Progress in Brain Research Volume 04 Growth and Maturation of Brain

P R O G R E S S IN B R A I N R E S E A R C H VOLUME 4 GROWTH A N D MATURATION O F THE BRAIN

PROGRESS I N BRAIN RESEARCH

ADVISORY BOARD W. Bargmann

E. De Robertis

J. C. Eccles J. D. French

H. HydCn

J. Ariens Kappers

S. A. Sarkisov

Kiel Buenos Aires Canberra

Los Angeles Goteborg Amsterdam Moscow

J. P. SchadC

Amsterdam

T. Tokizane

Tokyo

H. Waelsch N. Wiener J. Z. Young

New York Cambridge (U.S.A.) London

PROGRESS I N BRAIN RESEARCH VOLUME 4

GROWTH AND MATURATION O F THE BRAIN EDITED B Y

D O M I N I C K P. P U R P U R A College ofphysicians and Surgeons, Columbia University, New York, N . Y. (U.S.A.) AND

J. P. S C H A D 6 Ceniral Institute for Brain Research, Amsterdam (The Netherlancis)

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

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LONDON

1964

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NEW YORK

ELSEVIER PUBLISHING COMPANY

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JAN VAN GALENSTRAAT, P.O.BoX

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This volume contain3 a series of lectures delivered during an interdisciplinary workshop on G R O W T H A N D M A T U R A T I O N OF T H E B R A I N

which was held at the castle “De Hooge Vuursche” from 7-9 September, 1962 This meeting was organized by the Department of Developmental Neurology from the Central Imtitute for Brain Research, Amsterdam (The Netherlands)

L I B R A R Y OF C O N G R E S S C A T A L O G C A R D N U M B E R

WITH

180

ILLUSTRATIONS AND

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63-19890

TABLES

ALL RIGHTS RESERVED T H I S B O O K 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 A N Y FORM, I N C L U D I N G P H O T O S T A T I C O R M I C R O F I L M FORM, W I T H O U T WRITTEN PERMISSION FROM THE PUBLISHERS

List of Contributors

V. BONAVITA, Department of Neurology, University of Palermo, Palermo (Italy).

K. BRIZZEE,Department of Obstetrics and Gynecology, University of Nebraska, College of Medicine, Omaha, Nebr. (U.S.A.). E. COLON,Central Institute for Brain Research, Amsterdam (The Netherlands).

M. HIEROWSKI, Department of Neurology and Department of Physiological Chemistry, Medical Academy, Poznan (Poland).

E. M. HOUSEPIAN, Departments of Anatomy and Neurological Surgery, College of Physicians and Surgeons, Columbia University, New York. T. HUMPHREY, Department of Anatomy, University of Pittsburgh, Pennsylvania (U.S.A.). X. KHARETCHKO, Department of Anatomy, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.). R. LEVI-MONTALCINI, Washington University, St. Louis, Mo. (U.S.A.). S. L~VTRUP, Institute of Neurobiology, University of Goteborg, Goteborg (Sweden).

R. MARTY,Centre de Recherches neurophysiologiques de l'Association Claude Bernard, Paris. C. R. NOBACK,Departments of Anatomy and Neurological Surgery, College of Physicians and Surgeons, Columbia University, New York. G. D. PAPPAS,Departments of Anatomy and Neurological Surgery, College of Physicians and Surgeons, Columbia University, New York. D. P. PURPURA, Departments of Anatomy and Neurological Surgery, College of Physicians and Surgeons, Columbia University, New York. . TH. RABINOWICZ, Laboratory

of Neuropathology, Institute of Pathology, Lausanne

(Switzerland). S. SARKISOV, Brain Institute, Moscow. J. P. SCHADI?, Central Institute for Brain Research, Amsterdam (The Netherlands).

J. SCHERRER, Institut National d'Hygikne, H8pital de la Salpstrikre, Paris. R. J. SHOFER, Departments of Anatomy and Neurological Surgery, College of Physicians and Surgeons, Columbia University, New York.

H. VANBACKER, Central Institute for Brain Research, Amsterdam (The Netherlands). J. VOGT,Department of Electrical Engineering and Graduate College, University of Nebraska, Lincoln, Nebr. (U.S.A.). M. WENDER, Department of Neurology and Department of Physiological Chemistry, Medical Academy, Poznah (Poland).

Other volumes in this series:

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

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

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

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

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

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

Volume 8: Biogenic Amines Edited by Harold Himwich and Williamina Hirnwich

Volume 9: The Developing Brain Edited by Williamina Himwich and Harold Himwich Volume 10: Structure and Function of the Epiphysis Cerebri Edited by J . Ariens Kappers and J. P. Schadk Volume 11 : Organization of the Spinal Cord Edited by J . C. Eccles and J. P. Schadk Volume 12: Physiology of Spinal Neurons Edited by J . C. Eccles and J. P. Schade

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

Volume 1 4 : Degeneration Patterns in the Nervous System Edited by M. Singer and J. P. Schade

Preface

No single mode of attack upon problems of morphogenesis is adequate. Experimental methods yield the most decisive evidence, and these require adequate knowledge of anatomical structure. The last is the contribution of comparative anatomy and comparative embryology, and both of these must be functionally interpreted to be fruitful.

C . J. HERRICK: The Bruin of the Tiger Salamander

TWOfactors have contributed to the renaissance of interest in the developing nervous system in the past decade: (1) the application of new operational methods in neurocytology, neurochemistry and neurophysiology to studies of the immature brain; and (2) the growing recognition of the value of ontogenesis as an analytical tool in neurobiological research. The expansion of investigative work in the phylogenesis and ontogenesis of the nervous system has brought into sharp focus the inevitable problem of establishing effective channels of communication within and across various disciplines. Considerable progress in this direction was made at a symposium sponsored by the Netherlands Central Institute for Brain Research. The site selected for this meeting was as much an inspiration of Dr. SchadC and his associates as was the meeting itself. Undoubtedly the all-to-few days spent at the castle ‘De Hooge Vuursche’ outside of Amsterdam will continue to conjure up pleasant memories for all participants long after the details of experimental data are forgotten. But that some of the record of the symposium be preserved is one of the purposes of this volume. The reports and discussions presented during ‘working-sessions’ of this symposium held from September 7-10, 1962 are published here in the hope that the casual reader as well as the specialist will glean from them something of the scope of the meeting. Unfortunately nothing can be done to retrieve the record of the many hours of informal discussions which greatly contributed to the success of the conference. This success is not to be measured solely in the character of formal reports. The success of any conference is to be measured by the degree to which participants allow themselves to become intellectually and emotionally involved in the work and enthusiasm of others. In this respect the Amsterdam meeting was entirely successful. The reader will appreciate that the several topics in this volume are illustrative of the validity of Herrick‘s dictum that ‘no single mode of attack upon problems of morphogenesis is adequate.’ Thus studies of brain mitochrondria and the molecular evolution of enzyme systems are as much a part of the story of brain maturation as the migration of neurons, development of dendrites, glia, synapses or reflex activities and behavior patterns. The difference is in the level of organization. Clearly the key problem remains to define the pathways by which successive levels of organization, molecular to multicellular may be satisfactorily approached, one from the other. And when such pathways are specified, one may be flanked by trim hedges and luxurient gardens and lead to an old castle a few miles from Amsterdam. August 1963

DOMINICK P. PURPURA

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Contents

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V

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VII

List of Contributors Preface

Events in the developing nervous system Rita Levi-Montalcini (St. Louis, Mo.)

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1

The evolutionary aspect of the integrative function of the cortex and subcortex of the brain S. Sarkisov (Moscow) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30

The cerebral cortex of the premature infant of the 8th month Th. Rabinowicz (Lausanne, Switzerland) . . . . . . . . .

39

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Some correlations between the appearance of human fetal reflexes and the development of the nervous system 93 Tryphena Humphrey (Pittsburgh, Pa.) . . . . . . . . . . . . . . . . . . . . . . . . Postnatal changes in glia/neuron index with a comparison of methods of cell enumeration in the white rat K. R. Brizzee, J. Vogt and X. Kharetchko (Omaha, Nebr.) . . . . . . . . . . . . . . . 136 Quantitative analysis of neuronal parameters in the maturing cerebral cortex J. P. Schade, H. Van Backer and E. Colon (Amsterdam) . . . . . . . . Electron microscopy of immature human and feline neocortex G. D. Pappas and D. P. Purpura (New York) . . . . . .

. . . . . . . . 150

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Comparative ontogenesis of structure-function relations in cerebral and cerebellar cortex D. P. Purpura, R. J. Shofer, E. M. Housepian and C. R. Noback (New York) . . . . . Critkrcs de maturation des systkmes affkrents corticaux R. Marty et J. Scherrer (Paris) . . . . . . . . . . Brain mitochondria Ssren Lsvtrup (Goteborg, Sweden)

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187

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Molecular evolution of lactate dehydrogenase in the developing nervous tissue Vincenzo Bonavita (Palermo, Italy) . . . . . . . . . . . . . . . . . . .

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Enzymatic mechanism of the protein biosynthesis in the developing nervous system M. Wender and M. Hierowski (Poznan, Poland) . . . . . . . . . . . . . . .

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281

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Author index. Subject index

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Events in the Developing Nervous System R I T A LEVI-MONTALCINI Washington University, St. Louis, Mo. (US.A . )

Ever since the most talented and perceptive of all neurologists, Ramon y Cajal, made available the silver technique for the selective impregnation of nerve cells, the nervous system of all phyla from celenterates to primates has become the object of an extensive and everlasting exploration for generations of neurologists. If the study of the different phyla provided a key for the understanding of the complexity of this system in higher forms, man included, it was the study of the developing nervous system which provided the Ariadne’s thread to unraveling the labyrinthic complexity of nerve centers and nerve fiber tracts in the mature brain. Again it was the intuitive mind of Cajal which realized the enormous potentialities of this approach. To him and to his students we owe the most penetrating and rigorous series of investigations on the developing vertebrate nervous system. This analysis served as the basis for Cajal’s fundamental work, The Histology of the Nervous System, which still represents today the most complete and authoritative study of this type (Cajal, 1909). At the same time as Cajal and co-workers concentrated their studies on the embryonic nervous system, other brilliant scholars such as Edinger, Ariens Kappers, Herrick, Crosby, and their students concentrated on the comparative analysis of the brain structures in the vertebrate phyla. Between the end of the past century and the first decades of this century, such a wealth of facts became known on the developing nervous system, as well as on its evolutionary changes, as to make one wonder if this field of investigation had not now been exhausted. It was with this outlook and a feeling of awe and curiosity for this most complex system that we first approached it two decades ago, shortly after the voice of the great master Cajal had become silent and while other outstanding scholars such as Ariens Kappers, Herrick and Crosby were still actively pursuing their search on the evolution of brain structures. The object of our first analysis was the chick embryo. As we became more and more familiar with this object and scrutinized its developing nervous system day after day and week after week, we became aware of the fact that this system had still not revealed more than a few of its many facets and that the essence of its developing mechanisms remained to be uncovered. Today, 20 years later, with the experience which comes from daily contact with the object of interest and with the dispassionate approach of the mature mind, the writer feels thatzwe have still taken little more than a first glance at the endless complexity and intrkacy of the differentiating nervous system. Thanks to the joined efforts of a large number of investigators, we have References p . 25/26

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possibly acquired a more precise knowledge of the sequence of events which take place during the growth and differentiation of the nervous system, but little progress has been accomplished in the understanding of the mechanisms which control these events. It is with this feeling of inadequacy and the awareness of the number of unsolved problems still ahead of us, that I propose to discuss some of the events which take place in the developing nervous system. I will limit myself to the presentation and discussion of some of the aspects which we explored and attempted to analyze in the past years. They will be considered in reference to the time pattern of their occurrence and also in reference to the opportunity they offer to obtain some information on the agents which control and direct growth and development of nerve cells and nerve centers. Two main processes which take place in early phases of the differentiation of the nervous system will be considered, namely the migration and degeneration of dzfferentiating nerve cells. We will limit our presentation to these areas not because we believe that the events to be presented may be of more interest than other events taking place in the developing nervous system, but because of our personal experience and interest in these aspects of the nervous system. Most of the observations to be reported are based on the analysis of these developmental processes in the chick embryo. They were supplemented by a few observations in mammals, specifically in the mouse embryo. Hence when not specifically stated, we refer to the chick embryo. We will then briefly consider the results of some experimental work aimed at the analysis of the above processes. M I G R A T O R Y P A T T E R N S I N T H E D E V E L O P I N G N E R V O U S SYSTEM

The capacity of differentiating nerve cells to migrate during the early stages of their differentiation has been known for a long time. Little, however, has been known about the extent and magnitude of these processes and the role they play in molding nerve centers and wiring them together. We will consider here some of these migratory movements which were the object of a close inspection in the chick embryo. The first to be described take place outside the central nervous system. They concern the cells which will form some of the cephalic ganglia and have their origin in the dorso-lateral and epibranchial placodes. Other important migratory movements occur at the same time outside of the spinal cord. These consist of the migration from the neural crest of the precursors of sensory spinal and sympathetic nerve cells. These migratory movements were considered at length during the past years and are well-known in all their aspects (Van Campenhout, 1931; Horstadius, 1950; Tello, 1925; Yntema and Hammond, 1947); for this reason they will not be further considered here. Next we will consider the active displacements which occur in the central nervous system. Migration from dorso-lateral and epibranchial placodes The origin of some of the cephalic ganglia from ectodermal placodes has been as-

THE D E V E L O P I N G N E R V O U S SYSTEM

3

certained through a number of investigations (Yntema, 1942, 1944; Levi-Montalcini, 1946). It is not within the aim of this presentation to report on the experimental evidence which provided the basis for the present knowledge of the origin of these ganglia. We refer the reader to the literature in this field and, in particular, to the extensive and exhaustive experimental analysis performed recently by Hamburger (1961) on the dual origin of the trigeminal ganglia in the chick embryo. Here we will consider some aspects of these migratory movements which give origin to 3 cephalic ganglia: the geniculate ganglion of the VIIth nerve, the petrosum and the nodosum which belong respectively to the IXth and Xth nerve. The observations to be reported below are based on the inspection of a large number of 2- to 6-day-old chick embryos stained with the De Castro modification of the Cajal technique (Levi-Montalcini, 1949) and sectioned serially in transversal, sagittal, and frontal sections. Although we referred many times during the past years to these studies, they were never the object of a detailed description. The first evidence for active cell migration from the dorso-lateral and epibranchial placodes can be obtained in embryos 2+ to 3 days of age. Since the maturation of the

Fig. 1. Epibranchial placode:(P) giving origin to the ganglion nodosum in a 3-day-old chick embryo. The migrating neuroblasts appear darkly stained and have short processes at opposite poles. 9

embryonic structures takes place along a rostro-caudal gradient, the migration from the dorso-lateral placode which gives origin to the geniculate ganglion precedes to some extent the migration from the 4th and 5th epibranchial placodes which give origin to the petrosum and nodosum ganglia. It was the migratory movements of these last two ganglia which offered the best condition for a close inspection. Immediately beneath the epithelial thickening which represents the epibranchial placodes, one sees in 23- and 33-day-old embryos a large number of swarming cells which show intense silver affinity. They have two short opposito-polar prolongations which also appear intensely stained with silver (Figs. 1, 17). Between the end of the 3rd and the 4th day, these cells move more and more away from the epithelial placodes where they were first seen and aggregate in two large References p . 25/26

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oval-shaped agglomerateswhich can be easily identified as the petrosum and nodosum ganglia (Fig. 18). At the end of the 4th day the two ganglia are well-delimited and stand out against the pale background of surrounding mesenchymal cells. One gets the impression that all neuroblasts have now assembled together and that the further size increase of these ganglia results from development and growth of individual nerve

d

Fig. 2. The drawings illustrate the morphological changes of the cerebellar Purkinje cells in successive developmental stages of the chick embryo: (a) 8 days, (b) 10 days, (c) 14 days, (d) 17 days.

cells rather than from addition of new units, And yet all around the ganglia one sees a large number of scattered cells intensely stained with silver, with one or two short, dark filament-like prolongations. In the subsequent days the silver affinity of these cells weakens and eventually one cannot recognize them from the surrounding mesenchymal cells. The question arises as to the fate of these cells and their identity. If one has to judge on the basis of the affinity for silver, one would consider these cells as neuroblasts but this view finds no support in the observations mentioned above. Two alternatives should be considered. The first is that not all cells which react positively to silver should be identified as nerve cells. In accepting this viewpoint we would, however, deny any significance to the intense silver affinity of nerve cells as contrasted with the lack of affinity of adjacent mesenchymal cells. Also we would have to deny any value to the opposito-polar prolongations of these cells which further add to the notion that these are indeed nerve cells. The inspection of hundreds of

5

T H E D E V E L O P I N G N E R V O U S SYSTEM

embryos impregnated with the Cajal-De Castro silver technique confirmed us in the belief that nerve cells, but not mesenchymal cells, react positively to silver. The second alternative would be that neuroblasts are produced in excess by the ectodermal placodes. Once the ganglia have reached a given size, they become incapsulated and the neuroblasts still present in the area no longer find access to the main ganglionic

T6

Fig. 3. Diagrammatic representation of the segregation and subsequent migration of the thoracic visceral column in the chick embryo. TI, 3-day-old chick embryo. The somato-motor and the visceromotor cells form a compact column. Tz,44-day-old embryo. Beginning of migration of preganglionic neurons. TB,T4, Ts, Ts illustrate the shift of the migrating cells from a ventro-lateral to a mediodorsal position in embryos of 5 (Ta),6 (T4), 7 (Ts) and 8 (Ts)days of incubation. (From R. LeviMontalcini, J . Morph., 86, 1950.)

agglomerates. They become dispersed and eventually die off, or possibly are otherwise utilized. Neither the first nor the second alternative were submitted to test and at present we must say that the problem of the identity and fate of these cells remains to be established. Migratory movements in the central nervous system

Between the 4th and the 9th day of incubation, the developing nervous system of the chick embryo is the stage of the most extensive, elaborate and complex cell movements. While the brain vesicles and the spinal cord increase considerably in size, but do not undergo radical changes in shape, neuroblasts in the thousands engage in most sectors of the neural tube in long migrations which last for 2-3 days. To the student who is still not familiar with this object, these complex migratory movements appear hopelessly complicated and chaotic. It is only the hour after hour inspection of the developReferences p . 25/26

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ing neural tube and higher brain centers in transversal, frontal and sagittal sections which persuade the observer that these movements are not at all chaotic but on the contrary are highly organized and rigidly patterned like complicated military maneuvers displacing hundreds of moving units along different routes, under the control of the central headquarters. Like soldiers, or like armies of ants or termites, the

BR

TH-L

LS

a

b

C

Fig. 4. Diagrammatic representation of the migratory movement of the thoracic visceral column as it appears in frontal sections of chick embryos fixed at (a) 4 days, (b) 6 days, (c) 7 days, (d) 8 days. BR, brachial segment; LS, lumbo-sacral segment; TH-L, thoraco-lumbar segment. (From R. LeviMontalcini, J. Morph., 86, 1950.)

neuroblasts move in compact rows one after the other in long trails where each slender and spindle-shaped cell follows the other. A characteristic common to all migrating nerve cells is their intense silver affinity and the elongated and slender shape of their bodies which stand out sharply against the pale background which forms the dense matrix of the developing nervous system. Each cell is provided with a short apical, and a long caudal filament which trails behind the body and actually represents the axon of the migrating cell. As a large number of cell units move simultaneously in different and many times opposite directions, one sees in many sectors of the brain stem or higher brain centers lines of cells crossing each other and then pursuing their own path which might bring them to entirely different areas of the developing nervous system. This intense traffic reaches its maximal expression toward the end of the 5th and the beginning of the 6th day

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THE DEVELOPING NERVOUS S Y S T E M

(q-\-] .

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...-., ...... r

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Fig. 5. Diagrammatic frontal and transversal representation of the segregation of the accessory nucleus from the main nucleus of the VIth nerve in the chick embryo. (a), (b), (c): stereographic views of the VIth nuclei at 5,6. and 12 days. In (a), the nuclei appear as two columns; in (b), the arrows indicate the migration of neuroblasts from the main nuclei in a lateral direction; in (c), the migration is completed and the two accessory columns are formed. (a’), (b), (c’): the same process as viewed in transversal sections. VI A, main nucleus; VI B, accessory nucleus. (From The Nature of Biological Diversity, edited by Dr. J. Allen. Copyright, 1963. McGraw-Hill Book Co. Used by permission.)

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of incubation. Immediately after, most of the migrating cells have reached their destination and settle down in the area where they will be lodged for their life. As the cells become sedentary and lose the capacity for migrating, they undergo significant morphological changes. The previously elongated cells with two opposito-polar prolongations round up and the apical filament is reabsorbed or transformed into a number of short dendrites which vary in number and length depending on the cell type. The most impressive of these transformations of the short apical filament is illustrated by the cerebellar Purkinje cells. Even before their long migratory movements became known (Uzman, 1960; Levi-Montalcini, 1963) Cajal was impressed by the sudden transformation of these cells once they have settled in the cerebellar cortex (Cajal, 1929). Figs. 2, 9-11 give an idea of these complex morphological changes which transform the immature nerve cells into full fledged nerve units. Once a migratory group has reached its terminal settlement it rounds up in most instances in a compact cell agglomerate. An exception to this rule is represented by the abovementioned cerebellar Purkinje cells (Fig. 6). These cells in fact do not aggregate, but display a distribution in a single row where each cell is parallel to the neighboring cells. In all other instances which we explored in detail, such as the formation of the preganglionic columns, the segregation of the cephalic motor nuclei, the shaping of the telencephalic centers, the cells aggregate in columns or in round or oval nuclei as mentioned above. It is not in the purpose of this paper to present a detailed description of all, nor for that matter of a number, of the migratory movements taking place in the developing nervous system. They were already the object of previous reports (Levi-Montalcini, 1942, 1950) and they were presented in a current review article (Levi-Montalcini, 1963). Here we will consider only two of these migratory movements which are particularly impressive and exemplify the main characteristics of these processes. The first occurs in the neural tube while the second takes place in the brain stem. Migration of the preganglionic viscero-motor column in birds and mammals

If one inspects the spinal cord of a chick embryo between the stages of 3 and 4 days, one sees a slender column of nerve cells in the ventro-lateral aspect of the tube extending from the cervical to the lumbo-sacral segments. Observations performed in previous stages showed that this column, easily identifiable as the motor column, results from the migration of cells which move laterally from their first site of origin in the ventral part of the basal plate of the ependyma which lines the central canal. At 4 days, the column presents almost the same size from the cervical to the lumbosacral segment of the spinal cord (Fig. 31). It is toward the middle of the 4th day that differences become evident in the cervical, brachial, thoracic, lumbar and sacral segments of the spinal cord. The developmental patterns characteristic of each segment were the object of a detailed report in a previous paper (Levi-Montalcini, 1950). Here we will consider only the thoracic segment, while in a subsequent section we will consider the differentiation of the same column in the cervical segment of the neural tube.

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Until the middle of the 4th day, the motor column in the thoracic segment of the neural tube appears to consist of a rather homogeneous cell population. If one traces, however, the peripheral distribution of motor fibers which leave the neural tube in this trunk segment, one realizes the composite nature of this apparently uniform cell population. About 9 of the nerve fibers emerging with the thoracic ventral roots make

E CELLS

KlNJE CELLS

BDAYS

IIDAYS

ELLS INJE CELLS

14 DAYS

19 DAYS

Fig. 6. Diagrammatic representation of the migratory movements of the Purkinje cells in the cerebellum of the chick embryo. (From The Nature of Biological Diversity, edited by Dr. J. Allen. Copyright, 1963. McGraw-Hill Book Co. Used by permission.)

connection with the primary sympathetic trunks which at this stage are still located in close apposition to the aorta. The residual t sends fibers to the trunk muscles of the same level. Hence about 2 of the motor nerve cells in the thoracic segment of the neural tube differ from the other 4 in their peripheral distribution. From the above connections with the sympathetic chain ganglia they were identified as the preganglionic visceral cells. At 44 days these nerve cells sharply segregate from the other motor cells. The segregation of the two cell components is foreshadowed by a loosening of the compact texture of the motor column. Soon after, the of the entire group shift to a more medial position. Between the end of the 4th and the 7th day, the cells which have segregated from the residual of the motor column, move in compact cell rows in a ventro-dorsal direction. During the migration the cells are elongated

+

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in shape and exhibit the intense silver affinity characteristic of migrating nerve cells. It is thanks to the dark, almost black shade of the neuroblasts during their entire displacement that it is possible to inspect closely each of the thousand units during the subsequent phases of their migration. Since this process presents in fact identical features in all embryos, one can reconstruct it by examining embryos fixed at slightly

b Fig. 7. The illustration of the medio-dorsal (MD) and the ventro-lateral (VL) cell populations in a brachial ganglion (a) and in a thoracic ganglion (b) of a 5-day-old chick embryo. The arrow in (b) points to some of the degenerating neurons in the ventro-lateral sector of this ganglion.

different developmental stages between the 4th and the 7th day. We refer to the drawings and to the series of microphotographs (Figs. 3,4,19-24) for a visualization of this impressive migratory movement which reminds one of the migration of termites or ants in its orderly fashion and in the close contact which keeps the individual units in reciprocal connection all along the route they cover in the 23-day period.

Fig. 8. The illustration of the transplantation of cervical spinal cord to the thoracic level. (A) donor with cervical spinal cord s e p e n t including the notochord removed; (B) transplant; (C) host with its thoracic segment removed and ready to receive the transplant. N, notochord; N.T., neural tube; S, somites. (From P. Shieh, J. exp. Zoo/., 117, 1951.)

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One wonders whether any of the migrating cells acts as ‘leader’ of these cells or if each embarks on the same migration under the directing forces of some agent extrinsic to the cells. During the entire migration period, each cell trails behind its long axon, which has already established synaptic contact with one or the other of the sympathetic nerve cells in the chain ganglia. Once the preganglionic motor cells have reached their permanent location adjacent to the dorsal aspect of the central canal, they gather together in two dense and compact columns. At this stage, namely at the end of the 7th day of incubation, they can be easily identified in the two columns which were first described by Terni as the preganglionic viscero-motor columns. In honor of their discoverer they are also at present known to neurologists as the ‘Terni columns’ (Terni, 1931). In mammals the preganglionic motor cells undergo a similar migratory movement but their final location is in the medio-lateral aspect of the neural tube instead of the dorso-ventral position adjacent to the central canal as in birds. The mass movement of the young nerve cells was examined in mouse embryos. it is represented in Figs. 25, 26. Migratory movements in the brain stem The brain stem of the chick embryo between 5 and 8 days of incubation shows even more than any other sector of the central nervous system the dynamic aspects of the differentiating nerve centers. It is only through the studies of this brain segment in transversal, frontal and sagittal sections that one can reconstruct the simultaneous and many times opposite movements of nerve cell populations in their way toward their different destinations. As shown in Figs. 12-14, some of these cells form compact columns, while others are loosely arranged and detach sharply on account of their dark color on the pale background of the brain stem texture, and still others are oriented in long rows which bridge the distance between some of the cell aggregates. i t was again the close inspection of this material hour after hour and day after day which revealed a number of highly organized and patterned mass movements in this apparently chaotic distribution of differentiating nerve cells. Tt was in fact found that nearly all the nuclei of the cephalic motor nerves do not form in the areas where they will be eventually located in the mature organism, but start to differentiate in other sectors of the brain stem and reach their end statioli through long and sometimes complex routes. In so doing, neuroblasts belonging (0different nuclei crisscross each other and i n this way one gets the impression of a chaotic and random cell distribution, while the individual units, on the other hand, move along precise and rigorously organized paths which invariably bring them io their final station. Here we will briefly describe only one 0: these migratory movements which first came to our attention many years ago (Levi-Montalcini, 1942) and which presents many suggestive, although still unexpla‘ned, facets. The migratory movement under consideration takes place in the brain stem of embryos between the end of the 5th and the 8th day of incubation. It consists of the segregation of about half the population which builds the nucleus of origin of the VIth nerve and results in the formation of the References p. 25/26

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Figs. 9-14. For legend see p. 13.

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accessory nucleus of the VIth nerve which provides for the innervation of the 3rd lid and of the two small muscles bursalis and quadratus. Up to the end of the 5th day, the main and accessory nuclei form a unique compact nucleus on the floor of the 4th ventricle. The right and left nuclei are located in close apposition to the two longitudinal fasciculi (Fig. 5a). They are easily identified on the basis of the axons of the cells which leave the brain stem in two compact nerve bundles immediately lateral to the midline (Fig. 5a’). At this time one sees a few neuroblasts detach from the lateral aspect of the two nuclei and move in a ventro-lateral direction. The neuroblasts trail their axon behind and one can therefore follow the migrating cells along the long route which will bring them from the nucleus of origin to their final destination in the ventro-lateral sector of the brain stem. During the subsequent days, hundreds of darkly-stained and spindle-shaped neuroblasts detach in turn from the two main nuclei and move along the same trail followed by the first migrating nerve cells (Fig. 5b’). When the migration comes to an end on the 8th day of incubation, one sees two compact nuclei in the ventro-lateral areas of the brain stem connected through their axons to the main nuclei which retain their position on the floor of the 4th ventricle. The axons of the main and of the two accessory nuclei leave the brain stem together in two compact nerve bundles which form the VIth nerves (Fig. 5c’). Even before this impressive mass movement of the accessory nucleus became known, its position far away from the main nucleus had been the object of interest by neurologists such as Ariens Kappers (1911) and Terni (1921). The latter considered the position of the accessory nuclei in the brain stem as the most convincing evidence of the validity of Ariens Kappers’ theory of neurobiotaxis. This theory, as is well-known, claims that the position of nerve centers is dictated by the influence exerted upon them by ‘the greater number or the more dominant impulses which reach the cells’ (Ariens Kappers et al., 1936). The location of the accessory nucleus of the VIth nerve, in close apposition to the descending root of the Vth nerve, is indeed most favorable for the discharge of impulses which, in turn, call for the closure of the lid when the eye or adjacent areas are endangered by noxic stimuli entering the field innervated

Figs. 9-14. All microphotographs from silver-stained, unretouched material, at different magnifications. Figs. 9-11 illustrate the transformation of the Purkinje cells (P) in embryos of 10 days (Fig. 9), 14 days (Fig. 10) and 17 days (Fig. 11). In Fig. 9, the neuroblasts are densely packed in cell groups, and the individual units cannot be recognized. In Fig. 10, the Purkinje cells appear in one single row. The cells show many dendrites (compare with drawing (c) in Fig. 2). In Fig. 1 1 , the apical dendrite characteristic of the Purkinje cell is formed (compare with drawing (d) in Fig. 2). Figs. 12-14 show 3 frontal sections of 8-day-old (Figs. 12, 14) and 7-day-old (Fig. 13) chick embryos. Fig. 12, V, main motor trigeminal nucleus, VIa and VIm, show the accessory and main nuclei respectively of the abducens nerve. The accessory nucleus has completed its migration. In subsequent stages this nucleus becomes more compact. Fig. 13 shows the dorsal motor nucleus of the Xth nerve (X)and the cells of the nucleus intermedius (I) as they move from a medial to a lateral position. Fig. 14 shows the two motor nuclei of the vagus complex (dorsal motor nucleus and nucleus ambiguus and the nucleus intermedius (I) in their final location. See also the long columnar nucleus of the XIIth nerve (XII). References p . 25/26]

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by the Vth nerve. While there is no way of testing the validity of this principle as directing force in the evolution of this center, we can object to its validity as acting force during the ontogenesis. We tested this hypothesis by extirpating the rostra1 part of the rhombencephalon in 2-day-old embryos. In this way, the Vth nerve and its descending root were absent while the caudal part of the rhombencephalon was not affected-byrthe ' , operation. In these embryos lacking the descending root of the Vth

Figs. 15-1 8. All microphotographs from silver-stained, unretouched material, at different magnifications. Fig. 15 shows a large sensory nerve bundle entering the lateral part of the mesonephros from an adjacent sensory ganglion in a 4+-day-old chick embryo. T, mesonephric tubule. Fig. 16 shows the same nerve bundle in the process of disintegration in a 54-day-old embryo. Arrow points to the nerve bundle. T, mesonephric tubule. Fig. 17 shows the epibranchial placode which gives origin to the ganglion nodosum in a 3-day-old chick embryo. P, placode. Compare with Fig. 1. Fig. 18 shows the ganglion nodosum (N) in a 43-day-old chick embryo.

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nerve, the mass migration of the accessory nucleus took place as in control embryos (Levi-Montalcini, 1942). Migratory movements in higher brain centers Although these movements are no less impressive than the ones which take place in the spinal cord and in the brain stem, they will be only mentioned here since they were already the object of extensive reports in previous publications (Jones and Levi-Montalcini, 1958; Levi-Montalcini, 1963). Extensive mass migration of neuroblasts occurs in the cerebellum where the immature Purkinje cells cover long distances in order to reach their final location in the cerebellar cortex (Fig. 6). During the migratory phase the nerve cells exhibit the characteristic elongated spindle-shaped form which is common to all nerve cells in the way of active displacement from the original to the final location (Uzman, 1960; Levi-Montalcini, 1963). A massive migratory movement takes place in the telencephalon (Jones and LeviMontalcini, 1958). Also in this case we followed the displacement of thousands of nerve cells and their final aggregation in the nucleus epibasalis centralis. By way of conclusion one can say that active displacement and migration of nerve cells for long distances in the developing nervous system is the rule rather than the exception. The orderly fashion of these movements and their time pattern which is ‘n all instances so rigorous as to permit prediction of when the first neuroblasts will start their long journey, leaves no doubt as to the existence of directing and guiding forces of these movement. It is for future work to attempt to identify these agents which are at present entirely unknown. D E G E N E R A T I V E PROCESSES I N T H E D E V E L O P I N G N E R V O U S SYSTEM

Death of isolated units or entire cell populations is a normal occurrence in morphogenesis and is so familiar to students of developmental processes as to be considered of no more interest than all other aspects of development. During metamorphic processes cell degeneration occurs in the central nervous system (or in ganglia in invertebrates) as well as in a number of other organs and body tissues (Glucksmann, 1951). Localized degenerative processes are described in mutants as in the well-known case of mice exhibiting the Waltzer-Shaker syndrome as a consequence of degenerative changes in the cochlea and vestibular apparatus (Gruneberg, 1956). The analysis of the extensive literature on cell death led Saunders and co-workers to state that ‘the onset and pace of degenerative changes during development are ultimately under genetic control‘ (Saunders et a[., 1962, p. 173) and we cannot but agree with this viewpoint which receives ample support from the numerous instances of massive cell degeneration taking place during developmental processes. We also agree with the above-quoted authors that in placing on the genes the burden for the sudden death of thousands of cells during development, we still leave unsolved the problem of why these cells should undergo early differentiation and then die when the ‘death clock‘ is set in mot’on. References p. 25/26

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Figs. 19-24. All figures from silver stained, unretouched material, at different magnifications. Figs. 19-24 illustrate the formation and migration of the preganglionic thoracic column in the spinal cord of the chick embryo between 3 and 11 days of incubation. Transversal sections of embryos at the same trunk level. Fig. 19, 4-day-old embryo. Fig. 20, 4%-day-oldembryo. Fig. 21, 6-day-old embryo. Fig. 22, 7-day-old embryo. Fig. 23, 8-day-old embryo. Fig. 24, 1 1-day-old embryo. Notice the progressive reduction of the central canal from 4 to 11 days. Arrows point to the preganglionic neurons.

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In the following we will briefly describe one of the most striking instances of these massive degenerative processes which takes place in the developing spinal cord and was described by us some time ago. We refer the reader for a more detailed analysis of this process to our original paper (Levi-Montalcini, 1950). We will then consider similar degenerative processes which occur in some of the sensory spinal ganglia. Mass cell degeneration in the cervical level of the spinal cord Till the beginning of the 4th day of incubation the motor column in the cervical segment of the spinal cord appears as a rather dense and compact cell population of about the same size as in the brachial, thoracic and lumbo-sacral segments of the cord. Toward the middle of the 4th day, a massive degenerative process sets in in the cervical segment and in a few hours at least 3 of the cell popu’ation in this region are reduced to cellular debris and pyknotic cells, scattered throughout the whole area. One sees in such preparations a large number of deeply stained round bodies scattered among the healthy nerve cells. A close inspection of these ‘dark bodies’ shows that they are the remnants of pyknotic or disintegrating nerve cells During the period between 43 and 5 days, the number of these pyknotic cells is so large as to obscure the presence of intact cells (Fig. 32). Occasionally, smaller granules are found collected in clusters. They suggest an advanced stage of cellular disintegration. Besides the pyknotic cells, one notices a large number of macrophages. The vital staining technique used in such embryos and described in detail in our original paper (Levi-Montalcini, 1950) clearly indicates that these cells are present in large numbers in the area where degenerative processes take place. It is possibly the activity of these ‘scavengers’, which engulf the remnants of the damaged and dead cells, which accounts for the rapid disappearance of pyknotic cells from this segment of the neural tube. A few hours later in fact (the beginning of the 5th day) neither degenerating cells nor macrophages are detectable in this area. The cervical motor column is, however, reduced in width to less than f its original size. The cells spared from death gather in a slender column, easily identifiable as the medial motor column which provides for the innervation of the axial muscles of the cervical region. We suggested in our original report that the cells undergoing early death could be part of an abortive visceral system. This suggestion was based on the observation that in stages prior to the massive degenerative processes, one sees rami communicantes in every segment of the cervical level. They are directed toward the primary sympathetic ganglia which are in the process of forming in this, as in more caudal levels of the trunk. These rami communicantes disappear at the same time as the large-scale cell degeneration takes place at 43 days of incubation. Experiments to be briefly mentioned in a following section gave support to the above hypothesis which will be again discussed after presentat:on of the results. Degenerative processes in the cervical and thoracic spinal ganglia While thousands of cells undergo sudden death and disappear from the cervical References p . 25/26

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segment of the developing spinal cord, an equally impressive degenerative process takes place in the cervical and thoracic spinal ganglia. In only one aspect do the degenerative processes in the neural tube and in the spinal ganglia show differences. While death hits most of the cells in the motor column of the cervical spinal cord, and dead cells are evenly scattered among the living ones in this area, in the cervical

Figs. 25-28. The Figs. 25 and 26 are from unretouched silver material, at different magnifications. Figs. 25 and 26 show the beginning and the end of the migration of the preganglionic thoracic motor cells in the mouse. Fig. 25,13-day-oId embryo. This visceral-motor (vm) cells start to segregate from the somato-motor (sm) cells. Fig. 26, 17-day-old mouse embryo. The visceral-motor (vm) cells are now located in their permanent dorso-lateral position. The somato-motor cells (sm) have retained their ventral position. Fig. 27 shows the degeneration of sympathetic neuroblasts in the superior cervical ganglion of a 4-day-old mouse, following daily injections of antiserum to the NGF. Arrows point to some of the degenerating cells. Toluidin blue stain. Fig. 28, control.

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and thoracic ganglia the disintegrating cells are confined to only one sector of the ganglia. It is in the ventro-lateral but not in the medio-dorsal part of these ganglia that massive degeneration takes place. The localization of pyknotic and dead cells is in fact so sharp that one could draw a line between the two parts of the.ganglia where this process does or does not occur. Wk described in previous papers (Levi-Montalcini and Levi, 1942; Hamburger and Levi-Montalcini, 1949) the morphological differences between the cells which lodge in the dorso-medial and in the ventro-lateral sectors of these ganglia. We also reported in subsequent papers on the different behavior of these two cell types when confronted with given experimental situations (Levi-Montalcini and Hamburger, 1951). It is not the purpose of the present study to consider these differences. Here we will only mention that while the dorso-medial cells undergo normal differentiation, a large number of the ventro-lateral cells undergo sudden death and are wiped out by macrophages which invade the area (Fig. 7a,b). What is the nature and the function, if any, of these cells doomed to die before their differentiation is fully achieved? Their number, as in the case of the motor cells in the cervical spinal cord, can be calculated in the thousands. Their early history does not differ, at least as far as one can say on the basis of their morphological analysis, from the history of neighboring cells which instead undergo normal differentiation and have a life expectancy of the same length as the organism to which they belong. Although we are not in a position to offer an answer to this question, we believe that it may be of more than passing interest to consider some parallel findings which may offer a clue to the identification of these cells and to their function. At 44 days, just before the degenerative processes set in in the ventro-lateral sectors of thoracic spinal ganglia, we noted that each of these ganglia contribute 2 to 3 rather large nerve bundles to the adjacent mesonephroi (Fig. 15). Since the ganglia which contribute these nerves are in the number of 6 to 7 for each side, the total number of these nerves is between 15 and 18. As the nerves reach the lateral-most part of the developing mesonephroi, they resolve themselves in individual nerve fibers and each fiber establishes contact with the epithelium lining some of the lateral tubules of the mesonephros. In the subsequent developmental stages, starting at the middle of the 4th day, the thickness of these nerve bundles decreases and toward the end of the 6th day nothing is left but remnants of the nerve bundles (Fig. 16). Also these residual nerve fibers eventually disappear altogether and no nerve can be seen to make connection with the mesonephroi in these or in subsequent developmental stages. Since the ganglia contributing nerves to the mesonephroi are exactly the same as those which show the massive cell degeneration described above, it is very tempting to correlate the two observations and identify the dying cells as the ones which establish a temporary contact through their nerve fibers with the mesonephroi. We are well aware of the fact that this observation leaves the main problem unanswered: what is the function of these nerve cells and why should the mesonephros (itself an organ doomed to a short life) receive a ‘temporary’ innervation from adjacent sensory ganglia? Also we must admit that we have no explanation for the equally impressive cell degeneration which occurs in the cervical ganglia. Their peripheral field was not the object of a close inspection and we do not know the termination of the axons of References p . 25/26

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these nerve cells which disintegrate between the middle of the 4th and the 5th day of incubation. As in the other instances described by Saunders, the rigid time pattern of these processes which present the same features in all specimens, clearly indicates that these massive cell degenerations are under genetic control in the same way as the mass migratory movements in other nerve cell populations. One may now ask if these developmental processes are amenable to experimental analysis. The results to be briefly reported in the following pages indicate that this is indeed possible. In at least one instance we have evidence that nerve cells which would not, under normal circumstances, undergo migration, can be induced to migrate in a given experimental condition. We will also show that massive cell degeneration can be experimentally produced in cell populations which under normal circumstances are not doomed to die in their early differentiative stages. The similarity between normally occurring and experimentally produced cell disintegration is in some instances so striking as to raise the question whether the same or similar mechanisms might not operate in both cases. We shaU consider this aspect of the problem after presentation of the results. Mass cell migration experimentally produced in the cervical spinal column

The hypothesis that degenerative nerve cells in the cervical spinal cord could represen. an abortive preganglionic visceral system was tested by Paul Shieh in our laboratorty He transplanted the cervical segment of the spinal cord of 9-25 somite chick embryos to the thoracic level of hosts of the same age which had been previously operated on for the extirpation of the thoracic segment of the spinal cord (Fig. 8). The cervical segment was then made to fit in the place of the extirpated thoracic segment. This operation is rather difficult and for this reason only a few cases survived up to the 7th-8th day. In all instances Shieh found that the transplanted segment of the cervical spinal cord underwent a differentiation similar to the differentiation which is characteristic of the thoracic segment (Shieh, 1951). In fact a slender column of cells detaches from the other motor cells of the transplant. In some instances the migration follows the same path as described in the thoracic segment of normal embryos (Figs. 33,34). In other instances the migration is more atypical but also in these cases it resulst in the building of a cell column adjacent to the dorsal aspect of the central canal (Figs. 35, 36). Nerves emerging from these cells were traced to the adjacent paravertebral sympathetic ganglia. The author states that not all cells which normally undergo degeneration are spared this fate in the transplanted segment of the spinal cord. On the contrary, the degeneration is still well apparent in the rostra1 part of the transplant. It is only from the caudal-most part of the transplant that migration actually occurs. These results, while giving support to the hypothesis that the degenerating cells in the cervical spinal cord might indeed represent an abortive preganglionic visceral column, also indicate that in a favorable environmental condition such as the one provided by the trunk region, motor nerve cells can undergo a massive migration and build a preganglionic motor column which establishes synaptic contact with adjacent sympathetic ganglia.

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Figs. 29-32. Fig. 29 shows the ventro-lateral cells in a normal brachial ganglion of a 6-day-old chick embryo. Fig. 30 shows the same area in the brachial ganglion following removal of the adjacent wing bud at 3 days. Arrows point to disintegrating nerve cells. Heidenhain hematoxylin stain. Fig. 31, sagittal section of a 5-day-old chick embryo. Note the uniform size of the motor column (two arrows point at the co!umn in the brachial and thoracic levels). L, leg; SC, spinal cord; W, wing. Silver stain. Fig. 32, 4)-day-old chick embryo. Transverse section at the cervical level of the spinal cord. Heidenhain hematoxylin. Degenerating cells appear as black spherical bodies scattered throughout the motor column. (Figs. 29,30 from V. Hamburger and R. Levi-Montalcini, J . exp. 2001.. 111, 1949. Figs. 31, 32 from R. Levi-Montalcini, J. Morph., 86, 1950.)

References p . 25/26

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Mass cell degeneration experimentally produced in spinal ganglia and in the neural tube Degeneration never occurs under physiological conditions in the brachial and lumbosacral ganglia nor does it take place in the corresponding segments of the neural tube. One can however produce a massive cell degeneration in the ventro-lateral sectors of these ganglia and in the motor column of the brachial and lumbo-sacral segments of the neural tube simply by extirpating the anterior or posterior limb buds in 2-day-old embryos (Hamburger and Levi-Montalcini, 1949). Two aspects of these experimentally produced cell degenerations are of interest : (I) degeneration does not follow a direct injury of motor or sensory nerve cells, nor of their axons. The extirpation of the limb buds is in fact performed long before sensory or motor nerve fibers branch in the limb bud; (2) the degenerative processes in the spinal ganglia and in the neural tube exhibit the same features as in the sensory ganglia and neural tube of normal embryos. The only difference between 'physiological' and experimentally produced cell degenerations is to be found in the regional areas where these processes take place. Physiological mass degeneration takes place in the cervical neural tube and in the cervical and thoracic ganglia, while the experimentallyproduced mass degeneration takes place in the brachial and lumbo-sacral segments of the neural tube and adjacent ganglia. We will first consider the degenerative processes which take place in the brachial motor column and were the object of a detailed analysis by Hamburger (1958). The extirpation of the anterior limb bud deprives these cells of their peripheral field of innervation and a sudden and abrupt degenerative process wipes out most of the cells of this area which provide for the innervation of the wing. Hence the extirpation of the limb bud, while not causing direct injury to these cells, has results fatal to them. In the sensory ganglia a massive cell degeneration takes place in the ventro-lateral cells and a slow and progressive atrophy sets in in the dorso-medial cells (Figs. 29, 30). Eventually both cell populations are dramatically reduced in size if the embryos are permitted to survive until the end of the incubation period (Levi-Montalcini and Levi, 1942). Evidence presented in a previous paper seems to indicate that the cells which undergo abrupt death are the proprioceptive cells, while the cells which undergo slow atrophy are the exteroceptive cells (LeviMontalcini, 1963). While we have no explanation to offer for the behavioral differences between the two sensory cell populations, it is of interest to consider the close similarities between the degenerative processes in the neural tube in normal and experimental conditions and the similarities of these processes in the ventro-lateral cells of the thoracic and the brachial and lumbo-sacral ganglia in normal and experimental conditions. We suggested that death in the motor column of the cervical neural tube follows the failure of these cells to establish synaptic contact with the sympathetic gangIia (Levi-Montalcini, 1950). In the thoracic ganglia we noticed the sudden withdrawal of nerve fibers which had succeeded in earlier stages in establishing contact with the adjacent mesonephroi. In the experimental material, the deprivation of the peripheral field of innervation is fatal to the same types of nerve cells. As a working hypothesis one could therefore suggest that in both normal and experimentally produced mass cell degeneration, death follows the withdrawal from the medium

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Figs. 33-36. All microphotographs from silver-stained, unretouched material, at different magnifications. Fig. 33. cross section through the medial part of normal thoracic spinal cord of 6-day-old chick embryo. Arrow points to preganglionic column in process of migration. N, notochord; S, paravertebral sympathetic ganglia. Fig. 34, cross section at the same level as Fig. 33 through transplanted cervical segment of the spinal cord. Note similar migration of motor cells as in Fig. 33. Arrow points to migrating cells. N,, notochord. Figs. 35, 36 show two other experimental cases. In Fig. 35 the migration is atypical. Arrow points to the migrating cells in this and in Fig. 36. Note 2 notochords (N) from donor and host in the experimental cases. (All figuresfrom P. Shieh, J. exp. Zool., 117,1951.)

of some agent which is essential to the very life of these cells. The experimental results by Paul Shieh would add support to this point. In that particular instance in fact, survival and migration of nerve cells doomed to die under normal conditions, were obtained when these cells were able to establish a permanent synaptic contact with the sympathetic ganglia at the thoracic tmnk level (Shieh, 1951). In the instances of References p . 25/26

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the latero-motor column of the brachial and lumbar spinal cord and of the ventrolateral sensory cells, this factor would be normally supplied by the growing limb bud. It might be of interest, in connection with the above problem, to consider how one can produce massive cell degeneration in other sectors of the nervous system by depleting the nerve cells of some essential factor by chemical rather than surgical procedures. We will consider these results in the following section. Massive degeneration of the sympathetic nerve cells by an ‘Anti-Nerve Growth Factor’ During recent years most of our work was devoted to the analysis of a specific protein isolated from mouse salivary glands. Upon injection of this protein in newborn and adult mammals, one obtains a striking size increase of the sympathetic nerve cells of injected animals (Levi-Montalcini and Booker, 1960a). This factor is now known as the Nerve Growth Factor (NGF). An antiserum to this NGF was produced by injecting the purified protein in rabbits. As reported in previous publications (LeviMontalcini and Booker, 1960a,b; Levi-Montalcini and Angeletti, 1961; Cohen, 1960), the antiserum injected in newborn mammals results in the sudden and massive death of almost the totality of the sympathetk nerve cells (Figs. 27, 28). This devastating effect is similar to the massive degenerative processes described above. Only in the last instance, however, do we have precise indication as to the cause of death. This follows the destruction of a protein which plays an essential role in the life of the receptive nerve cells. It is tempting to draw a parallel between this degenerative process and the ones considered above in normal embryos and in embryos deprived of some parts of their peripheral fields of innervation. ‘Physiological death’ of some nerve cell populations could be due to the sudden depiivation of an essential factor. Death in other nerve cell populations follows the surgical extirpation of their field of innervation. The above considerations, if substantiated by more extensive studies, could shed light on some more general principles of nerve cell differentiation. Each cell population might be dependent for its growth and survival on some specific nerve growth factor, which is normally supplied by the environment. In only the last mentioned instance was this factor identified and isolated. It remains to future work to seek for such factors and attempt to correlate the observations reported above with the depletion of these ‘essential factors’. ACKNOWLEDGEMENTS

The work described in this paper was supported by grants from the National Institute of Neurological Disease and Blindness, Public Health Service (B 1602-C-C& and from the National Science Foundation (G 13946) and by a contribution from the American Cancer Society (C 1801-C-C2) to Washington University. SUMMARY

This review deals with two developmental mechanisms which play an important role in neurogenesis : migration and massive degeneration of large cell populations. Both

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processes take place during the early differentiation of the central nervous system and of the sensory ganglia. They were investigated under normal and experimental conditions in the chick embryo, which provides a most favorable material for such analysis. The significance of these mechanisms is discussed on the basis of experimentally produced migration and degeneration of nerve cell populations.

REFERENCES ARIBNSKAPPERS, C. U., (191 I); The migrations of the motor root-cells of the vagus group, and the phylogenetic differentiation of the hypoglossus nucleus from the spino-occipital system. Psychiat. neurol. Bl. (Amst.), 15, 408427. ARIENSKAPPERS, C. U., HUBER,G. c., AND CROSBY, E. c.,(1936); The Comparative Anatomy of the Nervous System of Vertebrates, including Man. Vol. I. New York, Macmillan Company (p. 76). CAJAL,R. S., (1909); Histologie du SysrPme Nerveux de I’Homme et des Vertkbrtfs. Vol. 1. Paris, Maloine. CAJAL,R. S., (1929); Etudes sur la Neurogenhe de quelques VertPbrPs. Madrid. COHEN,S . , (1960); Purification of a nerve-growth promoting protein from the mouse salivary gland and its neurocytotoxic antiserum. Proc. nut. Acad. Sci. (Wash.), 46, 302-311. GLUCKSMANN, A., (1951); Cell deaths in normal vertebrate ontogeny. Bid. Rev., 26, 59-86. GRUNEBERG, H., (1956); Hereditary lesions of the labyrinth in the mouse. Brit. med. J., 12, 153-157. HAMBURGER, V., (1958); Regression versus peripheral control in motor hypoplasia. Amer. J. Anaf., 102, 365410. HAMBURGER, V., (1961); Experimental analysis of the dual origin of the trigeminal ganglion in the chick embryo. J. exp. Zool., 148, 91-124. HAMBURGER, V., AND LEVI-MONTALCINI, R., (1949); Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J. exp. Zool., 111,457-502. HORSTADIUS, S . , (1950); The Neural Crest. London, New York, Toronto, Oxford University Press. JONES, A. W., AND LEVI-MONTALCINI, R., (1958); Patterns of differentiation of the nerve centers and fiber tracts in the avian cerebral hemispheres. Arch. itul. Biol.,96, 231-284. LEVI-MONTALCINI, R., (1942); Origine ed evoluzione del nucleo accessorio del nervo abducente nell’ embrione di pollo. Acta Pontij. Acad. Sci.,6, 335-345. LEVI-MONTALCINI, R., (1946); Ricerca sperimentale sull’origine dei gangli del glosso-faringeo e del vago nell’embrione di pollo. Atti Accud. naz. Lincei, 1, 1349-1352. LEVI-MONTALCINI, R., (1949); The development of the acoustico-vestibular centers in the chickembryo in the absence of the afferent root fibers and of descending fiber tracts. J. comp. Neurof.,91,209-242. LEVI-MONTALCINI, R., (1950); The origin and development of the visceral system in the spinal cord of the chick embryo. J. Morph., 86, 253-284. LEVI-MONTALCINI, R., (1963); Growth and differentiation in the nervous system. The Nature qf Biological Diversity. J. Allen, Editor. New York, McGraw-Hill Book Company. P. U., (1961); Growth control of the sympathetic system by LEVI-MONTALCINI, R., AND ANGELETTI, a specific protein factor. Quart. Rev. Biol.,36, 99-108. LEVI-MONTALCINI, R., AND BOOKER, B., (1960a); Excessive growth of the sympathetic ganglia evoked by a protein isolated from mouse salivary glands. Proc. nut. Acad. Sci. (Wash.), 46, 373-384. LEVI-MONTALCINI, R., AND BOOKER, B., (1960b); Destruction of the sympathetic ganglia in mammals by an antiserum to a nerve-growth protein. Proc. naf. Acud. Sci. (Wash.), 46, 384-391. LEVI-MONTALCINI, R., AND HAMBURGER, V., (1951); Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the ch~ckembryo. J. exp. Zool., 116, 321-362. LEVI-MONTALCINI, R., AND LEVI,G., (1942); Les consequences de la destruction d‘un territoire d’innervation pkripherique sur le developpement des centres nerveux correspondants dans l’embryon de poulet. Arch. Biol.,53, 537-545. SAUNDERS, J. W., JR., GASSELING, M. T., AND SAUNDERS, L. C., (1962); Cellular death in morphogenesis of the avian wing. Develop. Biol.,5, 147-178. SHIEH,P., (1951); The neoformation of cells of preganglionic type in the cervical spinal cord of the chick embryo following its transplantation to the thoracic level. J. exp. Zool., 117, 359-395.

.

26

DISCUSSION

TELLO, J . F., (1925); Sur la formation des chaines primaires et secondaires du grand sympathique dans l'embryon de poulet. Trav. Lab. Rech. biol. Univ. Madrid,23, 1-28. TERNI, T., (1921); Ricerche sul nervo abducente e in special mod0 intorno al significato del suo nucleo accessorio d'origine. Folia neuro-biol., XII, n. 2. TERNI,T., (1931); I1 simpatico cervicale degli amnioti (Ricerche di morfologia comparata). Z . Anat. Entwick1.-Gesch., 96, 289426. UZMAN, L. L., (1960); The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. 1.comp. Neurol., 114, 137-160. VAN CAMPENHOUT, E., (1931); Le dkveloppement du systeme nerveux sympathique chez le poulet. Arch. Biol. (Paris), 42, 479-507. YNTEMA, C. L., (1942); Experiments on the origin of some of the sensory cranial ganglia in the chick. Anat. Rec., 82, 455. YNTEMA, C. L., (1944); Experiments on the origin of the sensory ganglia of the facial nerve in the chick. J . comp. Neurol., 81, 147-167. c. L., AND HAMMOND, W. s., (1947); The development of the autonomic nervous system. YNTEMA, Biol. Rev., 22, 344-359.

DISCUSSION

WAELSCH: Is there anything known about the chemical make-up of the cells before and after migration? LEVI-MONTALCINI: Not that I know. The only difference I am aware of is that the migrating cells react much more intensively to silver stain than the stationary cells and this may well reflect biochemical differences, which would be most interesting to investigate. WAELSCH: I am very much interested to know how these cells move. LEVI-MONTALCINI : It is a n amoeboid type of movement. The cell which is preparing to migrate becomes spindle-shaped and elongated. The cells assume a fish-like appearance and this, in our own experience, indicates that they will soon migrate. WAELSCH : I wanted to ask something in regard to some work which Dorothea Rudnick did in our laboratory about enzyme development. An interesting relationship was found between the development of the enzymes in the yolksac and those in the embryo. They showed some kind of a mirror image. A certain enzyme which was very high in the yolksac decreased when it went up in the embryo. I wonder whether there are in the yolksac or in the embryo substances which have a blocking or inhibiting effect? LEVI-MONTALCINI: I would not be able to answer this question. WAELSCH:Could you say something more about the general significance of your growth factor? LEVI-MONTALCINI: Although I am not in a position to give a satisfactory answer to your question, I must say that we do believe that the growth factor is normally present in the embryo as well as in the mature organism. It is quite possible that during development and differentiation the nerve growth factor is present in the peripheral field and acts upon the nerve centers which become associated with this periphery. This is, however, only a hypothesis and may be very difficult to prove. Also we believe that every nerve cell has its own specificgrowth factor. We are planning to investigate this point in detail in the future and search for other specific nerve growth factors. SCHADI?: In conjunction with some earlier work we have done on the development

THE DEVELOPING N E R V O U S S Y S T E M

27

of unit activity in the cerebral cortex, I would like to ask you whether you have any information regarding the functional activity of the cells in the oculomotor nucleus. We have detected spike-activity in neurons of the prenatal rabbit cortex, which showed with routine histological techniques hardly any sizable basal dendrites and a very short apical dendrite. LEVI-MONTALCINI : I regret to say we have no particular information regarding the early functional activity of the oculomotor nucleus. SCHADB:I am curious to know whether these moving cells process any number of dendrites or just like in the early differentiation of pyramids, have a single dendrite opposite to the axon? LEVI-MONTALCINI: They are just spindle-shaped and have a single process or dendrite opposite to the axon like the differentiating pyramid cells, which you mention. There seem to be no other dendrites growing out from the cell bodies as long as the cell is in the process of migrating. SCHADB:I have one more question regarding your very interesting experiments. Looking from your slides I saw that in the sympathetic ganglia, treated with growth factor, there was also an increase of glial cells. Do you have any idea why this is? LEVI-MONTALCINI: It may be that you are right, but I never studied this point in detail. I can, however, tell you something about the effect of the antiserum to the nerve growth factor. Upon injection of the antiserum, the glial cell population is not affected for the first few days but then suddenly decreases and then disappears almost completely. I assume that the opposite situation occurs when the growth factor is injected, namely that the glial cells increase in number. In this case the effect of the growth factor would be an indirect one, that is, it would affect the nerve cells and this, in turn, would affect the population of satellite and glial cells. PURPURA: The assumption that nerve centers are under the influence of growth factors implies inhibiting as well as excitatory substances. Do you believe these factors are operating in the effects observed in favor of injury to cortical nerves as originally described by Cajal and confirmed in some of our recent studies? LEVI-MONTALCINI: It could well be, but I regret to say that I am not able to answer this question. PURPURA: Do you believe that the phenomenon of neurobiotaxis is explainable in terms of the release of different and specific growth factors by different centers? LEVI-MONTALCINI: I would not say that we have evidence for it. It was indeed a very interesting hypothesis offered by the late C . U. Ariens Kappers that nerve centers shift position in different phyla and become located in close apposition to nerve fibers which establish a synaptic contact with them. I must say, however, that I was not able to prove the validity of this principle as acting force during the differentiation of embryonic nerve centers. I refer to the experiment I reported on the extirpation of the rostra1 part of the medulla in the 2-day-old chick embryo. As a consequence of this operation, the descendant root of the Vth nerve did not form but the accessory center of the abducens underwent migration as normal, thucdisproving that its migration is dependent upon a direct influence of the descendant root of the Vth nerve.

28

DISCUSSION

BONAVITA: You said that the growth factor can be isolated from several sources. Can you make a comparison in chemical as well as in biological terms? In other words, can you say that there is a relative species specificity of the factor? LEVI-MONTALCINI: The factor was isolated first from mouse sarcomas 180 and 37, then from snake venom and salivary gland, and more recently from experimentally produced granuloma in several mammalian species. As found by Dr. Cohen, the factors from the snake venom and salivary gland differ in molecular weight, which is 20,000 for the snake venom and 44,000 for the salivary factor. There are also other differences as to their antigenic properties. Biologically all the above nerve growth factors call forth the same effect. That is, they all affect embryonic sensory and embryonic and mature sympathetic nerve cells. BRIZZEE:What was the earliest developmental stage at which growth factor was employed? LEVI-MONTALCINI: When the sarcoma 180 is implanted in the 3-day-old chick embryo, the first reaction in the sensory nerve cells is apparent at 6 days. Of the two cell populations which we described in the sensory ganglia, it is only the medio-dorsal but not the ventro-lateral which is affected by the tumor nerve growth factor. The snake venom and salivary gland factors were instead tested only in embryos 7-9 days old and in all instances the medio-dorsal sensory nerve cells and the sympathetic nerve cells showed a growth response to these agents. In subsequent developmental stages the sensory nerve cells lose their reactivity to the nerve growth factor while instead the sympathetic nerve cells remain reactive throughout all their life cycle. BRIZZEE:Do cells in the central nervous system invariably migrate in a direction opposite to axonal localisation? LEVI-MONTALCINI: Yes, in the central nervous system the cells migrate, as far as I could see, always in a direction opposite to the axonal outgrowth. WENDER : I wanted to know if there are morphological or histochemical differences between the cells which migrate and those which degenerate. LEVI-MONTALCINI: No, from a morphological point of view I was not able to see any difference in cells which would eventually migrate or degenerate. WENDER:Is it possible to say that this migration process is a very active process? LEVI-MONTALCINI: Yes, the process is certainly a n active one and is due to amoeboid movement of individual' nerve cells. In order to reach the terminal location many times they have to cross a fiber-barrier or to move in a dense texture of the central nervous system.'The hour after hour and day after day analysis gave us positive evidence that we are dealing with a n active displacement and not a passive one. BONAVITA: Do neurons show the silver reaction at a different degree before differentiation? In the absence of histochemical data this could tentatively indicate the existence of chemical differences 'at a n early stage. LEVI-MONTALCINI: Yes, neuroblasts show a different silver affinity from one group to another and also the affinity in the same cell population changes from one stage of differentiation to another. I certainly agree with you that this may reflect histochemical differences. WAELSCH:May I ask you another question, Rita, which I wanted to ask you in

THE DEVELOPING NERVOUS SYSTEM

29

the beginning. You said that in the adult your growth factor leads to an increase in size without cell division. This is probably a very important phenomenon from a biochemical point of view. Some investigators have postulated that also in the adult nervous system you can show incorporation of tritiated thymidine into the nuclei, or what is meant into the DNA of the adult nucleus. Others like Maurer and collaborators in Germany were unable to show this. Your experiment would be an ideal case to test whether without cell division in growth there would be an activity of DNA. And this would explain something which starts now to be very puzzling: does the nucleus in the adult nerve cell have a metabolic activity? LEVI-MONTALCINI: I fully agree with you that our material could indeed be most suitable for investigation of this problem.

30

The Evolutionary Aspect of the Integrative Function of the Cortex and Subcortex of the Brain S. S A R K I S O V Brain Insiiiuie, Moscow (U.S.S.R.)

Contemporary success of physics, chemistry and electronics open up great possibilities for the study of functional peculiarities of the brain as a whole and of its individual structural formations. Electroencephalography and acute and chronic macro and micro electrode methods are well-known in the study of function localization. The possibilities of such methods of investigation used in the study of the structure and functions of the brain, combined with the conditioned reflex method, are not yet exhausted. Although these methods are far from being perfect, they permit to show the integrative role of separate formations of the central nervous system in the activity of the brain taken as a whole. Naturally, the clinical picture of lesions of the higher regions of the central nervous system occupies an important place in the study of these questions. In addition to the study of the structure and function of the cortex and subcortex in the definitive condition great importance should be attached to their study in the process of evolution. Detection of laws of their phylogenesis and ontogenesis considerably enrich our notions of the main structural and functional peculiarities of the brain in man and animal (Brodmann, 1909; Rose, 1926; Conel, 1939-1959; Filimonoff, 1949; Polyakov, 1961). This is especially true for the integrative functions of the cortex of the brain. The purpose of this paper is to present some results of investigations by members of the Moscow Brain Institute which permit to speak of the highest degree of integration in the brain cortex. Study of evolution shows, that progressive development in vertebrates is characterized by greater and greater transfer of priority by cortical centres as compared to the other segments of the central nervous system (Fig. 1). This conception is confirmed by numerous anatomical studies carried out by earlier as well as by contemporary authors. In mammals the cortex of the brain is much larger than the subcortical formations. In addition to qualitative changes, quantitative modifications are very significant. In man, about 96% of the surface of the hemispheres is covered by the neopallium, in dogs 84 %, in rabbits 56 %, in hedgehogs only 32 %, the comparative size being even smaller in reptiles (Filimonoff, 1949). Surfaces of the cortical regions which, according to the clinical data, play an

I N T E G R A T I V E F U N C T I O N O F T H E C E R E B R A L C OR TEX

31

B

c 1

\

Fig. 1 . The development of the telencephalon of vertebrates. Black = cerebral cortex. (A) brain of fish: 1 = midbrain; 2 = cerebellum; (B) brain of lizard: 3 = medulla oblongata; (C) rabbit brain: 4 = telencephalon; (D) human brain: 1 = hemisphere/telencephalon,4 = midbrain, 5 = thalamus.

Fig. 2. The development of the surface of 4 regions of the cerebral cortex in relation to the surface of the whole hemisphere in primates. References p . 36-38

32

S. S A R K I S O V

especially important role in complex functions - in the first place in speech, praxis, gnosis - increase comparatively to the whole surface of the hemispheres in Primates, from the hapale to man (Fig. 2). These are, for example, the frontal region in the hapale 4%, in the chimpanzee 13%, and in man 24% (Kononova, 1948, 1961), the lower parietal region in the hapale 0.4 %, in the chimpanzee 3 %, and in man 8 %. At the same time, cortical surfaces of other regions decrease in process of phylogenesis as compared to the whole surface of the cortex. The precentral (motor) region, field 4 according to Kukuyev’s (1952, 1961) experiments, occupies 8.9% of the whole cortex in the hapale, 4.5 % in cercopithecus, 3.6 % in orang outang and only 1.7 % in man. The surface of field 17 of the visual (occipital) cortex decreases as compared to the surface of the whole hemisphere from 8.5 % in orang outang to 3 % in man (Filimonoff, 1932, 1933). Studies on the evolution of the brain show certain trends of development not only of separate regions of the brain: motor, auditory, visual, etc., but also of its different layers. Structural peculiarities of separate regions and layers of the brain are detected even during the intra-uterine period of development. The stratification of the neopallium, formation of neurons, peculiarities of the rate of their development continue also after birth, according to Sarkisov’s (1929, 1956) studies in dogs (fields 4, 6, 17), in man by Filimonoff (1932, 1958, 1960) (occipital and precentral regions), Kononova (1940, 1948, 1957) (frontal region), Preobrazhenskaya (1948, 1959, 1960) (occipital region), Stankevich (1961) (lower parietal region), Minayeva (1948, 1959) (upper parietal region). Morphological, architectonic and electrophysiological investigations demonstrate that the development and complication of the brain structure correspond to perfection of the forms of behaviour, complication of the analysis and synthesis of stimuli reaching the body, which already in the first years of life are mediated through speech. The verbal system reflects the internal and external life of man and ensures the higher nervous activity of man in cooperation with the first signal system. The works of Krasnogorsky (1939, who studied the ontogenetic development of speech, show that verbal reactions in children are elicited through imitation and intensified as a result of reflex repetition (physiological ecology). The difference between man and animal is that in the former all the newly acquired cortex reactions, conditioned inhibitory reactions, all the signals of relations of strength and rate - accelerative and decelerative, increasing and decreasing stimulations - are mediated through speech, beginning with the first years of life, and are then integrated in the verbal-auditory analysor with verbal signals ; when received by the verbal-motor analysor they acquire kinaesthetic expression and enter into the child’s vocabulary. With the development of the function of speech the brain, and mainly the cerebral cortex acquires a new and very important property of developing specific speech generalization by way of forming verbal stimuli of different degrees and levels of integration. Abstract thinking especially helps to develop wide generalization by means of forming words in ontogenesis, which are generalizers in the highest degree of integration. The inclusion of word-generalizers of this or that degree in speech increases its power of generalization on a corresponding integration level. Simultaneously with these studies of the structure of the frontal

TABLE I

Hc $

T H E D E V E L O P M E N T O F A R E A S 44 A N D 45 O F T H E F R O N T A L R E G I O N O F M A N The development of these areas, parts of the speech-motion analyser, is not uniform. The development of area 44 is gradual and only in the brain of 7-year-old children is a great increase in its surface observed. Area 45 develops differently; in the prenatal period it is smaller than area 44, after birth the surface of area 45 begins to grow unevenly and exceeds area 44 in size.

k

l4

Area 44 percentage Absolute size of the area

6 months before birth 8 months before birth Newborn

14 days after birth 6 months after birth 1 year

2 years 3 years and 3 months 7 years 12 years Adult

95 85 188 204 224 215 315 337.5 341 440 448 636 587 569 42 1 457 1200 1218 1167 1025 1084 926

with With With With respect respect respect Absolute respect to the to (he to the size of the J r . ofthe J r . ofthe whole the area given Jr’ newborn adult hemisphere

10.3 8.6 10.2 11.4 8.2 8.2 5.5 6.8 4.1 5.7 4.8 6.3 3.9 3.9 3.3 3.5 7.3 7.1 5.9 5.4 5.2 4.4

z

Area 45 percentage

8.5 9 12.3 21 20.6 23 29 42.8 28.7 47.5 41.3 68.7 54.1 64.6 38.8 49.3 110.7 131.5 107.6 110.8

-

-

1.1

1.5 -

-

1.o 1.3 0.9 0.9

-

1.6 1.6

-

1.2 1.o

46 45 112 166 237 264 527 726 722 676 69 1 810 1599 1631 1387 1467 1544 1593 1852 1693 1837 1485

With With With With respect respect respect respect to the to the to the to the J r . ofthe J r . of the whole given J r . newborn adult hemisphere

5 4.5 6 9.3 8.5 10

9.4 12.6 8.6 8.7 7.2 8.1 10.4 10 10.9 10.9 9.4 10.7 8.3 8.4 8.8 8.1

20 17 47.2 63 -

-

2.5 3 6 11.2 12.8 17.8 28.9 48.8 38.8 45.5 37.2 48 86.1 89.8 74.7 87.6 83 94.5 100 96.7 -

-

-

-

-

-

1.9 2.1 1.5 1.7 2.6 2.6 -

, 2.1 2.2 2.1 1.7

w w

34

S. S A R K I S O V

cortex, which is most important for speech, it is shown how in children of corresponding ages, according to the works of Kononova (1961) the cortex gradually develops and differentiates with the development of speech (Table I). We shall give here some data about investigations on the formation of the nervous activity in the pre- and postnatal periods. These investigations show, that the formation trends of higher nervous activity in children in early postnatal ontogenesis is determined mainly by the degree of maturity of the structural elements of the cortex and the subcortical formations of the brain. These investigations prove, that premature children of the same age, but differing as to their degree of prematurity, show a great variability in their reflex activity. In 2.5-3-month-old premature children subcortical reflexes (posture of foetus, automatic actions, labyrinthine reflexes) prevail during the first 2 months. Clinical data show, that the functions of a whole number of organs and systems (metabolism, thermoregulation, respiration, nutrition) still correspond to a great extent to the intra-uterine functions and manifest a certain insufficiency of adaptation to the new environment. Stimuli (light, sound, smell) bring about a generalized reaction in the form of generalized motor restlessness:tremors, increase of mimic actions, nystagmus which are always accompanied by changes of respiration and of cardiac activity. These reactions are not orientation reflexes and do not have an adaptive character, since they are constantly repeated after numerous stimulations. In this early period of development it is impossible to elicit conditioned reflexes even after hundreds of combinations. As the brain cortex matures, the character of the reactions changes. Already towards the end of the second or the beginning of the third month of life reactions to the same stimuli in the same children become more localized, specialized. A typically orientative reaction appears, and disappears when the stimulus is applied repeatedly. From this age one can elicit food, motor and defense-preservation conditioned reflexes (combinations : light-milk, bell-milk, sound-ammonium vapour). These data suggest that the brain cortex in less premature children is functionally more developed than in more premature children of the same postnatal age. Some aspects of correlation between the activities of the cortex and subcortex in children in early ontogenesis are revealed by analysis of the relations of the vegetative and somatic components in the process of formation of conditioned reflexes. For example, the formation of the conditioned defense-preservation reflex elicited by combining sound (T 500 Hz) and smell of ammonium vapour shows that a specialized defense-preservationconditioned reflex is not formed immediately. In morecpremature children practically no conditioned reflex is formed during the first 2 months. Only towards the end of the second month, after many hundreds of combinations, do individual positive reactions appear from time to time in the form of changes of respiration and cardiac activity dissociated in time. These reactions, which become more and more frequent, prove that the vegetative components become involved in temporary connection. Only some time later (from 10 to 50 combinations) do motor components of the conditioned reaction appear too. Earlier appearance of the vegetative components of the conditioned defense-pre-

INTEGRATIVE F U N C T I O N OF T H E CEREBRAL CORTEX

35

servation reflex in premature children and the dissociation in the respiratory and cardiac activity reactions observed in them show that the necessary coordination between the vegetative and somatic components is lacking in the early stages of ontogenesis of man. Apparently, this is due to the fact that immature cortex structures of premature children at this stage of development are not yet in a condition to provide the integration of an adaptive act so complicated in its manifestation as is the conditioned defense-preservationreflex. The dependence of the formation of a specialized conditioned reflex and of the degree of its integration on the development of the brain cortex function is illustrated by data pointing to a considerable decrease or absence of dissociation in vegetative and somatic components when the formation of conditioned reflexes has just initiated in premature children of an older age, when the brain cortex becomes more mature. This is also confirmed by data showing that in less premature children dissociation in vegetative and motor components is considerably less marked and disappears rapidly. The motor component and further specialization of the conditioned reflex, the integration of which is, apparently, performed at a higher cortex level, appears at an earlier age than in more premature children. In birds the cerebellum is highly developed, since it is an organ of the coordination of movements and equilibrium; so are the subcortical centres with numerous regions (paleostriatum, archistriaturn, mesostriatum, hyperstriatum, neostriatum) and especially the reticular formation. Subcortical centres in birds carry out complex chain reactions of the instinctive type and become the main, more complex and highly organized apparatus which regulates the motor activity through a system which controls the lower-lying formations. The reticular formation of the brain stem plays a very important integrative role. In mammals the brain cortex is especially welldeveloped being the main substratum of conditioned reflexes. The brain cortex develops and becomes more complex not only quantitatively, but also qualitatively, at the expense of perfection of the nervous elements and intraneural connections themselves. If axosomatic connections predominate in the subcortical formations, axodendritic connections ensuring a great number of different variants of contacts on one neuron are found to occupy a first place in the brain cortex (Sarkisov, 1929, 1960). However, in the process of development subcortical formations do not remain immutable, as was formerly believed, but also become more complex and grow, though at a slower pace than the brain cortex (Kukuyev, 1959; Preobrazhenskaya, 1959, 1960; Amunts, 1959; Dzugayeva, 1959, 1960; etc.). The role of the subcortex formations, and, especiallythe coordinative and integrative role of the reticular formation, becomes more and more complicated not only in motor functions, but also in their effect on the brain cortex, ensuring together with main afferent analyser systems, a ready response of the whole cortex to stimuli from the external and internal environments of the body. The latest morphological and electrophysiological studies suggest that different regions of the cerebral cortex do not develop uniformly so far as their structure and References p. 36-38

36

S. SARKISOV

motor activity are concerned, and as a leading element of morphophysiological processes reflect the growth and greater complexity of the structure of the neopallium. The neopallium is the bearer of the highest forms of behaviour and processes of integration of brain functions, while the palaeocortex, archicortex and cortex intermedius (Filimonoff, 1947,1960)represent a lower level of integration, which is proved by modern experiments of stimulation and destruction of the hippocampus and amygdala. On an even lower integration level are the formations of the subcortical structures and mechanisms, including the reticular formation. One of the main causes of the recent overestimation of its role in the integrative activity of the brain is, undoubtedly, the underestimation of the laws of evolution of the central nervous system and of its place and role of its separate formations in the general activity of the brain. Recent data about the cellular and myelin structures of the brain cortex, extremely complicated and intimate mechanisms of neurons and their correlations, variety of axosomatic and axodendritic connections which become more and more complex in the process of development,clearly demonstrate the highest integrative function of the cerebral cortex. SUMMARY

The purpose of this paper is to present some results of investigations of the Moscow Brain Institute on the ontogenesis of the brain and especially of the cortex, which represents the highest degree of integration. The cortex plays an extremely important role in complex functions, as speech, praxis, gnosis etc. In a series of Primates from hapale to man the size of the cortex increases comparatively to the whole surface of the hemispheres, e.g. in the frontal and parietal regions. Cortical surfaces of other regions relatively decrease at the same time, e.g. in the precentral area. Studies on the evolution of the brain show the trends of development not only of the cortex, but also of its different layers, during the intra-uterine period and after birth. The development and complication of the structure and function of the cortex correspond to perfection in the forms of behaviour, complication of the analysis and synthesis of stimuli reaching the body, which, in cooperation with the first signal system, ensure the nervous activity of man : speech. The formation of higher nervous activity in children is determined by the degree of maturity of the cortex and the subcortical formations. The subcortical formations grow and become more complex, but at a slower rate than the cortex. The neopallium becomes more and more complicated and appears as the bearer of new patterns of behaviour and processes of integration of the cortical functions, while the palaeocortex, archicortex and cortex represent a lower level of integration. An even lower integration level is represented by the subcortical formations including the reticular formation. REFERENCES AMUNTS, V. V., (1959); Cytoarchitectonics of the reticular formation of the brain of certain mammals. Structure and Function of the Reticular Formation and its Place in the Analyser System. MOSCOW, (p. 27-40).

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BRODMANN, K., (1909); Vergleichende Lokalisationslehre der Grosshirnrinde. Leipzig, Ambr. Barth. CONEL,J. LEROY.,(1939, 1941, 1947, 1951, 1959); The Postnatal Development of the Human Cerebral Cortex. Vol. I-VI. Oxford. S. B., (1959); Topographico-anatomical interrelationships of the reticular formation and DZUGAYEVA, the afferent pathways of the brain in the context of comparative anatomy. Structure and Function of the Reticular Formation and its Place in the Analyser System. Moscow, (p. 12-26). DZUGAYEVA, S. B., (1960); Unresolved problems in the study of the afferent pathways of the brain. Some Theoretical Questions regarding the Formation and Activity of the Brain. S. A. Sarkisov et at., Editors. Moscow, Medgiz. FILIMONOFF, I. N., (1929); Zur embryonalen und postembryonalen Entwicklung der Grosshirnrinde des Menschen. J. Psychol. u. Neurol., 39, 323-389. FILIMONOFF, I. N., (1932); Regio occipitalis heim erwachsenen Menschen. J. Psychol. u. Neurol., 44, H. f, 1-96. FILIMONOFF, I. N., (1933); Regio occipitalis bei hoheren und niederen Affen. J. Psychol. u. Neurol., 45, H. 3, 69-137. FILIMONOFF, I. N., (1947); A rational subdivision of the cerebral cortex. Arch. Neurol. a . Psychiat., 58, 3, 296-316. FILIMONOFF, I. N., (1949); Comparative Anatomy of the Cerebral Cortex in Mammals. Moscow, U.S.S.R. Academy of Medical Sciences Issue. FILIMONOFF, I. N., (1958); Formation of the corpus callosum in man and its transformation in the process of ontogenesis and phylogenesis. U.S.S.R. Academy of Medical Sciences Bulietin, 5 , 37-47. I. N., (1960); General conformities in the development of the cerebrum. Neurological FILIMONOFF, Problems. Collection of papers dedicated to Prof. S. N. Davidenkov on his 80th birthday. Leningrad, Medgiz (p. 93-101). KONONOVA, E. P., (1940); Development of the frontal region at and after birth. Transactions of the Brain Institute, Moscow, 5, 73-124. KONONOVA, E. P., (1948); Development of the frontal region of the human brain in the foetal period. Transactions of the Brain Institute, Moscow, 6, 8114. KONONOVA, E. P., (1957); Frontal region of the human cerebral cortex and its place in the general system of the cortical ends of the analysers. Korsakov J. Neuropathol. Psychiat., 57, No. 2,1383-1 394. KONONOVA, E. P., (1961); Development of certain areas of the frontal region related to the speechactivating analysers (areas 44 and 45). Structure and Function of the Analysers in Human Ontogenesis. S. A. Sarkisov, Editor. Moscow, Medgiz (p. 237-245). KRASNOGORSKY, N. I., (1958); Higher Nervous Activity in the Child. Leningrad, Medgiz. L. A., (1952); Development of the striopallidurn in ontogenesis and phylogenesis. NeuroKUKUYEV, pathol. Psychiat., 16,No. 5, 3 8 4 . KUKUYEV, L. A., (1959); Details of the development of the subcortical formations of the motoranalyser. Development of the Central Nervous System. Moscow, Medgiz (p. 102-109). KUKUYEV, L. A., (1961); Development of the motor-analyser in human ontogenesis. Structure and Function of the Analysers in Human Ontogenesis. Moscow, Medgiz (p. 257-263). MINAYEVA, V. M., (1948); Postnatal development of the superior sincipital region of the human brain. Transactions of the Brain Institute, Moscow, Medgiz, 6, 77-107. V. M., (1959); Development of the superior sincipital region of the human brain in the MINAYEVA, prenatal period. Development of the Central Nervous System. Moscow, Medgiz (p. 55-71). PILIPENKO, V. I., (1961); Central regulation of the sensory impulses in the light of certain general conformities between the structural and functional organization of the central nervous system. Pavlov J. higher nervous Activ., 2, No. 5 , 884-894. POLYAKOV, G. I., (1961); Some results of research into the development of the cortical ends of the analysers in man. J. comp. Neurol., 117,No. 2, 197-212. PREOBRAZHENSKAYA, N. S., (1948); Postnatal development of the occipital region of the human brain. Transactions of the Brain Institute, Moscow, Medgiz, 6, 4476. PREOBRAZHENSKAYA, N. S., (1959); Prenatal development of the cortical end of the optical analyser in man. Development of the Central Nervous System. S. A. Sarkisov and N. S. Preobrazhenskaya, Editors. Moscow, Medgiz (p. 27-39). PREOBRAZHENSKAYA, N. S., (1960); Structural development of different links in the human optical analyser and their functional significance. Structure and Function of the Nervous System. S. A. Sarkisov, Editor. Moscow, Medgiz (p. 58-62). ROSE,M., (1926); uber das histogenetische Prinzip der Einteilung der Grosshirnrinde. J . Psychol. u. Neurol., 32, H.3, 97-160.

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S . SARKISOV

ROSE,M., (1926); Allocortex bei Tier und Mensch. J. Psychol. u. Neurol., 34, H. 1-30 u. 42-76. SARKISOV, S. A., (1929); ifber die postnatale Entwicklung einzelner cytoarchitektonischer Felder beim Hunde. J. Psychol. Neurol., 39,486505. SARKISOV, S . A., (1935); Variability of the anterior region of the human cerebral cortex (areas 4, 6 and 8). Neuropathol. Psychiat., Psychohygime, 4, No. 9/10, 271-282. SARKISOV, S. A., (1948); Particulars of the Formation of the Neuron Conrmunicationsof the Cerebral Cortex. Moscow, U.S.S.R. Academy of Medical Sciences Issue. SARKISOV, S. A., (1956); Relationship between structure and function of the cerebrum. Proc. 20th Intern. Congr. Physiol., Brussels. Moscow, U.S.S.R. Academy of Sciences Issue (p 127-141). SARKISOV, S. A., (1960); Functional interpretation of certain morphological formations of the cortex of the cerebral hemispheres from the evolutionary aspect. Korsakov J. Neuropathol. Psychiut., 60, NO.6, 645-651. SARKISOV, S. A., (1960); The functional interpretation of certain morphological structures of the cortex of the brain in the evolutionary aspect. Structure and Function of the Cerebral Cortex. D. B. Tower and J. P. Schadk, Editors. Amsterdam. Elsevier (p. 81-87). STANKEVICH, I. A., (1961); Development of the inferior sincipital region in man and its place in the system of cortical ends of the analysers. Structure and Function of the Analysers in Human Ontogenesis. Moscow, Medgiz (p- 246256).

39

The Cerebral Cortex of the Premature Infant of the 8th Month TH. RABINOWICZ Laboratory of Neuropathology, Institute of Pathology, Lausanne (Switzerland)

The present systematic study has been undertaken in order to establish an atlas of the cerebral cortex of the premature infant of the 8th month. This work is the first step to a greater study of the development and the building up of the elements of the cerebral cortex from the beginning of cortical differentiation, that is to say from approximately the 5th foetal month till birth. From this age on, we have Conel's atlases, which cover the newborn at term up till the child of 4 years old. In the same way as for the 8th month premature and using the same methods as Conel, we shall also prepare atlases for the cerebral cortex of the premature infants of the 7th and of the 6th month. In our work, 44 areas of the cortex were topographically and cytoarchitectonically chosen according to Von Economo and Koskinas' definitions (1925). The cytoarchitectonic study of each area is based on the following elements: (I) The thickness of the cortex and of each layer in each area. (2) The number of neurons in each unit of Von Economo. One knows that this unit is theoretically composed of a cube whose sides have a length of 100p. As the counting of the cells is not possible on slides 100 p thick, slides of 25 ,u are used, the figures found being multiplied by 4. In addition, the differentiated and undifferentiated cells were counted separately in each layer. (3) The number of other cells, that means mostly neurons or granular cells coming from the adjacent layers, of the layer under consideration. (4) The number of astrocytes in each layer of each area. (5) The number of Cajal cells in the molecular layer of each area. (6) The number of neurons and astrocytes of the white matter, 0.3 mm under each area. (7) The length and the width of the neurons. (8) The condition of the Nissl bodies, if there were any, of the chromophil substance, and the differentiation of the cellular body. (9) The condition of the neurofibrils, if we found any, and also of the axons. (10) The development of the dendrites and of their bulbs and thorns. (11) The degree of myelinization, where it existed. The numerical results are given in Tables. The drawings of each area were made out of Golgi-Cox impregnations at the same References p . 86

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diameters and in the same way as in Conel's atlas of the newborn at term so as to be directly comparable. IDENTIFICATION

Inevitably, with the measurement and counting of the neurons we had to face the problem of their identification, that is to say in which group the neurons should be classified (Fig. 1).

Fig. 1 . Drawings of the main types of cells as seen in Cresyl violet stained sections, magnified by lo00 diameters.

In the cerebral cortex of the premature infant of the 8th month, a great number of the nerve cells are still not quite clearly differentiated. We have called them undifferentiated cells or neuroblasts; their nucleus is round with a thick nuclear membrane containing a large amount of chromatin and two or three, even four nucleoli. Between the elements of the chromatin network, there is a diffuse substance probably made up of nucleoproteins, which causes the basophilia of the nucleus. The latter is surrounded

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41

by a slight quantity of protoplasm which does not yet have the clear triangular form that we see later. These are the elements which are given in our Tables as undifferentiated cells and their number is compared to that of the more developed elements of the same origin.

Fig. 2. Sup. picture = photograph of lateral aspect of the cerebral left hemisphere of a premature infant of the 8th month; the length of the formalin-fixed brain is 10.5 cm. Inf. picture = photograph of dorsal aspect of the cerebral left hemisphere; the frontal lobe is on the left. References p . 86

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We call them neurons, although we realize that they are still far from being matured. These neurons already possess a triangular protoplasm ; they are still basophilic, have a short dendrite and do not contain any Nissl body. Their nucleus is still round and has slightly less chromatin than the neuroblasts or what we call undifferentiated cells. The granular cells in the premature infant greatly resemble the lymphocytes. They consist of a dense nucleus, which is smaller than that of the neuroblasts; generally, they have one or more nucleoli and very little protoplasm around them. The spindle cells and the micro- and oligoglial cells are already recognizable, The astrocytes are found in great quantity in all the layers of the cortex and sometimes their differentiation is quite difficult. The astrocyte has a pale nucleus, whose irregular border is oval or round. It has a thin nuclear membrane and thin chromatin network with several nucleoli. Very little nucleoprotein is found between the chromatin network, which gives the nucleus a white base and which contrasts readily with that of the neurons or the neuroblasts. M ACROSC OPY

Before getting to the histological generalities and detailed description of some areas, we shall describe the macroscopic aspect of the brain (Figs. 2 and 3). Compared to the brain of the newborn at term, the premature brain is approximately 1.5 cm smaller in all its dimensions (length 11 cm, width 8 cm, height 7 cm). The weight of the brain averages 270 g, whereas the brain of the newborn at term is 335 g (Conel, 1939). Another finding is that the circumference of the premature infant’s head is on the average about 28.5 em. If we now examine the exterior aspect of the brain, we notice the width of the lateral fissure of Sylvius, which does not yet completely cover the insula. The main circumvolutions are well designed in all lobes, while the secondary ones are but slightly formed. In the frontal lobes, the fissures are less deep in the regions corresponding to FEm (frontal pole), in the orbital circumvolutions and the gyrus rectus. They correspond to Von Economo’s FDp, FDL, FF, FH. As one proceeds posteriorly in the frontal lobe, the fissures become deeper and the circumvolutions are better designed and begin to subdivide into secondary circumvolutions. Generally, compared to that of the full-term newborn brain, the radius of the crown of the circumvolution is smaller in the premature, the fissures are wider and the circumvolutions are designed in a more simple manner. In the temporal lobe, the superior temporal gyrus is the most developed while the inferior temporal gyms is the least developed. Thus TA of Von Economo is more developed than TE. In the parietal lobe, the posterior central gyrus areas PB and PC are clearly more developed than PG, that is to say the angular gyrus. In the occipital lobe, the area around the calcarine fissure, that is OC, is clearly more developed than OA, which largely corresponds to the anterior external part of the occipital lobe. One thing we also note is the greater width of the parieto-occipital fissure and the relative delay in the development of the precuneus and of the cuneus. The olfactory lobe is definitely much wider and is almost as large as that of the newborn at term.

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Fig. 3. Sup. picture

=

medial aspect of the right hemisphere. Inf. picture hemisphere; the frontal lobe is on the right.

=

43

ventral aspect of the left

Finally, the consistency of the premature brain is that of custard, which is probably due to its increased water content, and causes many technical difficulties, especially with the embedding procedures. Before giving further particulars of some of the areas, that is to say the numeration of the various elements, we shall speak about their morphology. To do this, we followed the scheme of the newborn a t term’s atlas, so as to have a comparative study. References p . 86

44

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T H E C Y T O A R C H I T E C TO N I C S T R U C T U R E

The cytoarchitectonic structure as shown by Cresyl violet staining is already established in the premature infant of the 8th month as it is in the newborn at term but it is not so clearly designed. Beside the marginal zones and some parts of the rhinence-

Fig. 4. Area FA?. Posterior wall of gyms centralis anterior in the region of the trunk. On the left: Cresyl violet stained section 12 ,u thick, magnified by 1 0 0 diameters. On the right: Conel’s modification of Cajal silver impregnation magnified by 1 0 0 diameters.

phalon, the cerebral cortex is formed of 6 layers. A layer of pyramidal cells is found in all the areas of the cortex. These cells form layer V in the isocortex and the pyramidal layers in the allocortex. Most of the time, these cells are irregularly distributed, some-

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45

Fig. 5 . Area FAy. Posterior wall of gyrus centralis anterior in the region of the trunk. Golgi-Cox impregnations, celloidin embedding, magnified by 100 diameters. In the molecular layer, some very short horizontal fibres. I n the third, fourth and fifth layers, the neuropil is well developed. The drawing of this section is to be seen on the right side of Fig. 6 . References p . 86

TH. R A B l N O W l C Z

46

times in clusters and sometimes in the form of a stairway in the preceiitral gyrus. In the IIIrd layer and depending on the region, their number can be from 1.5 to 6 times as great as in the newborn at term. In layers V and VI, this increase is not as great.

.

.

, .-. .* . :.*

* * 1 :*

.’.. .’

0

:*.

*

I ‘ v * ,

Fig. 6. For legend see p. 47.

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47

The pyramidal cells are more numerous in the insula and in the uncus. In the hippocampus, they can even form a compact rank, which corresponds to what can be seen one month later. With the Cresyl violet stain, their protoplasm colours slightly basophilic, but in the hippocampus as well as in the fascia dentata it is slightly darker. The extra large pyramidal cells are the largest cells found in the precentral gyrus where they constitute the cells of Betz. From this region and as we go towards the frontal lobe or backwards to the parietal lobe, the size of the extra large pyramidal cells diminishes. They become slightly larger in the area striata, in the insula and in the superior temporal gyrus, as is found in the newborn at term. We have never found any mitosis in the neurons. But we have found few of them in astrocytes, in area FAy layer I and in the parietal region. Nor have we ever found Nissl bodies in the pyramidal cells but a violet dust on Cresyl violet coloration in the Betz cells as if there were very thin Nissl bodies. We have never found any neurofibrils with the Cajal silver method, which gives a powdery impregnation in the neurons of the premature infant. As had already been seen (Angevine and Sidman, 1961) in the mouse, using tritiated thymidine labelled cells, the various types of neurons, especially pyramidals, can be found above or below their ‘assigned’ layers in what we call ‘Ie ballet des neurones’, in other words their dynamic development. The granular areas constituting the coniocortex are well recognizable with all the methods we employed. However, the layers are less clearly defined than in the newborn at term. With Golgi-Cox impregnation, which gives remarkable results in the premature, we have found Golgi type I1 cells in all areas of the cortex. In general, the impregnation of these cells is less well defined, and sometimes these cells are smaller, less well designed and less numerous than in the newborn at term. The network of fibres of layers 111, IV and V as described by Conel, is also visible in the premature but is less dense. The coniocortex is also clearly defined in the regions known as such by Von Economo, that is to say: PB, OC, TC, LE and HD. The horizontal cells of layer I were sometimes recognizable as such, and on the other hand in layers I1 and I11 they were very rarely seen. In layer V, and especially in layer VI, a certain number were found; but it seems that there are less of them in the premature infant than in the newborn at term.

Fig. 6. On the left: Area FAy. Posterior wall of gyrus centralis anterior in the region of thz legs. On the right: Area FAy. Posterior wall of gyrus centralis anterior in the region of the trunk. Drawings from Golgi-Cox impregnations: the drawings are made with a camera lucida and magnified by 100 diameters from sections 150 p thick. The cells are drawn and magnified by 250 diameters. At this magnification, the entire length of the apical dendrites of the pyramidal cells cannot be drawn; therefore they are shortened in order to make them end a t the level in the cortex where they actually terminate in the sections. The branches of the apical dendrites, however, as well as the basal dendrites and the axons, are drawn in their entirety excepting where they extend beyond the boundaries of the drawing. Representative cells are drawn in each layer of the cortex. The vertical, exogenous and horizontal fibres are not shown except in the molecular layer if there were any. The upper dots indicate the surface of the cortex. As the drawings‘have been made from celloidin-embedded material, the shrinkage is about 25% less than in the.paraffin-embedded Cresyl violet stained sections. References p , 86

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In layer VI, in all areas of the isocortex, we found some small and large spindle cells, as did Conel. In this layer, we also frequently found pyramidal cells in oblique, horizontal or inversed position. The inverted cells have a n axon which rises over a short distance towards the surface, then returns down into the white matter. It seems that some pyramidal cells have not yet completed their rotation at the time of their penetration into the cortex. This is especially seen in Golgi-Cox impregnations. The granular cells are slightly triangular and the Golgi-Cox type I1 cells are present in layer IV, in all the isocortex. It seems to us that the variation of the size in the Golgi type I1 cells is not very great in the premature and it is consequently difficult to distinguish large from small cells, which is not the case one month later. Not only does the width of layer I1 vary greatly, as is also the case one month later, but the layer is often sinuous and has many more cells. In the newborn at term it takes a linear aspect. The limits of layer I1 are also less well defined. As we shall see in the drawings made out of the Golgi-Cox impregnations, the degree of development of the dendrites varies greatly. Generally, we can say that the dendrites are less wide-spread in the premature and possess far fewer bulbs, thorns and ramifications. The apical dendrite never grows as high. On the other hand, the extra large pyramidal cells of layer V of the isocortex are really more mature than the others and have already a long axon, as is found in the newborn at term. However, we noticed that, using the Golgi-Cox impregnation, the body of the extra large pyramidal cells is almost as large as that of the newborn but with shorter and less numerous dendrites and their axon has generally no collaterals. The small and large pyramidal cells of layers V and VI are also differentiated, but this development is slower than that of the extra large pyramidal cells. As Conel has already remarked, the pyramidal cells of layer 111 develop relatively later than those of layers V and VI. We also noticed that the pyramidal cells are more developed and better impregnated in the deeper parts and in the walls of the circumvolutions than in the crown of the adjacent less well developed circumvolutions. DEGREE OF DIFFERENTIATION

We also tried to evaluate the degree of differentiation in the different parts of the cortex according to the size and the length of the neurons and their dendrites, the number of branches and the number of bulbs, as well as the number of exogenous and horizontal fibres and the degree of myelinization of the cortex. Frontal lobe Using these criteria, we noticed that in the frontal lobe the precentral gyrus (area gigantopyramidalis FAy) (Figs. 4,5,6and 7) is the most differentiated and developed of a11 areas. We also found regional variations: in the motor area for the legs, using Golgi’s technique, several horizontal fibres in layer I and a greater number of fibres in layer V, mostly exogenous fibres, were seen. As found in the newborn at term, the

THE CORTEX OF THE PREMATURE I N F A N T

49

Fig. 7. Same techniques as in Fig. 6. On the left: Area FAy. Posterior wall of gyrus centralis anterior in the region of the hand. On the right: Area FAY. Posterior wall of gyrus centralis anterior in the region of the head.

References p . 86

50

TH. R A B I N O W I C Z 3

:.';. . .. ...-.

-.-.-..... *. . ..

Fig. 8. Area FCBm. Pars opercularis of gyrus frontalis inferior. On the left: Cresyl violet stained section 12 ,u thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

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51

motor region FAY for the trunk is the most developed area of the precentral gyms. In this region, the apical dendrites of the Betz cells are at their greatest length and have the greatest number of bulbs, while the basal dendrites are the most numerous

Fig. 9. Area FDA. Anterior region of gyms frontalis medius. On the left: Cresyl violet stained section 12 p thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations. References p . 86

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TH. R A B I N O W I C Z

and the longest. The network of fibres in layer I is clearly denser than in the highest part of the precentral gyrus. Similarly, the network of exogenous fibres in layers V and VI is denser. In the motor area for the arms and the hands, the extra large pyramidal cells are less developed than in the motor area for the trunk. So,the motor area for the hands

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

..

8

I

Fig. 10. Area FE. Crown of gyrus frontalis superior at the frontal pole. On the left: Cresyl violet stained section 12 ,u thick. magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

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,

53

- . - . . . . - - . . .. . . I

i..

Flg. 11. Area PB. Upper two-thirds of the anterior wall of gyrus centralis posterior in the region of the trunk. On the left: Crespl violet stained section 12 ,u thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

Rejhrunces y . 86

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TH. R A B I N O W I C Z

seems to be less developed than the motor area for the legs. The motor area for the head is the least developed. In this area, the Betz cells are at their smallest, in their body as well as in their dendrites; the fibres of layers I, V and VI are less numerous. We frequently found neurons with two apical dendrites leaving the cell bodies side by side; this is probably a sign of lack of maturity. Contrary to what is found in the newborn at term, where the development of the motor area for the arms and that of

.

.

. .. - _. . . . : .. . . . . -. .. - .

Fig. 12. Area PC. Crown of gyms centralis posterior in the region of the trunk. On the left: Cresyl violet stained section 12 p thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

T H E CORTEX OF T H E P R E M A T U R E I N F A N T

55

the legs is approximately the same, we found in the premature infant that the motor area for the arms is less well developed than that of the legs. In the other regions of the frontal lobe (Figs. 8 , 9 and lo), the tangential fibres are practically non-existent in layer I. The fibres of layer V are slightly more numerous in F D than in FC. In the superior part of area FD, they are slightly more developed than in the inferior region. The degree of development is almost the same in F C as in FB. On the other hand, the neurons are definitely much smaller in F D and its various subdivisions, those of FE being the least well developed. However, if we consider the Golgi-Cox impregnations of area FF, we find that the neurons are slightly larger than in the other anterior regions of the frontal lobe and that their basal and apical dendrites are slightly more developed. Using Weigert and Weigert-Pal's methods for the myelinized fibres, none of the latter was found in the frontal cortex. On the other hand, the white matter contains some myelin sheaths, thin, solitary or in bundles. These sheaths are most numerous below the precentral gyrus, and in a general way are distributed according to the map of Flechsig. Parietal lobe In the area PB of the coniocortex (Fig. 1 l), we find that the degree of development is relatively less advanced than that of the precentral gyrus at the same level. It seems to us that there is a greater difference in the premature between the parietal region and the frontal region at the same level. Area PB of the coniocortex has approximately the same degree of development as area PC (Fig. 12) of the isocortex so far as the extension of the neurons, their dendrites, and the extension of the fibrillary network in layer V are concerned. Moreover, it appears that the large neurons of area PB are slightly more developed than those of PC. If we examine the other parietal areas using the meshwork of horizontal fibres and the neuronal size as criteria, we find that PH (temporal intermediate zone) is but slightly developed, while areas PG and PE are at about the same degree of development. Thus we find that the slightly differentiated areas are more numerous in the parietal lobe in the premature infant than in the full-term newborn. In PB and PC the neuropil network is relatively slightly developed. The neuropils are more noticeable in areas PH and PF (Fig. 13). The number of fibres in the white matter and the number of vertical fibres in the cortex in layers V and VI are much less numerous than in the frontal areas. The network of fibres in layer IV in the full-term newborn is quite noticeable, but in the premature infant it is more visible in PF than in PB. In layer V, the neuropil network is not as well developed as in the full-term newborn, in the parietal lobe. There are more tangential fibres in layer I of P F than in PH, which has more of them than area PG, which only has rare fragments (Fig. 14). Area PE has a moderate amount of fibres but its neuropil in layers IV and V is relatively poor. Later we shall see that numeration gives a better account of the degree of differentiation of these areas than the Golgi-Cox impregnation, which gives variable results. References p . 86

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TH. R A B I N O W I C Z

Fig. 13. Area PF. Crown of gyrus supramarginalis. Photomicrograph of Golgi-Cox impregnations.

T H E CORTEX OF T H E PREMATURE INFANT

57

Weigert’s method did not show any myelinized fibres in the parietal cortex. On the other hand, the myelinized fibres in the white matter are less numerous than those found in the frontal regions.

i

Fig. 14. Area PG. Crown of gyrus angularis. On the left: Cresyl violet stained section 12 ,u thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations. References p . 86

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Occipitd lobe

In the occipital cortex, we find that the calcarine region (OC) (Fig. 17) is far more developed than area OB, which in turn is more developed than area OA. In Cresyl violet sections the limit between OB and OC (limen OBy) (Fig. 16) is already clearly visible in the premature. It has the same appearance as that found in the full-term newborn. The lack of columnar formation of the neurons in area OA was quite notice,

-

- .

. - . .. --.- .. .-• ..-.* /"

I

Fig. 15. Area peristriata, OAl of lobus occipitalis. On the left: Cresyl violet stained section 12 ,u thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

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59

Fig. 16. Transition region between area parastriata, OB, of lobus occipitalis, area OBy, limen parastriatus gigantopyramidalis of lobus occipitalis, and area striata, OC, of lobus occipitalis. The subdivision of the 4th layer is well visible. Cresyl violet stained section 12 /A thick, magnified by 100 diameters.

able. This clearly shows that the differentiation of OA (Fig. 15) is much less advanced in the premature. The vertical columnar formation is due to the presence of vertical fibres situated between the cells but in our opinion is more likely to be due to the development of the lateral dendrites of the neurons. The development of these lateral dendrites will separate the neurons from each other. This and the absence of mitosis suggest that the cerebral cortex in a general way develops mostly by formation of dendrites and not by the increase in the number of neurons. The meshwork of horiReferences p . 86

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T H. R A B I N O W I C Z

.. . . . - .-., ;

’;

*‘.*...

..* I

7’ Fig. 17. Area striata, OC, of lobus occipitalis. On the left: Cresyl violet stained section 12 p thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

zontal fibres in layer IV of OC is fairly dense and can readily be seen with the naked eye. This network has a sharp limit from the beginning of area OC beside OBy. It is also less dense in layer V of OC than is found in the newborn. In areas OB and OA these networks are less dense than in the newborn. It also appears to us that the network of the fibres in layer IV is denser in the coniocortex PB than in OC. The diminution of density of these networks is great between OC and OB.

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. .. . .

. ...

I

,

. . ,. .. . ... . .. .

Fig. 18. Area TA. Crown of gyrus temporalis superior. On the left: Cresyl violet stained section 1 2 p thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

References p . 86

62

TH. R A B I N O W I C Z

...

- .

Fig. 19. Area TG. Polus temporalis. On the left: Cresyl violet stained section 12 ,u thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

T H E C O R T E X OF T H E P R E M A T U R E I N F A N T

63

We found no vertical or horizontal myelinized fibres in the occipital cortex.

Temporal lobe In the temporal lobe, if the extensions of the neurons are longer in TC, we notice that TB is almost at the same stage of development, except for the basal dendrites, which are slightly shorter. The tangential fibres are fragmented and rare in TB and TC in layer I. There are almost none in TA (Fig. 18), TE and TF. The last three areas are all at approximately the same degree of development, that is to say relatively immature.

I

Fig. 20. Area precentralis insulae, IA. On the left: Cresyl violet stained section 12 /I thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations. References p .

86

64

TH. R A B I N O W I C Z

Very few horizontal fibres are found in layer V. These fibres are especially noticeable in TC, TE and TB. Sections of TG (temporal pole) (Fig. 19) show that it is remarkably well developed: in this area the apical dendrites reach layer I1 and have many bulbs and thorns; the lateral dendrites are well developed, there are many tangential fibres, and the exogenous fibres of layer V are more numerous than in TC; there are also more fibres in the white matter than in TC. Thus it appears that TG is more developed than TC, which corresponds to what is found in the full-term newborn.

. ... - _ ...,.*..- ..... . .. - . .. .

I

Fig. 21. Area postcentralis insulae, IB. On the left: Cresyl violet stained section 12 ,u thick. magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

65

THE CORTEX OF T H E P R E M A T U R E I N F A N T

insula The two areas of the insula, IA and IB, differ considerably in their degree of development. IA (Fig. 20) is clearly more developed and has longer dendrites than IB (Fig. 21). The neurons, especially the pyramidal ones, are much more developed in IA. Furthermore, we found numerous horizontal fibres in layers 111, IV, V and VI. The axons in the white matter are also more numerous in IA. There are fewer horizontal fibres in

.. .-. ., .. ... ...*. ... .

. * .

. I

-

Fig. 22. Area LA. Anterior region of gyrus cinguli. On the left: Cresyl violet stained section 12 p thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations. References p . 86

66

TH. R A B I N O W I C Z

IB in layers V and VI, where they d o not yet form a clear network. The horizontal fibres in the molecular layer are few and fragmented both in IA and IB. Gyms cinguli Similar to the newborn at term, the posterior part of the gyrus cinguli is more developed than the anterior one. However, this difference is less pronounced and LD

.. . ...-...

0

- . ..

-

1

ii

111

V

Fig. 23. Area LC. Posterior region of gyms cinguli. On the left: Cresyl violet stained section 12 p thick, magnified by 100 diameters. On the right: Drawings from Golgi-Cox impregnations.

T H E C O R T E X OF T H E P R E M A T U R E I N F A N T . 1

..

... .. . .. . ..

*'-*-

..

- - - /

-.

-.._ _ _ .

? ;

__

...

-.

A-

Fig. 24. Area HA. The uncus gyri hippocampi. On the left: Cresyl violet stained section 12 ,u thick, magnified by 100 diameters; two glomeruli are visible in the upper part of the cliche. On the right: Drawings from Golgi-Cox impregnations; one inverted pyramidal cell is visible on the left inferior comer. References p . 86

23 I MON IBV X ‘ H I

89

THE CORTEX OF THE PREMATURE INFANT

69

and LE are almost as well developed as LA (Fig. 22). Also in the latter, the dendrites have not grown as high. Generally, in the limbic lobe the different layers are iess well defined with Cresyl violet stainings. LC (Fig. 23) is the least developed area. LE and LD are better developed and have more differentiated middle and deep layers. The tangential fibres of layer I are rare and fragmented, while the horizontal fibres of layer V are poorly developed throughout the limbic lobe. There are few axons and myelinized fibres in the white matter. Ammon’s horn

In the cornu Ammonis (Figs. 24, 25 and 26) the different structures are well differentiated and it is possible to recognize all the areas described by Von Economo. In areas HE and HD, we can still find the superficial cells of Obersteiner. Small round embryonic cells are also found just outside the cells of Giacomini’s band. The only elements which are less well differentiated are the glomeruli. The pyramidal layers are well developed throughout. The development of the pyramidal cells in Ammon’s horn is a rather peculiar one because of the exceptionally well developed apical dendrites and cell bodies, while the basal dendrites and the fine branchings of the apical dendrites are very poorly visible. This can especially be observed in the neurons of Giacomini’s band and of the glomeruli. One month later, there will be many more basal dendrites and branchings of the apical ones, but the apical dendrites will not have grown much longer. Thus, this special type of pyramidal cells seems to have a pace of development different from the others. Golgi-Cox preparations show also that there are some horizontal fibres, but the real networks are seen only in the glomeruli of H E l a and in the central part of HETj3. The horizontal network is clearly visible around the cells of Giacomini’s band and in area H F itself. Some tangential fibres are found in the superficial layer of HE2 and HA. No fibres of Golgi type I1 cells were found in area HD, in accordance to what is seen in the newborn. Fewer horizontal fibres are found than in the newbornat term, in the entire area HE1 p. Even in the premature, there is a definite difference b:tween HEla and HElP. The Golgi type I1 cells are clearly visible and the network of horizontal fibres is well defined at the limits of HEIP. Outside this area the horizontal fibres are much less numerous; they are quite dense in HE 2, as is also found in the newborn at term. Summary

FAy is the most developed area. Both PB and PC are advanced areas of development but clearly less than in the full-term newborn, and less than FAY. HD and HE are almost as well differentiated as OC, which is less developed than PB. TG and TC are more differentiated than the areas of the limbic lobe. The latter is more differentiated in its posterior areas, the central and anterior areas being less well differentiated. Thus the foci of development in the premature are about the same as those of the full-term newborn, but relatively less well developed areas are more numerous. References p . 86

70

TH. R A B I N O W I C Z

Fig. 26. Area pyramidalis arnmonica, HE2, and area dentata, HF. Golgi-Cox impregnation of the same region given on Fig. 25 with Cresyl violet staining. Area HF occupies the right part of the superior third of the picture. The whole inferior third of the picture is occupied by area HE2.

T H E CORTEX OF T H E P R E M A T U R E I N F A N T

71

NUMERICAL CRITERIA

To establish these criteria, the counting of the cells was effected on slides 25 ,u thick, using Cresyl violet stained sections. A surface of 10,000 square was counted with the aid of a high definition 100 times magnifying oil immersion objective associated with an 8 times compensation ocular having designed squares in its field. The countings were done in exactly the same way as those of Conel, that means on the crown of the circumvolution in the middle of each layer and sublayer, taking an average of 10 surfaces and multiplying the result by 4. We counted the differentiated neurons whose apical dendrites were well evidenced by the Cresyl violet stainings separately from those having very little protoplasm and called undifferentiated cells or neuroblasts. We also counted separately the granular cells and the astrocytes. The cells belonging to the adjacent layers but being found in the layer under consideration were counted either separately or together with the rare fusiform microand oligoglial ones and designed as ‘other cells’ in the Tables. Thus the Tables are constituted as follows: In the left hand column are the figures given by Conel (1939) for the newborn at term. In the other columns are our results. Our total number of cells is compared to that of Conel; this is called the equivalent count, which is obtained by adding the underlined numbers; this allows the same type of cells as mentioned by Conel to be compared directly. In the other columns, we have the figures of the differentiated pyramidal cells subdivided into small, large and extra large cells, as was done for the newborn at term. A supplementary column indicates the undifferentiated cells which we have previously spoken about. By comparing this number with the number of differentiated pyramidal cells, we can appreciate the degree of maturation of a layer or sublayer. We counted the number of granular cells that were found outside their own layer. As we also found pyramidal cells outside their assigned layer, we decided to count every cell that was not yet in its proper place. This too allows us to appreciate the degree of maturity of each layer. In the right hand column is shown the number of astrocytes found in each layer. In a general way, we have been struck by the relative increase of the number of cells per unit of volume compared to that of the newborn a t term and that of the adult. Thus, in area FE of layer I1 of the adult one finds 92 granular cells (Von Economo and Koskinas, 1925). In the newborn at term, the same area contains 650 of them (Conel, 1939). In the premature infant of the 8th month there are 3,200 of them. This can be seen in each area in a more or less pronounced manner. Thus, we may conclude that the maturer a n area is, the fewer neurons are found per unit of volume. This basic rule has been used to interpret the figures obtained by our countings. Under the number of neurons we always give the cellular dimensions, the length and the width. In layer I, the number of cells found was 4 times that found in the full-term newborn. In layer I of all areas of the cortex, we found many Cajal cells, having a variable distribution and almost always isolated. When they are found in clusters we noted it in our Tables, but this was not done when they appeared singly. The Cajal cells in the premature brain were generally too heavily impregnated by the Golgi-Cox method, Rejerences p . 86

TABLE I CELL T Y P E S

4 h,

Newborn

8 Months premature

Results of Conel

Equivalent count

I

120

41 1

247 8147317

312 814

I1

360 313 1015

2721

153 815-1117

2568 413 615

IIIa

100 1618 23/13

606

226 lO/c15/8

380 615-817

568

IIIb

80 1618 26/13

407

300 816 -1711 1

107 615-817

311

52

IIIC

110 1618 29/14

479

358 816-1719

121 615-817

306

62

IV

180 313 1216

Layer

Va

-

-

S. 90 1316 1618

-

Small

-

Extra large

Undifferentiated

Granular

Spindle

Astrocyte

-

164 812-1413

26 513 1118

-

w

143 -

437

260 816-1218

34 12/8-20/13

216 816-1218

31 31 121879112 22111132115

-

49

-

54

273

64

220

56

Vb

S. 80 L. 16 Ex.L. 12 13/6-1618 23!12-33/16 48113- 83/18

398

VIa

py. 140 sp. 1316 33/16 2316 36/13

628

270 9/5>0/8

180 -

150 -

28 712-1213

63

VIb

1316 33/16

496

205 816-1819

138 -

119 -

34 712-1213

70

White matter

-

-

Py. 80 Sp. 2316 - 36/13

120 -

165 816 - 20110

313 - 715

> E z

2ii N

349 413-715

558 917 T5110

907

L. 30 26/13 33/16

Pyramidal Large

97

THE CORTEX OF T H E P R E M A T U R E I N F A N T

13

but were clearly shown by the Cresyl violet stain. Swellings are sometimes found on their dendrites which terminate in the form of a drop, from which point the dendrites continue, as it has already been shown in the full-term newborn., The motor area of the trunk, FAY (Table I ) The pyramidal and granular cells represent half the total amount of cells in layer I; spindle cells (sometimes of astrocytic nature) in a smaller quantity have also been found. The number of astrocytes, as in most of the regions of the cortex, is less numerous in layer I. Layer I1 presents the greatest growth and the greatest cell concentration of all the frontal layers. Here, there are approximately 7 times as many cells as in the newborn at term. Among the granules we found some pyramidal cells (153), which probably arise from layer 111. This implies that some pyramidal cells may go too far in their migration. The three sublayers of layer 111 show considerable variation. Layer IIIa has 6 times as many cells as the full-term newborn. This increase is primarily due to non-differentiated cells, which are much more numerous than the differentiated ones. If we compare these figures with those of layers IIIb and IIIc, we can conclude that these layers are clearly more developed because most cells are differentiated. Thus the differential count allows us to show the variation in the development of the sublayers. These findings appear important to us because they indicate that not only some layers are more developed than others, but that there is also a n inequality of maturation within the sublayers of the more organized layers. In layers IIIa and IIIc, cells not belonging to these layers were quite frequently found, which is probably due to the greater concentration of cells in layers I1 and IV. From layer I11 on, the number of astrocytes seems to remain stable at approximately 60 elements per unit. In layer IV there is a substantial increase in the number of cells with Iriore pyramidals than granules. In layer V the increase in cells is not quite 4 times that of the full-term newborn. The majority of the differentiated pyramidaI ceIls are small and we noted a slight delay of maturation in layer Va as compared to Vb, as the former has more undifferentiated cells. The other cells found in this layer consist of granular and spindle cells and form a considerable proportion of the total number found herein. Thus, the total number of cells, not shown here, is 711 elements, of which 273 do not belong to this layer. In layers VIa and VIb, which are difficult to differentiate from one another, we found that VIa has a smaller increase in the total number of cells as compared to VIb. VIb contains more undifferentiated cells than VIa. There is already a considerable number of spindle cells which approximate those found in the newborn at term. On the other hand, there is an increase in the number of astrocytes in VIb, which reaches a maximum in the white matter. This is true for all areas of the cortex. In the white matter, 165 neurons are found, which number is less than that found under FE, a less mature area. References p. 86

TABLE I1 C E L L TYPES

4 P

Newborn

8 Months premature

Results of Conel

Equivalent count

I

160

802

I1

650 313 1015

3196

IIIa

115 814 1618

180

48 7/5713/7

732 5147115

120

56

IIIb

100 814 1819

-

120

96 1 / 4 2117

624

600

48

IIIC

120 814 20110

580

112 8l577/1

468 -

608

52

IV

414 - 1015

Layer

-

-

-

Pyramidal Large

Small

(Cajal: 2) 1416 32/13

-

-

114 1311

s. 100 814 1015

Vb

-

L. 16 Ex.L. 20 1216 1411 22110-24/12

S. 80 814 1015

-

-

VIa

Py. 200 sp. 1214 - 2218 1015 1316

VIb

1015 25/10

White matter

-

-

Astrocyte

120412 - 514 -

80 7/2-1213

514 241217

3116 412 614

-

-

48

828 4127614

24

508

112 9147216

12 1216- 11/7

3 84

624

36

520

120 3147216

24 1315 17/8-18/1- 25/10

376

644

36

-

-

1128

914 1111

1044

914 1717

Py. 120 sp. 1214 - 2218

Spindle

668 1/4-1317

1496

L. 16 1216 1417

-

Granular

20 1 / 4 3216

360

Va

UndifferExtra large

-

100 -

664 -

514 - 715

408

514 - 715

360 -

208 715 - 1117

20 1/270/3

16

20 7/270/3

56 56

T H E CORTEX OF T H E P R E M A T U R E I N F A N T

75

The frontal pole, FE (Table II) In area FE, which we will compare to FA, we find more Cajal cells in layer I. There are 5 times as many cells as in the newborn in layer I of FE, while FAy has 4 times as many cells. Most of the cells are undifferentiated (approximately 720), while the neurons in the prefrontal gyrus are much better differentiated and can be classified as pyramidal cells. The same degree in differentiation is also seen in the spindle cells, which are less numerous than in the prefrontal gyrus. There are also few astrocytes, approximately half the number found in the other areas. Another point is the few pyramidal cells found among the granular cells of layer 11; 20 are found here as compared to 153 found in FA. The number of granules is also increased. It is almost impossible to differentiate between mature and immature granular cells. The three sublayers of layer I11 are already visible in FE, but there is a remarkable increase in the number of undifferentiated cells. There are 732 undifferentiated cells against only 48 pyramidal cells recognized as such in layer IIIa, in comparison to FA, where we found only 1.5 times as many undifferentiated cells as differentiated ones. The increase in cells is greater in sublayer IIIa than in IIIb or IIIc, the latter being the most differentiated. In layer IV there are 1.5 times as many cells in FE as in FAy with approximately 6 pyramidal or undifferentiated cells for 8 granules. There are also very few astrocytes, which is probably due to the slow myelinization in this area. In layer V, the situation is entirely different from that of FAy. In the latter, there are approximately twice as many differentiated pyramidal cells as compared to the undifferentiated ones. In Va the differentiated pyramidal cells are only about one half the number of undifferentiated ones. This proportion is maintained in layer Vb. We also note a certain number of extra large pyramidal cells whose bodies are of approximately the same size as those found in the newborn at term. However, on closer scrutiny, the apical dendrite leaves the neuron’s body with a much larger base in the premature. Thus the cell body appears much bigger than it is in reality due to the difficulty to determine its actual limit. But if we consider the width of the cells we notice that they are smaller than those found in the newborn at term. Once again in layer VI, we can see some elements of immaturity: there are more cells and it is almost impossible to distinguish between differentiated and undifferentiated cells, so that we give over-all figures. If there are 6 times as many cells in layer VIa compared to the newborn, there are 9 times as many in VIb; thus there is a greater degree of undifferentiation in VIb. The number of heterotopic neurons found in the white matter is increased, which emphasizes the degree of immaturity of this area. Parietal lobe, PC and PH (Tables III and IV) If we now compare PC at the level of the trunk to a less mature area PH, we find that the development follows similar lines. In layer I there are almost twice as many References p. 86

T A B L E I11 CELL TYPES

__

Equivalent count

I

100

670

I1

600 414 816

-

2436

IIIa

120 1216 20110

-

568

152 8/4y7/7

416 514-816

540

IIIb

85 1216 23/12

512

236 10/577/8

216 -

344

ILIc

100 1216 26/13

-

568

312 10/578/8

256 -

380

IV

450 414 - 8/6

I565

Va

S. 90 L. 30 Ex. L. 12 1015- 1316 1618- 20112 30112 - 50120

632

240 8/472/7

40 12/7-17/10

8 1819 -25112

344 -

260

40

255 914-1316

28 13/6717/10

4-

217

173

40

-

Small

Pyramidal Large

-

Results of Conel

Layer

(Cajal: 2) 1718 33/15

-

714 - 1617

Undifer-

Extra large 584 -

36 814 7 2 1 5

304 315-1818

Py. 160 Sp. 1216 - 2311 1 18/6 2818

759

244 814-1718

404 514-816

527

164 814-1718

243 5/4-8/6

White matter

sp. 18/6 - 2818

-

84 712-1213

514 917

44

-

28 36

1

5

20 11/5-20/8

N

-

VIa

py. 75

Astrocyte

1264 412 614

504

1216 - 23/11

o\

5d

L. 16 1618 - 23/11

-

Spindle

2400 __ 412 - 714

S. 85 1015 1216

-

Granular

312 614

Vb

VIb

4

8 Months premature

Newborn

48

99

12 712-1113

64

104

16 71271113

64

72 312-614

96

TABLE I V CELL TYPES

h

D

2

8 Months premature

Newborn

0

P

g Layer

Results of Conel

Equivalent count

I

140

1166

I1

600 414 816

-

3512

IIIa

120 1015 1618

-

756

128 914-1516

IIIb

115 1015 1819

688

IIIC

120 1015 20110

-

836

IV

560 414 816

-

2347

S. 90 L.20 Ex.L. 10 814 1015 1618 20110 23/12 26113

816

128 814-1216

12 1316- 15/7-17/7-25/9

676 -

440

36

640

116 8147216

4 1316-1918

520 -

348

32

Va

-

-

-

-

Vb

Py. 160 Sp.

VIa VIb White matter

-

1015 1216

-

-

1213 2516

Py. 70 Sp.

1015 1216

-

1213 2516

1078 440

Small

7!4

Pyramidal Large

Extra large

Undi'erentiated

(Cajal: 2) 2018 25/15

- 1617

-

16 814 5 2 1 6

Granular

Spindle

Astrocyte

1132 312-614

32 712-1213

24 413 - 916

3496 312 614

-

32

628 514-715

736

20

128 1215 - 2016

560 -

672

16

88 12/5-2017

748 -

636

24

115 lO/c18/7

2172 312 614

-

60 8157816

768 514-616

24

248 514-516

815-1816

212 10/5-22/7

28

236

14 7/270/3

52

148

20 7/2?0/3

32 52

1

2

78

TH. R A B I N O W I C Z

cells in PH as in PC. The number of astrocytes found in PH is among the lowest in the cortex. The situation is about the same in layer 11. The less mature area contains many more granular cells than the differentiated area. There are 4 times as many granules in PC and 6 times as many granules in PH as are found in the newborn at term. In layer 111 there are again more undifferentiated cells in PH than in PC; however, PC has 3 times as many undifferentiated cells as pyramidal cells in layer IIIa. The numeration and the histological findings show that PC trunk is much less differentiated than FAY trunk. In layer IIIb of PC there are almost as many differentiated cells as undifferentiated ones. In IIIc there are clearly many more differentiated cells. Once again we notice the difference between IIIa and the two remaining sublayers. However, in PH, IIIc is the least differentiated layer, because it has the least number of differentiated cells as compared to undifferentiated ones. As in the other layers we have now seen, layer IV has more granules in PH than in PC, that is to say PH is less mature. In comparison to the newborn at term, there are 5 times as many granules in PH and also in PC. On the other hand, the number of pyramidal cells coming from other layers is greater in PC than in PH. In layers V and VI of PH there is a n appreciable increase in the number of undifferentiated cells as compared to differentiated ones : there are almost twice the number of undifferentiated cells in PC and 4 times as many undifferentiated cells in PH. In layer VI of PC there are about twice as many undifferentiated cells, whereas PH has 8 times as many. There is also a considerable difference in the white matter: in PC there are relatively few heterotopic pyramidal cells and more granular cells, but the total number of pyramidal and granular cells in PH is about twice that found in PC. There are also twice as many in PH as compared to the two frontal areas we have just seen. PH has about half the number of astrocytes as found in PC. Occipital lobe, OC and OA (Tables V and VI) In the two areas we have chosen to study in the occipital lobe, layers I, 11,111and IVa and b of OC have twice as many cells as found in the newborn at term. On the other hand, there are only 1.3 times as many cells in IVc; therefore we can assume that IVc is more developed than IVa and IVb. As elsewhere in the cortex, there is only a slight increase in the number of cells in layers V and VI, following the rule that layers V and VI are sooner mature than the other layers. In OA, the same general principles already seen still hold: that is to say, the less mature a n area is, the more cells one finds there. In 1ayer.I of OA, there are almost 4 times as many cells, while there are only twice as many cells in OC. In layer I1 there are 3 times as many cells in OA and twice as many cells in OC. Inlayer 111,thereare4to 5 timesasmanycellsinOAandnot quitetwiceas manyinOC. In layer IV, which is not subdivided here, there are twice as many cells, while this increase is not quite reached in the calcarine area.

References p . 86

TABLE V CELL TYPES

3 L,

P

Newborn

8 Months premature

0 h

61 -

2028 413 - 116

IVb

300 414 616

-

1913

59 -

1854 413 716

IVC

800 414 - 818

3200

105 -

3095 413 - 816

m

56

o

\

691

112

-

151

48

63 7/2-2015

49

80 7/2-2015

54

120

79

120 1315 22/12

595 -

W

21 1 413-715

66

d

4/3-1/5

D

108 -

w

317

444

560

\

1814 - 2616

41

*

14 1519-23117

Py. 100 sp.

616 20112

-

-

-

31

m

1210

-

36 12/7-1718

.r

Py. 520 Sp. 616 2011 2 1814 2616

136 7/4-1217

955

-

3

503

690 514-616

3

g

VIb

White matter

I .

CI I

VIa

-

-

29

\

2089

94 914-2016

-

THE CORTEX O F THE P R E M A T U R E I N F A N T

-

450 414 - 616

M

IVa

S. 180 L. 30 Ex. L. 12 1015- 1216 1618 20110 26113 30115

26 514 1217 Q N

m

r m m

I I

360 815 - 1517

V

171 1/275/3

I-. .

U

111

784

Astrocyte

3510 __ 413 - 614

63 913 5 0 1 6

3573

Spindle

N

600 313 - 515

528 412 - 614

(Cajal: 2) 1317 - 23/15

1014 - 18/10

Granular

.

I1

UndifferExtra large

-,

70 1

Pyramidal Large

w N

120

Small

z m

I

d

Equivalent count

$

Results of Conel

Layer

TABLE V1

80

CELL TYPES

W

0

Newborn

8 Months premature

Equivalent cuont

I

100

375

I1

650 414 - 816

1952

IIIa

100 814 1216

-

474

39 814-1316

5/47-15

355

106

IIIb

80 814 1618

459

96 8/51] 518

363 -

250

98

IIIC

110 814 18/Y

-

505

147 1015 1819

358 -

247

78

IV

720 414 816

1328

4

Results of Conel

L

B

Layer

371

96

369 -

81

84

418 514-716

2 712 913

-

81

187

4 1/21913

80

67/4:5/ 56

64

TH. R A B I N O W I C Z

19 715-1 719

'0

210

38 715 7 1 1 9

344 -

3

Py. 75 sp. 1213 2518 1014 1618

2

-

108

m

VIb White matter

-

Nl

458

-

124

1325 412 614

8 1016: 1718

Py. 180 Sp. 1213 2518 1014 1618

-

3 814 -1417

35 7/4110/6

VIa

-

-

10 6 1 2 / 6 ~ 1 1 / 1 1717 -22113

414

61

435

25 7/4:10/6

L. 30 1618 - 1819

-

-

385

814 1015

-

-

Aslrocyte

d

s. loo

1952 412 614

? 813 1316

-

Vb

m

-

22 7 / 2 7 113

312 - 614

. -

L. 30 Ex. L. 12 1618 1819 24/12 26113

Spindle

N

S. 80 814 - 1015

(Cajal : 17) 336 10/6>5/ I2

-

814 1215

Granular

r-

-

UndiflerExtra large

M: '1 .

Va

-

Pyramidal Large

Small

THE CORTEX OF THE PREMATURE I N F A N T

81

Even in layers V and VI there is an increase in cells, 3 times and twice, respectively, in OA and OC. In layer VI there are more than twice as many cells in OA and only 1.2 in OC. Note the large number of astrocytes found in OA, which is about the largest number found in the cortex. Limbic lobe, LA (Table VII) In the anterior part of the limbic lobe, and contrary to what we find in the occipital region, the number of Cajal cells is relatively low. There are 5 times as many cells in layer I as in the full-term newborn. There are more than 6 times as many cells in layer 11, 5 to 6 times as many in layer 111, 4 times as many in layer IV, 4 times as many in layer Va, 5 times as many in Vb, 5 times as many in layer VIa and more than 10 times as many cells in VIb. This shows that the limbic lobe is immature and is in accordance with what we have seen in the histological sections. A study of the degree of development within the layers themselves reveals that the number of undifferentiated cells is very great in layer IIIa, whereas IIIb and IIIc are much maturer. Note the size of the cells in layers V and VI. The neurons in these layers have almost attained the size that is found in the full-term newborn for the larger neurons, but there exist many small neurons, even smaller than those noticed by Conel. In layers VIa and VIb, the cells are principally undifferentiated and are of a much smaller size. T h s shows that while the cells in layer V have attained the size of those of the full-term newborn, the neurons of layer VI are much slower in their development. By comparing the dimensions of the neurons of layer VI, it can be seen that the spindle cells are much less mature than the pyramidal cells in comparison with those of the full-term newborn. There are many heterotopic neurons in the white matter and many more astrocytes there than in the cortex. Temporal lobe, TA (Table VIII) We will end this comparative study with area TA. We find 5 times as many cells as in the full-term newborn in layers I to IIIb, 7 times as many in IIIc, which is much less mature than are other layers. Layer IV is also very immature, because there are 7 times as many cells here as in the newborn at term. In layers VIa and b there is a 7 to 8 times increase in cells. There are many heterotopic neurons, approximately 200, in the white matter which have not yet reached their respective layers. This is more than seen elsewhere. About 40 astrocytes are distributed throughout this area even in the white matter. Individual variation Some variation exists in the amount of neurons between one brain and anothef. We saw a variation in the cell counts of more or less 10%. When the structure of the cortex is less well developed, there are fewer columns and more horizontal lines in References p . 86

TABLE VII CELL TYPES

00 h,

Newborn

8 Months premature

Results of Conel

Equivalent count

Small

I

100

488

915 1811

I1

410 414 1216

-

2840

IIIa

120 1216 20110

744

128 10/4i20/7

IIIb

60 1216 20110

468

196 10/6-23/8

IIIC

100 1216 20110

476

204 10/6-25/9

IV

215 414 1115

Layer

Va

-

-

L. 33 23/10 33/12

-

-

485 336

Py. 120 sp.

-

VIa

1316 1819

VIb

1316 1819

White matter

-

Spindle

Astrocyte

-

52 712 -1113

12 414 917

312 614 2840 412 714

-

-

914 1117 616 5/5-7/6

-

36 448

36

272 -

448

44

272

332

24

524 13/5-2219

820

60 1216 20110

Vb

(Cajal: 4) 432 12/1-23/13

-

Granular

0

-

-

UndifferExtra large

Q

-

s. 80 1216 20110

Pyramidal Large

1615 - 30110

-

17 13/7-3311 1

12 -

-

1213 2018

44 -

24

348 -

25 1

20

220 -

268

24

612

1015 1718

-

428 514-716

528

1015 1718

-

384 5/4-7/6

Py. 50 sp.

1615 30110

120 10/5-13/7

N

296 4127614

116 915 - 2219

164 128 -

20 7/270/3

32

16 7/2110/3

32 148

TABLE VIII CELL TYPES

z

9 2

Newborn

8 Months premature

Results of Conel

Equivalent count

Small

146

734

8/6- 13/11

I

-

IIIa

120 1015 20110

IIIb

92 1216 20110

IIIC

91 1216 20110

IV

329 414 616

Va

-

-

-

Vb

-

1216 20110

770 662

120 815-1818

698

56 815-1 8/8

-

60 1216 - 20110

547 464

122 7/5-11 17

Py. 67 VIb White matter

1416

-

2015 3218

413 - 614

161 712-1213

57 513 1217

-

E

3224 413 614

-

73

n 0 F

1

m

690 5/5-7/7

770

49

z

542 -

526

56

T m

642 -

799

X

a 0

-

m

52

C F

41

>

454 -

601

331 -

332

1

40

735 816-1819

164 5/<7/7

46 6/2>0/3

39

505

349 8/6-1819

120 5/5-7/7

36 612 7 0 1 3

39

209 815 1718

-

-2 m

2:

11

1217 18111-

r> 4

1985 413 - 615

- 25/12

51

945

Sy.

2015 - 3218

Astrocyte

4

13 1217 17/10-17/10

-

Spindle

~

214 12/5-20/8 80 7/672/7

Granular

4

-

2199

L. 12 32/12 40116

1416

cwtiated

1015 1517 80 8/577/7

Py. 121 sp. VIa

Extra large

(Cajal: 4) 569 1 8 / 9 2 0 / I7

3224

-

S. 90

Undiffeer-

?

545 414 816

I1

Pyramidal Large

00

w

-

313 614

58

84

TH. R A B I N O W I C Z

the cellular arrangement. But even when the number of cells in two brains is approximately identical, the structural pattern of the cortex can differ by its degree of development. Thus the number of cells is not the only criterion of differentiation. The number of astrocytes in any one particular area of different brains may vary from 40 to 90 elements per unit. To a slight degree, one may also find variations in the relationship of differentiated to undifferentiated cells. Summary

The foci of development are almost the same as those found in the full-term newborn, that is by decreasing degree, FA, PB, OC, HB, LE and TC. These foci are more wide-spread and less differentiated in the premature. TAy trunk is the most developed area of the brain. PB is almost as well differentiated as PC. The differentiation is almost the same throughout the ammonic region, which is relatively well differentiated. There are less noticeable differences in the limbic lobe; however, the posterior part is clearly more developed in the newborn at term. Generally, layers V and VI are more developed than the others, but VI is less well developed than V. In layer 111, sublayer IIIa is clearly less well developed than the other sublayers. We think that these differences are very important in relation to the timing of the future functioning of the neurons in their respective layers. Although the cortical myelinization is practically non-existent in all areas, the number of astrocytes found gives a n approximative indication of the amount of myelinization which will be found there later. The number of heterotopic neurons found in the white matter varies from one region to another. This number is greater under the less mature areas and is thus another criterion for the appreciation of the degree of maturity. Therefore we think that the study of the premature brain is essential if we want to understand the structure of the different layers and how the functional relationship that exists between the layers is built up. GENERAL SUMMARY A N D CONCLUSIONS

The present systematic study of the cerebral cortex of the premature infant of the 8th month was made in order to establish a mainly cytoarchitectonic atlas. 44 areas of the cerebral cortex were chosen topographically and cytoarchitectonically according to Von Economo’s map and studied in the same way as is done in Conel’s atlas of the full-term newborn infant. Several elements were established in each area and in each layer. Cresyl violet stained sections were used for the cell countings. Weigert and WeigertPal‘s techniques were utilized for the study of the myelinization on blocks of each area and on sections of the whole brain. Cajal silver and Golgi-Cox impregnations were done of each area. Drawings from the Golgi-Cox impregnations were made in such a way as to be directly comparable to those obtained by Cone1 in the full-term newborn infant.

THE C O R T E X O F T H E P R E M A T U R E I N F A N T

85

In a premature infant of the 8th month, the structure of the cerebral cortex is clearly outlined in each area. Some regions have a less advanced differentiation; especially the anterior areas of the frontal lobe are much less advanced and developed than the precentral gyrus; in the latter, we find very noticeable differences according to the region: while the motor areas for the feet and for the lower extremities are rather well developed, those for the trunk are the most well developed. The motor area for the upper extremities is less well developed. The least developed among them all is the motor region for the head and the neck. This distribution of the different degrees of development, the primary motor or sensory areas being sooner matured than the association areas, is to be found in all cerebral lobes. Thus, in the parietal lobe, the postcentral gyrus is much more mature one month before birth than the posterior areas, for instance, the angular gyrus, which is an association region. In the occipital lobe, the calcarine is bctter built up than the visual association areas. In the temporal lobe, things go quite differently in that the whole ammonic region is structurally well differentiated, the uncus being the most advanced, while the central and posterior regions of the temporal lobe are much less so. The limbic lobe is better developed in its central part than in its anterior one. Finally, the insula is maturer in its anterior part (IA) than in its posterior areas (area IB of Von Economo). Some other facts were noticed along this study. Thus, even one month before birth, we no longer find mitosis in the neurons; this implies that most probably the multiplying phase is over. The Nissl bodies and the neurofibrils are not yet visible, not even in the larger neurons. In a general way, the lateral dendrites of the pyramidal cells are less well developed and the apical dendrite grows less high in the cortex of the premature infant than in that of the full-term newborn. The axon of the large pyramidal cells is well developed but has very few collateral branches. These different elements show that the functions of association are less developed even at the cellular level. Myelinization is very poor in the cortex but is visible in the underlying white matter, where it is slightly developed though the motor and ammonic regions show more myelinization in the subjacent white matter than in the other areas of the brain. The non-myelinized segment at the emergence of the axon from the body of the neuron seems to be longer. Thus, the covering of the axon is less well advanced in its first part, that is in the cortex, than it is in a full-term newborn. This peculiarity as well as the shorter dendrites of the pyramidal cells could, apart from many other factors, explain the outline of the electroencephalograms of premature infants. Another important fact is that quite a number of neurons do not figure yet in their 'assigned' layer. This could be explained by the slower migration of some neurons, which thus come much later to their proper places. While migrating, the neurons, which first have their apical dendrite oriented towards the interior of the brain, rotate on themselves and then have their apical dendrite going towards the exterior. Some of them, however, take more time to do so or are stopped in the white matter. This References p . 86

86

TH. R A B I N O W I C Z

urged us to count those neurons in the white matter at a precise distance from the cortex, in order to obtain numerical statements. However, there are not only slow migrating neurons. Some proceed too fast and are found in a layer above their own, where they do not belong to. This showed us that in the cortex the neurons are doing a kind of ‘ballet’, going very slowly up and down before getting settled in their proper places. However, the cellular movement is much slower in the cortex than it has been described in the settling of some nuclei of the brain stem. Another noticeable point is the difference in maturation of the six layers of the brain: nearly everywhere in the isocortex, the fifth layer is the first to be matured and the best differentiated. Then come by decreasing order the sixth, the third, the fourth and the second, the first layer being inappreciable. The granular cells of the fourth and second layers seem to come more slowly to maturation. Not only should these differences between the layers explain some physiological peculiarities, but they could as well explain the selective sensitivity to pathological processes. We abstain from discussing the different results presented herein. This we shall do in the atlas which we are now preparing and which will give the description of the whole cerebral cortex. On the other hand, the numerical results here given have been obtained from one brain only whose values compared to those of other brains are means. The final figures will be obtained from the countings of 4 to 5 brains and the average as well as the statistical study of those results will be given in the atlas. ACKNOWLEDGEMENTS

This work was made possible by a grant (M 1561 Cl-C4) from the National Institute of Mental Health, National Institutes of Health, U.S. Public Health Service, Department of Health, Education and Welfare, Bethesda, Maryland. I am greatly indebted to Dr. J. LeRoy Cone1 for his friendly help and encouragement, for his advice in the preparation of the microscopical sections and the counting of the cells. I am also very grateful to him for suggesting a separate counting of the different types of cells. I wish to thank Dr. C. Bozic for providing us with the material and Miss A. Messmer, who helped us with the drawings. The late Miss J. Kojchen, Mrs. F. Gutberlet-Plumettaz and Miss A. Balmas did the counting of the cells and the microscopical sections. Dr. M. Ballon did the translation from the French and Miss M. Wiist helped in reviewing the text. REFERENCES ANGEVINE, J. B., JR., AND SIDMAN, R. L., (1961); Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature (Lond.), 192, 766-768. CONEL, J. L., (1939); The postnatal development of the human cerebral cortex, I. The Cortex of rhe Newborn. Cambridge, Mass., Harvard University Press. VONECONOMO, C., UND KOSKINAS, G. N., (1925); Die Cyroarchifektonik der Hirnrinde des erwuchsenen Menschen. Wien und Berlin, Verlag Julius Springer.

T H E CORTEX O F T H E P R E M A T U R E I N F A N T

87

DISCUSSION

WAELSCH:The neurochemist is becoming more and more interested in dynamic problems and is going to use minute amounts of tissue. Therefore he needs this type of information more than at the time when he took the whole brain for analysis. In this respect I want to ask you a question. You made a remark on the Nissl granules. On the basis of the work of Palay and Palade, we are nowadays used to equate the Nissl granules in the central nervous system with endoplasmic reticulum. You mentioned that in the prenatal cerebral cortex you only have what we are used to call the ‘basophilic dust’. These are the nucleoproteins, without the membranes of the endoplasmic reticulum. This raises the following interesting question: Is the development of the Nissl granule in any way in time connected with the development of a steady-state metabolism? And is for the growing organ this non-developed endoplasmic reticulum characteristic? My question is: Do you have exact data as to the time when a Nissl granule as such appears in the development of the brain? RABINOWICZ: This is a problem with several angles. First of all, it depends quite a lot on the type of stain you are using. Thus with some Nissl-staining methods, you do not even find a basophilic dust in the largest pyramidal cells. We are using an old prewar Grubler cresyl-fast-violet which gives the best results and which stains the protoplasm of some cells that other cresyls do not even evidence. In the 8-month-old prenatal brain, we can only observe basophilic dust in the large Betz cells but no Nissl bodies. These cells are well developed and the most differentiated of all cortical cells. In the 3-month-old infant, some Nissl bodies can be seen in the Betz cells. The large pyramidal cells have well-developed Nissl bodies at the age of about one year but many of the smaller pyramidal cells will never have any Nissl bodies, the protoplasm remaining filled with basophilic dust. WAELSCH: Do these small pyramidal cells always stay at the level of this basophilic dust? RABINOWICZ : Yes, with the kind of technique we are using, there are many smaller cells which do not show clear Nissl bodies, not even in the older infants. It depends of course on the area and the type of cells you are looking at. SCHADI?: In addition to the points made by Rabinowicz I would like to discuss some of the results of the relationship between basophilic dust and Nissl bodies obtained in our laboratory. We have some quantitative data of the cerebral cortex in rabbit and man. All are data obtained with the light-microscope on preparations stained with gallocyanin. In the frontal cortex of rabbit, Nissl bodies are first seen in relatively few cells in 10-day-oldanimals (3 %) in the postnatal period; 46 % of the cells showed dust-like basophilia. The number of cells with Nissl bodies increases very fast between 10 and 20 days after birth. In 20-day-old animals 34% of the cells showed dust-like basophilia and 52 % Nissl bodies in a particular area of the frontal cortex. In 30-day-old animals the percentages were 32 and 63, respectively. Even in adult animals about 30% of the cells (in general the smallest) showed dust-like basophilia. The appearance of a high percentage of Nissl bodies coincides with the fast increase in number and s;ze of the cell processes and the cessation of growth of the nucleus. Peters and Flexner (1950) found also a remarkable increase in the quantity

88

DISCUSSION

of Nissl substance in a relatively short period (from the 41st to the 44th day of gestation in the guinea-pig). In the human frontal cortex (middle frontal gyrus) the picture is about the same. No Nissl bodies are present in any cells in this area in the newborn. Dust-like basophilia and a suggestion of agglutination of the cytoplasm is seen in some of the largest cells in layer V. In the 3 and 6 months’ infants the basophilic substance is more advanced in differentiation. Clumps of basophilic material are now clustered around and on the membrane of the nucleus. Comparing the 3 and 6 months’ preparations a substantial increase was found in basophilic substance, especially in large cells in layer V and 111. In the 15 months’ infant many neurons in layer V and 111show large amorphous clumps of basophilic material resembling true Nissl bodies. In preparations of adults a considerable number of cells (usually between 10 and 15 %) shows the absence of true Nissl bodies. This is particularly the case for the small pyramidal cells. The fact, however, that the smallest have no Nissl bodies could be caused by some peculiarity of the stain or the staining technique. PURPURA: There is a big difference here in equating the Nissl body by the lightand the electron-microscope. In the electron-microscope we have seen in the human foetus a fairly well developed endoplasmic reticulum. They have different characteristics, they are not as elongated and tortuous but the RNP particles are associated with these membranes. It appears from this kind of analysis that the internal metabolic machinery is well developed at 6 to 8 months. In the cat we see the same kind of things. From the morphological standpoint a n electron-microscopist can not tell the endoplasmic reticulum of a l-day-old kitten from that of a 7-day-old cat. They look different in terms of the piling up of the material but we cannot really say anything, even with respect to the mitochondria. LEVI-MONTALCINI: Could you say a few words about the way the branches of the neurons develop? Do you see something in the way of a moving of cells? RABINOWICZ : The protoplasm of the neurons develops itself with several buds going into different directions. Then, while the neuroblast is migrating, some buds grow faster than the others. While approaching the cortex, the neuroblast rotates slowly on itself and sends its one or more apical dendrites towards the exterior. Thus, one month before birth, one can observe in layer VI some neurons which are finishing their rotation, but most of them, however, are already well orientated. Ulteriorly, the apical dendrite develops itself and if there are more than one, one becomes the definitive apical dendrite and the others the basal ones. One month before the fullterm birth, the thorns and bulbs are already visible but much less numerous than later on. From the Golgi-Cox impregnations I showed you, one can see, in layer VI, neurons completing their rotation (Fig. 24) and neurons with two parallel apical dendrites (Fig. 19). Afterwards, this kind of neurons disappear almost completely. In the premature infant of the 8th month, the axons often are well developed, especially in large pyramidal cells and can be traced deep into the white matter; but they have only few collaterals and branchings. We have never seen any synapses with the methods we are using. In spite of the very incomplete myelinization in the white matter and the lack of it in the cortex, the axons are well visible especially with Cajal’s method .

T H E C O R T E X O F THE P R E M A T U R E I N F A N T

89

As far as the moving of the cells is concerned, I a m speaking mostly froma numerical point of view. During the last month preceding the birth at full term, we do see tremendous changes in the density of the cells which varies from one area to the others, as well as from one layer to other ones. A striking example is that in the premature frontal isocortex, in the midst of layer V, we can observe many small granules which most probably belong to layer IV. In the full-term newborn cortex, almost all these granules have disappeared; so we may assume that they have moved to their proper layer. S C H A DI~ would : like to comment on the determination of the packing density of cells. We have developed a rather simple method for the calculation of the number of cells per unit of volume of cortex. The perikarya are counted in a column of cerebral cortex as long as the thickness of a particular layer (or as long as the thickness of the whole cerebral cortex) and as deep as the thickness of the preparations, the width of the column was usually 300 p. Two thicknesses of preparations were used: 20 and 40 p. It frequently occurs that a cell body does not lie completely within the limits of the column, either in the plane of the section or in the plane rectangular to the section. The errors made in the plane of the section can be avoided by counting all the nerve cell bodies, which are completely or partly located at one boundary, and leave out all those at the other boundary. The other error, counting parts of cell bodies in a plane rectangular to the section is eliminated by computing the cells in two adjacent sections of different thicknesses (e.g. 20 and 40 p for the rabbit cortex). In each of these sections all the nerve cell bodies visible within the 300 p limits are counted. The number found in the 20 p section is subtracted from the number in the 40 p section. The difference is considered to be the number of nerve cell bodies present in a n imaginary section, 20 p thick (the difference between the thicknesses of the two sections used). In each section errors are made in its upper and lower surface. These errors are independent of the distance between the two surfaces, since the thickness of the section i s greater than the diameter of the individual nerve cell bodies. When counting a sufficiently large number of cells the errors made will be the same in each section and by deducting the two values these errors will be eliminated. By dividing the number of cells by the calculated number of the section in which they are counted the packing density of cells in the area can be calculated. In the postnatal development of rabbit and human frontal cortex we always found a decrease in the packing density. The values are the following: newborn rabbit, 55.0 * lo4 per mm3; in adult rabbit, 5.8 . 104. In newborn and adult preparations of layer I11 in the middle frontal gyrus (human) the values are respectively: 99.0 and 12.5 * 103 per mm3. WAELSCH: I think what you say is also brought out by chemical analysis. If you do DNA determinations in the developing brain you find that, irrespective of the dehydration of the brain during this period, the DNA content drops per unit weight or whatever you want to do, g or 100 mg. So this means that the cells are pushed, so to say, apart. RABINOWICZ : I must precise a few things about our countings. These latters being very long to do in the premature, we have by now counted the cells of only 4 brains.

90

DISCUSSION

We believe that it is necessary to count those of about 7 brains in all. The countings have been made in the same way for all the brains we have chosen. To determine the cellular density, we have utilized the old method of Von Economo, the one used by Conel from the onset of his research in this field. Of course, we know that this way of counting does not give results corresponding to the entire reality, but a correction can be made by the method of Haug* which allows us, knowing the dimensions of the cells and the thickness of the sections, to calculate the real number of neurons. However, we had to use Von Economo’s method to obtain figures comparable to those found by Conel in his study of the postnatal development of the cerebral cortex. Another problem is that with the premature infant the density of the cells can reach 10 times the one of the newborn at term. We also know that in the premature infant of the 7th month this density is even heavier. Thus to establish our intended atlas of the cerebral cortex of the premature infant of the 7th month we shall have to use 12 ,u thick sections instead of 25 p thick ones. This is why the countings for the premature infant of the 8th month are also done on 12 p thick slices allowing, for the future, comparative figures with those of younger premature infants. MARTY:Selon les critkes de dkveloppement que vous venez d‘exposer, quelle aire nCo-corticale prtsente, aux divers moments de la prkmaturitt, le plus grand degrk de diffkrentiation? RABINOWICZ: The most advanced area, from the morphological point of view, is FA y, the motor area in the precentral gyrus. LINDSLEY: I would like to know whether there is any order in the development of the sensory areas. RABINOWICZ: As just said, the most advanced area is FA y, but the postcentral gyrus is much less developed. So PB and PC that we have studied at the same different levels as FA y are always less mature than the corresponding motor areas. The other sensory areas are less well developed. PG (angular gyrus) is not a well developed area and PH (posterior part of the middle temporal gyrus) is the least developed of all the sensory areas. The limbic lobe is at about the same level of development as the least well developed parietal areas. LINDSLEY: What about the visual areas OA, OB and OC? RABINOWICZ: There exists a great difference between those three areas: the least well developed is OA; OB is better developed than OA while OC is a rather far advanced area with well developed neurons. The optic pathways being already built up, it might most probably be possible for a premature infant of the 8th month to see things without, however, understanding what it sees. SCHERRER: I would like to ask you the following question. It is a bit complicated. Very often in electrophysiology we do see a phenomenon which appears and which grows with the development. After growing for a certain time it becomes stationary and the development, from a physiological point of view, is stable. I would like

* HAUG,H., (1953); Der Grauzelkoeffizientdes Stirnhirnes der Mammalia in einer phylogenetischen Betrachtung.Acru anal. (Barel), 19/I, p. 60-100; 19/11, p. 153-190; 19/III, p. 239-270.

THE CORTEX OF THE PREMATURE I N F A N T

91

to know if from the histological point of view you think it is possible to make a kind of statistical distribution of maturation. I mean the following. Let us say one can analyse that a cell is mature when it is at 90 % of the maturation level. Then one notices the day the first cells are at 90% of maturation and then later on one notices the number of cells which are maturating per day of development. In this way you get a kind of curve of the number of cells which are mature at a given day. You ought to find something which might be a Gaussian distribution. Do you have any data on this kind of analysis? RABINOWICZ: We have tried to determine the degree of maturation of the various layers by counting the number of cells in all the layers and sublayers, and by knowing the ratio between differentiated and undifferentiated cells of each type of cells in every layer and sublayer. Looking at the aspect of the cells and studying the degree of maturation of the layers and sublayers from the numerical results, it appears that the layers are not ready to function all at the same time. Morphologically, layer V is the first one to be matured, has the least number of undifferentiated cells, and consequently should be the first one to function. Inside this layer, we presume that the large neurons function before the small ones whose degree of maturation is proportionally quite delayed. Then comes layer VI, and later on layer 111. So there must exist great differences between the moment a layer starts to function and the onset of functioning of a neighbouring one. The Figures and the Tables of the present work support this assumption. However, using our criteria only it is not possible to know how many cells are at 90 % of their maturation. We have divided the pyramidal cells into non-differentiated and differentiated cells. The non-differentiated ones are certainly not yet functioning because their morphological aspect is still embryonic and their axon and dendrites still very short. Among the differentiated ones, most of them are probably not far from functioning. But technically, it is almost impossible to establish an exact percentage of these cells. We have to use morphological and numerical criteria that might not always correspond exactly to physiological ones. SCHERRER: The point I was making was not only regarding the type of the curve. I imagine that there could be two types of maturation. In one type you have a very narrow Gaussian distribution, that means that almost all cells mature at the same day, and in another type you have a very large Gaussian distribution, that means that very slowly some cells are arriving to maturation, and so forth. And this type of numerical data would be very interesting to correlate with functional electrophysiological data. RABINOWICZ: I think this could only be possible if in the atlasses of the newborn at term and the infants of 3, 6 months old and so on, the ratio of non-mature to mature cells had been counted. But this has not been done, so that it should be necessary to complete these atlasses in this direction. PURPURA: I would not like this discussion to end without going on record saying that I do not believe in lamina analysis of function. The notion that different functions might be attributable to different layers of cortex progressively is very unlikely. Most available data would indicate that regions of cortex e.g. that you d o not consider very much of with these analyses are functioning in the near term foetal and at least

92

DISCUSSION

newborn, immature born cat. The superficial neuropils e.g. of these animals in neocortex are quite prominently developed as I think that data of Scherrer and our own laboratory would indicate. Functioning, at least functioning from electrophysiological standpoint, has not anything to do with the layer of the cell and where it is located. With respect to the question of the number of processes at birth: in the cat in particular the basilar regions of the pyramidal neuron region have far many more protoplasmic processes than are eventually going to give rise to basilar dendrites. There is a resorption phase, as there is of course in the Cajal-Retzius cells, and certainly in the Purkinje cells which we have pictures of in the cat which I will show. So I do not believe that one can carry the layer problem with respect to there being more maturation deeper than superficially. All evidence at least points to the fact that axo-dendritic or dendritic synaptic activities are prominent first, before there are much in the way of axo-synaptic type activities. The cell body in other words tends to want to grow out its basilar dendrites before it puts synapses on the cell.

93

Some Correlations between the Appearance of Human Fetal Reflexes and the Development of the Nervous System* TRYPHENA HUMPHREY** Department of Anatomy, University of Pittsburgh, Pennsylvania ( U.S.A.)

Human behavior has its beginning very early in prenatal life. Indeed, both Fitzgerald and Windle (1942) and Hooker (1952, 1954, 1958) found that the human embryo is capable of movement by the time that it has attained a length of only 20 to 21 mm (Fitzgerald and Windle, 20.0 mm; Hooker, 20.7 mm). The embryo is then only about 7.5 weeks of menstrual age (about 5 weeks of fertilization age; Patten, 1953). Only rarely does a mother report ‘feeling life’ by 12 weeks and it is said that, in favorable cases, movements may be noted by the obstetrician by the 12th week (Greenhill, 1960), that is, at approximately 14 weeks of menstrual age***. By this age, however, the fetus is capable of a wide variety of reflex movements (see Tables V-IX and XII). Direct observations of fetal movements are possible only when some mischance results in the loss of the fetus, or the life or health of the mother necessitates a therapeutic termination of pregnancy by Caesarean section. Isolated observations of fetal movements and records for a relatively small number of fetuses, such as the reports of Fitzgerald and Windle (1942) and the work of Russian observers (Golubewa et al., 1959; Mavrinskaya, 1960) have been more numerous than the more extensive, organized studies such as those of Bolaffio and Artom (1924), Minkowski (1923, 1928) and Hooker (1938, 1939, 1944, 1952, 1954, 1958). Although moving picture recording was used by Fitzgerald and Windle (1942), cinematographic records have been employed

* The physiological and morphological studies on human prenatal development, of which this paper is publication no. 37, have been aided by Grant B-394 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. Support for these studies was also obtained earlier from the Penrose Fund of the American Philosophical Society, the Carnegie Corporation of New York and the University of Pittsburgh. The preliminary morphologic data included in this paper were compiled and presented in connection with the author’s part of a University Lecture at the University of Michigan entitled ‘Human fetal activity and its morphological basis’ given jointly by Davenport Hooker and Tryphena Humphrey, October 10, 1957, at Ann Arbor, Michigan under the auspices of The Laboratory of Neurosurgery Research. * * Present address: Department of Anatomy, University of Alabama Medical Center, 1919 Seventh Avenue South, Birmingham 3, Alabama. *** The fetal ages given throughout this paper are menstrual ages as estimated from the tables of Streeter (1920). References p . 130-133

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T. H U M P H R E Y

extensively only by Hooker, whose pictures have been widely used by psychologists and various other authors (GeseIl, 1945; Dennis, 1951;Munn, 1954,1955;Carmichael, 1960; Flanagan, 1962). In connection with the discussion which follows, the fetal reflex activity mentioned, unless otherwise stated, is from the data of Hooker (1938, 1939, 1944,1952,1954, 1958) and Humphrey and Hooker (1959, 1961a). Some additional data have been used from unpublished material in the protocols of Dr. Hooker and the author is very grateful for the opportunity of consulting these records in connection with various specific points in question. For the most part the fetal reflexes which will be discussed have been elicited by stimulation of the cutaneous (and sometimes mucous membrane) surfaces by touching, or usually by stroking, them with a hair tipped with a smooth, inert material to prevent penetration of the epithelium and cause direct muscle stimulation. These hair esthesiometers were graded by Hooker (1938, 1952) to exert different strengths of stimulation. Non-viable fetuses were observed in an isotonic bath at normal body temperature. Fetuses old enough for respiration to be established temporarily were observed in premature beds, as were also all viable premature infants. Further details concerning the methods used are given in the various papers of Hooker. In Hooker’s series of premature infants, one of 27 weeks of menstrual age (25 weeks of fertilization age) was the youngest found capable of maintaining life. Some observers have elicited reflexes from young fetuses by tapping on the amnion or moving a limb to exert stretch on the tendons and muscles (Fitzgerald and Windle, 1942). Since the nature and strength of the stimuli are difficult to evaluate correlations with cutaneous stimulations are not possible and these reflexes will not be discussed here. Only the reflexes that follow cutaneous (or mucous membrane) stimulation during fetal life will be considered. T Y P E S OF R E F L E X E S E L I C I T E D B Y C U T A N E O U S S T I M U L A T I O N

The first reflexes obtained from stimulation of areas supplied by the trigeminal nerve have been considered by Hooker (1944, 1952, 1958) as comparable to the ‘total pattern’ movements described by Coghill (1929) for amphibians. In these reflexes all of the neuromuscular mechanisms sufficiently developed to participate are believed to take part in the reflex. Thus Coghill (1929) found that when the extremities developed they first moved in conjunction with the trunk and only later acted independently. Coghill referred to these later developing movements of separate body parts as partial patterns (Coghill, 1929). A reflex action limited to movement in a restricted area has been termed a specific or local reflex (Hooker, 1952, 1954, 1958; Humphrey, 1953, 1954; Humphrey and Hooker, 1959). In the development of human fetal reflexes, the early total pattern type of activity is the first response to sensory trigeminal stimulation (Table I). These reflexes are stereotyped (Hooker, 1952, 1958) and repetition of the stimulus elicits essentially the same response. Local reflexes following trigeminal stimulation begin to appear by 9.5 weeks and as they increase in number the total pattern type of activity begins to

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disappear and is seen only as anoxia begins to suppress reflex activity (Hooker, 1952, 1958). For the most part, as the movements become less stereotyped the same stimulus may produce a local reflex, before anoxia appears, and the early total pattern type of action, as anoxia progresses. After 12 weeks of menstrual age, combinations of two or more local reflexes, or of a local reflex with the total pattern type of head and trunk movement, may be seen. Such combinations appear in sequences which will become functional postnatally. For example, mouth closure is combined with swallowing TABLE I P E R I O D S O F C H A N G E I N T H E C H A R A C T E R OF F E T A L REFLEXES E L I C I T E D B Y S T I M U L A T I N G SKIN AREAS SUPPLIED BY THE TRIGEMINAL NERVE*

Menstrual age

7.5-9.5 Weeks 9.5-1 2 weeks

12 weeks and later

13-14 weeks and later

Nature of the reflexes observed

‘Total pattern’ type of movements only. Movements stereotyped and repetition of a stimulus gives essentially an identical response. ‘Total pattern’ type of movements disappearing and seen only with the onset of anoxia. Appearance of various local reflexes on stimulation of sensory trigeminal fibres. Newly appearing reflexes remain stereotyped for a time after first elicited, but repetition of a stimulus no longer always gives the same response. Combinations of reflexes appear (e.g., ventral head flexion, mouth closure and swallowing) or local reflexes combine with such movements as lateral trunk flexion or ventral head flexion. Reflexes no longer at all stereotyped. Repeated stimulation may produce different reflexes or combinations of reflexes. Movements are graceful and flowing in nature. Local reflexes dominate the activity, either singly or in combinations.

* Based on the data of Hooker (1952, 1958). (Table VI) and, later, mouth closure, tongue movements, swallowing and the appropriate head movements may appear together. After 13-14 weeks of menstrual age the local or specific reflexes, singly or in combinations, dominate the fetal reflex activity (Table I), and a repeated stimulus often evokes a different response. In addition, the movements become ‘graceful and flowing’ (Hooker, 1952) and lose the more jerky appearance often seen earlier. The change from a stereotyped to a varied reflex response is also seen on stimulation of the palm of the hand. The quick, partial closure of all fingers, first elicited at 10 to 10.5 weeks, may involve only part of the fingers at later ages (Table IX). A comparable picture is found for genital stimulation, for repeated stimulation at 10.5 to 11 weeks results in identical responses whereas at 18.5 weeks there is some variation (Table XII). On stimulation of the sole of the foot, however, the initial plantar flexion of the toes is soon partially replaced by dorsiflexion and fanning of the great toe so that identical stereotyped responses are lost almost at once. References p . 130-133

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T. H U M P H R E Y SEQUENCE OF DEVELOPMENT O F C U T A N E O U S SENSITIVITY

The first surface area to become sensitive to stimulation is the region of the lips known as the rima oris or the vermilion border (Table 11). Although limited to the perioral region at first (Hooker, 1952,1958),the sensitivity spreads as the fetus becomes older so that by 8.5 weeks of menstrual age it includes the region of the alae of the nose, the area about the mouth, and the chin (Hooker, 1952, 1958). Probably the mucous membrane of the inside of the mouth and over the tongue becomes sensitive early also, but the mouth is usually closed and so tests cannot be made (Hooker, 1958). Possibly the upper lip responds to stimulation before the lower lip, but the evidence is not conclusive. All of these areas are supplied by the maxillary and the mandibular divisions of the trigeminal nerve. The region of the upper eyelid, supplied by the ophthalmic division of the trigeminal nerve, does not become sensitive to stimulation until 10 to 10.5 weeks of menstrual age. By 11.5weeks sensitivity has extended peripherally to involve all but the most peripheral part of the face (Hooker, 1952, 1958). T A B L E I1 T H E S E Q U E N C E O F D E V E L O P M E N T OF C U T A N E O U S S E N S I T I V I T Y *

Menstrual ape [weeks) 7.5 8-9.5 10-10.5 10.5

10.5-1 1 11

11.5 11-12 13 14

15 17 32

Skin area demonstrated sensitive to stimulation

About the mouth (perioral - maxillary and mandibular fibres of the trigeminal nerve) Extension of perioral area to include the alae of the nose and the chin Eyelids (ophthalmic division of the trigeminal nerve) Palms of the hands Shoulder Genital area* * Anal area** Genitofemoral sulcus** Soles of the feet Eyebrow and forehead Upper arm and forearm Entire face now sensitive, except peripherally Upper chest Thighs and legs Remaining chest areas Tongue* * * Back, side of trunk and over scapula Abdomen Buttocks** Inside of thighs

* Based on the data of Hooker (1952, 1958) and Humphrey and Hooker (1961) with some added information from unpublished protocols. ** Observations at this age are from dictated records only. *** The tongue area is probably sensitive to stimulation earlier, but cannot be tested, as a rule, because the mouth is usually closed. Since the area tested is of ectodermal origin, the mucous membrane of the tongue is included here with the skin. 6 This region also is probably sensitive earlier, but at this age, and possibly earlier, its stimulation elicits the cremasteric reflex (see Table XII).

97

H U M A N F E T A L REFLEXES

By the time that the facial areas supplied by the ophthalmic division of the trigeminal nerve have become sensitive to stimulation, other areas of the skin, supplied by spinal nerves, have also become sensitive (or reflexogenous; Hooker, 1958). The first of these may be the palms of the hands (Table 11) which become sensitive by 10.5 weeks or possibly earlier. Rarely the shoulder has responded to stimulation at this time also (Hooker, 1958). Recently stimulation of the anal and genital areas of a 10.5-week fetus (44.0 mm CR) has evoked reflexes but no moving picture records were obtained and the observations need corroboration. At the present time, then, it is not possible to say which of these cutaneous areas (palmar, anal or genital) first becomes sensitive to stimulation. It is not unlikely that sensitivity about the anal and genital orifices of the body may follow oral and perioral sensitivity in the order of appearance. By 11 weeks, and for some fetuses earlier, the sole of the foot responds to stimulation (Table 11). Other areas of the skin respond to stimulation still later. Table 11provides the data thus far available, but it should be borne in mind that further observations may change the sequences given here. Nevertheless, it should be evident from the data given in this table that, although sensitivity of the skin begins about the mouth, there is no regular progressive spread from the mouth t o more distal regions. Instead, the cutaneous areas that next become clearly sensitive to stimulation are those which, in postnatal life, develop the greatest numbers and varieties of specialized receptors, that is, the genital region, the palm of the hand, and the sole of the foot. EFFECTS O F A N O X I A ON F E T A L REFLEXES

Before respiration can be established permanently, there is a progressive increase in anoxia (and asphyxia) following placental separation. Various authors (Angulo, 1930; Barcroft and Barron, 1937; Barron, 1941; Windle, 1944, 1950; Windle and Becker, 1940; Windle et al., 1942) have considered that the initial increase in carbon dioxide in the blood of the fetus facilitates the elicitation of reflexes. Certainly, as Windle and others (see Humphrey, 1953) have pointed out, there is usually a brief interval TABLE I11 E X A M P L E O F T H E E F F E C T S O F A N O X I A O N R E F L E X E S ELICITED B Y C U T A N E O U S S T I M U L A T I O N *

This table illustrates the sequence for the suppression of fetal reflexes (that are elicited by cutaneous stimulation) with the onset of anoxia and asphyxia. Reflex

Neck and trunk flexion to the opposite side with rotation of the pelvis and extension of the arms at the shoulders Finger closure Foot-sole response Elevate angle of mouth

*

Based on the data of Hooker (1952, 1958).

References p . 130-133

First appearance (weeks)

Order of suppression

8.5

Last to disappear

10.5 11 13-14

Third Second First to disappear

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T. H U M P H R E Y

after delivery of the fetus before any reflexes can be secured. As anoxia progresses, however, the reflexes are suppressed. The reflexes which disappear earliest, at any age, are those that have appeared most recently in development. Thus, if, as an example, a sequence of four typical reflexes is chosen in the order of their age of development, as in Table 111, it will be noted that the first reflex to disappear, as oxygen deprivation begins, is the latest to have appeared. Likewise, the reflex which can still be elicited, after the others have been suppressed by lack of oxygen, will be the reflex that appeared earliest in the course of development. The effects of anoxia on reflexes elicited by stretching muscles may differ in fetuses, as in the adult, from the sequence observed for cutaneous reflexes (see Humphrey, 1953) and will not be discussed here. REFLEXES ELICITED BY TRIGEMINAL STIMULATION*

The reflexes that have been elicited on stimulation of cutaneous (and mucous membrane) areas supplied by the trigeminal nerve may be considered on a chronologic basis, as in Tables I and I1 of Hooker (1958). They may also be grouped into those related to the development of feeding postnatally (as mentioned by Hooker and Humphrey, 1954) and those which could serve, potentially at least, an avoiding or protective function (Hooker, 1952, 1958; Hooker and Humphrey, 1954). In the use of the term ‘avoiding reflex’ (Hooker and Humphrey, 1954), there is no implication on the part of observers of fetal activity of denoting ‘intent’ on the part of the fetus T A B L E IV RELATION OF THE D I R E C T I O N O F H E A D A N D T R U N K REFLEXES T O T H E AREA O F THE F A C E STIMULATED*

Reflex elicited

Menstrual age for appearance of reflex (weeks) Away from stimulus

Lateral flexion of head (neck muscle contraction) and trunk Midline head (and trunk) movements: Head extension (face moved away from stimulus) Ventral head flexion (face moved toward stimulus) Rotation of face, accompanied by lateral trunk flexion or head extension Rotation of face alone (i.e.,without lateral trunk flexion)

*

** ***

7.5

Toward stimulus 8

(neck muscles only) 9.5

-

-

lo**

10.5** 13.5-14

11-1 1 3 * *

Newborn infants***

The table is based largely on data from the papers of Hooker (1952, 1958). Unpublished data from the protocols of Dr. Hooker. From Prechtl, 1958.

* In the presentation of this paper on Sept. 8, 1962, motion picture sequences were used to illustrate many of the reflexes discussed here, including reflexes from stimulation of the face, the palm of the hand, the sole of the foot and the genital areas.

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as might be the interpretation of many psychiatrists. The word ‘avoiding’ is simply used to indicate that the reflex in question separates the site stimulated from the stimulating agent and so reflexly avoids a possibly or potentially harmful contact. For amphibian embryos in an aquatic environment, the protective or avoiding function of such reflexes is real and obvious. For the human embryo in utero, comparable reflexes, when they occur, can have no comparable function although they may prevent prolonged contact with amniotic surfaces or release a kink in the umbilical cord. In the present discussion, I would like to consider the reflexes elicited by sensory trigeminal stimulation in the two groups just mentioned, that is, ( I ) those potentially protective and/or avoiding and (2) those which become part of the postnatal reflex feeding pattern. In each category, the total pattern type of reflex, the local reflex, and combinations of reflexes are found. For reflexes involving solely or primarily neck and trunk muscles it will be noted (Table IV) that reflex movement away from the stimulus always appears before the comparable movement toward the stimulus, whether the action is by lateral flexion of the neck and trunk, by midline head movements - ventral head flexion or extension of the head - or by head rotation, either combined with trunk movements or by rotation of the head alone. Trigeminal refexes of an avoiding andlor protective type The earliest reflex that has been elicited by cutaneous stimulation of the human embryo follows light stroking of the perioral region with a hair (Hooker, 1952, 1958) at 7.5 weeks of menstrual age (embryo 20.7 mm long). This reflex, which has also been seen following tapping on the amnion (Fitzgerald and Windle, 1942; embryo 20.0 nim long), consists of contraction of the neck muscles on the side opposite the stimulation (Table V). Thus stroking the perioral region on the right side results in contraction of the neck muscles on the left and consequent bending of the head to the left so that the surface touched is moved away from the stimulator. This reflex, referred to by Hooker as contralateral flexion in the neck region, has been observed only a few times at 7.5 weeks and no ipsilateral response has been noted at this early age as yet (Table IV). A caudal extension of this contralateral neck flexion reflex, to include upper trunk flexion as well, has been seen by 8 weeks (Hooker, 1958) and by 8.5 weeks (Fig. 1A and B) perioral stimulation results ir, a reflex consisting of (a) contralateral neck and trunk flexion, (b) extension of both arms at the shoulders, and (c) rotation of the pelvis to the contralateral side (Hooker, 1952, 1958). At 9.5 weeks of menstrual age the perioral region of the face (supplied by the maxillary and mandibular branches of the trigeminal nerve) is still the only cutaneous area sensitive to stimulation (Table 11) although the sensitive zone about the mouth has increased in area (Hooker, 1958). Until 9.5 weeks, also, no local reflexes have been observed (Hooker, 1954, 1958). The essential change in activity between 8.5 and 9.5 weeks, then, is mainly an increased caudal extension of the contralateral trunk flexion reflex with all elements of the reaction becoming more pronounced (Hooker, 1958). References p . 130-133

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T. H U M P H R E Y

Thus throughout the first two weeks of fetal activity, up to 9.5 weeks (Table I), all of the reflexes elicited by stimulation of the skin surfaces supplied by the trigeminal nerve are of the total pattern type of Coghill (1929), where the movement is of the whole fetus. Up to this time also, the cutaneous areas supplied by the trigeminal nerve are the only ones which, as yet, have been demonstrated to be sensitive to

Fig. 1. Four frames from a moving picture sequence showing the reflex action following stimulation of the perioral region of a human fetus of 8.5 weeks of menstrual age (26.0 mm CR, No. 24). From A preliminary Atlas of early humanfetal Activity (1939)through the courtesy of Dr. Davenport Hooker. A and B (sequences 2 and 4 from p. 19) illustrate the contralateral flexion of the trunk and neck with backward movement of both arms at the shoulders and slight rump rotation. (A) The position of the fetus at the beginning of the reflex. (B) The position during the height of the reflex. In this instance, without further stimulation, the trunk and neck muscles then contracted on the side ipsilateral to the stimulus. C and D (sequences 8 and 11 from p. 19) illustrate the ipsilateral reflex. (C) The position at the height of the ipsilateral movement. (D) The position upon returning to the resting posture. With the ipsilateral reflex there was little movement of the upper extremities such as is seen with the contralateral reflex, but the rotation of the pelvis as well as the ipsilateral trunk and neck flexion movements are easily seen.

stimulation (Table 11). Other reflexes of an avoiding type which involve both head and trunk are the neck and trunk extension reflex and head rotation combined with lateral flexion of the trunk (Table IV), resulting in turning the face away from the stimulus. In addition to the total pattern type of avoiding reflexes (contralateral flexion of the neck and trunk, head and trunk extension, rotation of the face contralateral to the stimulus combined with trunk flexion contralaterally or with trunk extension), a more localized type of reflex, consisting of contralateral rotation of the face only, i.e., without trunk movement, appears by 13.5 to 14 weeks (Table IV). Even earlier (10 to 10.5 weeks), however, stimulation of the upper eyelid area results in contraction of the ipsilateral orbicularis oculi muscle to give a ‘squint’ type of action (Fig. 2) and slightly later stimulation of either the eyelid or the eyebrow areas may result in corrugator supercilii contraction to produce a ‘scowl’ type of reflex (Table V). Although the ‘squint’ reflex, when first elicited, follows eyelid stimulation, the area of sensitivity spreads and this reflex has even been seen following stimulation over the mandibular area (Hooker, 1952, 1958). The ‘squint’ reflex is sometimes combined with contralateral trunk flexion movements or trunk extension during the early period,

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but later again occurs without trunk movements. After first appearing separately, the ‘squint’ and ‘scowl‘ reflexes also may occur together. Sometimes, but rarely, a ‘squint’ may be combined with mouth closure and head extension (Table V). The ‘squint’ reflex, 2s an isolated response, is comparable to the corneal and conjunctival reflexes of the adult and by 14.5 weeks sometimes occurs bilaterally as do these latter

Fig. 2. Two frames from a moving picture sequence following stimulation of the face from the angle of the jaw across the right cheek and eyelids to the forehead of a human fetus of 13 weeks of menstrual age (88.5 mm CR, No. 13). From A preliminary Atlas of early humm fetal Activity (1939) through the courtesy of Dr. Davenport Hooker. A and B are sequences 2 and 6 from p. 75. At A, the stimulator is just touching the face. In B, the contraction of the orbicularis oculi muscle (‘squint’) and the shift in the position of the head to a contralaterally flexed and extended position, is seen easily. At the same time the upper lip, angle of the mouth and ala of the nose were also raised, but these movements are less evident from the two figures shown.

reflexes in the adult. As the local reflexes come to dominate fetal reflex activity to a greater and greater degree at 13 to 14 weeks, stimulation of the upper lip and ala of the nose sometimes produces an elevation of the angle of the mouth and ala of the nose to provide a ‘sneer’ like reflex ipsilaterallq (Table V). A high-pitched cry follows resuscitation as early as 23.5 weeks (Hooker, 1952). According to Golubewa et 01. (1959), stimulation of the nostrils may result in sneezing with fetuses as young as 24 weeks. These reflexes have been grouped as avoiding and/or protective types of reflexes because they either serve to remove the area stimulated from a potentially noxious stimulating agent or, as in the case of the ‘squint’ and ‘scowl’ reflexes serve postnatally to protect the eye from possible damage. In the case of the ‘sneer’ type of reflex, such action, when bilateral, bares the teeth, a characteristic expression of adult mammals when danger appears. It is interesting that mouth closure, which is grouped here with reflexes related to feeding (Table VI) may be combined with such protective reflexes as the ‘squint’ or orbicularis oculi contraction and sometimes head extension (Table V) since reflex mouth closure could serve also to shut out potentially harmful material from passing into the mouth as well as constitute part of the essential action for the intake of food. References p . 130-133

T. H U M P H R E Y

102

TABLE V SEQUENCE OF DEVELOPMENT OF REFLEXES O F A N A V O I D I N G A N D / O R PROTECTIVE NATURE, O N STIMULATION O F A R E A S SUPPLIED B Y SENSORY FIBRES OF T H E T R I G E M I N A L NERVE*

Menstrual age (weeks)

1.5 8.5 9.5 10-10.5

10.5 10.5 11

12 12-12.5 12.5 13-14 13.5-14 13-16.5

Area stimulated

Reflexes observed

Contralateral flexion in neck region, i.e., away from stimulus (avoiding reflex) Contralateral flexion in neck and trunk region, i.e., away Perioral from stimulus, coupled with shoulder movement and pelvic rotation Head and trunk extension (movement away from Perioral (particularly stimulus) common from midline) Upper eyelid (spreading Orbicularis oculi contraction (‘squint’ type of reflex) to include even upper and lower lips by 13 weeks)

Perioral

Bridge of nose and below Rotation of face to contralateral side (movement away from stimulus), usually with lateral neck and trunk eyelid flexion Contralateral trunk flexion with or without extension of Lower eyelid brachia Upper eyelid or eyebrow Corrugator supercilii contraction (‘scowl’ type reflex) area Upper eyelid or eyebrow ‘Squint’ and ‘scowl’ reflexes may be combined area Downward rotation of eyeballs Upper eyelid Lips, unilaterally Head extension with mouth opening and closing Upper lip and ala of nose Elevation of angle of mouth and ala of nose (‘sneer’ type reflex) Contralateral rotation of face without trunk movement; Perioral face area sometimes with ‘sneer’ type reflex Upper eyelid and eye- Local reflexes such as ‘squint’ are combined with neck and trunk extension or with rotation of the face away brows; later, lower eyefrom the stimulus lid, cheek region, lips and inside of mouth

14.5 15.5

Upper lip bilaterally Lips at rima oris

16

Nose and upper lip

23.5

Use of respirator**

24 25

Inside of nostrils**** Tapping eyelids gently

Bilateral ‘squint’ type reflex ‘Squint’ may be combined with mouth closure and/or head extension ‘Squint’, ‘scowl’ and ‘sneer’ combined with head extension Phonation (high pitched cry***) on establishing respiration Sneezing** * * Palpebral reflex

* Based on the records of Hooker (1952, 1958) and on unpublished data from the protocols of Dr. Hooker except for the material indicated by asterisks. ** Stimulation is more extensive than the areas supplied by the trigeminal nerve, but the reflex is included since these areas are stimulated also. *** Spontaneous crying has also been noted by Hooker at 25 weeks (protocols). Spontaneous crying was reported by Golubewa et al., 1959, but the age of its first appearance is not clear from the report. **** Golubewa et al., 1959.

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103

Trigeminal reflexes related to feeding

Turning now to the reflexes that enter into the activity pattern for feeding (Table VJ), it is at once evident that the earliest of these reflexes, i.e., lateral flexion toward the stimulating agent (Fig. 1C and D), begins a half week later than the avoiding type of reflex of flexion away from the stimulus. Moreover, when these two types of lateral flexion reflexes are tabulated for the 8 fetuses observed at 7.5 to 8.5 weeks of menstrual age, it was found that 56 out of a total of 62 reflexes (90.3 %) were toward the contralateral side (away from the stimulus) and only 6 (9.7%) to the irsilateral side, i.e., toward the stimulus. For the 5 fetuses observed at 9.5 weeks of menstrual age, only 2 of the total of 63 such reflexes were toward the stimulating agent (3.1 %). At 9.5 weeks extension of the head and trunk appears and with the increasing number of local reflexes after 10 weeks fewer reflexes of the total pattern type are seen. Those observed, however, are almost entirely of the avoiding type, i.e., away from the stimulus. In considering the total pattern type of reflexes resulting in movement toward the stimulus, it will be noted also that ventral head (and trunk) flexion (movement toward the stimulus) and ipsilateral rotation of the face (toward the stimulus) all begin later than the corresponding avoiding type of reflex, i.e., head extension and contralateral rotation of the face (movement away from the stimulus) respectively (Table IV). Although the earliest developing trunk and head reflex is away from the stimulus, the first local reflex to appear on stimulation of areas supplied by the trigeminal nerve is one of the reflexes essential for feeding. Other related local reflexes follow in sequence with swallowing appearing next, then lip closure. Tongue movements probably appear early, but have not been seen before 14 weeks (see footnote * * * Table VI). As seen from Table VI, protrusion of the lips, pursing the lips and sucking begin much later. Only relatively few combinations of reflexes appear during fetal life in the series of avoiding or protective reflexes (Table V). For the development of adequate feeding reflexes, however, it is essential that the various local reflexes (Table VI) not only be combined, but also that the combinations take place in functional sequences. Thus, mouth opening must precede swallowing, mouth closure and tongue movements. And although head extension may appear as a part of the swallowing sequence of movements, it precedes the movement of the larynx, which is accompanied by ventral head flexion. Postnatally, also, swallowing is not accomplished with the head back in the extended position although the mouth may open, but only when the head is in the normal erect position or with ventral head flexion. For the survival of the newborn infant, pursing the lips and sucking activity of adequate strength must be combined in the proper sequence with tongue movements and swallowing and integrated with respiratory movements as well. In addition, the rotation or otherwise turning of the face toward the stimulus (Tables IV and V1) aids in finding the nipple (Prechtl, 1958) and so also constitutes part of the postnatal sequence of reflex activity for feeding. In one instance stimulation of the left palm (fetus No. 104,13 weeks) caused mouth opening, mouth closure, swallowing and ventral head flexion as well as partial finger References p. 130-133

T. H U M P H R E Y

104

T A B L E VI S E Q U E N C E OF D E V E L O P M E N T O F F E E D I N G RE FLEX ES E L I C I T E D BY S T I M U L A T I O N OF A R EA S S U P P L I E D B Y S E N S O R Y F I B R E S OF T H E T R I G E M I N A L ( A N D C E R V I C A L ) N E R V E S *

Menstrual age (weeks) 8

9.5 10 11-11.5

12-1 2.5

12.5 13 13 14 14 17 20 22 24

29

Area stimulated Perioral Edge of lower lip Lower lip and over mandible Perioral

Reflexes observed Ipsilateral flexion of neck and trunk, Le., flexion toward the stimulus Mouth opening by lowering of mandible Ventral head flexion, i.e., toward the stimulus

Ipsilateral rotation of face accompanied by lateral trunk flexion or head extension, is.,movement toward the stimulus Lips** and/or tongue*** Momentary lip closure and swallowing on repetition of stimulus Ventral head flexion and swallowing Lips, unilaterally Lips * * Maintained lip closure Mouth opening, closure, swallowing,ventral head flexion Palm of hand and partial finger closure Inside of lips Upper lip** Upper lip * * Lower lip * * Lips** Mouth region' Lips* *

Tongue movements*** Lip closure with head flexion and swallowing Protrusion of upper lip Protrusion of lower lip Simultaneous protrusion and pursing of both lips Sucking1 Audible sucking

(or earlier) Prematures and Pressure on palms of both Mouth opening with rotation of face to midline and elevation of tongue (Babkin reflex)2 newborn infants hands simultaneously2 Newborn About lips, outside of Head movements bringing mouth toward stimulus by rotation of head, ventral head flexion or head exinfants rima oris3 tension3

* ** *** 1

The functional data, except where otherwise indicated, are from the papers of Hooker (1952, 1958) and from as yet unpublished data from Dr. Hooker's protocols. These stimulations of the lips are primarily of the so-called rima oris or vermilion border. Stimulation of the tongue is not usually possible since the mouth is generally closed. Tongue movements probably occur near the time that mouth opening begins, as was found for the rat by Angulo (1932). Golubewa et al., 1959; Parmelee, 1963a; Prechtl, 1958.

closure. Stimulation along the arm between the shoulder and elbow has been followed by mouth opening and head extension as well as flexion of the arm (fetus # 50, 16 weeks). The mouth opening, face rotation and tongue elevation reflex of Babkin (Babkin, 1953; see Lippman, 1958, and Parmelee, 1963a) following pressure on the palms of the hands of premature infants has points in common with the fetal reflexes just mentioned as does also the palmomental reflex (Marinesco and Radovici, 1920; Blake and Kunkle, 1951; August and Miller, 1952; Parmelee, 1963b). It seems probable that all of these reflexes are dependent upon the close relationship between the sensory fibres of cervical nerves from the hand and the caudally ending fibres of

H U M A N F E T A L REFLEXES

105

the spinal tract of the trigeminal nerve (Humphrey, 1952, 1954, 1955). Because of the obvious overlap of incoming cervical and sensory trigeminal fibres (Humphrey, 1955; Crosby, Humphrey and Lauer, 1962) no doubt some interneurons interrelate the incoming cervical sensory impulses with the motor neurons for mouth opening, mouth closure and swallowing (facial and motor trigeminal nuclei and nucleus ambiguus of the brain stem) in addition to forming the many interconnections with the motor neurons innervating the upper extremity muscles (the ventral horn cells of the cervical enlargement). In premature infants particularly (Parmelee, 1963b), but also in some normal adults and more often with brain damage (August and Miller, 1952), the palmomental reflex may be seen. As suggested by Parmelee (1963a,b), and evidenced by the counterparts of these reflexes in the first half of fetal life, the Babkin and palmomental reflexes are undoubtedly mediated through brain stem and upper spinal cord levels. M O K P H O L O G I C C H A N G E S I N T H E N E R V O U S SYSTEM C O R R E L A T E D W I T H T H E A P P E A R A N C E O F REFLEXES FROM T R I G E M I N A L S T I M U L A T I O N

A survey of the developmental changes taking place in the trigeminal nerve receptors peripherally, of the growth and termination of its fibres in the nervous system centrally, of the related sensory trigeminal nuclear complex, and of the various motor cell groups which obviously participate in the reflexes elicited demonstrates progressive differentiation throughout these structures as functional activity develops and becomes more complex. In each case it is possible to correlate the appearance of specific reflexes and combinations of reflexes with morphologic changes. Development of the peripheral receptors of the trigeminal nerve When the first reflex appears at 7.5 weeks, after stimulation about the mouth (Fig. 1 and Table V), the growing nerve tips (Table VII) are well below the surface epithelium. A week later, when the reflex has increased in extent and may be elicited repeatedly, a few fibres may be very close to the epithelium. During this period all of the reflexes elicited are of the total pattern type (Table I). It is not until some nerve tips reach the basement membrane of the epithelium in the lip region that the first local reflex, mouth opening, appears. As it becomes possible to elicit other local reflexes such as swallowing and mouth closure many nerve fibres have reached the basement membrane (Table VII) and some have pierced it to end on epithelial cells (Hogg, 1941). These terminations on epithelial cells constitute ‘crude discs which resemble touch corpuscles of Merkel’ (Hogg, 1941). Such endings on epithelial cells thus constitute the first specialization of receptors. They are soon followed by the appearance of nerve fibres ending on the hair follicles of the superciliary ridges, the upper lip and the zygomatic areas (13-14 weeks; Hogg, 1941). It should be noted that no local reflexes appear until nerve fibres reach the basement membrane of the region stimulated. As primitive Merkel’s end discs appear and nerve fibres terminate on hair follicles, the number of local reflexes increases and the reflexes become more variable. References p . 130-133

106

T. H U M P H R E Y

T A B L E VII D E V E L O P M E N T O F P E R I P H E R A L S E N S O R Y T R I G E M I N A L R E C E P T O R S C O R R E L A T E D W I T H F E T AL

R E F L E X E S ELICITED B Y T H E I R S T I M U L A T I O N *

Menstrual age (weeks)

7.5 8 8.5

9.5

10-1 1

12-1 2.5

13-14

20

Status of nerve endings

Activity on trigeminal stimulation

Nerve plexus in corium of lips1 Contralateral flexion in neck region and tongue2 Nerve fibres approaching epi- Contralateral neck and upper trunk flexion. Very thelium of lips3 rare ipsilateral neck and upper trunk flexion Undifferentiated nerve tips most- Contralateral neck and upper trunk flexion now ly a 5 0 y from epithelial cells accompanied by extension at shoulders and of lips; a few only 5 y distant3 rotation of pelvis to opposite side Comparable ipsilateral reflex rarely seen A few nerve tips reach the base- First local reflex, lowering of mandible ment membrane of the epi- Contralateral trunk flexion extends farther thelium of the lips3 caudalward Extension of trunk and neck An increasing number of nerve Local reflex of swallowing appears fibresreach the basement mem- Contralateral trunk flexion combined with robrane of the lips but do not tation of face contralaterally appear to pierce it3 Trunk and neck extension combined with roRich nerve network just below tation of pelvis epithelium of tonguez Orbicularis oculi contraction (‘squint’) and corNerve fibres approach epithelium rugator supercilii contraction (‘scowl’) on eyebetween hair follicles of superlid and eyebrow or eyelid stimulation alone ciliary ridge3 Some nerve fibres pierce the base- Lip closure appears and may be accompanied by ment membrane of the lips and swallowing spread onto nearest epithelial Ventral head flexion, with rotation of the face cells to form ‘crude discs which ipsilaterally and medial rotation of both arms resemble touch corpuscles of Merkel’, expanding in a single large growth cone3 Downward rotation of eyeballs on eyelid stimulation Nerve fibres now in sheaths of Consistent orbicularis oculi contraction (‘squint’) hair follicles on medial side of often accompanied by action of corrugator superciliaryridges and a few supercitii (‘scowl’) on upper lip3 Many nerve fibres have pierced Mouth closure and swallowing with marked the basement membrane of the ventral head flexion lips and reach the epithelial Tongue movements * * cells3 Nerve fibres end on hair follicles Elevation of angle of mouth and ala of nose of upper lip and cheek region, (‘sneer’ type of reflex) with rotation of face some with collateral branches; away from stimulus about alae and upper lip those of hair follicles of super- Orbicularis oculi contraction (‘squint’) combined ciliary ridge and upper lip with neck and trunk extension better developed than those of cheek and lower lip3 Free nerve fibres in deeper layers Closure and protrusion of both lips (upper lip, of epidermis2 17 weeks; lower lip, 20 weeks) Tongue papillae richly innervated but no taste buds2

H U M A N FETAL REFLEXES

After 20

Newborn infant

Network of fibres among epithelial cells of lip on either side of midline4 Intraepithelialfibres are frequent at 24 weeks and better developed at margins of lips5 An abundance of differentiated end organs are present6

107

Pursing of lips (22 weeks) Spontaneous sucking (22-23 weeks7) Side to side head turning (22-23 weeks7; 28 weekss) Sneezing on stimulating nostrils (24 weeks7) Audible sucking (29 weeks, perhaps earlier) Turning face toward stimulation on touching around the lipss

* The functional data are from the records of Hooker (1952, 1958) except for the reflexes indicated by superscript numbers 7 and 8. ** Tongue movements probably occur earlier, but the mouth is usually closed and so they cannot be observed readily. The superscript numbers indicate the source of the data quoted: Humphrey, unpublished observations; Hewer, 1935; Hogg, 1941; Piepex, 1947; Suga, 1951; Becker, 1933; Golubewa et UI., 1959; 8 Prechtl, 1958. No data have been found on the appearance of Meissner’s corpuscles in the lip or tongue regions, but it might be reasonably assumed that they are present in the lips as early as they are found in the fingers, where they have been identified by 24 weeks (Szymonowicz, 1933; 28 weeks, Hewer, 1935; see Table IX). During the second half of pregnancy, then, when pursing of the lips and effective sucking appear, the tactile corpuscles of Meissner are probably present in the lip region. In the full term newborn infant, when sucking is usually of adequate strength and the face is uniformly turned toward the object touching it about the lips (Prechtl, 1958), there is an abundance of specialized receptors in the lips (Becker, 1933, p. 267). Development of the spinal tract of the trigeminal nerve and the sensory trigeminal nuclear complex In embryos shortly before the appearance of any reflexes, for example in one of 6.5 weeks of menstrual age (14.0 mm long, Humphrey, 1954), only a few scattered descending axons of the trigeminal nerve (descending root or spinal tract of the trigeminal) reach the first cervical level of the spinal cord (Fig. 3 and Humphrey, 1954, Figs. 17A and 18A). These are probably primarily the so-called pioneering fibres of Harrison (1910) and others. At this time also differentiation is only just beginning at the first cervical level of the spinal cord (Table VIII) in the subnucleus caudalis (terminology of Crosby and Yoss, 1954) of the nucleus of the spinal tract of the trigeminal nerve (Brown, 1958). A week later, at 7.5 weeks, when perioral stimulation first elicits a reflex, the fibres of the spinal tract of the trigeminal nerve (Fig. 4) have grown well into the second cervical segment of the spinal cord (Humphrey, 1954, Figs. 17B and 18B) and many more of them are then present. At this age differentiation of subnucleus caudalis (Table VIII) has also progressed so that its pars gelatinosa and pars magnocellularis are now differentiable at caudal levels (Brown, 1958) although not in the frontal region of this subnucleus. Slightly later, for fetuses of about 8 weeks of menstrual age, when the contralateral flexion reflex is expanding somewhat, the number of fibres in the spinal tract of the References p . 130-133

T. H U M P H R E Y

108

Ependyma

Dorsal funiculus

c1

bular

[I

/

C2

c3 .Ophthalmic flbers

c4

Appearance at middle of C1

Greatest caudal extent

Fig. 3. Diagrams to illustrate the degree of development of the fibres in the spinal tract of the trigeminal nerve of a fetus of 6.5 weeks of menstrual age (14.0 mm CR, No. 113), one week before the earliest reflex from perioral stimulation. At the left, the few fibres in the spinal tract of the trigeminal nerve are represented by dots. In the diagram at the right, the caudal extent of the tract is shown in the lower part of the fkst cervical segment. The exact point of termination could not be determined with certainty (Humphrey, 1954). The magnification is the same as for Figs. 4-6.

Fasciculus grocills Fasciculus cuneotus.

Moxillomondibulor

fibers Ependyma

-

Ophthalmic fibers

c4

Appearance a t middle of C1

Greatest caudal extent

Fig. 4. Diagrams to illustrate the degree of development of the fibres in the spinal tract of the trigeminal nerve of a fetus of 7.5 weeks of menstrual age (20.7 mm CR,No. 93A). This is one of the youngest fetuses for which perioral stimulation was followed by contralateral flexion in the neck region (Hooker, 1954). As indicated at the left in the diagram of a transverse section at the middle of C1, the area occupied by the spinal tract of V, although small, is now well filled in with fibres. By this time also the tract extends well into the second cervical segment of the spinal cord, as shown at the right. The magnification for this Fig. is the same as for Figs. 3, 5 and 6, in order that these Figs. can be compared.

trigeminal is greater and some of them may have entered even the third cervical segment of the spinal cord (Humphrey, 1952, 1954, Figs. 17C and D, 18C, 20 and 22). At this time subnucleus caudalis of the nucleus of the spinal tract of N. V has also

109

H U M A N F E T A L REFLEXES

differentiated farther cephalad and its magnocellular and gelatinous parts are larger and show more cytological differentiation (Brown, 1958). By 8.5 weeks some of the descending maxillomandibular fibres in the spinal tract of the trigeminal nerve (Fig. 5) have been shown to reach the upper part of the fourth Fasciculus aracilis asciculus cuneatus

Maxil lomandibular

Ependyma

c3 fibers

Appearance at middle of C1

Greatest caudal extent

Fig. 5 . Diagrams showing the development of the fibres in the spinal tract of the trigeminal nerve of a fetus of 8.5 weeks of menstrual age (26.5 mm CR, No. 19). Note the increase in size of the area occupied by the spinal tract of the trigeminal nerve in transverse sections (diagram at the left), the markedly greater caudal extent of the tract by this age (diagram at the right) and the more extensive movement resulting from pericral stimulation. The magnification is the same as for Figs. 3,4 and 6. Perioral stimulation: (a) contralateral neck and upper trunk flexion; (b) extension of both arms at the shoulders; (c) rotation of pelvis to contralateral side; (d) rarely, homolateral flexion of neck and upper trunk.

cervical cord segment (Humphrey, 1952, 1954, Figs. 17E, 21 and 22). At this time also the gelatinous and magnocellular parts of the subnucleus caudalis (Table VIII) are distinguishable nearly throughout the extent of subnucleus caudalis (Brown, 1958). A week later, at 9.5 weeks, when the first local reflex from sensory trigeniinal stimulation has been noted (Hooker, 1954, 1958), no additional caudal growth of the fibres in the spinal tract of the trigeminal was found (Humphrey, 1954, Figs. 21, 22 and 24). In fact, for the fetus suitable for this study (Fig. 6 ) the fibres were not identified so far caudally in the spinal cord as for the one of 9.5 weeks of menstrual age. Undoubtedly, as indicated earlier (Humphrey, 1954, p. 134) this lesser caudal extent of the spinal tract of the trigeminal at 9.5 weeks, as compared with the greater caudal level found for an 8.5-week fetus, probably indicates two things. First, the adult level of caudal growth of the tract has very likely been attained by 8.5 to 9.5 weeks of fetal life, although the full number of fibres found postnatally may not be present. Second, the differences in termination level at 8.5 and 9.5 weeks of fetal life probably reflect the variations which may be seen in adult brains (Jimenez Gonzales, 1944). At 9.5 weeks, there is additional differentiation in the nucleus of the spinal tract of the trigeminal nerve (Table VIII) for the marginal part of the subnucleus caudalis, as well as pars gelatinosa and pars magnocellularis, becomes identifiable (Brown, 1962). References p . 130-133

110

T. H U M P H R E Y

Between 9.5 and 14 weeks differentiation of the subnucleus interpolaris takes place and subnucleus rostralis undergoes its initial changes (Brown, 1962). It is at this time that many of the local reflexes in response to sensory trigeminal stimulation are first seen and that these reflexes begin to combine into functional sequences (Table VIII). /

b

c

Fosciculus grocilis

i

c

u

l

u

s cuneoius Maxillomondibular fibers

Appearance at middle of

CI

Greatest caudal extent

Fig. 6. Diagrams to show the area occupied by the spinal tract of the trigeminal nerve in a fetus of 9.5 weeks of menstrual age (34.3 mm CR, No. 134). Although the spinal tract of the trigeminal nerve occupies a slightly greater area in this fetus (diagram a t the left), it could not be followed as far caudalward (diagram at the right) as at 8.5 weeks (see also p. 109). The increased growth is accompanied by additional types of reflex activity: (a) contralateral flexion reflex farther caudalward ; (b) extension of neck and trunk; (c) first local reflex, lowering of mandible. The magnification is the same as for Figs. 3-5.

As subnucleus rostralis completes its differentiation from the common area frontal to subnucleus caudalis (Brown, 1962) and the chief sensory nucleus increases in size to attain its adult position (Brown, 1960) protrusion and pursing of the lips appear (Table VIII). It is only after all subdivisions of the sensory trigeminal nuclear complex have differentiated, however, and additional growth and differentiation of the secondary sensory neurons is taking place that the necessary reflex elements of mouth opening, mouth closure, tongue movements, swallowing and respiratory movements (Hooker, 1952, 1958) are integrated into an adequately functional suctorial reflex. The reflex pathway f o r the contralateral neck and trunk flexion reflex

On the basis of the anatomical data just presented on the caudal growth of the spinal tract of the trigeminal nerve, it was suggested earlier (Humphrey, 1952, 1953) that the primary reflex pathway for the first contralateral flexion reflex in response to perioral stimulation is probably merely a simple 3-neuron type of reflex arc such as is represented in Fig. 7. The commissural fibres, which connect the afferent and

HUMAN FETAL REFLEXES

111

T A B L E VIII R E L A T I O N OF D I F F E R E N T I A T I O N OF S E N S O R Y T R I G E M I N A L N U C L E A R C O M P L E X T O T H E DE-

V E L O P M E N T O F H U M A N FETAL R E F L E X E S M E D I A T E D T H R O U G H T H I S N U C L E U S ”

Menstrual age (weeks)

Reflex activity

Status of sensory trigeminal nuclear complex

Differentiation of cell types is beginning in C1 and caudal medulla

6.5

None

7.5

Contralateral flexion in neck Pars gelatinosa and pars magnocellularis of subnucleus caudalis are first recognizable at caudal region levels of this subnucleus

8.5

Contralateral neck flexion reflex Pars gelatinosa and pars magnocellularis are distinguishable nearly throughout the future exhas extended to include movement of arms at shoulders and tent of subnucleus caudalis rotation of pelvis contralaterally Ipsilateral flexion reflex of this type is seen rarely

9.5

Neck and trunk extension ap- Marginal part of subnucleus caudalis is first identifiable, so completing differentiation of pears First local reflex of mouth openall parts of subnucleus caudalis ing, by lowering of mandible, Anlage of subnuclei interpolaris and rostralis appears frontal to subnucleus caudalis first seen (9 weeks)

10-10.5-1 1 Orbicularis oculi contraction Differentiation of subnuclei interpolaris and rostralis is beginning in the ‘common frontal’ (‘squint’ reflex) Rotation of face away from stiregion or anlage of these nuclei (11 weeks) mulus combined with lateral Definitive chief sensory nucleus is now identifitrunk flexion able (10.5 weeks) Ventral head flexion 12-14

Added local reflexes of swallow- Basic adult structure of subnucleus interpolaris completed and subnucleus rostralis is partly ing, lip closure, tongue movements, elevation of angle of differentiated mouth Combinations of local reflexes such as lip closure and swallowing or ‘squint’ and ‘scowl’ Combined local reflexes with trunk and head movements

17-20

Protrusion of upper (17 weeks) Subnucleus rostralis is differentiated throughout its extent by 13.5 weeks and lower (20 weeks) lips Chief sensory nucleus has attained its adult position (18.5 weeks)

20-22

Protrusion of both lips and purs- Added maturation and growth of neurons in sensory trigeminal nuclear complex ing of lips (22 weeks)

* Functional data are from the papers of Hooker (1952, 1958). Morphologic data are from the papers of Brown (1956, 1958, 1960, 1962). References p . 130-133

112

T. H U M P H R E Y

efferent limbs of this reflex arc, develop early. Indeed, even as early as 7 mm, long before any reflexes appear, Windle and Fitzgerald (1937, p. 495) found the ventral (or anterior) commissure of the human embryo ‘well formed’ in the cervical region of the spinal cord. With the inclusion of trunk movements in this lateral flexion reflex CEREBEh

8 WEEKS EMBRYO

1

PONS

cn PL

IV

STIMULATION OF L E F T MAX OR MAN. V

CONTRACTION OF AXIbL MUSCLES AREA ON RIGHT

IN CERVICAL

Fig. 7. A diagram to illustrate the probable pathway for the nervous impulses resulting in the early contralateral flexion in the cervical region following perioral stimulation. Based on Fig. 4 from the 1952 paper and Fig. 1 from the 1953 paper of Humphrey.

the utilization of the spino-spinal fibres in fasciculusproprius (Humphrey and Hooker, 1959) is undoubtedly essential for the spread of the action. Other phylogenetically old and early developing pathways, such as some of the reticulospinal tracts, probably participate also, as has been suggested earlier (Barcroft and Barron, 1937; Barron, 1941; Humphrey, 1952) for internuclear connections probably reach cells of the reticular formation early in development, since the reticular formation is an old system phylogenetically (Ariens Kappers, Huber and Crosby, 1936). Like the ventral white commissure, the ventral nerve roots are present throughout the spinal cord long before the first fetal reflex appears. By 8.5 weeks, at least, the ventromedial cell column and the spinal accessory nucleus are clearly recognizable in

H U M A N FETAL REFLEXES

113

the figures of upper cervical spinal cord levels (Pearson, 1938). Obviously, by the time that the first lateral neck flexion reflex appears at 7.5 weeks, differentiation of the motor neurons at upper cervical cord levels has reached a developmental stage at which function is possible. Differentiation of other structures related to refexes from trigeminal stimulation The differentiation of the cranial nerve motor nuclei, through which the various local reflexes and combinations of reflexes take place, is less well known than the development of the sensory trigeminal nuclear complex. Although there is some evidence that, in general, cranial nerve motor nuclei in some animals may have a tendency to develop in a cervicorostral sequence as does the sensory trigeminal nuclear complex (Brown, 1956, 1962), and such a general sequence was suggested for human fetuses (Humphrey, 1954), differentiation of the human motor trigeminal and facial nuclear masses has been found to progress in a different fashion (Jacobs, 1959). For these nuclei, the first part to differentiate is the region where the two nuclei first separate from each other. From this level differentiation progresses cephalad through the posterior trigeminal and main trigeminal motor nuclear complex and caudalward throughout the accessory facial nucleus and motor facial complex (Jacobs, 1959). Cell groups within the facial nucleus were not identified by Pearson (1946) until the end of the third or beginning of the fourth month (about 80 mm CR, Pearson, 1946) and not all of the cell groups are present until later. By 10.5 weeks (45 mm CR) the hypoglossal nucleus has differentiated into most of the cell groups found in the adult (Pearson, 1939). Tongue movements have not been observed until some time later (14 weeks, Table VI), but probably would be noted earlier if the mouth were open more often so that observations could be more readily made. In view of the fact that rudimentary motor endings are found early in tongue muscle (about 12 weeks, Hewer, 1935; see Table X) and the nerve fibres come in contact with the developing tongue muscle fibres much earlier (by 7 weeks, Hewer, 1935), it seems likely that tongue movements begin soon after mouth opening, as they do in some other mammals (Angulo, 1932, rat). At the age when the first reflex follows perioral stimulation (Table V) there is no evidence for myelination of any cranial or spinal nerve fibres, peripherally or centrally (Langworthy, 1933; Lucas Keene and Hewer, 1931). Evidently the beginning of myelin formation is not necessary for the functioning of the reflex arcs which first appear. Since myelin has appeared in late developing tracts such as the corticospinal system long before there is any evidence of function (Humphrey, 1960), it is of particular interest that the fibres utilized by these early appearing reflexes do not myelinate until some time after they have begun functioning. REFLEXES FROM C U T A N E O U S STIMULATION O F THE U P P E R EXTREMITY

The palmar surface of the hand is the most sensitive cutaneous area of the upper extremity of fetuses as well as of the adult. Sensitivity appears at 10.5 weeks, or References p . 130-133

114

T. H U M P H R E Y

perhaps a little earlier (Hooker, 1958), at a time when extension at the shoulder has also been seen following stimulation over this area. At first the reflex from palmar stimulation consists of a quick partial finger closure (Fig. 8), usually without any

Fig. 8. Drawings of the hand from moving picture frames illustrating finger closure following palmar stimulation of a fetus of 11 weeks of menstrual age (48.5 mm CR, No. 26). (A) The position of the digits at rest. (B) The position of the fingers after stimulation of the palm of the hand and partial finger closure. (C) Drawings A and B superimposed with the position of the fingers at rest indicated by the shaded areas. Figs. 8 through 11 were made available through the courtesy of Dr. Davenport Hooker. These Figs., hitherto unpublished, were used in connection with the presentation of a paper on the origin of grasping before the American Philosophical Society in 1938.

movement of the thumb (Hooker, 1938, 1958). Shortly after this time, by I 1 weeks, the partial finger closure may be accompanied by wrist and sometimes elbow flexion, medial rotation of the brachium and forearm pronation. By 13-14 weeks, finger closure becomes complete and shortly thereafter (15-15.5 weeks) the closure is maintained for an appreciable time. Between 15.5 and 18.5 weeks, a weak but true grasp appears and by 27 weeks Hooker (1958) found sufficient grasp present in one premature infant ‘almost to support the body weight momentarily’. The thumb only rarely flexes even a week after partial finger closure first appears (Hooker, 1938, 1939, 1952, 1958), and at first all of the fingers move to essentially the same degree (Table IX and Fig. 8). Finger flexion gradually becomes more complete and at 12 weeks and later (Fig. 9), the movement of some of the digits may be greater (Hooker, 1939) than that of certain others. More variability in response appears by 13.5 weeks (Hooker, 1938). With these changes the position of the fingers alters so that the hand is partly open at rest (Fig. 10) with the index finger and thumb (and sometimes the ring finger) showing the most extension (Hooker, 1939). From 16 weeks

HUMAN FETAL REFLEXES

115

Fig. 9. Drawings of the hand from moving picture frames illustratingfinger movements after palmar stimulation of a fetus of 15 weeks of menstrual age (102 mm CR, No. 32). (A) The position of the digits at rest. (B) The position of the fingers after stimulation. (C) Drawings A and B combined with the original position of the fingers before stimulation indicated by the shading.

onward the fingers are usually closed with the hand at rest (Fig. 1l), but this closure is tightened and the wrist flexed also if the ‘heel’ of the hand is stimulated (Hooker, 1938). Although the thumb may flex actively before that time and opposition may occur as early as 12.5 weeks (Hooker, 1958), the thumb does not respond regularly until 15 weeks and has no active part in grasping until 25 weeks, when it is often caught under the index finger (Fig. 11) and sometimes the middle finger as well (Hooker, 1939). Extension of the fingers has also been seen on stimulating the palm of the hand as early as 11.5 and 12 weeks of menstrual age in the double simultaneous stimulation studies of palm and sole (Hooker, 1954; Humphrey and Hooker, 1959) and again at 19.5 weeks (data from the protocols). It was also seen following palmar stimulation of two fetuses of 17 weeks (No. 151), one when the temperature of the isotonic bath was several degrees below normal body temperature. Stimulation of the back of the hand may evoke finger extension, accompanied by wrist extension. This reflex has been seen at 18.5 weeks on brushing against the back of the hand and probably occurs References p . 130-133

116

T. H U M P H R E Y r

Fig. 10. Drawings of the hand from moving picture frames to show the movements of the fingers and thumb following palmar stimulation of a fetus of 17 weeks of menstrual age (124.5 mm CR, No. 7). (A) The position of the hand at rest. (B) The position after movement has bogun. (C) The final position of the digits after the movement was completed. (D) Drawings A and C superimposed with the original position of the digits indicated by the shaded areas.

Fig. 11. Drawings of the hand from moving picture frames to show the movements of the thumb and fingers following palmar stimulation of a fetus of 22 weeks of menstrual age (190 mm CR, No. 15). (A) The position of the digits a t rest. (B) The position of the thumb and fingers after the reflex occurs. At this age, as at 17 weeks, the thumb participates in the movement.

HUMAN FETAL REFLEXES

117

earlier. This reflex was noted again at 27 weeks when it followed movement of the back of the closed hand along the surface of the bed. Until 12 weeks, at least, the movements in partial finger closure are stereotyped (Hooker, 1939). As spontaneous movements appear (13.5 weeks) and finger closure becomes more complete, the reflexes are more variable. T A B L E IX C O R R E L A T I O N OF DIFFERENTIATION OF S E N S O R Y N E R V E E N D I N G S OF T H E H A N D WITH THE D E V E L O P M E N T OF G R A S P I N G *

Menstrual age (weeks) 10.5

12-12.5 12.5 13-14

14.5-15

16.5-23.5

27

Newborn infant

Reflex observed

Status of nerve endkgs

Beginning of incomplete closure of all fingers Consistent incomplete finger closure of all fingers Thumb flexion occasionally Finger closure may include only part of digits Rare thumb opposition Finger closure complete, but momentary

Nerve branches ‘very closely related to epidermis’ (9 to 11.5 weeks)‘ Pacinian corpuscles begin development (along nerve trunks)2 Nerve fibres make contact with epithelial cells3

Nerve fibres ’retreat’ and send new branches forming meshwork in corium (12-14.5 weeks)’ Nerve fibres penetrate epithelium of palms between papillary ridges3 Maintained finger closure, some- Increase in free nerve terminals in epithelium indicated by additional reports of their presence times with thumb action; be(end of 4th month)4 ginning grasp Often fingers no longer move alike

Effective but weak true grasp Additional reports of free nerve terminals in epithelium (20 weeks5; 24 weeks6) with or without thumb parDefinitive Merkel’s corpuscles (end 4th month)4 ticipation (by 18.5 weeks) Definitive Meissner’s corpuscles (24 weeks4; 28 weeks5) Cholinesterase activity in nerves and terminal arcades under epidermis (4 month^)^ Strong cholinesterase activity in inner core of Pacinian corpuscles (24 weeks)7 Maintained grasp with ability to Inner (as well as outer) bulb lamellae of Pacinian corpuscles developed8 support almost entire body Meissner’scorpuscles show cholinesterase activity weight momentarily at 28 weeks7 Grasp weak but becomes strong- All nerve endings found in adult are now represented but Meissner’s corpuscles are not as er (even tonic) upon suckingg complicated as in adult5 Capsule of Meissner’s corpuscle not yet developedlO

* The functional data are from the papers of Hooker (1939, 1944, 1952, 1958) except for the movement indicated by the superscript number 9 (see below). ‘Cauna and Mannon, 1961; 2Cauna and Mannon, 1959; 3Hogg, 1941; 4Szymonowicz, 1933; Hewer, 1935; 6 Perdz and Perbz, 1932; Beckett, Bourne and Montagna, 1956; Takashi, 1957; Prechtl, 1953; l o Cauna, 1956. References p . 130-133

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T. H U M P H R E Y

Certain Russian observers (Shulejkina, 1958, 1959) have studied the development of the motor neurons at C5 and C8 and made correlations with the development of the grasp reflex. Shulejkina reports that up to 9 weeks (probably menstrual age, but not so stated) there is no difference in the degree of development of the various motor cell groups of the ventral horn of the spinal cord. From 11 to 28 weeks, however, Shulejkina (1959) found the cells supplying the flexors of the fingers and the intrinsic muscles of the hand better differentiated than the other cell groups of the ventral horn. This observation is of interest since the grasp reflex (Table IX), which involves the action of these muscles, is undergoing maturation during this period. After 28 weeks, according to Shulejkina (1959) there are no longer differences in the degree of differentiation of the cell groups at these levels of the spinal cord. Before and at the time that the palmar surface of the hand becomes sensitive, Cauna and Mannon (1961) found the nerve fibres ‘very closely related to the epidermis’. Such data are comparable to the condition found for the lips (Table VII) by Hogg (1941) who observed the nerve fibres approaching the epithelium shortly after the lips become sensitive to stimulation. By 12.5 weeks some nerve fibres have reached the epithelial cells of the palms (Hogg, 1941). At 15 weeks, when finger closure is first maintained, additional nerve terminals have penetrated the epithelium (Table IX). By 16.5 weeks, when grasp begins to appear, definitive corpuscles of Merkel are present (Szymonowicz, 1933). By 24 weeks, Pacinian corpuscles, which were found beginning to develop along nerve trunks as early as 12.5 weeks (Cauna and Mannon, 1959) have been shown to have abundant cholinesterase activity (Beckett, Bourne and Montagna, 1956). Both the inner and outer bulb lamellae of the Pacinian corpuscles are developed by the time that grasp is sufficiently strong to practically support the weight of the fetus (27 weeks, Hooker, 1952, 1958) and Meissner’s corpuscles also show cholinesterase activity (Table IX). At birth all types of specialized receptors are present but are less complicated than in the adult (Hewer, 1935) and Meissner’s corpuscles still have no capsule (Cauna, 1956). Although little information is available concerning the development of motor nerve endings (Table X) in the extremities, it is evident that finger closure begins well before there is differentiation of motor end plates, for only knob-like terminals are present by 12 weeks (Hewer, 1935). Cuajunco (1942) found that motor fibres made contact with developing muscle cells of the biceps brachii by 11 weeks, however, and according to Klishov (1960) nerve terminals appear in the forearm before they are found in the shoulder region. According to Klishov (1960) receptors make their appearance in muscles before the effector endings are found. However, Cuajunco (1940, 1942) found that motor and sensory endings could be differentiated at the same time in the biceps brachii of the human fetus, i.e., at 11 weeks. If, as Klishov (1960) states, nerve terminals appear in the forearm muscles before they are present in more proximal muscles, sensory receptors certainly would be developing in the forearm muscles by 11 weeks. Free endings are present about joints by the end of the fourth month (Table XI) and encapsulated endings appear shortly thereafter (Hromada, 1960). According to both Hewer (1935) and Hromada (1960) mature corpuscles are found about joints at

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birth (Table XI). Both exteroceptive receptors (Table IX) and proprioceptive receptors (Table XI) are more mature in the newborn infant than are the motor terminals in skeletal muscle (Table X), according to Hewer (1935). TABLE X D E V E L O P M E N T O F MOTOR E N D P L A T E S I N S K E L E T A L M U S C L E S OF H U M A N F ETU SES

Menstrual age (weeks)

7

7-8 11

12

12+ 14

2628 Newborn infant

1

Hewer, 1935;

2

Status of motor nerve endings

Rich nerve supply in tongue with many terminal swellings in relation with finely striated developing muscle fibres‘ Primitive motor endings in intercostal muscles2 Contact with muscle cells having striations (biceps brachii)3 ‘Exploring fibres with knob-like terminals are very numerous’ and ‘extend to extremities of fingers and toes’’ Doyere’s eminence beginning to develop in biceps brachiis Beginning rudimentary motor endings in tongue1 Myelin sheath appears peripherally3 Motor endings in forearm muscles just beginning to differentiate1 Motor endings of diaphragm and intercostal muscles are highly differentiated1 Motor endings of tongue are highly differentiated Motor endings of limbs, particularly foot and lez, are not yet nearly completed’

Mavrinskaya, 1960;

Cuajunco, 1942.

Although emphasizing that dorsal thalamic regions, in the opinion of the author, undoubtedly do not function during the first half of fetal life, at least, it is interesting to note that the thalamic nucleus related to the reception of sensation from the extremities, nucleus ventralis posterolateralis, has differentiated from nucleus ventralis posteromedialis (the arcuate nucleus) by 12.5 to 14.5 weeks of fetal life (Cooper, 1950; see also p. 127), an age somewhat earlier than Merkel’s and Meissner’s corpuscles were found for the hand (Table IX), but approximately the same age (12 weeks, Hogg, 1941) that nerve fibres reach the epithelium of the palm and that crude Merkel’s discs were found for the lips (Hogg, 1941). Here again, then, we see an example of differentiation progressing concomitantly in related peripheral, spinal cord and thalamic regions. REFLEXES FROM C U T A N E O U S STIMULATION O F T H E LOWER EXTREMITY

The first cutaneous area of the lower extremity to respond to stimulation is the sole of the foot, at 10.5 to 11 weeks. The reflex elicited at that time is plantar flexion of the toes (Hooker, 1952, 1958). At first no other movements are noted following plantar stimulation. By 11.5 weeks, the character of the movements has changed and there may be either plantar flexion of the toes or dorsiflexion of the great toe and fanning of the other toes. Either type of toe movement may be accompanied by knee References p . 130-133

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flexion (which may be followed by extension to give a kick) and flexion at the hip with rotation of the thigh inward or outward or abduction of the thigh.

Fig. 12. A drawing from moving picture frames to show the movements of the lower extremity following stimulation of the sole of the foot of a fetus of 14 weeks of menstrual age (88.5 mm CR, No. 13). Sequences 1 and 4 from page 90 of A preliminary atlas of early humanfetal activity (Hooker, 1939) were made available through the courtesy of Dr. Davenport Hooker. The sole of the right foot was stimulated by stroking it with a hair esthesiometer. The shaded right lower extremity, superimposed upon the right lower extremity at rest, illustrates the dorsiflexion of the great toe, the fanning of the other toes, and the flexion at ankle, knee and hip joints that usually follow such stirnulation at this age.

By 12.5 weeks, stimulation of the sole of the foot is followed primarily by dorsiflexion of the great toe and toe fanning. This reflex is a Babinski-like reaction (Hooker, 1958), so designated because it is found during development whereas the reflex described by Babinski occurs with pathology in the central nervous system. The reflex

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may be accompanied by dorsiflexion of the foot, flexion at the knee and flexion at the hip. By 13.5 weeks, if not earlier (Hooker, 1958), dorsiflexion of all the toes may occur on plantar stimulation. Although plantar flexion of the toes has largely disappeared by 12.5 weeks, it may occur at any later age of fetal development, although only rarely (Hooker, 1958). Following the appearance of reflex action from stimulation of the sole of the foot, no other area of the skin of the lower extremity has been found sensitive to stimulation up to 17 weeks, when stroking over the buttock area elicited ipsilateral extension at both knee and hip joints. Possibly additional cutaneous areas of the lower extremity may also become sensitive to stimulation and further tests should be made for such areas as the dorsum of the foot, for example. T A B L E XI DIFFERENTIATION O F PROPRIOCEPTIVE NERVE E N D I N G S I N MUSCLES, TENDONS A N D ABOUT JOINTS OF H U M A N FETUSES

Menstrual age (weeks)

7-8 10 11 up to 4th month 4th month

4.5 months 20 22 26-28

Newborn infant

Receptors and their degree of development

Primitive endings in intercostal muscles* ‘Exploratory fibres with their enlarged endings’ become ‘related to the large muscle cells’2 Contact made with muscle cells in biceps brachii3 Beginning muscle spindle formation in biceps brachii with sensory endings distinguishable from motor3 Only free endings about joints4 Anlage of encapsulated endings along blood vessels and nerves of carpal and cubital joints4 Spindles in practically all muscles, but not in diaphragm or in tongue5 First mature corpuscles about joints with concentric rings forming capsule4 Spindle type and Golgi body type endings in mand leg2 Tendon spindles in external abdominal oblique muscle near aponeurosis are quite well differentiated5 Sensory endings about joints still have ‘terminal knobs and are not yet completed‘2 Sensory endings have finished look2 3/4 of alI corpuscles about joints are mature4 Most corpuscles are Golgi-Mauoni type; typical VaterPacinian corpuscles are rare about joints4

Based on the data of various authors as follows: 1940; Hromada, 1960; Stilwell, 1957.

1

Mavrinskaya, 1960; Hewer, 1935; Cuajunco,

The data available concerning the differentiation of the motor and sensory nerve endings of the lower extremity indicate little difference in their time of appearance for the two extremities. According to Hewer (1935) and Klishov (1960) the development of sensory nerve terminals in the upper extremity is in advance of that for the lower extremity. Likewise, Hromada (1960) found that the receptors around the joints of the upper extremity differentiate before those of the lower extremity. These findings are in harmony with the appearance of sensitivity in the palm and sole, for plantar References p. 13LL133

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reflexes can be elicited almost as early as the earliest partial finger closure occurs (Hooker, 1952). REFLEXES F R O M S T I M U L A T I O N OF THE G E N I T A L A N D A N A L REGIONS

Reflexes from stimulation of the genital area have been reported for three fetuses of 11, 17 and 18.5 weeks of menstrual age (Humphrey and Hooker, 1961a,b). Reflexes were also observed more recently after stimulation of the genital area of a fetus of 10.5 weeks of menstrual age. Moving picture records were obtained froni only the oldest fetus but dictated records are available for the others. Although many reflexes were observed, without cinematographic records it is still possible that some aspects of the reflex movements may have been missed. At 10.5 and 11 weeks the reflex noted was a bilateral flexion of the thighs on the pelvis, when the stimulation was over the genital area (Table XII). For a very few stimuli along the genitofemoral suIcus, an

Fig. 13. Photographic prints from moving picture frames to illustrate the movements following stimulation of the penis of an 18.5-week fetus with a hair esthesiometer (140 mm CR, No. 153). (A) The position at the time of stimulation. (B) The position at the peak of the reflex action. This reflex followed the 14th stimulation of the fetus and consisted of ‘tonic flexion of both thighs, tonic extension of both knees, sharp dorsiflexion of both ankles, marked fanning of all toes, some flexion of pelvis on trunk and pelvic rotation to the right’ (Humphrey and Hooker, 1961a). The apparent rotation of the chin to the left was caused by the operator’s hand holding the fetus within the photographic field.

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ipsilateral flexion of the thigh on the pelvis was seen at 10.5 weeks and two ipsilateral reflexes were noted at I1 weeks, probably when the stimulus was unilateral. At 17 weeks movement at the knee as well as flexion of the thigh was seen (Table XII). The stimuli were all unilateral and the responses were ipsilateral. A week and one-half later (18.5 weeks), 12 of the 28 stimuli tried produced reflexes. Stimulation of the midline genital area gave a bilateral response (Fig. 13); if the stimulus was on one side, an ipsilateral reflex appeared. Stimulation along the side of the penis and scrotum produced a more extensive ipsiIatera1 reflex than stimulation of the genitofemoral sulcus (Table XII). A truly localized reflex has been observed only much later when stimulation of the inside of the thigh produces a cremasteric reflex. This has been seen as early as 32 weeks, but probably occurs earlier (Hooker, 1952, 1958). Thus far only very few reflexes have been obtained from anal stimulation by the use of a hair esthesiometer. One of these was at 10.5 weeks and the action noted consisted of flexion of the pelvis on the trunk. Two other reflexes were observed at 17 weeks with the fetus in a prone position and included flexion of both lower extremities at the hips and knees. With one stimulus there was also flexion of the pelvis on the trunk. Moving pictures were not secured. T A B L E XI1 R E F L E X E S F R O M S T I M U L A T I O N OF G E N I T A L A R E A S *

Menstrual a,oe (weeks) and sex 10.5**

Female 11**

Male 17** Male 18.5 Male

32, probably earlier Male

* **

Area stimulated

Reflexes observed

Bilateral flexion of thighs on pelvis Genital area Ipsilateral flexion of thigh on pelvis Genitofemoral sulcus Bilateral flexion of thighs on pelvis Penis and scrotum (probably bilaterally) Ipsilateral flexion of thigh on pelvis Penis and scrotum (probably unilaterally) Ipsilateral flexion of thigh and leg Scrotum and penis unilaterally Penis stroked down- Bilateral reflex consisting of tonic flexion of both thighs, ward, probably biextension of both knees, sharp dorsiflexion of both laterally ankles, marked fanning of all toes, some flexion of pelvis on trunk and pelvic rotation to right Scrotum and penis uni- Ipsilateral reflex consisting of quick double kick of leg ending with flexion of both thighs, extension of both laterally knees, dorsiflexion of both feet, very marked fanning of all toes, ventral flexion of lower back and rotation of pelvis ipsilaterally Genitofemoral sulcus Ipsilateral reflexes which varied from (a) flexion of thigh (Summary from 8 alone to (b) flexion of pelvis on trunk with or without stimuli) thigh flexion, (c) flexion of thigh, leg and foot with toe fanning or (d) flexion of thigh and double (or single) extension of knee, with or without toe fanning Inside of thigh Cremasteric reflex

Compiled from the data of Humphrey and Hooker, 1961, and as yet unpublished protocols. Records from dictated observations only.

References p. 130-133

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T. H U M P H R E Y

Although the number of reflexes obtained from genital and anal stimulation is limited, several things of interest are evident. Perhaps the most significant is the fact that the earliest genital and anal reflexes have been seen at the same age as the first reflex in response to palmar stimulation, and earlier than reflexes are usually obtainable from plantar stimulation. Since the reflexes from genital stimulation at 10.5 weeks were secured repeatedly and until well toward the end of the period of reflex activity, it seems probable that a response to stimulation may be obtained at an even younger age. If this should prove to be the case, then sensitivity of these cutaneous areas would follow that of the perioral region and precede that of both the palm of the hand and sole of the foot. Further data are needed to determine this point, however. The reflexes from genital stimulation at 10.5 and 11 weeks apparently involve only the proximal joints of the lower extremities even though flexion of the toes occurs at 11 weeks after plantar stimulation. At 17 and 18.5 weeks, however, the reflex following genital stimulation has extended distally and at 18.5 weeks it included dorsiflexion of the foot with fanning of all toes. Thus the reflexes obtained from genital stimulation involve proximal joints and muscles at the earlier age period, whereas the first reflex from stimulating the sole of the foot consists of toe movements alone and only later includes the more proximal muscles and joints of the lower extremity. The lower extremity and pelvic reflex movements from genital stimulation reported here have been referred to as a withdrawal type of reaction by Rioch (see Humphrey and Hooker, 1961a, p. 160). The basic relation to movements of sexual activity was also mentioned (Humphrey and Hooker, 1961a.,b). Of particular interest is the fact that the early movements following genital stimulation involve both the lower extremities and the trunk together. Only later in development does a specific local reflex, like the cremasteric reflex, appear. Although reflexes from anal stimulation have been observed with only two fetuses, here again activity is evidently of a general type at first since it involves trunk flexion and movement of the proximal joints of the lower extremities. By 12 weeks, according to Hewer (1935), the fetal subepithelial nerve plexus is ‘very complex’ and ‘particularly well marked below the surface of the penis’. Free endings were found in the epithelium of the phallus of a 150-mmfetus (Ohmori, 1924), i.e., a fetus of about 19 weeks of menstrual age if the crown-rumplengthis the measurement given. At this age also (150-mm fetus, CR length) Calabrisi (1956) found no specialized endings in the genitalia of a female fetus. In the newborn infant, thinly encapsulated nerve endings are present in the penis (Crosby, Humphrey and Lauer, 1962, Fig. 25) and Pacinian corpuscles with inner and outer bulbs also have been described by Becker (1933) in the penis and about the base of the clitoris. F E T A L REFLEXES FROM D O U B L E S I M U L T A N E O U S S T I M U L A T I O N

Two series of simultaneous stimulations, one of face and palm stimuli and another for palm and sole, have been used in studying fetal reflexes (Hooker, 1954; Humphrey

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125

and Hooker, 1959). These observations demonstrate that, up to and including 13.5 weeks of menstrual age, there is a tendency for the appearance of only the typical reflex evoked by stimulation of face areas, when both the palm and the face are stimulated at the same time. Although local reflexes, such as orbicularis oculi contraction, can be elicited during this age period, when simultaneous stimuli of face and palm elicit a reflex only from stimulation of the face, this reflex is of the total pattern type, i.e., contralateral body flexion or neck and trunk extension. Opposed to this tendency for suppression of the palmar reflex is the appearance, after 14 weeks of menstrual age, of reflexes from both the facial and palmar stimuli, when both sites are stimulated simultaneously. It is significant that, by 14 weeks, local reflexes from sensory trigeminal stimulation have largely replaced the earlier total pattern type of response (Tables V-VI), for when reflexes from both stimuli were secured, the reflex obtained by facial stimulation was usually a local reflex alone, or such a reflex combined with another local reflex. The total pattern type of reflex appeared only when the head or trunk extension or the contralateral trunk flexion was also combined with a local reflex. From these findings it was suggested (Humphrey and Hooker, 1959) that, until sufficient connections are established in the brain stem with cranial nerve motor nuclei for the local reflexes, enough impulses pass caudally over the spinal tract of the trigeminal nerve both to elicit the contralateral flexion reflex and inhibit the ipsilateral palmar reflex. After the local reflexes dominate the activity, however, the impulses set up by facial stimulation are distributed through internuclear fibres to cranial nerve motor nuclei and only rarely pass caudalward into the spinal cord to inhibit the palmar reflex or produce a trunk extension or lateral flexion reflex in conjunction with the local reflex. As a consequence, the reflex pattern has changed by 14.5 weeks so that both palmar and facial reflexes tend to appear following simultaneous stimulation of the two regions. When palm and sole areas were tested simultaneously with two equal stimuli the same trends were found to occur (Humphrey and Hooker, 1959). For fetuses up to and including 13.5 weeks of menstrual age, there was a tendency for only the palmar reflex to appear. At 14.5 weeks and later, both palmar and plantar reflexes tend to appear, when these two areas were stimulated simultaneously. Here the change in the reflexes secured accompanies the appearance of more complete finger closure and active thumb participation. These added movements probably necessitate a wider spread of the sensory impulses at cervical enlargement levels and consequently a frequent loss of transmission to caudal levels. As a result the inhibitory influence over the plantar reflex, mediated through the lumbosacral enlargement, is lost. DISCUSSION

As has been pointed out many times in the past, the first reflexes following cutaneous stimulation of human fetuses constitute, by whatever name one wishes to designate them, the total pattern type of reflex as the term was used by Coghill (1929). In other words, the fetus responds as a whole, insofar as the neuromuscular development at References p. 130-133

126

T. H U M P H R E Y

the time enables movement to take place. As growth and differentiation progress, more of the neuromuscular system participates so that the upper extremities move with the trunk and the lower extremities are also moved as the pelvis is rotated. Consequently the fetus continues to react as an entity. Only later do movements of a single extremity, or reflexes such as opening or closing the mouth, appear. These reactions have been designated local reflexes. A comparable ipilateral neck and trunk flexion reflex appears slightly later than the contralateral lateral flexion reflex (Tables IV, V and VI). Thus the first reflex movement of the human fetus is a negative reaction to stimulation - movement away from the stimulus - rather than a positive reaction - movement toward the stimulus. The latter reflex not only appears a half week later, but has been observed much less often (p. 103). Whether or not a weak stimulus results in movement toward and a strong stimulus cPuses movement away from the stimulus (Carmichael, 1960, p. 141), it is impossible to say. With animal self-stimulation, Olds and Olds (1963) more often found negative reactions from strong and positive ones from weak stimuli. As development progresses and other total pattern type reflexes appear, such as head and trunk extension and ventral head flexion (Table IV), in each case the reflex away from the stimulus precedes the comparable movement toward the stimulus. Even when head rotation without trunk movement is first seen, the face is rotated away from the stimulus before being turned toward it (p. 98). When considered on the basis of the positive or negative character of the reflexes, those following sensory trigeminal stimulation fall into two distinct groups, the negative responses being of an avoiding or protective type and the positive reactions constituting the reflexes related to eating or feeding (Tables V and VI). Although less clearly so, perhaps, the withdrawal reflexes from anal, genital, palmar and plantar stimulation also represent negative reactions. Apparently a total pattern type of reflex action is not limited to stimulation of the sensory trigeminal areas, however. At least the earliest reflexes observed in response to stimulation of the cutaneous areas supplied by the most caudal sacral nerves, the genital and anal regions, include pelvic flexion and bilateral flexion of both lower extremities. If stimulation of genital and/or anal areas is found to be effective even earlier, it may be that the reflex action will involve the trunk region farther cephalad. In any case, at 18.5 weeks the reflex still includes pelvic movements; local reflex action (the cremasteric reflex) begins much later (Table XII). In correlating these total pattern type reflexes with the degree of differentiation of the peripheral receptors, it is interesting to note that these reflexes, both from stimulation of the face and the anogenital areas, appear at a time when no specialized sensory receptors have developed. Indeed, in both instances (Table VII and p. 124), when the earliest reflexes have been seen the growing nerve tips are still well below the surface of the epithelium. Such cones of growth would constitute even more primitive receptors than the so-called free endings that often have been classed as nociceptive (or noxireceptive) and that give rise (Sherrington, 1906) Frimarily to ‘negative rejexes’ (Ariens Kappers, Huber and Crosby, 1936) although positive reactions also may occur.

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127

During the time that the totd pattern type of reflex constitutes the only activity that has been seen following cutaneous stimulation of human fetuses, little differentiation has occurred in the central nervous system as well as peripherally. In the sensory trigeminal nuclear complex, only the subnucleus caudalis, the subdivision of this nuclear complex related to pain and general tactile sensations, is differentiated out of the common sensory trigeminal nuclear gray (Table VIII). Up to this time also (Gilbert, 1935; Cooper, 1950) there is little differentiation at the higher levels, but differentiation in the doIsal thalamus begins by 30 mm CR (Gilbert, 1935; Cooper, 1950) and differentiation of nucleus ventralis posterior into its pars posteromedialis (arcuate nucleus) and pars posterolateralis is possible by 70-80 mm CR (Cooper, 1950). It is in this area that the developing secondary sensory systems terminate. By the time that the earliest specific, localized reflex has made its appearance at 9.5 weeks (Table VII), some nerve tips have reached the basement membrane of the epithelium of the lips. As these specific reflexes are elicited more often and the total pattern types of movement become less frequent, differentiation continues to progress in the sensory receptors peripherally (Tables V11, IX and XI), in the sensory trigeminal nuclear complex (Table VIII), and in the dorsal thalamus (p. 119). We see here, then, differentiation taking place at all levels throughout the nervous system embryologically, just as it does phylogenetically (Ariens Kappers, Huber and Crosby, 1936; Crosby and Yoss, 1954). Much stress has been placed on the appearance of the local reflexes, or as stated by Coghill (1929), the individuation of partial patterns (Hooker, 1952, 1958; Carmichael, 1960). Less attention has been devoted to the combination of local reflexes with each other into functional sequences, some of which, at least, will be utilized postnatally. This aspect of the development of reflex activity was pointed out by Pavlov (1932) when he mentioned the ‘initial decomposition of the whole into its parts or units, and then the gradual reconstruction of the whole from these units or elements’. The combination of reflexes during fetal development has been stressed more recently by other Russian workers in the field of fetal development (Golubewa et al., 1959). This combination of local reflexes on stimulation of the face begins at 12 weeks when ‘squint’ and ‘scowl’ reflexes appear together (Table V) and by 12.5 weeks (Table VI) includes the combination of ventral head flexion with swallowing, and head extension with mouth opening and closing (Table VI). Such combinations of reflexes increase in number and variety as development proceeds. Isolated reflexes disappear for the most part before the age of viability (at 24 weeks, according to Golubewa et al., 1959), but orbicularis oculi contraction alone (‘squint’) may be elicited after the other local reflexes have disappeared (Table V). In connection with the combination of reflexes it should be mentioned that reference is made here only to reflexes elicited by cutaneous stimulation. Tendon reflexes are evidently of localized nature during fetal life, just as they are in the adult (see Humphrey, 1953). From the observations thus far reported, cutaneous sensitivity to stimulation develops in the sequence indicated in Table 11. Adequate tests have not been possible, as yet, to determine whether or not anal and/or genital areas may become sensitive to stimulation after the oral (and perioral) regions and earlier than the palm of the References p . 130433

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T. H U M P H R E Y

hand. The double stimulation tests of Bender (1952) and his associates (see also Hooker, 1954, p. 110-1 11) indicate that the genital region of young children is dominant over both the hand and the foot. Whether or not the palms of the hands and soles of the feet were used for these tests was not stated, although it is the palmar and plantar areas that are the most sensitive. The observations on genital and anal reflexes reported here (Tables I1 and XII) indicate that perhaps both anal and genital areas may become sensitiveto stimulation before either the palmar or plantar surfaces, although up to the present time these reflexes have been seen only at the same ages. If that should be the case, the postnatal observations of Bender for young children would represent a repetition of the fetal sequence for the development of skin sensitivity. Although the earliest reactions to oral stimulation are of the total pattern type, and the anal and genital reflexes have at least some comparable characteristics, the first reflexes elicited by palmar and plantar stimulation involve only movements of the digits (p. 114and 119).These limited flexion movements of the digits are soon followed by flexion at more proximaljoints so that the entire ipsilateral extremity may participate in the reflex. By the age at which respiration can be established temporarily, upper and lower extremity movements h?ve been integrated into the spontaneous movement combinations which form the basis for postnatal crawling and later walking movements. The early plantar flexion reflex, like the finger closure following palmar stimulation, constitutes the initial step in the development of a grasp reflex. The plantar flexion reflex, although present occasionally later in fetal life, has largely disappeared by 12.5 weeks (p. 120). However, it is sometimes seen in newborn infants, where it has been referred to as a foot grasping reflex (Stirnimann, 1940; Galant, 1931). It undoubtedly represents an equivalent of the foot-grasp reflex found phylogenetically in birds and some arboreal mammals. The Babinski-like reflex that replaces the plantar flexion reflex (11.5 weeks) and the finger extension which sometimes follows palmar stimulation (1 1.5 weeks) are withdrawal reflexes. Both reactions begin before nerve fibres have reached the surface epithelium (Table IX and p. 115). The finger closure reflex is stereotyped at first. As the peripheral nerve fibres reach the epithelial cells and end there in greater numbers (at 12.5 to 14 weeks, Table IX), the finger movements following palmar stimulation become more variable. Likewise, at this time the motor end plates are probably beginning their differentiation in the muscles involved in the action (Table X and p. 118) and muscle spindle differentiation also should have commenced (Table XI and p. 118). Here again, then, we have an example of variability and selectivity in reflex action accompanying morphologic differentiation. In the adult, the greatest numoer and variety of specialized sensory nerve endings, or receptors, are found in the lips, the external genitalia, the finger tips, and the plantar surface of the foot. If these facts are compared with the now known (Table 11) and probable (p. 97) sequence of development of cutaneous sensitivity for the human fetus (oral, genitoanal, palmar and plantar) then it becomes evident that it is the areas that first become sensitive to stimulation that develop the greatest number of the most highly specialized receptors.

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129

At various times in the past, different authors (Crosby and Woodburne, 1938; Crosby and Henderson, 1948; Schemm and Kahn, 1960; Schemm, 1961; Crosby, Humphrey and Lauer, 1962) have pointed out that the somatotopic patterns for higher nervous system centers depend on the morphologic relations that develop earlier at lower nervous system levels and, in turn, the relations established peripherally. Functionally, also, the higher levels of the nervous system are dependent upon the lower centers, as evidenced by the many postnatal repetitions of prenatal activity sequences (Hooker and Humphrey, 1954; Humphrey, 1954, 1960; Hooker, 1958; Humphrey and Hooker, 1959). In view of these facts it is of some interest that the negative and positive types of fetal reflexes evidently are represented at various higher nervous system levels in the avoidance (or punishment) and approach (or reward) centers demonstrated by the self-stimulation studies of Olds (1960a, by 1963), Brady (1960), McGeer (1962) and others. Such nervous system centers have been found at tegmental, hypothalamic, and septa1 levels (Olds, 1960a) and even for the cortex (McGeer, 1962). At the early fetal level of development, the preponderance of reflex activity is of the avoidance (and protective) or escape type. A preponderance of negative reactions was obtained on self-stimulation in the midbrain, particularly in the tegmentum (Olds and Olds, 1963), but self-stimulation at higher nervous system levels (which develop later), yielded a greater number of positive than of negative reactions. Although the two fundamental types of fetal reactions at the reflex level are represented at progressively higher nervous system levels, including the cortex, the reactions from the avoiding areas increase in complexity and intensity. The nature of the effect from self-stimulation of the reward areas is indicated only by the fact that continued stimulation is chosen in preference to water or food (Brady, 1960)even if the animal is starved (McGeer, 1962). Obviously, even for the primitive negative and positive reactions which constitute the first fetal reflexes there is a reappearance of the activity pattern at higher nervous system levels, although with many modifications. ACKNOWLEDGEMENTS

The cooperation of the Department of Obstetrics and Gynecology, University of Pittsburgh, and the Elizabeth Steel Magee Hospital, in carrying on this research, is acknowledged with gratitude. SUMMARY

The characteristics of human fetal reflexes at different developmental periods are discussed and the sequence for the development of cutaneous sensitivity is reviewed and tabulated. Human fetal reflexes resulting from the stimulation of sensory trigeminal areas are classified into ( I ) avoiding and/or protective reflexes and (2) those later functioning in feeding. The sequence of development of reflexes of each type, with the area from which it is elicited, is tabulated. The development of the spinal tract and the peripheral receptors of the trigeminal References p. 130-133

130

T. H U M P H R E Y

nerve and the differentiation of the sensory trigeminal nuclear complex are correlated with the appearance of the various fetal reflexes elicitable by stimulation of this nerve. The known developmental relations for the motor neurons involved in these reflexes are considered and the probable reflex arc for the first reflexes from trigeminal stimulation is discussed. Reflexes obtained by stimulating the upper and lower extremities (particularly palmar and plantar surfaces) are discussed and correlated with known nervous system development. Correlations with the development of peripheral exteroceptive and proprioceptive receptors and with motor end plate differentiation are tabulated. The reflexes elicited by anal and genital stimulation are discussed and the latter correlated with the differentiation of the peripheral receptors. The differences in the reflexes obtained on double simultaneous stimulation before and after 14 weeks of menstrual age are discussed. The significance of the ‘total pattern’ reflexes, the various local reflexes and the combination of reflexes into functional sequences is considered. The frequent postnatal repetition of fetal reflex sequences is mentioned. The negative or positive nature of different reflexes is discussed and related to the reward and punishment centers of the nervous system. REFERENCES ANGUMY GONZALES, A. W., (1930); Endogenous stimulation of albino rat fetuses. Proc. SOC.exp. Biol. (N. Y.), 27, 579. ANGULO Y GONZALES, A. W., (1932); The prenatal development of behavior in the albino rat. J. comp. Neurol., 55, 395-442. ARIENSKAPPERS, C. U., HUBER,G. C., AND CROSBY, E. C., (1936); The Comparative Anatomy of the Nervous System of Vertebrates, including Man. New York, The Macmillan Company. (Reproduced without revision in 1960 by Hafner Publishing Co., New York.) AUGUST, B., AND MILLER,R. B., (1952); Clinical value of the palmomental reflex. J. Amer. med. Ass., 148, 120-121. BABKIN,P. S., (1953); The establishment of reflex activity in early postnatal life. Central nervous system and behavior. Translations from the Russian Medical Literature. The Josiah Macy Jr. Foundation and the National Science Foundation, 1960. BARCROFT, J., AND BARRON, D. H., (1937); Movements in midfoetal life in the sheep embryo. J. Physiol. (Lond.), 91, 329-351. BARRON, D. H., (1941); The functional development of some neuromuscular mechanisms. Biol. Rev., 16, 1-33. BECKER, J., (1933); Uber periphere Nervenendigungen in den ausseren Genitalien von Neugeborenen. Z. Kinderheilk., 55, 266268. BECKETT,E. B., BOURNE, G. H., AND MONTAGNA, W., (1956); Histology and cytochemistry of human skin. The distribution of cholinesterase in the finger of the embryo and the adult. J.Physiol. (Lond.), 134, 202-206. BENDER, M. B., (1952); Disorders in Perception. Springfield, Illinois, Charles C. Thomas. BLAKE,J. R., AND KUNKLE,E. C., (1951); The palmomental reflex: a physiological and clinical analysis. Arch. Neurol. Psychiat. (Chic.), 65, 337-345. BOLAFFIO, M., AND ARTOM,G., (1924); Richerche sulla fisiologia del sistema nervosa de1:feto umano. Arch. Sci. bioi. (Bologna), 5,457-487. BRADY, J. V., (1960); Temporal and emotional effects related to intracranial electrical self-stimulation. Electrical Studies on the Unanesthetized Brain. E. R. Ramey and D. S. ODoherty, Editors. New York, Paul B. Hoeber, Inc. (p. 52-77). BROWN,J. W., (1956); The development of the nucleus of the spinal tract of V in human fetuses of 14 to 21 weeks of menstrual age. J. comp. Neurol., 106, 393-424.

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BROWN, J. W., (1958); The development of subnucleus caudalis of the nucleus of the spinal tract of V. J. comp. Neurol., 110, 105-134. BROWN,J. W., (1960); The development of the chief sensory nucleus of V in the human fetus. Anar. Rec., 136, 171 (Abstract). BROWN, J. W., (1962); Differentiation of the human subnucleus interpolaris and subnucleus rostralis of the nucleus of the spinal tract of the trigeminal nerve. J. comp. Neurof., 119, 55-76. CALABRISI, P., (1956); The nerve supply of the erectile cavernous tissue of the genitalia in the human embryo and fetus. Anat. Rec., 125, 713-723. CARMICHAEL, L., (1960); Manuel of Child Psychology. New York and London, John Wiley and Sons, Inc. CAUNA, N., (1956); Structure and origin of the capsule of Meissner’s corpuscle. Anat. Rec., 124,77-94. CAUNA,N., AND MANNON, G., (1959); Development and postnatal changes of digital Pacinian corpuscles (corpuscula lamellosa) in the human hand. J. Anat. (Lond.), 93,271-286. CAUNA,N., AND MANNON, G., (1961); Organization and development of the preterminal nerve pattern in the palmar digital tissues of man. J. comp. Neurol., 117, 309-328. G. E., (1929); Anatomy and the Problem of Behavior. Cambridge, England, Cambridge COGHILL, University Press. COOPER, E. R. A., (1950); The development of the thalamus. Acta anat. (Busel), 9, 201-226. CROSBY, E. C., AND HENDERSON, J. W., (1948); The mammalian midbrain and isthmus region. 11. Fiber connections of the superior colliculus. B. Pathways concerned in automatic eye movements. J . comp. Neurol., 88, 53-91. CROSBY, E. C., HUMPHREY, T., AND LAUER, E. W., (1962); Correlarive Anatomy of the Nervous System. New York, The Macmillan Company. E. C., AND WOODBURNE, R. T., (1938); Certain major trends in the development of the CROSBY, efferent systems of the brain and spinal cord. Univ. Mich. med. Bull., 4, 125-128. CROSBY, E. C., AND Yoss, R. E., (1954); The phylogenetic continuity of neural mechanisms as illustrated by the spinal tract of V and its nucleus. Res. Publ. Ass. nerv. ment. Dis., 33, 174-208. CUAJUNCO, F., (1940); Development of the neuromuscular spindle in human fetuses. Contr. Embryol. Carneg. Znstn, 28, 95-128. CUAJUNCO, F., (1942); Development of the human motor end plate. Contr. Embryol. Carneg. Instn, 30, 127-152. DENNIS,W., (1951); Readings in Child Psychology. New York, Prentice-Hall, Inc. FITZGERALD, G. E., AND WINDLE,W., (1942); Some observations on early human fetal movements. J . comp. Neurol., 76, 159-167. FLANAGAN, G. L., (1962); The First Nine Months of Life. New York, Simon and Schuster. GALANT, J. S., (1931); Uber die rudimentaren neuropsychischen Functionen der Sauglinge. Jb. Kinderheilk., 133, 104-108. GESELL, A., (1945); The Embryology ofBehavior. New York and London, Harper and Brothers. GILBERT,M. S., (1935); The early development of the human diencephalon. J. comp. Neurol., 62, 81-115. GOLUBEWA, E. L., SHULEJKINA, K. V., AND VAINSTEIN, I. I., (1959); The development of reflex and spontaneous activity of the human fetus during embryogenesis. Obstet. Gynecol. (U.S.S.R.), 3, 59-62. GREENHILL, J. P., (1960); Obstetrics. Philadelphia and London, W. B. Saunders Company. R. G., (1910); The outgrowth of the nerve fiber as a mode of protoplasmic movement. HARRISON, J. exp. Zool., 9, 787-848. HEWER, E. E., (1935); The development of nerve endings in the human fetus. J. Anat. (Lond.), 69, 369-379. HOGG,I. D., (1941); Sensory nerves and associated structures in the skin of human fetuses of 8 to 14 weeks of menstrual age correlated with functional capability. J . comp. Neurol., 75, 371410. HOOKER, D., (1938); The origin of the grasping movement in man. Proc. Amer. Phil. SOC.,79,597-606. HOOKER, D., (1939); A Preliminary Atlas of Early Human Fetal Activity. Pittsburgh, Pennsylvania, U.S.A. Privately published by the author. HOOKER, D., (1944); The Origin of Overt Behavior. Ann Arbor, University of Michigan Press. HOOKER, D., (1952); The Prenatal Origin of Behavior. 18th Porter Lecture. Lawrence, Kansas, University of Kansas Press. HOOKER, D., (1954); Early human fetal behavior, with a preliminary note on double simultaneous fetal stimulation. Res. Publ. Ass. nerv. ment. Dis., 33, 98-113.

132

T. H U M P H R E Y

HOOKER,D., (1958); Evidence of prenatal function of the central nervous system in man. James Arthus M u r e on The evolution of the human brain for 1957. New York, American Museum of Natural History. HOOKER, D., AND HUMPHREY, T., (1954); Some results and deductions from a study of the development of human fetal behavior. Gaz. mid. port., 7, 189-197. HROMADA, J., (1960); Beitrag zur Kenntnis der Entwicklung und der Variabilitiit der Lamellenkorperchen in der Gelenkkapsel und im periartikuliiren Gewebe beim menschlichen Fetus. Acta anat. (Basel), 40, 2140. HUMPHREY, T., (1952); The spinal tract of the trigeminal nerve in human embryos between 7.5 and 8.5 weeks of menstrual age and its relation to early fetal behavior. J. comp. Neurol., 97, 143-210. HUMPHREY, T., (1953); The relation of oxygen deprivation to fetal reflex arcs and the development of fetal behavior. J. Psychol., 35, 3-43. HUMPHREY, T., (1954); The trigeminal nerve in relation to early human fetal activity. Res. Publ. Ass. nerv. rnenf. Dis., 33, 127-154. HUMPHREY, T., (1955); Pattern formed a t upper cervical spinal cord levels by sensory fibers of spinal and cranial nerves. Relation of this pattern to associated gray matter. Arch. Neurol. Psychfat. (Chic.), 73, 3-6. HUMPHREY, T., (1960); The development of the pyramidal tracts in human fetuses, correlated with cortical differentiation. Structure andFunction ofthe Cerebral Cortex. D. B. Tower and J. P. SchadB, Editors. Proceedings of the Second International Meeting of Neurobiologists. Amsterdam, Elsevier (p. 94-103). HUMPHREY, T., AND HOOKER, D., (1959); Double simultaneous stimulation of human fetuses and the anatomical patterns underlying the reflexes elicited. J. comp. Neurol., 112, 75-102. HUMPHREY, T., AND HOOKER, D., (1961a); Reflexes elicited by stimulating perineal and adjacent areas of human fetuses. Trans. Amer. neurol. Ass., 86,147-152. HUMPHREY, T., AND HOOKER, D., (1961b); H u m fetal reflexes elicited by genital stimulation. Proceedings of the Vllth International Congress of Neurology, Rome, 2,473476. JACOBS, M. J., (1959); The Development of the Motor Trigeminal Nucleus (and certain closely associated Cell Groups) in Human Fetuses. Thesis, University of Pittsburgh. JIMENEZ GONZALES, L., (1944); Topografia del ‘tractus spinalis nervi trigemini’ en relaci6n con la operaci6n de la tractotomia. Rev. esp. Oto-neuro-oft&., 1, 127-133. KLISHOV, A. A., (1960); Embryohistogenesis of nerve elements of the somatic musculature in man. Arkh. Anaf. Gisfol.Embriol., 39, 7 6 8 7 (Quoted from abstract). LANGWORTHY, 0. R., (1933); Development of behavior patterns and myelinization of the nervous system in the human fetus and infant. Contr. Embryol. Carneg. Instn., 24, 1-57. LIPPMAN, K., (1958); Uber den Babkinschen Reflex. Arch. Kinderheilk., 157,234-238. LUCASKEENE, M. F., AND HEWER,E. E., (1931); Some observations on myelination in the human central nervous system. J. Anat. (Lond.) ,66, 1-1 3. A., (1920); Sur un rtflexe cutant nouveau A rkflexe palmomentonnier. MARINESCO,G., AND RADOWCI, Rev. neurol., 27,231-240. MAVRINSKAYA, L. F., (1960); On correlation of development of skeletal muscle nerve endings with appearance of motor activity in human embryo. Arkh. Anat. Gistol. Embriol., 38,61-68. MCGEER,P. L., (1962); Mind, drugs, and behavior. Amer. Scientist, 50, 322-338. MINKOWSKI, M., (1923); Zur Entwicklungsgexhichte, Lokalisation und Klinik des Fusssohlenreflexes. Schweiz. Arch. Neurol. Psychiat., 13,475-514. MINKOWSKI, M., (1928); Neurobiologische Studien am menschlichen Foetus. Handbuch der Biologischen Arbeitsmefhoden. E. Abderhalden, Editor. Abt. 5, Teil 5B, Heft 5, Lief 253, S. 511-618. MUNN,N. L., (1954); Psychology, the Fundamentalsof Human Adjusfment. Boston and New York, Houghton Mifflin Company. MUNN,N. L., (1955); The Evolution andGrowth of Human Behavior. Boston and New York, Houghton Mifflin Company. OHMORI,D., (1924); Uber die Entwicklung der Innervation der Genitalapparate als peripheren Aufnahmeapparat der genitalen Reflexe. 2. Anat. Entwickl.-Gesch., 70,347410. OLDS,J., (1960a); Approach-avoidance dissociations in rat brain. Amer. J. Physiol., 199, 965-968. OLDS,J., (1960b); Differentiation of reward systems in the brain by self-stimulation technics. Electrical Studies on the UnanesfhetizedBrain. E. R.Ramey and D. S.O’Doherty, Editors. New York, Paul B. Hoeber, Inc. (p. 17-51). OLDS, M. E., AND OLDS,J., (1963); Approach-avoidance analysis of rat diencephalon. J. comp. Neurol., 120,259-295.

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PARMELEE, A. H., JR., (1963a); The hand-mouth reflex of Babkin in premature infants. Pediatrics, 31, 734-740. PARMELEE, A. H., JR., (1963b); The palmomental reflex in premature infants. Develop. Med. Child Neurol., 5, In the press. PATTEN, B. M., (1953); Human Embryology. 2nd ed. New York, Blakiston. I. P., (1932); The reply of a physiologist to psychologists. Psychol. Rev., 39, 91-127. PAVLOV, PEARSON, A. A., (1938); The spinal accessory nerve in human embryos. J. comp. Neurol., 68,243-266. PEARSON, A. A., (1939); The hypoglossal nerve in human embryos. J. comp. Neurol., 71, 21-39. PEARSON, A. A., (1946); The development of the motor nuclei of the facial nerve in man. J. comp. Neurol., 85, 461476. PERBZ,R. M., AND PEREZ,A. P. R., (1932); L'Bvolution des terminaisons nerveuses de la peau humaine. Trav. Lab. Rech. biol. Univ. Madrid, 28, 61-73. A., (1947); Uber besondere Nervenbildungen im Epithel der beim Saugakt des Sauglings bePEEPER, teiligten Mundpartien. Anat. Anz., %, 210-220. PRATT, K. C., (1960); The neonate. Manual of Child Psychology. L. Carmichael, Editor. New York, John Wiley and Sons, Inc. PRECHTL, H. F. R., (1953); uber die Koppelung von Saugen und Greifreflex beim Saugling. Naturwissenschaften, 12, 347-348. PRECHTL, H. F. R., (1958); The directed head turning response and allied movements of the human baby. Behaviour, 13,212-242. SCHEMM, G. W., (1961); The pattern of cortical localization following cranial nerve cross anastomosis. J. Neurosurg., 18, 593-596. G. W., AND KAHN,E. A., (1960); The effect of peripheral nerve anastomosis on the cortical SCHEMM, localization pattern. Trans. Amer. neurol. Ass., 85, 225-226. SHERRINGTON, C. S., (1906); The Integrative Action of the Nervous System. New Haven, Connecticut, Yale University Press. K. V., (1958); The role of irregular maturation of embryonic structures in the formation SHULWKINA, of normal functions in the newborn. Obstet. and Gynecol. (U.S.S.R.), 4, 49-52. SHULEJKINA, K. V., (1959); Comparative characteristics of the development of motor centers in the cervical segments of the spinal cord in man. Arkh. Anat. Gistol. Embriol., 36, 42-54. L., JR..(1957); The innervation of tendons and aponeuroses. Amer. J. Anat., 100,289-317. STILWELL,D. STIRNIMANN, F., (1 940); Psychologie des Neugeborenen Kindes. Zurich, Rascher (Quoted from Pratt, 1960). STREETER, G. L., (1920); Weight, sitting height, head size, foot length, and menstrual age of the human embryo. Contr. Embryol. Carneg. Znstn., 111, 143-170. SUGA,Y., (1951); Histology of the lip and its innervation in human embryos studied by Seto's silver impregnation (Japanese text). Tohoku med. J., 45, 437-446 (From Excerpta med. (Amst.), Sect. I , 1952, No. 2303, p. 565). SZYMONOWICZ, W., (1933); ifber die Entwicklung der Nervenendigungen in der Haut des Menschen. Z . Zelvorsch., 19, 356-382. TAKASHI, M., (1957); On the development of the complex pattern of Pacinian corpuscles distributed in the human retroperitoneum. Anat. Rec., 128, 665-678. WINDLE,W. F., (1944); Genesis of somatic motor function in mammalian embryos: a synthesizing article. Physiol. Zool., 17, 247-260. WINDLE,W. F., (1950); Reflexes of mammalian embryos and fetuses. Genetic Neurology. P. Weiss, Editor. Chicago, Illinois, University of Chicago Press. WINDLE, W. F., AND BECKER, R. F., (1940); Relation of anoxemia to early activity in the fetal nervous system. Arch. Neurol. Psychiat. (Chic.), 43, 90-101. WINDLE,W. F., BECKER, R. F., m m s , R., AND COWGILL, E. J., (1942); Effect of anoxia on trigeminal reflexes in cat fetuses. Physiol. Zool., 15, 375-382. WINDLE, W. F., AND FITZGERALD, J. E., (1937); Development of the spinal reflex mechanism in human embryos. J. comp. Neurol., 67, 493-509. DISCUSSION

LINDSLEY: Do I understand you correctly that it requires some kind of continuing stimulation or repeated stimulation to elicit these reflexes? I think you said at one point that just punctate stimulation was not sufficient.

134

DISCUSSION

HUMPHREY: It takes repeated punctate stimulations to give a response. The stroking movements seem to bring in spatial summation and give a response more readily. When a fetus is first delivered, often there is no activity for a short period. The suggestion has been made repeatedly that as carbon dioxide accumulates it offers a source of stimulation itself. Certainly you often see that activity begins only after a little time has elapsed. LINDSLEY: If you progress from the earlier weeks to the later ones of your period of observation, could you notice any decrease in latency of the responsiveness to stimulation? HUMPHREY: No. There has been no real effort made to do this. The time involved could be determined by the number of frames that you could count on the moving picture record, I think. Probably it could be done. PURPURA: As far as I could see it looked that the more complex response showed a greater refractory period. I did not see any refractoriness in the entire sequence. In the experiment which you demonstrated on the genital stimulation, was the failure to respond the second time due to refractoriness of the whole system? In other words: there were times when on repeated stimulation there was no response at all, or an abortive response. No efforts, I take it, were made to study the relationshp between serial stimulations and the kind of response one obtained? HUMPHREY: No, but we did do something which I did not get to discuss here, that is, we used double stimulations simultaneously so that the face and the palm were stimulated simultaneously, or the palm and sole. The observation showed that, often, till about 14 weeks of menstrual age there was a trend towards the appearance only of the face reflex if face and palm were stimulated together, or of the palm reflex if palm and sole were stimulated at the same time. But after 14 weeks of menstrual age there was a tendency for both reflexes to make their appearance. This suggests, of course, that one was suppressed or inhibited by some mechanism. LINDSLEY: Am I right in stating that the predominant posture was one of flexion? And then when you stimulate the tendency is to produce extension? HUMPHREY: Yes, up to a certain age level particularly, midline stimulation of the face gives extension, and then flexion will take over more, predominantly. In other words, perhaps we have here with these reflexes the forerunners of the avoidance areas in the brain and the positive reinforcing areas, or the negative reinforcing and the self stimulation for pleasure. We have here, at least, the 2 opposing situations, almost from the very beginning of the onset of reflex activity. LEVI-MONTALCINI: What strikes me is the difference with lower forms like birds. In these animals motility precedes the reflex. Here you did not see any motility which preceded the reflex? HUMPHREY: No. There are no spontaneous movements until some time after reflexes may be obtained from stimulation. SCHERRER: I would like to ask the following question. Did you have an opportunity to study thermal stimuli in your fetuses? I am asking this question because as you know it was shown recently that the non-myelinated fibres are not, as was thought some years ago, only pain fibres. These fibres are at the same time tactile and thermal

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fibres. So as i n these fetuses you have a non-myelinated system, I would like to know if a thermal stimulus might already be effective. HUMPHREY: In certain cases we had an opportunity to try, but only to a very limited extent. In one of the fetuses a stream of warm water was run over the chest at the same time that the sole of the foot was stimulated. We noticed no variation from what you might ordinarily see and such attempts were given u p in that case. For the fetuses that I have been studying in connection with genital stimulation, because there was not much help, I did not succeed in maintaining a normal body temperature of the isotonic bath. Instead the temperature was down to 78-82' F, quite a bit below the normal body temperature. Then one of the things that I did notice was that in stimulating the palm of the hand, whereas one ordinarily gets a finger flexion, we saw several extensions instead of flexion movements. This suggested that the lower temperature may have been a factor in producing the reflex seen.

136

Postnatal Changes in Glia/Neuron Index with a Comparison of Methods of Cell Enumeration in the White Rat K E N N E T H R. BRIZZEE, JEARY VOGT

AND

X E N I A KHARETCHKO

Department of Obstetrics and Gynecology, University of Nebraska College of Medicine, Omaha. Nebraska; Department of Electrical Engineering and Graduate College, University of Nebraska, Lincoln, Nebraska; Department of Anatomy, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.)

INTRODUCTION

It has become increasingly evident in recent years that a clear understanding of the chemical and metabolic properties of nervous tissues is dependent to a very great extent on quantitative evaluations of the basic cellular composition of the tissues. In recognition of the importance of relating chemical data to the proper morphological substrates, some workers have given attention to development of exquisitely fine methods for quantitative determinations of such parameters as single cell (neuron) weight and volume together with comparable analytic chemical technics. On the other hand a number of investigators have been interested in differential analyses of various tissues of the nervous system, emphasizing the metabolic properties of cell aggregates rather than individual units. Laboratories most active in investigations at the single cell level are those of Lowry and his associates (1957), HydCn and his co-workers (1953, 1958, 1960, 1962), and Edstrom (1953a,b, 1956a,b, 1957a,b, 1960). Of those workers who have focused attention on various sites or structures in the nervous system the work of Elliott and Heller (1957) and of Korey and Orchen (1959) is especially notable. Both of these latter two groups have been concerned especially with the problem of the relative contribution of the glial, as opposed to the neuronal components of nerve tissues, to the respiratory activity of the tissues as a whole. In relating their chemical findings to structural make-up of the tissue, Elliott and Heller based their estimates on the quantitative histological data of Heller and Elliott (1954). Korey and Orchen (1959) on the other hand computed their values for relative respiratory activity of glia, as compared with neurons, on the basis of the quantitative histological data of Nurnberger and Gordon (1957). It is particularly interesting to note that differences in values obtained by these two groups of workers and their final conclusions were the result of differences in the basic histological data as well as in chemical determinations. This points up the need for a closer look at methods currently employed in quantitative histology and constitutes the basis of the present report.

CHANGES I N GLIA/NEURON INDEX

137

It is our purpose in this study to compare results obtained with three different methods of cell enumeration, with particular emphasis on obtaining values for neuroglial packing density and the neuroglia/neuron index at certain post-natal stages of development. METHODS A N D RESULTS

The three methods of cell enumeration employed in this work are (a) the Chalkley method (1943, 1949) as it relates to cell packing density, (b) the DNAP method of Gray and DeLuca (1956), and (c) the cell maceration method of Nurnberger and Gordon (1957). In the first of these methods the cerebral cortical tissues were fixed in situ by perfusion with isotonic saline followed by Bouin’s fluid, and were then imbedded in paraffin and stained with thionin. Animals were sacrificed at 10, 20, 50, 100, 200, 300, and 730 days of age, with from 4-6 animals at ezch age level. Cell counts were made by focusing a grid reticule through a given depth of tissue in the appropriate sections of cortex and differentially enumerating all cells observed. Corrections were made for cells bisected by all of the edges or borders of the ‘counting chamber’ and the cell population was thus estimated as number of cells/mm3 of tissue. The basic technique of cell enumeration was carried out as described previously (Brizzee and Jacobs, 1959a). Employing this method in tissue from area 2 of the rat cerebral cortex (Krieg, 1946), it was found that the neuron packing density (Fig. 1) dropped from a value of about

0 5

.

pdos1’ , Y P 0 2

.

2

./

_ --- _ -__-.--___--__ Imrogjin

1-

I 1

2 403.,;‘

g

01-’

’

145,000 cells/mm3 in 10-day animals to about 110,000 at 20 and 50 days with a subsequent slow decrease to about 85,000-90,000 at 100 days. This approximate level was maintained even in 2-year-old animals. The neuroglial packing density increased from about 30,000 cells/mm3 in 10-day animals to 50,000 at 50 and 100 days with a subsequent slow increase to about 85,000 cells/mm3 in 2-year-olds. The glia/neuron index in the same period (i.e. 10 days to 2 years) increased from a value of about 0.2 to 0.95. Total brain weight in the rat attained a value of about 0.65 g at 20 days, 0.95 g at References p. 145/146

138

K. R. B R I Z Z E E , J. V O G T AND X. K H A R E T C H K O

50 days and about 1.9 g at 200 days, maintaining this level in 2-year-old animals. It will be noted from these data that the glial density and glia/neuron index in the rat cortex, as was the case in the cat (Brizzee and Jacobs, 1959b), increases well beyond the period at which brain weight becomes relatively stable. From these two studies in both cat and rat it appears that neuroglial packing density in cerebral cortex is not a critical factor, ontogenetically at least, in determining brain weight and does not appear to be greatly influenced by brain weight. In order to establish a direct comparison between values for cell packing densities in fixed-stained tissues and in fresh tissues, the relatively new method of Nurnberger and Gordon (1957) was employed. Animals were grouped according to age at 5, 10, 20, 50, and 100 days. With this technique, freshly dissected cerebral cortical tissues (400 mg) were placed in 10-20 ml of a ‘macerating’ solution of KCl-CaC12 buffered to a pH of 6.5. The preparation was placed in a 125-ml pyrex bottle together with a standard number (20 g) of 5-mm glass beads. The bottle with its contents was placed in heavy duty agitator in a cold room and agitated at the rate of 125 cycles/min for periods up to two hours. Aliquots of the cell suspension thus produced were drawn into white cell pipettes and diluted by a factor of 10 with a solution of 0.1 % methylene blue in the standard KCI-CaC12 solution. After drawing the sample and diluting with methylene blue solution each white cell pipette was placed on a mechanical pipette shaker for 1 min. This constituted the period of staining of the nuclei in the suspension. After this staining period a blood cell counting chamber was filled with the cell suspension and a total of 10 squares in 2 chambers was counted, this totalling one cubic ml of suspension. In most of the determinations aliquots were drawn every min for the first 5 min, every 2 min for the next 10 min and every 15 min thereafter for periods up to about 2 h in some instances. The number of neuronal and neuroglial nuclei were counted for each aliquot, employing the 40 x (high dry) objective of a binocular microscope equipped with 12.5 x compensating oculars. The number of neurons and neuroglia at each time interval were plotted separately and the data were subjected to mathematical treatment to determine the cell packing density of the fresh tissue sample. This derivation, which has been worked out by one of the authors (J.V.) is based on the method of Nurnberger and Gordon (1957). It will result in an equation relating the total number of cells in a neural tissue sample to the number of observed stained nuclei and the time of each observation. It is assumed that two successive reactions occur in the breakdown of a given cell by action of the glass beads: ( I ) The cell membrane is broken releasing the intact nucleus into the solution. (2) The nucleus is destroyed by the action of the glass beads. The steps are, diagrammatically : intact neural cell

& Iiberated nucleus 2 destroyed nucleus.

It is also assumed that the rate of the above reactions is proportional to the concentration of the reactant. At any instant during the counting procedure : n=A+B+C

(1)

CHANGES I N GLIA/NEURON INDEX

139

where n = initial number of neural cells in the counting sample A = number of intact neural cells in the counting sample B = number of stained nuclei observed in counting sampIe C = number of nuclei destroyed by action of beads. From the assumption that the rate of reaction is proportional to the concentration of reactant: -A' _ -- KIA dt

A In - = - K1t n -Kit A = ne

(3)

similarly dc dt

-- KzB

(4)

at any instant the rate of change of stained nucleus concentration is the sum of the formation rate and the destruction rate, thus : dB dt

-dA dc dt dt = KIA-KzB -

= Kine-K1t- KzB

dB dt

+ KzB

=

K1rzeCKit

equation (6) is a first order linear differential equation of the form ~

dY f M(x)y dx

=

N(x)

where y=B x=t M(x)= Kz

N ( x ) = Kine-K1t

The general solution for equation (7) is: y = e -sMdxlNeJMdxdx

+

Ke-JMdx

so B=e

-Kit SKzdt

+ +

-$Kzdt

SKzdtJK1ne e dt Ke - Kzt 1 (Kz- Kx)t Ke- Kzt = Klne e KZ- Ki

References p. i45Ii46

-

140

K. R. BRIZ Z E E , J. V O G T A N D X. K H A R E T C H K O

K is evaluated by the boundary conditions, i.e., at t = 0, B = 0

O=

-KiO -&O Ki ne +Ke Kz - Ki -Ki K= n K2 - Ki

thus B=

Ki KZ- Ki

n=

ne

-Kit

-

KI K2 - K1

ne

-Kzt

K I - KZ -K

e

t

2-e

-Klt

the experimental values for n included in this presentation were found by evaluation of K1 and K2 for each set of experimental data. A plot of B-count vs. time is necessary B

A

Fig. 2. Plot of experimental data with tangent line drawn.

for the method to be described (Fig. 2). It will be noted that for times greater than the time at maximum B-count, tm, equation (5) reduces to* : dB dt

- = -KzB

In terms of a tangent line drawn to the data curve: K z z - - 1 B:!-B1 Bt tz - tl where Bt = point of tangency

B1,tl and 8 2 , tz are points on the tangent line. An additional equation must be derived to find K1 and K2. At the point of maximum B-count, Bm, the slope of the B curve is zero;

* For K I > KZ and t > tmthe contribution of the term KIA = Klne-Klt is negligible. Experimental resuits have shown KI to be greater than KZ in each case.

141

C H A N G E S I N G L I A ~ N E U R O NI N D E X

mathematically ~

dB = 0 at B = Bm, t dt

=

tm

from equation (9)

dB dt

-=-

d dt

Ki [Kl-Kz Kle

n(e

-&tm -

-Kzt

Kze

-e

-K2tm

-Kit

)]

=

o

=o

K1 (K1-Kz)frn _ _ -- e K2 (K1 - Kz)tm = In K i - In Kz 1 1 Kl=-lnKl +Kz--InKz tm trn Equation (12) may be solved graphically or by trial and error for K1*. Equation (10) may now be solved using K1, Kz, and any desired B, t vaIue including the point Bnl, tm. The initial rate of release of nuclei with their ultimate destruction is illustrated for both neurons and neuroglia in the cerebral cortex of a 100-day animal in Fig. 3. In

-

,1200

-

"0

r

k 100

sl

s

z

10

20

30

40

50

60

70

ao

90

100

Time (min)

Fig. 3. Curves illustrating rate of liberation and destruction of free nuclei in cerebral cortex of 100-day-old rats. 0 Neuron + neuroglia nuclei, x Neuroglia nuclei, 0 Neuron nuclei.

this particular case aliquots were drawn only at 1, 3, 5 , and 15 min and every 15 min thereafter to 90 min, with one additional determination at 100 min. In Fig. 4 the same phenomena are illustrated for a 5-day animal showing the more rapid release and destruction in the younger tissue. In this case the samples of cortex were pooled from 5 animals to obtain 400 mg of tissue. Aliquots were drawn at more frequent intervals in this case than in counts in tissues from 100-day animals. Applying the mathematical treatment outlined by Gordon and modified as

*

Trial and error methods have been used by the authors due to relative ease of estimating Ki.

References p . 1451146

142

K. R. BRIZZEE, J. VOGT A N D X. K H A R E T C H K O 400r

Fig. 4. Curves illustrating rate of liberation and destruction of free nuclei in cerebral cortex of 5-day-old rats. 0 Neuron f neuroglia nuclei, X Neuroglia nuclei, 0 Neuron nuclei.

noted above to our data, it was found that neuron packing density decreased from a value of about 60,000cells/mm3 at 5 days to 20,000 cells/mm3 at 100 days (Fig. 5). In Fig. 24 of the original publication of Nurnberger and Gordon (1957) the value for

"'"C 11.0

\

Total (neural)

g 6.0 4.0

2.0 1.0 I

I

10

20

I

I

I

I

I

I

I

I

30 40 50 60 70 80 90 100 Tirne(doys)

Fig. 5. Mean values of neuron, neuroglia and total neural cell packing density in fresh tissues of rat cerebral cortex.

neuron density in the dorsal and medial part of the cortex is 21,700 cells/mm3. Values for neuroglial density in our studies on the other hand fluctuated between about 58,000 and 63,000 cells/mm3, maintaining an average level of a little over 55,000/mm3 between 5 days and 100 days. The total neural cell (neuroglia plus neurons) count thus is about 75,000 cells/mm3fresh tissue in 100-day animals.

CHANGES IN GLIA/NEURON INDEX

143

The glia/neuron index in this period, mainly as a result of the decreasing neuronal density, increased from a value of 1.0 at 5 days to a value of about 3.5 at 100 days (Fig. 6). It will be noted that values for neuron packing density are markedly lower

10

20

30 40 50 60 7 0 00 90 100 Time (days)

Fig. 6. Mean values of glia/neuron index in fresh tissues of rat cerebral cortex.

in fresh tissue determinations than in the fixed-tissue method even if a correction is made for a volume shrinkage of 52% in fixed tissues. On the other hand the value for neuroglia packing density in 100-day animals in fresh tissues is higher than in fixed tissues, especially when the shrinkage correction is applied to the latter. This obviously results in a much higher glia/neuron index. The direction or trend of the curves, however, is very similar with the two methods. A third method of cell enumeration (Gray and DeLuca, 1956) involves the estimation of DNAP with calculation of number of cells in the tissue mass from the quantitative DNAP determination. In this study a comparison was made in 100-dayold rats between total cell counts made directly on thionin stained paraffin sections as described previously (Brizzee and Jacobs, 1959a) and estimates of total cell density computed from quantitative determination of DNAP - employing the SchmidtThannhauser method (1945) - according to the procedure and formula employed by Gray and DeLuca (1956). The data from this study are summarized in Table I. In fixed-stained tissues of the somato-sensory area a mean total cell packing density of about 230,000 cells/mm3 was obtained. It will be recalled that neuron density alone in the previous study on fixed-thionin stained tissues from 100-day rats was about 90,00O/mrn3 with a neuroglial density of 50,000/mm3. The amount of tissue shrinkage during the dehydration and paraflin embedding procedure was determined to be 52 %. The shrinkage determinations were carried out by the weight-specific gravity method (Swinyard, 1939) combined with planimeter determinations of the volume of tissues fixed in Bouin’s fluid and stained with thionin as noted above. Applying a correction for this amount of shrinkage and taking into account the specific gravity of the fresh cortex, the corrected mean total cell density per mg fresh tissue was found to be 106,000 cells/mm3. References p . 1451146

144

K. R. B R I Z Z E E , J. V O G T A N D X. K H A R E T C H K O

TABLE 1 C O M P A R I S O N O F CELL PACKING DENSITY DETERMINED B Y D N A P METHOD DELUCA,1956) A N D C H A L K L EMYE T H O D (1943, 1949)

(GRAYA N D

Coniparison of chemical data and total cell counts

No. of animals used for DNAP determination No. of animals used for total cell counts Age (days) No. of sections used for counts (per animal) No. of cylinders per section Volume of the cylinder &3)

*

9 5 100 10 22 26,590

Mean total cell density (per mm3 of section) Shrinkage of tissue during preparation Specific gravity of fresh tissue Calculatedmean total cell density per mg fresh tissue pg DNAP/mg fresh tissue Total cell density per mg fresh tissue calculated from formula of Gray and DeLuca*

23 1,298 52 %

1.016

106,140 0.0728 112,000

pg DNAP/g wet weight tissue 65 . 10-8 pg DNAP/nucleus (cell)

Determining the DNAP/mg of fresh cortical tissue from the somato-sensory area and applying the formula of Gray and DeLuca in which the amount of DNAP was determined by the Schmidt-Thannhauser method, we obtained a value of 112,000 cells/mm3 fresh tissue. With the Nurnberger and Gordon method, it will be recalled, our findings for total neural cell density (including both neurons and neuroglia but no other cellular elements) were about 75,000 cells/mm3 wet weight of tissue in 100-day-old rats. Nurnberger and Gordon themselves obtained a value of 119,000cells/mm3for total number of cells including endothelial elements in an area of rat cerebral cortex closely adjacent to the area studied. The difference between these values is of the same order as the difference between the values for total cell counts (230,000) and total neural cell counts (140,OOO) in the fixed-stained tissues as described above. DISCUSSION

From these findings, it appears that all three of the methods compared in this study are in fairly good agreement insofar as total cell packing density is concerned and that the proportion of non-neural cells in rat cerebral cortex is between 30 and 35 % in young adult animals, as determined in either fresh and fixed tissue methods. Considering the problem of differential counts of neurons and neuroglia in fixedstained tissues as compared with fresh tissues, however, it is at once apparent that values obtained with the two methods are quite different. Values for neuron density are somewhat higher in fixed preparations than those obtained with fresh tissues with the Nurnberger and Gordon method, while glial densities are higher in the latter. The question as to why one obtains a different proportion of neurons and glia in the two principal methods used in this work, however, remains a very difficult one to

CHANGES I N GLIA/NEURON INDEX

145

answer. There is, of course, no apparent reason why the proportions of any given cell types should change markedly with different methods of tissue fixation or any degree of tissue shrinkage. The question then centers about a consideration of criteria for identifying neuroglia vs. neurons. It is the opinion of the authors from experience with both methods, that the criteria for identifying and distinguishing these elements are much better in fixed than in fresh tissue since one has the characteristic structural features of the neuronal cell body as well as nuclear morphology to assist in identification of neurons, even in cases in which nucleolar form or configuration may not be typical or ideal. Nurnberger and Gordon state :‘It is possible that the nuclei of relatively small neurons e.g. granular cells, might have been misidentified as glial elements in these counts, so that the neuronal counts reported are, if anything, on the low side’. In view of this difficulty in accurately distinguishing between small granule-cell neurons and larger neuroglia we are of the opinion that the proportional counts of neurons and neuroglia obtained from fixed-stained tissues in this investigation are much more accurate than those from the fresh tissue technique. While this remains a serious drawback to the fresh tissue method, it should be noted that the method offers the great advantage of permitting an accurate enumeration of total cell content in whole brain or any region of the brain and has the added advantage of being very much simpler and requiring much less time than the older methcds. A C K N 0 W LE D G E ME N TS

This investigation was supported in part by a PHS research grant (NB-03608-02) from the Institute of Neurological Diseases and Blindness and PHS Traineeship 5T 1 GM631. SUMMARY

The glia/neuron index in rat cerebral cortex increased throughout the periods studied as determined in both fresh and fixed-stained tissues. The index in fixed tissues increased beyond the period at which brain weight became stable (200 days). Total cell counts were in good agreement in all 3 methods employed. Relating our findings to those of Nurnberger and Gordon (1957), the proportion of non-neural cells appeared to be about equal as determined in both fresh and fixed-stained tissues. Differential counts of neuroglia and neurons in our studies, however, revealed a higher glia/neuron index in fresh tissue than in fixed-stained preparations. Since the differential counts of neuroglia and neurons made in fixed-stained tissues are based on more definite morphological criteria than in fresh tissue, the fixed tissue differential counts are considered more accurate. For total cell counts whether in whole brain or specific sites the fresh-tissue method offers the advantages of speed, accuracy, and simplicity. REFERENCES

BRIZZEE, K. R., AND JACOBS, L. A., (1959a); Early postnatal changes in neuron packing density and volumetric relationships in the cerebral cortex of the white rat. Growth, 23, 337-347.

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BRIZZEE,K. R., AND JACOBS,L. A., (1959b); The glia-neuron index in the submolecular layers of the motor cortex in the cat. Anat. Rec., 134,97-106. H. W., (1943); Method for quantitative morphologic analysis of tissues. J. nut. Cancer CHALKLEY, Inst., 4, 47. CHALKLEY, H. W., (1949); Method for the measurement of average nuclear volumes independent of shape. Anat. Rec., 103, 433. EDSTROM, J. E., (1953a); Quantitative determination of ribonucleic acid from individual nerve cells. Biochim. biophys. Acta (Amst.), 11, 3W301. EDSTROM,J. E., (1953b); Ribonucleic acid mass and concentration in individual nerve cells. A new method for quantitative determination. Biochim. biophys. Acta (Amdt.), 12, 361-386. J. E., (1956a); The content and the concentration of ribonucleic acid in motor anterior EDSTROM. horn cells from the rabbit. J. Neurochem., 1, 159-165. J. E., (195613); Separation and determination of purines and pyrimidine nucleotides in EDSTROM, picogram amounts. Biochim. biophys. Acta (Amst.), 22, 378-388. J. E., (1957a); Necessary conditions for the separation of compounds at quantitative cell EDSTROM, analyses. Exp. Cell Res. (Cytochemical methods with quantitative aims), Suppl. 4, 264-268. EDSTROM, J. E., (1957b); Effects of increased motor activity on the dimensions and the staining properties of the neuron soma. J. comp. Neurol., 107, 295-304. J. E., (1960); Extraction, hydrolysis, and electrophoretic analysis of ribonucleic acid from EDSTROM, microscopic tissue units (microphoresis). J. biophys. biochem. Cytol., 8, 3946. ELLIOTT,I<. A. C., AND HELLER,1. V., (1957); Metabolism of neurons and glia. Metabolism of the Nervous System. New York, Pergamon Press (Roc. Second Int. Neurochem. Symp., Aarhuus, 1956), p. 286-290. GRAY,D. E., AND DELUCA,H. A., (1956); Use of desoxyribonucleic acid as a reference standard in metabolic experiments. Amer. J. Physiol., 184, 301-303. I. V., AND ELLIOTT,K. A. C., (1954); Desoxyribonucleic acid content and cell density in HELLER, brain and human brain tumors. Canud. J. Biochem.., 32., 584-592. HYDEN,H., (1953); Determination of mass of nerve-cell components. J. Embryol. exp. Morph., 13, 315-3 17. HYDEN,H., AND LANGE,P. W., (1962); A kinetic study of the neuron-glia relationship. J. Cell Biol., 13, 233-237. HYDEN,H., L~VTRUP, S., AND PIGON, A., (1958); Cytochrome oxidase and succinoxidase activities in spinal ganglion cells and glial capsule cells. J. Neurochem., 2, 304-311. HYDEN,H., AND PIGON, A., (1960); A cytophysiological study of the functional relationship between oligodendroglial cells and nerve cells of Deiter’s nucleus. J. Neurochem., 6, 57-72. KOREY, S. R., AND ORCHEN, M., (1959); Relative respiration of neuronal and glialcells. J. Neurochem., 3,277-285. KRIEG, W. J. S., (1946); Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas. J. comp. Neurol., 84, 221-275. LOWRY,0. H., (1957); Quantitative analysis of single nerve cell bodies. Progress in Neurobiology. ZZ. Ultrastructure and Cellular Chemistry of Neural Tissue, H. Waelsch, Editor. New York, Hoeber (p. 69-82). NURNBERGER, J. I., AND GORDON,M. W., (1957); The cell density of neural tissues: direct counting method and possible applications as a biologic referent. Progress in Neurobiology. 11. Ultrastructure and Cellular Chemistry of Neural Tissue. H. Waelsch, Editor. New York, Hoeber (p. 1W138). SCHMIDT, G., AND THANNHAUSER, S., (1945); A method for the determination of desoxyribonucleic acid and phosphoproteins in animal tissue. J. biol. Chem., 161, 83. SWINYARD, C. A., (1939); Methods for volumetric determination of fresh endocrine glands. Anat. Rec., 74, 71-78.

C H A N G E S I N C L I A ~ N E U R O NI N D E X

147

DISCUSSION

WAELSCH: The troubles for the biochemist only start with the evaluation of the cell counts. As you know people have measured respiration and tried to calculate from there the relative contribution of neurons and glia. Heller and Elliott as well as Korey took as respiration of glia, glioma’s or the corpus callosum. This is under the supposition that all glia have the same type of respiration whether they are in the glioma or in the corpus callosum, and that one can equate from there back to the respiration of the glia in the cortex. I think this is probably the major error made in estimating the relative respiratory contribution of glia in the cortex. The other problem which is still more serious is of course the metabolism of the glia itself. We were used to the thinking that the enzymatic make-up of the glia is an indication of a low oxidative metabolism, and this goes back to the data of Marinesco I think, which he published in 1916. He found a very low indophenol-oxidase activity. Recently, due to the work of HydCn and his group, it turns out that the glia - and I think it was the glia in the Deiters cells - have a very high cytochrome-oxidase content and a high succinic-oxidase content. If you take the in vitro measurements of the concentration of the enzyme as a parameter of what happens in vivo these glia cells should have a very high oxidative metabolism. I have to point out, however, that this is a special species of glia, the glia of the Deiters cells, and of course we always translate in vitro measurements under optimal conditions for the measurements of the enzyme to in vivo calculations. But, as I said in the beginning, I think the problems of the biochemist are more serious than those of the histologist. Whether the count of Nurnberger or Korey varies by 20 % or so, the error the biochemist makes is much greater. BRIZZEE:In considerations such as this one parameter has been ignored or at least given far too little attention. This is the importance of cell surface, more specifically the dendritic surface area in the case of neurons, in establishing the metabolic characteristics of the cell. WAELSCH: If I may correct you, Oliver Lowry once stated very clearly that the metabolism of the cortex is the metabolism of the dendrites. S C H A DBaxter ~: and I published in 1960 in Experimental Neurology a method for the calculation of the surface area of dendrites and cell body. The surface area of the basal dendrites of the pyramids in the motor cortex of the rabbit increased in a 25-day period (5 to 30 days after birth) more than ten-fold. The total surface area of the cell bodies per unit volume of cerebral cortex decreased from 0 to 5 days after birth to remain almost constant till the adult stage. BRIZZEE: And have you related this to metabolic and chemical data? S C H A D In ~ : the initial series of experiments we related the morphological data to the levels of glutamic acid decarboxylase and a number of amino acids, such as GABA, glutamic acid, aspartic acid, etc. The development of GAD activity coincided most closely with the development of the surface area of the dendrites. L ~ V T R U: PConsidering the surface area I may mention that Dr. Eastern has found that the amount of RNA is more related to the surface area than to the volume of the neuron.

148

DISCUSSION

On the whole I think it is almost impossible to know for a neurochemist which morphological reference he should use. One thing that has been stressed by Hydtn and his coworkers is the fact that it is important to compare the levels of different enzymes. It is more important to find the relation between two enzymes in different types of cells (neurons and glia) than to correlate the amount of enzyme with the volume, dry weight or something else. WAELSCH: You are absolutely right in saying that we really have no decent basis to compare this. Nor the DNA, nor the volume, nor the protein content or whatever you take, makes anyone happy. Surface area is probably the most reasonable basis for comparison, but how to do this in a reliable way is another question. So we are left with very unhappy choices of a reference for biochemical parameters. In this connection I would like to ask Dr. Brizzee: is the relation between neurons a n d g l i a 3 : 1 or 1 : l ? BRIZZEE: Actually, if you accept the findings in fresh-tissue method, it is about 3 : 1; that is what we found. But if you accept the findings with the fixed tissue in the rat cortex it is about 1 : 1. LBVTRUP: What consequences does this finding have with respect to DNA determinat:ons? Half of the total number of cells would disappear if the ratio goes down f r o m 3 : 1 to 1 : 1. BRIZZEE: I think it is merely a matter of differential identification of cellular elements. The agreement in total cell counts in the three different methods appears to be fairly good. LBVTRUP:So you think that the total number is the same with all methods, but the difficulty in establishing the ratio neurons-glia lies in the identification. SCHADB:I believe that with the Niirnberger method 3 is very hard to distinguish between smaller neurons and glia cells. I would like to ask in this connection: was it possible to do a differential glia count, I mean to distinguish between astroglia, oligo’s and microglia? BRIZZEE:We are contemplating this but have not done it as yet. LBVTRUP: What is the exact origin of the increase of the glial cells? BRIZZEE:This is an extremely difficult question. They simply are there, we count them. It was interesting recently to note that Brownson at Virginia Medical College found that perineural satellite cells in cerebral cortex increased in numbers with normal aging. This is essentially what we have found: In our counts the greatest number of cells among the glia, certainly was in the satellite position. However, we have not yet studied the process of cell division in these elements. LIZIVTRUP: It is interesting that you found an increase in the number of glial cells during the latest stages so to speak, because I did - that is now many years ago some DNA determinations in brains of growing rats. I went up to 4 months and there was never any significant increase in DNA from the weight of 50 g up to 4 months later from that stage. You could not find any significant DNA increase? BRIZZEE : There is a n increase in glia cells, although it is rather a small one in earlier stages such as those which you studied. The greater increase appears in the later stages of aging.

C H A N G E S I N G L I A ~ N E U R O NI N D E X

149

L0VTRUP: Is it possible that some other cells are transformed? But what kind of cells should that be? BRIZZEE: It is interesting to note that in 1936 a German worker, Muhlmann, postulated a reversal of the glia/neuron index from neural preponderance to glial preponderance with age but with no over-all change in total cell packing density. This would seem to suggest a transformation from one cell type to another. In our own studies, however, we found that the neuron packing density remained constant in the older animals while the glial density increased.

150

Quantitative Analysis of Neuronal Parameters in the Maturing Cerebral Cortex J. P. SCHADE, H. V A N BACKER

AND

E. COLON

Cenfral Institute for Brain Research, Amsterdam

At the present time it is still impossible to formulate a completely satisfactory theory of neuronal organization in structures such as the cortical and cerebellar gray substance, for there is as yet no adequate method for an exhaustive analysis of such systems. Dr. Sholl, of the University College in London, was one of the first who, between 1950 and 1960, systematically applied the idea that it is only possible to think of neuronal organization in statistical terms. His untimely death in 1960 was a great loss to the small group of neurobiologists and neurologists who were working on the formulation of a statistical and mathematical language for the description of the cerebral cortex. He was not only interested in a quantitative approach but he could also painstakingly trace all the fine ramifications of an axon to show the widespread distribution of the endings (Fig. I). In September 1959 he gave his last major lecture during a conference on the structure and function of the cerebral cortex and we had

Fig. 1. A figure taken from Sholl’s publication in 1960: ‘An example of the more extensive terminations found in some efferent axons to the pericruciate cortex of the cat’.

N E U R O N A L P A R A M E T E R S OF T H E C O R T E X

151

a final opportunity to discuss a number of problems of mutual interest. This paper is dedicated to his memory and will follow up a few lines of approach to the quantitative analysis of the cerebral cortex originally set out in one of his earlier papers on this subject. The analysis of measurable parameters of the cerebral cortex seems only fruitful if it clarifies functional and histological relationships that were previously unknown. Sholl (1956) has listed three categories or sets of variables to which the quantitative histologist should direct his attention: (a) the total number of neurons present in the cortex, (b) the afferent connective surface of these neurons and (c) the patterns of connectivity. The advance in the employment of quantitative histological methods during the last five years is demonstrated by a discussion of these factors in the following three sections of this paper. Section I deals with the quantitative parameters of nerve cell bodies and the packing density of neurons; section I1 is concerned with the surface area of cell bodies and dendrites and section I11 regards the patterns of maturation and organization of the dendrites. In the last section specific attention will be drawn to the applicability of the measurements of dendritic organization to mathematical models of nervous function. The addition of ontogenetic (the analysis of the postnatal development of the packing density of neurons and the dendritic organization in various mammals) and experimental results (the influence of antimetabolites on the quantifiable parameters of the cerebral cortex) has provided significant complementary data for the understanding of the structural and functional organization of the cerebral cortex. Our approach has also a multidisciplinary character, since the correlation of the anatomical, physiological and chemical changes with the development of behavioural patterns will indicate the degree of development and maturation required for various reflexogenic and higher functions in the cerebral cortex. A profound understanding of the normal maturation patterns of the constituents of the mammalian cerebral cortex is essential if we are to study in experimental animals the neurological and psychiatric disorders that are found in the human brain. I . Q U A N T I T A T I V E P A R A M E T E R S OF N E R V E C E L L B O D I E S

Three characteristic features of the cell bodies have been determined: (a) the size (the mean volume of the perikaryon), (b) the packing density, and (c) the gray cell coefficient. This last factor indicates the relation between the proportional volume of nerve cell bodies and the proportional volume of other structures in the cortex such as : dendrites and axons, glia cells, blood vessels and intercellular substance proper. This group of structures can also be defined as comprising the extraperikaryal space. Comparative and neuropathological studies in recent years (Haug, 1958, 1959) have stressed the importance of this coefficient in analyzing differences between various animal species and between normal and pathological brains. (a) The size of cell bodies. The dimensions of the cell bodies were measured in sections stained with toluidine-blue or gallocyanin (SchadC, 1959a,b; SchadC and Van References p . 1741175

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J. P. SCHADI?. H. V A N B A C K E R A N D E. C O L O N

Groenigen, 1961). The determinations were made throughout the thickness of the cortex in 300 p wide cortical strips. Two dimensions were taken of each cell body: (a) the maximum length, and (b) the maximum width. The dimension perpendicular to the plane of section was considered to be equal to the width of the cell body. The volume was calculated by treating the cell body as a spheroid, using the equation 1/6 nab2. The gray cell coefficient was determined with the ‘hit’ method (see for a description the next section of this report). The results of the observations on the cell bodies in layers 111, IV and V of the middle frontal gyrus during the postnatal development of the cerebral cortex are summarized in Table 1 and Fig. 2. In the third layer of the cortex mean perikaryal volumes were found to increase from a value of 240 p3 at birth to 1040 p3 in the adult stage. A fast increase of the cell body volume occurs until about 2 months after birth. A decrease in the rate of cell body growth was observed hereafter. Only a small difference was found between 2-year-old and adult preparations. The graph reflecting mean values for the cell body volume of pyramidal cells in layer V was very similar (Fig. 2). Starting with a value of 460 p3 at birth a rapid

4.0

Log volue body weght lg)

II o

3

6

9

ism24

Age (months)

4.5

Adult

Fig. 2. Graph of the cell body volume of pyramidal cells in the middle frontal gyrus. In the graph is plotted on the ordinate the log value of the cell bodies in p3. On the upper abscissa is plotted the log value of the body weight and on the lower abscissa an alogarithmical time scale. The small vertical line through the points indicates the standard error of the determinations.

increase was notea until about 1 month with a slower increase hereatter. 1he highest average value, 2505 p3, for the perikarya of adult pyramidal cells was found in this area. The graph illustrating the development of the cell body volumes in layer IV shows a slightly different aspect. A steady increase from 43 p3 at birth to 115 ,$ in adult was found but there was no indication of a differentiation in a rapid and a slow growth phase. In area 2/3 of the cerebral cortex in rabbit the growth of the cell bodies of the pyramidal cells shows a similar trend (Figs. 3 and 4). In 2-day-old animals a mean

I53

N E U R O N A L P A R A M E T E R S OF THE CORTEX

Fig. 3. Photomicrograph of Nissl-stained preparations to show the decrease in cell density during postnatal development in the rabbit. Cortical layers 2/3. A: 2-day-old animals. B: 10-day-old animals. C : 20-day-old animals. D: Adult rabbit. The calibration line indicates 25 p.

value of the cell body volume of 272 p3 was found. The speed of growth is rather fast until about 10 days after birth. At this age a nerve cell body volume of 715 p3 was found. Afterwards a decrease in the rate of cell body growth was observed, at 21 days

I /

20

23

26

29

32

25

Fig. 4. Graph of the development of the cell body volume of neurons in area 2 of the rabbit cortex. In the graph is plotted on the ordinate the log value of the mean volume of the cell bodies in p3. On the lower abscissa is plotted the log value of the body weight and on the upper abscissa an alogarithmical time scale. The small vertical line through the points indicates the standard error of the determinations.

an average value of 920 p3 was reached. The value found in 30-day-old animals (1 156 p3) did not differ statistically from that found in adult preparations (1 193 p3). Two distinct periods with a different rate of growth can be recognized. During the References p . 1741175

154

J. P. S C H A D E ,

H. V A N B A C K E R A N D E. C OLON

first period a mean increase of 54 p3 per day was calculated, during the second period (12-30 days after birth) a mean increase of 22 p3 was observed. (b) Packing density of pyramidal cells. This parameter was determined at various ages in Nissl stained preparations. The cell bodies were counted in a column of cerebral cortex as long as the thickness of the cerebral cortex, as deep as the thickness of the preparations and 300 p wide. A special counting technique was employed to avoid various errors inherent in a number of classical methods for counting packing densities of cells (SchadC, 1959a,b; SchadC and Van Groenigen, 1961).Already at birth in the human cortex the definite features of packing density of neurons as revealed by Nissl staining is established in a normal infant. Layer IV is characterized by the highest packing density and layer V by the lowest (Table I). The density of neurons TABLE I NEURODENSITY

(ND)

A N D G R A Y C E L L COEFFICIENT* ( G Y R U S FRONT. MED.)

( G C C ) OF

PYRAMIDAL CELLS

~

Newborn Layer 111 ND ( x 103/mm3) GCC Layer IV ND GCC Layer V ND GCC

* **

6 Months

24 Months

Adult

30.5 f 3.1 53

20.1 f 1.7 55

12.5 f0.5 77

444.1 f 24.2 55

151.0 f 9.7 95

59.8 f 3.9 180

35.0 f 2.7 250

60.5 f 5.7 36

16.1 f0.9 37

8.9 f 0.9 52

6.0 & 0.7 66

99.0 Z!C 6.1** 41

volume of griseum only calculated for pyramidal cells, not for all volume of nerve cells contained in it’ neurons in the various layers. Mean & standard error.

GCC

=

considerably lessens between 0 and 6 months of age. A value of 12.5 x lo3 cell bodies per mm3 in layer I11 was found in adult preparations, and of 35 and 6 x lo3 respectively in layers IV and v. In the cortex of the rabbit the number of nerve cell bodies per mm3 decreased from 550 to 99 x lo3 in the period from 0 to 10 days after birth. In adult preparations this value is 58 x lo3 per mm3. As could be expected from its place on the phylogenetic scale, the cerebral cortex of the rabbit shows a much higher packing density of neurons. As will be shown in the last section of this report, however, the dendritic field of the neurons is more elaborate in human beings than in rabbits. Although the time scale and the specific rate of growth are obviously different, the graphs illustrating the postnatal development of the cell bodies in rabbit and man show similar trends. Our histological data suggest that the rabbit cortex reflects an accelerated ‘telescopic’ picture of the human cerebral cortex. The gray cell coefficient (Table I) shows an analogous trend during the postnatal

N E U R O N A L P A R A M E T E R S O F T H E CORTEX

155

development of the human brain. This value, which is lowest in layer V, shows an almost twofold increase from birth to adulthood in layers V and 111, but a nearly fivefold increase in layer IV. On the abscissa of the graphs (Figs. 2 and 4) are plotted not only a time scale but also the logarithmic values of the body weight. The relation between body weight on the one hand and cell body volume on the other hand, during development, can be expressed as the power function y = axk. In this equation y is the dimension of a n organ or cellular constituent and x the body weight. The factor a has no biological significance. The factor k is the ratio of specific growth rates; it remains the same for certain phases in the development but changes at definite points. Such a change may take various lengths of time and is known as an interphase or critical development period. It seems unrealistic to define a critical development period for the whole cerebral cortex on the basis of the cell body volume, since an immeasurable number of histological, physiological, chemical and behavioural parameters change during the postnatal development. However, it is a suitable landmark to indicate similarities and dissimilarities in the development and maturation of different species. The differential growth equations are linear when plotted on log paper and the interphases are then shown as breaks in the graph (cf. Ariens Kappers, 1936, for the relationship between body weight and brain weight in man). The postnatal maturation of the nerve cell body volume in the rabbit shows three periods of growth separated by two interphases. The first interphase occurs when the mean body weight is 175 g, which is approximately between 10 and 12 days after birth. This interphase coincides with the period in which the electroencephalogram becomes mature (SchadC, 1959a,b). The second interphase occurs when the body weight is 425 g (about 30 days after birth). The k of the first phase is 1.21, that of the second 0.45. The k factors were determined with the method of the least squares. The equation was also found to apply to the relation between body weight and cell body volume in the human cerebral cortex. Fig. 2 shows that during various growth periods straight lines can be drawn through the points of observation. The maturation of the nerve cell body volumes in layers I11 and V (middle frontal gyrus) shows from birth to 24 months of age two periods of growth separated by an interphase. The interphase for the pyramidal cells occurs at about 1 month, and for the pyramids in layer I11 at about 2 months. Not enough data were available to determine a second interphase, which probably lies somewhere between 24 months and adulthood. For this reason the values of these age groups are connected with a broken line. 11. S U R F A C E A R E A O F CELL B O D I E S A N D D E N D R I T E S

In view of the heterogeneity of the cellular constituents of the cerebral cortex it seems hardly possible to determine the surface area of neurons in a direct way. Therefore an indirect method was developed measuring the proportional volume of the constituents of cortical neurons and their diameter. From these data the surface area can be computed. To measure the dimensions of nerve cells and dendrites histological preparations References p..I74\1?5

156

J. P.

SCHADB,

H. V A N B A C K E R A N D E. C O L O N

had to be made in which the fluid distribution between neuronal and extraneuronal compartments of the cerebral cortex was represented as faithfully as possible. A freeze-substitution technique was employed similar to that used for the determination of the value of cell bodies. For the study of cell bodies a gallocyanin stain was used and for dendrites method B of Ram6n y Cajal(l936). The proportional volume of nerve cell bodies (total volume per mm3 of cerebral cortex) and of apical and basal dendrites was determined according to a modification of the method of Chalkley (1943, 1949) with the corrections of Haug (1953, 1956). According to this method the relative volume occupied by a specific constituent of a tissue is proportional to the number of times a point, placed at random throughout the tissue, will hit the constituent of the tissue. This measurement was accomplished by cementing four tungsten wires with points of 1 to 2 ,u to the diaphragm of the ocular of the microscope (Schadt and Weiler, 1959). The wires appear in the focal plane. A section of cortex is moved at random under the objective of the microscope. A contact of the image of the point of the wire with the clearly observed image of the tissue constituent is counted as a hit. The number of hits made in this way on nerve cell bodies, apical, and basal dendrites are recorded. Under the heading ‘basal dendrites’ all dendritic branches are counted (basal dendrites, branches apical dendrites, dendrites of stellate cells, etc.) except the unbranched portions of the apical dendrites, because in Cajal-stained preparations the smaller dendrites are undistinguishable from each other. Observations were made using a 20 x ocular and a 2 mm oil immersion objective. With the aid of a camera lucida the diameters of cell bodies and the dendrites were measured. The magnification of the system was 3000 x . Whenever a constituent was hit with the tungsten wire the diameter of this element was also measured on the camera lucida screen. Cell bodies and dendrites were subdivided on the basis of measured diameters, each subdivision representing a difference of 1/3 p (equivalent to 1 mm on the camera lucida screen). A television-camera-monitor-calculator system is being developed to automatize the measurements and calculations. Rabbits (Alaska F1 mongrels) of various age groups were used exclusively for this investigation. Twelve histological sections taken from area 2/3, 25 ,u thick (SchadC, 1959a, b), from each animal were investigated. Four series of animals (litter mates) were used. After measuring the standard deviation of the first series of samples it was found that 2000 to 3000 hits per section of cortex gave a sufficient degree of accuracy. (a) Calculations. The total surface area of cell bodies and dendrites per unit volume was calculated as follows: nerve cell bodies

3v r

A=--.

in which A = the surface area per 1111113 of cerebral cortex, V = the percentage of cortex which is nerve cell body, measured by the ‘hit’ method, and = the mean radius of the cell bodies. In this equation the cell bodies are treated as spheres. As an independent check, individual nerve cell bodies were measured and the surface area was calculated by

I57

N E U R O N A L PARAMETERS OF THE CORTEX

treating the cell bodies as prolate spheroids by using the equation: A

=

2nb2

. + 2n a6 sin-1 e e -

in which A = the surface area of the individual cell body, a of the nerve cell body, b = half of the minor axis, and

=

half of the major axis

2v

A==

dendrites

r

in which A = the surface area of the dendrites per mm3 of cerebral cortex, V = the percentage which is occupied by the apical or ‘basal’ dendrites measured by the ‘hit’ method and = the mean radius of the apical or basal dendrites. In this equation the surface is calculated as the curved surface of a cylinder. The results are given in four Tables (11-V) and six Figures (5 through 10 inclusive). Examples of the counts (average values with standard deviations) and the measurements of the diameter of the dendrites are shown; range values of four series of experiments are plotted in graphs against age. TABLE I1 PERCENTAGE OF CORTEX OCCUPIED BY NERVE CELL BODIES A N D THEIR CALCULATED SURFACE AREA*

Age

(days)

Animal

A3 B3

10 10 10 10 30 30 30

c3

D3

A7 B7 c7

30 300 300 300

D7

A10 B10

c10

300

D10

Proportional volume

S.D.**

7.1 6.8 5.9 7.2 7.1 5.6 5.4 7.2 6.7 6.4 7.0 6.8

0.47 0.46 0.43 0.48 0.57 0.52 0.50 0.58 0.56 0.55 0.57 0.56

Mean radius (P)

5.5 6.0 6.0 5.4

6.5 6.I 7.1

6.8 7.0 6.5 6.4 6.1

S.D . * * *

0.41

0.34 0.42 0.38 0.73 0.84 0.69 0.82 0.67 0.53 0.74 0.80

Surface area per mm2 of cortex ( x 107~2)

3.9 4.1 3.O 4.8 3.3 2.8 2.3 3.2 2.9

3.0 3.3 3.3

* To avoid lengthy tables only the data of three ‘key’ age groups are given. The data of the groups of 0, 5, 15,20,25,45,and 65 days of age are omitted. The range values for all age groups are given in the figures.

**

S.D. =

V

p(loo-pp) n

References p. 1741175

in which p is the percentage and n the number of hits.

158

J. P.

SCHADB,

H. V A N B A C K E R A N D E. COLON

(b) Percentage of cortex occupied by nerve cell bodies, apical and basal dendrites. In newborn rabbits between 12.9 and 15.2% (range values) of the cortex is occupied by nerve cell bodies (Figs. 5 and 7). The proportional volume of the nerve cell bodies decreases during the first 5 days after birth from an average of 14.2 % to an average value of 7.5 %. This change can be attributed to the fast decrease in packing density of the neurons (SchadC, 1959a,b). During later stages of the development the volume occupied by the nerve cell bodies remains practically constant and in adult animals an average value of 6.7 % is found.

0

5

10

15

20

25

30

300

65

AGE I O A Y S I

Fig. 5. Changes during growth in a proportional volume of the nerve cell bodies in area 2/3 of the rabbit cortex. The points indicate the mean values, the small vertical line the range values.

J I0 + e 7 J o 0

5

15

20

25

30

AGE IDAYSI

Fig. 6. Changes during growth in a proportional volume of the basal dendrites in area 2/3 of the rabbit cortex. The points indicate the mean values, the small vertical line the range values. Apical and basal dendrites were measured together in preparations, 5 days of age. The dotted line indicates the interpolated value for the basal dendrites if we consider an even distribution of apical and basal dendrites.

159

N E U R O N A L PARAMETERS OF T H E C O R T E X

At 5 days of age the total volume occupied by the dendrites in the cortex is between 1.2 and 1.6%. The volume of the dendrites increases sharply during the next 15 days (Figs. 6, 7 and Table 111). In the adult animals between 6.6 and 7.7 % of the cortex is occupied by the unbranched apical dendrites and between 7.5 and 8.4% by 'basal' dendrites. In living tissue, however, this percentage of apical and basal dendrites may be much higher, as the smaller branches of the dendrites cannot be observed with light microscopy and probably are not stained. Pappas and Purpura (1961) (see also

Cell bodies

AGE IN DAYS

Fig. 7. The percentage distribution in volume of nerve cell bodies and of apical and basal dendrites during postnatal development in area 2/3 of rabbit cortex.

T A B L E 111 PERCENTAGE OF CORTEX O C C U P I E D B Y BASAL DENDRITES A N D THEIR C A L C U L A T E D S U R F A C E AREA

Age (days)

10 10 10 10 30 30 30 30 300 300 300 300 References p . 1741175

A3 B3 c3 D3 A7 B7 c7 D7 A10 B10 c10 D10

Proportional volume

S.D.

Mean radius (P)

s.D .

Surface area per mm3 of cortex ( x l07p2)

1.9 2.3 2.1 1.8 6.9 8.3 6.7 7.5 7.9 8.4 8.1 1.5

0.25 0.27 0.30 0.24 0.57 0.62 0.56 0.59 0.60 0.62 0.61 0.59

0.24 0.32 0.28 0.3 1 0.39 0.43 0.40 0.41 0.31 0.45 0.44 0.38

0.08 0.06 0.09 0.10 0.09 0.08 0.09 0.10 0.12 0.09 0.08 0.11

15.8 14.4 16.8 11.6 35.4 38.6 35.5 36.6 51.0 37.3 36.8 39.5

J. P. S C H A D ~ .H. V A N B A C K E R A N D E. C O L O N

160

their contribution in this volume), in studying the immature neocortex of the cat with the electron microscope, have revealed that dendritic branches as small as 300 A can be found in the neuropil. Apical dendrites are seen at birth in Golgi-Cox preparations as thin threads. This finding is consistent with the observations of Eayrs and Goodhead (1959) on the postnatal development of the rat cortex. Noback and Purpura (1961) are of the opinion that the apical dendrites are much more extensively developed in the immature cortex of the cat. However, the Golgi method has many modifications and the results differ from one laboratory to another. Our modification (Van der Loos, 1959) gives very consistent results in both rabbit and man. To check the validity of the results obtained by the ‘hit’ method the axes of eight hundred nerve cells were measured in the same preparations. The percentage of cortex occupied by the nerve cell bodies was calculated on the basis of these measurements and the previously determined packing densities of the cells (SchadC, 1959a,b). TABLE IV PERCENTAGE O F CEREBRAL CORTEX O C C U P I E D B Y NERVE CELL BODIES C A L C U L A T E D O N T H E BASIS OF AXIS MEASUREMENTS

Age (days)

300 300 300 300

* **

Volume average nerve cell body (Z) Animal

A B C D

Proportional volume

Prolate spheroids

(I*)

(II**)

Spheres 1423 1318 1298 141 1

1257 1128 1075 1287

8.25 7.64 7.52 8.18

7.29 6.54 6.23 7.46

This value is obtained by multiplying the packing density of the nerve cells and the volume of the average cell body (treated as a sphere). This value is also obtained by multiplying the packing density of the nerve cells and the volume of the average nerve cell body (treated as a prolate spheroid).

Results are shown in Table IV and are in close agreement with those shown in Table 11. The slightly different values may be the result of subjective errors in measuring the length of the axes of the nerve cell bodies. The difference in values obtained by the ‘hit’ method and by axis measurements was also observed by Peters and Flexner (1950) in studying the frontal cortex of the guinea-pig. With a different technique, the length and diameter of the apical dendrites of 1359 pyramidal cells, in a known volume of cerebral cortex, was determined (SchadC and Van Backer, to be published). Consequently the proportional volume of the apical dendrites could be calculated. A value of 7.5 f 0.9 (S.D.) was found, which is in close concordance with the results obtained:by the ‘hit’ method. Comparison of the results obtained by the two different methods indicates that the ‘hit’ method seems a reliable technique to determine the relative volume of cortex occupied by nerve cell constituents as observed by the light microscope.

161

NEUKONAL PARAMETERS OF THE CORTEX

( c ) Tlze surface area of cell bodies and dendvitesper unit volume. In newborn animals an average surface area for the nerve cell bodies of 12.7 x lo7 ,dwas calculated per 1 1111113 of cerebral cortex. The total surface area of the cell bodies per unit volume of cerebral cortex decreases very fast from 0 to 5 days after birth (Figs. 8 and 10). The

0

5

10

75

20

25

30

300

AGE 1OAYSJ

Fig. 8. Changes during growth in surface area of nerve cell bodies. Same legend as Fig. 5.

O - e -

AGF I D A I300 S)

Fig. 9. Changes during growth in surface area of basal gendrites. Same legend as Fig. 6.

surface area of the apical and basal dendrites increases markedly 5 days after birth. The surface area of the basal dendrites and branches of apical dendrites increases in a 25-day period (5 to 30 days after birth) more than tenfold (Fig. 9). Table V shows calculations €or the surface area of the average nerve cell body and dendritic plexus. The data were obtained by dividing the data of Table LI and Table 111 (percentage References p. 1741175

162

J. P.

SCHADB,

H. V A N B A C K E R A N D E. C O L O N

TABLE V V O L U M E A N D S U R F A C E AREA O F T H E AVERAGE NERVE C E L L B O D Y A N D THE AVERAGE DENDRITIC PLEXUS

Volume

Surface area

Age (days)

Animal

Average nerve cell body ( p 5 )

Average dendritic plexus ( p a )

Average nerve cell body ( p 2 )

Average dendritic plexus (pa)

300 300 300 300

A B C D

1155 1103 1206 1173

2586 2637 2534 2620

500 517 568 568

11400* 8880 9090 9660

* On the basis of these values the ratio cell body surface area :dendritic plexus surface area could be calculated. This ratio is respectively for A-1 : 22.8, B-1 : 17.2, C-1 : 16.0, D-1 : 17.0. values) by the packing densities of the cells in area 2/3 of the rabbit cortex. In this way a value for the surface of an average neuron in a particular field of the rabbit cerebral cortex can be obtained.

Apical dendrites

BH

Bas01 dendriles

8.

Cell bodies

10

15

20

30

65

300

AGE IN DAYS

Fig. 10. The percentage distribution of the surface area of nerve cell bodies and of apical and basal dendrites during postnatal development in area 2/3 of rabbit cortex.

Recently a number of investigators have reported values for the volume occupied by nerve cell bodies in the cerebral cortex. Using the Chalkley technique, Peters and Flexner (1950) found this value to be about 12 % in the frontal cortex of the guinea-pig. Haug (1953, 1956) calculated the gray cell coefficient of the rabbit as 0.045 (i.e. 4.5 % of the frontal cortex is occupied by cell bodies). Values for the percentage of cortex occupied by cell bodies in a number of other mammals range from about 2 to 20% (Haug, 1956; Shariff, 1953).

NEURONAL PARAMETERS OF THE CORTEX

I63

Apart from two preliminary communications by Sholl(l955, 1956) no other recent detailed studies of surface area measurements have been reported. Although Sholl's absolute values are considerably higher (his analyses concern neurons in the cat cortex) than those reported here, his observation that the dendritic surface forms approximately 90 to 95% of the receptive surface of the neuron, is in remarkably good agreement with our estimates. It was found (Table V) that for the average neuron in area 2/3 of the rabbit cortex the dendritic surface contributed to 9 4 9 6 % of the receptive area of the neuron. In view of recent electron microscopic data (Pappas and Purpura, 1961) it seems reasonable to assume that the contribution of the cell body to the total receptive pole of the neuron is less than 1 %. For a correlation of the biochemical composition of the cerebral cortex with histological parameters, a number of criteria might be used. The levels of y-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) were chosen as biochemical parameters because of the unique presence of GAD and GABA in the central nervous system (cf. SchadC and Baxter, 1960). No relationship could be demonstrated in the cerebral cortex between the number of cell bodies per unit volume and the levels of GABA and GAD (Baxter et al., 1960). Those histological measurements which appeared to bear some kind of relationship to the above-mentioned biochemical constituents are listed below. The greatest increase in the level of GABA in area 213 coincided in time with the sharpest decrease in the proportional volume of the nerve cell bodies and an increase in the proportional volume of the dendrites. This might point to a n accumulation of GABA in the dendrites. The rapid increase of GABA levels also marked the beginning of growth of basal dendritic plexuses and branches of the apical dendrites. According to earlier studies it was also correlated with a change of dustlike basophilia into true Nissl bodies (Roberts e l al., 1951; SchadC, 1959a, b). The time at which adult GABA levels were observed coincided with the morphological maturation of the cell body nucleus and the attainment of adult EEG patterns (SchadC, 1959a,b). The greatest increase of GAD activity coincided most closely with the fastest growth of the surface area of the dendrites. GAD apoenzymes increased almost ninefold in the first 20 days of life and did not attain adult levels until 30 days after birth (SchadC and Baxter, 1960). However, a numerical correlation between this surface area and the activity of the apoenzyme could not be demonstrated. It might be anticipated that the activity of enzyme systems associated with cytoplasmic membranes and the cytoplasm of nerve cells will be correlated most closely with the measurements of proportional surface area and volume. The activity of particulate bound enzyme systems such as those associated with mitochondria, might be expected to correlate more closely with the proportional surface and volume of particulate structures. Some opinions were expressed at recent neurochemistry meetings on the current high status of chemical studies on nervous tissue and the decline of anatomical studies. It seems realistic to assume that for the interpretation of a number of chemical studies one is dependent on the availability of quantitative histological data. References p. 1741175

164

J. P. S C H A U ~ ,H. V A N B A C K E R A N D E. C O L O N

111. M A T U R A T I O N A N D O R G A N I Z A T I O N O F D E N D R I T E S

To gain a better understanding of the function of the cerebral cortex it is necessary to have not only a good insight into the total extent of the neuronal membranes, the number of synapses etc., but also a clear picture of the organization of the individual neuron. As has been pointed out in a number of previous papers (SchadC and Van

Fig. 1 1 . Photomicrograph of Golgi-stained cells in the cerebral cortex of the middle frontal gyrus. A and C are preparations fromnewborn animals; B and D, preparations from 3-month-old animals.

N E U R O N A L P A R A M E T E R S OF T H E C O R T E X

165

Fig. 12. Photomicrograph of Golgi-stained neurons in the middle frontal gyms to show the increase in dendritic branching during postnatal development in layer 11. A: newborn, B: preparations from 6-month-old animals. References p . 1741175

166

I. P. S C H A D I ~ H. , V A N BACKER A N D E. C O L O N

Groenigen, 1961; Schadk and Meeter, 1963) the Golgi-Cox method seems the only one available for a quantitative analysis of dendritic patterns in a grey substance. Sholl's (1953, 1955, 1956) careful analysis of neurons in the visual and motor cortices of the cat enabled him to draw up a number of general rules for the mode of branching of pyramidal and stellate cells in the cerebral cortex. With the cooperation of the physicist Uttley (Sholl and Uttley, 1953) a tentative programme was mapped out for the application of the histological data to the design of a conditional probability machine. (a) Disadvantages of the Golgi-Cox method. Several objections can be put forward to the use of Golgi methods for the quantitative study of dendrites. Noback and Purpura (1961) are perfectly right when they criticize the value of the Golgi method, pointing to the great variations between the results of investigations from different laboratories. Their conclusion, however, that only qualitative studies should be done seems unwarranted. The results of qualitative studies can only be conveyed in a narrative way, from which sufficient information is difficult to obtain. Quantitative histological studies, in general, compel to a higher degree of preciseness. Of course, quantitative studies always involve a loss of information of the data of individual cells. Although the exact chemical nature of the impregnation process in the Golgi-Cox method is largely unknown, there are sufficient indications to assume that the staining is primarily due to mercuric chloride. In comparison with other staining techniques for dendrites such as the reduced silver methods, the Golgi-Cox method differs not only in its staining ability (it stains only 2 to 4% of the neurons present) but also in its mode of staining. Preliminary comparative studies have shown that silver salts form a chemical complex with some compound in the neuronal membrane, but that mercuric salts form a thick layer of material also outside the boundaries of the membranes. Thus, the former method gives a fair indication of the thickness of the dendrite, but measurements have shown that using the latter method the dendrite gives the impression of being at least twice as thick. Sholl (1953) found an average diameter of 1.5 ,u for the basal dendrites of pyramidal cells in the motor cortex of the cat employing one of the modifications of the Golgi method. Schad6 and Baxter (1960), on the other hand, report an average diameter of 0.8 ,u for the basal dendrites in the motor area of the cortex of the rabbit, using a reduced silver technique. A final objection which can be mentioned and which is not specific of the Golgi method but relates to the use of the light microscope is that dendrites smaller than the resolving power of the light microscope (generally about 0.5 p ) are not seen. With the electron microscope (Pappas and Purpura, 1961, and their contribution in this volume) dendritic endings with a diameter of 300 A and a length of 0.5 to 1 ,u have been observed. They seem to turn abruptly into dendrites of about 0.1 to 0.5 ,u. When we make sure that from the cell body the dendrites taper gradually to a diameter of 0.1 ,u a reconstruction of the dendritic tree could be carried out combining submicroscopic with light microscopic data. Notwithstanding a number of disadvantages of the Golgi technique we feel at present that continued use of the method for the quantitative evaluation of the

N E U R O N A L P A R A M E T E R S O F T H E CORTEX

167

dendrites is justified and the data obtained are reliable for the interpretation of growth and maturation of the receptive area of the neuron. ( b ) The dendritic organization of the cerebral cortex during development. The changes in the dendritic organization during development will be illustrated with the results obtained from the data of the analysis of the middle frontal gyrus in the human cerebral cortex (Figs. 11, 12). Measurements were made of the basal dendritic field of pyramidal cells in layers 111, IV and V. To facilitate the measurements, the image of each perikaryon was projected onto the centre of a target and all dendrites of the basal plexus were drawn into 12 concentrically arranged zones each of a width which represented a distance of 25 p in the histological section. The methods are described in detail elsewhere (SchadC and Van Groenigen, 1961; SchadC and Meeter, 1963). layerlll fayerlll

IayerlV

1ayerV

DISTANCE FROM CENTRE OF PERIKARVON 1P)

Fig. 13. Distribution of intersections and branching points in relation to the distance from the centre of the perikaryon. Comparison between pyramidal cells in layers 111, IV and V of the middle frontal gyrus. The black portion of the histogram gives the value for the branching points, the white portion for the intersections. References p. 1741175

168

J. P.

SCHADB,

H. V A N B A C K E R A N D E. COLON

The structural features mentioned here in comparing the quantitative degree of differentiation in the various layers of the cortex are: the number of dendrites arising from the perikaryon; the number of intersections, which is characteristic of the extent of the dendritic field; the branching pattern of the basal ramifications of the pyramids, and the total length of the dendrites of a n ‘average’ neuron. The intersections, which are the crossing-points of the dendrites with the concentrical zones, are graphically presented in such a way (Fig. 13) that the dendritic segments intersecting a zone e.g. at 100 p from the centre of the perikaryon are placed in the histogram in the class of 75-100 p. This value is said to be at 100 p from the centre of the perikaryon of this class and is also the modal value of the histogram. The following general conclusions can be drawn regarding the intersections and branching points of the basal dendrites of the pyramids in layers Ill, IV and V of the middle frontal gyrus : (a) The distribution of the intersections of any one of the cortical pyramids is quite similar, whether they are localized in layer 111, IV or V. (b) The histograms illustrating the mode of distribution of the intersections show rather sharp modal values. (c) Temporal rather than spatial differences can be observed in the development of the dendritic plexus of the various neurons. For the pyramids in layer I11 the modal value (peak of the histogram) of the intersections was at birth 25 p from the centre of the perikaryon. A fast increase is noticed up to 6 months of age, when a modal value is found of 75 p. Then the rate of growth slows down to such an extent that this value is also found in the 24-month preparations. In the adult the peak of the graph is shifted towards 100 p and observable is a general increase in the density of the dendritic plexus. The position of the peak in the histogram is an indication of the highest packing density of the dendrites around the perikaryon. A rise in the histogram denotes that the branching points of the dendrites exceed in number the points of ending; a fall in the histogram has the reverse meaning. The modal values for the distribution of the branching patterns are, as could be expected, localized nearer to the centre of the perikaryon. A sharp increase is seen in branching points from newborn to 6 months of age, reaching at that stage a modal value of 50 p ; only in the adult is a shift to a larger value seen. Another indicator of T A B L E VI DENDRITIC PARAMETERS OF PYRAMIDAL CELLS I N N E W B O R N ( G Y R U S F R O N T A L I S MEDIUS)

Layer III

Number of dendrites arising from perikaryon Number of intersections Number of branching points Estimate of total length of dendrites

*

Mean f standard error.

*

6.7 0.8* 5.8 f 1.5 3.1 f 0.6 203 51

*

Layer IV

Layer V

4.8 0.8 0.8 f 0.6 0.4 & 0.2 28 f 21

8.4 1.4 24.5 f 7.0 12.0 & 2.7 858 f 245

169

NEURONAL PARAMETERS OF THE CORTEX

the growth of dendritic plexus is the total length of the dendrites. A comparison of the data in Tables VI and VII shows very marked differences in the various dendritic parameters. TABLE VII DENDRITIC PARAMETERS OF PYRAMIDAL CELLS I N A D U L T

( G Y R U S F R O N T A L I S ME DIUS)

Number of dendrites arising from perikaryon Number of intersections Number of branching points Estimate of total length of dendrites

Layer III

Layer IV

Layer V

7.6 f 0.9 195.3 19.8 40.8 4.7 6836 & 693

4.9 f 0.9 81.7 f 8.3 14.7 f 2.8 2860 f 291

8.2 & 1.2 216.8 21.6 44.4 4.1 7558 756

*

**

The dendrites of the pyramidal cells in layer IV are the least developed at birth (Fig. 13). Although the histograms illustrating the mode of distribution of the intersections and branching points show the same characteristic features as for the pyramidal cells in layers 111 and V, the modal values are less sharp. An explanation may be the presence of neurons in layer IV of a very divergent size or a different branching pattern of the dendrites. The smallness of the neurons is characterized by the fact that up to 24 months the dendrites rarely extend more than 200 ,u from the perikaryon. The dendritic plexus of the pyramidal cells in layer V is most advanced at birth. A

Fig. 14. Logarithmic representation of the number of intersections per pz in relation to the distance from the centre of the perikaryon. Comparison between pyramidal cells of newborn in layers 111, IV and V of the middle frontal gyrus. A = layer 111; A = layer IV; 0 = layer V. References p . 1741175

I70

J. P. S C H A D ~ ,H. V A N B A C K E R A N D E. C O L O N

sizable plexus is present extending to 100-125 p from the perikaryon. A fast growth takes place between 0 and 6 months, reaching a modal value of 100 p ; the dendrites now extend already to over 250 ,u from the perikaryon. The modal value at 6 months is the same as found in the adult preparations. After 6 months the growth of the dendritic plexus is thus mainly caused by an increase in the length of the most peripheral branches and by the addition of a few peripheral bifurcations. The expansions of the dendritic branches of a given neuron can be attributed to at least two factors: an increase in the number of dendrites arising from the perikaryon and the branching of existing dendrites. The first process does not seem to play an important role postnatally in the neurons just described. The average number of dendrites arising from the perikaryon in the newborn does not differ significantly from the number in adult preparations. The maturation of the dendritic field is thus due to growth and ramification of peripheral branches, which is clearly shown by the increase in the number of branches and the total length of the dendrites (Tables VI and VII). (c) Logarithmic representation of the dendriticfield. When plotting the logarithmic values of the number of intersections per pz against the distance from the perikaryon a distinct linear relationship is obtained in a correlation diagram (Figs. 14 and 15). This implies that the number of intersections per pz decreases exponentially with the distance. Such a relationship can be described by the following equation: y

=a - k z

where y represents the number of intersections per p2 (in the graphs of Figs. 14 and 15 this value is multiplied by lo6), x is the distance to the cell body and both a and k

Fig. 15. Same legend as Fig. 14. Pyramidal cells of preparations from 24-month-old animals.

NEURONAL PARAMETERS OF THE CORTEX

171

are functions of the type of neuron, the age and the location in the cortex. This relationship could be established for all age groups. It must be stated, however, that the parameters a and k have been calculated for the various age groups using the method of the least squares starting from the average values. This was done because che significance of the straight line was evident and no test for the straightness of this line was required. In this case complete linearity of the regression line is impossible, as the number of intersections becomes zero rather abruptly. For a complete linearity the number of intersections should approach zero asymptotically. As a consequence a greater deviation from the exponential relationship occurs as the number of intersections becomes smaller. Statistically no changes could be demonstrated in the number of dendritic trunks arising from the perikaryon when comparing the various age groups (Tables VI and VII, and Schadt and Van Groenigen, 1961). Therefore the changes in the regression coefficient of the logarithmic graph indicate the course of the postnatal development of the dendritic plexus. A dendritic field factor (dff) was introduced to characterize with a single parameter the expansion of the dendritic field. dff = l / k log e, in which k is the regression coefficient. The application of this method to the establishment of differences between normal and pathological brains has already shown promising results (Schadt and Meeter, 1963). I n section 11of this paper an estimate was given of the total surface area of dendrites and cell bodies per unit volume of cerebral cortex by the light microscope. It seemed interesting to attempt to calculate from the data obtained by the Golgi method the surface area of the dendrites and to compare these data with the results acquired with the random hit method. Using as point of departure for the basal dendrites of the pyramidal cells the exponential equation: y

=

ae-k.2

(1)

then the total number of iiitersections (N) through the branching-sphere of the dendritic plexus equals : N

. 2nx2

= ae-kx

(2)

A factor equalling 4 2 is introduced as correction for the winding of the dendrites, because the dendrites are not running straight from one zone to the other. The equation now becomes: N

=

a c k X. 2n ( . \ / 2 . x ) ~

(3)

The total length of the dendrites (Lba) of a given basal dendritic plexus can be obtained in the following way: Lbd =

r

d o

ae-kx. 2n ( d 2 .x ) ~

(4)

m =

References p . 1741175

4na

e - k x . x2dx

(5)

172

J. P. SCHADE, H . V A N B A C K E R A N D E. C O L O N

from which follows by partial integration: =

(1/,4)3

=

8na (dff)3

For a simple calculation of the surface area we now must assume that the diameter of the dendrites is constant throughout the whole plexus. The surface area of the basal dendritic plexuses (A) per unit volume of cerebral cortex (mm3) can be written as A

=

87~a(dff)~ 2nr. n

(8)

=

157.7 ~ ( d f f rn )~

(9)

in which r = the mean radius of the dendrites and n = the packing density of neurons. Equation (9) has been applied to the data of the basal dendrites in the middle frontal gyrus of the human cortex. Table VIII shows the results and it can be concluded that the order of magnitude is about the same for the two methods (data of Fig. 9 and Table VIII). The close resemblance between the two sets of data justifies TABLE VIII L O G A R I T H M I C V A L U E OF T H E S U R F A C E AREA OF T H E B A S A L D E N D R I T I C P L E X U S PER

mm3 ( P Y R A M I D A L

CELLS, M I D D L E F R O N T A L G Y R U S )

Layer ZZZ

Newborn 6 Months 24 Months Adult *r

=

8.0 8.6 8.5 8.9

+ log r* + log r + log r + log r

Layer ZV 7.9 9.1 8.7 8.7

+ log r + log r + log r + log r

Layer V

8.4 8.5 8.2 8.4

+ log r + log r + log r + log r

Mean radius of the dendrites at a given age and in a given layer.

the application of surface area measurements for comparative studies. Edstrom (1958) and Edstrom and Pigon (1958) have already made attempts to relate the surface area of spinal ganglion cells with the ribonucleic acid content. They conclude that the surface area is a factor determining the RNA content of these cells and probably also of other kinds of true nerve cells. The study of this type of relationships in populations of neurons and glia cells such as the cerebral cortex now comes within the realm of possibilities. Neurocyberneticists have only recently become interested in the axonal and dendritic branching patterns of cortical neurons. Sholl and Uttley (1953) and Uttley (1955) proposed a theory of pattern perception related to a classification machine and used some of the data found by quantitative analysis of the cerebral cortex. Braitenberg and his coworkers (1960,1961, 1963) use Golgi material in an attempt to translate the structure of the gray substance of cerebral and cerebellar cortex into sets of neuronic equations. He envisages a method of translating structural descriptions of nerve nets directly into functional schemes and therefore he is particularly interested in the branching pattern of individual neurons (Braitenberg, 1963).

N E U R O N A L PARAMETERS OF THE CORTEX

173

One of the programmes of our laboratory (cf. Smit and Schadt, 1963) is directed towards a statistical approach to the study of the cortical organization, to be described in terms of probability theory. We use as starting-point the classification and conditional probability model of Uttley (1954, 1955, 1956). If we assume that the mammaGan cortex with regard to classification and determination of conditional probabilities behaves as the model for conditional probabilities, then we can try to interpret the data about maturation of the dendrites on the basis of this model. In a forthcoming paper (Smit and Schade, 1963) we will discuss in detail some aspects regarding the comparison between the structural organization of the cerebral cortex and the classification and probability model. In conclusion we would like to say that rapid advance is made in the methodology of quantitative brain research, which may open new avenues for multidisciplinary brain research. As closing remark we would like to quote Sholl (1956): ‘If progress is to be made in the search for those anatomical parameters of the cortex that will lead to a better understanding of its organization, then we must avail ourselves of information derived from all the techniques at our disposal, and attempt to make our criteria and judgments quantitative’. ACKNOWLEDGEMENTS

This investigation was supported by research grants from the National lnstitute of Mental Health ( M H 6825) and the National Institute of Neurological Diseases and Blindness (B 3048), Bethesda, USA. The authors are indebted to Mr. G. Rijskamp and Miss N. M. van der Kleyn for histological preparations and to Miss J. Sels and Miss L. Voermans for typing the manuscript. SUMMARY

This report concerns the quantitative analysis of a number of parameters of the maturing cerebral cortex. Section I deals with the determination of the volume of nerve cell bodies and the packing density during postnatal development. In Section I1 a method is discussed for measuring the surface area of cortical nerve cells. The proportional volume and surface area of the perikaryal membranes decrease during the first 5 days after birth in the rabbit and then remain constant during development. The proportional volume and surface area of the apical and basal dendrites increase markedly from 5 to 30 days after birth. The data are discussed in relation to physiological and biochemical development of the cerebral cortex. Section III1deals with the organization of dendritic patterns during postnatal development. Special attention is drawn to the applicability of the measurements of dendritic organization to mathematical models of nervous function.

References p . 174/175

174

J. P.

SCHADB,

H. V A N B A C K E R A N D E. C O L O N

REFERENCES BAXTER, C. F., SCHADE, J. P., AND ROBERTS, E., (1960); Maturational changes in the cerebral cortex. 11. Levels of glutamic acid decarboxylase, y-aminobutyric acid and some related amino acids. Inhibition in the nervous system and GABA. E. Roberts, Editor. London, Pergamon Press (p. 213-220). BRAITENBERG, V., (1961); Funktionelle Deutung von Strukturen in der grauen Substanz des Nervensystems. Naturwissenschaften, 48, 489496. BRAITENBERG, V., (1 963); Histology, Histonomy, Histologic. Progress in Brain Research. Vol. 2. N. Wiener and J. P. Schadk, Editors. Amsterdam, Elsevier (p. 160-176). BRAITENBERG, V., AND LAURIA, F., (1960); Toward a mathematical description of the grey substance of nervous systems. Nuovo Cimento, Suppl. 2, 18, 149-165. CAJAL,RAMONY, S., (1936); Degeneration and Regeneration in the Nervous System. R. M. May, Translator. London. Oxford Univ. Press. CHALKLEY, H. W., (1943); Method for the quantitative morphologic analysis of tissues. J. nut. Cancer Inst., 4, 47-53. CHALKLEY, H. W., (1949); A method for the measurement of average nuclear volumes independent of shape. Anat. Rec., 103,433. EAYRS,J. T., AND GOODHEAD, B., (1959); Postnatal development of the cerebral cortex of the rat. J. Anat. (Lond.), 93,385402. J. E., (1958); Quantitative determination of ribonucleic acid in the micromicrogram range. EDSTROM, J. Neurochem., 3, 100-106. EDSTROM, J. E., AND PIGON,A., (1958); Relation between surface, ribonucleic acid content and nuclear volume in encapsulated spinal ganglion cells. J. Neurochem., 3,95-99. HAUG,H., (1953); Der Grauzellkoeffizient des Stirnhirnes der Mammalia in einer phylogenetischen Betrachtung. Acta anat. (Basel), 19,239-270. HAUG,H., (1956); Remarks on the determination and significanceof the gray cell coefficient. J . comp. Neurol., 104, 473492. HAUG,H., (1958); Quantitative Untersuchungen an der Sehrinde. Die individuelle Schwankungsbreite beim Menschen verbunden mit einigen Bemerkungen uber die Schizophrenie. Die Entwicklung der menschlichen Sehrinde. Die Volumenverhaltnisse bei einigen Marnmalia. Stuttgart, Thieme Verlag. HAUG,H., (1959); Die Zelldichte und ihre Bedeutung fur die Hirnrinde und ihre Areale. Dtsch. Z. Nervenheilk., 178,648-667. KAPPERS, ARIENS,J., (1936); Brain bodyweight relation in human ontogenesis. Proc. kon. ned. Akad. Wet., 39,3-12. CH.,AND PURP~RA, D. P., (1961); Postnatal ontogenesis of neurons in cat neocortex. J . NOBACK, comp. Neurol., 117. 291-307. PAPPAS,G. D., AND PURPURA, D. P., (1961); Fine structure of dendrites in the superficial neocortical neuropil. Exp. Neurol., 4, 507-530. V. B., AND FLEXNER, L. B., (1950); Quantitative morphologic studies on developing cerebral PETERS, cortex of the fetal guinea-pig. Amer. J. Anat., 86, 133-157. ROBERTS, E., HARMAN, P. J., AND FRANKEL, S., (1951); y-Aminobutyric acid content and glutamic decarboxylase activity in developing mouse brain. Proc. SOC.exp. Biol., 78,799-808. J. P., (1959a); Maturational aspects of EEG and of spreading depression in rabbit. J. NeuroSCHADE, physiol., 22, 245-257. SCHADE, J. P., (1959b); A histological and histochemical analysis of the developing cerebral cortex. Proc. kon. ned. Akad. Wet., 62,445-460. SCHADB, J. P., AND BAXTER, C. F., (1960); Changes during growth in the volume and surface area of cortical neurons in the rabbit. Exp. Neurol., 2, 158-178. SCHADB, J. P., AND MEETER, K., (1963); Neuronal and dendritic patterns in the uncinate area of the human hippocampus. Progress in Brain Research, Vol. 3. W. Bargmann and J. P. Schade, Editors. Amsterdam, Elsevier (p. 89). SCHADE, J. P., AND VANBACKER, H., (1963); Changes during growth in the volume and surface area of dendrites in the cerebral cortex. In the press. SCHADE,J. P., AND VANGROENIGEN, W. B., (1961); Structural organization of the human cerebral cortex. 1. Maturation of the middle frontal gyrus. Acta anat. (Basel), 47,74-1 11. SCHADB, J. P., AND WEILER, L. J., (1959); Electroencephalographic patterns of the goldfish (Carassius auratus L). J . exp. Biol., 36,435452. SHARIFF, G.A., (1953); Cell counts in the primate cerebral cortex. J. comp. Neurol., 98,381400.

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SHOLL,D. A., (1953); Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat. (Lond.), 87, 387406. SHOLL,D. A., (1955); The surface area of cortical neurons. J. Anat. (Lond.), 89, 571-572. SHOLL,D. A., (1956); The measurable parameters of the cerebral cortex and their significance in its organization. Progress in Neurobiology. J . Ariens Kappers, Editor. Amsterdam, Elsevier (p. 324333). SHOLL, D. A., AND UTTLEY, A. M., (1953); Pattern discrimination in the visual cortex. Nature (Lond.), 171, 387-388. SMIT,G. J., AND SCHADF,J. P., (1963); Maturation of dendritic patterns in the cerebral cortex in relation to a brain model. In the press. UTTLEY, A. M., (1954); The classification of signals in the nervous system. Electroenceph. clin. Neurophysiol., 6, 479494. UTTLEY, A. M., (1955); The probability of neural connections. Proc. roy. Soc. B, 144, 229-240. UTTLEY,A. M., (1956); A theory of the mechanism of learning based on the computation of conditional probabilities. 1st Znt. Congr. Cyb. Namur. (p. 83Ck-856). VANDERLoos, H., (1959); Dendro-dendritische verbindingen in de schors van de grote hersenen. Thesis. University of Amsterdam.

I76

Electron Microscopy of Immature Human and Feline Neocortex G. D. PAPPAS

AND

D. P. P U R P U R A

Departments of Anatomy and Neurological Surgery, College of Physicians and Surgeons, Columbia University, New York

INTRODUCTION

The morphogenesis of the mammalian cerebral cortex has been well documented in light microscope studies of the structural features of neural and non-neural elements at different developmental stages (Cajal, 1911; Conel, 1939-1959; Eayrs and Goodhead, 1959; Noback and Purpura, 1961; SchadC and Baxter, 1960). These studies have been useful in providing information on the major sequential changes in neurons during the cytoarchitectural differentiation of the cerebral cortex. The overt changes described in these light microscope studies reflect developmental alterations at subcellular as well as cellular levels of organization. It is clear, therefore, that an analysis of fine structural alterations is fundamental to a n adequate understanding of cortical ontogenesis. The present report summarizes some observations on the fine structure of neural and non-neural processes in the cerebral cortex. Since previous studies have focused on the problem of developmental synaptology (Voeller et al., 1963) this aspect of neuronal ontogenesis is only briefly considered here. Emphasis is placed in this report on the salient features of the comparative development of neural-vascular and neuralglial relations in immature human and feline neocortex. The findings extend earlier observation on the ontogenesis of fine structure of elements of the neuropil and permit some evaluation of the contribution of glial-vascular membranes to the development of the blood-brain barrier. METHODS

Human cortical tissue was obtained from three 12-week-old fetuses and from two 16-week-old fetuses. Fetal age was determined by crown-to-rump measurements and available clinical data. Near-term kitten fetuses were obtained by caesarian section. As in the case with adult cats, thin pieces of cortical tissue (0.5 mm or less) were fixed for 30 min to 1 h in 2 % Os04, buffered with Verona1 acetate a t p H 8.2 to which 0.01 mg per mi CaClz had been added. Specimens were subsequently embedded in Epon 812. All the sections were stained with uranyl acetate, then carbon coated, and examined in an electron microscope (RCA-EMU 3 F).

Fig. 1. An area of superficial neocortex from a 12-week-old human fetus. Portions of three cells with prominent nuclei (N) and thin rim of cytoplasm are present. These cells are in close apposition without any intervening processes. Clusters of ribonucleoprotein granules and vacuolar structures as well as occasional mitochondria may be found in the cytoplasm, x 32,000. References p . 186

Fig. 2. A portion of capillary in the superficial neocortex of a 12-week-old human fetus. A red cell (R) is found in the lumen. Terminal bars (T) can be seen at the junction of the endothelial cells. The endothelial cells are surrounded by a basement membrane (BM) about 300 A thick. A cell body with a prominent nucleus (N) surrounds the basement membrane. At this age no smaller intervening processes are generally present between the basement membrane and the cell bodies. M = mitochondria, x 32,000.

I M M A T U R E HUMAN A N D F E L I N E NEOCORTEX

179

OBSERVATIONS

Light microscope studies have indicated that in the 12-week-old human fetus the brain has attained its general features (p. 106 in Arey, 1954). Electron microscope observations confirm the extraordinarily dense packing of cell bodies at virtually all levels of the cerebral cortex. These cells, presumably neurons, have a thin rim of cytoplasm and their membranes are in close relationship (Fig. 1). No intervening processes are detectable throughout large areas of apposition. Cytoplasmic organelles are similar to those described in embryonic and fetal tissue (Tennyson and Pappas, 1961; Voeller et al., 1963). The mitochondria have few cristae, and membranous elements of the endoplasmic reticulum appear as large vacuoles dispersed in the cytoplasm (Fig. 2). Scattered clusters of ribonucleoprotein (RNP) granules are arranged in rosettes and are usually unattached to membranes. Very few RNP granules are found to be intimately related to vacuoles of the endoplasmic reticulum at this developmental stage. The paucity of fine processes in cellular layers in the neuropil is a n outstanding characteristic of the human fetal cortex as well as fetal cortex in the kitten. Evidence has been presented elsewhere that the thick processes encountered in the feline fetal neocortex are apical dendrites of immature pyramidal neurons and dendrites of stellate cells (Voeller et al., 1963). Similar thick processes are observed in the superficial neocortex of the 16-week-old human fetus (Fig. 3). The homologous characteristics of dendrites in the fetal near-term kitten and 16-week-old human neocortex is emphasized in the micrographs of Figs. 3 and 4. The dendritic processes of the human fetal neocortex have typical tubular structures (Gray, 1959; Pappas and Purpura, 1961) but some of these are less sharply defined in fetal neocortex especially in longitudinal profiles. No clearly recognizable glial processes are found between the dendrites (Figs. 3 and 4). The relationship between neural elements and capillaries is especially noteworthy in human and feline fetal neocortex. Capillaries in 12-week-old human fetal cortex have a continuous basement membrane that surrounds the endothelial cell lining (Fig. 2). The thickness of the basement membrane in 12-week-old human fetal cortex is approximately 300 A. The cytoplasmic membrane of neurons is immediately external to the basement membrane of capillaries at this developmental stage (Fig. 2). No smaller intervening processes are observed between basement membrane neuronal contacts. During the 4th month of antenatal development, a number of changes are observed in the human fetal neocortex. The basement membrane surrounding the endothelium of capillaries is thicker (450 A) and more dense at 16 weeks (Fig. 5) than it is in the 12-week-old fetus (Fig. 2). A large proportion of the area external to the basement membrane is surrounded by smaller cell processes (Fig. 5), many of which are identifiable as dendrites by virtue of their characteristic tubules. The striking difference between elements of the immature and mature neuropil is illustrated in Figs. 6 and 7. The basement membrane of the capillaries of the superficial neocortex in the adult cat is about 750-1000 A thick, or approximately 2-3 times References p . 186

180

G. D. P A P P A S A N D D. P. P U R P U R A

Fig. 3. An electron micrograph of an area of superficial neocortex from a 16-week-old human fetus. Portions of four cell bodies with prominent nuclei (N) and thin rim of cytoplasm are seen. By this age (cf. Fig. 1 ) many thick dendritic processes (D) containing profiles of dendritic tubules are found dispersed between cell bodies. No recognizable glial processes are present, x 29,OOO.

IMMATURE H U M A N AND FELINE NEOCORTEX

181

Fig. 4. An area of superficial neocortex from a near-term fetal kitten. Two cell bodies with prominent nuclei (N) each with a very thin rim of cytoplasm and intervening thick dendritic processes (D) are seen. The appearance of this cortical tissue from a near-term fetal cat is similar to that ofa 16-week-old human fetus shown in Fig. 3, x 32,000. References p. 186

c. 00 h)

0

P

a k a a

*

01

k

z

b b

a d

c 'p

a C

w

k

Fig. 5. Electron micrograph of a portion of a capillary from a 16-week-oldhuman fetus. The basement membrane (BM) surrounding the endothelial cell (E) is thicker (450 A) and more dense than that shown in Fig. 2. While a cell body with a small portion of its nucleus (N) included in the micrograph is found next to the basement membrane in the lower left, most of the immediate area is covered by cell processes. Profiles of dendritic tubules may be found in most of these processes, x 29,000.

IMMATURE H U M A N A N D FELINE NEOCORTEX

183

Fig. 6. Electron micrograph of the neuropil of the superficial neocortex (Suprasylvian gyrus) of an adult cat. On the left edge a small portion of an endothelial cell of a capillary (E) surrounded by thick basement membrane (BM) can be found. Just external to the basement membrane are glial processes. An axodendritic synapse may be found in the neuropil. A multivesicular (MV)body is present in the dendrite near the synaptic junction, x 36,000.

thicker than that of the earliest stages described here. Also in contrast to the fetal cortex, fine processes of the neuropil are external to the basement membrane of the capillaries. Identification of these fine processes is facilitated in many instances by the presence of oriented fibrils which exhibit fundamentally different characteristics from those of dendritic tubules (Fig. 7). Although typical glial processes are in contact with a large portion of the basement membrane in mature cortex, complete envelopment of cortical capillaries by glial processes may not occur (Maynard et al., 1957). Perivascular glial processes are in turn surrounded by various elements of the neuropil. These include axons, axon-terminals, finedendritic processes and axodendritic synapses. References p . 186

184

G . D. P A P P A S A N D D. P. P U R P U R A

Fig. 7. Electron micrograph of a portion of a capillary in the cortex of an adult cat. A glial cell process (G) is resting on the basement membrane (BM) which surrounds the endothelium of the capillary. Longitudinal arrays of filaments can be found in this glial process. These filaments differ from the dendritic tubules present in the nearby dendrite (D). Note that some of the dendritic tubules show areas of dilatation in the dendrite (DT) at the bottom of the micrograph, X 36,000.

An example of an axodendritic synapse containing a multivesicular body in the dendritic terminal is shown in Fig. 6. COMMENT

Prior to the appearance of recognizable cytoarchitectural lamination, neurons in the human fetal neocortex are in immediate contact with the thin basement membrane of capillaries. Not until several weeks later are recognizable small processes interposed

IMMATURE H U M A N A N D FELINE NEOCORTEX

185

between basement membrane and neurons. Although identification of these small processes is not complete, evidence has been presented that many are dendritic in nature. Others have characteristics similar to the glial processes observed in adult cortex. It has been inferred from several varieties of experimental studies that the complex of membranes constituting the vascular-glial system is involved in the maintenance of the blood-brain barrier (Luse, 1962). The finding that perivascular glial processes are not developed in early fetal cortex may be related to the observations suggesting that at this stage the blood-brain barrier is similarly poorly developed. However it must be recalled that one of the changes observed in the present study is an increase in the thickness of the basement membrane. Whether this developmental change in the basement membrane is a major factor in the maturation of the bloodbrain barrier (Dempsey and Wislocki, 1955) cannot be evaluated from the limited material studied here. Developmental changes in capillaries similar to those observed in human and cat cortex have been analyzed in more detail in the rat (Donahue and Pappas, 1961). These and other observations suggest that the basement membrane alterations play a role in the establishment of the blood-brain barrier. One of the chief postnatal developmental changes in feline neocortex may be viewed as a transition from appositional relationships between large elements to smaller elements of the neuropil. Cell body-to-cell body and cell body-to-large dendrite appositions are characteristically observed in antenatal and neonatal preparations. Elaboration of fine dendritic processes from large dendrites as well as the development of small glial and axonal elements is a postnatal event. Thus the interposition of fine processes between cell bodies and cell body dendrite appositions results in the basic architectural characteristics of the adult cortical neuropil. An obvious consequence of this is the development of a considerable increase in the total volume of space between different elements (Horstmann and Meves, 1959). Hence, although the distance between different processes remains the same (150-200 A), it can be expected that the development of a variety of fine processes will be accompanied by an expansion in the extracellular space. The problems of the extracellular space of brain and its functional relationship to the blood-brain barrier remains largely unresolved. A new approach to this problem may be afforded by the findings described here pertaining to the parallel development of extracellular space and those membrane elements primarily involved in the operation of the blood-brain barrier. The increase in the number of fine processes of the neuropil during cortical ontogenesis is associated with an increase in the number and types o f synapticinterrelations (Voeller et al., 1963). It follows from this that the determinants of synaptogenesis must be related to the growth and elaboration of fine processes leading to the establishment of versatile functional connections between neurons over distances. Some of the physiological and pharmacological consequences o f the latter ontogenetic changes are discussed in detail elsewhere in this volume (Purpura et al., 1963). ACKNOWLEDGEMENTS

Supported in part by National Institutes of Health grants NB-03448, NB-02314, and References p . I86

186

G . D. P A P P A S A N D D. P. P U R P U R A

NB-01312-07, the Life Insurance Medical Research Fund, and the United Cerebral Palsy Research and Educational Foundation, R- 135-62C. SUMMARY

The paucity of fine processes in the cellular layers in the neuropil is an outstanding characteristic of the human fetal neocortex as well as that of the cat. The cell membrane of neuron cell bodies is immediately external to the basement membrane of capillaries at the early fetal stages. No smaller intervening processes are observed between basement membrane-neuronal contacts. In the 16-week-old human fetus a larger proportion of the area external to the basement membrane of the capillary endothelium has become surrounded by smaller cell processes many of which are presumably dendrites. The basement membrane of the capillaries of the superficial neocortex in the adult cat is about 2 to 3 times thicker than that of the earliest stages described in this paper. Also, in contrast to the fetal cortex, fine processes (principally glial) of the neuropil are just external to the basement membrane of the capillaries. REFERENCES AREY,L. B., (1954); Development anatomy. Philadelphia, Saunders (p. 106107). CAJAL,RAMONY, S., (1911); Histologie du Systtke nerveux de 1’Homme et des Vertibris. Madrid, Consejo Superior de Investigaciones Cientificas. CONEL,J. L., (1939-1955); The postnatal Development of the human cerebral Cortex. Vols. I-VI. Cambridge, Mass., Harvard University Press. DEMPSEY, E. W.,AND WISLOCKI,G. B., (1955); An electron microscopic study of the blood-brain barrier in the rat employing silver nitrate as a vital stain. J. biophys. biochem. Cytol., 1, 245-256. DONAHUE, S., AND PAPPAS, G. D., (1961); The fine structure of capillaries in the cerebral cortex of the rat a t various stages of development. Amer. J. Anat., 108, 331-348. EAYRS,J. T., AND GOODHEAD, B., (1959); Postnatal development of the cerebral cortex of the rat. J. Anat. (Lond.), 93, 385402. GRAY,E. G., (1959); Axo-somatic and axo-dendritic synapses of the cerebral cortex. An electron microscopic study. J. Anat. (Lond.), 93, 4 2 M 3 3 . HORSTMANN, E., UND MEVES,H., (1959); Die Feinstruktur des molecularen Rindengraues und ihre physiologische Bedeutung. Z . Zelvorsch., 49, 469404. LUSE,S. A., (1962); Ultrastructure of the brain and its relation to transport of metabolites. Ultrastructure and Metabolism of the Nervous System. Baltimore, Williams and Wilkins (p. 1-26). MAYNARD, E. A., SCHULTZ, R. L., AND PEASE, D. C., (1957); Electron microscopy of the vascular bed of the rat cerebral cortex. Amer. J. Anat., 100,409-433. NOBACK, C. R., AND PURPURA, D. P., (1961); Postnatal ontogenesis of neurons in cat neocortex. J. comp. Neurol., 117, 291-307. PAPPAS, G. D., AND PURPURA, D. P., (1961); Fine structure of dendrites in the superficial neocortical neuropil. Exp. Neurol., 4, 507-530. PURPURA, D. P., SHOFER, R. J., HOUSEPIAN, E. M., AND NOBACK, C. R., (1963); Comparative ontogenesis of structurefunction relations in cerebral and cerebellar cortex. Progress in Brain Research, Vol. 4 . J. P. Schade and D. P. Purpura, Editors. Amsterdam, Elsevier (p. 187). SCHADB, J. P., AND BAXTER, C., (1960); Changes during growth in the volume and surface area of the cortical neurons in the rabbit. Exp. Neurol., 2, 158-178. TENNYSON, V. M., AND PAPPAS,G. D., (1961); Electron microscopic studies of the developing telencephalic choroid plexus in normal and hydrocephalic rabbits. Disorders of the Developing Nervous System. W. S . Fields and M. M. Desmond, Editors. Springfield, Ill., Charles C. Thomas. VOELLER, K., PAPPAS, G. D., AND PURPURA, D. P., (1963); Electron microscopic study of development of cat superficial neocortex. Exp. Neurol., 7 , 107-130.

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Comparative Ontogenesis of Structure-Function Relations in Cerebral and Cerebellar Cortex D. P. P U R P U R A , R. J. SHOFER, E. M. H O U S E P I A N A N D C. R. N O B A C K Departmenis of NeuroIogical Surgery and Anatomy, College of Physicians and Surgeons, Columbia University, New York, N . Y. (U.S.A.)

INTRODUCTION

Ontogenetic studies have long been recognized as powerful analytical approaches to the understanding of cortical organization. For the most part, however, such studies have emphasized sequential changes in one type of cortex without reference to the comparative ontogenetic development of phylogenetically different types of cortex in the same species. Several problems may be profitably examined by comparative ontogenetic studies of cerebral and cerebellar cortex. Of some interest is the question of whether the phylogenetically older cerebellar cortex undergoes an earlier ontogenetic development than the later evolving neocortex in the mammal. Considerably more importance attaches to the problem of the general pattern of postnatal development of the two types of cortex. In this context it is of interest to define the manner in which different patterns of morphogenesis are reflected in differences in functional activity. Such information may be of value in facilitating interpretations of structureactivity relations in mature cerebral and cerebellar cortex. Data on differential development may also provide important clues relevant to the problem of the susceptibility of different structures to various antenatal and postnatal traumatic or metabolic insults to the nervous system. This report surveys several types of investigations carried out in the past four years on the morphological and electrophysiological properties of cerebral and cerebellar cortical neuronal organizations during postnatal ontogenesis in the kitten. The major objective in these studies has been to characterize electrocortical activities (Purpura, 1959) in terms of their probable morphological substrates. The data obtainedZon the time-course of cerebral and cerebellar cortical development indicate that the fundamentally different patterns of neuronal morphogenesis in these structures satisfactorily account for the different electrographic characteristics of evoked potentials observed in immature cerebral and cerebellar cortex. I. D E V E L O P M E N T A L S T R U C T U R E - A C T I V I T Y R E L A T I O N S

I N F E L I N E N EOC OR TEX

( A ) Postnatal Ontogenesis of Neocortical Neurons (Light Microscopy)

Two types of studies have been particularly useful in providing a detailed picture of References p . 219-221

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Figs. 1-12. For legend see p. 189.

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the morphogenesis of neocortical neurons and synapses in the kitten: analysis of Golgi-Cox preparations and electron microscope studies (Noback and Purpura, 1961; Voeller et al., 1963). The latter studies are summarized elsewhere in this volume and will be referred to here only to indicate wherein they complement electrophysiological and light microscope observations. The criteria utilized to determine the maturational status of neurons in the mammalian cerebral cortex include: (I) the number of dendrites and their branches; (2) the size, caliber, and length of these processes; (3) the presence of dendritic spines (pedunculated bulbs, thorns or gemmules); and (4) the number and length of axoncollateral branches. In view of the fact that axons are usually incompletely revealed in Golgi-Cox preparations (Conel, 1939-1955), studies on pyramidal axon maturation were carried out separately (vide infru). For descriptive purposes, cortical neurons may be classified into three basic cell

Figs. 1-12 are untouched microphotographs of neurons revealed in 200 p-thick Golgi-Cox sections taken from the dorsolateral aspect of neocortex (suprasylvian and pericruciate cortex) in neonatal cat; birth to three days after birth. Pial surface of cortex is in upper part of or above each figure. (From: Noback and Purpura, 1961.) Figs, 1-7 are of neurons in the molecular (plexiform or first) layer. All are Retzius-Cajal or modified Retzius-Cajal cells (Cajal, 1959). Cells and processes are restricted to the molecular layer. Figs. I and 2. Retzius-Cajal cells with long horizontal dendrites extending parallel to the cortical surface. In Fig. 1 the length of the horizontal process from the cell body to its termination is 425 p. Note the collateral branches that extend at right angles from the dendrites toward the pial surface. Fig. 2, ~ 4 4 0 .Fig. 3. Modified Retzius-Cajal cell (marginal cell of Cajal, 1959, Fig. 173) with cell body at pial surface, x 440. Fig. 4. Retzius-Cajal cell illustrating the spherule (arrow) at the end of a collateral branch terminating at the pial surface, x 440. Figs. 5, 6 and 7. Retzius-Cajal cells, x440. Fig. 8. ‘Stellate’-type cell in molecular layer (Ca,jal, 191 1 and 1959) that may represent a modified Retzius-Cajal cell, x 220. Fig. 9. Pyramidal cell in sub-molecular layer. Note axon, arborization of apical dendrite and absence of basilar dendrites, x 220. Fig. 10. Pyramidal cell in submolecular layer. Apical dendrite terminates in several branches in the molecular layer. Several short, thin collateral branches of the apical dendrite are visible. Fine basilar dendrites and axon are demonstrated, x 220. Fig. I I . Pyramidal cell in the submolecular layer with two apical dendrites extending from cell body. Note axon and a few short basilar dendrites, x 600. Fig. 12. Pyramidal cell in deep pyramidal layer. The apical dendrite that extends to the molecular layer terminates in two branches that have a lateral spread that is less than the lateral spread of the pyramidal cells of the submolecular layer, x 140. The following observations pertain to Figs. 8-21. (1) The wide lateral spread (100 to 200 p) in layer I of apical dendritic branches of pyramidal cells of the submolecular layer is illustrated in Figs. 9, 10 and 14. The lateral spread of apical dendritic

branches of pyramidal cells of the deep pyramidal layer (Figs. 12,16) is less than that of the pyramidal cells in the superficial layers. (2) In general, the basilar dendrites and the collateral branches of the apical dendrites appear to be further developed in the superficial pyramidal cells of the superficial layer than in the deep pyramidal cells (compare Figs. 13 and 16). (3) The sequential changes in the maturation of cortical neurons from birth to 21 days are summarized as follows: (a) enlargement of cell body and elongation of cell processes; (b) increase in number of basilar dendritic branches; (c) increase in number of the collateral branches of apical dendrites; (d) increase in number of dendritic spines from a few in the 8-day-old to numerous in the 21-day-old cat, (4) The morphology of the pyramidal cells in a 21-day-old cat is similar to that in the neocortex of adult cat. (5) The dendrites of stellate cells lengthen and possess more spines as the interval from birth to 21 days progresses (Figs. 19, 20 and 21). References p . 219-221

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types : pyramidal neurons, stellate cells, and horizontal cells of Retzius-Cajal (Sholl, 1956). The maturation of these basic cell types may be summarized with reference to four stages: birth to 3 days, 8, 14, and 21 days postnatally. From the time of their initial, development until their morphological maturation during the first postnatal month, pyramidal neurons in the neocortex maintain their radial orientation with axons directed into the white matter and apical dendrites extending distally toward the molecular layer. Differentiation of the collateral branches of axons and of apical dendrites and basilar dendrites to produce tangential or lateral extensions is essentially a postnatal event in the cat. During the first postnatal month, neuronal density decreases due largely to the invasion and development of non-neural elements and overall increases in neuronal volume (Cajal, 1959; Brizzee and Jacobs, 1959; Eayrs and Goodhead, 1959; SchadC and Baxter, 1960, and elsewhere in this volume).

( I ) Birth to 3 days postnatally Neurons described at this stage include: (I) cells of Retzius-Cajal (Figs. 1-7); (2) pyramidal cells (Figs. 9-12); and (3) stellate cells (Fig. 8). Molecular layer: The cells of Retzius-Cajal are the horizontal cells in layer I (Figs. 1-7). These neurons whose cell bodies are located in the middle and lower third of the molecular layer have two long dendrites that extend parallel to the cortical surface, but in opposite directions. Their morphology in the newborn cat is similar to that in the newborn human infant (Cajal, 1911, 1959). Dendrites of these cells may exceed 1 mm and their axons probably extend for even greater distances (Cajal, 1959). Their branches emerge at right angles from tangentially oriented dendrites of RetziusCajal cells. These branches, 5-10 p apart, arise from small varicosities on the main dendrite. Other cells, probably modified-Retzius-Cajal cells, are also.prominent{These include: (I) neurons with their cell bodies at the pial surface (Fig. 3); and (2) ‘stellate’ cells (Fig. 8), Cajal’s (1959) triangular cells with short axon. Sub-molecular layer: Each pyramidal cell in this layer has its apical dendrites terminating and arborizing in the molecular layer (Figs. 9 and 10). The terminal portions of the apical dendrites extend to about 25 ,u of the pial surface. The arborization consists of two to‘four branches whose distal terminations have a lateral spread of up to 100-200 p. Most cells possess these short and unbranched basilar dendrites although some cells are devoid of basilar dendrites (Fig. 9). Spines and collateral branches are absent on apical dendrites but occasionally minute, bulbous dilatations are observed especially on the distal portions. Deep pyramidal layer: Pyramidal-cells in this layer exhibit features similar to those more superficially located. Aconspicuous feature of the newborn cat’s cortex is that the basilar dendrites of the deep pyramidal cells are not as differentiated as the cells of the sub-molecular layer. Many of the deep pyramidal cells have thin, short, unbranched (up to 20 p) basilar dendrites (Fig. 12), and several short apical dendritic collateral branches in layers 2, 3, and 4. Dendritic spines are absent on pyramidal neurons at this stage. Stellate cells exhibit somewhat greater differentiation in neonatal kittens. Dendrites

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Figs. 13-21, Figures are untouched microphotographs of cells in Golgi-Cox preparations from the neocortex of the dorsolateral aspect (suprasylvian and pericruciate cortex) of the cerebrum of young cats. Pial surface is above each figure. All figures at same magnification ( x 220). Axons are visible on all pyramidal cells. Pyramidal cells of the sub-molecular layer are from neocortex of an 8-day-old cat (Fig. 13), of a 14-day-old cat (Fig. 14), and of a 21-day-old cat (Fig. 15). Pyramidal cells of the deep pyramidal layer are from neocortex of an 8-day-old cat (Fig. 16), of a 14-day-old cat (Fig. 18), and of a 21-day-old cat (Fig. 17). Stellate cells are from the neocortex of an 8-day-old cat (Fig. 19), of a 14-day-old cat (Fig. 20), and of a 21-day-old cat (Fig. 21). For further observations see at the bottom of p. 189. References p . 219-221

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are conspicuous and appear as thin radially oriented elements directed mainly apically and basally from the cell body. (2) 8-day-old kitten (Figs. 13, 16 and 19) Molecular layer: The Retzius-Cajal cells, as illustrated in the neonatal stage, are not observed. Bipolar cells with horizontally oriented processes and stellate cells are noted occasionally but dendritic processes of these cells do not possess spines. Sub-molecular layer: The pyramidal cells (Fig. 13) at this stage are considerably more differentiated than at birth. The terminal arborizations of the apical dendrites in the molecular layer are apparently completed with the addition of a few branches in some neurons. Several branched collateral processes are noted on apical dendrites which are longer and thicker than in the neonatal period. All cells have a full complement of basilar dendrites, which are longer, thicker and more branched than at birth. Short axon-collateral branches are observed on pyramidal neurons at this stage. Deep pyramidal layer: Dendrites of the large pyramidal cells are thicker than at birth (Fig. 16). No additional branchings of the fine terminal arborizations in the molecular layer are present on the apical dendrites, but collateral branches are slightly more numerous on the proximal portions of apical dendrites close to the cell body. Basilar dendrites are more numerous and branched than at birth. Spines are present on apical dendrites, but none are observed on basilar dendrites. ( 3 ) 14-day-old kitten (Figs. 14, 18 and 20) The dendrites of all cells are thicker and longer (Figs. 14 and 18). Spines are present on main stem dendrites and on some large collateral branches. Basilar dendrites of sub-molecular layer pyramidal cells are longer and more branched. Those of the deep pyramidal cells (Fig. 18) are thick, long, branched and possess spines while short, blunt processes are lacking. Stellate cells (Fig. 20) are considerably advanced in their development. Their dendrites are thicker, longer and more branched than those in the previous stage. The thickest dendrites have spines. ( 4 ) 21-day-old kitten (Figs. 15, 17 and 21) The general morphological characteristics of neurons in the neocortex of the 3-week-old kitten do not significantly differ from those of the fully mature cat. Cells appear to have their full complement of dendritic processes. Pyramidal neuron basilar and apical dendrites and their collateral branches and stellate cell dendrites are longer, thicker and more branched. A marked increase in the number of dendritic spines is apparent except in the vicinity of cell bodies where they are sparse even in the cells of adult cats. ( B ) Postnatal Maturation of Pyramidal Neuron Axons Studies on the development of pyramidal neuron axons were carried out on elements contributing to the corticospinal tract. Histological studies of the medullary pyramidal tract of kittens at various ages were carried out on sections stained by the Bodian

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protargol method for axons and the Weigert and Kluver and Barrera technique for myelin sheaths.

Fig. 22. Microphotographs of medullary pyramidal tracts of kittens at various postnatal deveiopmental stages. A = 16 days; B = 28 days; C = 51 days; D = 100 days. Kluver and Barrera (1953) preparations. Some of the largest axons are shown (at arrows) in typical fields surveyed for construction of the histograms of Fig. 23, x 880.

Pyramidal tract axons in a standard high-power field were counted and classified in terms of overall axon-diameter at four developmental stages (Fig. 22). The results were compared with those obtained in the adult cat (Fig. 23). (1) Newborn kitten: All axons in the medullary pyramidal tract are unmyelinated and less than 2 p in diameter in the neonatal kitten. References p. 219-221

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(2) 10-16-day-old kitten: During this period early myelination is detectable in a few axons but no fibers exceed 2 p in diameter (Fig. 22A). (3) 4-week-old kitten: At this time pyramidal axons exhibit minor variations in size and degree of myelination (Fig. 22B). A standard high-power field contained 1240fibers of which only 3 % were greater than 2 ,u in diameter (Fig. 23). (4) 8-week-old kitten: A total of 885 axons were counted in a comparable high-power field; 96% of these were less than 2 p ; 2 4 % were 2-4 p ; and 1.7% were 4-6 p (Fig. 23). (5) 14-week-old kitten: 790 axons were counted of which 2 % were 2-4 p ; 1.6 % were 4-6 p ; and 1.2% were 6-10 ,u (Figs. 22 and 23).

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(6) Adult: After 14 weeks the major change in the pyramidal tract histogram consisted in the inclusion of a small number of 10-16 p fibers. Less than 1 % of 710 fibers in a standard high-power field were 10-16 p in diameter (Fig. 23). SUMMARY

The ontogenesis of pyramidal neuron dendrites in general is as follows: ( I ) apical dendrites; (2) branches of apical dendrites in the molecular layer; (3) and (4) primary basilar dendrites and collateral branches of apical dendrites near the cell bodies; (5) and (6) the branching of basilar dendrites and collateral branches of apical dendrites and differentiation of new collateral branches of apical dendrites; (7) appearance of dendritic spines and increase in length and caliber as well as number. Pyramidal neuron axons undergo relatively delayed growth and myelination at least with respect to differentiation of dendrites. Thus the morphogenesis of neocortical pyramidal neurons in the kitten may be viewed as a three-phase partially overlapping sequence of events involving first the development of apical dendrites, secondly a phase of cell body and basilar dendritic development in which apical dendritic development continues to completion and finally a period of axon growth and myelination (Fig. 24).

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( C ) Electron niicroscope studies of immature neocortex As described previously (Voeller et a/., 1963) and elsewhere in this volume, electron microscopy has provided additional details of neuronal organization during the postnatal ontogenesis of cat neocortex. For present purposes it is sufficient to point out BIRTH

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Fig. 24. General features of morphogenesis of large pyramidal neurons in feline neocortex. Dendritic ramifications were not added in 14-week and adult animals since there is little change in dendrites after the 1st month. Below, representation of axon-diameters of largest pyramidal fibers and their conduction velocities at various ages.

that cell bodies and dendrites with characteristic tubules are densely packed in superficial regions of neocortex in the immediate neonatal period. Close apposition (150-200 A) of dendrites and cell bodies is commonly seen at this developmental stage. Dendritic processes in the neuropil are relatively large and homogenous in fetal and newborn kittens. Dendritic terminals 300 A to 400 A in diameter are not observed until the sixth to ninth postnatal day. Of particular importance is the observation that axodendritic synapses with characteristics similar to those observed in adult animals (Gray, 1959; Pappas and Purpura, 1961) are seen in neonatal kittens. In contrast, axosomatic synapses are rarely observed until the end of the first postnatal week. These observations taken together with light microscope studies indicate a differential development of axodendritic synaptic pathways related to apical dendrites in the immediate neonatal period. By the fifth to seventh postnatal day, however, axosomatic synaptic pathways become well established. Further elaboration of axodendritic synapses on apical and basilar dendrites occurs during the entire second and third postnatal week. References p . 219-221

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( D ) Electrophysiological properties of immature neocortex ( I ) Activities generated by local stimulation of superfkial synaptic pathways The characteristic response recorded from the neocortical surface following weak local cortical stimulation in near-term fetal and newborn kittens consists of a surface negativity of variable duration whose magnitude is dependent on stimulus strength (Purpura, Carmichael and Housepian, 1960). The minimal duration of these superficial cortical responses (Adrian, 1936) (SCR) is 12-20 msec when recorded monopolarly with a fine wire electrode virtually in contact with the pair of wire electrodes employed for stimulation (Fig. 25). At recording distances greater than 1 mm from the site of

Fig. 25. Left: Characteristics of long-duration superficial cortical responses (SCR) recorded 1.5 mm from stimulating electrodes on suprasylvian gyrus in a near-term fetal kitten. Stimulus frequency 0.5/sec; six superposed responses at different stimulus strengths. Negativity upwards in this and all subsequent figures. Right: 5-h-old kitten. Comparison of SCR recorded 0.5 mm from stimulating electrodes with a large (0.5 mm) ball-tipped electrode (upper channel) and SCR recorded ‘at site of stimulation’ with 0.1 mm tip-diameter wire electrode. When recorded with wire electrode at site of stimulation, SCR has characteristics identical to those recorded in the adult animal. Calibrations I00 cycles/sec; 0.1 mV. (From : Purpura, 1 96 la.)

stimulation the SCR may be of relatively longer duration due to summation of additional 10-20 msec surface negative components (Purpura, 1961b ; Purpura, Carmichael and Housepian, 1960). The SCR is detectable 5-7 mm from the site of stimulation in neonatal kittens, whereas it is recordable at greater distances (8-10 mm)

Fig. 26. Typical responses recorded with a 0.5 mm tip-diameter electrode placed 1-2 mm from stimulating wire electrodes in locally anesthetized, paralyzed kittens, aged: 1 = 8 h ; 2 = 12 h ; 3 = 18 h ; 4 = 1.5 days; 5 = 2 days; 6 = 2.5 days; I = 4.5 days; 8 = 7 days; 9 and 10 = 9 days; 11 = 5 weeks; 12 = 6 weeks. Note long duration of SCRs and variable relationship of early and late components. Calibrations 100 cycles/sec. (From : Purpura, Carmichael and Housepian, 1960.)

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after the first postnatal week. Linear increases in latency of distant responses are observed at all stages of development when care is taken to insure that distant responses are not compounded of prior surface-positivity. In agreement with similar findings in adult animals (Brooks and Enger, 1959), SCR’s recorded at distant sites from the locus of stimulation in 2-3-week-old kittens may be of greater magnitude than those recorded close to the stimulating electrodes. Examples of S C R s recorded 1.5 to 2.0 mm from stimulating electrodes in kittens ranging in age from 8 h to 9 days are shown in Fig. 26 (1-10) arranged in order of increasing age. S C R s evoked in 5- and 6-week-old kittens are shown in Fig. 26 (1 1,12). All the responses were recorded from mid-suprasylvian gyrus with the same stimulating-recording electrode combination. Attention is directed to the variability in the overt characteristics of S C R s as well as their long duration in neonatal preparations. Clear dissociation between the initial 10-15 msec surface negative component in the SCR and later components is seen as early as the third postnatal week and by the 4th to 5th weeks the adult pattern is fully established (Fig. 26, 10). The characteristics of the SCR evoked in neonatal kittens are readily accounted for by the presence of relatively well-developed superficial axodendritic synaptic pathways as described above. The major locus of action of a weak surface stimulus such as that employed in the experiments of Figs. 25 and 26 is undoubtedly confined to the superficial regions of cortex (Adrian, 1936; Burns, 1958; Chang, 1951; Clare and Bishop, 1955; Ochs, 1962; Purpura, Girado and Grundfest, 1960; Purpura and Grundfest, 1956). Such a stimulus initiates conductile activity in horizontally oriented axons and postsynaptic potentials are generated in closely approximated apical dendrites (Eccles, 1951 ; Purpura and Grundfest, 1956). The long duration of the SCR recorded

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Fig. 27. Early phases of near and distant SCR’s in a 12-h-old-kitten. 1 and 2 = responses recorded 1.5 mm from stimulating electrodes. In 1 , C: T; both responses shown in isolation and superposed. Broken line drawn through peak of C response. Increment in C response detectable with 5 msec C-T stimulus interval. In 2, C : T; both responses shown in isolation and superposed. Broken line drawn through peak of T response. Reduction in T response with 5 msec C-T interval is attributable to residual depression of elements activated by C stimulus. Considerable recovery seen at 6 msec C-T intervals. 3 = SCR’s recorded 5 mm from stimulatingelectrode. Conditioning SCR shown in isolation as first response of series. With C = T, first significant increment is detectable at 4.5 msec stimulus intervals; example of further temporal summation with 1 1 msec C-T interval. Calibrations 0.1 mV; 100 cycles/sec in 1 and 2; lo00 cycles/sec in 3. (From: Purpura, Carmichael and Housepian, 1960.) References p . 219-221

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at sites remote from the locus of stimulation is thus referable to summation of PSP’s in dense populations of dendritic elements (Pappas and Purpura, elsewhere in this volume). It follows from the known morphological properties of dendrites in the newborn kitten that the hypothesis concerning propagation of the SCR in dendrites to account for the spread of this response outward from the site of stimulation (Chang, 1951) is rendered untenable by the fact that dendrites of pyramidal neurons in the neonatal kitten do not have tangential branches that are longer than 50-100 p (Figs. 10 and 12). Although the ontogenetic data on the SCR are indicative of the operation of functional synaptic pathways in the superficial neuropil of newborn kittens, fundamental differences in the properties and organizations of these pathways in immature and mature animals may be inferred from additional studies. Activity cycles of SCR’s in newborn kittens reveal relatively prolonged absolute unresponsive periods (Fig. 27). Delayed synthesis of transmitter(s) coupled with refractoriness in the presynaptic conductile pathway might account for this period of unresponsiveness. The absolute unresponsive period shortens to less than 1.5 msec by the end of the first postnatal week, at a time when no apparent change is detectable in the conductile properties of presynaptic elements (Fig. 28). This change in excitability reflected in shortening of

Fig. 28. Comparison of early phase of activity cycles of near (1.5 mm) and distant (5.0:mm) SCRs in a 10-day-old kitten. Conditioning response (C) larger than testing response (T). Numbers above records indicate stimulus interval. Minimal increment in both near and distant responses at 1.5 msec stimulus intervals, marked increment at 3 to 10 msec intervals. Note extraordinary magnitude of smoothly summated distant responses at 10 msec C T stimulus interval. Delayed recovery still evident at 16 msec. Calibrations 100 cycles/sec; 0.3 mV. (From: Pupura, Carmichael and Housepian, 1960.)

absolute responsive periods, and occurs at a time when distant reinforced SCRs are detectable, i.e., after the first postnatal week. This suggests that temporal summation, as well as spatial summation of PSP’s generated in apical dendrites may contribute to the development of distant responses of great magnitude. These findings also suggest that during the first week there is a marked acceleration of enzymatic mechanisms subserving the manufacture, storage and release of transmitters, as well as the production of new synaptic contacts on apical dendrites. Changes occurring in the intracytoplasmic organelles of neurons are indicative of the high rate of metabolic activity of these elements during the first postnatal week (Voeller et al., 1963). Additional support for this notion may be found in the literature on the biochemistry of the developingnervous system (Flexner, 1955; Roberts, 1960;and elsewherein this volume)

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and in recent studies on amino acid metabolism of developing kitten neocortex (Berl and Purpura, 1963). The different effects of pharmacological agents on SCRs evoked in newborn and older kittens are also suggestive of the changes occurring in organizations of superficial axodendritic synaptic pathways during the first three postnatal weeks. Particularly noteworthy are differencesobserved with respect to the actions of topically applied aliphatic w-amino acids which produce rapid augmentation of SCRs evoked in mature neocortex (Purpura, 1960; Purpura et al., 1959). Topical application of Iong chain ‘convulsant’w-amino acids in newborn animals produces minimal depressionof SCRs despite the fact that there is some augmentation in background electrocorticalactivity. Towards the end of the third postnatal week the depressant effects of long chain w-amino acids are no longer observed. Comparison of the different effects of &-amino caproic acid (c6)and w-amino caprylic acid (CS)on near SCRs evoked in an 8-day-old animal, and of c6 on near and 4 mm distant responses in a 3-week-old litter-mate is shown in Fig. 29. In the 8-day-old animal, c6 depressed all components of the SCR, whereas c8 depressed early and augmented late components and induced convulsant activity. In the 21-day-old litter-mate, c6 rapidly augmented all components of the near SCR, but did not significantly alter distant responses. Augmentation of the second, late negativity in near SCRs was more prominent than the increase in the early negativity. The onset of paroxysmal discharges after CS resulted in marked fluctuations in late components of near responses and depression of distant SCRs. Paroxysmal discharges, apparently triggered by the surface stimulus and initiated close to the site of stimulation were propagated to distant sites at a velocity similar to that of distant SCRs (Fig. 29). By the fourth postnatal week, both c6 and c8 rapidly augmented all components of near SCRs as in mature animals (Purpura et al., 1959). Interpretation of the different effects of long chain w-amino aliphatic acids on S C R s evoked in newborn and 34-week-old kittens follows from their presumed 6 days

21 days

Fig. 29. Comparative effects of &-aminocaproic acid (CS)on SCRs in 8- and 21-day-old litter-mates. 1 and 2 = control SCRs recorded 1.0 mm from site of stimulation; 3 and 4 = 2 min after topical c6; 5 and 6 = after complete recovery from depressant effects of c6, and 2 min after w-amino caprylic acid (CS);differences in the actions of c6 and CS are apparent; note in particular changes in late components of SCR after C S ;7 and 8 = control near (upper channel) and 4 mm distant (lower channel) SCRs; 9 and 10 = immediate effects of c6; rapid augmentation of early and late components of near SCR with more pronounced action on late components; distant response unaffected; 11 and 12 = onset of C6-induced convulsant activity. Fast sweep 100 cycles/sec; slow 10 cycles/sec. (From: Purpura, 1961b.) References p . 219-221

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mode of action observed in studies of mature animals. Suffice it to say that the convulsant effects of these components have been attributed to blockade of inhibitory PSP’s generated in apical dendrites (Purpura et a/., 1959). The failure to observe augmentation of SCRs in newborn kitten following topical application of long chain w-amino acids may be ascribed to the absence of a substrate of inhibition in superficially activated pathways in the immediate neonatal period. Viewed in this fashion the data suggest that functional maturation of some inhibitory axodendritic synaptic pathways in superficial neuropil of cat neocortex proceeds at a considerably slower rate than maturation of excitatory synaptic pathways. That sustained states of paroxysmal activity are not more commonly observed in newborn cat neocortex (Grossman, 1955) may be due to the relative paucity of neuronal interconnections and the restrictions imposed by the prolonged post-excitatory depression of processes operating at superficial axodendritic synapses in the neonatal period. The phase of maximum neocortical neuronal development occurs during the second postnatal week and is heralded by a significant increase in the capacity for temporal and spatial summation of excitatory PSP’s in superficialdendrites. This period corresponds to the time during which inhibitory synaptic activities in the SCR become demonstrable with long chain w-amino acids. Thus, despite initial differences in the temporal pattern of development of inhibitory and excitatory processes, both appear to attain full expression by the third to fourth postnatal week. (2) Activities generated in immature neocortex by corticipetal path ways The differential development of superficial axodendritic synaptic pathways in the 1

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Fig. 30. A = Primary responses evoked by supramaximal contralateral sciatic nerve stimulation in locally-anesthetized, paralyzed newborn kitten before (1) and after (2) topically applied GABA. 3 = Recovery after rinsing cortex with warm Ringer’s solution. B = Same sequence as in A, but primary responses evoked by weak sciatic nerve stimulation. Note minimal late triphasic potential following the initial surfacenegativity (Bl) and development of late components immediately after recovery from GABA action (B3). C = Same sequence as in A, but after injection of 15 mg/kg pentobarbital sodium. Initial surface-negativity is augmented and late components are abolished in transition from unanesthetized state. D = Primary responses evoked by contralateral sciatic nerve stimulation in a 2-week-old kitten. Topical application of GABA eliminates surface-negativity and increases the amplitude and duration of surface-positivity (D2). D = Early phase of recovery from GABA action. Note enhancement of long-latency slow negativity during partial recovery of the negative component of the diphasic primary. Calibrations A-C, 200 cycles/sec; D, 100 cycles/sec. (From: Purpura, 1962.)

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neonatal period adequately accounts for the striking features of primary evoked potentials in somesthetic (Scherrer and Oeconomos, 1955) and visual cortex in newborn kittens (Ellingson and Wilcott, 1960) and rabbits (Hunt and Goldring, 1951). As is now well established through several lines of investigation (Anokhin, 1961; Marty, 1962; Marty and Scherrer, in this volume; Purpura, 1961a), primary responses evoked in newborn animals are predominantly surface-negative and lack the initial surface-positive component characteristic of responses elicited in mature animals. In the case of sciatic nerve stimulation the prominent surface-negativities generated in somesthetic cortex of newborn kittens are often succeeded by additional complex potentials that are exquisitely sensitive to barbiturates (Fig. 30). Early surface-positivity is detectable in primary responses of kitten somesthetic cortex by the 5th to 6th day at which time the overt configuration of such responses is fundamentally similar to that of the adult animal except in latency and duration of both surface-positive and -negative phases (Fig. 31). Analysis of the sciatic evoked responses in somesthetic cortex of newborn kittens indicates that all components of responses including late multiphasic potentials are reversibly eliminated by topical application of 1 % y-aminobutyric acid (GABA) (Fig. 30). In contrast the effects of GABA on specific evoked responses in 1- to 2-week-old kittens are similar to those described in mature cat (Iwama and Jasper, 1957; Purpura et al., 1959). Results obtained in the kitten essentially confirm those reported in the newborn rabbit (Anokhin, 1961). The explanation of the differences in the electrographic characteristics of specific responses of somesthetic cortex in newborn and 1- and 2-week-old kittens may be sought in the rapid development of synaptic pathways related to basilar dendrites and

Fig. 31. Ontogenetic changes in primary responses evoked by contralateral sciatic nerve stimulation. Initial surface-negativityis succeeded by a triphasic potential in 1-day-old kitten (time bar, 50 msec). In a 6-day-old kitten initial surface-positivity is small (time bar, 100 msec). By 2 weeks latency decreases and diphasic configuration of the primary response is fully established (time bar, 50 msec). At later stages (5 weeks) the initial surface-positivitymay be of greater magnitudethan the subsequent negativity (time bar, 100 msec). (From: Purpura, 1962.)

cell bodies of neurons in the cortical depths (vide supra). The rapid and reversible elimination of surface-negativity by topical GABA suggests that in the immediate neonatal period thalamocortical pathways activated by sciatic nerve stimulation are distributed primarily in relation to superficial dendritic elements (Anokhin, 1961; Purpura, 196la) most likely through small cortical interneurons synaptically linked to apical dendrites. The surface-positivity of the specific evoked response is generally held to be a reflection of postsynaptic activity of a large population of deep-lying References p . 219-221

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cortical elements (Purpura, 1959; Purpura et al., 1963). This component of the primary response becomes prominent in kittens at a time when elements in the cortical depths have developed relatively large surface areas for contacts with thalamocortical afferents. On the basis of the ontogenetic data the sequence of events involved in the production of the characteristic diphasic positive-negative response of primary projection cortex may be visualized as a series of synaptic activities independently generated in deep and superficial neuropil. The two-stage activation process thus reflects the operation of two species of specific thalamocortical afferents : one, early developing but slower conducting, that terminates in relation to superficial dendritic elements; the other later developing and faster conducting, that terminates in relation to elements comprising the deep cortical neuropil. The fact that the prominent surface-negativity of the specific evoked response of somesthetic cortex in the newborn kitten is devoid of prior surface-positivity clearly indicates that the surface-negativity is not referable to antidromic invasion of apical dendrites by a wave of depolarization originating in the soma and basilar dendrites of deep-lying pyramidal neurons (Bishop and Clare, 1952). Despite the apparent simplicity of primary responses in newborn kittens, it cannot be assumed that only excitatory axodendritic synaptic activities are generated in superficial neuropil by peripheral nerve stimulation. Involvement of more complex activities involving inhibitory pathways terminating at different loci on cortical pyramidal neurons and interneurons must also be recognized. This follows from the combination of the effects of pharmacological agents on responses elicited in somesthetic cortex of neonatal animals by lateral thalamic stimulation (Fig. 32). Under these conditions a wide variety of additional components are revealed indicating the operation of several inhibitory and excitatory synaptic pathways as in the case of

Fig. 32. Effects of topical GABA and strychnine on ‘primary’ response evoked in posterior sigmoid gyrus following lateral thalamic stimulation (stimulus frequency O.S/sec); 2-day-old kitten. 1 = weak and 2 = strong stimulation; 3 and 4 = stimulus as in 1 and 2 after topical GABA; 5 and 6 = superposed tracing of control response as in 1 and 2 and responses after GABA as in 3 and 4; 7 and 8 = early stages of recovery from GABA-effect; 9 and 10 = full recovery; 11-14 = genesis of changes induced by topical strychnine (0.1 %) in response to strong stimulation; 15 and 16 = immediate effect of GABA applied to strychninized cortex, weak and strong stimulation as in 3 and 4; 17 and 18 = recovery from GABA effects and reappearance of strychnine action. Sweep duration 150 msec in all records. (From: Purpura, 1961b.)

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mature neocortex (Purpura, Girado and Grundfest et al., 1960; Purpura et al., 1963). The employ of additional analytical methods including intracellular recording techniques may be expected to further define the contribution of excitatory and inhibitory PSP’s to primary responses at various developmental stages. The hypothesis that synaptic pathways related to apical dendrites of cortical pyramidal neurons are primarily involved in the production of a major portion of the spontaneous and evoked electrocortical activities observed in the newborn kitten would appear to account satisfactorily for a number of observations on the characteristics of non-specific evoked activities elicited at this developmental stage. Such activities are not only predominantly surface-negative in overt configuration, but exhibit a laminar distribution that is consistent with the interpretation of their origin in vertically oriented elements subtending the upper half of cortex in the newborn kitten (Purpura, 1962). A description of the characteristics of these non-specific neocortical evoked activities elicited by midline thalamic and ponto-mesencephalic reticular stimulation in neonatal kittens is beyond the scope of the present survey. It has been pointed out elsewhere (Purpura, 1961a) that the wide variety of changes in non-specific evoked activities including the generation of 10-14/sec hypersynchronous activity during high-frequency reticular stimulation in newborn kittens (Fig. 33) are

Fig. 33. Electrocorticalactivation patterns recorded from suprasylvian gyrus following 50/sec (A-E) and 1OO/sec (F) stimulation of medial ponto-mesencephalicregions. Locally anesthetized, paralyzed newborn kitten. Records taken from a continuous strip show pattern of development of 10-14/sec synchronous activity initiated by high-frequency brain stem stimulation. (From : Purpura, 1962.)

to be considered reflections of the complex organization of excitatory and inhibitory synaptic pathways in neocortical as well as subcortical neuronal organizations in neonatal animals. (3) Functional maturation of neocortical pyramidal axons and axon-collaterals The foregoing sections have reviewed data indicating that the increase in synaptic receptor surface of pyramidal neurons during the first three postnatal weeks is associated with well-defined changes in evoked cortical activities. After the 3rd week little alteration in the overt electrographic characteristics of a variety of evoked activities is observed. There are, however, marked changes in the latency of most References p . 219-221

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types of evoked potentials after the 3rd week. Such latency changes are obviously related to growth and myelination of central (Grafstein, 1963; Langworthy, 1929; Marty, 1962; Ulett et al., 1944; and Windle et al., 1934) and peripheral pathways (Eccles et al., 1963; Hursh, 1939; Skoglund, 1960; and Wilson, 1962). To obtain a more complete picture of the development of pyramidal neurons requires examination of the time-course of functional as well as anatomical (Fig. 22) changes in their axons. Analysis of the general characteristics of responses evoked in the medullary pyramidal tract by motor cortex stimulation has provided this information. The fact that such stimulation also elicits activity in corticofugal projections to brain stem has also permitted some evaluation of the relative contribution of pyramidal and extrapyramidal activities to cortically evoked motor responses in neonatal kittens (Henry and Woolsey, 1943). (a) Directly evoked responses in medullary pyramidal tract. The earliest detectable responses recorded in the medullary pyramidal tract following pericruciate cortex stimulation in neonatal kittens consisted of diphasic or triphasic discharges whose latencies varied from 20-40 msec (Fig. 34A). Estimates of the length of the conductile

W/MhV/$FJ Fig. 34. Ontogenetic changes in evoked medullary pyramidal tract activity. Motor cortex stimulation at O.S/sec 3-10 superimposed sweeps of responses recorded with saline-filled micropipettes of 100 p. Teflon-coated silver wires in ventral pyramid. Ages of kittens as follows: A = 1 day; B = 10 days; C = 25 days; D = 33 days; E = 43 days; F = 57 days; G = 98 days. Calibrations: A-C = 100 cycles/sec; D = 500 cycles/sec; E-H = LOO0 cycles/sec. Long-latency (25-30 msec) triphasic responses are recorded during the first few weeks. The general features of evoked pyramidal tract activity similar to those observed in adult animals are seen in 45-week-old kittens. Latencies of directly initiated responses less than 1 msec are not recorded in the medullary pyramidal tract until after the 4th month. Further explanation in text.

pathway from pericruciate cortex to ventral medulla (hereafter referred to as PC-VM) during this period ranged from 2&25 mm. The conduction velocity of corticospinal axons giving rise to the prominent compound action potentials recorded in the ventral medulla was 0.8-1.5 m/sec. Towards the end of the second week the earliest detectable responses in the ventral medullary pyramidal tract varied from 15-18 msec (conduction velocities about 2 m/sec). During the 2nd and 3rd postnatal weeks little change occurred in the conduction velocity of fibers giving rise to the shortest latency response recorded in the medullary pyramidal tract. However, significant increases in conduction velocity occurred during

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the fourth and fifth weeks (Fig. 34C). Latencies of 2-4 msec and conduction velocities of 12-20 m/sec were consistently recorded. By the end of the first month medullary pyramidal tract activity was electrographically similar to that recorded in adult animals. One exception was the finding of prominent discontinuities on the initial positivity of the evoked response (Fig. 34D) suggesting residual temporal dispersion in axons contributing to initial direct responses. During the 2nd to 4th month progressive shortening of the latency of the earliest direct response occurred (Fig. 34, E-H). The discontinuity on the initial phase of the earliest response became less prominent and was no longer obvious after the 8th week. Towards the end of the 4th to 5th month the latency of the initial component of the medullary pyramidal tract response was 0.7-0.9 msec. The response was succeeded by several positive deflections repeating at 2.0-2.5 msec intervals as in adult animals (Patton and Amassian, 1960; Purpura and Grundfest, 1956). Results similar to those shown in Fig. 34 are summarized in Fig. 35. Emphasis is placed on the relatively 90

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Fig. 35. Changes in conduction velocity of largest fibers in the kitten corticospinal tract during postnatal ontogenesis. Note rapid increase in conduction velocity during the 4th-6th weeks, then slow, progressive increases up to the end of the 5th month.

minor change in the conduction velocity of corticospinal axons giving rise to the earliest detectable response in the medullary pyramidal tract during the first two weeks. Attention is also directed to the rapid change in conduction velocity of these elements during the 4th and 5th weeks and the further slow increase in conduction velocity during the subsequent 8 to 10 weeks. (b) Indirectly relayed responses in corticospinal axons. It is generally held that the initial component of the repetitive sequence recorded in the pyramidal tract in adult or young cats (Fig. 34, G and H) represents conductile activity initiated by direct stimulation of corticospinal neuron cell bodies or axons whereas the second and all subsequent components are indirect or synaptically relayed responses of corticospinal neurons (Patton and Amassian, 1960; Purpura and Grundfest, 1956). Were it possible to determine the developmental stage at which indirectly relayed responses are initiated by pericruciate cortex stimulation data would be provided on the maturational status of axon-collateral-interneuronal organizations synaptically linked to corticospinal neurons. One way in which this information may be obtained takes advantage of References p. 219-221

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findings that during low frequency repetitive motor cortex stimulation indirectly relayed responses are augmented, whereas during post-stimulation phases indirect responses exhibit marked variations in amplitude and duration in association with cortical seizure activity (Purpura, Girado and Grundfest, 1960). No evidence has been obtained in animals less than three weeks old that late components of the medullary pyramidal tract responses to strong cortical stimulation represent indirectly relayed tract activity. Strong low-frequency repetitive cortical stimulation occasionally induced a minor increase in late components of the evoked pyramidal tract response in 34week-old kittens. Unequivocal augmentation of late components of repetitively evoked tract responses was observed after the 4th week (Fig. 36). Then single shock stimulation of pericruciate cortex elicited a 2 msec latency

Fig. 36. Effects of repetitive motor cortex stimulation on directly and indirectly evoked activity in medullary pyramidal tract of a 30-day-old kitten. Arrow in A indicates synaptically induced (indirect) responses. A = 0.5/sec stimulation. Augmentation of late component occurs at 25/sec stimulation (B), whereas failure of this response is seen at 50/sec stimulation (0.D-F = responses elicited by 0.5/sec motor cortex stimulation 5-30 sec after 50/sec stimulation. Facilitation of the late positive component of tract activity occurs immediately after termination of motor cortex tetanization (D). A prolonged period of depression of all responses ensues, then recovery of early phases but persisting depression of late positivity (G).

direct response with a prominent discontinuity and a second positive component (Fig. 35A). The latter was followed by a longer duration positivity that augmented during 25/sec stimulation (Fig. 36B). During 50/sec stimulation late components were abolished (Fig. 36C), but during the post-tetanic period marked enhancement of O.S/sec evoked responses was observed (Fig. 36D). This phase of facilitation was succeeded by depression of all components of the evoked medullary pyramidal tract activity (Fig. 36, E-F) then gradual recovery of responsiveness. Similar results were obtained in all kittens over 5 weeks of age tested in this fashion. It is inferred from these data that intracortical axon-collaterals of corticospinal neurons established powerful synaptic contacts with interneurons synaptically related to Betz cells after the 3rd week. Since these interneurons exert both recurrent excitatory and inhibitory actions on corticospinal neurons (Purpura and Grundfest, 1956)it may be argued that only after the 3rd to 4th week do efferent discharges from motor cortex have temporal and spatial characteristics qualitatively similar to those of adult animals. This has also been established with respect to relayed volleys in the pyramidal tract subsequent

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to stimulation of specific and non-specific thalamocortical projections to motor cortex. Such relayed volleys in 5-6-week-old kittens are, however, of longer latency and more temporally dispersed than relayed volleys observed in adult animals (Brookhart and Zanchetti, 1956; Purpura and Housepian, 1961). (c) Evoked corticofugalactivity in brain stem synaptic organizations. Several varieties of responses to pericruciate cortex stimulation have been recorded in medulla, pons and lower mesencephalon during exploration of these areas prior to placement of electrodes in the medullary pyramidal tract. The most frequently encountered activity in neonatal kittens consisted of long-latency (50-70 msec) long-duration responses of relatively large amplitude. These responses were detectable throughout the entire extent of the exposed brain stem. Some examples of ipsilateral evoked responses recorded dorsal to the medullary pyramidal tract in kittens of various ages are shown in Fig. 37. The latency and overt configuration of these responses were essentially

Fig. 37. Responses recorded in medial medullary reticular regions following motor cortex stimulation. Ages of kittens as follows: A = 5 days; B = 21 days; C = 28 days. Latencies ranging from 40-70 nisec were observed prior to the 4th week. Thereafter latency decreases were associated with responses exhibiting more complex configurations than those recorded in 1-2-week-old kittens. D-F = focally recorded activity in medial bulbar regions following motor cortex stimulation in a 22-day-old kitten. D = saline-filled micropipette located in region of prominent focal negativity. E = 50 ,u downward displacement of recording electrode and F = another 50 ,u downward rnove.%ent. The different responses recorded in D and F are indicative of activities of different reticular organizations as evidenced by the lack of relationship between focally recorded negative and positive components. Calibrations A-C = 50 cycles/sec; D-F = 100 cycles/sec.

unchanged up to the 3rd postnatal week. Considerable shortening of latency was noted during the 4th-5th week. In kittens of all ages responses might be initially positive in polarity or exhibit prominent initial negative components (Fig. 37D). In the latter instance advancement of the recording microelectrode 50-100 p frequently resulted in prompt 'inversion' of focally recorded responses (Fig. 37, E and F). The extent of cortex giving rise to projection pathways involved in the production of prominent brain stem activities in young kittens was established by comparing responses evoked by weak stimulation of different cortical areas. In kittens less than 3 weeks old maximal effects were observed following stimulation of pericruciate cortex, whereas little or no activity was recorded in the brain stem during stimulation of other areas of rostra1 neocortex (Fig. 38, A-D). Focally recorded slow waves in the brain stem of 5-7-day-old kittens often exhibited multi-unit discharges on their various phases (Fig. 38E). These discharges along with Referenws p. 219-221

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the slow components of the response were eliminated following intravenous administration of 10-15 mg/kg of pentobarbital sodium (Fig. 38F). Recovery from this amount of barbiturate was usually not complete after 1 h (Fig. 38G).

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Fig. 38. A-D = 12-day-oldkitten, 0.5/sec stimulation of motor cortex recordingfrom medial pontine reticular region. A and B = responses evoked by supramaximal and threshold stimulation of motor cortex respectively. Stimulus strength identical to that used in B was employed in stimulation of posterior sigmoid gyrus (C) and anterior suprasylvian gyrus (D). Origin of projection pathways to medial pontine reticular region was confined to pericruciate cortex in young kittens. E-G = 7-day-old kitten, recording from contralateral midbrain reticular region. Stimulation of pericruciate cortex elicited multiphasic response with superimposed spike-like deflections (E). F = immediately after 15 mg/kg pentobarbital sodium. G = 1 h after F. Calibrations 100 cycles/sec.

The foregoing results establish that in neonatal kittens corticofugal activity originates largely in rostral cortical areas and is conducted to brain stem and spinal cord in pathways with conduction velocities less than 1 m/sec. The fact that motor cortex stimulation in newborn kittens is capable of activating topographically related muscle groups (Henry and Woolsey, 1943) or restricted movements (Weed and Langworthy, 1926) clearly indicates that the effects of such volleys on spinal interneuron-motoneuron organizations are by no means insignificant. The present results favor the view that in newborn kittens effects exerted by pathways arising in rostral neocortex on spinal reflex activities are probably mediated by extrapyramidal outflows from the brain stem. This follows from the relative potency of synaptic drives evoked in midbrain and pontobulbar organizations by motor cortex stimulation in newborn kittens in contrast to the activity directly conducted in the medullary pyramidal tract. In this connection it is of interest that the motor performance observed in adult cats is achieved in kittens during the 2nd month (Windle et al., 1934). It is now established that by the 3rd postnatal week motoneurons of kittens have similar properties and connections as motoneurons in adult cats (Eccles et al., 1963). Differences in reflex activities are at this time attributable solely to delayed conduction in afferent and efferent fibers (Skoglund, 1960; Wilson, 1962). These differences are likely to be important in determining the speed and precision of movements in young kittens. It is also probable that the repertory of movements characteristic of kittens during the 2nd month (Windle et al., 1934) is dependent on the development of temporo-spatial patterns of impulses in corticofugal and bulbospinal pathways. These patterns could result from the elaboration of intracortical synaptic connections of pyramidal neuron axon-collaterals and interneurons as well as further development of brain stem and

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cerebellar organizations. At least it is clear that the earliest developmental changes in electrocortical potentials and overt behavioral activities are related to the maturation of local synaptic interrelations between neurons, whereas later ontogenetic changes result from maturation of conductile pathways which relate synaptic organizations at various neuraxial sites. SUMMARY

Neocortical activities evoked by local subcortical or peripheral nerve stimulation in neonatal animals are characterized by prominent surface-negative components of variable duration. These responses are assignable to postsynaptic activities of apical dendrites and probably synaptic activities of dendrites of stellate cells located at various cortical depths. Remarkably complex electrocortical potentials may be elicited in neonatal animals indicating the operation of powerful synaptic systems in the superficial neuropil. Additional complexities in evoked responses are referable to the increase in receptor surface of neurons provided by the growth and differentiation of deep cortical neuropil as well as changes in the overall effectiveness of excitatory and inhibitory synaptic drives. Elaboration of synaptic organizations related to dendrites and cell bodies precedes the period during which pyramidal neuron axons exhibit msrked changes in their conductile properties and intracortical synaptic distribution. After the 1st month the major neocortical maturation event is related to an increase in conduction velocity of pyramidal neuron axons. The processes of growth and myelination which underlie changes in physiological properties of pyramidal neuron axons proceed slowly to completion during the 4th to 5th month. 11. D E V E L O P M E N T A L S T R U C T U R E - A C T I V l T Y R E L A T I O N S I N

FELINE CEREBELLAR CORTEX

( A ) Postnatal Ontogenesis of Purkinje Cells The histogenesis of the mammalian cerebellum has been described in different species (Addison, 1911 ; Cajal, 191 1; Chiarugi and Pompeiano, 1954) and has been investigated more recently with histochemical and autoradiographic techniques (Sidman and Miale, 1961; Uzman, 1960). Two general features of the maturation of cerebellar cortex in the kitten are described here in connection with electrophysiological studies : the changing morphologicsrl characteristics of Purkinje cells and the relationship of Purkinje cell dendrites to elements constituting the external granular layer (EGL) (cf. Dow and Moruzzi, 1958). ( I ) 1st week: Purkinje cells in neonatal kittens are readily identified in Golgi-Cox preparations. These elements have a well-developed main stem axon which can be traced into the underlying white matter. Numerous small highly branched axoncollaterals are observed apparently distributing in relation to adjacent Purkinje cells and other neurons. The cell body does not have a smooth contour such as is1observed in Purkinje cells of slightly older kitten. Numerous short, protoplasmic projections References p . 219-221

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Fig. 39. For legend see p. 211.

are observed which give the appearance of thin perisomatic dendritic ramifications similar to those seen in pyramidal neurons of neonatal neocortex (Fig. 39A). These perisomatic processes appear to be resorbed within 4-5 days after birth. The main

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dendritic trunk(s) is frequently not much more prominent than the perisomatic extensions. This trunk is generally bifid and is covered with numerous small protuberances. The tips of these dendrites terminate at the lower border of the EGL. This relationship is particularly well demonstrated in 200 p-thick Golgi-Cox preparations with a Nissl counterstain (Fig. 40A). The EGL is approximately 10-12 cell layers thick

Fig. 40. Microphotographs of Purkinje cells revealed in 200 p-thick Golgi-Cox sections of cerebellum counterstained with Nissl to show relationship of superficial dendritic terminals to external granular layer. Ages of kittens as follows: A = 2 days; B and C = 8 days; D = 20 days; E = 30 days; F = 42 days; G = 57 days. Horizontal bar is 100 p for all microphotographs.

Fig. 39. Microphotographs of Purkinje cells revealed in 200 p-thick Golgi-Cox sections of cerebellum in kittens of various ages. A = 2-day-old kitten. Main stem dendrites are short, occasionally branched and have several protuberances. Dendrite-like ramifications are seen emerging from the cell body. B and C = 2 different stages of Purkinje cell development seen in the same section but from different parts of the cerebellum in an 8-day-old kitten. Note in B: terminal portions of dendrites do not penetrate into lower border of external granular layer. C = possible 'transitional' stage of Purkinje cell with smoothly contoured primary, secondary and tertiary rami. D = 20-day-old kitten; E = 42-day-old kitten. Note that during the 2nd month a marked increase in length of dendrites occurs but not in density of tertiary rami and spiny branchlets. Further description of A-E in text. Horizontal bar below each microphotograph is 50 p. References p . 219-221

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in the immediate neonatal period (Chiarugi and Pompeiano, 1954). At this time lamination is detectable and more superficially located granular cells appear more basophilic in Nissl preparations. ( 2 ) 2nd week: During the early part of the second week (8-12 days) a remarkable expansion of the dendrites of Purkinje cells is detectable. Two types of Purkinje cells have been observed at this time. In one type primary and secondary rami are prominent and smoothly contoured. Such elements appear to have been ‘stripped’ of the spiny protuberances seen in the neonatal preparations (Fig. 39C). Most Purkinje cells have well-developed primary, secondary and tertiary rami. The delicate distal tertiary branches give rise to spiny branchlets (Fig. 39B). Differences in the two types of Purkinje cells seen at this stage are shown more clearly in Fig. 41 which compares

Fig. 41. Details of developmentalchanges in Purkinje cell dendrites during the first month. A = main stem dendrite from cell in Fig. 39A (2-day-old kitten). B = distal portion of left main stem dendrite of cell shown in Fig. 39C. C = distal portion of dendrite of Purkinje cell from same section as in B. B and C = from 8-day-old kitten. D = 30-day-old kitten. Tertiary branch with spiny branchlets from cell adjacent to that shown in Fig. 40E. Further explanation in text.

superficial dendritic terminals of 8-day-old animals with those observed in neonatal and 30-day-old kittens. As in earlier stages, dendritic processes terminate at the lower border of the EGL. The EGL appears less densely packed than in neonatal animals, but is still 4-8 cell layers thick (Fig. 40, B-D). (3) 3 to 6 weeks: During this period there is further elaboration of Purkinje cell dendrites with considerable elongation of main stem dendritic trunks (Fig. 39, D and E). Tertiary branchescontaining spiny branchlets are seen on all Purkinje cells by the 3rd week. Throughout the 3rd-6th weeks there is progressive thinning of the EGL. This is brought about by inward migration of cells as described in detail by Cajal (1911, 1959). This inward migration is reflected in an increase in cellularity of the molecular layer. Towards the end of this period the EGL is only one to two cell layers thick but the presence of these elements apparently prevents the terminal portions of Purkinje cell dendrites from attaining the pial surface of the folium (Fig. 40, E and F).

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( 4 ) 2 months: The full splendor of the Purkinje cell is clearly seen in kittens by the end of the 2nd month. At this time the EGL has virtually disappeared although in some areas a single-layered remnant is detectable immediately below the pial surface. For the most part, however, the distal portions of Purkinje cell dendrites have spiny branchlets that are contiguous with this single layer or actually in 'contact' with the pial membrane.

SUMMARY

The postnatal development of Purkinje cells may be visualized as involving first resorption of fine perisomatic dendritic-like processes in the immediate neonatal period then rapid expansion of main stem dendrites. A phase of 'dendrite-stripping' is observed during the 2nd postnatal week preceding the elaboration of secondary and tertiary rami and spiny branchlets. Purkinje cell dendritic development is not complete until the end of the 2nd month. Prior to this time distal portions of dendrites extend to the lower border of the external granular layer (EGL) which progressively attenuates in association with inward migration of its constituent elements. (B) Electrophysiological Properties of Immature Cerebellar Cortex

( 1 ) Activities generated by local folial stimulation Weak stimulation of the folial surface of the cerebellum in adult cats elicits a 10-15 msec surface-negative response which is usually detectable up to 5-6 mm from the site of stimulation (Dow, 1949). This response, hereafter referred to as the superficial cerebellar cortical response (SCbR), is confined to the folium stimulated and is maximal at a locus corresponding to the longitudinal axis of the folium (Dow, 1949). Attempts to evoke the SCbR in neonatal kittens were unsuccessful. Stimulation of

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Fig. 42. Development of superficial cerebellar responses (SCbR) to local folial stimulation in kittens of various ages as shown. Stimulating electrodes consisted of a pair of 100 p-Teflon-coated silver wires cemented together as in studies of SCR of neocortex (cf. Figs. 25-29). Recording electrode placed 1-3 mm away from site of stimulation and parallel to the long axis of the folium. Spike-like responses seen in first week are succeeded by 'transitional' responses with minimal negativity and late surface-positivity. SCbR clearly detectable by the end of the 3rd week. Surface-negative SCbRs evoked during the2nd month are similar to those recorded in adult animals. Calibrations 100 cycles/sec. References p. 219-221

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the folialSurface of the posterior vermis or paramedial lobule in 1-2-day-old kittens elicited diphasic or triphasic spike-like responses which were detectable 2-4 mm from the site of stimulation (Fig. 42). The duration of spike-like components ranged from 3-10 msec. In older kittens spikes were succeeded by surface-positive waves of 30-40 msec duration. These responses attained maximal amplitude during the 2nd week but this was usually preceded by spike components and succeeded by prolonged positivenegative waves. SCbR's similar to those recorded in adult cats were obtained during the 3rd to 4th postnatal weeks. After this period S C b R s were readily elicited and were identical to responses evoked in mature cerebellar cortex (Fig. 42) (Dow, 1949; Purpura and Grundfest, 1956; Purpura, Girado and Grundfest, 1959). The possibility that low-amplitude surface-negativities might be masked in the prominent positive waves evoked by folial stimulation in 1-2-week-old kittens was examined by comparing responses to progressively stronger stimuli. During the developmental period in which surface-positivity constituted the predominant response to strong folial stimulation, no other responses were detectable with weak stimuli (Fig. 43A). Stronger stimulation resulted in a decrease in latency of surface-

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Fig. 43. Characteristics of cerebellar surface responses to graded folial stimulation. Stimulating and recording conditions as in Fig. 42. A = 8-day-old kitten; weak or strong stimulation elicits prolonged surface-positivity.B = 19-day-oldkitten. Weak stimulation elicits 10-20 msec low-amplitude surfacenegative SCbR, but this is swamped by succeeding positivity elicited by strong surface stimulation. C = 2-month-old kitten. Typical SCbRs are recorded during weak and strong stimulation. Calibrations 1 0 0 cycles/sec.

positivity and development of prior spike-like responses. At transition stages corresponding to the earliest time of SCbR appearance, weak surface stimulation evoked low-amplitude 10-20 msec surface-negativity. This activity was swamped by the succeeding surface-positivity associated with stronger surface stimulation (Fig. 43B). With the development of typical S C b R s graded surface-negativities were readily elicited (Fig. 43C). In these preparations no surface-positivity was observed even with folial stimulation strong enough to initiate high-frequency after-discharge (Fig. 43C). The effects of repetitive stimulation of the cerebellar cortex surface provided additional information on the properties of elements involved in local surface responses at different postnatal developmental stages. Responses evoked in young kittens that consisted largely of slow positive waves were markedly attenuated during low-frequen-

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cy surface stimulation (Fig. 42). The latency of early spikes was increased during 25/sec stimulation, at which frequency all secondary positivity was eliminated (Fig. 44A 3). At the end of the transitional period (3 weeks), when adult-like SCbRs were elicited, such surface-negativitiesfailed to follow S/sec stimulation (Fig. 44B 6 ) . Depression of surface-negativity under conditions of repetitive stimulation was accompanied by unmasking and possibly facilitation of brief spike-like components which were barely detectable on the rising phase of the surface-negativityin Fig. 44B 5.

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Fig. 44. Effects of repetitivefolial stimulation on locally evoked cerebellar responses. A = 12-day-old kitten. 1 = O.S/sec; 2 = 5/sec; 3 = 25/sec; and 4 = recovery, O.S/sec stimulation. Note depression of late positivity. B = 21-day-oldkitten. 5-8 = stimulus frequencies as in A. Surface-negativeSCbR is markedly depressed at 5/sec and eliminated at 25/sec stimulation. C = 50-day-old kitten. 9 = O.S/sec; 10 = 5/sec; and 11 = 25/sec surface stimulation. Potentiation of SCbR's is prominent with repetitive stimulation. Calibrations 100 cycles/sec.

All responsiveness was lost during continued high-frequency stimulation (Fig. 44B 7) An entirely different situation was encountered in 7-8-week-old kittens (Fig. 42C). Then SCbRs were facilitated during low-frequency repetitive stimulation and continued to exhibit augmentation for long periods of high-frequency surface stimulation, as in adult cats (Purpura and Grundfest, 1957). ( 2 ) Evoked activity in an aflerent cerebral-cerebellar pathway Pericruciate cortex stimulation in adult cats elicits short-latency multiphasic responses of variable duration from relatively restricted loci in the contralateral paramedian lobule (Dow and Moruzzi, 1958). In locally anesthetized, paralyzed adult cats a double surface-positivity succeeded by a longer duration negativity is usually recorded from sites of maximal projection (Purpura, Girado and Grundfest, 1959). Diphasic positive-negative responses were readily recorded from the paramedian lobule of neonatal kittens following contralateral pericruciate cortex stimulation (Fig. 45). These responses, like those reported in similar studies on newborn rabbits (Ulett et al., 19441,were of extraordinary latency (50-70 msec) and failed to follow stimulation frequencies in excess of 2/sec. As in adult animals, responses observed in neonatal kittens were restricted in distribution to two or three folia of the paramedian lobule. It is of interest that stimulation of more caudal areas of neocortex References p . 219-221

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failed to evoke activity in paramedian lobule or other exposed regions of the posterior cerebellum in newborn kittens. The relatively simple configuration of paramedian evoked responses was unaltered during the first postnatal month. However, dramatic changes in latency were observed throughout this period. During the 5th-7th week additional complexitieswere detected in paramedian evoked responses. These consisted in the appearance of slow and fast components, the latter frequently appearing as a 10 msec diphasic or triphasic spikelike sequence superimposedon a slower surface-positivity.Severalvarieties of responses were elicited after the 2nd month, most typical of which are shown in Fig. 45. The 6d

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Fig. 45. Changing characteristics of paramedian lobule evoked responses to contralateral motor cortex stimulation in kittens of various ages as shown. Further explanation in text. Calibrations 100 cycleslsec.

response recorded in an 84-day-old kitten illustrates the short-latency slow positivity succeeded by longer latency, short-duration positivity typical of responses recorded in adult animals (Jansen, 1957; Purpura, Girado and Grundfest, 1959). It should be noted that although paramedian evoked responses in 3-month-old kittens were similar in electrographic characteristics to those observed in mature cerebellar cortex, latency differences were still detectable. No attempt was made to establish the time at which minimum latencies were achieved. But in view of findings on ontogenetic changes in conduction velocity of corticospinal axons (see Fig. 35), it is reasonable to suspect that conductile components of the cortico-ponto-cerebellar pathway acquire mature characteristics during the 4th-5th postnatal month. The development of short and long-latency surface-positive components in paramedian evoked responses was further analyzed in terms of the relationship of both responses to stimulus strength. Fig. 46 summarizes the pertinent data on this point. Prior to the appearance of short-latency surface-positivities no difference was noted in the latency of evoked responses at any strength of stimulation. When long-latency positivity was prominent in responses to supramaximal pericruciate cortex stimulation marked shifts in latency were observed in association with multiphasic early positivities (Fig. 44, B and C ) . The foregoing results permit several tentative conclusionsregarding the relationship of evoked potentials to the morphogenetic features of cerebellar cortex. The failure to elicit 10-20 msec surface-negative responses following local folial stimulation is undoubtedly related to the absence of subpial neuropil in cerebellar cortex of newborn kittens. The superficial half of the molecular layer of the cerebellar cortex in the

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immediate neonatal period consists of a dense multilaminated sheet of granule cells which are interposed between surface stimulating electrodes and Purkinje cells and related pathways (Chiarugi and Pompeiano, 1954). It is questionable whether external granule cells contribute to the spike-like responses observed in neonatal preparations or for that matter whether these primitive elements are excitable at all. What is

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Fig. 46. Alterations in paramedian lobule evoked responses to stimulation of contralateral motor cortex with increasing strengths’ of stimulation. A = 10-day-old kitten. Surface-positivenegative response increases in amplitude during progressively stronger (1-3) motor cortex stimulation. B = 60-day-old kitten. Weak motor cortex stimulation elicits long-latency surfacepositivity whereas marked shortening in latency and increasing complexity of overt response occurs with strong stimulation. C = 80-day-old kitten. Same results as in B. Calibrations 1 0 0 cycles/sec; that for B and C shown in B.

particularly important is that the earliest detectable responses of cerebellar cortex to local folial stimulation are compounded of spike-like responses and surface-positivity rather than the surface-negativity of adult animals. The surface-positivity, which augments with progressively stronger surface stimulation, is inferred to represent synaptic activation of neurons in the depths of the cerebellar cortex (Purpura, 1959; Purpura, Girado and Grundfest, 1959). The implication here is that synaptic activities generated in elements of the internal granule layer, or Purkinje cell bodies are prominent during the immediate neonatal period. This is supported by findings on the surface-positive characteristics of paramedian lobule evoked responses to motor cortex stimulation. These responses are largely reflections of focal negativities in the depths of the cerebellar cortex in young kittens (Shofer and Purpura, unpublished observations) as in the case of mature animals (Jansen, 1957; Purpura, Girado and Grundfest, 1959). The ease with which 10-20 msec surface-negative SCbRs are evoked by the end of the 3rd week appears to be mainly attributable to the development of synaptic contacts on superficial portions of Purkinje cell dendrites. During the 2nd and 3rd week a marked general expansion of Purkinje cell,dendrites occurs along with inward migration of cells of the EGL. The EGL remains formidable in 3-week-old kittens despite the fact that a large number of external granule cells have migrated inward, differentiated, and contributed to the development of the parallel fiber system which effects synaptic contact with fine dendritic processes of Purkinje cells (Cajal, 1911, 1959; Fox and Barnard, 1957; Gray, 1961). The finding that typical SCbR’s are detectable while the EGL is prominent suggests that the major factor responsible for the absence References p . 219-221

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of surface-negative SCbRs in newborn kittens is the relatively poor development of parallel fiber-Purkinje dendrite-synaptic pathways rather than the presence of an EGL per se. Concerning the development of the parallel fiber system it is of interest that during the period of rapid expansion of Purkinje cell dendritic surface area (8-12 days) some dendrites may be relatively devoid of spines for variable periods of time. This could come about if the proliferation of dendrites greatly exceeded the rate of development of parallel fibers effecting appropriate synaptic contacts. Further information along these lines may be forthcoming when electron microscope studies of mature cerebellar cortex (Gray, 1961) are extended to immature animals (Pappas, Shofer and Purpura, in preparation). It will be recalled that the studies on cerebral-cerebellar evoked activity in neonatal rabbits (Ulett et al., 1944) clearly established that functional activity in complex systems preceded myelination of conductile pathways. These observations have been repeatedly confirmed in numerous other types of studies including those described or cited in this report. The long latency of paramedian lobule evoked responses in newborn kittens is entirely referable to delayed conduction in cortico-ponto-cerebellar pathways. Although decreases in latency closely parallel increases in conduction velocity of corticofugal projections, it must be allowed that latency changes in paramedian lobule evoked responses observed in older kittens may also be attributable to activation of different synaptic pathways as in the case of primary evoked responses of immature neocortex. SUMMARY

Purkinje cells undergo a prolonged period of postnatal development that extends well into the 2nd month in the kitten. In neonatal animals Purkinje cell dendrites are poorly developed and less than 25-50 p in length. Progressive elaboration of dendrites and axodendritic synaptic pathways occursparipassu with alteration and inward migration of cells of the external granular layer. In neonatal animals surface-negativity is not detectable by local folial stimulation and afferent stimulation elicits prominent surfacepositive activity. It is inferred from this that development of synaptic organizations in the depths of the cerebellar cortex precedes maturation of superficial axodendritic synaptic pathways including the parallel fiber-Purkinje dendrite-synaptic system. The relatively late development of the latter system corresponds to the appearance of superficial negative responses to local folial stimulation. These data support the view that such responses are postsynaptically generated in superficial dendrites of Purkinje cells (Dow, 1949; Purpura, 1959; Purpura and Grundfest, 1956, 1957). CONCLUSIONS

Fundamentally different postnatal ontogenetic patterns are observed in feline cerebral neocortex and cerebellar cortex. Elaboration of axodendritic synaptic pathways in superficial neocortical neuropil precedes the development of axosomatic and axodendritic pathways in the cortical depths. An inverse morphogenetic pattern is seen

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in cerebellar cortex where axodendritic synaptic pathways involving Purkinje cell spiny branchlets exhibit delayed development with respect to deep-lying synaptic organizations. The consequences of these patterns of postnatal development are reflected in two major differences in cerebral and cerebellar potentials evoked by local surface or afferent stimulation in newborn kittens: ( I ) Local stimulation of neocortex in neonatal kittens elicits surface-negative responses similar to those observed in adult animals, whereas such responses are not demonstrable in cerebellar cortex until about 2 weeks postnatally; (2) Specific afferent responses of neocortex in newborn animals lack initial surface-positivity and do not acquire this component until the’end of the 1st week. In contrast, afferent responses of cerebellar cortex have prominent surfacepositivity in newborn kittens as in adult animals. Subcortical elements interposed in the major projection pathways are capable of elaborating a wide variety of complex electrical activities in newborn kittens. For the most part these activities are initiated by volleys in axons with conduction velocities less than 1-2 m/sec. The postnatal development of synaptic organizations in cerebral and cerebellar cortex is completed prior to the maturation (increase in axon diameter and myelination) of conductile pathways arising in neocortex and distributing to brain stem and spinal cord. Myelination of these pathways is completed by the end of the 4th month at which time the latency and electrographic characteristics of corticofugal discharges and the responses they elicit in related organizations are identical to those observed in adult cats. ACKNOWLEDGEMENTS

These studies were supported in part by the United Cerebral Palsy Research and Educational Foundation (R-135-62 C) and the National Institute of Neurological Diseases and Blindness (NB-01312-07 and NB-03473-02). Dr. Purpura is a Research Career Development Fellow, National Institute of Neurological Diseases and Blindness (NB-K3-5280-C1) and Dr. Shofer is a Special Fellow, National Institute of Neurological Diseases and Blindness (BT-999). REFERENCES ADDISON, W. H. F., (1911); The development of the Purkinje cells and of the cortical layers in the cerebellum of the albino rat. J . comp. Neurol., 21,459-488. ADRIAN,E. D., (1936); The spread of activity in the cerebral cortex. J. Physiol. (Lond.), 88, 127-161. ANOKHIN, P. K., (1961); Electroencephalographicanalysis of cortico-subcortical relations in positive and negative conditioned reactions. Ann. N . Y. Acad. Sci., 92, 899-938. D. P., (1963); Postnatal changes in amino acid content of kitten cerebral BERL,S . , AND PLJRPURA, cortex. J . Neurochem., 10, 237-240. BISHOP, G. H., AND CLARE, M. H., (1952); Sites of origin of electric potentials in striate cortex. J. Neurophysiol., 15, 201-220. BRIZZEE, K. R., AND JACOBS,L. A., (1959); Postnatal changes in volumetric and density relationships of neurons in cerebral cortex of cat. Acta anat. (Basel), 38, 291-303. J. M., AND ZANCHETTI, A., (1956); The relation between electrocortical waves and BROOKHART, responsiveness of the cortico-spinal system. Electroenceph. elin. Neurophysiol., 8, 427-449. V. B., AND ENGER, P. S., (1959); Spread of directly evoked responses in the cat’s cerebral BROOKS, cortex. J . gen. Physiol., 42, 761-777.

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BURNS, B. D., (1958); The Mammalian Cerebral Cortex. London, Edward Arnold. CAJAL,R A M ~Y,NS., (1911); Histologie du SystPme nerveux de I'Homme et des Vertkbrks. Madrid, Consejo Superior de Investigaciones Cientificas. CAJAL, RAMON Y,S., (1959); Studies on Vertebrate Neurogenesis. Springfield, Ill., Charles C. Thomas. CHANG, H.-T., (1951); Dendritic potential of cortical neurones as produced by direct stimulation of the cerebral cortex. J. Neurophysiol., 14, 1-21. O., (1954); Sui rapporti fra istogenesi ed eccitabilitk del lobus anterior CHIARUGI, E., AND POMPEIANO, nel gatto neonato. Arch. ital. Biol., 38,493-531. CLARE, M. H., AND BISHOP,G. H., (1955); Properties of dendrites; apical dendrites of the cat cortex. Electroenceph. clin. Neurophysiol., 7, 85-98. CONEL, J. L., (1939-1955); The Postnatal Development of the Human Cerebral Cortex. Vols. I-VI. Cambridge, Mass., Harvard University Press. Dow, R. S., (1942); Cerebellar action potentials in response to stimulation of the cerebral cortex in monkeys and cats. J. Neurophysiol., 5, 121-136. Dow, R. S., (1949); Action potentials of cerebellar cortex in response to local electrical stimulation. J. Neurophysiol., 12, 245-256. Dow, R. S., AND MORUZzl, G., (1958): The Physiology andpathology of the Cerebellum. Minneapolis, University of Minnesota Press. EAYRS, J. T., AND GOODHEAD, B., (1959); Postnatal development of the cerebral cortex in the rat. J. Anat. (Lond.), 93, 385402. ECCLES, J. C., (1951); Interpretation of action potentials evoked in the cerebral cortex. Electroenceph. din. Neurophysiol., 3, 449-464. ECCLES, R. M.,SHEALY, C. N., AND WILLIS,W-. D., (1963); Patterns of innervation of kitten motoneurons. J. Physiol. (Lond.), 165, 392402. ELLINGSON, R. J., AND WILCOTT,R. C., (1960); Development of evoked responses in visual and auditory cortices of kittens. J. Neurophysiol., 23, 363-375. FLEXNER, L. B., (1955); Enzymatic and functional patterns of the developing mammalian brain. Biochemistry ofthe Developing Nervous System. H. Waelsch, Editor. New York, Academic Press. Fox, C. A., AND BARNARD, J. W., (1957); A quantitative study of the Purkinje cell dendritic branchlets and their relationship to afferent fibers. J. Anat. (Lond.), 91, 299-313. GRAFSTEIN, B., (1963); Postnatal development of the transcallosal evoked response in the cerebral cortex of the cat. J. Neurophysiol., 24, 79-99. GRAY,E. G., (1959); Axo-somatic and axo-dendritic synapses of the cerebral cortex. An electron microscope study. J. Anat. (Lond.), 93,42&433. GRAY,E. G., (1961); The granule cells, mossy synapses and Purkinje spine synapses of the cerebellum, light and electron microscope observations. J. Anat. (Lond.), 95, 345-356. GROSSMAN, C., (1955); Electro-ontogenesis of cerebral activity. Arch. Neurol. Psychiat. (Chic.), 74, 136-202. HENRY, E. W., AND WOOLSEY, C. N., (1943); Somatic motor responses produced by electrical stimulation of the cerebral cortex of newborn and young kittens. Fed. Proc., 2, 21. HUNT,W. E., AND GOLDRING, S., (1951); Maturation of evoked response of the visual cortex in the postnatal rabbit. Electroenceph. clin. Neurophysiol., 3, 465471. HURSH,J. B., (1939); The properties of growing nerve fibers. Amer. J. Physiol., 127, 140-153. IWAMA, K., AND JASPER, H. H., (1957); The action of gamma-amino butyric acid upon cortical electrical activity. J. Physiol. (Lond.), 138, 365-380. JANSEN, J., JR., (1957); Afferent impulses to the cerebellar hemispheres from the cerebral cortex and certain subcortical nuclei. Acta physiol. scand., Suppl. 143, 41, 1-99. KLUVER,H., AND BARRERA, E., (1953); A method for the combined staining of cells and fibers in the nervous system. J. Neuropath. exp. Neurol., 12, 400-403. LANGWORTHY, 0. R., (1929); A correlated study of the development of reflex activity in fetal and young kittens and the myelinization of tracts in the nervous system. Contr. Embryol. Carneg. Znstn., 20, 127-172. MARTY, R., (1 962) ; Dkveloppement post-natal des rkponses sensorielles du cortex ckrkbral chez le chat et le lapin. Arch. Anat. micr. Morph. exp., 51, 129-264. D. P., (1961); Postnatal ontogenesisof cat neocortex. J . comp. Neurol., NOBACK, C. R., AND PURPURA, 117, 291-308. OCHS, S., (1962); Analysis of cellular mechanisms of direct cortical responses. Fed. Proc., 21,642-647.

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PAPPAS, G. D., AND PURPURA, D. P., (1961);Fine structure of dendrites in the superficial neocortical neuropil. Exp. Neurol., 4,507-530. PATTON,H. D., AND AMASSIAN, V. E., (1960); The pyramidal tract: its excitation and functions. Handbook of Physiology, Vol. II. J. Field, H. W. Magoun and V. E. Hall, Editors. American Physiology Society (p. 837). PURPURA, D. P., (1959);Nature of electrocortical potentials and synaptic organizations in cerebral and cerebellar cortex. International Review of Neurobiology, Vol. I. C. C. Pfeiffer and J. R. Smythies, Editors. New York, Academic Press. PURPURA, D. P., (1960);Pharmacological actions of o-amino acid drugs on different cortical synaptic organizations. Inhibitions of the Nervous System and y-Aminobutyric Acid. E. Roberts, Editor. New York, Pergamon Press (p. 495-514). PURPURA, D. P., (1961a);Analysis of axodendritic synaptic organizations in immature cerebral cortex. Ann. N . Y. Acad, Sci., 94,604-654. PURPURA, D. P., (1961b);Ontogenetic analysis of some evoked synaptic activities in superficial neocortical neuropil. International Symposium on Nervous Inhibition. E. Florey, Editor. New York, Pergamon Press (p. 424446). PURPURA, D. P., (1962);Synaptic organization of immature cerebral cortex. Wld Neurol., 3, 275-298. PURPURA, D. P., CARMICHAEL, M. W., AND HOUSEPIAN, E. M., (1960); Physiological and anatomical studies of development of superficial axodendritic synaptic pathways in neocortex. Exp. Neurol., 2, 324-347. PURPURA, D. P., GIRADO,M., AND GRUNDFEST,H., (1959); Synaptic components of cerebellar electrocortical activity evoked by various afferent pathways. J . gen. Physiol., 42, 1037-1066. PURPURA, D. P., GIRADO,M., AND GRUNDFEST, H., (1960); Components of evoked potentials in cerebral cortex. Electroenceph. clin. Neurophysiol., 12,95-1 10. PURPURA, D. P., GIRADO,M., SMITH,T. G., CALLAN, D., AND GRUNDWST, H., (1959); Structurcactivity determinants of pharmacological effects of amino acids and related compounds on central synapses. J. Neurochem., 3, 238-268. PURPURA, D. P., AND GRUNDFEST, H., (1956);The nature of dendritic potentials and synaptic mechanisms of cat cerebral cortex. J. Neurophysiol., 19, 573-595. PURPURA, D. P., AND GRUNDFEST, H., (1 957); Physiological and pharmacological consequences of different synaptic organizations in cerebral and cerebellar cortex. J . Neurophysiol., 20, 494-522. PURPURA, D. P., AND HOUSEPIAN, E. M., (1961);Alteiations in corticospinalneuron activity associated with thalamocortical recruiting responses. Electroenceph. clin. Neurophysiol., 13, 365-381. PURPURA, D. P., SHOFER, R. J., AND MUSGRAVE, F. S., (1963);Intracellular potentials of cortical neurons during augmenting and recruiting responses. Fed. Proc., 22, 457. ROBERTS, E.,(1960); Third Conference on Central Nervous System and Behavior. M. A. B. Brazier, Editor. New York, Josiah Macy, Jr. Foundation. SCHAD!~, J. P., AND BAXTER, C. F., (1960);Changes during growth in the volume and surface area of cortical neurons in the rabbit. Exp. Neurol., 2, 158-178. SCHERRER, J., AND OECONOMOS, D., (1955); Reponses C v o q u b corticales somesthetiques des man-.mifkres adulte et nouveau-nt. Les Grandes Activitb du Lobe Temporal. Paris, Masson. SHOLL,D. A., (1956); The Organization of the Cerebral Cortex. London, Methuen. SIDMAN, R, L., AND MIALE,I. L., (1961); An autoradiographic analysis of histogenesis in mouse cerebellum. Exp. Neurol., 4, 277-297. SKOGLUND, S., (1960);The spinal transmission of proprioceptive reflexes and the postnatal developmentof conductionvelocity ofdifferent hindlimbnervesin thekitten. Actaphysiol.scand.,49,313-329. ULETT,G., Dow, R. S., AND LARSELL, O., (1944); Inception of conduction in the corpus callosum and the corticoponto-cerebellar pathway in young rabbits with reference. to myelinization. J. comp. Neurol., 80, 1-10. UZMAN,L.L.,(1960);The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. J. comp. Neurol., 114, 137-159. VOELLER, K.,PAPPAS,G. D., AND PURPURA, D. P., (1963);Electron microscope study of development of cat superficial neocortex. Exp. Neurol., 7 , 107-1 30. WEED,L. H.,AND LANGWORTHY, 0. R., (1926);Physiological study of cortical motor areas in young kittens and in adult cats. Contr. Embryol. Carneg. Znstn., 17,89-106. WILSON, V. J., (1962);Reflex transmission in the kitten. J . Neurophysiol., 25, 263-276. WINDLE,W.F., FISH, M. W., AND ODONNELL, J. E., (1934); Myelogeny of the cat as related to development of fiber tracts and prenatal behavior patterns. J. comp. Neurol., 59, 139-165.

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J. SCHERRER

Centre de Recherches neurophysiologiques de I'dssociation Claude Bernard et de I'lnstitut National d'Hygihe, H6pital de la SalpltriPre, Paris

Pour apprtcier la maturation des systkmes afftrents corticaux, on peut ttudier avec la niajoritt des auteurs le comportement des animaux et l'tvolution histologique du systkme nerveux en recherchant tventuellement une relation anatomo-fonctionnelle entre les deux catkgories d'informations. L'utilisation de critereselectrophysiologiques substituts au comportement prtsente un certain nombre d'avantages. En effet, ces critkres, plus aistment mesurables, permettent de suivre de faCon plus Ctroitel'tvolution de la maturation dans une espkce animale, de comparer des espkces entre elles et de confronter, dks lors, le dtveloppement de la fonction avec celui des structures anatomiques. Dans l'ttat actuel de nos connaissances, nous proposons les crittres tlectrophysiologiques suivants: ( I ) La vitesse de propagation des messages afftrents de la ptriphkrie jusqu'a l'tcorce ckrtbrale. (2) Les caractkres tlectriques de la rkponse corticale localiste. (3) Le retentissement des messages afferents sur l'activitt tlectro-corticale gkntralisee. L'&tude simultante de la mytlinisation de certaines voies afferentes et celle du dtveloppement du cortex ctrtbral 21 l'tchelle neuronique permettent de connaitre avec une certaine rigueur le substratum des deux premiers de ces critkres. Nous venons d'indiquer ci-dessus la perspective gtntrale d'un certain nombre d'investigations sur la maturation du systkmenervew poursuivies au Centre de Recherches neurophysiologiques de la Salpztriere depuis 1953 (Scherrer et Oeconomos, 1954) et qui ont abouti rtcemment 21 un travail d'ensemble (Marty, 1962). Ces travaux, effectuts chez le chat et le lapin, seront analysts ici en tenant compte des recherches consacrtes par d'autres auteurs a ce meme probltme, en particulier Hunt et Goldring (1951) et Rose et aE. (1957). PROPAGATION DES MESSAGES

AFFBRENTS

CORTICAUX

Les recherches dans ce domaine ont portt sur les trois principaux systbmes affkrents: la somesthtsie, l'audition et surtout la vision.

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CORTICAUX

Le ddai global de propagation des trois types de messages aflkrents (a) Pour tvaluer la vitesse de propagation des messages le long des voies afftrentes,

on a mesurt le dtlai ntcessaire au parcours de ces voies sur toute leur longueur, de la ptriphtrie jusqu’a l’ecorce ctrtbrale, c’est h dire I’intervalle de temps stparant I’instauration de la stimulation de l’apparition d‘une reponse corticale localiste ; il s’agit, en d’autres termes, de la latence globale du potentiel tvoqut cortical. La mesure de celle-ci ne constitue cependant qu’une apprtciation incomplkte de la vitesse de propagation puisque les fibres nerveuses subissent, a cette ptriode du dtveloppement, une croissance en longueur dont l’ttude quantitative est encore trks fragmentaire. Afin de prtciser I’evolution de ce dtlai de propagation dans les trois systkmes afftrents, on peut utiliser des stimulations de type physiologique (stimulations mtcaniques des ttguments, clics, eclairs). On peut tgalement mettre en parallkle la stimulation Clectrique de la peau ou du nerf optique avec la stimulation auditive par clics, cette dernikre modalitt de stimulation apparaissant dans une certaine mesure comparable a l’excitation directe des fibres afftrentes. La Fig. 1 reprtsente l’tvolution des dtlais des rtponses corticales lors d’une mise

Lapin

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Fig. 1. Cornparaison du delai de propagation des messages dans les trois systernes afferents. 8 :stimulation electrique des teguments, x : stimulation sonore (clics), 0: stimulation electrique du nerf optique. Le delai de propagation des messages, ou latence globale des reponses, est calculeen multiples de la valeur de la latence definitive, egale a 1. La representation graphique est arrctee au moment oh le delai de propagation a le double de sa valeur definitive. Noter la prkession evolutive de la somesthesie sur l’audition, la vision n’atteignant ce critere qu’en derniere position. Rernarquer egalement que l’evolution est plus rapide chez le lapin que chez le chat. Bibliographic p . 234

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en jeu des afftrences somesthtsiques, auditives et visuelles. Afin de permettre une nieilleure comparaison des rtsultats, on a reprtsentt sur cette figure, non pas les latences elles-mCmes, mais leur valeur exprimte sous la forme d’un multiple de la latence definitive; les mesures ont ttt arrCttes pour une valeur double de la latence definitive. La reprtsentation choisie fait ressortir I’existence d’une hierarchie chronologique entre les trois systbmes afftrents. La somesthtsie occupe la prernikre place, prtctdant I’audition; la vision n’occupe que la troisibme position. La pente des courbes confirme cette hierarchie tvolutive que montrait dtja leur position sur le graphique. Elle souligne en outre que Ie lapin, plus immature h la naissaiice que le chat, btntficiera d’une evolution plus rapide que ce dernier. L‘inttrCt de cette hitrarchie Cvolutive pour comparer la maturation des trois systkmes doit Ctre soulignte car, sur le plan fonctionnel, elle est plus rtelle qu’une classification strictement baste sur la date d’apparition des premibres rCponses tlectrocorticales a la stimulation du rtcepteur. En effet, si l’on adopte cette date comme base de classification, l’antCriorit6 de la somesthtsie se trouve confirmke mais, chez le chat tout au moins, on note que les rtponses visuelles apparaissent avant les rtponses auditives. Ces faits demandent 5 Etre bribvement discutts. La somesthtsie manifeste indubitablement la premikre son activite i l’kchelon cortical, et ceci dbs la ptriode foetale. La prtsence de rtponses tlectro-corticales i la stimulation des teguments chez des chats nts avant terme le dtmontre. Le potentiel tvoqut auditif, lit A un clic et visuel, lit h un flash, n’apparaissent qu’aprbs la naissance et, chez le chat, les riponses 5 la stimulation photique s’exttriorisent avant les rtponses auditives. Or, au moment OG les rtponses visuelles peuvent Ctre mises en evidence pour la premibre fois, l’animal a encore les yeux ferrnts. On ne peut donc tirer de cette prtcession aucune conclusion valable sur le plan fonctionnel. L‘Cvolution du delai de propagation au contraire met bien en tvidence la chronologie du fonctionnement des trois systkmes telle que des ttudes de comportement le laissaient prevoir (voir ci-dessous), la fonction auditive prtctdant la fonction visuelle. La date relativement tardive d’exttriorisation des premikres rtponses auditives s’explique par le dtveloppement retard6 des rCcepteurs dans ce systbme. Les caractbres des rtponses dks leur apparition le suggeraient dtji, la stimulation Clectrique de la cochlte le prouve (Marty et Thomas, 1963). L’anttrioritt de la somesthtsie a l’tchelon cortical ne doit pas surprendre. Les etudes de comportement effectutes chez divers mammifkres et en particulier chez l’homme ont en effet montrP que les premibres reactions motrices observables au cours de la vie foetale dtpendaient de la stimulation des ttgunients et tout sptcialement du territoire trigemink (Windle et Griffin, 1931; Humphrey et Hooker, 1959). Dans la periode nto-natale, ces etudes confirment la chronologie fonctionnelle que nous avons exposte ci-dessus (Tihiey et Casaniajor, 1924). Les mEmes conclusions seniblent pouvoir &treadmises chez I’honime. Quelques investigations tlectrophysiologiques, notamment celles d’Ellingson (1958) sur les reponses corticales visuelles, sont a signaler ici. (b) La prtcession si marquee de la somesthtsie sur les deux autres niodalitCs affkrentes relbve de plusieurs facteurs parmi lesquels les caractkres particuliers de la

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mise en jeu des rkcepteurs. Une distinction fondamentale s’impose en effet sur ce plan entre la somesthCsie d’une part et l’audition et la vision de I’autre. L‘audition et la vision sont tributaires de rtcepteurs complexes, les ttlCricepteurs de Sherrington, dont la maturation est indispensable au fonctionnement du systkme affirent. La somesthksie au contraire peut exercer son activite a partir de ‘terminaisons libres’. Chez l’adulte, ces terminaisons reprtsentent un contingent important de I’innervation cutanCe. Au cows de la vie foetale, leur prCcocitC d’apparition est remarquable d’autant qu’on est en droit de considCrer Cgalement comme terminaisons libres, B cette Cpoque du developpement, des terminaisons nerveuses appeltes ulttrieurement 21 participer a la constitution d‘organes recepteurs plus complexes. Chez le lapin, on voit ainsi apparaitre les rtseaux nerveux p&ri-folliculaires avant les follicules pileux auxquels ils sont destints (Winkelmann, 1960). Chez le foetus humain, des rtponses motrices peuvent &tre obtenues par la stimulation de la peau ii une Cpoque oh il n’existe encore aucune formation spCcialisCe dCcelable (Hogg, 1941). Cette modalit6 particulikrenient simple de la rtception des messages apparait comme l’un des facteurs d’antCrioritC de la somesthksie. La maturation retardte des ttltrkepteurs tant au point de vue anatoniique que fonctionnel doit &treconsidtrt cornme UII fait acquis. En ce qui concerne la vision, les rksultats de la stimulation Clectrique du nerf optique le prouvent, qui provoque en effet rkgulikrenient des rtponses corticales d t s la naissance, a un 2ge oh la stimulation photique est encore dtpourvue d’effets Clectro-corticaux (Hunt et Goldring, 1951; Marty, 1962). Les voies de conduction, les synapses et les centres optiques se rtvklent donc capables de fonctionner avant leurs rtcepteurs. Les m&mesconstatations valent pour l’audition (Marty et Thomas, 1963). Analyse du ddai global de propagation des messages (a) Qu’il s’agisse de stimulations physiologiques ou Clectriques, la latence globale de la reponse associe iiCcessaireinent le dClai de transmission synaptique au temps de conduction le long des fibres nerveuses. La valeur du dtlai syiiaptique par rapport a la latence globale n’a pas etC apprCcite pour les systkmes affkrents. On est cependant tent6 de le corisiderer conime nkgligeable en se basant sur les rCsultats d’autres explorations du systkme nerveux dans la ptriode nCo-natale. C‘est ainsi par exemple que, chez le chat, la lenteur de la conduction le long des fibres suffit a rendre compte de la plus grande partie du dtlai de propagation dans l’arc moiiosyiiaptique spinal (Wilson, 1962). (b) Lorsque l’on utilise une stimulation de caracttre physiologiyue, I’ttude du dtlai de propagation necessite I’evaluation prkalable du r61e revenant aux rtcepteurs. I1 est doric interessant de comparer l’tvolution de la lateiice des reponses provoquCes par une stimulation physiologique et par la stimulation electrique. Dam le systkme visuel du chat, au cours de la preniikre semaine nto-natale, cette Cvolution n’est pas identique pour les deux variktb de stimulation (Fig. 2). Durant les premiers jours, on observe la rtgression rapide de la lateiice globale des rCponses corticales a la stimulation photique, cependant que le dClai de rCponse a la stimulation Bibliographir p. 234

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I

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Fig. 2. Evolution comparee de la latence des rkponses la stimulation photique et des rtponses a la stimulation du nerf optique chez le chat. La courbe superieure ( 0 )represente 1’6volution de la latence des reponses electro-corticales a la stimulation photique (flash); la courbe inferieure ( O ) , celle des rCponses a la stimulation Blectrique du nerf optique. La mesure porte sur la latence du sommet de l’accident le plus prkoce. Noter, les premiers jours, la diminution rapide de la latence des rCponses A la stimulation photique, et la stabilite de la latence des rtponses 5 la stimulation du nerf optique.

directe du nerf optique reste constant. Dans un temps ultbieur, au contraire, la diminution de latence se manifeste de faqon comparable pour 1es deux categories de rtponses. Ceci signifie que la diminution de laterice observCe les premiers jours pour les seules reponses A la stimulation photique est de nature exclusivemeiit rCtinienne ; les progrbs de la conduction dans les voies nerveuses ne se nianifestent qu’ensuite. Le phknomkne qui vient d’&tredtcrit semble particulier A l’appareil visuel. Ce fait tient peut-&treA ce que le fonctiorinement des rtcepteurs est bask sur d’importantes reactions photo-chimiques ayant leur maturation propre, sous la forme d’apparition d’enzymes par exemple. Substratum histologique du gain de conduction L‘accClCration de la conduction le long des fibres nerveuses dans le systkme visuel apparait comme un facteur important et gtntral dans la rtduction du dtlai de propagation des messages. L‘ttude histologique des systkmes afftrents confirme que la mydinisation des,fibres constitue selon toute vraisemblance le substratum anatoniique principal de la modification fonctionnelle. Les caractkres proprement histologiques de la mydinisation, que nous avons particulibrement Ctudite sur les voies visuelles, doivent tout d’abord Ctre prCcists. (a) La inytlinisation de la voie visuelle s’effectue selon une cinetique trks particulikre. DCbutant stir la bandelette optique dans la partie voisine du chiasma, elle gagne ensuite, de part et d’autre de celui-ci, le corps genouillC lateral et le nerf optique en

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direction de la rttine. Cependant qu’elle progresse vers la lame criblte du rlerf optique, une nouvelle rtgion de mytlinisation apparait sur le segment thalamo-cortical de la voie visuelle; elle tvolue, ti ce niveau, d‘une faCon orthodromique. Ce processus de mytlinisation s’accompagne d’importantes variations de la densitt des cellules nkvrogliques dont le r61e dans la mytlinogenbse est maintenant bien dtmontrt. Au point de vue chronologique, la mytlinisation, dtjja commencte la naissance, inttresse l’ensemble de la voie visuelle vers la fin du premier mois. (b) L‘influence capitale de la mytlinisation sur les performances de conduction est une notion classique dont la dtmonstration a t t t faite A de nombreuses reprises au niveau des fibres nerveuses ptriphtriques. Dans le systbme visuel, dont toutes les parties constituantes dependent du systbme nerveux central, cette influence ne parait pas s’exercer nettement dans la ptriode initiale de la mytlinisation, si inattendue qu’apparaisse cette constatation. En effet, durant la premibre ptriode de la vie extrauttrine du chat, le processus de mytlinisation, dtjia Cbaucht B la naissance, s’ttend sans diminution apprtciable de la latence des rtponses obtenues par stimulation nerveuse directe. Un facteur anatomique pourrait expliquer ce paradoxe, la croissance en longueur de la voie visuelle, qui viendrait compenser les progrks de la conduction acquis par la mydinisation sur une courte distance. Le degrt atteint A cette Cpoque par le processus de mytlinogenkse pourrait tgalement &re discutt ;la gaine de mytline, bien que presente, ne possbderait pas encore une tpaisseur suffisante pour modifier de facon decisive le mtcanisme de la conduction. Dbs qu’ils sont devenus tvidents, les progrks de la conduction se poursuivent pour aboutir, vers 1 mois 1/2 A 2 mois, A doter le systbme afferent d’un dtlai de propagation identique 2 celui de l’animal adulte. Cette date est postirieure a la fin apparente de la mydinisation. I1 faut admettre que Ies effets de l’augmerztation du diamgtre des Jibres nerveuses, difficiles a apprtcier tant que se poursuit la mytlinisation, jouent un r61e notable dbs que la mytlinisation est acquise. Au deli de 2 mois d’2ge, elle permet a la latence de la rtponse corticale pour stimulation nerveuse de rester constante malgrC une croissance en longueur de la voie visuelle. Le chat n’est en effet adulte qu’h I’fige de 12 mois. Hursh (1939) a d‘ailleurs montrk chez ce m&me animal, au cows de la croissance post-natale, I’existence d’une relation lintaire entre la vitesse de conduction et l’augmentation progressive du diambtre des fibres mytlinistes du nerf saphbne. C A R A C T ~ R E SB L E C T R I Q U E S

DE L A REPONSE

CORTICALE

LOCALISBE

La diminution du dtlai de propagation des messages affkrents constitue I’un des tCmoins de l’tvolution des rkponses corticales. La modification de leurs caracttres tlectriques en est un autre. Pour la somesthtsie et la vision, les rCponses, de signe tlectrique ntgatif les premiers jours de leur apparition, acquikrent progressivement une phase positive initiale (Fig. 3). Dans le systbme auditif, les potentiels tvoquts, apparus relativement tard, ont d’eniblte une phase positive initiale dont l’aniplitude augmente rapideinent (Fig. 4). Cette tvolution survient a une date difftrente pour chaque syst2me et confkre finalenlent aux rCponses leur aspect classique d’onde diphasique A signe positif initial. L’acquisition dz ces caractbres tlectriques dtfinitifs est Bibliographic p . 234

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contemporaine de profondes modifications de la structure du cortex cerebral, qui ont dtjk fait l’objet de nonibreuses publications; celles de Ramdn y Cajal (1909-191 1) restent parmi les plus demonstratives.

Fig. 3. Evolution de la reponse electro-corticale a la stimulation photique chez le lapin. Anesthesie legere (Nembutal ou urethane). Stimulation photique (flash). Lapins iiges respectivement de 9 jours (A), 1 1 jours (B), 13 jours (C) et 17 jours (D). Temps: 100 msec (A et B) et 50 msec (C et D). Amplitude: 200 pV.

La recherche de correlations anatomo-physiologiques prtcises justifiait cependant la reprise d’une ttude cyto-architectonique, en utilisant conjointement la mtthode de Nissl et la mtthode de Golgi, et en confrontant les rksultats obtenus avec ceux des investigations neurophysiologiques. Les modifications cyto-architectoniques observables dbs la naissance dans le cortex ctrtbral sont multiples : accroissement de I’tpaisseur de l’Ccorce, diminution de la densit6 cellulaire, augmentation de volume du soma des cellules pyramidales, apparition relativement tardive des neurones stellaires en reprtsentent quelques aspects. Mais le caractkre dominant de cette tvolution structurale apparait &tre le dkveloppement du champ dendritique basilaire des neurones pyramidaux. L‘ttude chronologique de l’aire visuelle du lapin, prise k titre d’exemple, le montre. A la naissance, la majorite des cellules pyramidales dans cette aire possbdent une dendrite apicale bien d6veloppie mais sont k peu prbs dtpourvues de dendrites basilaires (Fig. 5). Celles-ci apparaissent dans les jours qui suivent la naissance et se

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Fig, 4. RBponse tlectro-corticale a la stimulation sonore chez le chat. Chat de 20 jours. Nembutal. Stimulation sonore (ciic). Sur tous les trads inferieurs figure la rkponse focale enregistrk a i’endroit reprisente sur le schema par un triangle. Le signe electrique des riponses et leur topographie sont identiques, 2 cet gge, B ceux de I’animal adulte.

ramifient progressivement. En m2me temps, le soma cellulaire augniente de voluint et les collatCrales de la dendrite apicale se multiplient. Vers le 1Oeme jour de la vie nCo-natale, l’epanouissement du champ dendritique basilaire est dCj2 tres prononck (Fig. 6 ) . A la fin de la troisitme semaine, les czllules pyramidales de l’aire visuelle d u Lapin ont un aspect identique a celui des neurones adultes. Cette dissociation entre le dCveloppement de la dendrite apicale et celui des dendrites basilaires n’est pas une constatation originale puisqu’on la trouve dkjja dkrite par Ram6n y Cajal (1909-191 1); mais sa chronologie n’avait pas semble-t-il CtC prCcisCe jusqu’ici. Au point de vue anatomique, la proliferation des dendrites basilaires determine directement la diminution de la densite cellulaire dans l’kcorce ctrtbrale, mais surtout elle accroit la surface rCceptrice des neurones pyramidaux dans dcs proportions considkrables. Les mesures effectutes par Schad& et Baxter (1960) chez le Lapin en donnent Line idte precise. A l’lge adulte, la surface dendritique representc pres de 95 p. 100 de la surface totale du neurone dont 70 p. 100 reviennent aux dendrites basilaires. Au dixiknie jour de la vie nCo-natale, date B laquelle nos imprkgnations soulignent l’importance de l’epanouissement des dendrites basilaires (Fig. 6), ces dernieres occupent dCjB 60 p. 100 de la surface totale cellulaire (SchadC et Baxter). Cet accroissement considkrable de la surface rkceptrice du neurone s’accompagne Biblingraphie p . 234

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Fig. 5. Developpement morphologique de I’ecorce visuelle cerebrale du lapin. Lapin de 48 h. Cellules pyramidales de l‘6corce visuelle. Les dendrites basilaires sont a peine Cbauchks. La dendrite apicale est bien dCveloppQ. Col. : methode de Golgi-Cox. Gr. : x 900.

peut-Etre d’une multiplication des contacts synaptiques, mais l’organisation et l’importance de ce champ synaptique basilaire restent 5 demontrer. De toutes faGons, l’augmentation de la surface du neurone permet I’acquisition par la cellule de modalitks nouvelles d’excitabilitb. Or, il apparait que l’apparition du signe Clectrique dkfinitif des rkponses corticales coincide avec l’kpanouissementdu champ dendritique basilaire des neurones pyramidaux (Marty et Chevreau, 1961) et se situe aux environs de la date a laquelle la surface dendritique basilaire pourrait representer 50 p. 100 de la surface totale. D’autres auteurs avant nous n’avaient pas manque d’evoquer le r61e possible de la proliferation des dendrites basilaires dans l’apparition du signe ClCctrique definitif des rCponses corticales (Purpura et al., 1960). On peut aujourd’hui Ctablir cette corrtlation fonctionnelle sur des bases chronologiques prkcises. L‘Ctude chronologiyue d‘un systtme afferent dans une esptce la dtcrit nettement, comme on vient de le voir (Figs. 3 et 6 ) ; la comparaison du degrC d’evolution respectif du territoire cortical et des rCponses dans deux syst6mes diffkrents d’affkrences chez le mEme animal la con-

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Fig. 6. DBveloppement morphologique de I’ecorce visuelle ckrebrale du lapin. Lapin de 10 jours. Cellule pyramidale de l’korce visuelle. Noter par rapport a la Fig. 5 I’augmentation de volume du corps cellulaire, I’importance des ramifications de la dendrite apicale et surtout la proliferation des dendrites basilaires. Col.: methode de Golgi-Cox. Gr. : x 900.

firme. Enfin, cette correlation se retrouve aussi bien ;hez le lapin et chez le chat. Chez l’animal adulte, nous rappellerons que Bishop et Clare avaient dkjja soulignk en 1952 le r81e des dendrites basilaires des neurones pyramidaux dans la genbse de la phase positive initiale des potentiels enregistrts A la surface de I’tcorce ckrkbrale. Purpura et al. (1960) ont Ctabli de leur c8t6, chez le chat nouveau-nk, les relations existant au cours du dkveloppement entre la structure d u cortex ctrtbral et l’tvolution des rkponses i la stimulation corticale directe. La prtsence dks la naissance de nombreuses synapses axo-dendritiques dans la couche superficielle de l’tcorce explique le signe Clectrique ntgatif des reponses locales obtenues ja cet 2ge. Purpura (1961) pense mEme que ces synapses pourraient &treen cause dans l’tlaboration de la rtponse par une stimulation d’affkrence. En tout ttat de cause, ce n’est pas ntcessairement le m&meagrkgat neuronique qui doit Etre tenu pour responsable de la reponse surface nkgative et des deux composantes de la rkponse diphasique qui lui succbde dans le temps, tout comme il.n’est pas Bibiiographte p. 234

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actuellenient acquis que ces deux composantes rtsultent de I’activitt des nihies cellules corticales.

AFFBRENTS S U R 6 L E CTRO- C O R T I C A L E GBNBR A L I S B E

RETENTISSEMENT DES MESSAGES

L’ACTIVIT~

Dans la ptriode nto-natale, I’activitt Clectro-corticale spontanee s’organise progressivement. L‘apparition des premibres rtactions d’arrCt (arousal reaction) en constitue I’ttape tvolutive principale. Chez le lapin, on observe ces reactions pour la preniikre fois au dtbut de la 2eme semaine, peu aprks le moment ou l’activitt electro-corticale spontante est devenue continue. A cette tpoque, elles surviennent A la suite de mouvements de l’animal ou sont dtclenchtes par des stimulations de caractkre nociceptif. Au cours de la deuxikme semaine, les autres modalitts sensitivo-sensorielles se montrent a leur tour capables de provoquer une reaction d’arrCt mais leur intervention se fait dans un ordre bien dtfini. En effet, les stimulatiam tactiles et olfactives sont actives avant les stimulations auditives qui prtctdent elles-mCmes les excitations visuelles. Ainsi se trouve confirm6 par une autre mtthode tlectrophysiologique I’ordre d’entrte en fonction des principaux systtmes afftrents se projetant sur l’tcorce ctrtbrale (Schemer, Contamin et Verley, 1960). La recherche d’un substratum histologique en ce domaine est difficile car elle doit ntcessairement s’ttendre aux structures du tronc ctrtbral encore insuffisamnient connues. L‘apparition des rtactions d’arrCt va de pair avec d’importantes modifications de la rtactivitt motrice qu’il est inttressant de rappeler. Les premiers jours, l’animal prtsente systtmatiquement une rtaction motrice gkntraliste en rtponse 5 toutes les stimulations, ce qui s’observe encore lorsque les premikres rtactions d’arrCt surviennent. Puis progressivement, les rtactions motrices se localisent B certains groupes musculaires, cependant que les rtactions d’arrCt conservent leur caractkres. On assiste ainsi, au cours des premieres semaines de la vie nto-natale, B une rtduction de l’activitk motrice contemporaine de l’organisation de la rtactivitt corticale globale (Verley et Schemer, 1960). RESUME

La maturation post-natale des systkmes affkrents corticaux a CtC CtudiCe chez le chat et le lapin. Trois crittres tlectrophysiologiques,la vitesse de propagation des messages, les caracttres tlectriques de la rkponse corticale locale et le retentissement des messages sur l’activitt tlectro-corticale gtntraliste ont ttC proposts. Des recherches histologiques ont complttt ces investigations. (1) L‘ttude de la propagation des messages afftrents met en tvidence l’anttrioritt fonctionnelle de la somesthtsie sur l’audition et la vision. L‘activitt des systkmes auditif et visuel est en effet subordonnte a l‘entrte en jeu de rtcepteurs complexes, comme le prouve ]’existence de rkponses corticales B la stimulation tlectrique du nerf cochleaire et du nerf optique a un ige ou il n’existe pas encore de rtponses aux stimulations naturelles.

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En ce qui concerne la vitesse de propagation, la mytlinisation des fibres affkrentes apparait Etre Ie substratum histologique principal du gain de conduction. (2) Les caractbres tlectriques de la rtponse corticale locale se modifient au cours de la maturation. A une date diffkrente pour chaque systbme, la rCponse acquiert ses caracttristiques Clectriques definitives. Ce phCnom5ne est contemporain de profondes modifications de la structure du cortex cCrtbral. Parmi celles-ci, l’accroissement considtrable de la surface rtceptrice des neurones pyramidaux, di3 en grande partie a la proliferation du champ dendritique basilaire, semble jouer un r81e dtterminant dans 1’Cvolution du signe tlectrique des rCponses. (3) Dans la pCriode post-natale, I’activitt tlectro-corticale spontanee s’organise progressivement. L‘apparition des premibes reactions d‘arrEt en constitue l’ttape tvolutive principale. La chronologie d’apparitjon des reactions d’arret en fonction des diverses modalitts afferentes est identique a celle que I’on observe pour les rtponses corticales locales.

SUMMARY MATURATION OF

THE

CORTICAL

AFFERENT

SYSTEMS

The post-natal maturation of the cortical afferent systems has been studied in the cat and the rabbit. Three electrophysiological criteria have been proposed : propagation speed of the messages, electrical characters of the local cortical response and repercussion of the messages on the general electro-cortical activity. Histological research has completed these investigations. (1) The study of the propagation of the afferent messages reveals the functional anteriority of the somesthesia over audition and vision. The activity of the auditory and visual systems depends in fact on the coming into play of complex receptors, as has been proved by the existence of cortical responses to the electrical stimulation of the cochlear and optic nerves at a n age when natural stimulations are not yet receptive to response. As to propagation speed, the myelinization of the afferent fibres seems to be the main histological base for speed increase. ( 2 ) The electrical characters of the local response change during maturation. At different ages, according to each system, the response acquires its definitive electrical characteristics. This phenomenon is contemporaneous with deep modifications in the structure of the cerebral cortex. Among them, considerable increase of the receptive surface of the pyramidal neurons, due for the most part to the proliferation of the basilar dendritic field, seems to play a determinant part in the evolution of the electrical sign of the responses. (3) In the post-natal period, the spontaneous electro-cortical activity organizes itself progressively. The appearance of the first arousal reactions constitutes its main evolutive stage. The chronology of appearance of the arousal reactions, according to the various afferent modalities, is identical to that observed in evoked cortical responses. BibIiogrophie p. 234

234

DISCUSSION

BIBLIOGRAPHIE BISHOP,G. H., ET CLARE,M. H., (1952); Sites of origin of electric potentials in striate cortex. J. Neurophysiol., 15, 201-220. CAIAL,S. R A M ~Y,N(1909-191 1); Histologie du systhme nerveux de I'Homme et des Vertibrks. 2 Vol. Paris, Maloine. ELLINGSON, R. J., (1958); Electroencephalograms of normal, full-term newborns immediately after birth with observations on arousal and visual evoked responses. Electroenceph. clin. Neurophysiol., 10, 31-50. HOGG,L. D., (1941); Sensory nerves and associated structures in the skin of human fetuses 8 to 14 weeks of menstrual age, correlated with functional capability. J. comp. Neurol., 75, 371410. HUMPHREY, T., ET HOOKER, D., (1959); Double simultaneous stimulation of human fetuses and the anatomical patterns underlying the reflexes elicited. J. comp. Neurol., 112, 75-102. S., (1951); Maturation of evoked response of the visual cortex in the HUNT,W. E., ET GOLDRING, postnatal Rabbit. Electroeneeph. clin. Neurophysiol., 3, 465471. HURSH, J. B., (1939); The properties of growing nerve fibers. Amer. J. Physiol., 127, 140-153. MARTY,R., (1962); Developpement post-natal des reponses sensorielles du cortex cerebral chez le chat et le lapin. Aspects physiologiques et histologiques. Arch. Anat. micr. Morph. exp., 51, 129-264. J., (1961); Maturation des rkponses visuelles et edification du neuropile MARTY,R., ET CHEVREAU, cortical. C . R. SOC.Biol. (Paris), 155, 705-707. MARTY,R., ET THOMAS, J., (1963); Reponse electro-corticale A la stimulation du nerf cochleaire chez le chat nouveau-ne. J. Physiol. (Paris), 55, 165-166. PURPURA, D. P., (1961); Analysis of axodendritic synaptic organisations in immature cerebral cortex. Ann. N . Y. Acad. Sci., 94, 604-654. M. W., ET HOUSEPIAN, E. M., (1960); Physiological and anatomical PURPURA, D. P., CARMICHAEL, studies of development of superficial axodendritic synaptic pathways in neocortex. Exp. Neurol., 2, 324-347. G., (1957); Electrical signs of maturation in the auditory ROSE,J. E., ADRIAN,H., ET SANTIBAN~Z, system of the Kitten. Acta neurol. 1at.-amer., 3, 133-143. J. P., ET BAXTER, C. F., (1960); Changes during growth in the volume and surface area of SCHAD~, cortical neurons in the Rabbit. Exp. Neurol,, 2, 158-178. J., ET OECONOMOS, D., (1 954); Reponses corticales somesthesiques du Mammifere nouveau~CHERRER, ne cornparks celles de l'animal adulte. Erud. nio-natal., 3, 199-216. J., CONTAMIN, F., ET VERLEY, R., (1960); Maturation comparie des rkponses Plectro-corticales SCHERRER, et des activiiis motrices et neuro-vkgitatives chez les Mamm$hres (Primate3 excepiiee). Rapports ler Congrtts europ. Pedo-psychiatrie, Paris, p. 61-76. L., (1924); Myelogeny as applied to the study of behavior. Arch. Neurol. TILNEY,F., ET CASAMAJOR, Psychiat. (Chic.), 12, 1-66. J., (1960); Etude experimentale des relations entre reactions motrices et VERLEY, R., ET SCHERRER, reactions electrocorticales aux stimulations au cours du developpement postnatal du lapin. Rev. neurol., 102, 311-315. WILSON,V. J., (1962); Reflex transmission in the Kitten. J . Neurophysiol, 25, 263-276. A. M., (1931); Observations on embryonic and fetal movements of the WINDLE,W. F., ET GRIFFIN, Cat. J. comp. Neurol., 52, 149-188. WINKELMANN, R. K., (1960); Similarities in cutaneous nerve end-organs. Advances in biology of' skin. Yol. 1. Cutaneous innervation. W. Montagna, Editeur. Oxford, Pergamon Press (p. 48-62). DISCUSSION

PURPURA: There are a number of points that occurred to me and I will take the prerogative as chairman to make some remarks. The lack of relationship apparently between the appearance of early myelination and the lack of a latency change of any significance is identical to what we found in the cortico-spinal system. In other words, there is early appearance of myelination in the cortico-spinal tract and yet the conduction velocity of the largest fibres does not

S Y S T ~ M E SAFFBRENTS

CORTICAUX

235

significantly change. The values are scattered still between 1.5 to 2.5 m per sec. If you go from 1.5 to 2.5 m per sec that is obviously a tremendous change, percentagewise, but it is unsignificant in terms of the total conduction velocity. So latency-wise if you have 30 msec to go from motor cortex to medullary pyramid and now you are going at 28 msec, even in 2 weeks there is not that much change. Perhaps there is a minimal amount of myelin that is required to give a significant change in conduction velocity. This brings to mind some of Skoglund’s investigations, that there was to be a change in axis cylinder diameter beyond which one starts to see a change in conduction velocity. There is obviously something here with respect to the actual ionic movements which we really don’t quite understand. The second point I would like to make before 1 turn the discussion over is the question of the relationship between the cholinesterase. We are not surprised that perhaps one has a few percentile of cholinesterase remaining and we can still get function. The question I raise is whether there is any relationship between the amount of cholinesterase that is present and any activity anyway. Curtis and coworkers attempted with their multi-barrel technique to apply acetylcholine to neurons in the superior colliculus and they met with absolute failure in all respects to activate cells in the colliculus. This area is known to have a tremendous amount of cholinesterase. I feel it is hardly possible to make any relationships between the postsynaptic events and acetylcholine. At least, I don’t know where the acetylcholine is, is it in the neuron or is it in the glia? WAELSCH: I fully agree with what you have found. I am not a cholinesterase addict and among things which always impressed me very much was one picture which Koelle published many years ago, which I found was most elucidating for those people interested in the cholinesterase problem. The paper appeared at the time when there was a long discussion about cholinesterase in the optic nerve. Koelle applied his method to the optic nerve and he found only a few fibres to carry cholinesterase while the majority of the fibres were negative. It is not only the question that you find it but also where you find it. SCHERRER : The relationship between conduction velocity and myelination is certainly a very interesting question. As you all know electronmicroscopists have shown rotating phenomena in the development of myelin around the axon. One would expect then even 2 or 3 rotations would be enough or more or less enough to make some isolation and to have a tremendous increase in speed. However, there is a real discrepancy between what we expected and the results of the experiments. We think that we found a slight increase perhaps because we do not have a decrease, and we ought to have a decrease because the nerve gets longer. We have a small increase but it seems to be insufficient. There is still no agreement between the electronmicroscopists and the neurophysiologists, who try to explain the changes in conduction velocity during maturation of the brain. WAELSCH: In the non-myelinated nerve before myelination there are of course the satellite cells. Is there any indication of the presence of nodes of Ranvier before myelination starts?

236

DISCUSSION

SCHADB: In preparations made according to the Haggqvist method for myelinated fibres one can detect very small amounts of myelin. In our experience there should always be a minimal amount of myelin to show an indication of a node of Ranvier. This minimal amount could very well be less than 0.1 of the final amount of myelin around the mature nerve fibre. PURPURA: But then you have properties of membrane to consider which probably are playing a role here, but that is only speculation. The question of the association response of course is something peculiar. You don’t obviously see this second long latency response to sciatic stimulation. Only on visual stimulation you find the fused activation of the cortex in the 1-day-old kitten. SCHERRER: We have no explanation for this difference. This second long latency response is only found on visual stimulation and not on sciatic or any other peripheral stimulus. We are dealing with anesthetized animals. SCHAD~: I am particularly interested in the contribution of the reticular formation to this type of response. Is it possible to differentiate the influence of the reticular formation in this response in the 1-day-old kitten and the adult cat? SCHERRER: We do not know what is likely to be the influence of the reticular formation on the blocking reactions. Blocking reactions occur in the rabbit on the 9th or 10th day. We think that the reticular formation is already active at that time. In the cat it is a bit later. PURPURA: I believe it is present at birth and that there are reticular components to specific activation. The sciatic stimulation under very favourable conditions in the 1-day-old cat will evoke not only the long latency negative response but multiple active responses which are very selectively knocked out by small amounts of nembutal. The multiple activities are selectively very sensitive to nembutal. I would suspect that these are not going along the specific projection system. This was observed in the 1-day-old kitten. LEVI-MONTALCINI: I have difficulties in imagining the onset of myelination. In the rotation theory: does the whole axon rotate or only parts of it? SCHERRER: We have only data about the onset of myelinization in the optic system. In the optic system the myelination starts in the medial plane, that is at the optic chiasm. Dr. Marty emphasized the importance of glia proliferation before myelination in the whole system. The presence of big vessels at the point where myelination starts is of importance.

237

*-

.

-

f

r’*

Brain Mitochondria S 0 R E N L0VTRUP Institute of Neurobiology, University of Goteborg, Goteborg (Sweden)

After the extensive studies on the properties of liver mitochondria the interest in mitochondria from other tissues has gradually increased. Although all mitochondria unquestionably have many basic features in common, some tissue specificity might expectedly obtain. The establishment of such differences may furnish important information about mitochondria1 activity in general, and also about various tissue specific activities. Work with brain mitochondria is particularly challenging because of the possibility it opens to discover properties, physical or chemical, which are peculiar to the brain. If such be found, they may eventually contribute to our endeavours to reach an understanding of the mechanism of brain action. Work on brain mitochondris was initiated by Brody and Bain (1952), but progress in the field has been rather slow. Even studies on the effects of psychopharmaca like amytal, chlorpromazine, etc., have been carried out to a major extent on liver mitochondria. The reason for this fact seems to be that brain mitochondria, isolated by procedures identical to those used for preparation of liver mitochondria, generally do not pass the established tests of morphological and chemical purity. This circumstance was considered a challenge, which has led to the development of a modified method for preparation of brain mitochondria (Lervtrup and Zelander, 1962). The method may not give mitochondria which meet maximum requirements, but it will be shown below that they conform to standard demands with respect to morphological and chemical properties. ISOLATION PROCEDURE

Rat brain is excised, and dura, larger blood vessels, and the major part of the white matter are removed. A 10 per cent homogenate is prepared in 0.44 A4 sucrose. This concentration turned out to give much purer preparations than 0.25 M . Several homogenizers have been tried, but the only one giving satisfactory results was the Dual1 tissue grinder supplied by Kontes Glass Company, New Jersey. The piston is connected with an electric motor through a flexible cable. In this way, it is possible to fix the homogenizer in an ice-cold bath. During the homogenization the piston rotates at a speed of about 1000 rev./min. After a homogeneous suspension has been obtained, the piston is slowly pulled up and down about 15 times. The whole homogenization procedure lasts about 7 min. References p . 252

238

S 0 R E N LPJVTRUP

Fig. I . Survey of a section through the pellet of a mitochondria1 fraction, showing the fairly even distribution of mitochondria (M)and two myelin fragments (I;)to the left, x 12,000.

BRAIN MITOCHONDRIA

239

The resulting homogenate is centrifuged for 10 min in the Spinco centrifuge (rotor SW 25-1) at 5000 rev./min (average 2100 x g). The supernatant is decanted and saved. If maximum yield is desired, the precipitate may be resuspended and centrifuged again under identical conditions. The combined supernatants are centrifuged at 13,000 rev./min (average 14,500 x g) for 15 min. The resuspended precipitate is washed with 0.44 M sucrose and centrifuged at 9000 rev./min (average 7000 x g) for 15 min. This operation is repeated twice, but 0.22 M (or 0.25 M ) sucrose is used as suspension medium, because this change was found to increase considerably the rate of oxidation (cf. Lnrvtrup and Svennerholm, 1962). The last precipitate, resuspended in 1 ml of 0.22 M sucrose per g fresh weight of brain, is employed in the experiments. It is very important to notice that the only satisfactory way of resuspending the mitochondria is to transfer the mitochondrial pellet in sucrose solution to the homogenizer, and homogenize gently by hand. M O R P H O L O G I C A L PROPERTIES

The mitochondria prepared according to the modified isolation procedure were investigated electron-microscopically (Larvtrup and Zelander, 1962). The preparations were found to contain three morphologically distinguishable kinds of structures. The evenly distributed, round or elongated mitochondria are predominant. A few, usually circular and split fragments of myelin are also seen (Fig. 1). Finally some round, elongated, or less regularly shaped bodies are observed, outlined by a single membrane and with a homogeneously dense internal substance. These dense bodies are of the same order of size as the smaller mitochondria (Fig. 2). Aggregation between mitochondria or between mitochondria and other structures is exceptional, and very few microsomal structures are seen. Occasionally two or three mitochondria are located in a common body interpreted as a small piece of cytoplasm encircled by a membrane. Evidently the morphological preservation of the mitochondria varies, but it is sufficient for easy identification. Changes are noted in the isolated mitochondria compared to those in brain tissue sections. Dilatations of the light layer of the three-ply outer mitochondrial membrane, interruptions in the same structure and broadening of the low density areas between inner membranes are the changes usually observed. The mitochondrial matrix has a very variable density, which in some mitochondria is abnormally low (Fig. 2). Some of these changes are undoubtedly a result of the fixation procedure, others may have arisen during the long isolation procedure prior to fixation. The purity of the mitochondrial fractions may vary somewhat from one preparation to another. As a measure of the purity the number of mitochondria per myelin fragment and per dense body may be counted from the micrographs. In the best fractions so far studied there are 300-500 mitochondria per myelin fragment and 50-100 mitochondria per dense body. These ratios are valid for the plane of the section. Presuming a statistical distribution of the impurities, the ratios in a given References p . 252

240

S 0 R E N L0VTRUP

Fig. 2. Two types of morphological changes of the mitochondria1 structure are seen, (A) widening of the internal membranes (cristae) and (B) decreased density of mitochondrial matrix. A dense body @B) is seen in the upper left-hand corner, x 71,000.

BRAIN MITOCHONDRIA

24 1

volume will be 5000 to 10,000 mitochondria per myelin fragment and 350 to 1000 mitochondria per dense body. An impression of the purity is also to be gained by the experiments on the subcellular localization of glutamic decarboxylase in brain (Lervtrup, 1961). The results showed that about 4 per cent of the total enzyme activity (5 per cent of the recovered activity) was present in the mitochondrial fraction. Assuming that this enzyme is absent in mitochondria, the observed activity will be an index of the presence of impurities. The contamination on the basis of dry weight or nitrogen will of course depend on the specific enzyme activity of the impurities, and may well be more than 4 per cent. One may also assume, of course, that the enzyme is present in the matrix of intact mitochondria, but is lost to the suspension medium during the fractionation procedure, and thus recovered in the supernatant. Since this fraction contains about 34 per cent of the total or 41 per cent of the recovered activity, it follows that 7 out of 8 mitochondria should have lost all enzyme from the mitochondrial matrix. Unless such loss can occur without any swelling or derangement of the mitochondrial structure, this possibility seems unlikely considering the reasonably well preserved structure shown in Figs. 1 and 2. Furthermore, loss of one enzyme without simultaneous loss of others present in the matrix, is not very probable. The enzymes located here are believed to include all those involved in the substrate-level transformations of the Krebs’ cycle. The very high specific activity of oxidation of brain mitochondria, higher than for liver mitochondria (cfi Lervtrup and Svennerholm, 1962), seems difficult to reconcile with an extensive loss of enzymes from the mitochondrial matrix. The morphological and chemical evidence taken together thus suggests that the brain mitochondria isolated according to the modified procedure are reasonably pure. CHEMICAL PROPERTIES

Composition of the incubation medium Different substrates may require minor variations in the incubation medium in order to ensure maximum rate of oxidation. It is, however, expedient to use a standard medium for all substrates, even if the oxygen uptake is somewhat below maximum for certain substrates. The inorganic components of the medium used in my laboratory for the manometric work with brain mitochondria were the following: 100 pmols KCI, 13 pmols MgS04, 27 pmols K-phosphate buffer, PH 7.4. By changing the concentrations of these components, in either direction, increases in the rate of oxidation were observed in some cases, more often the changes were insignificant, and in some cases very drastic decreases occurred. It was thus concluded that the composition given above was acceptable for a standard medium. In these experiments, the effect of K+ was investigated by increasing and decreasing the KC1 concentration three times. If the mitochondrial activity is to be determined in the absence of K+, the buffer must also be changed. An attempt to demonstrate the K+ effect is shown in Table I, where in one series Naf-phosphate buffer was used. It is seen that within each of the series the effect of K+ is rather slight, the complete References p . 252

242

S0REN L0VTRUP

TABLE I I N F L U E N C E OF

K+

Na+

pmols

0 33 100 81 148 348

48 48 48 0 0 0

K+ ON

T H E R A T E OF O X I D A T I O N

Pyruvaie f malaie paioms

0 2 per

19 21 22 71 73 69

Succinaie

h and nig N

43 49 49 66 72 59

At PH 7.4,4 parts secondary and 1 part primary phosphate is mixed. The concentration of K+ or 4 x 2 x 0.133 1 x 0.133 = 0.24 M . Thus 200 p1 0.133 M P-buffer contains Na+ therefore is

+

5

48 pmols of cation.

omission of potassium leading only to a 10 per cent loss in activity. The oxidation rates are much lower in the presence of sodium. The experiments were made on different mitochondria1 preparations, and part of the difference may be a result of variation between batches, but the difference is great, and a certain inhibition by Na+ is therefore indicated. The striking stimulation of oxygen uptake by K+ observed with brain slices (cf. Quastel, 1962) is thus not observed with brain mitochondria. The presence of Na+ is necessary for the maintenance of brain slice respiration (Hertz and Schou, 1962), but apparently not for the oxidation in brain mitochondria. Finally, it may be mentioned that no inhibition of slice respiration by Na+ is observed except at very high concentrations. Besides the inorganic components the medium contains 26 pmols substrate, a phosphate trap (2 ymols ATP, 56 pmols glucose, and 0.75 mg hexokinase), and mitochondria corresponding to 0.5 g fresh weight of brain in 500 pl0.22 M sucrose. The reaction volume was made up to 2 ml with 0.22 M sucrose, generally 700 p1 was added. The addition ofcytochrome c and DPN led to significant increases (10-20 per cent) in the rate of oxygen uptake, but in spite of that it was decided not to include these substances in the standard medium. Concentration of substrates and mitochondria In the work on oxidation we have added 26 ymols substrate per vessel, in a total reaction volume of 2 ml. The resulting concentration is actually too low to ensure maximum respiration with pyruvate, glutamate, and a-ketoglutarate; only with succinate is maximum rate of oxygen consumption observed (Larvtrup and Svennerholm, 1962). It may therefore be reccmmended to increase the addition to 50 pmols. The rate of oxaloacetate oxidation is very sensitive to changes in the concentration.

BRAIN MITOCHONDRIA

243

TABLE I 1 R A T E OF O X I D A T I O N OF V A R I O U S S U B S T R A T E S

patoms per h and mg N ~~

+

Pymvate malate Succinate Glutamate a-Ketoglutarate Oxaloacetate Glycerol-1-phosphate Fumarate Glucose-6-phosphate /?-Hydroxybutyrate Isocitrate Citrate y-Aminobutyrate Aspartate

56.2 48.7 22.8 20.2 8.6 7.0 6.6

6.3 5.2 2.7 0

60.3 38.9 18.4 21.6 8.2 7.3 5.8 3.2 2.6 0.6 0

As shown in Table I1 a value of about 70 is obtained with the addition of 26 pmols oxaloacetate; with the same niitochondria and with an addition of 13 pmols the rate of oxidation was 33. In a number of experiments where the substrate addition was 37 pmols values around 20 were obtained. Oxidation of various substrates

Among 15 substrates tested, only 5 were oxidized at high rates, viz. pyruvate, succinate, glutamate, a-ketoglutarate, and oxaloacetate (Table 11). The average values for the rate of oxidation of these substrates lie in the range 45-75 patoms 0 2 per h and mg N at 37". Expressed as QOZ(pl per h and mg protein) the corresponding values are 80-135. These values compare favourably with QOZvalues reported for other mitochondria. As an arbitrary example it may be mentioned that Azzone and Carafoli (1960) found somewhat lower values for liver mitochondria (27-55), an observation which has been confirmed in my laboratory, whereas the value for muscle mitochondria reported by these authors (33-157) is in the same range as that found for brain mitochondria. However, these results were obtained at 25", the corresponding values at 37" will be somewhat higher. In niitochondria with low oxaloacetate decarboxylase activity an accumulation of oxaloacetate may occur after incubation with succinate, resulting in a low rate of succinate oxidation (e.g. rat and human muscle mitochondria, Azzone et al., 1961). Like rat liver and pigeon breast muscle mitochondria those from brain can oxidize succinate at a quite high rate, indicating the presence of a fairly active oxaloacetate decarboxylase. Amytal will increase succinate oxidation by muscle mitochondria, because it prevents oxaloacetate formation by inhibiting malate oxidation. No effect on succinate oxidation could be observed with brain mitochondria, in complete agreement with expectation. It is seen from Table I1 that y-aminobutyric acid is not oxidized by brain mitoReferences p. 252

244

S B R E N LPJVTRUP

chondria. The Krebs’ cycle shunt: a-ketoglutarate +glutamate + y-aminobutyrate + succinic semialdehyde + succinate, which has been found in brain (Bessman et al., 1953; Roberts and Bregoff, 1953), cannot therefore be located in the mitochondria. This conclusion is corroborated by the fact that only a small fraction of the total glutamic decarboxylase activity is recovered in the mitochondrial fraction (Larvtrup, 1961). Glutamic dehydrogenase activity cannot be demonstrated in brain tissue. It therefore seems that all glutamate in brain mitochondria is oxidized via the glutamicaspartic cycle (Krebs and Bellamy, 1960; Borst and Slater, 1960’. Brain mitochondria are similar to muscle mitochondria in oxidizing glycerol-1-phosphate at a rather high, and fatty acids (p-hydroxybutyrate) at a rather low rate (cJ Klingenberg and Wcher, 1960; Azzone et al., 1961). Oxidation of glucose by brain mitochondria has been reported by several authors, suggesting that by possessing glycolytic capacity they are able to oxidize glucose aerobically (cf- Hesselbach and duBuy, 1953; Gallagher et al., 1956; Balhzs, 1959; Abood et al., 1959). In order to approximate conditions as closely as possible to such under which glucose oxidation has been reported to occur, the incubation medium was fortified with versene and nicotinamide. The results of such an experiment are shown in Table 111. Considering first the effect of versene-nicotinamide, there seems to be a slightly TABLE I11 OXIDATION OF GLUCOSE

patoms per h and

Versene and nicotinamide Hexokinase Glucose

+ -

i g

N

+ + - + - i 7.4 6.9

6.4 0

6.0 6.4

5.8 3.0

enhancing effect on the rate of oxygen uptake in the presence of glucose or hexokinase, or both, whereas the endogenous oxygen uptake is completely inhibited. Addition of glucose, hexokinase, or in combinatioii also increases the rate of oxygen uptake, and to the same extent. It seems very difficult from these experiments to decide definitely whether or not glucose is oxidized by brain mitochondria. However, if it is oxidized, it occurs at a very low rate, so low in fact that it reasonably may be attributed to impurities in the mitochondrial preparations. Respiratory control

Determinations of respiratory control are shown in Table IV. The values are seen to be comparable to those obtained with mitochondria from other tissues, also with respect to the low value obtained with succinate. The determination of respiratory control is most convincingly carried out polaro-

245

B R A I N MLTOCHONDRIA

TABLE I V RESPIRATORY CONTROL

patoms per h and mg N Pyruvare ma

~

No addition ATP hexokinase Respiratory control

+

+ glucose

10.4 74.6 7.2

9.7 60.4 6.2

Succinate

43.1 66.0 1.5

47.8 59.0

1.2

Glutanrare 4.8 53.3 11.1

3.7 54.0 14.6

a-Ketoglutarate

2.8 50.2 17.9

3.2 43.4 13.6

graphically, by estimating the increase in oxygen uptake upon addition of ADP. Such experiments were performed*, but it was found that, with the exception of succinate, the increase in rate of oxygen consumption upon substrate addition was very low, sometimes barely perceptible. The same result was obtained with mitochondria treated with 0.22 M and 0.44 M sucrose. If, however, 0.22 M mitochondria after the final centrifugatio? were suspended in 0. I 1 M sucrose, both endogenous and exogenous oxygen uptakes, as measured polarographically, were substantially increased. Respiratory control on these mitochondria was about 3, thus considerably lower than that observed in the Warburg manometer. One possibility to explain this difference is that the mitochondria are partly uncoupled in the hypotonic solution. This was excluded experimentally by determining the respiratory control of 0.1 I M mitochondria in the Warburg apparatus. The following values were found: 13.5; 1.5; 18.6; and 12.4 for the 4 substrates in the same order as in Table IV. Both these values and the values for the rate of oxygen uptake, were within the normal range. The failure of the mitochondria to respond to ADP addition in the polarograph may be compared with the similar observation by Bellamy and Bartley (1960) in their study of differences between manometric and polarographic methods. Influence of D N P on oxidation and phosphorylation

In Fig. 3 is shown the effect on the oxidation observed upon the addition of 2,4dinitrophenol (DNP), when maximal respiration is secured by the addition of ATP, hexokinase and glucose. With a-ketoglutarate and glutamate no stimulation occurs, and at concentrations above 0.01 m M oxidation is inhibited. Withpyruvate (+ malate) and with succinate a slight stimulation is seen at concentrations around 0.01 mM, but at higher concentrations the rate of oxidation decreases. In the absence of the phosphate-trapping system similar results were obtained, the relative stimulation of pyruvate oxidation being rather high in this case (Lnvtrup and Svennerholm, 1962). It may be noted that the abscissa in the corresponding figure (Fig. 1) in that paper represents the concentration of the added solution of

*

The help of Dr. Joseph T. Cummins in these experiments is gratefully acknowledged.

References p. 252

246

S0REN L0VTRUP

DNP, which is 10 times higher than the final concentration, represented in the abscissa in Fig. 3. Phosphorylation

0 .w L

c

$

50-

U L 0,

IX

25-

0 .......... -

5

4

3

m

5

0

3

CI (M) Fig. 3. The influence of 2,4-dinitrophenol on oxidation and phosphorylation in the presence of various substrates. The vertical bars indicate the standard error of the mean. The results on oxidation represent 3 experiments with pyruvate, and 2 with each of the other three substrates. The results on phosphorylation represent 2 experiments with pyruvate and with glutamate, and one with succinate and with a-ketoglutarate. A = pyruvate. 0 = succinate; A = glutamate; 0 = a-ketoglutarate. -Log

As shown by this figure, phosphorylation is more sensitive to DNP than oxidation, and the effect is almost independent of the substrate. The influence of Ca2+ Ca2+ has a very deleterious effect on the respiration and phosphorylation of liver mitochondria. Thus, in the experiments published by Ernster (1956), the absolute rate of oxygen uptake decreased with increasing Ca2+ concentration, and a continuous decrease occurred during the experiment so that no straight lines could be obtained. In the presence of 0.25 mM Ca2+the rate of oxidation by liver mitochondria was reduced to about 20% of the control, and with 0.5 mM the rate was less than 10%.

c - 0

2

0

0.25

0.50

Ca2' (mM)

Fig. 4. The influence of Ca2+on the oxidation in the presence of various substrates. malate; 0 = succinate; A = glutamate; x = a-ketoglutarate.

=

pyruvate

+

247

BRAIN MITOCHONDRIA

As shown in Fig. 4 brain mitochondria are much more resistant to Ca2+.At a concentration of 0.25 mM 80-90% of the oxygen uptake remains, and even at 0.5 mM the uptake was about 50 ”/,. In all cases normal straight lines were obtained. Phosphory lation In Table V have been compiled mean and standard deviations for the P/O ratio for 5 experimepts in which phosphorylatioa was determined on 4 substrates at the same TABLE V PHOSPHORYLATION

Pyruvate malate

P/O

+

2.5 f 0.3

Succinate

Glutamate

a-Ketoglutarate

2.0 f 0.1

2.4 f0.2

2.5 f 0.3

time. Onlythe value for succinateagrees with the theoretical expectation.The theoretical value of 4 for a-ketoglutarate was never approached. It should be mentioned that values close to 3 were often obtained. Influence of serum albumin on the rate of oxygen uptake and on phosphorylation Serum albumin is known to exert a protective action on oxidative phosphorylation (Berger and Harman, 1955). The effect of this substance on the rate of oxygen uptake and on phosphorylation of brain mitochondria was also ascertained. The results in Table VI show that a striking increase in the rate of glutamate and a-ketoglutarate TABLE V I I N F L U E N C E O F S E R U M A L B U M I N ON T H E R A T E O F O X I D A T I O N A N D O N P H O S P H O R Y L A T I O N

Albumin (final concentration 0.2 %)

+

patoms Oa per h and mg N

-

PI0

-

+

Pyruvate malate

Succinate

Glutamate

a-Ketoglutarate

86.0 81.2 2.7 2.4

89.6 78.8 2.0 1.9

81.5 60.0 2.6 2.3

71.8 49.5 2.8

+

2.4

oxidation occurs, whereas the effect on the two other substrates, although present, is less marked. Also the P/O ratios are improved, but the effect is not so great as that reported for pigeon breast muscles by Azzone et al. (1960). References p . 252

248

S 0 R E N LBVTRUP

DNP-activated ATPase

From the results shown in Fig. 5 it is seen that the enzyme activity in 0.44 M mitochondria is extremely low. The addition of DNP has hardly any effect. If the mito1.5

a

5 -Log M DNP

Fig. 5. The inlluence of DNP on the ATPase activity of various preparations of brain mitochondria. The modification consisted in using 0.22 M sucrose, and (or) adding ATP to the isolation medium during the two last centrifugations in the isolation procedure. - = mitochondria in 0.22 M sucrose; - - - - mitochondria in 0.44M sucrose; x = no ATP; 0 = 1 m M ATP.

chondria are treated with a sucrose solution containing ATP (1 mM) in the last 2-3 steps of the isolation procedure an increase in enzyme activity is observed (Jobsis, personal communication). A better response to the addition of DNP is also observed (Fig. 5). A still higher activity is obtained with 0.22 M mitochondria, but this activity is but little influenced by DNP. It is interesting to note the very slight response of 0.22 M mitochondria to the addition of ATP. The maximal activity, about 1 pg P liberated by 100 mg fresh weight equivalent mitochondria is only 10% of that observed with liver mitochondria (cf. Low, 1959). Mg2+-activated ATPase

Brain mitochondria are maximally activated at a concentration of 1 mM of Mg2+. Since the DNP-stimulated ATPase activity in brain is rather low, there is very little

2

.o

a

f

1.0

0.5

0

. . . . . . . . . . . . . 1

2

3

4

5

6

?

Mg2+ (mM)

Fig. 6. The influence of Mg2+ on the ATPase activity in the presence and absence of DNP and DOC. - = mitochondria in 0.22 m M sucrose; - - - = mitochondria in 0.22 mM sucrose 0.1 % DOC; x = no DNP; 0 = 0.67 m M DNP.

-

+

249

BRAIN MITOCHONDRIA

differencebetween the curves at low Mg2+concentrations (Fig. 6). At higher Mg2+ concentrations the enzyme activity in the presence of DNP is inhibited, while it remains constant in the absence of this substance, thus making the DNP curve the lowest. Treatment with DOC is seen to have no effect on the Mg2+-activatedATPase (Fig. 6). In the absence of Mg2+ the enzyme activity is considerably reduced by DOC-treatment. The 2 curves ( 5 DNP) are identical for the DOC-treated mitochondria, which may mean that under the experimental conditions there is no DNP-stimulated ATPase activity. The ATPase activity in mitochondria from different rat tissues varies considerably in several respects. The data compiled in Table VII illustrate this fact very clearly. TABLE V I I V A R I A T I O N I N T H E C H A R A C T E R I S T I C S O F T H E ATPASEA C T I V I T Y I N M I T O C H O N D R I A F R O M V A R I O U S T I S S U E S OF T H E R A T

Liver

Activation of fresh mitochondria

DNP (+) Mg2+(-)

Activation of DOCtreated mitochondria Activating Mg2+ concentration

DNP (-) Mg2+( High (4 mM)

+)

Muscle

Heart

DNP (+) MgZ+(+)

DNP (-) D N P (-) Mg2+(+) Mgz+(+) DNP with Mg2+

(+I

Low (1 mM)

Brain

D N P (-)* Mg2+(--1 Low

D N P (-) Mgz+(+> Low High concentration of MgZt inhibits in the presence of DNP

*

Human skeletal muscle. The data are collected from Myers and Slater (1957); Holton et al. (1957); Azzone et al. (1960); Azzone et aZ. (1961); Lsvtrup and Svennerholm (1962). The signs and - represent significant, resp. slight or no activation.

+

Liver mitochondria are seen to occupy a rather unique position but even the mitochondria from the other tissues may be distinguished chemically on the basis of the properties of the ATPase activity. Lipid composition of brain mitochondria

It is evident from the results in Table VIII that phospholipids constitute the predominating fraction of the mitochondria1 lipids. The relative distribution of cephalins and lecithins is very similar in both types of mitochondria, but the relative sphingomyelin content is considerably lower (about 60 %) in liver mitochondria. The contents of cerebrosides was considerably higher in brain mitochondria. In these 20-25 % of the cerebrosides occurred as the sulphate ester (sulphatides). In the liver preparation the sulphate ester content was probably somewhat lower, but due References p. 252

250

S0REN L0VTRUP

to the low concentration the result must be very approximate. The results for cholesterol are remarkably low in both preparations, but it should be noted that the ratio of cholesterol to total phospholipids is about 5 times higher in the brain mitochondria. In both sources all cholesterol was in the free form. TABLE VIII R E L A T I V E L I P I D C O M P O S I T I O N OF B R A I N A N D LIVER M I T O C H O N D R I A

Brain mitochondria Liver mitochondria Percentage of total lipids 76.7 91.3 18.0 7.1 5.0 1.3 0 0 0 0

Phospholipids Cerebrosides Cholesterol Triglycerides Gangliosides

Percentage of phospholipids 54.0 59.0 40.1 39.7 6.2 3.6

Cephalins Lecithins Sphingomyelins

It is also important to note that no gangliosides could be detected in any of the preparations, although large samples were assayed. SWELLING

According to Tapley and Cooper (1956), rat brain mitochondria are incapable of swelling, an observation which has been confirmed with my preparations. Swelling ensues, however, if they are treated with Triton X-100, as suggested by Spirtes and Guth (1961), even though these authors observed swelling even in phosphate-water. The degree of swelling is not very high, compared with the swelling of liver mito-

0.013

0.017

n

0.2

-0 0

5

10

15

20

Time (min)

Fig. 7. The influence of Triton X-100 on the swelling of brain mitochondria.The numbers represent the final concentration of the detergent in per cent (w/v).

BRAIN MITOCHONDRIA

25 1

chondria, even in the presence of Triton X-100 (Fig. 7; Tapley, 1956). DNP is a powerful inhibitor of the swelling of liver mitochondria, but has no influence upon brain mitochondria (Fig. 8). The swelling of liver mitochondria is enhanced by Ca2+ (Tapley, 1956), but this substance inhibits the process in brain mitochondria (Fig. 9).

0

5

10

15

20

Time (min)

Fig. 8. The influence of DNP on the swelling of brain mitochondria. The numbers represent the final concentration of DNP in mM.

0.50

s c

0.4

2

0.3

.\

0.25 0

The swelling behaviour of brain mitochondria is thus remarkably different from that of liver mitochondria. It is worth noting in this connection that phosphatides (especially phosphatidic acid and cardiolipin) may be of importance for the swelling process (cf. Lehninger, 1962). As was shown above (Table VIII), liver mitochondria contain much more phosphatides than brain mitochondria, and it was furthermore possible to establish that in the phosphatide fraction from liver mitochondria a considerable part is either phosphatidic acid or cardiolipin (Lsvtrup and Svennerholm, unpublished results, cf. MacFarlane, Gray and Wheeldon (1960) who have shown that the substance is cardiolipin). In brain mitochondria these substances could not be demonstrated. References p . 252

252

SBREN L 0 V T R U P

REFERENCES ABOOD, L. G., BRUNNGRABER, E., AND TAYLOR, M., (1959); Glycolytic and oxidative phosphorylative studies with intact and disrupted rat brain mitochondria. J. biol. Chem., 234, 1307-1 3 1 1. AZZONE,G. F., AND CARAFOLI,E., (1960); Biochemical properties of skeletal muscle mitochondria. I. Oxidative phosphorylation. Exp. Cell Res., 21, 447455. AZZONE,G. F., CARAFOLI, E., AND MUSCATELLO, U., (1960); Biochemical properties of skeletal muscle mitochondria. 11. The ATPase activity and the albumin effect. Exp. Cell Res., 21,456-467. AZZONE, G. F., EEG-OLOFSSON, O., ERNSTER, L.,LUFT,R., AND SZABOLCSI, G., (1961); Studies on isolated human skeletal muscle mitochondria. Exp. Cell Res., 22, 41 5 4 3 6 . BALAZS,R., (1959); The point of the aerobic inhibition of glycolytic activity associated with brain mitochondria. Biochem. J., 72, 561-574. BELLAMY, D., AND BARTLEY, W,, (1960); The effect of the incubation conditions on the respiration of mitochondria. Biochem. J., 76, 78-87. BERGER,M., AND HARMAN, J. W., (1955); Comparative cytology and function of skeletal muscle; oxidative phosphorylation in pigeon skeletal muscle. Amer. J. phys. Med., 34, 467476. BESSMAN, S. P., ROSSEN, J., AND LAYNE, E. G., (1953); y-Aminobutyric acid-glutamic acid transamination in brain. J. biol. Chem., 201, 385-391. BORST,P., AND SLATER, E. C., (1960); The oxidation of glutamate by rat-heart sarcosomes. Biochim. biophys. Acta (Amst.), 41, 170-171. BRODY,T. M., AND BAIN,J. A., (1952); A mitochondrial preparation from mammalian brain. J. bid. Chem., 195, 685496. ERNdTEk, L., (1956); Organization of mitochondrial DPN-linked systems. I. Reversible uncoupling of oxidative phosphorylation. Exp. Cell Res., 10, 704-720. GAL LAG HE^, C. H., JUDAH, J. D., AND REES,K. R., (1956); Glucose oxidation by brain mitochondria. Biochem. J., 62, 436-440. HERTZ,L., AND SCHOU,M., (1962); Univalent cations and the respiration of brain cortex slices. Biochem. J., 85, 93-104. HESSELBACH, M. L., AND DUBUY,H. G., (1953); Localization of glycolytic and respiratory enzyme systems on isolated mouse brain mitochondria. Proc. SOC.exp. Biol. ( N . Y . ) , 83, 62-65. HOLTON,F. A., HULSMANN, W. C., MYERS,D. K., AND SLATER, E. C., (1957); A comparison of the properties of mitochondria isolated from liver and heart. Biochem. J., 67, 579-594. KLINGENBERG, M., AND BUCHER,T., (1960); Biological oxidations. Ann. Rev. Biochem., 29, 669-708. KREBS,H. A., AND BELLAMY, D., (1960); The interconversion of glutamic acid and aspartic acid in respiring tissues. Biochem. J., 75, 523-529. LEHNINGER, A. L., (1962); Water uptake and extrusion by mitochondria in relation to oxidative phosphorylation. Physiol. Rev., 42, 467-517, L0VTRUP, S., (1961); The subcellular localization of glutamic decarboxylase in rat brain. J. Neurochem., 8, 243-245. L~VTRUP, s., AND SVENNERHOLM, L., (1963); Chemical properties of brain mitochondria. Exp. Cell Res., 29, 298-313. LSVTRUP,S . , AND ZELANDER, T., (1962); Isolation of brain mitochondria. Exp. Cell Res., 27,468473. Low, H., (1959); On the participation of flavin in mitochondrial adenosine triphosphate reactions. Biochim. biophys. Aeta (Amst.), 32, 1-10. MACFARLANE, M. G., GRAY,C. M., AND WHEELDON, L. W., (1960); Fatty acid composition of phospholipids from subcellular particles of rat liver. Biochem. J., 77, 626631. MYERS,D. K., AND SLATER, E. C., (1957); The enzymatic hydrolysis of adenosine triphosphate by liver mitochondria. 2. Effect of inhibitors and added cofactors. Biochem. J., 67, 572-578. QUASTEL, J. H., (1962); Effects of electrolytes on brain metabolism. Neurochemistry, 2nd Ed., K. A. C. Elliot, Irvine H. Page and J. H. Quastel, Editors. Springfield, Charles Thomas (pp. 226-237). ROBERTS, E., AND BRECOFF,H. M., (1953); Transamination of y-aminobutyric acid and B-alanine in brain and liver. J. biol. Chem., 201, 393-398. SPIRTES,M. A., AND GUTH, P. S., (1961); An effect of chlorpromazine on rat-mitochondria1 membranes. Nature (Lond.), 190, 274-275. TAPLEY, D. F., (1956); Effect of thyroxine and other substances on swelling of rat liver mitochondria. J. biol. Chem., 222, 325-339. TAPLEY, D. F., AND COOPER, C., (1956); Effect of thyroxine on the swelling of mitochondria isolated from various tissues of the rat. Nature, 178, 11 19.

BRAIN MITOCHONDRIA

253

DISCUSSION

WAELSCH: I have more notes than I can discuss, but I only want to say how important this type of work is about these brain mitochondria. I think this work is really characterized by its great clarity of experimental approach. There are a few interesting points which I would like to discuss. You relate the hydroxybutyrate oxidation to the fact that the fatty acids are probably not oxidized in the mitochondria. From data which we published more than 20 years ago, it could be concluded that the half life time of the fatty acids in the brain is about 2 weeks compared with the liver where it is less than 1 day. With all the reservation one has about relating enzyme concentration directly to metabolism these results might be an expression of the very slow turnover of fatty acids in the central nervous system. I was glad to hear what you said about the GABA. Tower and his colleagues thought that the GABA pathway was a major pathway for the metabolism. Your results show no direct evidence for this. It looks to me from your evidence that this would not be a major pathway and the isotope data surely don’t show it either. According to isotope data the GABA pathway can only be a minor one. I have another question about the use of chlorpromazine. Does chlorpromazine uncouple your preparation? LOVTRUP:Yes it does, but only to a slight extent. The effect is partly masked because oxygen uptake also is inhibited. SCHERRER: I would like to ask a question which is perhaps what English people would call a wild one. In neurophysiology there is a lot of talk about the function of neuroglia. In this respect I would like to know if from the biochemical point of view very important differences exist between neuroglia and neurons. Is it possible to use gliomata for the study of differences between glia and neurons? LBVTRUP: I may first say that we began with the notion, which we had got from the literature, that mitochondria are something which differ very much from tissue to tissue. We thought that some of the particular characters of brain metabolism might be reflected in the brain mitochondria. But the more we worked on this problem the less we believed this idea. Regarding the second part of your question: we have also discussed in Goteborg the problem of using gliomata. Every time it is given up because people always say: The glia you are going to use is abnormal. I believe that at the moment there is only one way to attack the problem as asked by you, namely by isolating nerve cells and glia cells and consider these as small bags of mitochondria. It is of course not pure mitochondria and you must get side-effects which may make the results difficult to interpret. In Goteborg, Dr. Hamberger is dissecting out glia cells and nerve cells and in the microdiver he adds various mitochondria substrates and measures the rate of oxidation. Sofar he has published only one paper in which he showed that the glutamic acid is oxidized relatively at a higher rate by nerve cells than by glia cells, whereas for some other of these substrates it was the reverse. WAELSCH: There of course is a fascinating paper from your laboratory about the different ATPases from glia and neurons which came out in Biochim. biophys. Acta (Amst.) a few months ago.

254

Molecular Evolution of Lactate Dehydrogenase in the Developing Nervous Tissue * VINCENZO BONAVITA Department of Neurology, University of Palermo, Palerrno (Italy)

It was a commonplace of classic enzymology to say that proteins catalysing the same reaction are all the same, their source appearing as a somewhat secondary matter. In contrast, the studies of Wieland and Meiderer (1957), Markert and Moeller (1959), Kaplan et al. (1960) have shown most recently how DPN-dependent enzymes may differ electrophoretically or kinetically according to the tissue or to the species to which they belong. The present report describes both electrophoretic and catalytic studies on lactate dehydrogenase in the developing brain. Phylogenetic and ontogenetic observations have been made, and the term development will be used, therefore, in its widest sense. Lt has been found that even the evolution of LDH meets, within certain limits, the requirements of Haeckel's statement according to which ontogenesis is a shortened repetition of phylogenesis. ONTOGENETIC STUDIES

The immaturity and the lack of differentiation of the rat brain at birth was shown some 50 years ago by Sugita (1917) in a classic paper on the post-natal morphogenesis of the brain in this species. The sequence of physiological and biochemical changes which parallel the histological maturation of the rat brain has been investigated, however, only recently. Biochemical quantitative studies on brain LDH during the postnatal development of the rat were performed in 1956 by Kuhlman and Lowry, who showed that LDH doubles from birth to maturity in the whole rat brain. A different biochemical approach to the study of the developing brain was made in 1960 by Flexner et al. LDHs of cerebral cortex and liver from adult and newborn mice gave different elution patterns when chromatographed on DEAE cellulose. However, because of variability in loss of activity on the columns, it was impossible to obtain accurate relationships between total anionic and cationic components of the

* The abbreviations used are: LDH, lactate dehydrogenase; DPN, diphosphopyridine nucleotide; DPNH, reduced diphosphopyridine nucleotide; APDPN, 3-acetylpyridine-DPN; Py3AIDPN, pyridine-3-aldehyde-DPN; TNDPN, thionicotinamide-DPN; DeDPN, deamino-DPN ; EPKDPN, 3ethyl-pyridyl ketone-DPN ; PPKDPN, 3-propyl-pyridyl ketone-DPN.

LDH

IN THE DEVELOPING NERVOUS TISSUE

255

enzyme. By contrast, we have obtained quantitative data on the post-natal development of LDH isozymes in the whole bruin (in the following, the rat brain without the diencephalon will be referred to as whole bruin), the diencephalon and the retina of the rat. Agar and starch gel electrophoresis of LDH Fig. 1 shows the electrophoretic pattern of rat brain LDH as separated by agar gel electrophoresis according to Grabar and Williams (1955), and Table I points out the 100 90

s

2

1 -

80 80.

7070 6 00 -

40

30 20

10

il: E

-k

Fig. 1. LDH isozymes from adult rat brain as separated by agar gel electrophoresis.The starting point is shown below the graph. TABLE I PERCENTAGE MEAN VALUES A N D S T A N D A R D DEVIATIONS O F LDH I S O Z Y M E S I N T H E W H O L E B R A I N OF

T H E ADULT RAT*

Isozyme A Isozyme B Isozyme C Isozyme D Isozyme E

3.95 f0.75 19.45 f 2.90 19.20 f 2.80 19.80 f 2.60 37.60 & 3.34

~~~~~~

* These data have been calculated from the electrophoretic patterns of 12 brain extracts. narrow limits of variability in the percentage distribution of the isozymes of adult albino rats. Similar data were obtained when starch gel was used as supporting medium. Three out of the 5 LDH isozymes separated by agar or starch gel electrophoresis of whole brain extracts exhibit definite changes in their percentage values during post-natal development. It can be readily seen in Fig. 2 that the final pattern is attained only after the 50th day. In contrast with isozymes A, B and E, components C and D show only minor variations, which are at the borderline of significance. References p . 2711272

256

VINCENZO BONAVITA

AGE

(days)

Fig. 2. Changes in the percentage activity of 3 LDH isozymes from the whole rat bruin during the post-natal development. Each experimental point represents the mean of at least 3 determinations.

Bonavita and Guarneri (1963) have recently described regional differences in the percentage distribution of isozymes of beef brain LDH. In agreement with these data, rat diencephalon revealed consistent differences in the isozymatic composition of its LDH as compared to the whole brain, although the same 5 components were found. Isozymes A, B, C, D and E are present in the ratios of 2 : 28 : 17 : 21 : 32. Moreover, as reported in Fig. 3, the post-natal maturation of LDH in diencephalon is definitely

60

+

t

I I

I

i

i

7

i i i

I

10

20

30 40 50 A G E (days)

'L

Fig. 3. Changes in the percentage activity of 2 LDH isozyrnes from rat diencephalon during the postnatal development. Each experimental point represents the mean of at least 3 determinations on pools of 4 to 8 diencephalons.

faster. LDH components B and E are the fractions which undergo the major changes, and, in contrast with the whole brain, diencephalon already contains isozyme E at birth. Rat retina exhibits a quite different electrophoretic pattern. Fig. 4 shows the iso-

LDH

I N THE D E V E L O P I N G N E R V O U S TISSUE

257

zymatic composition of retina LDH on starch gel according to Plagemann et al. (1 960). In contrast with the brain enzyme, LDH from retina has been submitted unsuccessfully to electrophoretic migration on agar gel. Thus, we turned to starch gel on which brain LDH gave a pattern very similar to that obtained on agar gel. Futterman and

F

Fig. 4. LDH isozymes from the retina of adult albino rat as separated by starch gel electrophoresis. The starting point is shown below the graph.

Kinoshita (1959) were able to separate on starch paste 5 fractions from a partially purified preparation of cattle retina. Conversely, only 4 LDH components have been separated after starch gel electrophoresis of crude extracts of rat retina. Since only a partial recovery of the enzyme from starch was obtained (see Appendix on Techniques), it may be that a minor component has not been revealed, although carefully looked for. The most outstanding finding seems, however, the lack of post-natal changes in the isozymatic composition of retina as compared to brain. It will be noted how the component migrating to the cathode is quantitatively predominant in the retina, while it represents the smaller fraction in the whole brain and diencephalon. It is quite suggestive to correlate this finding to the peculiar metabolic features of retina in so far as anaerobic glycolysis is concerned (see Discussion). The post-natal development of retina has been investigated also in a rat strain which was described for the first time by Bourne et al. (1938). This strain exhibits a

Fig. 5. LDH isozymes from the retina of adult rats with hereditary retinal degeneration as separated by starch gel electrophoresis.The starting point is shown below the graph. References p. 271/272

258

VINCENZO BONAVITA

spontaneous degeneration of the retina which is inherited as a simple recessive. The histological retinal lesion usually appears 21 days after birth. Biochemical studies on anaerobic glycolysis have recently shown that a metabolic disturbance occurs as early as 12 days after birth (Brotherton, 1962), though major changes in glucose metabolism are observed only after the 21st day (Reading and Sorsby, 1962). Fig. 5 shows that LDH from the adult retina in this rat strain has a quite peculiar isozymatic composition, although the same 4 isozymes of normal rats are found. This is not so on the 2nd day after birth. At that time, normal rats and rats with retinal degeneration exhibit the same pattern of retina LDH. A progressive modification follows, as re-

Fig. 6. Changes in the percentage activity of 3 LDH isozymes-ftom the retina of rats with hereditary retinal degeneration during the post-natal development. Each experimental point represents the mean of at least 3 determinations on pools of 8 to 20 retinae.

ported in Fig. 6. The possibility of observing a different isozymatic pattern when a morphological alteration occurs in a tissue points out the plasticity of the multiple composition of enzymes, and may give a clue to the understanding of their biological significance. Catalytic studies Kaplan and Ciotti (1961) have shown definite differences in the reactivity of heart LDH of the adult and newborn rat with structural analogues of DPN. By contrast, they could not discriminate the LDH’s of adult and newborn skeletal muscle or the young and adult liver enzymes by the same catalytic techniques. While liver and skeletal muscle are characterized by a definite predominance of 1 LDH fraction (Wieland and Meiderer, 1961), both the heart and the brain enzyme reveal a true multiple composition after zone electrophoresis on starch or agar gel. It was thought, therefore, that the kinetic study of brain LDH with DPN analogues could reveal significant differences likewise similar catalytic studies on the heart. The kinetic characterization of LDH’s in the developing nervous tissues has been

LDH

I N THE DEVELOPING NERVOUS TISSUE

259

performed, however, also with other procedures. A description follows of several catalytic data. (a) The interaction with pyridine and purine analogues of DPN. The use of pyridine and purine analogues of DPN as a tool for discriminating LDH’s or other dehydrogenases may be misleading. There are 2 major pitfalls in studies with coenzyme analogues : (1) the possibility of considering operational artifacts as indicative of real differences; (2) the possibility of overlooking small differences. The first possibility was pointed out by Flexner et al. (1962) while this paper was in preparation. They showed that considerable variation is introduced in the ratio of reduction of DPN and APDPN by several purification procedures when applied to the enzyme from beef brain cortex. This finding confirms previous observations by Bonavita and Guarneri (1961, 1962) on changes of catalytic features of LDH components from beef brain after zone electrophoresis on agar gel according to Grabar and Williams (1955). The second pitfall can be overcome by carrying out a most extensive investigation with several analogues and comparing ratios between reaction rates in the presence of various coenzymes and at least 2 substrate concentrations. The experimental scheme for kinetic screening suggested by Kaplan et al. (1960) has proved to be very useful. The following conclusions can be drawn from the study of LDH reactivity with DPN analogues during post-natal neurogenesis : (1) LDH’s from the whole brain, TABLE I1 R A T I O S O F R E A C T I O N RATES M E A S U R E D W I T H T H E

LDH

FROM T H E W H O L E E R A I N A N D

D I E N C E P H A L O N OF N E W B O R N A N D A D U L T R A T S AFTER A D D I T I O N OF D I N U C L E O T I D E S A N D 2 CONCENTRATIONS O F L(+)LACTATE

Brain areas

Whole brain (2 days after birth) Whole brain (adult rat) Diencephalon (2 days after birth) Diencephalon (adult rat)

5

OXIDIZED

DPN(L)

APDPN(L) Py3AIDPN(L) TNDPN(L) DeDPN(L)

DPN(H)

APDPN(H) Py3AIDPN(H) T N D P N ( H ) DeDPN(H)

0.52

1.14

0.20

0.22

0.45

0.74

1.39

0.29

0.40

0.60

0.58

1.13

0.24

0.23

0.42

0.64

1.34

0.27

0.32

0.52

The data collected in this Table have been obtained by assaying the LDH activity with 2 concentrations of lac lactate (0.074 M and 0.0074 M , which are referred to as (H) and (L), respectively). Thus, according to Kaplan et al. (1960), DPN(L) symbolizes the rate of lactate dehydrogenation, when DPN is the coenzyme and lactate is 0.0074 M . All the data have been obtained in the presence of 0.7 pmoles of dinucleotide, and each value represents the mean of at least 3 determinations.

diencephalon and retina can be discriminated from ratios between the reduction rates of DPN and other coenzymes in the presence of 2 concentrations of L(+)lactic acid (Tables 11 and 111); (2) in agreement with the gradual modification of the isozymatic pattern, the enzyme from the whole brain and the diencephalon of the adult animal References p. 2711272

260

VINCENZO BONAVITA

can be easily differentiated from LDH of the newborn animal. It is worth noting in this connection that the kinetic discrimination is definitely better for the whole brain, and this is in agreement with the electrophoretic data (Table 11); (3) the enzyme from the adult and newborn retina cannot be discriminated by the use of DPN and its analogues (Table 111). TABLE I11 R A T I O S OF R E A C T I O N R A T E S M E A S U R E D W I T H T H E LDH FROM T H E R E T I N A OF N E W B O R N A N D A D U L T R A T S AFTER A D D I T I O N OF 5 O X I D I Z E D D I N U C L E O T I D E S A N D 2 CONCENTRATIONS O F L(+)LACTATE

For details, see Table I1

Retina (2 days after birth) Retina (adult rat)

DPN(L)

APDPN(L) Py3AlDPN(L) TNDPN(L) DeDPN(L)

DPN(H)

APDPN(H) Py3AlDPN(H) TNDPN(H) DeDPN(H)

0.38

1.23

0.20

0.20

0.25

0.39

1.24

0.20

0.20

0.28

With LDH from the whole brain 2 more analogues have been used, i.e. the ethylpyridyl ketone of DPN and the propyl-pyridyl ketone of DPN. These 2 coenzymes, however, did not show any appreciable differencebetween LDH of the newborn and adult brain. The calculated ratios are reported in Table IV. TABLE IV R A T I O S OF R E A C T I O N R A T E S M E A S U R E D W I T H LDH FROM T H E B R A I N O F N E W B O R N A N D A D U L T R A T AFTER A D D I T I O N OF 3 - E T H Y L - P Y R I D Y L K E T O N E - D P N A N D 3 - P R O P Y L - P Y R I D Y L KETONE-DPN A N D 2 CONCENTRATIONS O F L ( + ) L A C T A T E

For details, see Table I1

EPKDPN(L)

PPKDPN(L)

EPKDPN(L)

EPKDPN(H)

EPKDPN(H)

PPKDPN(H)

PPKDPN(L)

PPKDPN(H)

Whole brain (2 days after birth)

0.28

0.23

I .14

1.16

Whole brain

0.25

0.23

1.16

1.10

(adult rat)

(b) Other catalytic observations on LDH of developing nervous tissues. The enzyme from the whole brain, diencephalon and retina of the newborn and adult rat has been further characterized from a catalytic viewpoint. Michaelis constants for pyruvic acid and L(+)lactic acid were determined in the same experimental conditions with the enzyme from all these sources. A Km for pyruvate equal to 1.55 x 10-4 M was calculated with the enzyme from all sources at any stage of development. It will be noted, however, that this value is definitely higher than those calculated with purified

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26 1

LDHs from other tissues (Neilands, 1955; Winer, 1960; Bonavita and Guarneri, 1962). Also the affinity for lactate did not change during the post-natal maturation of brain and normal retina. No changes were found during the development of the whole brain, diencephalon and retina in so far as the pH-activity curve for lactate dehydrogenation was concerned. The maximal activity and the shape of the curve were the same at any stage of development. A typical curve for the adult brain enzyme is reported in Fig. 7, and

h 8.0

8.5

9.0

9.5

70.0

70.5

PH

Fig. 7. Lactate dehydrogenation with the enzyme from the adult rat brain as a function of the hydrogen ion concentration.

this same curve was found with LDH from diencephalon and retina of newborn and mature animals. By contrast, a slight shift of the optimal pH for pyruvate reduction occurs during the development of the brain, Fig. 8 shows the pH-activity curves obtained with brain LDH of the newborn and the adult rat. It can be readily seen that the pH-optimum

Fig. 8. Pyruvate reduction with the brain enzyme from newborn (A) and adult rat (B) as a function of the hydrogen ion concentration. References p. 271/272

262

VINCENZO BONAVITA

changes and the shape of the curve itself is modified. The enzyme from diencephalon behaved quite alike. Conversely, the normal retina did not exhibit a similar kinetic modification after birth, and this is in good agreement with the lack of post-natal modifications in the isozymatic pattern of retina LDH. This is not so with the enzyme from the retina of animals with hereditary retinal degeneration. As reported by Bonavita ef al. (1963), in this last instance the pH-activity curve with the enzyme from newborn retina is very similar to that observed with normal rats of the same age, and a subsequent modification occurs during the development. P H Y L O G E N E T I C STUDIES

Agar and starch gel electrophoresis have revealed many more peculiarities of LDH's from the brain of various vertebrates than the kinetic analysis of the unresolved enzyme. Thus electrophoretic studies will be described in detail. Agar and starch gel electrophoresis In a comparative analysis of brain LDH's from different species, it was necessary to use throughout a reference material which could be easily obtained and whose electrophoretic behaviour varied within narrow limits. The brain extract of adult albino rat was taken for this purpose. Since (with the exception of retina) the major part of our ontogenetic studies has been performed on agar gel, our first screening of LDH's in the vertebrate species was carried out using the same supporting medium. Although it is not the most sensitive technique for resolving LDH isozymes (see below), agar gel electrophoresis according to Grabar and Williams (1955) has given

!zzzl

PlUSTEL U S PlUSTELUS

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CARASSIUS COflNUNlS

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EPIPlYS R A T T U S

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n o n o Eza .

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,

'

.

'

.

.

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.

l

.

.

.

.

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1

.

.

.

.

.

.

.

.

.

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1

1

flUSCULUS

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.

.

CAPRA HIRCUS FELIS CATUS .

.

.

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Fig. 9. Diagrammatic representation of agar gel electrophoresis of brain LDH's of different vertebrates. The starting point is shown below the graph. Dashed areas indicate the major LDH component in species with several isozymes.

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a general scheme which has been confirmed by subsequent starch gel studies in the experimental conditions of Plagemann et al. (1960). Fig. 9 summarizes the major part of the electrophoretic observations on agar gel. It can be easily realized that several phenomena are going on during the phylogenesis: ( I ) an initial increase of the number of LDH's which can be separated by agar gel electrophoresis at pH 8.4; (2) the first appearance in the mammals of LDH isozymes migrating toward the anode; (3)the liminary position of LDH from the adult rat brain as far as the number of separated fractions ( 5 ) is concerned. This last finding may be an artifact, since the experimental conditions were chosen for the rat brain itself. LDH from adult rat brain holds, however, an interesting position in the progressive development of the enzyme during the phylogenesis, since in it 3 fractions migrate toward the cathode and 2 fractions toward the anode. In the brain of higher mammals, the number of detectable isozymes decreases again, but the components which disappear are those migrating toward the cathode. Another finding which seems worth mentioning is the great difference between Triturus cristatus carnifex and Discoglossus pictus. Such a findmg is in good agreement with the most recent zoological theories (Colbert, 1955) which consider Urodeles and Anurans as derived from different phyla (subclasses of Aspidospondyli and Lepospondyli, respectively). It will be noted that this is also the only instance in which starch gel electrophoresis has been less sensitive than the agar gel technique for discriminating enzymes of different sources (see Figs. 9 and 10).

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

tzzzzza

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FELlS CATUS

Fig. 10. Diagrammatic representation of starch gel electrophoresis of brain LDHs of different vertebrates (see Fig. 9). The starting point is shown below the graph. Dashed ,areas indicate the major enzyme components in each species.

When starch gel is used as the supporting medium, a very similar general pattern of LDH isozymes in vertebrates is obtained, although the number of separated isozymes is higher both in lower vertebrates and in mammals. Fig. 10 summarizes the data obtained on starch gel. References p . 2711272

264

VINCENZO BONAVITA

Starch gel electrophoresis has almost always resolved more fractions than agar gel. For instance, this has been so with fishes. As shown in Fig. 11, the whole brain of Mustelus mustelus (A) and Carassius communis (B) contains 3 forms of lactic de90

-

80

.

70

-

60

-

A

Fig. 11. LDH isozymes from the whole brain of Mustelus mustelus (a Selachian: A) and Curassius cornrnunis (a Teleost: B) as separated by starch gel electrophoresis.The dashed area below the graph indicates the starting point. The solid squares show the position of isozymes 1 and 4 of rat brain or retina taken as reference material.

hydrogenase, which are not separated by electrophoretic migration on agar gel, where the enzyme moves as a single band toward the cathode. Starch gel reveals a substantial difference between the 2 classes: the major fraction of LDH from Mustelus

Fig. 12. LDH isozymes from the whole brain of Testudo gruecu (a Chelonian) as separated by starch gel electrophoresis. For other details, see Fig. 11.

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mustelus (a Selachian) exhibits a net positive charge, while the major component from the brain of Carassius communis (a Teleost) moves to the anode. In Testudo graeca, only 2 components have been separated by starch gel electrophoresis (Fig. 12), instead of the 3 LDH fractions separated from the brain extracts of fishes. It is quite interesting, however, that the isozyme migrating toward the anode has almost the same mobility as the major fraction found in the brain of Carassius communis. Also on starch gel LDH from rat brain holds a very peculiar position in so far as the apparent electric charge of isozymes is concerned. By contrast, the enzyme from the brain of all the other mammals has not shown any component migrating to the cathode in the experimental conditions of the present investigation.

Fig. 13. LDH isozymes from the whole brain of Cupru hircus as separated by starch gel electrophoresis. For other details, see Fig. 11. Fractions indicated by dotted lines are to be multiplied by a factor of 10.

Fig. 13 shows the isozymatic pattern of brain LDH from Capra hircus. It can be taken as representative of the patterns found in all mammals more evolute than rodents. In all these species the slowest components are also definitely smaller. Catalytic studies

Several kinetic properties of the enzyme were investigated also in the phylogenetic studies. Although a kinetic analysis of relatively crude enzymes is open to criticisms, we think that the following data point out quite a number of interesting differences between LDH’s from various sources. (a) The interaction with pyridine and purine analogues of DPN. As reported in Table V, the velocity ratios for each coenzyme are not the same in all species. For instance, the values of DPN (L)/DPN(H) change from Mustelus mustelus (a Selachian) References p. 2711272

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VINCENZO BONAVITA

TABLE V R A T I O S OF R E A C T I O N R A T E S M E A S U R E D W I T H T H E LDH FROM T H E B R A I N O F S E V E R A L VERTEB R A T E S AFTER A D D I T I O N OF 5 O X I D I Z E D D I N U C L E O T I D E S A N D 2 CONCENTRATIONS O F L ( + ) L A C T A T E

For details, see Table I1 DPN(L)

Animal species

DPN(H) Mustelus mustelus Carassius communis Discoglossus pictus Triturus cristatus carnifex Testuab graeca GalIus gallus Epimys rattus Mus musculus Oryctolagus cuniculus Capra hircus Felix catus

0.39

0.44 0.53 0.58 0.40 0.51 0.73 0.82 0.72 0.71 0.85

APDPN(L) Py3AIDPN(L) TNDPN(L) DeDPN(L) APDPN(H) Py3AIDPN(H) TNDPN(H) DeDPN(H) 1 .oo I .20 1.20 1 .oo 1.25 1.65 1.40 1.25 1.40 1.30 1.35

0.30 0.25 0.34 0.45 0.30 0.42 0.40 0.46 0.45 0.43 0.41

0.16 0.14 0.15 0.17 0.17 0.30 0.30 0.36 0.27 0.29 0.28

0.32 0.45 0.42 0.39 0.46 0.31 0.60 0.53 0.45 0.50 0.52

to mammals, with a sharp increase between Gallus gallus and the mammals reported in the Table, When Py3AlDPN was used as a coenzyme, the increase was found between Testudo graeca and Callus gallus, while the ratios determined with APDPN, TNDPN and DeDPN did not discriminate the various proteins so well. (b) Other catalytic observations on LDH from the brain of vertebrates. Km values for pyruvate have been calculated for all the enzymes in the presence of DPNH, 7 x M. From this viewpoint, no clear-cut discrimination has been possible, Km values for the substrate being between 0.85 x M (Callus gallus) and 2 x M (Carassius communis). Since all the other data were found to be randomly distributed between these 2 values, it was not possible to give any significance to the differences observed. In the investigated range of pyruvate concentrations, however, a net discrimination was obtained between lower vertebrates and mammals in so far as the TABLE V I M I C H A E L I S C O N S T A N T S FOR L ( + ) L A C T A T E

Animal species Mustelus mustelus Carassius communis Discoglossus pictus Triturus cristatus carnifex Test& graeca Gallus gallus Epimys rattus Mus musculus Oryctolagus cuniculus Capra hircus Felix catus

Km valuesfor LDH 3.33 1.43 1.07 0.96 0.83 0.56 0.63 0.50 0.39 0.65 0.38

x 10-2 M 10-2M X 10-2 M x 10-2M X M X M X M x M x M X 10-2 M x M X

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inhibition by excess pyruvate is concerned. In fact, the ratios between velocities in the presence of 3.3 x M and 3.3 x M pyruvate gave values in fishes and amphibians twice as low as in mammals. The affinity for lactate has been investigated in the presence of DPN, 2.33 x M. As reported in Table VI, the Km values decrease gradually from Mustelus mustelus to mammals. Although these differences are not very great, they may indicate real differences among the various LDH’s. The possible metabolic significance of this finding will be discussed in the next section. Both reactions catalyzed by LDH were studied with the enzyme from the various sources at several pH values. In so far as the pyruvate reduction is concerned, a sharp limit was observed at the level of Gallus gallus, since all the species which are zoologically less evolute show the maximal activity around pH 7 or a little below, while mammals exhibit the maximum at slightly higher pH values (pH 7.4). Fig. 14 shows A

Fig. 14. Pyruvate reduction with the brain enzyme from Carassius communis (A) and Mus musmlus (B) as a function of the hydrogen ion concentration.

the pH-activity curves for pyruvate reduction as observed with the brain LDH of Carassius communis and Mus musculus. In contrast, curves obtained for lactate oxidation do not reveal gradual modifications through the investigated zoological range. It has been found in mammals that the maximal activity is measured when the pH is higher than 9.5 with only minor variations among the various species. In the lower species the maximal activity for lactate dehydrogenation lies between pH 9 (Mustelus mustelus, Carassius communis, Triturus cristatus carnifex, Testudo graeca) and 9.4 (Discoglossus pictus, Gallus gallus). However, although the pH of maximal activity varies considerably, the shape of the curve is substantially similar. G E N E R A L DISCUSSION

The first section of this paper describes data of different type on the molecular evolution of brain LDH in the post-natal development of the organ. It has been found that the enzyme exhibits at birth a composition which gradually changes to attain the final pattern observed in the adult. That LDH composition changes in the early developmental stages of several tissues had already been reported (Markert and References p. 271/272

268

VINCENZO BONAVITA

Moeller, 1959; Flexner et ul., 1960), but a quantitative study of these changes had not been described up till now. As shown by Sugita (1917), the first 10 days after birth represent a period of rapid growth of the rat brain, the thickness of the cortex increasing from 0.74 to 1.7 mm. The neurochemical findings on LDH isozymes parallel the rapid growth in the first days after birth. Isozyme E (Fig. l), which is not present in detectable amounts 2 days after birth, represents 15 per cent of the enzyme on the 22nd day. Similarly, the percentage decrease of isozyme B is quite steep at the beginning. Nevertheless, the final pattern is attained only at the end of the 2nd month, i.e. when the cerebral cortex is fully organized. The maturation of LDH from diencephalon follows a different time curve. As reported above, diencephalon exhibits also a different LDH pattern on starch or agar gel as compared to other brain areas, and the major difference is the lower level of fraction E and the higher level of fraction B. In this respect, the pattern of LDH from diencephalon may be assimilated to that of the whole brain before complete maturation. That diencephalon attains its final pattern in a shorter time than the whole brain could also be anticipated from the finding at birth of a considerable amount of isozyme E not detectable in the whole brain at the same stage. In fact, isozyme E and the other fastly migrating components are present in higher amounts at the last stages of maturation, and this is in good agreement with our own findings on the phylogenetic distribution of LDH isozymes in the brain of vertebrates. Thus, one would conclude that both in the phylogenetic and ontogenetic development of the nervous system 2 phenomena are going on: ( I ) the progressive decrease or even the disappearance of isozymes migrating toward the cathode ; (2) the appearance and the subsequent increase of isozymes fastly moving to the anode*. These 2 phenomena have not been observed during the post-natal development of retina in normal albino rats, although Detwiler (1932) has shown that the morphogenesis of this organ comes to completion only after birth**. Electrophoretic data on retina LDH do not suggest the same intimate correlation between molecular evolution and morphological development that findings on the whole brain and diencephalon strongly indicate. Also the observations on the developing retina of rats with hereditary degeneration do not point out an intimate parallelism between morphology and electrophoretic patterns of LDH. Lucas et ul. (1955) have reported that in the affected strain development proceeds normally up to 14 days or so, and only at this stage do the distal segments of rods suddenly appear to break down. As observed also by Rubino and Ponte (1961), bipolar and ganglion cells are much less altered than rods. Nevertheless,the isozymatic pattern of LDH begins to change on the 3rd or 4th day after birth, i.e. when the histological damage is not yet apparent. Observations by Bonavita and Guarneri

* The comparative analysis in the vertebrates has been made possible by an arbitrary selection of the species to be investigated. It is quite obvious, therefore, that the general significance of the findings described above can be questioned. ** It will be recalled in this connection that the bulk of retina LDH is in the layers 5 to 8 (Lowry et al., 1956) and that 2 days after birth ganglion cells are already of adult appearance (Noell, 1958a, b). These findings were obtained, however, in the rabbit and we do not know whether the same holds true for the rat.

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(1963) on the regional neurochemistry of LDH isozymes and findings by Bonavita, Guarneri and Beduschi (1963) on LDH patterns in some brain tumours give convincing evidence that the correlation between biochemical and morphological evolution is quite variable. In the attempt at interpreting experimental data on isozymes in the nervous tissues, the neurochemist is obviously faced with the problem of the physiological significance of isozymes in terms of general biology. If LDH isozymes are considered as tetramers of 2 different subunits (H or heart type and M or muscle type) in the 5 possible recombinations of 4, as shown by Appella and Markert (1961) and Cahn et al. (1962)*, the shift in the isozymatic composition of LDH during phylogenesis and ontogenesis must be considered as the result of the gradual predominance of one recombination on the others. The general conclusion from the present electrophoretic studies on agar and starch gel may be as follows: the ‘parental’ LDH of the heart type and the most closely related ‘hybrid’forms tend to prevail progressively in the phylogenesis and ontogenesis of nervous tissues with the exception of normal retina. Our view is that this trend is not an operational curiosity but a physiologically important indication of a gradual change in the metabolic set-up of the tissue. As stated by Cahn et al. (1962), the heart enzyme may be regarded ‘as a catalyst geared for activity in an aerobic environment, whereas the muscle enzyme functions in an anaerobic environment’. Thus, it may be supposed that during phylogenesis as well as in the ontogenesis of rat brain an increase in the dependence of normal function on aerobiosis occurs. That this is so in the ontogenesis was already known from physiological studies on the newborn rat. Soon after birth, the rat is extrexely resistant to anoxia and shows only a gradual decline in such a resistance during post-natal development (Fazekas et al., 1941). The lack of isozyme E (heart type) in the newborn animal and its increase after birth parallels the physiological decrease in the resistance to anoxia. On the other hand, the lack of isozyme E in the rat retina of newborn and adult animals is in keeping with the high resistance of the organ to anoxia (Noell, 1958b). The major part of the catalytic data described above represent further evidence of such a physiological trend. In so far as ontogenesis is concerned, Kmvalues for lactate and pyruvate as measured on crude enzyme preparations have not shown significant changes during the post-natal development. However, that a gradual change occurs in the kinetic properties of the enzyme from the whole brain and diencephalon is clearly pointed out by data on pH-activity curves for pyruvate reduction as well as by velocity ratios calculated with the structural analogues of DPN. When kinetic studies were carried out with LDH’s from various vertebrates, a significant and progressive decrease of Km values for L(+)lactate from fishes to mammals was found. The higher affinity for lactate in mammals and the stronger

* They have distinguished 2 ‘parental types’ of LDH, each composed of 4 identical subunits (HHHH and MMMM), and ‘hybrid forms’which differ in their subunitary arrangements (HHHM, HHMM, HMMM). References p . 2711272

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VINCENZO BONAVITA

inhibition by excess pyruvate seem to indicate that even small amounts of lactic acid can be rapidly oxidated owing to a fast removal of pyruvate through a more active Krebs' cycle. It has been stressed already that DPN and Py3AIDPN both revealed a phylogenetic sequence, although the absolute reactivity of this last coenzyme with LDH is quite low. The gradual increase of the ratios calculated both with DPN and its analogues shows that the ability of the protein to form the enzyme-coenzyme-substrate complex (the ternary complex) increases gradually from the less evolute to the higher vertebrates. This is in very good agreement with the decrease of Km values for L(+)lactic acid during phylogenesis. A final comment is worth making on the change of pH-activity curves for pyruvate oxidation during phylogenesis. The modification is quite similar to that observed in the post-natal development of the rat brain. Though a few data do not fit in the general picture of a parallelism between phylogenetic and ontogenetic maturation of LDH, the major part of the findings described above is in favour of such a conclusion. APPENDIX O N TECHNIQUES

LDH was extracted with a standard procedure: brains were quickly removed soon after the sacrifice of the animal by decapitation, washed in saline (0.9% NaCl) and homogenized in 3 volumes of cold distilled water. Centrifugation was carried out in a Lourdes refrigerated centrifuge mod. LRA at 16,000x g for 30 min at 4". The supernatant kept in ice was used within 2 h for electrophoretic or kinetic analyses. Preliminary experiments with beef and rat brains showed that the extraction with isotonic sucrose or 0.1 M sodium phosphate buffer pH 7 did not give a different isozymatic pattern. Agar gel electrophoresis was performed according to Grabar and Williams (1955), with a barbital-HC1 buffer, 0.05 M , pH 8.4. Gels were 18 x 12 x 0.4 cm and the current intensity was 45-48 mA. The migration time at 14-15" was 3.5 h. Elution of the enzymic proteins from agar (Bactor-Agar Difco) was performed as previously described (Bonavita and Guarneri, 1961, 1962), the recovery being quite satisfactory (90 to 95 per cent). Starch gel electrophoresis was carried out in the experimental conditions suggested by Plagemann et al. (1960) with only minor modifications. Gels were 30 x 12.5 x 0.5 cm and the migration time was 14.5 to 15 h at 4". Crystallized bovine haemoglobin was used as a visual indicator, and the electrophoresis carried out was interrupted when bovine haemoglobin had migrated a distance of 5.3-5.5 cm. The elution of the enzyme from starch gel was performed with the same technique as used for agar gel, but the recovery was quite low, ranging from 27 to 30 per cent. The good reproducibility of recoveries and the agreement with values obtained after treatment of starch gel with amylase (Plagemann et al., 1960), however, made the data on percentage activity of LDH isozymes worth considering. LDH activity was determined in the same experimental conditions as previously described for the beef brain enzyme (Bonavita and Guarneri, 1962). When APDPN

LDH

IN THE D E V E L O P I N G N E R V O U S T I S S U E

27 1

and Py3AlDPN were used, a tenfold amount of protein was added to the reaction mixture. All the kinetic analyses were performed with a Beckman DB spectrophotometer to which a Sargent recording unit (mod. SR) was connected. One LDH unit was defined as the amount of the enzyme which gives an optical density change at 340 m,u of 0.001 in 1 min at 25", when measuring pyruvate reduction in the experimental conditions previously described (Bonavita and Guarneri, 1962). ACKNOWLEDGEMENTS

The investigations described in this paper have been made possible by grants from the National Institute for Neurological Diseases and Blindness (Grant B-2917) and the Consiglio Nazionale delle Ricerche, Rome. The author gratefully acknowledges the collaboration of Dr. Giuseppe Amore, Dr. Rosa Guarneri and Dr. Francesco Ponte in various aspects of the present studies. REFERENCES

E., AND MARKERT, C. L., (1961); Dissociation of lactate dehydrogenase into subunits with APPELLA, guanidine hydrochloride. Biochem. biophys. Res. Commun., 6, 171-176. BONAVITA, V., AND GUARNERI, R., (1961); Sugli isoenzimi della latticodeidrogenasi di tessuto nervoso. 1. Reazione con analoghi piridinici del difosfopiridin-nucleotide ed affinita per I'acido piruvico, Rend. Accad. Naz. Lincei, 30, 754-760. BONAVITA, V., AND GUARNERI, R., (1962); Lactic dehydrogenase isozymes in the nervous tissue. I. The reaction with diphosphopyridinenucleotide analogues and the inhibition by sodium metabisulfite. Biochim. biophys. Acta, 59, 634442. BONAVITA, V., AND GUARNERI, R., (1963); Lactic dehydrogenase isozymes in the nervous tissue. 111. Regional distributicn in the beef brain. In preparation. BONAVITA, V., GUARNERI, R., AND BEDUSCHI, A., (1963); Unpublished results. BONAVITA, V., PONTE,F., AND AMORE,G., (1963); Neurochemical studies on the inherited retinal degeneration of the rat. I. Lactate dehydrogenase in the developing retina. Vision Res., submitted. M. C., CAMPBELL, D. A., AND TANSLEY, K., (1938); Hereditary degeneration of the rat retina, BOURNE, Brit. J . Ophthal., 22, 613-623. BROTHERTON, J., (1962); Studies on the metabolism of rat retina with special reference to retinitis pigmentosa. I. Anaerobic glycolysis. Exp. Eye Res., 1, 234245. CAHN,R. D., KAPLAN,N. O.,.LEVINE,L., AND ZWILLING, E., (1962); Nature and development of lactic dehydrogenases. Science, 136, 962-969. COLBERT, E. H., (1955); Evolution of the vertebrates. New York, John Wiley and Sons (p. 435). DETWILER, S. R., (1932); J . comp. Neurol., 55, 473. Cited by Noell, W.K., (1958a). FAZEKAS, J. F., ALEXANDER, F. A. D., AND HIMWCH,H. E., (1941); Am. J. Physiol., 134, 281. Cited in Neurochemistry. K. A. C . Elliott, I. H. Page, J. H. Quastel, Editors. Springfield, Thomas, 1955 (p. 254). FLEXNER, L. B., DE LA HABA,G., AND FLEXNER, J. B., (1962); Further studies on the components of lactic dehydrogenase of cerebral cortex. J . Neurochem., 9, 31 3-320. FLEXNER, L. B., FLEXNER, J. B., ROBERTS, R. B., AND DELAHABA,G., (1960); Lactic dehydrogenases of the developing cerebral cortex and liver of the mouse and guinea-pig. Develop. Eiol. 2,313-328. FUTTERMAN, S . , AND KINOSHITA, J. H., (1959); Metabolism of the retina. 11. Heterogeneity and properties of the lactic dehydrogenase of cattle retina. J . biol. Chem., 234, 3174-3178. GRABAR, P., AND WILLIAMS, C. A., (1 955) ; Methode immunodectrophorCtique d'analyse de melanges de substances antigkniques. Biochim. biophys. Acta, 17, 67-74. KAPLAN, N. O., AND C ~ o r n M. , M., (1961); Heterogeneity of the lactic dehydrogenases of the newborn and adult rat as determined with coenzyme analogs. Biochim. biophys. Acta, 49, 425426. KAPLAN, N. O., CIOTTI,M. M., HAMOLSKY, M., AND BIEBER, R. E., (1960); Molecular heterogeneity and evolution of enzymes. Science, 131, 392-397.

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KUHLMAN, R. E., AND LOWRY, 0. H., (1956); Quantitative histochemical changes during the development of the rat cerebral cortex. J. Neurochem., 1, 173-180. LOWRY,0. H., ROBERTS,N. R., AND LEWIS,CH.,(1956); The quantitative histochemistry of the retina. J. biol. Chem., 220, 879-892. LUCAS,D. R., AITFIELD,M., AND DAVEY,J. B.,(1955); Retinal dystrophy in the rat. J. Path. Bact., 70,469474.

MARKERT, C. L., AND MOELLER, F., (1959); Multiple forms of enzymes: tissue, ontogeneticand species specific patterns. Proc. nut. Acad. Sci. (Wash.), 45, 753-763. NEILANDS, J. B., (1955); Lactic dehydrogenaseof heart muscle. Methodsin Enzymology.S . P. Colowick and N. 0. Kaplan, Editors. New York, Academic Press, Vol. I (p. 449). NOELL,W. K., (1958a); Differentiation, metabolic organization and viability of the visual cell. Arch. Ophthal., 60,702-733. NOELL,W. K., (1958b); Studies on visual cell viability and differentiation. Ann. N. Y. Acad. Sci.. 74, 337-361. PLAGEMANN, P. G. W., GREGORY, K. F., AND WROBLEWSKI, F., (1960); The electrophoretically distinct forms of mammalian lactic dehydrogenase. I. Distribution of lactic dehydrogenases in rabbit and human tissues. J. Biol. Chem., 235, 2282-2287. READING, H. W., AND SORSBY, A., (1962); The metabolism of the dystrophic retina. I. Comparative studies on the glucose metabolism of the developing rat retina, normal and dystrophic. Vision Res., 2, 315-325.

RUBINO, A., AND PONTE,F., (1961); Alcuni aspetti istologici ed elettroretinografici della neuropatia eredofamiliare del ratto. G. ital. Ojial., 14, 147-160. SUGITA,N., (1917); J. comp. Neurol., 28, 495. Cited by Kuhlman, R. E. and Lowry, 0. H. G., (1957); Nachweis der Heterogeneitat von Milchsaure-dehydroWIELAND, TH., AND ~FLEIDERER, genasen verschiedenen Ursprungs durch Tragerelektrophor. Biochem. Z., 329, 112-1 16. TH.,AND PFLEIDERER, G., (1961); Chemical differences between multiple forms of lactic WIELAND, acid dehydrogenases. Ann. N. Y. Acad. Sci.,94, 691-700. WINER,A. D., (1960); Purification and some kinetic properties of ox brain lactic and malic dehydrogenase. Biochem. J., 76, 5P.

273

Enzymatic Mechanism of the Protein Biosynthesis in the Developing Nervous System M. WENDER

AND

M. HIEROWSKI

Department of Neurology and Department of Physiological Chemistry, Medical Academy, Poznan’ (Poiand)

The biosynthesis of proteins, especially its enzymatic mechanism is one of the basic biochemical problems with regard to the development. Since the problem of protein biosynthesis in its general biological aspect is only known in its outlines, the very problem pertaining to the development and maturation of the nervous system still remains open. Researches (Hoagland, 1955; Hoagland et al., 1956 and others) showed that the process of the protein biosynthesis may be presented as a three-stage schema: ( I ) This stage is the activation of amino acids, which starts the chain of reactions; (2) This stage is the transfer of activated amino acids to the soluble cytoplasmic fraction of ribonucleic acid (sRNA); (3) This stage is the incorporation of amino acids in the ribonucleoprotein particles, so-called ribosomes, and then the building of a space structure of the complete protein. The activation of the carboxyl group of amino acids as mentioned above, is the first stage in protein biosynthesis. In the reaction, in which ATP takes part, the anhydride of adenyl-amino acid combined with the activating enzyme (Fig. 1) is formed H

O H II I E+ATP+R-C-COOH+E-AMP-C-C-RRPP

1

I

I

NHz

NHz Fig. 1

as an intermediate product. The amino acid activating enzymes are present in the soluble fraction of cytoplasmic protein, their isoelectric point being about pH 5 (so called ‘pH 5 enzymes’). The activated amino acids form with hydroxylamine a hydroxamate (Fig. 2). The hydroxamate reacts in acid solution with ferric chloride, O

H

H

I1

I

I

E - AMP - C - C - R

+ NHzOH + E + AMP + R - C 1

I NHz Rrferences p . 279

Fig. 2

NHz

O

II

C - NHOH

274

M. W E N D E R A N D M. H I E R O W S K I

yielding a pink-brown colour. The intensity of the reaction, which can be measured colorimetrically, is proportional to the rate of amino acid activation. It is likely that this first stage of protein biosynthesis is closely connected, especially by the activating enzymes, with the second stage, which is the reaction of activated amino acids with sRNA. The intermediate amino-acyl-soluble RNA gives also a positive reaction in the test with hydroxylamine. On the other hand, little is known about the enzymatic mechanisms involved in the incorporation of amino acids in the ribosome particles. The present report deals with the activation of amino acids by pH 5 enzymes in the developing nervous system of guinea-pigs. In the course of development of the central nervous system there occur profound changes of the amino acid composition of proteins. It thereforc seems to be of interest to investigate whether these changes correlate with the rate of amino acid activation. M ATE RIALS A N D METHOD S

Our investigations were performed on the cerebral white and grey substances of guinea-pigs of the following age groups: foetuses in the 9th week of gestxtion, animals of 3 and 9 days, 2-month-old animals, and adults of 6-8 months. The animals were anaesthetized with ether and killed by cutting the heart; the brain was removed from the cranial vault as quickly as possible, and aliquots of white and grey substances were taken for chemical investigations. The samples of white substance included the corpus callosum with its radiation, the white substance of the cerebral hemispheres, the cerebral peduncles, cerebellar hemispheres and the macroscopically visible white substance of the brain stem. Samples of grey substanc2 were taken from all cerebral lobes by cutting the cortex. The samples were taken from the same topographical areas in the foetal specimens. The activation of amino acids in the nervous system was studied by the method of Hoagland (1955) in acetone powder of the nervous tissue. Preparation of acetone powder. The samples of the cerebral tissue were put in the ice-cooled homogeniser and homogenised for 1 min with 5 ml of -30" cold acetone. The homogenised cerebral tissue was washed twice with 10 ml of -30" cold acetone on a Buchner's funnel and then dried in vacuum for about 4 h. Enzymatic preparation. 5 ml of the 0.02 M Tris buffer of pH 7.76 were added to 250 mg of acetone powder. Ten min later, the sample was centrifuged at 0" for 30 min at 10,OOO xg. To the supernatant a saturated solution of ammonium sulphate was added until a 0.6 saturation was reached. The obtained mixture was adjusted to pH 5.0 with 0.2 N HCl and then centrifuged at 0" for 5 min at 10,000x g. The sediment obtained was dissolved in 0.2 M Tris buffer of pH 7.76 to a volume of 2.5 ml. The above method of enzymatic preparation eliminates free amino acids present in acetone powder (Szafranski and Sulkowski, 1959). Activation of amino acids. The individualactivation of 14 amino acids was studied: 1cysteine, I-lysine, 1-histidine, dl-aspartic acid, dl-glutamic acid, 1-arginine, I-glycine, 1histidine, I-isoleucine, m-leucine, I-hydroxyproline,I-proline, I-tryptophane,1-tyrosine.

THE B I O S Y N T H E S I S OF P R O T E I N S

275

To the incubation mixture were added successively 10 pmoles of MgC12, 0.2 ml of 0.2 M Tris buffer of pH 7.76, 10 pmoles of the Z-amino acids, 11 pmoles of KzATP, 1500 pmoles of NHgOH, 5 pg of pyrophosphatase and 0.1 ml of enzymatic preparation. The sample was incubated for 30 min at 37". The enzymatic reaction was stopped by adding 2.3 ml of Hoagland's reagent (50.5 g of CChCOOH, 25 ml of HCI, 60.0 g of FeCls made up in distilled water to a volume of 1000 ml). Thereafter the sample was shaken and centrifuged. The intensity of colour reaction in the supernatant was measured on the Unicam S.P. 500 spectrophotometer at 520 mp. The concentration of the hydroxamates formed was calculated by comparing with a standard curve prepared on glycine hydroxamate according to Safir and Williams (1952). The control experiments with inactivated pH 5 enzymes were carried out for each individual amino acid. The enzymes were inactivated by heating of the acetone powder during 2 min in boiling water. The results are expressed as pmole of hydroxamates/mg of protein/h. The protein content in the enzymatic preparation was determined by the biuret method (Kingsley, 1942). Samples for histological examination were fixed in 15 % (w/v) formalin. Slices were cut on a freezing microtome at 30 p and stained by the myelin method of Spielmeyer. Further slices were cut at 20 p and stained by the Nissl's cresyl violet method in the modification of Spielmeyer (1930). The state of myelination in the early stages of development was also determined in 5 p paraffin slices by the osm-haematoxylin method of Schultze (1906). R E S U LTS

In the foetal white substance 7 amino acids were found to be activated, viz. : cysteine, histidine, glutamic acid, glycine, tyrosine, proline and tryptophane. The rate of activation for glutamic acid was found to be much higher than for the other amino acids. The results in the group of 3-day-old animals were similar to those in the foetal group, and the small differences were statistically insignificant. In the cerebral white substance of the group examined next (9-day-old animals) the activation of cysteine disappeared. The rate of activation of glutamic acid increased, but the difference was not yet statistically significant. No other significant changes if compared with the previous group were noted. The results obtained from 2-month-old animals and adults (6-8 months of life) closely resembled those obtained for 9-day-old guinea-pigs, with one exception concerning glutamic acid, which showed an increased rate of activation. The differences in the rate of activation of glutamic acid in the group of 2-month-old and adult animals in comparison to foetal and 3-day-old guinea-pigs are significant. In the grey substance of the foetal brain the activation of cysteine, histidine, glutamic acid, glycine, tyrosine, proline and tryptophane was determined. The rate of activation of glutamic acid differs essentially from that of the other amino acids examined, which was several times greater. Rqferences p . 279

276

M. W E N D E R A N D M. H I E R O W S K I

During the extra-uterine development of the grey substance the activation of cysteine disappears. The rate of activation of glutamic acid markedly increases during development, so that in the adult grey substance of guinea-pigs the rate of activation of glutamic acid is almost twice as high as in the foetal tissue. The data presented above indicate that the activation of amino acids in the grey substance was found to be very similar to that established in the white substance, both qualitatively and quantitatively. Only the rates of activation of glutamic acid in the groups of 9-day-old, 2-month-old, and of adults were significantly higher in the grey substance than in the white substance. In the foetal grey substance and in the group of 3-day-old guinea-pigs the activation of glutamic acid was almost identical to that found in the white substance. The results of the experiments are presented in the Tables I and 11. DISCUSSION

During the last days of gestation and during the first days of extra-uterine life in the guinea-pigs there were no significant changes in the rate of amino acid activation by pH 5 enzymes in the cerebral white and grey substances. This means that the decisive period of myelination of the nerve fibres is not accompanied by parallel changes in the activity of the pH 5 enzymes examined by the hydroxamic method. The activation of cysteine disappeared and that of glutamic acid increased, but later on. When comparing the rates of activation of the individual amino acids with their content in the proteins of cerebral white and grey substances (Wender and Waligbra, 1961, 1962) it appears that there exists no correlation between them. Thus, the higher or lower content of an amino acid in the cerebral protein was independent of the higher or lower rate of activation. This was very clearly demonstrated with reference to glutamic acid, whose rate of activation in the cerebral grey substance and in the white substance was many times higher than that of the other amino acids examined, which did not correspond with the content of this amino acid in the cerebral proteins. Neither was any correlation found between the developmental changes of these values. Our investigations on the first stage of protein biosynthesis - the activation of amino acids by the pH 5 enzymes - and on the second stage of protein biosynthesis were performed with the use of the hydroxamic method. Another known test of amino acid activation is based on the exchange between adenosine triphosphate and radioactive pyrophosphate. The results obtained by the hydroxamic test and those obtained by the exchange method of 32Pwith ATP are essentially different. Some amino acids whose activation has been determined by the hydroxamic method, show a negative reaction in the test with radioactive pyrophosphate. There are also other amino acids, which, on the contrary, give a positive reaction in the exchange method, without any confirmation being found in the hydroxamic test. Also the positive reactions found by both methods are not correlated quantitatively (DeMoss and Novelli, 1956; Novelli, 1958). On the ground of these facts it was suggested, that the amino acids can be divided into 3 groups: those which catalyse only the hydroxamic reaction, those which are subject only to the exchangereaction and, lastly, those which

277

T H E B I O S Y N T H E S I S OF PROTEINS

TABLE I R A T E O F A M I N O A C I D A C T I V A T I O N I N T H E C E R E B R A L W H I T E S U B S T A N C E OF G U I N E A - P I G S D U R I N G DEVELOPMENT

The results are given as mean in pmole of hydroxamates/mg of protein/h f standard error. Number of animals in each group was 6

I-cysteine HC1 I 4 ysine 1-histidine dl-aspartic acid dl-glutamic acid I-arginine HC1 I-glycine 1-tyrosine 1-leucine I-isoleucine I-a-alanine I-hydroxyproline I-proline I-tryptophane

Foetuses (9th week of gestation)

3 days

0.015 f 0.004 O.OO0 0.010 f 0.003 O.OO0 0.500 f 0.024

0.015 f 0.002 O.OO0 0.010 f 0.002 O.OO0 0.515 f0.010

O.OO0

O.OO0

0.010 f 0.002 0.010 f 0.002 O.OO0 O.OO0 O.OO0 O.OO0 0.010 f0.003 0.010 f 0.002

0.010 f 0.002 0.005 f 0.002 O.OO0 O.OO0 O.Oo0 O.OO0 0.010 f0.003 0.005 f 0.002

9 days

2 months

8-10 months

0.000 O.OO0 O.OO0 0.000 O.OO0 O.OO0 0.020 f 0.005 0.015 f0.004 0.025 f 0.006 0.000 O.OO0 O.OO0 0.600 f 0.024 0.690 f0.030 0.700 f0.033 0.000 O.OO0 O.OO0 0.015 f0.005 0.015 f 0.002 0.020 f 0.002 0.010 f 0.003 0.005 f 0.001 0.010 f 0.004 0.000 O.OO0 O.OO0 0.000 O.OO0 O.OO0 O.OO0 0.000 O.OO0 0.000 O.OO0 O.OO0 0.020 f 0.003 0.015 f0.005 0.020 f 0.005 0.005 f 0.003 0.005 f 0.002 0.005 f 0.003

T A B L E 11 R A T E O F A M I N O A C I D A C T I V A T I O N I N T H E C E R E B R A L G R E Y S U B S T A N C E OF G U I N E A - P I G S D U R I N G DEVELOPMENT

The results are given as mean in pmole of hydroxamates/mg of protein/h f standard error. Number of animals in each group was 6

Foetuses (9th week of gestation) I-cysteine HCI I-lysine 1-histidine dl-aspartic acid dl-glutamic acid I-arginine HCI 1-g1ycine 1-tyrosine I-leucine I-isoleucine I-a-alanine I-hydroxyproline 1-proline I-tryptophane References p. 279

3 days

0.020 f 0.003 0.010 f 0.002 O.OO0 0.010 & 0.003 0.010 f 0.002 O.OO0 O.OO0 0.470 & 0.020 0.515 & 0.020 O.OO0 O.OO0 0.015 f 0.002 0.010 f 0.002 0.010 f 0.003 0.010 f 0.002 O.OO0 O.OO0 O.OO0 O.OO0 O.OO0 O.OO0 O.OO0 O.OO0 0.010 0.002 0.010 f 0.002 0.005 & 0.002 0.005 f 0.001

O.OO0

9 days

2 months

O.OO0 O.OO0 O.OO0 O.OO0 0.025 f0.005 0.020 f 0.004 0.o00 O.OO0 0.720 f0.010 0.830 i0.013 O.OO0 O.OO0 0.015 f0.002 0.020 f 0.002 0.010 ==! 0.002 0.010 f0.004 0.o00 O.OO0 O.OO0 O.OO0 O.OO0 O.OO0 O.OO0 O.OO0 0.020 f 0.005 0.015 f 0.002 0.010 f 0.002 0.005 f 0.004

8-10 months

0.000 0.000 0.020 f 0.003 0.000 0.870 f 0.014 0.000 0.015 f 0.002 0.015 f0.004 0.000 0.000 0.000 0.000 0.015 f 0.003 0.010 f 0.004

278

M. W E N D E R A N D M. HIE R OW SKI

catalyse both reactions. These arguments may point to the fact that both methods examine different reactions in the mechanism of activation of the carboxyl group and suggest besides that there exist amino acids which are activated indirectly in the process of protein biosynthesis by the transacylation with other already activated amino acids. As already stated, in our studies we could not establish any correlation between the content of individual amino acids in the cerebral proteins and the rate of activation of these amino acids. The results obtained are in accordance with the observations of Szafrahski and Sulkowski, who did not find a relationship between the rate of activation of individual amino acids and their content in the proteins of various organs of adult guinea-pigs either. The increase in protein biosynthesis, as it was shown for instance by Szafranski and Sulkowski in the case of regenerating rat liver, does not enhance the rate of amino acid activation either. The studies discussed suggest that the activity of pH 5 enzymes are not the only factor determining the rate of protein biosynthesis. It seems likely, therefore, that the activation of the carboxyl group of amino acid by pH 5 enzymes is not a unique, preliminary stage in the biosynthesis of proteins. The other way of protein biosynthesis is not yet well-known, even in its outline. There is only scanty information about a possible mechanism of the incorporation of amino acids in the proteins without previous activation. Of the utmost interest in this respect are perhaps the results of experiments of Beljahki and Ochoa (1 958), who succeeded in demonstrating in Alcaligenesfaecalis the enzyme responsible for the incorporation of amino acids into proteins without activation of carboxyl group (so-called ‘amino acid incorporation enzyme’). This problem in relation to the nervous system is still unsolved.

SUMMARY

The rate of activation of 14 amino acids by pH 5 enzymes, was studied in the developing nervous system of guinea-pigs. The estimations were performed by the hydroxamic method in the acetone powder of cerebral white and grey substance. The data found led to the following conclusions: (I) In the foetal white and grey substancesthe activation of 7 amino acids was found : cysteine, histidine, glutamic acid, glycine, tyrosine, proline and tryptophane. (2) The rate of activation in the cerebral tissue for glutamic acid was much higher than for the other amino acids. (3) During the extra-uterine development the activation of cysteine disappeared and the rate of activation of glutamic acid increased. ( 4 ) There exists no correlation between the rates of activation of the individual amino acids with their content in the proteins of cerebral white and grey substances. (5) The rate of activation of amino acids shows no significant differences between white and grey cerebral substances. (6) The activation of amino acids by pH 5 enzymes is probably not a unique mechanism of the peptide chain synthesis in the proteins of the nervous system.

THE B I O S Y N T H E S I S O F P R O T E I N S

279

REFERENCES B E L J A ~ KM., I , AND OCHOA,S., (1958); Protein biosynthesis by a cell free bacterial system. Proc. nut. Acad. Sci. (Wash.), 42, 494-501. DEMOS, J., AND NOVELLI,G., (1956); An amino acid dependent exchange between 32P labeled inorganic pyrophosphate and ATP in microbial extracts. Biochim. biophys. Acta, 22, 49-61. HOAGLAND, M.,(1955); An enzymatic mechanism for amino acid activation in animal tissues. Biochim. biophys. Acta, 16,288-289. J., (1956); Enzymatic carboxyl activation of amino HOAGLAND, M., KELLER,E., AND ZAMECNIK, acids. J. hiof. Chem., 218,345-358. KINGSLEY, M., (1942); The direct biuret method for the determination of serum proteins as applied to photoelectric and visual colorimetry. J. Lab. d i n . Med,, 27, 840-846. NOVELLI,G.,(1958); Some problems concerning the activation of amino acids. Proc. nut. Acad. Sci. (Wash.), 44,86-92. J., (1952); Synthesis of hydroxamic acid of glycine. J. Org. Chem., 17, SAFIR,S., AND WILLIAMS, 1298-1301. SCHULTZE, O., (1 906) ; Uber den fruhesten Nachweis Markscheidenfarbung im Nervengewebe. Sitzungsberichte der physiologisch-medizinischen Gesellschaft, Wiirzburg. SPIELMEYER, W., (1930); Technik der mikroskopischen Untersuchung des Nervensystems. Berlin, Springer-Verlag. SZAFRA~K P.,I , AND SULKOWSKI, E., (1959); Activation of amino acids in various organs of the guinea-pig. Acfu biochim. pol., 6, 133-141. WENDER, M., AND WALIGORA, Z., (1961); The content of amino acids in the proteins of the developing nervous system of the guinea-pig. I. Cerebral white matter. J. Neurochem., 7,259-263. WENDER, M., AND WALIGORA, Z., (1962); The content of amino acids in the proteins of the developing nervous system of the guinea-pig. 11. Cerebral grey matter. J. Neurochem., 9, 115-118.

DISCUSSION

WAELSCH: This is a very interesting paper for several reasons. In 1956 Fritz Lipmann presented some data on the amino acid activation in the newborn rat at the Second International Neurochemical Symposium*. He reported on a significant activation of tryptophane but this was very early in the game, where one did not really know how to handle the activating enzyme properly. I think that your data are particularly interesting because they show the activation of glutamic acid. This is a unique thing because the difficulty of showing activation of glutamic acid has gone through the whole field of amino acid activation. It has been suggested very recently that it is not glutamic acid which is activated and incorporated but glutamine, which then is deamidated in the protein to glutamic acid. It is extremely difficult to show a distinct activation of glutamic acid by the activating enzyme. I am somewhat more optimistic about the demonstration of amino acid incorporation into brain proteins, particularly since we have shown about 2 years ago that in the system amino acids are incorporated into brain ribosomes at a faster rate than they are incorporated into liver ribosomes. So not only there is in the brain the complete system but it is actually operating with ribosomes at a faster rate than in the liver. I want to ask you: is glutamic acid active or not in the pyrophosphate exchange?

* The paper is published in: Metabolism of the Nervous System, D. Richter, Editor. Pergamon Press, 1957.

280

DISCUSSION

WENDER: I don’t know. I have not studied this by the exchange method but by the hydroxamate method. WAELSCH: This is extremely interesting because it is really something which many people have tried, not with brain but with other tissue. The glutamic acid activation has always presented a great problem and has been really now demonstrated here very clearly and particularly for the brain where you have such a high glutamic acid concentration. BONAVITA: I have a very trivial question. Why did you choose guinea-pig for this kind of study? Would not the rat have been much better, because the guinea-pig has a well-developed brain at birth? WENDER:The rat brain would be too small to divide it up into white and grey matter. This is the only reason why we use guinea-pig. SCHAD~~: The rabbit may even be a more suitable animal than the guinea-pig. At birth the development of the brain is about at the same level as in the rat. The postnatal growth of the rabbit brain is impressive. The weight increases about 5 times, myelination starts and matures and many other developmental parameters such as Nissl bodies, dendritic plexus, axonal branching, show a fast increase in the postnatal period.

28 1

Author Index* Abood, L. G., 244 Addison, W. H. F., 209 Adrian, E. D., 196, 197, Adriin, H., 222 Alexander, F. A. D., 269 Amassian, V. E., 205 Amunts, V. V., 35 Angeletti, P. U., 24 Angevine, J. B., Jr., 47 Angulo y Gonzales, A. W., 97, 113 Anokhin, P. K., 201 Appella, E., 269 Arey, L. B., 179 Ariens Kappers, C. U., 13, 112, 126, 127 Ariens Kappers, J., 155 Artom, G., 93 Attfield, M., 268 August, B., 104, 105 Azzone, G. F., 243, 244, 217, 249 Babkin, P. S., 104 Bain, J. A,, 237 Balazs, R., 244 Barcroft, J., 97, 112 Barnard, J. W., 217 Barron, D. H., 97, 112 Bartley, W., 245 Baxter, C. F., 163, 166, 176, 190, 229 Becker, R. F., 97, 107, 124 Beckett, E. B., 117, 118 Beduschi, A., 262, 269 Beljahki, M., 278 Bellamy, D., 244, 245 Bender, M. B., 128 Berger, M., 246 Berl, S., 199 Bessman, S. P., 244 Bieber, R. B., 254, 259 Bishop, G. H., 197,202,231 Blake, J. R., 104 Bolaffio, M., 93 Bonavita, V., 254-272 Booker, B., 24 Borst, P., 244 Bourne, G. H., 117, 118 Bourne, M. C., 257 Brady, J. V., 129 Braitenberg, V., 172, 173

*

Bregoff, H. M., 244 Brizzee, K. R., 136-149, 190 Brodmann, K., 30 Brody, T. M., 237 Brookhart, J. M., 207 Brooks, V. B., 197 Brotherton, J., 258 Brown, J. W., 107, 109, 111, 113 Brunngraber, E., 244 Biicher, T., 244 Burns, B. D., 197 Cajal, S. R. y, 1, 8, 156, 176, 189, 190,209,212, 217,228,229 Calabrisi, P., 124 Callan, D., 199, 201 Calm, R. D., 269 Campbell, D. A., 257 Carafoli, E., 243, 247, 249 Carrnichael, L., 94, 126, 127, 196198, 230, 231 Casamajor, L., 224 Cauna, N., 117, 118 Chalkley, H. W., 137, 156 Chang, H. T., 197, 198 Chevreau, J., 230 Chiarugi, E., 209, 212 Ciotti, M. M., 254, 258, 259 Clare, M. H., 197, 202, 231 Coghill, G. E., 94, 100, 125, 127 Cohen, S., 24 Colon, E., 150-175 Conel, J. LeRoy, 30, 42, 71, 176, 189 Congill, E. J., 97 Contamin, F., 232 Cooper, C., 250 Cooper, E. R. A., 119, 127 Crosby, E. C., 13, 105, 107, 112, 124, 126, 127, 129 Cuajunco, F., 118, 119, 121 Davey, J. B., 268 De La Haba, G., 254,259, 268 DeLuca, H. A., 137, 143 DeMoss, J., 276 Dempsey, E. W., 185 Dennis, W., 94 Detwiler, S. R., 268 Donahue, S., 185

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

282

AUTHOR INDEX

DOW, R. S., 204, 209,213-215, 218 DuBuy, H. G., 244 Dzugayeva, S. B., 35 Eayrs, J. T., 160, 176, 190 Eccles, J. C., 197 Eccles, R. M., 204,208 Edstrom, J. E., 136, 172 Eeg-Olofsson, O., 243, 244, 249 Ellingson, R. J., 201,224 Elliott, K. A. C., 136 Enger, P. S., 197 Emster, L., 243, 244, 246, 249 Fazekas, J. F., 269 Filimonoff, I. N., 30, 32, 35 Fish, M. W., 204, 208 Fitzgerald, G. E., 93, 94, 99, 112 Flanagan, G. L., 94 Flexner, J. B., 254, 259, 268 Flexner, L. B., 160, 162, 198,254, 259, 268 Fox, C. A., 217 Frankel, S., 163 Futterman, S., 257 Galant, J. S., 128 Gallagher, C. H., 244 Gasseling, M. T., 15 Gesell, A., 94 Gilbert, M. S., 127 Girado, M., 197,199,200,203,206,214,215,217 Gliicksmann, A., 15 Goldring, S., 201, 222, 225 Golubewa, E. L., 93, 101, 102, 104, 107, 127 Goodhead, B., 160, 176, 190 Gordon, M. W., 136-138, 142 Grabar, P., 255, 259, 262, 270 Grafstein, B., 204 Gray, C. M., 251 Gray, D. E., 137, 143, 179, 195, 217,218 Greenhill, J. P., 93 Gregory, K. F., 257, 263, 270, 271 Griffin, A. M., 224 Grossman, C., 200 Grundfest, H., 197, 199, 201,203, 205, 206, 214, 215, 217,218 Griineberg, H., 15 Guarneri, R., 256,259,261, 262, 269, 270 Guth, P. S., 250 Hamburger, V., 3, 19, 21, 22 Hammond, W. S., 2 Hamolsky, M., 254, 259 Harman, P. J., 163, 246 Harrison, R. G., 107 Haug, H., 151, 156, 162 Heller, I. V., 136 Henderson, J. W., 129

Henry, E. W., 204, 208 Herz, L., 242 Hesselbach, M. L., 244 Hewer,E.E., 107, 113, 117-119, 121, 124 Hierowski, M., 273-280 Himwich, H. E., 269 Hoagland, M., 273,274 H o ~ I., D., 105, 107, 117-119,225 Holton, F. A., 219 Hooker, D.,93-102,104,107,109-112,114,115, 117-120, 122-125, 127-129, 224 Horstadius, S., 2 Horstmann, E., 185 Housepian, E. M., 185,187-221,230,231 Hromada, J., 118, 121 Huber, G. C., 13, 112, 126, 127 Hiilsmann, W. C., 249 Humphrey, T., 93-135, 224 Hunt, W. E., 201, 222, 225 Hursh, J. B., 204, 227 Hydkn, H., 136 Iwama, K., 201 Jacobs, M. J., 113, 137, 138, 190 Jansen, J., Jr., 217 Jasper, H. H., 201 Jimenez Gonzales, L., 109 Jones, A. W., 15 Judah, J. D., 244 Kahn, E. A., 129 Kaplan, N. O., 254,258, 259,269 Keller, E., 273 Kharetchko, X., 136-149 Kmgsley, M., 275 Kinoshita, J. H., 257 Klingenberg, M., 244 Klishov, A. A., 118, 121 Kononova, E. P., 32,34 Korey, S. R., 136 Koskinas, G. N., 39, 71 Krasnogorsky, N. I., 32 Krebs, H. A., 244 Krieg, W. J. S., 137 Kuhlman, R. E., 254 Kukuyev, L. A., 32, 35 Kunkle, E. C., 104 Lange, P. W., 136 Langworthy, 0.R., 113, 204,208 Larsell, O., 204, 215, 218 Lauer, E. W., 105, 124, 129 Lauria, F., 172 Layne, E. G., 244 Lehninger, A. L., 251 Levi, G., 19, 22 Levi-Montalcini, R., 1-29

AUTHOR INDEX

Levine, L., 269 Lippman, K., 104 Lervtrup, S., 136, 237-253 Low, H., 248 Lowry, 0. H., 254 Lucas, D. R., 268 Lucas Keene, M. F., 113 Luft, R., 243, 244, 249 Luse, S. A., 185 MacFarlane, M. G., 251 Mannon, G., 117, 118 Marinesco, G., 104 Markert, C. L., 254, 268, 269 Marty, R., 201, 204, 222-236 Mavrinskaya, L. F., 93, 119, 121 Maynard, E. A., 183 McGeer, P. L., 123 Meeter, K., 166, 167, 171 Meves, H., 185 Miale, I. L., 209 Miller, R. B., 104, 105 Minayeva, V. M., 32 Minkowski, M., 93 Moeller, F., 254, 268 Montagna, W., 117, 118 Moruzzi, G., 209, 215 Mum, N. L., 94 Muscatello, U., 247, 249 Musgrave, F. S., 202, 203 Myers, D. K., 249 Neilands, J. B., 261 Noback, C. H., 160, 166, 176, 185, 187-221 Noell, W. K., 269 Novelli, G., 276 Numberger, J. I., 136, 138, 142 Ochoa, S., 278 Ochs, S., 197 O’Donnell, J. E., 204, 208 Oeconomos, D., 201,222 Ohmori, D., 124 Olds, J., 126, 129 Olds, M. E., 126, 129 Orchen, M., 136 Pappas, G. D., 159, 163, 166, 176-186, 189, 195, 198,218 Parmelee, A. H., Jr., 104, 105 Patten, B. M., 93 Patton, H. D., 205 Pavlov, I. P., 127 Pearson, A. A., 113 Pease, D. C., 183 Pertz, A. P. R., 117 Pertz, R. M., 117 Peters, V. B., 160, 162

283

Meiderer, G., 254, 258 Pieper, A., 107 Pigon, A., 136, 172 Plagemann, P. G. W., 257, 263,270 Polyakov, G. I., 30 Pompeiano, O., 209, 212 Ponte, F.,268 Prechtl, H. F. R., 98, 103, 104, 107, 117 Preobrazhenskaya, N. S., 32, 35 Purpura, D. P., 159, 160, 163, 166, 176-186, 187-221, 230,231 Quastel, J. H., 242 Rabinowicz, Th., 39-92 Radovici, A., 104 Reading, H. W., 258 Rees, K. R., 244 Rhines, R., 97 Roberts, E., 163, 198, 244 Roberts, R. B., 254, 268 Rose, M., 30,222 Rossen, J., 244 Rubino, A., 268 Safir, S., 275 Santibankz, G., 222 Sarkisov, S., 30-38 Saunders, J. W., Jr., 15 Saunders, L. C., 15 Schadt, J. P., 150-175, 176, 190, 229 Schemm, G. W., 129 Scherrer, J., 201,222-236 Schmidt, G., 143 Schou, M., 242 Schultz, R. L., 183 Schultze, O., 275 Shariff, G. A., 162 Shealy, C. N., 204,208 Sherrington, C. S., 126 Shieh, P., 10, 20, 23 Shofer, R. J., 185, 187-221 Sholl, D. A., 151, 163, 166, 172, 173, 190 Shulejkina, K.V., 93,101,102,104,107,118,127 Sidman, R. L., 47,209 Skoglund, S., 204, 208 Slater, E. C., 244, 249 Smit, G. J., 173 Smith, T. G., 199, 201 Sorsby, A., 257 Spielmeyer, W., 275 Spirtes, M. A., 250 Stankevich, I. A., 32 Stilwell, D. L., Jr., 121 Stirnimann, F., 128 Suga, IJ., 107 Sugita, N., 254, 268 !Sulkowski, E., 274

284

A U T H O R INDEX

Svennerholm, L., 239, 241, 242, 245, 249, 255 Swinyard, C. A., 143 Szabolcsi, G., 243, 244,249 Szafra&lci, P., 274 Szymonowicz, W., 107, 117, 118 Takashi, M., 117 Tansley, K., 257 Tapley, D. F., 250,251 Taylor, M., 244 Tello, J. F., 2 Tennyson, V. M., 179 Terni, T., 11, 13 Thannhauser, S., 143 Thomas, J., 224, 225 Tilney, F., 224 Ulett, G., 204, 215, 218 Uttley, A. M., 166, 172, 173 Uzman, L. L., 8, 15, 209 Vainstein, I. I., 93, 101, 102, 104, 107, 127 Van Backer, H., 150475 Van Campenhout, E., 2 Van Der Loos, H., 160 Van Groenigen, W. B., 152,154,166,167, 171 Verley, R., 232 Voeller, K., 176, 179, 185, 189, 195, 198

VO@, J., 136-149 Von Economo, C., 39, 71 Waligora, Z., 276 Weed, L. H., 208 Weiler, L. J., 156 Wender, M., 273-289 Wheeldon, L. W., 251 Wieland, Th., 254, 258 Wilcott, R. C., 201 Williams, C. A., 255, 259, 262, 270 Williams, J., 275 Willis, W. D., 204, 208 Wilson, V. J., 204, 208, 225 Windle, W. F., 93, 94, 97, 99, 112, 204, 208, 224 Winer, A. D., 261 Winkelmann, R. K., 225 Wislocki, G. B., 185 Woodburne, R. T., 129 Woolsey, C. N., 204,208 Wroblewski, F., 257, 263, 270 Yntema, C. L., 2, 3 Yoss, R. E., 107, 127 Zamecnik, J., 273 Zanchetti, A., 207 Zelander, T., 237, 239 Zwilling, E., 269

285

Subject Index Abducens, accessory center of, 27 Accessory nucleus, facial, 113 migratory movement of, 13 spinal, 112 Acetone powder, preparation of, 274 Activation, of amino acids, 273, 275, 278 rate of, in cerebral tissue, 278 Activity, cortical seizure, 206 corticofugal, in brain stem synaptic organizations, 207 in rostral cortical areas, 208 medullary pyramidal tract, 206 neocortical, on subcortical or peripheral nerve stimulation, 209 postsynaptic, of apical dendrites, 209 ADP-addition, response of mitochondria to, 245 Afferents, thalamocortical, 202 Agar, elution of enzymic proteins from, 270 Alcaligenes faecalis, incorporation of amino acids into proteins, 278 Allocortex, pyramidal layers in, 44 Amino acids, activation of, 273, 275, 278 in peptides, 278 &-aminocaproic acid, cerebral response to, 199 o-amino caprylic acid, cerebral response to, 199 content of, in fetal brain, 275-278 incorporation of, in ribosomes, 273 in regenerating liver, 278 Ammon’s horn, structure of, in premature infant, 69 Amygdala, effect of stimulation of, on integration level of neocortex, 36 Amytal, effect of, on mitochondria, 237,243 Analysor, verbal-auditory, integration with verbal signals, 32 verbal-motor, integration with kinesthetic expression, 32

Anoxia, effects of, on fetal reflexes, 97 Area, cytoarchitectonic study of each, 39 primary motor, 85 structural organization of fetal brain, 48-55 rostral cortical, corticofugal activity in, 208 sensory, 85 structural organization of fetal brain, 48-55 surface, of cortical neurons, cell bodies and dendrites, 151-163 factor determining RNA content of cells, 172 visual, electro-ontogenesis of, 228 Astrocytes, development of, in cerebral cortex, 39 number of, in white matter, 81 ATP-addition, response of mitochondria to, 245, 248 ATPase, patterns of activations, 246249 Axodendritic synapse, development of, in feline cerebral cortex, 35 electronmicroscopy of, 195 predominance of, in cerebral cortex, 35 Axon, collateral branches, number and length, 189 corticospinal, conduction velocity in, 204, 205 pyramidal neuron, physiological properties of, 209 Axosomatic synapse, development of, in feline cerebral cortex, 35 electronmicroscopy of, 195 predominance of, in subcortical structure, 35 Basophilia, changes of, into Nissl bodies, 163 development of, 153 in immature neurons, 87 Betz cells, in fetal brain, 47, 87 Biosynthesis, of proteins, enzymatic mechanisms, 273-280 Blood-brain barrier, involvement of, basement membrane in, 185 Bodian protargol method, use of, in neonatal kittens, 193 Brain stem, cytoarchitectonic structure, in premature infant, 44 of embryos, 11 extrapyramidal outflows from, 208

286

SUBJECT INDEX

migratory movements, in chick embryo, 11 Brain weight, in newborn, 42 in premature infant, 42 Cajal cells, number of, in developing brain, 71, 228 Cajal-Retzius cells, degeneration of, during development, 92 Calcarine region, in premature infant, 58 Cephalin, relative distribution of, in mitochondria, 249 Cerebellar cortex, cell bodies, analysis of, 151-155 detection of electrical properties in, 217 electrophysiological properties of, immature, 213 gray cell coefficient, 151-155 packing density of neurons in, 151-155 response to folial stimulation, 213 size of cell bodies in, 151-155 subpial neuropil in newborn, 216 surface area, of cell bodies, 155-1 58 of dendrites, 161-163 Cerebral cortex, basement membrane in, mature, 183 dendritic organization of, during development, 167 electrical activity, cerebellum and, 231 evolution of integration, 30-38 extraperikaryal space, 113 frontal, structure of, 32 glutamic decarboxylase of, 241 integration level of, 30 of premature infant, 39-92 regression coefficient, dendritic development, 171 somesthetic, evoked potentials in, 201 stimulation of thalamocortical projections to, 207 structural organization of, 173 Chalkley, random hit method, in nervous tissue, 156 Chlorpromazine, effect of, on mitochondria, 237 Cholinesterase, activity, in developing cell bodies, 118 optic nerve, content of, 235 Cochlea, degenerative changes, 15 Conduction, fiber, histological substrate of, 226 Coniocortex, development of areas in, 55-57 granular area, 47 Connections, internuclear, of reticular formation, 112 intracortical synaptic, 208

Corpus callosum, analysis of, neural glia, 274 chemical analysis of, 274 Cysteine, activation, in fetal brain, 275 Cytochrome, addition of, to mitochondria, 242 Degeneration, massive cell, during development, 20, 24 in metamorphic processes, 15 and migration of differentiating nerve cells, 2 sympathetic nerve cells, 24 Dendrite, apical, 51, 156, 190 development, 159, 165, 229 percentage of cortex, 158 postsynaptic activation, 209 pyramidal neurons, immature, 179 ultrastructure of, 195 basal, 51, 156, 158, 192 development, 155-163 percentage of cortex, 159 ultrastructure, 176-1 86 criterion for maturation, 39-42, 189 number, 169, 189 Purkinje cell, superficial position of, 217 spines, development, 189 stellate cells, 156, 179 stripping, phase during maturation, 213 structural organization, 151, 164-1 73 Dendritic field, factor, as determinant of development, 171 logarithmic representation, 170 Diencephalon, lactic dehydrogenase, content of, 268 Dimension, neuronal constituent, power function, 155 Discoglossus pictus, LDH activity, 263 Disintegration, stage of neuronal, 17 DNA, activity, during growth, 29 determination in growing rat brain, 148 DNP, influence, on oxidation and phosphorylation, 245 DPN, interaction with pyridine and purine analogues of, 259 ketone derivatives of, 260 reduction rates, 259 Electronmicroscopy, feline cortex, neonatal, 181-185, 195 immature human cortex, 176-185 Electrophoresis, agar, method of enzyme studies, 262

SUBJECT INDEX

starch gel, method of enzyme studies, 262 Embryos, amphibian, fetal reflexes of, 99 appearance of reflexes in human, 94-104 development cell patterns in chick, 2, 8, 11 Fasciculus proprius, involvement in trunk movements, 112 Fissure, lateral, size during development, 42 parieto-occipital, in fetal brain, 42 Frontal lobe, development in premature infant, 48, 75 GABA, application to brain, 200, 201 content of, brain, 147, 163, 253 during development, 147, 163 pass-way in brain, 253 GAD, activity, increase during development, 147 Gallocyanin, sta’ning method, neurons, 151 Ganglion, geniculate, chick embryo, 3 spinal thoracic, 19 sympathetic, degeneration in, 27 synaptic contact, 23 Glutamic acid, during postnatal development, 147 rate of activation in brain, 275, 278 Hippocampus, effect of stimulation, on integration level of neocortex, 36 Histidine, concentrations in fetal brain, 275-278 incorporation in proteins, 276 rate of activation, 278 Hyperstriatum, division of bird brain, 35 Interneuron, cortical, synaptical activity, 201 synaptical linkage with pyramidal cells, 206 Intra-uterine period, development, 32 Isozymes, appearance and increase, during development, 268 fractions in retina, 257 LDH, number of, 263-269 number in rat brain. 258 a-Ketoglutarate, rate of oxidation, 242 Kreb’s cycle, significance of shunts in mitochondria, 244 substrate level transformations, 241

287

Latency, cortical evoked potential, 223, 225 potential in medullary pyramidal tract, 205 LDH, agar and starch gel electrophoresis, 255 biochemical techniques, determination, 270 diencephalon, content, 256, 268 isozymes, 255, 263, 269 molecular evolution, 254-272 retina, content, 268 Limbic system, in premature infant, structural organization, 8 1 Lobe, olfactory, macroscopic appearance, 42 Maceration method, cell count in cerebral cortex, 137 Matrix, structure in developing nervous system, 6 Medulla oblongata, recording of responses, 207 Membranes, cytoplasmic, and enzyme level, 163 Mesencephalon, recording of responses, 207 Mesostriatum, division of bird brain, 34 Metabolism, disturbance of glucose, 258 Microglial cells, immature human brain, 42 Migration, differentiating nerve cells, 2 Mitochondria, comparison of brain and liver, 242 influence of calcium, 246, 247 influence of magnesium, 248, 249 lipid composition, 249 modified isolation procedure, 239 morphological preservation of, 239 muscle, succinate oxidation, 250 oxidation of, 242, 244 swelling of, 251 Motoneurons, development of function, kittens, 208 electrical properties of, cats, 208 Motor area, development in human brain, 73 Motor column, migration of cells, in chick brain, 8 Myelin, development of, in central nervous system, 39 formation of, and conduction velocity, 205 formation, in visual system, 226, 227 maturation of, formation in pyramidal tract, 192, 194 process of, formation in cat, 227 sequence of, formation, 228 staining method, 275

288

S U B J E C T INDEX

Neocortex, development of amino acid metabolism, 199 electronmicroscopy of immature, 195 superficial neuropil in cat, 200 Neopallium, development of stratification, 32, 36 Neostriatum, division of bird brain, 35 Nerve cells, cytoplasm of, 163 fetal subepithelial plexus, 124 growth of nerve fibre, 227 migratory patteins, systems, 2 specialized sensory endings, 128 Neural tube, experimental cell degeneration, 22 Neurobiotaxis, mechanisms of, theory, 27 position of nuclei, during, 14 Neuroblasts, number in developing cortex, 40 Neurofibrils, criterion for brain development, 39 Neuroglia, deveiopment, 27 index, quantitative analysis, 137-145 involvement in blood-brain barrier, 185 number in cortical layer, 39 perivascular processes, 185 ultrastructure of processes, 179 Neuropil, axodendritic synaptic pathways, 209, 21 8 cerebral cortex, 185 electronmicroscopy during maturation, 179 Nucleus, epibasalis centralis, 15 ventralis, posterior, 127 posterolateralis, 119 posteromedialis (arcuate nucleus), 1 19 Olfactory lobe, macroscopic appearance, 42 Orang outang, size of premotor cortex, 32 Oxidation, mitochondria and rate of, 242, 245 Paleocortex, structure in bird brain, 36 Paleostriatum, structure in bird brain, 35 Pathway, afferent cerebral-rebellar, 21 5 corticofugal and bulbospinal, electrical activity, 208 corticopetal generation of electrical activity, 200

cortico-ponto-cerebellar,conduction, 216,218 rostra1 neocortex, fiber spectrum, 208 Pattern formation, axon and dendrite, 172 maturation of cortex, 151 Phosphorylation, influence of serum albumin, 247 Probability machine, relation to brain structure, 17CL173 Propagation, signals in nerve fibers, 225 Proteins, biosynthesis, 275-278 in cerebral tissue, 278 incorporation of amino acids into cytoplasmic urotein. 213 study by agar and starch gel electrophoresis, 262-265 Purine, interaction with pyridine, 259 Purkinje cell, expansion of dendrites, 212 migratory movements, 8 postnatal development, 209 Pyramidal cells, average value for perikarya, 152 axon and axon-collateral function, 203 development of apical dendrites, 203 intracortical axon collaterals, 206 lateral dendrites of, 85 morphogenesis in feline neocortex, 195 ontogenesis of dendrites, 164-169 postnatal development of axons, 192 relation to visual response, 227-230 size of cell bodies, 154 ultrastructure during development, 179, 185 value of cell body volume, 152 Pyramidal tract, electrical activity in medullary, 204, 207 Pyridine, interaction with purine, 259 Receptor, development of trigeminal nerve, 105 Reflex, appearance of cremasteric, 123, 126 conditioned, development of, 34 contralateral flexion, 99, 125, 126 development of, palmomental, 104 features of, subcortical, 34 mechanism of avoiding, 103 onset of mouth-opening, 105 pattern formation of, systems, 125 trigeminal, related to feeding, 103 trigeminal stimulation, 105 Response, appearance of first electrocortical, 224 characteristics,

SUBJECT INDEX

of cortical spinal axon, 204 of surface-positive, in cortex, 217 difference in cortical areas, 207 medullary pyramidal tract, 204 parameters of local cortical, 222, 227 superficial cerebellar cortical (SCbR), definition, 21 3 superficial cortical (SCR), definition, 196 Reticular formation, development of internuclear connections, 1 12 influence of, on blocking reactions, 236 role of, in cerebral activity level, 35 Retina, electrophoresis of enzymes, 25&258 isozymatic composition, 257 LDH activity, 256-261 postnatal development, 257 Rhinencephalon, development in fetal brain, 44 Ribosomes, incorporation of amino acids, 273 soluble cytoplasmic solution, 273 Sensitivity, sequence of development, cutaneous, 96 Speech, ontogenetic development, 32 Sphingomyelin, content in mitochondria, 249 Spinal cord, cervical transplanted segment, 20 degeneration at cervical level, 17

289

Spinal ganglia, degenerative processes, 17 experimental cell degeneration, 22 Spinal tract, development of, trigeminal nerve, 105 Synapse, axodendritic, of apical dendrites, 209 electronmicroscopy of, 195 in feline cerebral cortex, 183 predominants of, in cerebral cortex, 35 superficial pathways, 218 axosomatic, predominants, in subcortical structures, 35 structure organization of during, development, 179 Telencephalon, migratory movements of neurons, 15 Temporal lobe, structure in premature infant, 63, 81 Thalamus, differentiation of, dorsal, 127 Thymidine, incorporation into neuronal nuclei, 29 Trigeminal nerve, development of spinal tract, 107 sensory nuclear complex, 11 3, 1 17 spinal tract, 105 Vestibular apparatus, degenerative changes during development, 15

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