei, IPSP, 261, 262 IPSP, recording, 254 responses, 271-278 smooth branches, 196199 spiny branchlets, 192, 195-197 stimvlation, recording, 272-278 Rebound effects, and cerebellum, stimulation, 328 Red nucleus, and cerebellar influences, 304-312 and cerebellar projections, 287, 288 stimulation, and motoneurones, 304-309 Reptilians, cerebellum, comparative survey, 61-69 fibre connections, 67-69 structure, microscopical -, 65-67 Reticular formation, and Deiters’ nucleus, cerebellar inhibition, 299, 300 Sensory areas, cerebral, and cerebellum, 322-331 ablation studies, 325, 326 anatomical studies, 322-324 ascending systems, 322-331 functional studies, 324, 325 and reticular formation, 323 Spinal cord, and cerebellar projections, functional organization, 282-31 6 somatotopic organization, 283-288 Spinocerebellum, afferent connections, 141-147, 153-155 modes of ending, 163-166 efferent connections, 151-153, 156163 fibres, topography, 142-147 mammals, 140,147, 150, 156 Stellate cells, and cerebellum, molecular layer, 201-214 and Purkinje cells, inhibition, 253-258

S UB JE CT I N D E X

Teleostei, cerebellum, afferent systems, 4 3 4 6 histology, 4 3 4 6 morphogenesis,2 6 2 8 , 36-38, 4 I , 43 Urodela, cerebellum, afferent systems, 58, 59 efferent systems, 59

fibre connections, 57-59 Vertebrates, cerebellum, fibre connections, 7-9 Vestibulocerebellum, afferent connections, 141 efferent connections, 151-153 mammals, 140, 150

355

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Progress in Brain Research Volume 25 The Cerebellum

Progress in Brain Research Volume 175 Neurotherapy

Progress in Brain Research Volume 176 Attention

Progress in Brain Research Volume 157 Reprogramming the Brain

Progress in Brain Research Volume 09 The Developing Brain

Progress in Brain Research Volume 119 Advances in Brain Vasopressin

Progress in Brain Research Volume 29 Brain Barrier Systems

Progress in Brain Research Volume 47 Hypertension and Brain Mechanisms

Progress in Brain Research Volume 22 Brain Reflexes

Progress in Brain Research Volume 29 Brain Barrier Systems

Progress in Brain Research Volume 125 Volume Transmission Revisited

Progress in Brain Research Volume 44 Understanding the Stretch Reflex

Progress in Brain Research Volume 05 Lectures on the Diencephalon

Progress in Brain Research Volume 16 Horizons in Neuropsychopharmacology

Progress in Brain Research Volume 15 Biology of Neuroglia

Progress in Brain Research Volume 156 Understanding Emotions

Progress in Brain Research Volume 35 Cerebral Blood Flow

Progress in Brain Research Volume 13 Mechanism of Neural Regeneration

Progress in Brain Research Volume 132 Glial Cell Function

Progress in Brain Research Volume 19 Experimental Epilepsy

Nanoneuroscience and Nanoneuropharmacology (Progress in Brain Research, Volume 180)

Progress in Brain Research Volume 26 Developmental Neurology

Progress in Brain Research Volume 162 Neurobiology of Hyperthermia

Progress in Brain Research Volume 164 From Action to Cognition

Progress in Brain Research Volume 41 Integrative Hypothalamic Activity

Progress in Brain Research Volume 18 Sleep Mechanisms

Progress in Brain Research Volume 66 Peptides and Neurological Disease

Progress in Brain Research Volume 26A Correlative Neurosciences: Fundamental Mechanisms

Progress in Brain Research Volume 28 Anticholinergic Drugs

Genetic models of schizophrenia, Volume 179 (Progress in Brain Research)

Progress in Medicinal Chemistry, Volume 25

Progress in Brain Research Volume 25 The Cerebellum

Progress in Brain Research Volume 175 Neurotherapy

Progress in Brain Research Volume 176 Attention

Progress in Brain Research Volume 157 Reprogramming the Brain

Progress in Brain Research Volume 09 The Developing Brain

Progress in Brain Research Volume 119 Advances in Brain Vasopressin

Progress in Brain Research Volume 29 Brain Barrier Systems

Progress in Brain Research Volume 47 Hypertension and Brain Mechanisms

Progress in Brain Research Volume 22 Brain Reflexes

Progress in Brain Research Volume 29 Brain Barrier Systems

Progress in Brain Research Volume 125 Volume Transmission Revisited

Progress in Brain Research Volume 44 Understanding the Stretch Reflex

Progress in Brain Research Volume 05 Lectures on the Diencephalon

Progress in Brain Research Volume 16 Horizons in Neuropsychopharmacology

Progress in Brain Research Volume 15 Biology of Neuroglia

Progress in Brain Research Volume 156 Understanding Emotions

Progress in Brain Research Volume 35 Cerebral Blood Flow

Progress in Brain Research Volume 13 Mechanism of Neural Regeneration

Progress in Brain Research Volume 132 Glial Cell Function

Progress in Brain Research Volume 19 Experimental Epilepsy

Nanoneuroscience and Nanoneuropharmacology (Progress in Brain Research, Volume 180)

Progress in Brain Research Volume 26 Developmental Neurology

Progress in Brain Research Volume 162 Neurobiology of Hyperthermia

Progress in Brain Research Volume 164 From Action to Cognition

Progress in Brain Research Volume 41 Integrative Hypothalamic Activity

Progress in Brain Research Volume 18 Sleep Mechanisms

Progress in Brain Research Volume 66 Peptides and Neurological Disease

Progress in Brain Research Volume 26A Correlative Neurosciences: Fundamental Mechanisms

Progress in Brain Research Volume 28 Anticholinergic Drugs

Genetic models of schizophrenia, Volume 179 (Progress in Brain Research)

Progress in Medicinal Chemistry, Volume 25

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