Cellular Aspects of Neural Growth and Differentiation [Reprint 2020 ed.] 9780520331884

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UCLA FORUM IN M E D I C A L VICTOR E . H A L L , JUDITH H E L L E R , M A B T H A AXELROD,

SCIENCES

Editor

Assistant Editorial

Editor Assistant

EDITORIAL BOARD

Forrest H. Adams Mary A. B. Brazier Carmine D. d e m e n t e Louise M. Darling Morton I. Grossman

UNIVERSITY

OF

William P. Longmire H. W. Magoun Sidney Roberts Emil L. Smith Reidar F . Sognnaes

CALIFORNIA,

LOS

ANGELES

CELLULAR ASPECTS OF NEURAL GROWTH AND DIFFERENTIATION

UCLA FORUM IN MEDICAL SCIENCES NUMBER 14

CELLULAR ASPECTS OF NEURAL GROWTH AND DIFFERENTIATION Proceedings of a Conference held November, 1969 Sponsored by the School of Medicine and the Brain Research Institute University of California, Los Angeles

DANIEL C. PEASE EDITOR

UNIVERSITY OF CALIFORNIA BERKELEY

LOS ANGELES 1971

PRESS

LONDON

CITATION FORM

Pease, D. P. (Ed.), Cellular Aspects of Neural Growth and Differentiation. UCLA Forum Med. Sci. No. 14, Univ. of California Press, Los Angeles, 1971

University of California Press Berkeley and Los Angeles, California

University of California Press, Ltd. London, England

© 1971 by The Regents of the University of California ISBN: 0-520-01793-5 Library of Congress Catalog Card Number: 73-126760 Printed in the United States of America

PARTICIPANTS IN THE CONFERENCE D A N I E L C . P E A S E , Chairman and Editor Department of Anatomy, UCLA School of Medicine Los Angeles, California 90024

R O B E R T BALÄZS

MRC Neuropsychiatry Unit Medical Research Council Laboratories Carshalton, Surrey, England S T E P H E N C . BONDY

Division of Neurology University of Colorado Medical Center Denver, Colorado 80220 W.

JANN B R O W N

Department of Pathology, UCLA School of Medicine Los Angeles, California 90024 DAVID W .

CALEY

Department of Anatomy, University of Virginia Charlottesville, Virginia 22901 CARL COTMAN

Department of Psychobiology, University of California Irvine, California 92664 W.

MAXWELL

COWAN

Department of Anatomy, Washington University School of Medicine St. Louis, Missouri 63110 A. N.

DAVISON

Department of Biochemistry, Medical School Charing Cross Hospital London W.C. 2, England J E A N DE V E L L I S

Laboratory of Nuclear Medicine and Radiation Biology and the Department of Anatomy UCLA School of Medicine Los Angeles, California 90024

S A M U E L EIDUSON

Department of Biological Chemistry, UCLA School of Medicine Los Angeles, California 90024 EDWARD G E L L E R

Department of Psychiatry, UCLA School of Medicine Los Angeles, California 90024 ALAN D .

GRINNELL

Department of Zoology, University of California Los Angeles, California 90024 MAX

HAMBURGH

Department of Anatomy, Albert Einstein College of Medicine and Department of Biology, The City College of New York Bronx, New York 10461 ROBERT M .

HERNDON

Johns Hopkins Hospital Baltimore, Maryland 21205 HARVEY

HERSCHMAN

Laboratory of Nuclear Medicine and Radiation Biology, and Department of Biological Chemistry, UCLA School of Medicine Los Angeles, California 90024 T E R R Y C . JOHNSON

Department of Microbiology, Northwestern University Chicago, Illinois 60611 IRWIN KOPIN

Laboratory of Clinical Sciences National Institute of Mental Health Bethesda, Maryland 20014 LAWRENCE

KRUGER

Department of Anatomy, UCLA School of Medicine Los Angeles, California 90024 ABEL LAJTHA

New York State Research Institute for Neurochemistry and Drug Addiction New York, New York 10035 JAN

LANGMAN

Department of Anatomy, University of Virginia Charlottesville, Virginia 22901

LOWELL W .

LAPHAM

Division of Neuropathology, The University of Rochester Rochester, New York 14620 RITA

LEVI-MONTALCINI

Department of Biology, Washington University St. Louis, Missouri 63130 and Laboratory of Cell Biology, Consiglio Nazionale delle Ricerche Rome, Italy FRANK

MARGOLIS

Roche Institute of Molecular Biology Nutley, New Jersey 07110 DAVID S .

MAXWELL

Department of Anatomy, UCLA School of Medicine Los Angeles, California 90024 JAMES MEAD

Department of Nuclear Medicine and Radiation Biology, UCLA School of Medicine Los Angeles, California 90024 BLAKE W .

MOORE

Department of Psychiatry, Washington University St. Louis, Missouri 63110 ENRICO

MUGNAINI

Department of Anatomy, Harvard Medical School Boston, Massachusetts 02115 ALAN P E T E R S

Department of Anatomy, Boston University Boston, Massachusetts 02118 EUGENE ROBERTS

Division of Neurosciences, City of Hope Medical Center Duarte, California 91010 SIDNEY R O B E R T S

Department of Biological Chemistry, UCLA School of Medicine Los Angeles, California 90024 E R I C SHOOTER

Departments of Genetics and Biochemistry Stanford University School of Medicine Stanford, California 94305

SILVIO VARON

Department of Biology, University of California, San Diego La Jolla, California 92037 J A M E S VAUGHN

Division of Neurosciences City of Hope National Medical Center Duarte, California 91010 JAMES A.

WESTON

Department of Biology, University of Oregon Eugene, Oregon 97403 FRED J .

WOLFGRAM

Department of Medicine, UCLA School of Medicine Los Angeles, California 90024 ARTHUR YUWILER

Neurobiochemical Laboratory, UCLA Department of Psychiatry Veterans Administration Center Los Angeles, California 90073 STEPHEN ZAMENHOF

Departments of Medical Microbiology and Immunology and Biological Chemistry Brain Research Institute and UCLA School of Medicine Los Angeles, California 90024 CLAIRE

ZOMZELY-NEURATH

Department of Biological Chemistry, UCLA School of Medicine Los Angeles, California 90024

PREFACE This volume results from a small and limited conference concerned with the developing nervous system, which was held in November, 1969 in Los Angeles. The conference was sponsored by the Brain Research Institute of the University of California at Los Angeles which has as one of its objectives a policy of promoting interdisciplinary symposia of appropriate interest. The Organizing Committee included Drs. Lawrence Kruger, David Maxwell, and myself, all having anatomical interests. Biochemical and metabolic approaches were represented on this committee by the inclusion of Drs. Sidney Roberts, Steven Zamenhof and Samuel Eiduson. It was our collective thought to limit our objectives severely, and our intent was a presentation and discussion of the morphological and biochemical information that has resulted particularly from the technical advances of recent years. This includes at one extreme electron microscopy, and at the other, the effective use of radioactive tracers to demark and follow metabolic events. By now, of course, there exists at least a narrow technical bridge across the full width of this spectrum in electron microscopic autoradiography. Radioisotopes are also being used to mark cell populations involved in morphological events and changes. Tissue and organ cultures provide models and specimens for the biochemists as well as for the morphologists. Cell components separated by differential centrifugation permit refined chemical analysis, yet also require and receive exact anatomical characterization. Interdisciplinary approaches to problems are not only fashionable today, but often productive and essential. It is difficult for any one individual to be fully knowledgable and competent in the diverse areas of potential interest. Thus, it seemed pertinent to bring morphologists and chemists together in a favorable environment for instructive talks, and for the free exchange of ideas and problems relating to developmental studies of nervous tissue. Thirty eight investigators attended this conference (a list of participants and their affiliation is to be found on page vii). Fifteen of these presented major reports on diverse topics. In addition there were short formal presentations concerned with related topics, and much informal discussion. The latter was recorded, and is reproduced here after appropriate editing. The financing of the Conference as well as the publication of this volume has been made possible by the sponsorship of the UCLA Forum in Medical Sciences. Dr. Victor E. Hall is Editor-in-Chief of the latter's publications. His staff includes Mrs. Judith Heller, assisted by Mrs. Martha Axelrod, who have ably handled the large editorial task of collating all of the manuscripts, transcripts, and corrections into one meaningful whole. It also includes Mrs. Linda Short who diligently took care of all of the organizational detail concerned with the arrangement and progress of the Conference, and later served as typist for the editorial revisions. The services of these three young ladies were invaluable to me.

I must also express more fully my dependence upon the Organizing Committee mentioned above. It was the Committee making individual suggestions, but acting as a whole, that decided upon the topics to be considered and the list of participants who would be most appropriate. Inevitably, of course, some who were invited were unable to attend. It was with regret, but with our limitations in mind, that we were unable to invite others. Daniel C. Pease

CONTENTS

MORPHOLOGICAL

ASPECTS

1. NEURAL C R E S T C E L L MIGRATION AND DIFFERENTIATION

1

James A. Weston 2 . E F F E C T S OF CORTISOL AND EPINEPHRINE ON G L I A L C E L L S IN C U L T U R E

23

Jean de Vellis, Diane Inglish and Frank Galey 3 . FORMATION AND MIGRATION OF NEURORLASTS

33

Jan Langman, Morimi Shimada and Cheryl Haden 4.

POSTNATAL D E V E L O P M E N T OF TETRAPLOID D N A CONTENT IN THE P U R K I N J E NEURON OF THE R A T :

A N ASPECT OF CELLULAR DIFFERENTIATION

61

L. W. Lapham, R. D. Lentz, D. J. Woodward, B. J. Hoffer and C. J. Herman 5 . DIFFERENTIATION OF THE NEURAL E L E M E N T S OF THE CEREBRAL C O R T E X IN THE R A T

73

DAVID W.

CALEY

6 . T H E MORPHOLOGY AND D E V E L O P M E N T OF NEUROGLIAL C E L L S

103

JAMES E. VAUGHN AND ALAN PETERS 7 . DEVELOPMENTAL ASPECTS OF SYNAPTOLOGY WITH SPECIAL E M P H A S I S UPON THE CEREBELLAR C O R T E X ENRICO

141

MUGNAINI

8 . T H E INTERACTION OF AXONAL AND DENDRITIC E L E M E N T S IN THE D E V E L O P ING AND THE M A T U R E SYNAPSE ROBERT M.

167

HERNDON

9 . STUDIES ON THE D E V E L O P M E N T OF THE AVIAN VISUAL S Y S T E M

177

W. MAXWELL COWAN 1 0 . T H E INVESTIGATION OF N E U R A L D E V E L O P M E N T BY E X P E R I M E N T A L IN VITRO TECHNIQUES

223

SILVIO S. VARON

METABOLIC

ASPECTS

1 1 . T W O CONTROL MECHANISMS OF G R O W T H AND DIFFERENTIATION OF THE SYMPATHETIC NERVOUS S Y S T E M

R. Levi-Montalcini and P. U. Angeletti

253

1 2 . T H E BIOLOGICAL ACTIVITIES OF T H E SUBUNITS OF T H E 7 S N E B V E G R O W T H FACTOR PROTEIN

269

E. M. Shooter and Silvio Varon 1 3 . BIOCHEMICAL E F F E C T S OF THYROID H O R M O N E S IN THE DEVELOPING B R A I N

273

R. Balazs 1 4 . T H E THYROID AS A T I M E CLOCK IN THE DEVELOPING NERVOUS S Y S T E M

321

Max Hamburgh, Lorenzo A. Mendoza, John F. Burkart and Franklin Weil 1 5 . H O R M O N A L AND NUTRITIONAL ASPECTS OF PRENATAL B R A I N D E V E L O P M E N T

Stephen Zamenhof and Edith Van Marthens

329

1 6 . T H E E F F E C T OF V I S U A L DEPRIVATION ON THE DEVELOPING AVIAN O P T I C LOBE

361

Stephen C. Bondy and Frank L. Margolis 1 7 . LIPIDS AND B R A I N D E V E L O P M E N T

365

1 8 . BIOGENIC A M I N E S IN T H E DEVELOPING B R A I N

391

A. N. Davison

Samuel Eiduson 1 9 . ALTERATIONS R E L A T E D TO THE C E R E B R A L F R E E A M I N O ACID P O O L D U R I N G DEVELOPMENT

419

Abel Lajtha and F. Piccoli 2 0 . D E V E L O P M E N T A L ALTERATIONS IN C E R E B R A L RIBONUCLEIC ACID AND P R O TEIN SYNTHESIS

447

Sidney Roberts, Claire E. Zomzely and Stephen C. Bondy 2 1 . REGULATORY M E C H A N I S M S R E S P O N S I B L E FOR ALTERATIONS IN PROTEIN AND NUCLEIC ACID SYNTHESIS IN DEVELOPING B R A I N T I S S U E

473

Terry C. Johnson 2 2 . LEVELS

OF T W O

BRAIN-SPECIFIC

PROTEINS DURING DEGENERATION

AND

DEVELOPMENT

481

Blake W. Moore, T. J. Cicero, V. J. Perez, and W. M. Cowan 23. S-100

PROTEIN

ACCUMULATION

IN

CULTURES

DEVELOPING

ANIMALS

AND

CELL 491

Harvey R. Herschman N A M E INDEX

499

S U B J E C T INDEX

503

1. NEURAL CREST CELL MIGRATION AND DIFFERENTIATION* J A M E S A. W E S T O N f Case-Western Reserve University Cleveland, Ohio

Few embryonic structures account for as much diversity in development as does the neural crest. This diversity is both morphogenetic and phenotypic in that there are a variety of patterns of distribution of crest cells in the embryo and numerous different cellular phenotypes that originate from the neural crest. The levels at which this diversity arises and is controlled will be considered in this paper. DIVERSITY IN NEURAL CREST DEVELOPMENT

Patterns of Distribution The neural crest is a transient structure in vertebrate embryos that is established as the edges of the folding medullary plate meet and fuse. In general, the crest forms in an anterior to posterior sequence along the length of the embryonic axis, and its cells emigrate along various rather precise pathways. The crest itself remains as a recognizable entity only as long as it takes for its constituent cells to emigrate from it and disperse in the embryo. In the chicken embryo at any given axial level, this emigration probably takes 12 to 15 hours ( 3 9 ) . When the major migration is completed, cells of the neural crest may be found distributed in at least three distinct topographical ways in different regions of the embryo, ( a ) Cells may be dispersed in or on epithelial surfaces such as epidermal sheets, the lining of the coelom or on various structures like blood vessels or the spinal cord, ( b ) Cells may be aggregated as cohesive structures "embedded" within a mesenchyme matrix, ( c ) Crest cells themselves may form a loose mesenchymatous tissue. The morphogenetic events that lead to these distributions, usually occur before the crest cells can be distinguished morphologically from other cells constituting their environment in the embryo ( 3 7 ) . Diversity of Phenotype Later in development, the crest cells diversify phenotypically by various overt cellular differentiations. Here the striking heterogeneity of crest deriv° This research supported in part by USPHS Grant No. HD-03477. I Present affiliation: Department of Biology, University of Oregon, Eugene, Oregon 97403.

1

2

N E U R A L

G R O W T H

AND

D I F F E R E N T I A T I O N

atives becomes apparent both regionally within the embryo, and at the level of tissues and individual cells (37). Thus, some cells which have associated with the ectoderm or the coelomic lining give rise to populations of pigment cells. Other crest cells enter the somitic mesenchyme and undergo nervous differentiation as sensory or autonomic ganglia. In addition, after suitable developmental interactions with endodermal components, cells that were distributed as mesenchyme in the head give rise to pharyngeal cartilages, to various components of the skull including its base and presumably some membrane bones, and also contribute to the formation of teeth (6, 15, 16, 37). The neural crest which arises at the trunk region of the embryo apparently differs from cranial crest in that it does not form skeletal elements, while cranial crest apparently does not give rise to an extensive pigment cell population as trunk crest does. Both trunk and cranial neural crest, however, form peripheral nervous structures including the cranial sensory and autonomic (parasympathetic) ganglia in the cranial region, and sensory and sympathetic ganglia and the adrenal medulla in the trunk region (10, 15, 17, 35, 37). Both cranial and trunk crest may also contribute some supporting elements of the nervous system (glia and neurilemmal cells), but the evidence on the precise origin of these cells is equivocal at the present time. These phenotypic contributions of the crest are summarized in Table 1.1. The Relationship between Distribution and Phenotypic

Expression

There is a general correlation between phenotypic expression and spatial arrangement of crest cells. Neuronal differentiation apparently proceeds only under conditions where neural crest cells are associated closely in cohesive clusters. Neural derivatives, for example, do not arise when cells are dispersed on surfaces or as mesenchyme. On the contrary, when cells are dispersed they seem often to form pigment. The crucial problem here is to determine whether precocious covert phenotypic differentiation leads to specific differences in crest cell distribution or vice versa. This is central to our understanding not only of the control of the precise morphogenetic movements which crest cells undergo, but also the role of the environment in affecting the course of cellular differentiation. A variety of experimental analyses have not yet satisfactorily established the extent to which individual crest cells are restricted to specific developmental fates before or during migration. Conclusions concerning the control of the precise pattern of migration of these cells, therefore, depend largely on unverified assumptions that are made about the degree of crest cell determination (10,13, 14,37). L E V E L S OF CONTROL OF

MIGRATION

Basically, there are two levels at which the precise distribution of neural

NEURAL

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crest cells might be regulated during embryogenesis. First, control might reside specifically within individual crest cells. Alternatively, more general environmental factors might affect the pattern of crest cell migration. The consequences of each of these alternatives are unique. Intrinsic Control If we assume, for example, that specific phenotypic differences exist in the neural crest population prior to the onset of migration, then control of the pattern of distribution could reside largely in the crest cells themselves. Such specifically determined cells presumably would follow highly specific and directional cues and would undergo a "directed migration." This suggests that one component of the phenotypic differences that could exist among neural crest cells might be some differential susceptibility to specific environmental cues. In addition, the embryonic environment itself would be assumed to provide specific directional and locational information to the migrating crest cells. This assumption has been made—both explicitly and implicitly—in the case of both neuroblasts and melanoblasts. Some migrating crest cells, for example, ultimately form tightly coherent aggregates which we recognize morphologically as ganglia. Within these ganglia, there ultimately appear several different cell types—including various kinds of neurons and neuroblasts, and also glial elements (12, 22). After the ganglion first forms, it undergoes a period of enlargement, and by eight days of incubation in the chick embryo, at least two distinct cell types can be recognized. These two populations of cells can be distinguished in terms of their size, their silver staining properties, their location in the ganglion, and various histochemical criteria (12, 22, 29). It has been suggested (10,13) that the formation of the ganglion is the consequence of the directed migration of two spatially distinct populations of neuroblasts from the crest, and that this migration could be an expression of selective affinity between the migrating cells and specific regions of the embryo where ganglia form, and also between the prospective ganglion cells themselves. Another case in which it has been implicitly assumed that some neural crest cells are specifically determined was reported by Peterson and Murray (25). They found that when young sensory ganglia are cultured in vitro, pigment cells occasionally appeared in the cultures. Numerous pigment cells appeared in cultures of 4-day ganglia, fewer appeared in cultured 5- and 6-day ganglia, and pigment cells were absent altogether in cultures of older ganglia. These authors suggested that the cells that produce pigment were melanoblasts moving past the region of the ganglion at the time that it was removed from the embryo. As in the previous example, it is assumed that some crest cells are specifically determined, in this case, as melanoblasts during or prior to migration. Our ability to establish experimentally what developmental restrictions

NEURAL CREST

DIFFERENTIATION

5

have been imposed on cells is rather limited. One standard procedure, however, has been to observe what phenotypes can be expressed when a particular tissue is explanted in vitro. When neural folds are cultured in vitro, the cultures give rise to many pigment cells and few undifferentiated mesenchyme cells (9, 23, 30, 41). Using this response as a criterion for developmental capabilities, we are led implicitly to the conclusion that many cells of the crest are already specifically determined either as pigment cells or as mesenchyme. If this be true, however, then the major component of the neural crest apparently will be prospective pigment cells—a conclusion which is not consistent with the observations on the various known derivatives of the crest in vivo. It seems more likely that culture conditions favor the appearance of certain phenotypes (8). If the two cell types that are usually found in vitro represent an initial cellular heterogeneity in the newly formed crest, however, we should again be particularly concerned to establish when and under what conditions such differences in developmental capability arose in the cells of the neural crest. This would be a primary consideration in the analysis of intrinsic control of crest cell migration. Extrinsic Control As a consequence of primary embryonic induction, some restrictions have clearly been imposed on the ectodermal cells that give rise to the neural crest (24, 26, 28, 37). As we have suggested, however, the experiments designed to determine the extent to which specific phenotypic differences exist in the population of neural crest cells have given ambiguous results. Thus, it is not impossible that neural crest cells retain a degree of developmental latitude prior to their migration. If this were the case, the precise distribution of crest cells in the embryo could not be caused by a directed migration in which determined cells respond to specific directional and locational signals. On the contrary, if crest cells were pluripotent, the idea of intrinsic control of the pattern of migration would be untenable simply because such cells would probably not be able to respond differentially to such directional and locational cues. The precise distribution of these cells, however, might conceivably be governed by factors in the embryonic environment which are relatively nonspecific, in the sense that all migrating crest cells would respond similarly. If this were so, it would be useful to establish how the embryonic environment itself might effect the precise migratory pathways of crest cells and hence the pattern of neural crest cell distribution. We must further ask whether these nonspecific factors alone are sufficient to account for the precise migration and localization of neural crest cells (37, 39). It seems appropriate, therefore, to consider exactly how much (or how little) specificity is sufficient to account for the precise pattern of distribution which we observe. To this end, we must decide whether initial crest cell migration is a random process or whether preferred pathways of migration exist. If there are preferred routes, we must determine how the environment

6

NEURAL

GROWTH

AND

DIFFERENTIATION

might impose limitations on the paths of migration. In addition, we must learn how the orientation and direction of crest cell movement is established. Finally, we must try to establish what causes the cells, which were once condensed as a coherent tissue in the crest, to begin migrating as single cells throughout the embryo, and how these cells might be caused to stop migrating and aggregate in new precise locations in the embryo. By introducing these questions, and suggesting how they might be answered, I hope to focus attention on the degree of specificity inherent in, and necessary for the control of crest cell migration. INITIAL PATTERN OF N E U R A L CREST C E L L MIGRATION I N T R U N K OF THE CHICK

THE

EMBRYO

The early migration of neural crest cells occurs before these cells can be distinguished from their environment; therefore, some marker must be applied so that these cells may be recognized and followed during their migration. Various markers have been used, and all have limitations which must be considered when the experiments are interpreted (36). We have, however, been able to follow the early movement of crest cells using tritiated thymidine as a cell specific marker (35). Using this marking procedure, labeled neural crest cells can be observed to migrate in two well-defined streams. A dorsallateral stream exists closely associated with the ectoderm near the mid-dorsal line. Most of the labeled cells that migrate along this pathway in the chick come to lie within the ectodermal layer itself and the distribution of these cells is more or less uniform and unsegmented in the ectoderm sheet. The second stream migrates ventrally into the somite. This stream, too, remains unsegmented until it enters the somitic mesenchyme. Cells appear to migrate further within somites than between them, and as early as 12 hours after grafting, some crest cells have already migrated precociously to the level of the dorsal aorta (See Figure 1.1). We can conclude, therefore, that migration seems to be facilitated within an organized cellular environment such as the ectoderm or somitic mesenchyme, and in the chick, at least, migration on surfaces of solid structures such as the neural tube seems to be rather limitedcontrary to what has previously been supposed (15). The fact that migration is favored only within a tissue environment suggests that the segmental distribution of crest cells might simply be caused by discontinuities in the environment into which these cells migrate. This idea can be tested directly since the labeling procedure allows us to identify migrating cells positively wherever they may be found. Thus, when grafts of labeled neural crest are implanted heterotopically in unsegmented mesenchyme of the lateral plate, the labeled cells can be seen to leave the crest, migrate away from their source, but remain essentially unsegmented. Similar uniform distributions are seen, of course, in the surface ectoderm. Whenever labeled neural crest grafts are placed so that the migrating cells will ultimately enter segmented mesoderm, distribution will be segmented even

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AGE (DAYS) Figure 13.3. Effect of T 3 -treatment on the average content of RNA per cell (DNA) in the cerebrum (a) and cerebellum (b). o—Normal and •—T 3 -treated rats. (From Balâzs et al., 16.)

simple chemical parameters in assessing the effects of abnormal conditions during brain development. It has recently been shown that the reduction in brain weight due to neonatal thyroidectomy is related to a decrease in the average cell size, rather than a reduction in cell numbers (12, 45, 53, 96). It was observed that the DNA content per unit weight of tissue increased, indicating that the cell territory was reduced. Furthermore, the amounts of RNA and protein per cell were also less than in controls (cf. Table 13.2). On the other hand, the small brain size in T3-treated rats resulted from a reduction in total cell number (cf. Section I), and neither the packing density of cells, nor the average cellular content of RNA and protein respectively were affected (15, 16) (Figure 13.3). Another factor which contributes to the reduction in brain weight in thyroid deficient rats compared with controls has recently been reported by Balazs et al. (13) and Walravens & Chase (124). The deposition of

282

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c O i_

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Figure 13.4. Effect of neonatal thyroidectomy on the deposition of myelin assessed by cholesterol content (in mg for g wet wt of brain) of isolated myelin. Cerebral myelin of 3 hypothyroid and normal 43day-old rats, respectively, was isolated by the method of Cuzner & Davison ( 2 9 ) and the mean and S.E. are indicated. Myelin at the other ages was separated as described by Balazs & Cocks ( 1 0 ) . The data were taken from Balazs etal., ( 1 3 ) .

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myelin is reduced in the brain of thyroid deficient rats. Figure 13.4 shows that the amount of cholesterol combined in the isolated myelin fraction of rat brain was significantly reduced after neonatal thyroidectomy during the whole period of rapid myelination ( 1 3 ) . Cerebrosides may also be used as markers for myelin. The concentration of cerebrosides in the rat brain was reduced by about 30 per cent even in the 35-day-old thyroid deficient rats, whereas the concentration of other lipids showed only a transient reduction at an earlier age ( 1 3 ) . The data indicated that the rapid deposition of myelin started at about the same time in the thyroid deficient rats as in the controls, and the difference was only quantitative. The rate of myelin deposition was most markedly reduced compared with controls in the third week of life (cf. Figure 13.4), and the observations of Walravens & Chase (124) may be related to this effect. These authors have found that the age of peak incorporation of 35S into cerebral lipids is shifted from about the 17th to 21st day of life in thyroid deficient rats (Figure 13.5). These authors have also observed that the in vitro activation of inorganic sulphate to adenosine 3'-phosphate, 5'-phosphosulphate was significantly reduced in the

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AGE ( D A Y S ) Figure 13.5, Effect of T 3 -Treatment and thyroid deficiency on the incorporation of 35S into cerebral lipids. The results at 17 days represent the means of two separate experiments (5 experimental and 5 control rats in each); the other points are the mean of 4-5 rats each. The data for T 3 -treatment were taken from Balazs et al. (18); o—normal, and •—T 3 -treated rats. The data for the thyroid deficient rats ( A ) were taken from Walravens & Chase (124); the age-curve for the normal animals was similar to that found by Balazs et al., (18), and was therefore not reproduced separately.

brain of thyroid deficient rats (Table 13.4) and they have suggested that this finding may account for the decrease in sulphatide synthesis. Hamburgh (59, 60) has recently observed an increased rate of myelination in cultures of cerebellar tissue in the presence of thyroxine. Slight increases in the concentration of cerebral cholesterol (108) and of phospholipids (92) have been reported in young rats treated with thyroxine. We have therefore, investigated the effect of T3-treatment in infancy on myelination in rat brain (18). Cerebrosides were used as myelin markers (Fig-

284

NEURAL

GROWTH

II

AND

15

DIFFERENTIATION

19

23

A G E (DAYS) Figure 13.6. Effect of T 3 -treatment on the concentration of cerebrosides in the brain, o—Normal, •—T3-treated rats. (From Balazs et al. 18.)

ure 13.6). When the data were subjected to analysis of variance, the curve for the deposition of cerebrosides in the brain in the experimental animals was not significantly different from that of the controls. However, it appeared that there was a transient increase in the cerebroside concentration in the brain of Ts-treated rats during the initial period of myelination, which was most marked at 17 days of age (133 per cent, P < 0.02). Davison & Gregson (30) have observed that the incorporation of radioactive sulphate into brain lipids displays characteristic age-dependence. In the rat a sharp peak is observed at 15-17 days of age, which coincides with the initial phase of rapid myelination in this species. The incorporation of 35S into brain lipids was used as an index by Chase, Dorsey and McKhann (25) in order to assess the effects of undernutrition on myelination. Figure 13.5 shows the effect of the thyroid state of the animal on this index. The data for the thyroid deficient animals were taken from Walravens & Chase (124) and for T3-treated rats from our own studies (18). In contrast to

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thyroid deficiency, thyroid treatment apparently did not affect significantly the pattern of 35S incorporation into sulphatides. The peak incorporation occurred at 17 days of age as in the controls. Since the amount of [35S]lipids in the brain of T3-treated rats was reduced by about 30 per cent compared with controls, a more detailed analysis was undertaken at this age (Table 1 3 . 3 ) . In agreement with previous reports ( 2 5 , 3 0 ) , more than 9 0 per cent of the [35S]lipids were recovered in the sulphatides isolated by thin-layer chromatography. The concentration of sulphatides was 34 per cent higher in the brain of T3-treated than of normal rats, and the specific radioactivity was reduced by 40 per cent. In these experiments the distribution of 35S at 2 4 hours after the injection of 3 0 m C / 1 0 0 g body weight was also studied. It was evident that the effect of T3-treatment was not confined to lipids only, since the total 35S content of the tissue was reduced to 73 per cent of the control value. [35S] Sulphatides accounted for approximately 30 per cent of the total 35S of the tissue in the brain of both the experimental and the control rats. There was an appreciable reduction in the amount of inorganic [35S]CV- after T3-treatment. The specific radioactivity of sulphatides when expressed on the basis of the concentration of inorganic [35S]Oi2~ was not significantly different from the control values. These results suggest, therefore, that T3-treatment affected the availability of labeled sulphate to the brain rather than the rate of sulphatide synthesis: but further work is required to determine the flux of inorganic sulphate into brain tissue and the kinetics of incorporation of 35S into sulphatides. The results indicate however, that the incorporation of 35S into brain lipids at different ages cannot be taken as conclusive evidence of the rate of sulphatide synthesis. Walravens & Chase (124) have reported that the incorporation of 35S into brain lipids was significantly increased at five days after birth as a result of daily injection of thyroxine. In view of the present findings these data do not necessarily indicate that myelination is advanced by the treatment. In summary: Single biochemical parameters can serve as useful indices of assessing the effects of thyroid state on cerebral growth and maturation. Thyroid deficiency results in a decrease in mean cell size, with reduced RNA and protein contents per cell, whereas T3-treatment apparently does not affect the mean size of cells, or the contents of cellular constituents, but leads to a reduction in total cell number. Myelination is quantitatively reduced in thyroid deficient rats, whereas it has not been shown convincingly whether or not thyroid treatment in infancy has an effect on myelination in vivo. There are a number of abnormal conditions known whose effect on brain development has recently been mapped in detail. Besides the thyroid state we have included in Table 13.2 corticosteroid treatment and undernutrition. This last condition may always complicate the effects of the other treatments. Although further studies are required the data indicate that the effects of undernutrition in infancy are different from those of thyroid deficiency;

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the former leads to a reduction in cell number but does not affect irreversibly the average cell size. Further differences between the two conditions are indicated by observations probably related to myelination. It has been reported that the peak of incorporation of 35S into brain lipids of undernourished suckling animals occurs at the same age as in the controls ( 2 5 ) whereas a significant retardation is observed in thyroidectomized rats ( 1 2 4 ) . Some effects of treatment with thyroid and corticosteroids in infancy, when investigated at an age when the central nervous system is already functionally mature, are similar to those of undernutrition. The similarities are the reduction in total cell number, without a permanent effect on the average cell size. A characteristic feature of the hormone treatments and undernutrition in infancy is the greater reduction in postnatal cell formation in the cerebellum compared with the cerebrum (67A). This effect is related to differences in the timing of the maturation of the two organs (3, 88, 116). A relatively greater vulnerability of the cerebellum has also been observed during exposure to a wide variety of insults during early life ( 5 ) . The results indicate that the effects of the different treatments must be followed in detail, especially during the rapid phase of brain maturation. It was previously shown that treatment with thyroid hormone affects postnatal cell formation at a different age from that with corticosteroid (Figure 13.1), although the final result of the two conditions may appear to be similar. I I I . E F F E C T OF THYROID STATE ON E N Z Y M I C ACTIVITIES

It is known that the maturation of the brain is associated with characteristic changes in enzymic activities (47, 6 5 ) . In the previous section, data were presented which suggested that the maturation of the brain is influenced by the thyroid state of the animal. Further evidence is provided by studies in which enzymic activities were determined during development in thyroid deficient or thyroid treated animals. Results of thyroid deficiency are summarized in Table 13.4; much less information is available about the effects of thyroid treatment in infancy. In the adult, cerebral respiration rate does not appear to be influenced by the thyroid state (83, 113). In contrast, an abnormal thyroid state in infancy leads to significant changes in oxygen consumption when studied in excised brain tissue; thyroid treatment accelerates the age-dependent increase in respiration rate ( 4 6 ) , whereas thyroid deficiency results in significant retardation ( 6 0 ) . The results of further analysis of this effect ( 1 2 ) indicated that thyroid deficiency selectively influences the developmental increase in the activity of enzymes which seem to be concentrated in a fraction of brain homogenates containing detached nerve terminals (31, 125). Succinate dehydrogenase is a mitochondrial enzyme which is relatively concentrated in the mitochondria recovered in the synaptosomal fraction (9, 107), and the activity of this enzyme markedly increases with brain maturation (57, 102). The activity of this enzyme is strongly reduced

288

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DIFFERENTIATION 13.4

E F F E C T O F T H Y R O I D D E F I C I E N C Y I N I N F A N C Y ON T H E A C T I V I T Y O F S O M E E N Z Y M E S IN D E V E L O P I N G B R A I N T I S S U E

Effect f Enzymes associated with respiratory activity Overall r e s p i r a t i o n r a t e {in vitro) K + - s t i m u l a t e d respiration r a t e Succinate dehydrogenase C y t o c h r o m e oxidase Glutamate dehydrogenase

— — —

none none

Glycolytic enzymes: Enzymes associated with amino acid metabolism: Aspartate aminotransferase

none

Alanine a m i n o t r a n s f e r a s e Enzymes associated with the maintenance of ionic gradients Enzymes associated with the metabolism of putative transmitter compounds: G l u t a m a t e decarboxylase

none

Acetyl Cholinesterase Miscellaneous E n z y m e s involved in s u l p h a t e m e t a b o lism: Galactolipid s u l p h o t r a n s f e r a s e S u l p h a t e a c t i v a t i o n (sulphurylase p l u s Phosphokinase) Aspartate carbamoyltransferase

—

—

—

C o m m e n t s a n d References*

C e r e b r a l cortex slice (1) S t i m u l a t i o n r e t a r d e d (2) (3, 4); e n z y m e relatively c o n c e n t r a t e d in s y n a p t o s o m a l m i t o c h o n d r i a {5, 6) (3, 4) (7); e n z y m e relatively c o n c e n t r a t e d in n o n s y n a p t o s o m a l m i t o c h o n d r i a (5, (?) Aldolase {3); l a c t a t e d e h y d r o g e n a s e (7)

Only l a t e n t a c t i v i t y affected (8), which is associated w i t h m i t o c h o n d r i a a n d s y n a p t o s o m e s ; d i s t r i b u t i o n of a c t i v i t y on sucrose g r a d i e n t similar to t h a t of s u c c i n a t e d e h y d r o g e n a s e (6) (7) N a + - K + - A T P a s e a c t i v i t y specifically r e d u c e d {4, 9)

{4, 7); e n z y m e relatively c o n c e n t r a t e d in s y n a p t o p l a s m (5, 6) OD-t

none (10) —

+

I n c o n t r a s t t o t h e e n z y m e s listed before, t h e a c t i v i t y decreases w i t h b r a i n m a t u r a t i o n ; t h e age curve i n d i c a t e s ret a r d a t i o n in b r a i n m a t u r a t i o n (8)

* References: (1) H a m b u r g h (60); (¿8) G h i t t o n i & Gómez (56); (3) H a m b u r g h & F l e x n e r (61); (4) G a r c í a Argíz, P a s q u i n i , K a p l ú n & Gómez (51); (5) Salganicoff & D e R o b e r t i s (107); ¡6) Balázs, D a h l & H a r w o o d (9); (7) Balázs e t al. (12); (8) P a s q u i n i e t al. (96); (9) V a l c a n a & T i m i r a s (117); {10) W a l r a v e n s & C h a s e (124); Geel & T i m i r a s (53A). t R e d u c t i o n a n d increase in enzymic activities c o m p a r e d w i t h controls a r e i n d i c a t e d w i t h — a n d + respectively. t Cholinesterase activities are also reduced. A decrease in t o t a l Cholinesterase a c t i v i t y was previously observed b y H a m b u r g h & Flesner (61).

after neonatal thyroidectomy (61). In contrast, there is no change in the activity of glutamate dehydrogenase (12), an enzyme which is relatively concentrated in the nonsynaptosomal mitochondria (9, 94, 107). Eayrs (35, 36) has shown that the axonal density and dendritic ramification are reduced after neonatal thyroidectomy. It has been proposed by Balázs et al. (12, 15) that

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the significant reduction in synaptosomal marker enzymes such as succinate dehydrogenase (synaptosomal mitochondria) and glutamate decarboxylase (synaptoplasm) indicates a retardation in formation of nerve terminals. There is evidence that a high proportion of cerebral oxygen uptake is due to the metabolic activity of neuronal processes (cf. Section IV). Thyroid treatment in infancy advances brain maturation (cf. Sections IV and V), and it has been claimed that the formation of dendritic spines is also accelerated under these conditions (109). The effects of the thyroid state in infancy on cerebral oxygen uptake are therefore consistent with a selective influence of thyroid hormone on the development of neuronal processes. The substantial reduction in the activity of Na+-K+-ATPase observed by Garcia Argiz et al. (51) and by Valcana & Timiras (117) is consistent with this explanation. Thyroid hormones affect the maturation of the cells in the brain, and thus are involved in a series of reactions leading to structural, functional as well as biochemical changes. Their action in the developing brain is different from that in target organs for thyroid hormone in the adult animal. This is indicated, for example, by the observation that the activity of a-glycerophosphate dehydrogenase, an enzyme which is induced by thyroid hormone in the liver, is unaffected in brain tissue (111). The activity of enzymes utilizing pyridoxal phosphate as cofactor has been shown to be influenced in liver by the thyroid state of the animal, but thyroidectomy in adult animals has no effect on the activity of glutamate decarboxylase in brain (20). I V . EFFECT OF THYROID HORMONE ON THE COORDINATION OF METABOLIC PATHWAYS RELATED TO CEREBRAL ENERGY METABOLISM: CONVERSION OF GLUCOSE-CARBON INTO A M I N O ACIDS IN BRAIN TISSUE

The maturation of nervous tissues involves not only quantitative changes, such as changes in enzyme activities and deposition of substances, but it also involves important qualitative changes. During maturation the metabolic pattern of the undifferentiated cells is altered in a way which results in the development of the unique coordination of the different metabolic pathways which is characteristic of the differentiated adult cell. The change in metabolic pattern can be used to assess the effects of thyroid hormone on the maturation of nervous tissues. Glucose is the main substrate of cerebral energy metabolism under normal conditions [cf. Vrba et al. (122), for review Balazs (77)]. It has been shown that in the brain of the adult rat already at 20 minutes after the administration of [U-14C] glucose, 50-70 per cent of the total 14C has been converted into glutamate and other amino acids associated with the tricarboxylic acid cycle. The biochemical mechanisms underlying this phenomenon are related to the rapid breakdown of blood-born glucose in brain tissue into the initial intermediates of the tricarboxylic acid cycle

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Figure 13.7. Conversion of glucosecarbon into brain amino acids at 20 min after the i.p. injection of 10-20 HC of [U- 14 C]-glucose per 100 g body wt. The amino acids of the acid soluble fraction of brain tissue were separated from neutral compounds and carboxylic acids by ion exchange chromatography (See Gaitonde & Richter, 50). The 14 C combined in amino acids is expressed in percentage of the 14 C content of the acid soluble extract ( ± S.E. indicated), o—Controls, • —neonatal thyroidectomy, and A—T3-treatment. (From Cocks et al. 27.)

AGE (DAYS)

(a-oxoglutarate) with relatively little dilution with unlabelled carbon on the way (7, 8, 50, 62). The factors involve the relatively small glucose and glycogen pools in the brain, the rapid and predominantly one-directional flow of glucose-carbon in glycolysis, the relatively small contribution of non-glucose substrates to the total oxidation, and the relatively large glutamate pool. The specific distribution pattern of glucose-carbon reflects the final coordination of pathways of energy metabolism characteristic of the adult brain [cf. Balazs (7)]. Gaitonde and Richter (50) have shown that the pattern of glucose metabolism characteristic of the adult brain develops during the period of functional maturation. We have recently confirmed these results (26, 27) and found that at 20 minutes after injection of [U-14C]glucose in 10-day-old rats only about 20 per cent of the 14C in the brain is combined in amino acids whereas at the age of 19 days the value is approximately 60 per cent which is similar to the adult value (Figure 13.7). The developmental features which are probably associated with this phenomenon were analysed by Cocks etal. (27). (i) The rate of glucose utilization is low in the immature brain in vitro (64). The potential activity of the mitochondrial enzyme systems involved in the final oxidation of substrates is known to increase with maturation, and the developmental curves are similar to those for the conversion of glucose-carbon into amino acids (51, 57, 102). Lowry, Passonneau, Hasselberger and Schultz (78)

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have estimated that the glucose flux in brain tissue in vivo is about twice as high in the adult as in the immature mouse brain. (ii) Oxidation of substrates other than glucose is relatively more important in the immature than in the adult brain. This is indicated by the observation that the specific radioactivity of glutamate, which is related to the rate of entry of glucose-carbon into the tricarboxylic acid cycle, is about five times higher in the brain of 19day-old than of 10-day-old rats (Table 13.5). Since the glucose flux in the immature brain is half of that in the adult (78) these results indicate that the rate of glucose utilization is not the only factor involved in the limited conversion of glucose-carbon into amino acids. A dilution of glucose-carbon may take place at the level of acetyl-CoA as a result of oxidation of non-glucose substrates. There is also other evidence which points to a bigger contribution of these substrates to total oxidation in the immature compared with the adult brain. Ketone bodies (68, 71), amino acids (97, 99, 118) and probably fatty acids (118) are utilized at at least the same rate in the young as in the adult brain, while the oxidation of glucose is markedly lower. (iii) The cerebral content of glutamate per unit weight doubles in the period from 10 to 19 days of age (cf. also references 2, 19, 121, 123, Figure 13.8 and Patel & Balazs (99). Since the glutamate pool constitutes the most important single "trap" for the metabolized glucose-carbon in the brain (8, 62), the increase in glutamate concentration may contribute to the change in the distribution pattern of 14C with maturation. It seems from the previous discussion that the maturation of cerebral energy metabolism is characterized by both quantitative and qualitative changes: the flux through the tricarboxylic acid cycle is at least doubled, and the capacity for using a wide range of substrates is replaced by predominant (although not exclusive, cf. next section) utilization of glucose. These biochemical changes occur in the same period in which the rapid growth of' neuronal processes takes place (1, 42, 69). There is independent evidence for their inter-relationship. Quantitative ultrastructural studies have shown that the mitochondrial density is the highest in terminal dendrites and presynaptic endings (93). Furthermore, it has been demonstrated that the greater part of the energy-yielding metabolism is associated with the dendrites rather than with the cell bodies of neurons or with white matter (77, 84, 105). As a result of the development of the metabolic compartment associated with neuronal processes, the apparently homogenous metabolic pattern of brain tissue is replaced by the heterogenous pattern characteristic of the adult: this will be discussed in the following section. It may be mentioned here that there is evidence that the new metabolic compartment developed during maturation is associated with a glutamate pool in which

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Cb)

AGE (DAYS) Figure 13.8. Effects of thyroid state on the postnatal change in the concentration of some amino acids in brain. The amino acids were separated by ion exchange chromatography. a: T3-treatment. b: Neonatal thyroidectomy, o—Glutamate; A—glutamine; • — aspartate. The closed symbols represent the experimental animals and the open symbols littermate controls. Values from a different experimental series by Cocks et al., (27) are indicated ( V ) . The other data taken from Patel & Balazs ( 1 0 0 ) .

glutamate is labeled through the oxidation of [14C]glucose more than through oxidation of other substrates ["large" glutamate pool (17, 23, 52, 79, 119)]. There is evidence that the "large" glutamate pool is not homogeneous but contains a "sub-pool" which is apparently identified with the formation of the "synaptic" pool of GABA (17, 79). The present results indicate that a glutamate "sub-pool" of similar characteristics becomes apparent during the same period during which the adult pattern of cerebral energy metabolism is acquired. The heterogeneity of the glutamate pool is indicated again by the observations of Minard & Mushahwar (90) who showed that, after administration of [ 14 C] glucose in the adult rat, the GABA/glutamate specific radioactivity ratio, which is relatively low in vivo, increases rapidly post mortem in the brain. This means that the GABA formed under these conditions must come from a pool of glutamate whose specific radioactivity is relatively high. In the present experiments when post mortem changes were allowed to proceed in the brain of 10-day-old rats, the GABA/glutamate specific radioactivity ratio was less than 0.5, whereas at 19 days of age it

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was about 1.0 [the in vivo value at that age is about 0.4 (90)] (Table 13.5). The thyroid state of the experimental animal had a marked effect on the biochemical maturation of the brain as indicated by the fate of [14C] glucose in the brain (Figure 13.7). The age-curve for the conversion of glucosecarbon into brain amino acids was displaced to the left after T 3 -treatment, which indicates that the development of the pattern of cerebral energy metabolism characteristic of the adult was accelerated under these conditions. Neonatal thyroidectomy resulted in a significant reduction in the conversion of glucose-carbon into amino acids; at 19 days of age this index showed a maturational state equivalent to that of a 12-13-day-old rat. Since the whole period of brain maturation assessed by the index used covers a period of nine days, the results show that the retardation due to thyroid deficiency is a profound effect. More detailed analysis of the effect of the thyroid hormone was confined to a comparison of the "immature" with the "mature" brain (10 and 19 days of age respectively) (Table 13.5). The 14C content of the acid-soluble fraction, which contains about 95 per cent of the total !4C in the tissue, in 19 day-old normal rats was approximately twice the value observed at 10 days of age (Table 13.5). The difference was mainly due to the amount of 14C conserved in amino acids, since the " C content of the fraction containing glucose and carboxylic acids did not change much. The glutamate concentration was about 60 per cent higher at 19 than at 10 days, but the specific radioactivity of glutamate increased about five fold. At 10 days of age thyroid treatment had no significant effect on the concentration of glutamate (cf. also Figure 13.8), but the specific radioactvity of glutamate was twice the control value. In the 19-day-old thvroid deficient animals the specific radioactivity of glutamate was only 30 per cent of that in the controls although the glutamate concentration was 83 per cent. It is also shown in Table 13.5 that the GABA/glutamate specific radioactivity ratio was significantly lower than the control values in the brain of 19-day-old thyroid deficient animals. These results may be taken to indicate that after neonatal thyroidectomy the development of the "sub-pool" of glutamate associated with GABA formation is also retarded. Eayrs (36) has shown that thyroid deficiency in infancy leads to structural changes in the central nervous system, including a retardation of the formation of dendrites [cf. also Legrand (76) and Hamburgh (60).] Circumstantial evidence has previously been presented that the formation of nerve terminals is retarded under these conditions (12). It has also been indicated that the formation of the dendritic spines of pyramidal cells in the cerebral cortex is accelerated after thyroid treatment during development (109). The morphological observations suggest, therefore, that the formation of neuronal processes is influenced by thyroid hormone. The conclusions derived from the present biochemical studies are similar. The effect of the thyroid state on the morphological and biochemical maturation

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of brain tissue thus gives further support to the proposal that the metabolic compartment which is characterized by a high rate of glucose utilization and by the predominance of glucose as the substrate of oxidation is related to dendrites and nerve terminals (99, 27). V . E F F E C T OF THYROID STATE ON THE METABOLIC C O M P A B T M E N T A T I O N OF G L U T A M A T E

The developmental index described in Section IV may be considered as reflecting one aspect of the biochemical processes associated with the formation of neuronal structures. Another index appears to be related to maturational changes in the glia-neuronal relations. It was observed by Waelsch's group that at a short time after the intracisternal injection of glutamate the specific radioactivity of glutamine was higher than that of glutamate (22, 23, 119). Since glutamine is formed from glutamate, these findings indicate that a small and highly active pool of glutamate is the precursor of glutamine; that is, glutamate is compartmented in brain tissue. The original observations have been confirmed and extended in different laboratories including our own (17, 79, 80). It is now known that metabolic compartmentation can be demonstrated by the use of a number of different labeled metabolites, which are metabolized in the tricarboxylic acid cycle before the glutamate becomes labeled (reviews 23, 119). It follows that besides glutamate, compartmentation involves the complex enzyme systems associated with the mitochondria. Berl (21) has shown that the metabolic compartmentation of glutamate develops during the period of maturation in the cat brain; the development of metabolic compartmentation of glutamate has also been studied in the rat brain (97, 99). [U-14C]Leucine is one of the substrates which shows metabolic compartmentation in the brain of adult animals (cf. Roberts & Morelos 104); that is, after administration the specific radioactivity of glutamine relative to glutamate is greater than 1. In the brain [U-14C] leucine is converted into [14C]acetyl-CoA which enters the tricarboxylic acid cycle and subsequently labels the amino acids. In Figure 13.9 the specific radioactivity of glutamine relative to glutamate (R.S.A.) in the brain at 10 minutes after subcutaneous injection of 10 pC of [U-"C] leucine per 100 g body weight is plotted as a function of the age of the animal (97, 99). Figure 13.9 shows that metabolic compartmentation develops during the same period of maturation as does the rapid conversion of glucose carbon into amino acids (27, 50) (Figure 13.7). This observation facilitated the understanding of metabolic compartmentation in brain tissue. Metabolic compartmentation implies that the fate of a marker substance in a sizeable fraction of the tissue is significantly different from that in the bulk of the tissue. What do we know about the characteristics of the compartment in which the fate of [ ,4 C]leucine differs from that in the bulk of the brain tissue? (i) The pool size of glutamate in that compartment

NEURAL GROWTH AND

10

DIFFERENTIATION

15 20 AGE (DAYS)

25

Figure 13.9. Effect of thyroid state on metabolic compartmentation of glutamate in brain tissue. The ordinate represents the specific radioactivity of glutamine relative to that of glutamate at 10 min. after s.c. injection of 10 fiC of [U- 14 C]-leucine per 100 g body wt. Normal, T 3 -treated, and - • • - thyroid deficient animals. (From Patel & Balazs, 100.)

must be small, since otherwise the oxidation of [U-14C] leucine would not lead to a high glutamine/glutamate specific radioactivity ratio. The metabolism of a number of other labeled metabolites results in similar high ratios, and it has been shown that a population of brain mitochondria is enriched in most of the enzymes essential for the metabolism of the "compartmentation" substrates (9, 94, 107, 120). It follows that these substances are probably utilized at a common site, (ii) The compartment associated with the "small" glutamate pool is characterized by the ability to utilize a wide range of substrates for oxidation, including fatty acids and amino acids, (iii) The relation of the "small" glutamate pool to GABA differs from that to glutamine, since the GABA/glutamate specific radioactivity ratio was found to

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be low throughout development. ( iv ) The observations of Machiyama et al. (80) have indicated that the rate of oxidative reactions relative to glutamine synthesis is low in the "small" compartment. The data of Nicklas et al. (95) and Van den Berg et al. (120) may be taken to suggest that the rate of the tricarboxylic acid cycle associated with the small glutamate pool is relatively low. However, other interpretations of the data are also possible, including the continuous removal of glutamine from the "small" compartment. Computer simulation of experimental results from different laboratories indicated that the flux in the "small" tricarboxylic acid cycle is only about 30 per cent of the total flux ( 52A ). There are apparent similarities, therefore, between the metabolic properties of the immature brain and the "small" compartment in which "compartmentation" substrates label glutamate. These include the wide substrate spectrum for oxidation and the relatively low rate of oxidative reactions. In the previous section, the properties of the "large" metabolic compartment were described; these include the restriction to glucose as the main oxidative substrate and the high rate of oxidative reactions. The evidence which was summarized showed that the "large" metabolic compartment develops during maturation. These observations therefore agree with the view that the apparently homogenous metabolic pattern of the tissue changes as a result of the expansion of a compartment (neuronal processes) whose metabolic pattern is both quantitatively and qualitatively different from that characteristic of the immature tissue. Hence, although the experimental results show that maturation results in a "small" compartment becoming functionally manifested, the primary cause is the development of the "large" metabolic compartment. The analysis of the fate of [1- 14 C]GABA and [1-"C]-glutamate in adult brain tissue led to the conclusion that the "small" compartment cannot be associated with presynaptic endings and that it is probably not related to terminal dendrites (17, 80). This view is supported by the present studies which indicate that neuronal processes are associated with the "large compartment (see section IV and reference 27). It was previously suggested that the "small" metabolic compartment involves the glial cells (17, 80), and the present results are compatible with this view. The development of glia does not correlate with the maturation of metabolic compartmentation and this is to be expected if the manifestation of metabolic compartmentation results from the growth of neuronal structures. Glial cells can divide even in the adult brain (112); thus, these cells are probably less specialized than the neurons and they may be expected to have the ability to utilize a wide range of substrates. The "small" compartment ought to be associated with a relatively small glutamate pool, and there is evidence indicating that the concentration of glutamate in the glia is less than the mean value in the tissue (87). It has also been reported that within a fraction which is isolated from brain dispersions and contains glial cells

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and detached neuronal processes, the glutamine-glutamate specific radioactivity ratio is as high as it is in the initial suspension at short times after the administration of a "compartmentation" substrate, [14C] glutamate (106). It has recently been suggested that a model consisting of a minimum of three compartments is compatible with the experimental data (17). Each "compartment" probably results from the summation of a number of different units with more or less similar metabolic patterns. The combination constituting what is defined on a functional basis as a "small" or a "large" compartment will depend on the site of entry and metabolism of the substrate considered. The present studies ("compartmentation" substrate [U-14C]-leucine) suggest that the following morphological structures may be allocated to the three main compartments hitherto identified (the list is clearly incomplete): "small" consisting of (I) glial cells; "large" consisting of (II) nerve terminals, and (III) terminal dendrites (neuronal cell bodies?). The effect of thyroid state on the development of metabolic compartmentation has recently been investigated (98, 100). Figure 13.9 shows that the development of the metabolic heterogeneity of brain tissue was advanced by Ts-treatment and it was retarded after neonatal thyroidectomy. The effects of these experimental conditions were therefore similar to those observed when the development of the organization required for conversion of glucose carbon into amino acids was studied (Figure 13.7). The underlying mechanisms are probably identical; thyroid hormone plays a part in regulating the processes of cell differentiation, and especially the maturation of nerve cells. It is of interest that the marked effects of thyroid hormone on the maturation of brain tissue influenced only slightly the development of the amino acid profile in the brain. It has previously been reported that the concentrations of glutamate, aspartate, GABA and Nacetylasparate are reduced in the brain of thyroid deficient rats whereas the age-dependent decrease in taurine is retarded (86, 91, 103). Similar results were obtained in the present studies (27, 98) (Figure 13.8). It should be emphasized that the developmental changes in the amino acid content do take place under these conditions, and the concentration of glutamate does not differ significantly from controls at 25 days of age, when the development of the compartmentation of glutamate is strongly retarded (Figure 13.9). The age curves for both the conversion of glucose carbon into amino acids and the compartmentation of glutamate showed advancement after Ts-treatment, although the concentrations of the amino acids studied were little affected. It seems that the steady-state concentration of a tissue constituent is not necessarily a sensitive marker for metabolic changes in a tissue, although the particular constituent studied may show a well defined maturational curve. On the other hand, markers which reflect the dynamic properties of the tissue, such as the two introduced in Sections IV and V, are sensitive indices of brain development. Furthermore, these results suggest

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that the age-dependent increase of overall glutamate content is not intimately associated with the metabolic processes related to the development of the phenomena associated with these two indices. V I . PERIOD OF BRAIN D E V E L O P M E N T CHARACTERIZED BY SENSITIVITY TO T H Y R O I D H O R M O N E S : I S T H E A C T I O N D I R E C T OR I N D I R E C T ?

There is evidence indicating that the action of thyroid hormone on the developing brain differs from that on the adult brain. It is generally agreed that thyroidectomy performed on the adult rat is without significant effect on the capacity for learning whereas the performance of rats made athyroid at birth is considerably below that of littermates in close-field tests (41). It has already been mentioned that similar age dependence was observed when the oxygen consumption of brain tissue was studied (Section III). Again, it has been reported (54) and confirmed in the present studies (11, 15), that the rate of cerebral protein synthesis is reduced after neonatal thyroidectomy whereas in the adult this is apparently not affected by thyroid deficiency (15, 89). The observations of Gelber et al. (55) support the view that thyroid hormones influence protein synthesis only in the immature brain. There appears to be a limited period of brain differentiation during which the tissue is thyroid sensitive (37). In the rat the manifestations of thyroid deficiency in terms of changes in behaviour (39) and in neurochemical maturation (60, 61) are reversed only when replacement therapy is started before the 10th postnatal day of life. Thyroid treatment leads to permanent effects in terms of adaptive behaviour and the structure of the pituitary gland only if started before the 14th day after birth (40, 43). The observations from different laboratories suggest therefore that the thyroid is an important factor regulating cerebral development during a limited period extending from birth to the 10-14th day of life. Interdisciplinary studies indicate that this period may be characterized as a preparation of the system for extensive differentiation of the nerve cells (65, 81). It appears, therefore, that thyroid hormones act primarily in the regulation of these preparatory' events. It is not yet understood whether thyroid hormones affect brain development in a direct or indirect way. It has been shown that thyroid hormones are taken up by brain tissue, and some regions which selectively accumulate them have been identified (49). It has also been observed that the accumulation of the hormones is apparently related to the thyroid state of the animal (49). A direct action of thyroid hormones on the cells in the central nervous system is, therefore, possible. The possibility that the symptoms observed in neonatal thyroid deprivation result from aspects of the experimental procedure other than those related to thyroid deficiency can be excluded, since all the symptoms investigated until now are reversed by remedial treatment when started at

300

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the right time (13, 41, 54, 75). Undernutrition and hypothermia (60), however, are sometimes complicating factors, and their involvement is not excluded by the success of replacement treatment. However, there is evidence which indicates that there are apparent differences in the effects of undernutrition and thyroid deficiency both on the developmental pattern of brain constituents (cf. Section I I ) and on behavior (44). Secondary effects due to the influence of the thyroid state on the maturation of the neuroendocrine system as a whole may play an important part in the symptomatology of both thyroid deficiency and T 3 -treatment in infancy. Eayrs & Holmes (43) have shown that T 3 -treatment in infancy leads to a marked change in the size and structure of the anterior pituitary. It has therefore been investigated whether or not the effects of thyroid deficiency can be influenced by hormones other than thyroid. The effects of growth hormone have been studied in detail, but the results are still controversial. Eayrs (38) and Hamburgh (60) observed only slight improvement after the administration of growth hormone to hypothyroid animals in the parameters investigated, whereas Krawiec et al. (75) claimed a reversal similar in many respects to that observed after thyroid treatment. Since the hypothalamo-hypophysial system may be involved in the efects of thyroid dysfunction we have investigated the effects of T 3 -treatment on some aspects of the biochemical development of the hypothalamus and pituitary gland (73). These studies are still in progress, and therefore only the results obtained on the incorporation of [ 1 4 C] leucine into protein will be described. Figure 13.10 shows that the age curve of protein concentration in the hypothalamus is similar to that obtained previously for the cerebral cortex and for the whole cerebrum (15). The specific radioactivity of protein determined at 30 minutes after the subcutaneous injection of 20 |jC/100 g body weight of [1- 14 C] leucine was significantly reduced by T 3 treatment except at 50 days of age. When the values were expressed on the basis of the concentration of free [1- 14 C]leucine (R.S.A.) it was observed that the apparent incorporation rate reached a peak in the hypothalamus of normal rats at about 21 days of age. The age curve for the T 3 -treated rats was displaced towards the left: the peak incorporation occurred at 14 days of age and was followed by a premature fall. At 21-28 days of age the R.S.A. determined in the hypothalamus of T 3 -treated rats was similar to that observed in 50-day-old controls. As a consequence the rate of [ 1 4 C] leucine incorporation compared to controls was reduced by about 30 per cent in the period between 21 and 35 days. The apparent rate of protein synthesis was also affected in the pituitary of T 3 -treated rats (73). The specific radioactivity of protein was significantly reduced after T 3 -treatment during the whole experimental period (7 to 50 days of age). A permanent effect on protein metabolism was indicated by the observation that the R.S.A. of protein was about 65 per cent of the control value in the period extending from 35 to 50 days. These observations indicate that T 3 -treatment in infancy may affect the whole neuro-

T H Y R O I D H O R M O N E AND T H E

DEVELOPING

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301

PROTEIN (mg./g)

HYPOTHALAMUS

100r 9080-

7060-

50403020'— PROTEIN CRSA.ond S.A.) 9

8

m

/s-

' V '

7

6

•=CONTROL • = T5

R

5 4 3 2 t

AGE (DAYS) 7 ^ANIMALS

R S.A. x10 2

^

7/?

4

•

14

21

12/g

12/9

*

28 5/5

S.A. -3 x 10

•

35

50

5/5

5/5

Figure 13.10. Concentration of protein (upper graphs) and incorporation of [1- 14 C] leucine into protein (lower graphs) in the hypothalamus of T 3 -treated rats during development. In the lower graphs S.A. stands for the specific radioactivity (d.p.m./mg), and R.S.A. for the relative specific radioactivity of protein (S.A. per 14 C content of the acid soluble fraction in d.p.m./g wet wt.). Significant differences compared with controls are indicated by stars (upper graph, and S.A. in lower graphs), and by crosses. The data were taken from Kovacs et al. (73).

endocrine organization as a result of permanent changes in the hypothalamopituitary system. CONCLUSIONS

The effects of thyroid hormones on the developing brain have been summarized in Table 13.6. ( 1 ) THYROID D E F I C I E N C Y . The growth of the brain is reduced and this effect is evident mainly after the 14th day of life ( 1 2 ) . The smaller brainweight is not due to a reduction in the total number of cells (12, 53, 9 6 ) , although the rate of cell formation is apparently lower than in normals, especially in the

302

NEURAL

G R O W T H AND T A B L E

DIFFERENTIATION 13.6

E F F E C T S O F T H Y R O I D H O R M O N E S ON T H E D E V E L O P I N G

Thyroid Administration Brain weight Postnatal cell formation Cell size: (a) Neurons branching of dendrites formation of dendritic spines enzymes related to synaptosomes (b) Glia myelination R a t e of protein synthesis R a t e of nucleic acid synthesis: (a) D N A (b) R N A Organisation of metabolic reactions characteristic of the adult: (a) Energy metabolism (b) Development of "glia-neuronal relations" Neuroendocrine organization

Behavior: (a) Innately organized (b) Adaptive behavior

behavior

Reduced Premature termination No effect

* Accelerated —

(Advanced?) Advanced maturation (hypothalamus) Initial reduction and premature decline —

BRAIN

Thyroid Deficiency Reduced Delay (cerebellum) Reduced Reduced —

Reduced Reduced Reduced

—

No effect

Advanced

Retarded

Advanced Degranulation of acidophil cells and reduction of rate of protein synthesis in pituitary gland

Retarded

Advanced Impaired

Retarded Impaired

* — indicates observations not yet available.

cerebellum (12). However, the final cell number is comparable to that in controls, as a result of the prolonged presence of the external granular layer of the cerebellar cortex (60, 76). It is therefore probable that the thyroid is involved in regulating the rate of cell division and/or cell migration. The brain, besides being reduced in size, is changed in shape as a result of a differential effect of thyroid deficiency on the ossification of the base of the skull and the calvarium (45). The pattern of cerebral vascularity is also modified (34). Morphological and biochemical evidence both show that the smaller brain weight results from a decrease in average cell size, which is related to an effect on the development of the neurons, although glial cells are probably also involved. Both the axonal and dendritic components of the neuropil are reduced (15, 36, 76), and consequently the probability of axodendritic interaction between different neurons is markedly diminished (36, 41). A reduction in the number of nerve terminals is indicated by a selective decrease in the activity of enzymes associated with synaptic structures (12). That glial metabolism may also be influenced by thyroid deficiency is shown by the reduction in myelination (13, 124).

THYROID

HORMONE

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THE

DEVELOPING

BRAIN

303

However, this effect may result from a decrease in axonal density (36). A concomitant defect in the synthesis of cerebral protein (11, 15, 54) would seem to involve the translation step of the genetic message, since the rate of RNA synthesis is not correspondingly affected (11, 12, 15). Evidence are accumulating that, in other tissues also, hormones affect the processes of protein synthesis at this level (74, 114). Neurochemical maturation is retarded: the postnatal changes in the concentration of ions in the brain is retarded (117), and the enzymic activity ( Na+-K+-ATPase ) implicated in the maintenance of the uneven cation distribution in the adult cells is strongly reduced (51, 117). The biochemical changes which determine the transition from the immature to the adult metabolic pattern in brain tissue are dramatically retarded. The "maturational" indices used indicated that the glia-neuronal relations did not develop as in the normal animal: this resulted mainly from a retardation of the normal expansion of the metabolic compartments associated with the dendrites and nerve terminals (27, 98, 100). Thyroid deficiency in early life gives rise, in man, to severe mental retardation and in experimental animals to an impaired behavioural performance (41), which may result from the structural and biochemical changes associated with this condition. However, "cretinism" in man does not necessarily result from athyroidism alone, since it is commonly accompanied by other congenital defects ( for discussion cf. Eayrs, 39 ). (2) THYROID TREATMENT DURING INFANCY. The smaller brain resulting from treatment with thyroid hormones is due to a decrease in cell number rather than in cell size (14-16). In the cerebellum, an organ in which the final assembly of cells develops mainly after birth, thé effect of T.-.-treatment on the daily increment in DNA takes place during the "late" period of postnatal cell formation. It is suggested that the premature termination of cell proliferation, in the brain is related to the accelerated differentiation of the cells, since (a) the formation of dendritic spines is accelerated (109), (b) biochemical indices of brain maturation showed a significant advancement (26, 27, 98, 100), and (c) the functional maturation of the brain as shown by electrocortical activity and innately organized behavior was advanced (40,41,70,109,110). The development of the neuroendocrine organization is deeply affected by thyroid treatment in early life. Changes, apparently of a permanent kind, are seen in the pituitary, where they consist in an overall hypoplasia associated with a degranulation of the acidophil cells (43). A persistent thyroid hypofunction is also indicated by the observations that thyroid treatment in infancy leads in the adult animal to a reduced secretion of TSH (thyroid stimulating hormone) both in the resting state and following challenge with propylthiouracil (6). The rate of protein synthesis in the pituitary is apparently permanently reduced (73). This effect may be related to the observation that the protein synthesis-age curve for the hypo-

304

NEURAL

GROWTH

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DIFFERENTIATION

thalamus shows an advancement, which implies a smaller rate of protein synthesis compared to controls during a period when the rate of protein synthesis in the pituitary is still increasing under normal conditions ( 7 3 ) . The permanent changes in the neuroendocrine system may contribute to the impairment of adaptive behaviour observed in rats treated with thyroid hormone in early life (39, 109). Another factor may be the deficiency in postnatal cell formation. Altman ( 3 ) has reported that postnatal cell formation in the brain involves neurons as well as glial cells. He has suggested that the microneurons formed after birth—whose formation, therefore, may be affected by thyroid treatment at this stage—may modify the structural and functional organization of the developing nervous system to an extent depending on environmental influences ( 4 ) . It must also be considered that, if the accelerated maturation triggered off by thyroid treatment is not completely synchronized throughout the whole central nervous system, dysfunction may follow. The results summarized provide new evidence that thyroid hormones have a role in the normal differentiation of nervous tissues. The period in which the thyroid may influence the maturation of the brain is restricted mainly to the period preceding that in which the cells undergo extensive differentiation (from birth till the 14th day). The mechanism of action is still only partly understood. REFERENCES 1. 2. 3.

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nutrition on the postnatal development of the brain and cord in pigs. Proc. Roy. Soc. London B., 1967, 166: 396-407. DOBBING, J., The influence of early nutrition on the development and myelination of the brain. Proc. Roy. Soc. London B., 1964, 159 : 503-509. EAYRS, J. T., The vascularity of the cerebral cortex in normal and cretinous rats. J. Anat. (Lond.), 1954, 88: 164-173. , The status of the thyroid gland in relation to the development of the nervous system. Animal Behav., 1959, 7: 1-17. , The cerebral cortex of normal and hypothyroid rats. Acta Anat. (Basel), 1955, 25: 160-183. , Age as a factor determining the severity and reversibility of the effects of thyroid deprivation in the rat. J. Endocr., 1961, 22: 409-419. , Protein anabolism as a factor ameliorating the effects of early thyroid deficiency. Growth, 1961, 25: 175-189. , Effects of thyroid hormones on brain differentiation. In: Brain-Thyroid Relationships with Special Reference to Thyroid Disorder (M. P. Cameron, and M. O'Connor, Eds.). Churchill, London, 1964 : 60-71. , Effect of neonatal hyperthyroidism on maturation and learning in the rat. Anim. Behav., 1964, 12: 195-199. , Thyroid and central nervous development. Scientific Basis Med. Ann. Rev., Athlone Press, Lond., 1966: 317-339. EAYRS, J. T., and GOODHEAD, B., Postnatal development of the cerebral cortex in the rat. J. Anat. (Lond.), 1959, 93: 385-402. EAYRS, J. T., and HOLMES, R. L., Effect of neonatal hyperthyroidism on pituitary structure and function in the rat. J. Endocr., 1964, 29: 71-81.

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Hor-

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J., and K R E B S , H . A . , The metabolism of glutamate in homogenates and slices of brain cortex. Biochiem. J., 1963, 88 : 566-578. 6 3 . H E R M A N , C . J., and L A P H A M , L . W . , Neuronal polyploidy and nuclear volumes in the cat central nervous system. Brain Res., 1969,15: 35-48. 64. H I M W I C H , H . E., Brain Metabolism and Cerebral Disorders. Williams & Wilkins Co., Baltimore, 1951: 157-163. 65. H I M W I C H , W. A., Biochemical and neurophysiological development of the brain in the neonatal period. Int. Rev. Neurobiol., 1962, 4: 117-158. 66. HOWARD, E., Effects of corticosterone and food restriction on growth and on DNA, RNA and cholesterol contents of the brain and liver in infant mice. 7- Neurochem., 1965, 12: 181-191. 67. , Reduction in size and total DNA of cerebrum and cerebellum in adult mice after corticosterone treatment in infancy. Exp. Neurol., 1968, 22: 191-208. 6 7 A . HOWARD, E., and GRANOFF, D . M . , Effect of neonatal food restriction in mice on brain growth, DNA and cholesterol, and on adult delayed response learning. J. Nutr., 1 9 6 8 , 9 5 : 1 1 1 - 1 2 1 . 68. ITOH, T., and QUASTEL, J. H., Acetoacetate metabolism in adult and infant brain. Biochem. ]., 1970, 116 : 641-655. 6 9 . KARLSSON, U . , Observations on the postnatal development of neuronal structures in the lateral geniculate nucleus of the rat by electron microscopy J. Ultrastruct. Res., 1967, 17: 158-175. 70. K H A M S I , F., and E A Y R S , J. T., A study of the effects of thyroid hormones on growth and development. Growth, 1966, 30: 143-156. 71. K L E E , C. B., and SOKOLOFF, L., Changes in D ( —)-^-hydroxybutyric dehydrogenase activity during brain maturation in the rat. J. Biol. Chem., 1967, 242: 3880-3883. 72. KOVACS, S., COCKS, W. A., and BALAZS, R., Incorporation of [2- 1 4 C]thymidine into deoxyribonucleic acid of rat brain during postnatal development: effect of thyroid hormone. Biochem. J., 1969, 114 : 60P. 73 . , Effect of thyroid hormone administration on the incorporation of [1- 14 C] leucine into protein of rat pituitary and hypothalamus during development. J. Endocr., 1969, 45: ix-x. 74. KORNER, A., RNA and hormonal control of protein synthesis. Progr. Biophys., Mol. Biol., 1967, 17 : 61-98. 6 2 . HASLAM, R .

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S. Roberts: I would like to know, Dr. Balâzs, whether your conclusion can be expressed simply as being that the metabolic compartmentation is associated with glial development?

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Balazs: Not quite; the evidence indicates that the manifestation of metabolic compartmentation is related to maturational changes in the glianeuronal relations, rather than to developmental processes affecting individual components only. S. Roberts: I asked this question because Dr. Zomzely has studied the developmental changes in leucine conversion to dicarboxylic acids both in cerebral cortex and in medullary-hindbrain of the intact rat. In cerebral cortex, conversion rises from practically a zero value in the newborn, to almost a maximum at about the time you indicated; that is about 14 days. Actually, the rate is still increasing in the young adult animal. Cerebral white matter also shows a rapidly increasing conversion of leucine from the 14-day to the adult animal. Thus, if this is a sign of developing metabolic compartmentation, it does not seem to be restricted to any particular cell component in the brain, but is, perhaps, a more general characteristic of cell maturation. Balazs: My feeling is that metabolic compartmentation is the result of differences in the metabolic pattern of the different structural and associated biochemical compartments within the tissue. So far as the whole brain is concerned, metabolic compartmentation probably reflects the differences in the metabolic pattern of neurons and glial cells; however, I would not be surprised at all if future investigations with improved techniques of isolating individual cell types from the CNS would reveal marked differences in the metabolic pattern of, for example, different types of glial cells. E. Roberts: I would like to suggest the possibility that there is metabolic compartmentation even within a given neuron. There is, I think, considerable evidence that when pre- and post-synaptic endings of a given neuron are very far apart as in the Purkinje cells, there is no free and rapid communication between the regions. The permeability and the ionic properties of synaptosomes are quite different from those of the dendrites. There must be at least 20 pools that we are talking about, considering the different types of neurons, and glial and endothelial cells. There is another category that can be added. There is some evidence, both developmental and chemical, that inhibitory and excitatory neurons have a different chemistry. I think that inhibitory interneurons probably form rather late in development. Is there a possibility that the finding that your Trj treated animals behave abnormally, but develop normally chemically, may be attributable to the fact that there is an inhibition of the development of the interneuronal elements which might be coming from the socalled microneurons? I think the chemistry of inhibitory and excitatory neurons may be quite distinct and various pharmacological agents may be acting differently on the two classes of cells. The chemists have distinguished very clearly two gross pools metabolically, but I think we really have to be careful when we try to translate these into morphological terms. Balazs: I quite agree with you. In this complex field one has to simplify

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the system in terms of models. We have proposed just recently that a model consisting of a minimum of three compartments is compatible with the experimental data (4). Each compartment probably results from the summation of a number of different units with more or less similar metabolic patterns, and may be further specified as our knowledge about the metabolic properties of system improves. On the basis of circumstantial evidence, morphological structures may be allocated to the three main compartments hitherto identified (the list is obviously incomplete); a fraction of the nerve terminals involved in the production of GABA (and thus probably inhibitory in function) is associated with a metabolic compartment which is different from that which is associated with at least the terminal branches of the dendrites, i.e., these two are intracellular compartments (4, 18, 19, 23). Another compartment whose metabolic characteristics are different from those related to nerve terminals and dendrites respectively is apparently contained in a fraction of the glial cells. Thus, there are also heterocellular compartments in the system. Hamburgh: Unraveling the effects of a hormone on the nervous system is a little like peeling an onion. Not only does it bring tears to the eyes, but with each layer removed there is another one underneath. Historically, the sequence by which the effect of thyroid hormone on the differentiation of the CNS was studied, started with behavioral observations, continued with neurohistological studies, and now increasingly concerns itself with biochemical analysis of the type and refinement which Dr. Balazs presented. I agree with your suggestion that micro-neurons may be involved in the behavioral effects of T3-treatment. A priori, I think almost everyone believes that hypothyroidism ought to reduce myelin synthesis. This statement perpetuates itself in the literature like a statute in the law books, but whether it is true or not is far from being settled. There are arguments, for instance, by Myant and Cole (21) who found that the ratio of myelin to nonmyelin phospholipids in whole brain is not changed with T 3 injection. Myant and Cole also seemed to find no effect of T 3 on the relative rate of incorporation of P32 into each of the four classes of phospholipids. Is there not some evidence that there is such a thing as transitory myelin or bad myelin? Could it be that the effect of thyroid hormone may be on such myelin? I have stained brain sections of hypo-andhyperthyroid rats with laxol fast blue and found that the myelin picture really does not change very much between controls and these two groups. However, the presence or absence of thyroid hormone affects the myelin picture when the Marchi stain is used. The latter methods specifically make visible degenerating myelin. The finding that the activity or concentration of succinic dehydrogenase and glutamate decarboxylase (two enzymes mainly located in the synaptosomal fractions) are affected by lack of thyroid hormone, but not glutamate dehydrogenase activity (an enzyme concentrate mainly in the nonsynapto-

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somal fraction) is of great interest. I agree with Dr. Balazs that this finding supports the contention that one of the targets of the hormone in the developing brain is probably the formation of nerve terminals (3). Gomez and collaborators (12) have described the depression of synthesis of a number of enzymes in developing cerebral and cerebellar cortex of hypothyroid rats. Some of the enzymes affected by thyroidectomy listed by these authors were: succinic dehydrogenase, GABA transaminase and aspartate amino transferase. I am not sure which of these enzymes are known to be synaptosomal and which are concentrated mainly within the perikaryon of the neuron. These and other data require review before one can accept as proof Dr. Balazs' thesis that thyroid hormone specifically influences synaptosomal enzyme synthesis. The idea that hormones selectively affect enzymes at specific sites is, however, attractive because it makes sense to those of us who have worried in the past about what a reduction in enzyme activity of the usual magnitude of 15 per cent to 35 per cent (that is frequently encountered following experimental treatment) might really imply in relation to the functional integrity of a cell. Heinrich Waelsch used to say that there are only a few bottleneck enzymes. The non-bottleneck enzymes can be depressed without accompanying changes in a cell. If, as Dr. Balazs suggests, a hormone or lack of it influences only selected enzymes that are located at very specific sites (for example, synaptosomal enzymes), it might make many of the observations on enzyme inhibition by experimental methods more meaningful in terms of functional consequences. Compartmentation of glutamate as indicated by the observation that at some time during maturation of the brain a small and highly active pool of glutamate is converted to glutamine is interpreted by Dr. Balazs to reflect an increase of dendrites and of the nerve terminals. The two may go together in the rat brain, but in the cat this correlation does not hold. Shofer, Pappas and Purpura (26) have shown that the evolution of the dendritic tree in neocortex (but not hippocampus) in the cat brain is well advanced by 12 to 14 days of postnatal age. All that happens between the 14th day and the 21st day when the availability of glutamate for glutamine is changed (for example, when compartmentation can be shown to emerge in the cat brain) is a tremendous elaboration of the glial network. I am really not sufficiently sophisticated to discuss biochemical compartmentization, but sometimes fools walk where angels fear to tread. I just suggest that, perhaps, the effect of T 3 on the biochemical compartmentization might reflect changes in the availability of the glial element, as has been suggested by both Pesetsky (24) and Mitskevich (20). The question always remains whether changes in differentiation of a target resulting from manipulation with one endocrine represents responses to the direct action of the hormone molecule that is being tested, or whether they constitute second to third order reactions. Removal or addition of one

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hormone during ontogeny may disturb the remaining endocrine balance or initiate developmental changes as a result of upsetting either the physiology of the mother or the embryo or both. Effects on blood supply, nutritional state and others are examples that come to mind. Studies comparing the effect of hypothyroidism with undernutrition on cerebral maturation, carried out by Eayrs and Horn (9) and Legrand (18), provide convincing evidence that the effects ascribed to the former condition is not a consequence of the latter. The classical experiments by Kollross (14) showing acceleration of maturation of larval amphibian brain in response to local implants of thyroid pellets also suggest that the hormone exerts a direct effect on maturation of neuroblasts. Similar evidence comes from some tissue culture work showing that labeled thyroxine gains access to all cells of the explants: glia, neuroblast, and macrophages." Unfortunately, nerve tissue culture does not lend itself too well for studies of direct hormone action on developing nervous tissue. No synthetic medium in which embryonic nerve tissue can be successfully grown has yet been devised. Nerve tissue requires the presence of either placental or horse serum or embryo extract in the medium. Since any of these components contains most certainly thyroid hormone in unknown concentration, attempts to study the influence of this hormone on maturing CNS structure in cultures must limit itself to observing effects of excess hormone on development. Excess hormone may be toxic, and the more critical analysis of the effect of absence of thyroid hormone on neural maturation cannot yet be done in tissue culture. The last point I wish to make is to recommend the hypothyroid syndrome also to those neurobiologists whose primary interest is not in hormone action but in correlation between behavior, mental ability and the biological substrate. The cretinoid syndrome is extremely easy to reproduce experimentally and it results in an animal with low mental ability. Usually studies of this kind rely on comparing brain weight and brain chemistry of genetic strains that may differ in performance in some selected tests, and that are subsequently classified as "good" or "poor" learners. It invariably turns out that the "good" learner on one set of tests performs poorly on another test, and vice versa. So most of the time it is not the animal that is stupid, but the investigator. The biological differences correlated with level of mental activity are most certainly very subtle, so subtle in fact that it takes the kind of sophistication exhibited by Dr. Balazs' approach to begin to clarify these problems. Balazs: I should like to return to the question of metabolic compartmentation. The results shown in Figure 13.9 indicated that metabolic compartmentation becomes manifested with cerebral maturation. These observations agree with the view that the apparently homogeneous metabolic pattern of the tissue changes as a result of the expansion of a compartment " Unpublished observations.

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(neuronal processes) whose metabolic pattern is both quantitatively and qualitatively different from that dominant in the immature tissue. On functional basis we may distinguish between a "large" and a "small" metabolic compartment in the adult tissue, in view of the size of the related glutamate pool and the differences in metabolic characteristics. The metabolic pattern of the compartment which expands during the period of functional maturation of brain (neuronal processes) is compatible with that of the "large" compartment (4, 8, 23), whereas there are apparent similarities between the metabolic properties of the immature brain and "small" compartment (23). The experimental results (Figure 13.9) show that a "small" compartment becomes functionally manifested with maturation, but the primary cause is the development of the large metabolic compartment. Circumstantial evidence suggests that the "small" compartment is associated with glial cells (4). Glial development, at least in the rat, does not correlate with the maturation of compartmentation, which would be expected if the manifestation of metabolic compartmentation is the result of the growth of the neuronal processes. S. Roberts: I think this is one situation in which cell separation techniques may provide an answer which can probably not be definitively provided in other ways. Even in the studies that I mentioned in our laboratory it is impossible, of course, when one is dealing with the neuronal portions of the brain, to be certain that glial infiltration and growth is not responsible for the related biochemical changes seen during development. Balazs: I agree. When isolated cell types from the brain will become available as a result of improved techniques of cell separation, it will be possible to test our hypothesis, which predicts certain differences in the metabolic pattern of some types of glial cells compared with some types of neurones. E. Roberts: Since the subject was not brought up, I would like to discuss release of rate-limiting reactions or processes and their possible role in differentiation. The general problem of rate-limiting biochemical reactions in enhancement of function and growth cannot be discussed in detail, and only one aspect, most probably linked to the neuroendocrine system, will be mentioned here. Most reactions which occur in living systems may be considered to be potentially rate-limiting in terms of some cellular activity; but, in a particular instance only one reaction actually is likely to play a decisive role. One logical place to seek rate-limiting reactions would be among the fundamental energy- and precursor-yielding reactions (glycolysis and respiration) which are common to all cells. In this connection it is of particularly great interest that a central regulatory role has been uncovered for adenosine-S'^'-phosphate (cyclic-3',5'-AMP) in a number of cellular functions (25, 26). Cyclic3',5'-AMP has been observed to increase the amount of active phosphorylase and phosphofructokinase in tissues, thus promoting glycogenolysis and glycolysis. In this manner it opens the gateway for a more rapid flow of en-

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ergy and carbon precursors to particular cell types, enabling them to perform their specialized physical and chemical functions as well as those related to general maintenance and growth. It is of special interest that many membrane-active substances including hormones and neuroactive substances may exert their action on specific target cells by increasing the amount of this substance either by accelerating its formation or decreasing the rate of its destruction. Among such substances are various pituitary hormones. Also active are the catecholamines, glucagon, serotonin, and histamine. The complex interdigitation of the cyclic adenylic biochemical system with the neuroendocrine system can be illustrated for the thyroid. Cyclic AMP may be involved in the release of a hypothalmic factor which, in turn, causes release of thyroid-stimulating hormone (TSH) from the pituitary, since implantation of the dibutyryl derivative into hypothalamic sites causes hyperthermia, hyperphagia and increased locomotor activity (5). An in vitro study suggests that cyclic AMP may be the "second messenger" in the release of TSH from the pituitary (7). TSH probably causes an increase in the level of cyclic AMP in the thyroid gland (10, 13), which appears to release rate-limiting reactions in carbohydrate metabolism in thyroid tissue (22, 11) as well as the fixation of iodine into organic form, and synthesis of thyroid hormone (1). The action of the thyroid hormone, itself, on the target organ (for example, heart) may also be mediated by an activating effect on adenyl cyclase, the enzyme which synthesizes cyclic AMP from ATP (18). Among the substances increased in amount during the general increase in protein synthesis occurring during the release of rate-limiting reactions might be RNA polymerase, which could increase the rate of transcription of genetic information (for example, by increasing the number of ribosomes available for protein synthesis). I would like to propose that perhaps among the various elements which are changing and differentiating during development, normal or abnormal, as created either by giving thyroid too early or removing the thyroid, the fundamental place to look biochemically would be at the adenyl cyclase level of tissue which is differentiating or maturing. S. Roberts: You mentioned cyclic AMP and touched a responsive chord. The differential activation of adenyl cyclase in different cells by thyroid hormone—and for that matter in different parts of the same cell—may to a considerable extent explain some of the compartmentation phenomena that are observed. Certain adenyl cyclases appear to be sensitive to one influence in one part of the cell, while others are activated by different influences in another part of the cell. Cotman: I have two questions. First, I would like to know whether there has been any work done on taking the ratio of, say, the available substrate to the enzyme levels present during the different stages of development, to see whether in fact one can find a bottleneck or a rate-limiting enzyme. Second,

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I would like to know more detail concerning the different changes in the sucrose gradients with succinic dehydrogenase. I would like to know if you have looked at electron microscope pictures of the synaptosomal fraction, and whether if fact you do see any type of evident morphological change. Is it possible that there is contamination? What are the details of the gradient analysis? Balazs: As to the final question, a comprehensive study of the level of glycolytic intermediates and corresponding enzymic activities in the brain of 10-day-old and adult mice was done by Lowry's group (15,16), who also investigated the glucose flux in glycolysis under conditions of accelerated activity as a result of anoxia. Their results may be taken to indicate that the control of glycolysis is mediated by the same enzymic mechanisms in the brain of the immature and adult animal, although the absolute value of the glucose flux in the brain of the 10-day-old mouse is only about 50 per cent of that in the adult. One of their conclusions has some bearing on your question. They have found that along glycolytic pathway in mouse brain there is no step that is limited by the amount of enzyme present. As far as I know, similar comprehensive study relating to other metabolic pathways has not yet been done, but data from different laboratories are available for both the activities of a great number of relevant enzymes and the concentration of many intermediates during development (cf. 2). As to your second question, we have not studied the synaptosomal fraction with electron microscopic techniques. I agree that further work is required to establish the proper conditions for the separation of subcellular fractions from homogenates derived from brain tissue of different developmental stages. Johnson: Some people may be interested to know that Dr. Luttges, working in my laboratory, has been studying compartmentalization with regard to neural cell maturation in the mouse. He has looked at the uptake of GABA, choline, glutamic acid and amino acids like valine and phenylalanine by cortical slices and isolated nerve endings. Indeed, he does find active uptake of these precursors by both young and mature preparations, although the younger tissue is less able to compartmentalize these compounds into nerve endings. This is particularly evident with those precursors that are active in inhibitory and excitatory synapses, for example, GABA and choline. These techniques were applicable to following the maturation of inhibitory and stimulatory synapses during brain maturation of the mouse. E. Roberts: Which did he find comes first? Johnson: The excitatory seems to come first. However, it is quite complicated, and I do not remember all of the data, because he actually looked at several sites of the brain such as the hypothalamus, cerebral cortex and cerebellum.

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REFERENCES

1. AHN, C.-S., and ROSENBERG, I. N., Prompt stimulation of the organic binding

of iodine in the thyroid by adenosine 3',5'-phosphate in vivo. Proc. Nat. Acad. Sci. USA. 1968, 60: 830-835. 2. BALÂZS, R., Carbohydrate metabolism in the central nervous system. In: Handbook of Neurochemistry (A. Lajtha, Ed.), Plenum, New York, III, in press. 3 . BALÂZS, R . , KOVÂCS, S . , TEICHGRABER, P . , COCKS, W . A . , a n d EAYRS, J . T . , B i o -

chemical effects of thyroid deficiency on the developing brain. /. Neurochem., 1968, 15: 1335-1349. 4 . BALÂZS, R . , MACHIYAMA, Y . , HAMMOND, B . J., JULIAN, T . , a n d RICHTER, D . ,

5. 6. 7.

8.

9. 10.

11.

12.

13.

14.

The operation of the GABA bypath of the tricarboxylic acid cycle in brain tissue in vitro. Biochem. /., 1970, 116: 445-467. BRECKENRIDGE, B. M., and LISK, R. D., Cyclic adenylate and hypothalamic regulatory functions. Proc. Soc. Exp. Biol. Med., 1969 131: 934-935. BUTCHER, R. W., Role of cyclic AMP in hormone actions. New Eng. J. Med., 1968, 279: 1378-1384. CEHOVIC, G., Rôle de l'adénosine 3',5'-monophosphate-cyclique dans la libération de TSH hypophysaire. C.R. Acad. Sci. (Paris), 1969, 268 : 2929-2931. COCKS, J. A., BALÂZS, R., JOHNSON, A. L., and EAYRS, J. T., Effect of thyroid hormone on the biochemical maturation of rat brain: conversion of glucose-carbon into amino acids. J. Neurochem., 1970, 17: 1275-1285. EAYRS, J. T., and HORN, G., The development of the cerebral cortex in hypothyroid and starved rats. Anat. Rec., 1955, 121: 53-61. GILMAN, A. G., and RALL, T. W., Factors influencing adenosine 3',5'phosphate accumulation in bovine thyroid slices. J. Biol. Chem., 1968, 243 : 58675871. , The role of adenosine 3',5'phosphate in mediating effects of thyroidstimulating hormone on carbohydrate metabolism of bovine thyroid slices. J. Biol. Chem., 1968, 243: 5872-5881. GOMEZ, C., Hormonal influences of the biochemical differentiation of the rat cerebral cortex. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.). Appleton-Century-Crofts, New York, in press. KANEKO, T., ZOR, U., and FIELD, J. B., Thyroid-stimulating hormone and prostaglandin Ei stimulation of cyclic 3',5'-adenosine monophosphate in thyroid slices. Science, 1969, 163: 1061-1063. KOLLROSS, J. J., Localized maturation of lid closure reflex mechanism by thyroid implants into tadpole hind brain. Proc. Soc. Exp. Biol. Med., 1942, 49: 204-206.

1 5 . LOWRY, O . H . , PASSONNEAU, J . V . , HASSELBERGER, F . X . , a n d SCHULZ, D .

W.,

Effect of ischaemia on known substrates and cofactors of the glycolytic pathway in brain. /. Biol. Chem., 1964, 239: 18-30. 16. LOWRY, O. H., and PASSONNEAU, J. V., The relationship between substrates and enzymes of glycolysis in brain. J. Biol. Chem., 1964, 239: 31-42. 17. LEGRAND, J., Comparative effects of thyroid deficiency and undernutrition in maturation of the nervous system and particularly myelination in the

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young rat. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.), Appleton-Century-Crofts, New York, in press. 18. L E V E Y , G. S., and E P S T E I N , S. E . , Activation of cardiac adenyl cyclase by thyroid hormone. Biochem. Biophys. Res. Commun., 1968, 33: 990-995. 1 9 . MACHIYAMA,

20.

21. 22.

23. 24.

25. 26.

Y . , BALAZS, R . , HAMMOND, B . J . , JULIAN, T . , a n d RICHTER,

D.,

The metabolism of y-aminobutyrate and glucose in potassium ion stimulated brain tissue in vitro. Biochem. J., 1970, 116: 469-481. MITSKEVICH, M . S., and MOSKOVKIN, G . N . , Some effects of the thyroid hormone on development of cerebral nervous system in early ontogenesis. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.), Appleton-Century-Crofts, New York, in press. M Y A N T , N. B., and C O L E , L. A., Effect of thyroxine on the deposition of phospholipids in the brain in vivo and on the synthesis of phospholipids by brain slices. J. Neurochem., 1966, 13: 1299-1307. PASTAN, I., and MACCHIA, V., Mechanism of thyroid-stimulating hormone action. Studies with dibutyryl 3',5'-adenosine monophosphate and lecithinase C. J. Biol. Chem., 1967, 242: 5757-5761. PATEL, A . J . , and BALÂZS, R . , Manifestation of metabolic compartmentation during the maturation of the rat brain. J. Neurochem., 1970, 17: 955-971. PESETSKY, I., and MODEL, P . G . , Thyroxin-stimulated ultrastructural changes in ependymoglia of thyroprivic amphibian larvae. Exp. NeUr61., 1969, 25: 238-244. ROBISON, G. A., BUTCHER, R . W., and SUTHERLAND, E. W., Cyclic AMP. Ann. Rev. Biochem., 1968,37: 149-174. SHOFER, R. J., PAPPAS, G. D., and PURPURA, D. P . , Radiation-induced changes in morphological and physiological properties of immature cerebellar cortex. In: Response of the Nervous System to Ionizing Radiation (T. J. Haley and R. S. Snider, Eds.), Little, Brown, Boston, 1964, 476-508.

14. THE THYROID AS A TIME CLOCK IN THE DEVELOPING NERVOUS SYSTEM* MAX HAMBURGH, LORENZO A. MENDOZA,f JOHN F. BURKART and FRANKLIN WEIL City College of New York and Albert Einstein College of Medicine Bronx, New York

One of the most extensively investigated problems of developmental endocrinology is the relationship of the thyroid hormone to the developing nervous system (3, 4, 11, 12). Previous reports, some of which are reviewed in this symposium, have described transitory increases of DNA in developing whole brain of hypothyroid chicks (17), in developing cerebral cortex in young postnatal rats (1, 2, 7-9) and transitory increases in cell number in the maturing rat cerebellum (11, 14-16). These reports encouraged us to test the proposition that lack of thyroxin may prolong the proliferative phase in some parts of the developing central nervous system, and that both the transitory as well as the permanent changes imposed by thyroid deprivation on the maturing CNS may be a consequence of "errors in timing or phasing" of the proliferative phase. MATERIALS AND METHODS

Pregnant rats of the Charles River Breeding Laboratories Caesarian Derived (CD) strain were used for all experiments. Animals were housed individually in an air-conditioned room and provided with a Rockland mouse diet (ground) and water ad libitum. Experimental

Procedure

Hypothyroidism was induced through administration of propylthiouracil (PTU) beginning during intrauterine life and continued until weaning. Propylthiouracil treatment was initiated during the last week of gestation by offering to pregnant rats a goitrogenic diet containing 0.2 per cent propyl" This investigation was supported by a research grant from the National Institute of Neurological Diseases and Blindness, NIH B-1716, and by Training Grant 5T1 HD0016-02-04 of the National Institute of Child Health and Human Development. Grateful acknowledgment is made to Miss Cassandra Kirk for her technical assistance; and to Mr. M. Kurtz, medical photographer, for his assistance with the photographic work, f Hospital Vargas, Caracas, Venezuela. 321

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thiouracil mixed into powdered Rockland mouse diet, starting on day 15 of pregnancy and continued until weaning or beyond. Day 15 was chosen for initiation of the experimental treatment because available evidence suggests that, in the rat, the fetal thyroid does not mature functionally before the eighteenth day of fetal age, but that during the third trimester of the gestational period, maternal thyroxin passes the placental barrier (13). Controls consisted of pregnant rats maintained on an unsupplemented diet and receiving daily injections of 0.9 per cent alkaline saline from birth until weaning. Uptake of Hs Thymidine by Rat Cerebellum Unanesthetized 15 and 21 day old rats were injected subcutaneously with 1 (jcurie thymidine H3 per gm of body weight (Sp. Activity 27.0 c/mmole obtained from Schwartz BioResearch Inc.). Six hours after injection of the tritiated thymidine the rats were sacrificed by decapitation, and brains were rapidly removed and fixed in Bouin's solution. The tissue was washed in 70 per cent ethanol, dehydrated and embedded in paraffin. Sections were cut at 5 n, mounted on microscope slides and coated with Kodak, NTB 3 bulk emulsion. The emulsion was exposed to the tritium labeled tissue section for two weeks and stored at 4°C. After development in D 19, the emulsion slides were stained with hematoxylin and eosin, cleared in xylol and coverslips were applied with Permount. RESULTS

The development of the rat cerebellum during the first three weeks of age is characterized by extensive migration of cells derived from the transitory external zone past the molecular area to form the layer of granular cells of the cerebellum. In the rat the thickness of this external zone attains its maximum at about eight days after birth and subsequently diminishes progressively. It has completely disappeared at the twenty-first day, at which time the surface of the cerebellum has acquired the characteristic aspect of the adult. In the cerebellar cortex of rats, the most immediate and striking effect of thyroid deficiency initiated during late fetal period or immediately at or shortly after birth includes the persistence of the external granular zone long after this layer of cells has disappeared in normal littermates (14-16). When thymidine H3 was injected on postnatal day 14 and the animals killed six hours later, labeled cells were confined mainly to the outer rows of the external granular layer. Moderate numbers of labeled cells were scattered through the white matter and the granular layer proper. There was no difference in intensity of number of labeled cells between normal control rats and rats that were deprived of thyroid hormone since before birth. When thymidine H3 was injected on postnatal day 21 and the control animals killed six hours later, only few labeled cells were found in the deep white matter and very rare ones were in the granular layer (See Figures

THE

*

/

mm.

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CLOCK

323

7

M

W

Figure 14.1. Radioautograph of cerebellum of 21-day-old rat. Sacrificed 6 hrs after injection of 1 p.c/g body weight of tritiated thymidine and exposed for 2 weeks. Note absence of grains. E = external granular layer, M = molecular layer, G = internal granular layer. (From M. Hamburgh, J. Burkart and F . Weil, 12A.) X 4 4 0

14.1 and 14.3). The external granular zone had disappeared at that time and there were practically no labeled cells present in the periphery of the cerebellar cortex. A radically different picture was presented by cerebella obtained from hypothyroid rats. When thymidine H3 was injected on postnatal day 21 into rats that had been deprived of thyroxine since before birth, and, which were then killed 6 hours after administration of the tritiated thymidine, the cells of the external granular layer were still heavily labeled. In addition, many labeled cells were scattered in the granular layer, the molecular layer and the underlying white matter (Figures 14.2, 14.4). CONCLUSIONS

The persistence of the fetal external granular zone in the cerebella of hypothyroid rats could be the result of either (i) failure of cells to migrate away from their source of origin toward their final destination in the cerebellar cortex, (ii) failure of disposal of excess cells of the fetal external zone through cell death, or (iii) delay in the cessation of proliferation of cells derived from the fetal external layer. The results of the present study permit us to choose between these three alternatives. The radioautographs obtained from cerebella of normal and hypothyroid

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Figure 14.2. Radioautograph of cerebellum of 21-day-old hypothyroid rat. Hypothyroidism was induced by mixing propylthiouracil into powdered diet starting on the 15th day of pregnancy and continued until day 21 of postnatal age. Sacrificed 6 hrs after injection of 1 fic of tritiated thymidine per g of body weight and exposed for 2 weeks. Note high concentration of grains in the external granular zone. (From M. Hamburgh, J. Burkart and F. Weil, 12A.) x 4 4 0

young rats, indicate that the fetal external granular layer that still persists in hypothyroid rats after day 21, when it has completely disappeared in control littermates, consists of cells that are still actively taking up tritiated H 3 thymidine, and therefore are presumably engaged in DNA synthesis, most likelv preparatory to mitosis. On the basis of these observations, we propose the hypothesis that thyroxin may push cells into the "differentiation" phase by pulling them out of the proliferative or mitotic phase. By acting as a timer, the optimal amount of thyroxin may turn off proliferation and thus allow differentiation to begin. Excess amounts of thyroxin may turn off the "proliferative phase" too soon while thyroid deficiency prolongs the period of proliferation, and thus delays the onset of processes of differentiation. The wrong timing may then lead to both permanent as well as to transitory abnormalities under conditions of hypothyroidism as well as hyperthyroidism. A consequence of the prolonged proliferation of "extragranular" cells and their delayed migration through the molecular layer past the row of Pur-

T H E T H Y R O I D AS A T I M E

CLOCK

Figure 14.4. Radioautograph of cerebellum of animals treated as in Figure 14.2. (From M. Hamburgh, J. Burkart and F. Weil, 12A.) X440

325

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kinje cells, seems to be a resulting hypoplasia of the dendritic spread of the Purkinje network in cerebella of hypothyroid rats (14-16). It is conceivable that the differentiation of the Purkinje network depends on some "inductive" stimulus exerted by the granule cells as they migrate past the row of perikarya of Purkinje neurons prior to taking their proper place in the internal granular zone ( 1 0 , 1 5 ) . The idea that the effect exerted by thyroid hormone on the developing brain may be mediated by the hormone's interference in the timing of the proliferative phase may also fit observations reported by Zamenhof et al. (17), some of which are presented at this conference." The magnitude of the difference in total DNA between "control" and "hypothyroid" brains is admittedly small. Possibly a much greater difference in DNA content could have been revealed if such studies had focused on specific areas of the brain whose maturational period coincides with the critical period of thyroid sensitivity. For the rat, such sensitive periods for brain maturation have been identified by Eayrs and Taylor ( 5 ) , Eayrs and Horn ( 6 ) and by Hamburgh and Flexner (12). The hypothesis that thyroxin may push cells into the "differentiative" phase by pulling them out of the proliferative or mitotic phase is a very attractive one, particularly in view of the frequently advanced postulate that growth and differentiation are mutually exclusive events. Possibly the prolongation of the proliferative period leading to the transitory increase in DNA content might, in turn, delay the timing of RNA synthesis and thus delay the production of important enzyme proteins. SUMMARY

The hypothesis was tested that thyroxin influences the maturation of some components of the central nervous system by timing the proliferative phase and in this way controls the cell population. Tritiated H 3 thymidine was injected into young postnatal rats, and autoradiographs of cerebella of normal and hypothyroid 15-and-21-day-old rats were prepared six hours after injection of the label. The autoradiographs revealed that as judged by grain counts, many cells, particularly those of the fetal granular zone of 21day-old hypothyroid rats, were still engaged in DNA synthesis, presumably in preparation for mitosis, while DNA synthesis had completely stopped in cerebella of 21-day-old control rats, except for a few scattered cells, probably glia. Comparing these observations with other reports of transitory increases of DNA in cerebral cortex of hypothyroid animals and transitory decreases in DNA of brains of hyperthyroid animals, the hypothesis that optimum amounts of thyroid hormone may pus hsome neuroblasts into the differentiative phase by terminating the proliferative or mitotic phase is recommended for further testing. " See also Dr. Zamenhof's chapter, pp. 329-361.

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REFERENCES 1. BALAZS, R . , KOVACS, S., TECHGRABER, P . , COCKS, W . A . , a n d EAYRS, J . T . , B i o -

chemical effects of thyroid deficiency on the developing brain. J. Neurochem., 1968,15: 1335-1349. 2. , Biochemical effects of thyroid hormones on the developing brain. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.), Appleton-CenturyCrofts, New York, in press. 3. EAYRS, J. T., Role of thyroid hormone in the differentiation of the nervous system. In: Proceedings of the Second International Congress of Endocrinology (S. Taylor, Ed.). Excerpta Medica Found. Int. Congress, Amsterdam, 1964,779-784. 4. ,Thyroid and developing brain: anatomical and behavioral effects. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.). Appleton-CenturyCrofts, New York, in press. 5. EAYRS, J. T., and TAYLOR, S. H., The effect of thyroid deficiency induced by methyl thiouracil on the maturation of the central nervous system. J. Anat., 1951, 85: 350-358. 6. EAYRS, J. T., and HORN, G., The development of the cerebral cortex in hypothyroid and starved rats. Anat. Rec., 1955, 121: 53-61. 7. GEEL, S., and TIMIRAS, P., The influence of neonatal hypothyroidism and of thyroxine on the ribonucleic acid and desoxyribonucleic acid concentration of rat cerebral cortex. Brain Res., 1967, 4: 135-142. 8. G E E L , S. E., VALCANA, T., and TIMIRAS, P. S., Effect of neonatal hypothyroidism and of thyroxine on L-[14C] leucine incorporation in protein in vivo and the relationship to ionic levels in the developing brain of the rat. Brain Res., 1967, 4: 143-150. 9. G E E L , S. E., and TIMIRAS, P., The role of thyroid and growth hormones on RNA synthesis in the developing brain. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.). Appleton-Century-Crofts, New York, in press. 10. HAMBURGH, M., Analysis of the postnatal developmental effects of "reeler" a neurological mutation in mice. A study in developmental genetics. Develop. Biol, 1963, 8: 165-185. 11. , An analysis of the action of thyroid hormones on development based on in vivo and in vitro studies. Gen. Comp. Endocr., 1968, 10: 198-213. 12. HAMBURGH, M., and FLEXNER, L. B., Biochemical and physiological differentiation during morphogensis. XXI. Effect of hypothyroidism and hormone therapy on enzyme activities of the developing cerebral cortex of the rat. J. Neurochem., 1957, Is 279-288. 12A. HAMBURGH, M., BURKART, J., and W E I L , F., Thyroid sensitive targets in the central nervous system. In: Proc. of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.). Appleton-Century-Crofts, New York, in press. 13. HAMBURGH, M., SOBEL, E. H., KOBLIN, R., and RINESTONE, A., Passage of thy-

328

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15. 16.

17.

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roid hormone across the placenta in intact and hypophysectomized rats. Anat. Ree., 1962, 144 : 219-227. LEGRAND, J., Analyse de l'action morphogénétique des hormones thyroïdiennes sur le cervelet du jeune rat. Anat. Micr. Morph. Exp., 1967, 56: 205244. , Variations, en fonction de l'âge de la réponse du cervelet à l'action morphogénétique de la thyroide chez le rat. Arch. Anat. Micr. Morph. Exp., 1967, 56 : 291-308. , Comparative effects of thyroid deficiency and undernutrition on the maturation of the nervous system and particularly on myelination in the young rat. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, (Eds.). Appleton-Century-Crofts, New York, in press. ZAMENHOF, S., VAN MARTHENS, E., and BURSZTYN, H. The effect of hormone on DNA synthesis and cell number in the developing chick and rat brain. In: Proceedings of an International Symposium on Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.). Appleton-CenturyCrofts, New York, in press.

15. HORMONAL AND NUTRITIONAL ASPECTS OF PRENATAL BRAIN DEVELOPMENT 0 STEPHEN ZAMENHOF and EDITH VAN MARTHENS University of California L o s Angeles, California

This paper deals with the prenatal development of one organ only, the brain (cerebral hemispheres).! The reasons for choosing this organ are the prospects of correlations between alterations in brain development and behavior; the reason for studying such early development is the evidence that the number of neurons becomes final at or before birth. Several reports indicate that in the rat the number of cerebral neurons does not increase after birth (2, 3, 5, 27, 42, 68), with the possible exception of short-axoned neurons (2). In man this termination of neuron proliferation may occur as early as the fifth to seventh month of pregnancy (42) but little work has been done on this important subject. In the chick the only report indicates that this termination occurs on or before the 12th day of incubation (50). In all of these species the increase in total number of cells after birth is much slower and reflects the proliferation of glia cells and, later, possibly also the cells of the circulatory system. Normal neuron and glia cells at birth are essentially diploid and the amount of DNA per diploid cell of a given species is constant (61, 71). While there have been reports of polyploid neurons, they concern cerebellar Purkinje cells (35, 38), Betz cells of the motor cortex (26), or large pyramidal cells in the hippocampus of the mature brain (25), but not cells in the neonatal cerebral cortex. Thus, determination of neonatal brain DNA is a convenient and objective quantitative method for determination of total neonatal brain cell numbers. Such determinations (40, 71) will be used throughout this work. From the DNA values per brain, the actual total number of brain cells could be calculated by dividing by a (constant) DNA content per cell (6 X 10"° ug for the rat; 2 X 10 6 ug for the chick) (cf. 39, 61). The total amount of brain DNA in the developing chick and rat reaches " This investigation was supported by USPHS grants No. HD-01909, HD-04612 and NS-08723, American Cancer Society grant No. P-503A, Nutrition Foundation grant No. 409, and U.S. Atomic Energy Commission Contract No. AT ( 1 1 0 1 ) - 3 4 . f The term "brain," used hereafter, refers to cerebral hemispheres without cerebellum and olfactory lobes in the rat, and, in addition, without optic lobes in the chick. 329

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a transient plateau at hatching or birth (see Results). At the plateau this amount (that is, also total brain cell number) is one of the most constant parameters of the developing organism. Later in this paper it will be shown that this amount cannot be changed by any of the numerous factors that can change neonatal body weight or neonatal brain weight. Brain weight, the parameter most often reported in the literature, represents a composite of many factors [such as amount of water (80-85 per cent in the chick; 88 per cent in the neonatal rat); lipid; protein; etc.] and, for individual animals, is not correlated with the amount of brain DNA [0.1 per cent in the chick; 0.35 per cent in the neonatal rat or cell number (see Results) ]. The constancy of the amounts of DNA in neonatal brains suggests one or more stringent regulatory mechanisms that are not easily disturbed. The outcome must be the precise and controlled slowing down of cortical neuron DNA synthesis towards the end of pregnancy. What are the controlling factors? Can we experimentally interfere with them? The purpose of this report is first to take cognizance of the possible factors and mechanisms, and then to summarize our results in those areas that were accessible to experimentation. M A T E R I A L S AND M E T H O D S

Chick embryos: Fertile eggs, White Leghorn strain K137, were supplied bi-weekly by the Kimber Farms, Pomona, Calif. For the study of genetic differences only, strain K155 was also used. The hens' ages and egg weights vary, and for each group as a whole (though not for single individuals) a correlation was found between egg weight and neonatal brain DNA content (see Results). To obviate this difficulty, care was taken to use as controls only eggs of the same weight, laid in the same week by hens of the same age as the experimentáis. The eggs were incubated in the Jamesway incubator type 252. Rats: The strain used was derived from the Sprague-Dawley strain. The rats have been bred in our laboratory for at least 20 generations; the females were virgin, 3-months-old and weighted 200 to 260 g. The animals were maintained on a pelleted diet ad libitum (Wayne Mousebreeder Block, Allied Mills, Chicago, Illinois), except as noted. Thyroxine and thiourea were supplied by CalBiochem, Los Angeles, California. Bovine Growth Hormone preparation was prepared by Dr. A. E. Wilhelmi, Emory University, and supplied by the Endocrinology Study Section, National Institutes of Health. Standard Treatments C H I C K E M B R Y O S . On the indicated day of incubation (see Results), the eggs were candled, and 0.1 to 0.2 ml of sterile solutions of the substance tested in physiological saline were injected with 25 gauge needle into the albumen or into the yolk. The control animals received sterile physiological saline

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331

injected in the the same manner. After the injections the holes in the egg shell were sealed with paraffin. RAT. Pregnant females were injected subcutaneously (0.1 ml) or intravenously (tail vein; 1 ml) with aliquots of the solutions. The control animals received sterile physiological saline injected in the same manner. Special rat diets are described in the next section. Dissections CHICKS. The embryos were removed on the days indicated (see Results) or allowed to hatch, and then weighed. Cerebral hemispheres (that is, without cerebellum and olfactory and optic lobes) were removed, weighed and then frozen and stored at —15°C for subsequent DNA determination.

The newborn rats (natural delivery or Caesarian, if so indicated) were weighed and then killed by decapitation within six hours of delivery. The cerebral hemispheres (without cerebellum and olfactory lobes) were removed, weighed and frozen as above. RATS.

D N A DETERMINATION. The brains were individually homogenized and the total DNA in each brain was determined by a modification (40, 71) of the diphenylamine colorimetric method. RESULTS

Normal Brain DNA

Contents

The brain DNA contents as a function of pre- and postnatal age are represented in Figure 15.1 [chick; see also ( 4 0 ) ] and Figure 15.2 [rat ( 7 7 ) ] . It can be seen that in both species these amounts reach a transient plateau at hatching or birth. This time, then, is a convenient one for estimating DNA because a time difference of even half a day either way will not cause any significant error. As explained in the introduction, the postnatal increase in brain DNA is not due to an increase in the number of brain neurons. Within individual small batches of chicks (n < 11) (see Figure 15.3), a significant correlation between individual neonatal brain weights and neonatal brain DNA cannot be demonstrated (correlation coefficient r = 0.00, p > 0.1, for n = 9; r = 0.32, p > 0.1, for n = 11). However, for large batches or for all animals considered together, a significant correlation becomes apparent (r = 0.54; p < 0.001 for n = 32; r = 0.61, p < 0.001 for n = 59). A similar situation exists for rats: Within any one litter, a significant correlation between individual brain weights and brain DNA also cannot be demonstrated (Figure 15.4. r = 0.097, p > 0.1, for n = 8; r = 0.146, p > 0.1, for n = 9; r = 0.32, p > 0.1 for n = 6; r = 0.52, p > 0.1, for n =

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I N

'3

T H E

CO +1 g ^

CO

TO CVJ

in e CL) TU

0.4

o u

Q. 0.2

O

0

10

20

top

30

bottom Gradient effluent (ml)

Figure 20.2. Sucrose density gradient profiles of purified polyribosomes prepared from cerebral cortical gray matter and hindbrain-medullary white matter of the young adult rat. ( , gray matter; , white matter) See legend to Figure 20.1 for description of the methods employed for density-gradient analysis.

saturating concentrations of the polynucleotide with significant formation of polyphenylalanine. The response to polyuridylic acid is favored by the presence of ribosomal monomers and subunits (9, 27). These small ribosomal particles were formed progressively during incubation of the cerebral polyribosomes in the amino acid-incorporating system (Table 20.2). In contrast, little or no dissociation of large ribosomal aggregates was noted in the analogous hepatic system even though the ribonuclease content was considerably higher. The major source of ribonuclease in these amino-acid incorporating systems was the pH 5 enzyme preparation which supplied the amino-acid synthetases, transfer RNAs and certain other factors required for polypeptide synthesis (28). The data also revealed that basal amino acid incorporation by polyribosomes isolated from cerebral cortex of young adult rats was equal to that observed with the analogous hepatic preparation under optimal conditions of incubation (Figure 20.5). This finding was consistent with the observations that the template activity (Table 20.3) and hybridization capacity (see below) of nuclear and cytoplasmic RNA preparations from adult rat brain were at least as great as those of comparable fractions from liver (5, 6). Purified polyribosomes derived from hindbrain-medullary white matter and cerebral cortical gray matter of the adult rat incorporated phenylalanine into protein at similar rates (Figure 20.6b). Thus, variations in the

450

NEURAL

GROWTH

AND

DIFFERENTIATION

Incubation time(min) Figure 20.3. Ribonuclease activity of postmitochondrial supernatant fractions obtained from different tissues of the young adult rat. Measurements were made by the method of Barondes and Nirenberg ( 4 ) with the use of 50 m^moles of [ 1 4 C] poly U polynucleotide phosphorus. Each point represents the average of three closely-replicating analyses. The samples in the lower set of curves each contained approximately 0.2 mg of postmitochondrial supernatant protein: O , cerebral cortex; • , liver; A, spleen; A, kidney cortex; The samples in the upper curves each contained approximately 1.6 mg of postmitochondrial supernatant protein: • , cerebral cortex; liver. (From Zomzely et al., 28.)

C E R E B R A L

R I B O N U C L E I C

A C I D

A L T E R A T I O N S

451

TABLE 20.1 I N F L U E N C E OF I O N C O N C E N T R A T I O N S ON S E D I M E N T A T I O N P R O P E R T I E S OF C E R E B R A L A N D HEPATIC P O L Y R I B O S O M A L P R E P A R A T I O N S

Ribosomal species

Mg 2+

K+

Na+

(mM)

(mM)

(mM)

Cerebral cortex S20.W

10

100

40

%

Liver S20. W

%

71 100 123 152 172 >172

6 10 10 9 7 58

72 105 132 >132

136

1 1 2 96

1

25

56 71 103 129 145 >145

2 21 12 11 8 46

73 104 128 >128

6 1 1 92

Polyribosomes were isolated by the method of Munro, Jackson and Korner (16) in medium containing 10 mM-magnesium acetate, 40 mM-NaCl, 100 mM-KCl and 20 mM-TrisHC1 buffer (pH 7.6). The pellets were stored frozen at — 60°C. Just prior to analysis in the analytical ultracentrifuge, pellets equivalent to approximately 3 mg of ribosomal protein were gently suspended either in the original isolation medium or in 50 mM-Tris-HCl buffer (pH 7.4) containing 25 mM-KCl and either 5 or 1 mM-MgCl 2 as indicated. Reprinted from Zomzely et al. (26).

overall rate of protein synthesis which may exist in different cerebral regions of the mature animal in vivo were not related to differences in polyribosomal capacity. Although the rates of amino acid incorporation in vitro by cerebral and hepatic ribosomes from the adult rat were quite comparable, the cerebral preparations exhibited certain distinctive properties in protein synthesis. These properties appeared to be related to the unique instability of certain cerebral messenger RNA-ribosome complexes. The concentrations of enzymes, cofactors, amino acids and ions required for maximum incorporation of amino acids into protein of cerebral ribosomal systems were critical (21, 29). Endogenous amino acid-incorporating activity of cerebral mixed ribosomes was maximal in the presence of 10-12 mM Mg2+ and 80 to 100 mM K+ and was greatly reduced by alterations in these concentrations. The analogous

452

NEURAL

GROWTH

AND

(a) Cerebral cortex 0.03 AJg RNase

A

80si

\

DIFFERENTIATION 1— 1 1.0 - ' 8 0« s ' ' (c) Cerebral cortex |i 1 jug RNase ! l'\ 1 ' ^ 0.5 • A i A "80s1 fx

0.6

(b) Liver 0.03 jug RNase

(d) Liver Ijug RNase

0.4 - !

1

'80 s'/)

V h

! / 0.2 1 1 1/ // 30 BOTTOM

^ v

0 TOP

—

10

i

20

iV 30 BOTTOM

Gradient effluent (ml) Figure 20.4. Influence of pancreatic ribonuclease on cerebral and hepatic polyribosomes from young adult rats. Polyribosomes were suspended in medium containing 10 mM magnesium acetate, 40 mM NaCl, 100 mM KC1, and 20 mM Tris-HCl buffer, pH 7.6. Pancreatic RNase (Sigma) was added to some of the samples, which were then incubated under various conditions. Each sample was subsequently layered on a linear sucrose gradient containing the same buffer and salts as the suspension medium, and centrifuged in a Spinco S W 25 swinging bucket rotor at 25,000 rpm and 0°. Absorbance of the effluent collected from the top of the gradient was monitored continuously at 254 m/j,. ( a ) Cerebral polyribosomes, centrifuged for 1% hrs in a 25 to 5% linear sucrose gradient: , untreated; , incubated with 0.03 /J.g of RNase per mg of RNA for 30 min at 0°. ( b ) Hepatic polyribosomes from rats fasted for 18 hrs centrifuged for IK hrs in a 25 to 5% linear sucrose gradient: , untreated; , incubated with 0.03 /xg of RNase per mg of RNA for 3 0 min at 0°. ( c ) Cerebral polyribosomes, centrifuged for 1/2 hrs in a 35 to 15% linear sucrose gradient: , untreated; , incubated with 1 fig of RNase per mg of RNA for 30 min at 0°. (d) Hepatic polyribosomes, centrifuged for /2 hr in a 3 5 to 15% linear sucrose gradient: , untreated; , incubated with 1 /J.g of RNase per mg of RNA for 3 0 min at 0°. (From Zomzely et al., 28.) h e p a t i c p r e p a r a t i o n s w e r e m u c h less sensitive t o ionic variations. R e l a t i v e l y h i g h c o n c e n t r a t i o n s of M g 2 + m a y e x e r t t h e i r s t i m u l a t o r y effects on p r o t e i n synthesis in t h e m o r e labile c e r e b r a l s y s t e m s b o t h b y r e d u c i n g t h e dissociation of m e s s e n g e r R N A f r o m p o l y r i b o s o m e s a n d b y inhibiting r i b o n u c l e a s e activity. Striking differences w e r e n o t e d in t h e kinetics of i n c o r p o r a t i o n of [ 1 4 C ] p h e n y l a l a n i n e into p r o t e i n of different r i b o s o m a l species in p r e p a r a t i o n s isol a t e d f r o m c e r e b r a l c o r t e x a n d liver of t h e y o u n g a d u l t r a t ( 2 8 ) . A l t h o u g h l a r g e r i b o s o m a l a g g r e g a t e s w e r e p r e s e n t in p r e p a r a t i o n s of c e r e b r a l m i x e d r i b o s o m e s only in t r a c e a m o u n t s , t h e specific r a d i o a c t i v i t i e s of t h e s e a g g r e g a t e s w e r e r e l a t i v e l y q u i t e h i g h d u r i n g t h e e a r l y p h a s e s of i n c u b a t i o n ( F i g -

CEREBRAL

40 -

RIBONUCLEIC

ACID

ALTERATIONS

(a) Cerebral cortex

io i O

453

(b) Liver

"i

132

%

71 100 123 152 172 >172

6 10 10 9 7 58

0

46 62 89 114 153 >153

5 17 9 9 8 52

54 79 99 176»

< 1 < 1 1 98

15

63 88 110 130 147 >147

30 9 10 12 9 30

48 71 95 151»

< 1 < 1 1 98

31 10 9 8 42

58 76 101 152»

< 1 < 1 1 98

—

30

59 89 112 129 148»

< 1 < 1 1 98

Conditions were those described in the legend to Figure 20.4. In addition to the results of the incubation experiments, analytical data are also given for cerebral and hepatic polyribosomes suspended in medium not containing pH 5 enzymes or cofactors (-). "Average value for polyribosomes. Reprinted from Zomzely et al. (28).

protein synthesis in situ (Figure 20.8). However, even at this early time interval ribosomes lighter than the tetramer accounted for more than 60 per cent of the ribosomal-bound radioactivity. Moreover, after 15 minutes, most of the radioactivity was found at the top of the gradient where monoribosomes, ribosomal subunits, and soluble protein were located. A similar, though less dramatic, shift of radioactivity was observed in preparations from cerebral white matter of the young adult rat. In contrast, relatively large polyribosomes present in hepatic postmitochondrial supernatants from similar animals continued to incorporate most actively over the entire 15 min period following intraportal injection of labeled amino acid (Figure 20.8). These observations suggested that cytoplasmic ribosomal systems in the

CEREBRAL

RIBONUCLEIC TABLE

ACID

ALTERATIONS

455

20.3

STIMULATION OF THE Escherichia coli (S-30 FRACTION) AMINO ACID-INCORPORATING SYSTEM BY CEREBRAL AND HEPATIC R N A PREPARATIONS Amino acid incorporation (counts/ min./mg. of protein) 0 . 2 5 MC of L - [ U - " C ] p h e n y l a l a n i n e (392 mC/m-mole) None Cerebral nuclear R N A (51 Mg.) Cerebral ribosomal R N A (68 Mg.) Hepatic nuclear R N A (68 Mg.) 0 . 2 5 M C of L-[U- 1 4 C]lysine (247 mC/m-mole) None Cerebral nuclear R N A (51 Mg-) 0 . 0 5 M C of L-[U- 1 4 C]leucine (280 mC/m-mole) None Cerebral nuclear R N A (51 g.)

1480 4880 1842 3240 885 1723 1027 1920

T h e reaction mixture (0.25 ml.) contained 100 m M - T r i s - H C l (pH 7.8), 50 m M - K C l , 12 m M - m a g n e s i u m acetate, 1 m M - N a A T P , 0 . 0 1 5 m M - G T P (sodium salt), 10 m M - c r e a t i n e phosphate (sodium compound), 6 m M - m e r c a p t o e t h a n o l , 0.12 mg. of creatine phosphokinase, 0.1 m M concentrations of each of 20 L - a m i n o acids except the [ I 4 C]-labeled amino acid and 1.17 mg. of S-30 protein. I n c u b a t i o n was carried out for 40 min. at 37°. Reprinted from B o n d y and R o b e r t s (5).

brain of the mature rat possessed several unique characteristics: (i) unusual instability of certain large messenger RNA-ribosome complexes, (ii) significant activity of relatively small ribosomes in protein synthesis and (iii) rapid turnover of proteins assembled on certain large polyribosomes, coupled with slower formation of polypeptides on the smaller ribosomes. Further evidence for these postulates is presented below. DEVELOPMENTAL ALTERATIONS IN CEREBRAL PROTEIN-SYNTHESIZING SYSTEMS

The experiments described above indicated that ribosomal protein synthesis in the brain of the adult rat, as in other cells, was most active on the larger ribosomal aggregates. Murthy (17) noted that preparations of brain polyribosomes from newborn rats contained a higher proportion of heavy aggregates than similar fractions from adult animals. He suggested that this phenomenon was the basis for observations which indicated that the capacity of brain ribosomal systems for amino acid incorporation in vitro may diminish with age (3, 14, 17, 18, 25). However, Johnson and Belytschko (14) reported only minor alterations in sedimentation profiles of cerebral ribosomes isolated from mice at various stages of development. In the present studies, ribosomal aggregation in preparations from rat cerebral cortical gray matter of the rat decreased during maturation (Figure 20.9). A similar trend was observed in hindbrain-medullary white matter (C. E. Zomzely, S. Roberts, S. Peache and D. M. Brown, unpublished obser-

456

NEURAL

GROWTH

AND

DIFFERENTIATION

Incubation time (min) Figure 2 0 . 6 . Kinetics of incorporation of [L- 1 4 C ] phenylalanine by polyribosomes isolated from cerebral cortical gray and hindbrain-medullary white matter of 14-day-old and 42-day-old (adult) rats. The p H 5 enzyme fraction was derived from cerebral gray matter of adult animals. T h e incubation system contained 0 . 5 - 1 . 0 mg of ribosomal protein, 2-4 mg of p H 5 enzyme protein, 2 mM ATP (sodium salt), 0 . 2 5 mM GTP (sodium salt), 2 0 mM creatine phosphate (sodium salt), 0.1 mg of creatine phosphokinase, 5 0 mM Tris-HCl buffer ( p H 7 . 4 ) , 1 2 mM MgCl 2 , 1 0 0 mM KC1 and 1 U C , ( 1 0 s c p m ) of uniformly labeled [ L - 1 4 C ] phenylalanine. Final volumes were 1 ml. E a c h value represents the average ± S.E. of three analyses. • , polyribosomes from cerebral gray matter; O , polyribosomes from cerebral white matter.

vations). These differences were not associated with significant alterations in ribonuclease activity of the cerebral postmitochondrial supernatants from which the polyribosomes were prepared. As noted earlier, the purified polyribosomes per se were devoid of measureable ribonuclease activity (see 28). Nevertheless, developmental differences in ribosomal aggregation in the various cerebral preparations were accompanied by corresponding variations in polyribosomal stability both in vitro and in vivo. Thus, polyribosomes isolated from cerebral cortical gray matter of 14- or 21-day-old (immature) rats exhibited a degree of disaggregation in medium containing 1 mM Mg2+ which was intermediate between that of cerebral polyribosomes from infant rats (0 to 2 days old) and mature animals (42 days old).° Polyribosomes isolated from the hindbrain-medullary region at 14 days of age were quite stable at low concentrations of Mg2+. However, in the adult rat polyribosomes derived from either cerebral gray or white matter revealed similar properties. These data suggested that polyribosomal instability was related to cellular maturation in both neurons and glia. Polyribosomes isolated from cerebral cortical gray matter of rats varying " C. E. Zomzely, S. Roberts, S. Peache, and D. M. Brown, unpublished observations.

CEREBRAL

RIBONUCLEIC

ACID

Gradient effluent

ALTERATIONS

457

(ml)

Figure 20.7. Incorporation of [ 1 4 C] phenylalanine into different species of cerebral and hepatic mixed ribosomes obtained from young adult rats. The incubation systems were similar to those described in the legend for Figure 20.6. After incubation, approximately 0.5 mg of ribosomal protein, in 2 ml of medium, was layered on a linear sucrose gradient (25 to 5%) containing the same buffer and salts as the incubation medium. The samples were centrifuged for 3 hrs in a Spinco SW 25 swinging bucket rotor at 25,000 rpm and 0°. The effluent from the top of the gradient was monitored continuously for absorbance at 254 m/x and collected as 1-ml samples for determination of radioactivity. Specific activity profiles are shown. O, incubated for 15 min; # , incubated for 30 min. (From Zomzely et al., 28.)

in age from 0 to 24 days possessed similar capacities for the incorporation of amino acids into protein in vitro, particularly during the early phases (30-60 minutes) of incubation (Figure 20.10). However, after incubation for 60 to 90 minutes, amino acid incorporation by cerebral cortical polyribosomes from adult rats was depressed below that of the corresponding preparation from newborn rats. This delayed difference in incorporation might be related to variations in polyribosomal stability with age. Rates of amino acid incorporation by polyribosomal preparations from cerebral white matter were comparable to those observed with gray matter in both immature and adult animals (Figure 20.6). Investigations of developmental variations in cerebral protein synthesis in vivo were carried out with rats given L-[(4,5)- 3 H]leucine by intracisternal injection and killed 5 or 15 minutes later. The heaviest ribosomal aggregates in postmitochondrial supernatants prepared from cerebral cortical gray matter were initially the most active in rats of all ages (Figures 20.8 and 20.11). After 15 minutes most of the radioactivity in ribosomal protein was attached to lighter ribosomes in preparations from adult animals (Figure 20.8). Similar results were obtained in the 14-day-old animal. In con-

458

NEURAL

GROWTH

AND

DIFFERENTIATION

Gradient effluent(ml)

Figure 20.8. Protein-synthesizing activities of cerebral and hepatic polyribosomes of mature rats in vivo: relationship to state of aggregation. Cerebral postmitochondrial supernatants were prepared from young adult rats given an intracisternal injection of uniformly labeled L-[ 1 4 C] leucine (10 ju-C,) 5 or 15 min before autopsy. Hepatic postmitochondrial supernatants were prepared from rats given an injection via the portal vein of 20 /¿C^ of [ I4 C]-leucine 1 or 15 min earlier. Generally, the injections were given under ether anesthesia and the animals were killed by decapitation. Postmitochondrial supernatants were layered on linear sucrose density gradients (35 to 15%). The gradients were then centrifuged for 4 hrs in a Spinco SW 25 swinging bucket rotor at 25,000 rpm and 0°. The effluents from the top of the gradient were monitored continuously for absorbance at 254 m/i and collected as 1-ml samples for determination of radioactivity. Optical density, ; radioactivity • • . (From Zomzely et al., 28.)

trast, radioactivity from leucine remained associated with the heavy polyribosomes in cerebral cortical preparations from newborn animals even at the later time interval ( F i g u r e 2 0 . 1 1 ) . T h e kinetics of amino acid incorporation in vivo into ribosomes of hindbrain-medullary white m a t t e r of the mature rat were similar to those observed with analogous preparations from cerebral cortex.* However, a major portion of the ribosomal-bound radioactivity was still attached to heavy aggregates in cerebral white matter of 14* C. E. Zomzely, S. Roberts, S. Peache, and D. M. Brown, unpublished observations.

CEREBRAL

RIBONUCLEIC

ACID

ALTERATIONS

459

Figure 20.9. Sucrose density gradient profiles of polyribosomes isolated from cerebral cortical gray matter of immature and mature rats. Polyribosomes were analyzed as described in the legend to Figure 20.4. Fourteen-day-old rats, ; adult (42-day-old) rats, .

Gradient Effluent (ml) day-old animals 15 minutes after the injection of [3H]-leucine (Figure 20.12). Thus, the relatively high stability in vitro of the larger messenger RNA-ribosome complexes from cerebral cortex of newborn rats and from cerebral white matter of 14-day-old animals was reflected in the prolonged capacity of these polyribosomes for incorporation of amino acids in vivo. Although purified polyribosomes from cerebral cortical gray matter and hindbrain-medullary white matter at different stages of development in the rat exhibited remarkably similar rates of amino acid incorporation in vitro,

Incubation

time

(min)

Figure 20.10. Kinetics of incorporation of L-[ 1 4 C] phenylalanine by polyribosomes isolated from cerebral cortical gray matter of rats at different stages of development. The pH 5 enzyme preparation used in each case was derived from cerebral cortices of adult rats. See legend to Figure 20.5 for additional explanations. Polyribosomes from adult rats (42-day-old) are shown as open circles ( O ) in each case.

460

NEURAL

GROWTH

AND

Gradient E f f l u e n t (ml

DIFFERENTIATION Figure 20.11. Protein-synthesizing activities of polyribosomes of cerebral cortical gray matter of newborn rats in vivo: relationship to brain development and ribosomal aggregation. Postmitochondrial supernatants were prepared from cerebral cortices of rats given an intracisternal injection of 25 /J.C-, of L-[(4,5)- 3 H]leucine, 5 or 15 min before autopsy. The postmitochondrial supernatants were prepared, centrifuged in sucrose density gradients, and analyzed as described in the legend to Figure 20.8. Results are expressed in terms of specific activity; i.e., cpm per optical density unit at 254 m/x ( x 10~2). O, 5 min after injection; # , 15 min after injection.

differences in the capacity of these tissue for protein synthesis might exist in situ. Such differences could result from variations in polyribosomal stability, in the capacity of cerebral ribosomes for continued incorporation of amino acids, or in pools of precursor amino acids (10, 11, 20) and active ribosomes. The total pool of ribosomes in cerebral postmitochondrial supernatants was similar in all samples. However, concentrations of free amino acids varied greatly, partly as a result of striking differences in amino acid utilization in cerebral gray and white matter during development (Table 20.4). Differences in precursor pool size were minimized by relating leucine incorporation into protein in vivo to the specific activity of free leucine. On the basis of these relative incorporation values, cerebral cortical gray matter and hindbrain-medullary white matter from the adult rat appeared to be at least as active in protein synthesis as the analogous brain regions from younger ani-

Gradient Effluent(ml)

Figure 20.12. Protein-synthesizing activities of polyribosomes of hindbrain-medullary white matter of immature rats in vivo: relationship to brain development and ribosomal aggregation. Postmitochondrial supernatants were prepared from cerebral white matter of 14-day-old rats given an intracisternal injection of 25 fiCi of L-[ (4,5)- 3 H]leucine, 5 or 15 min before autopsy. See legends to Figures 20.8 and 20.11 for additional explanations. Results are expressed as cpm per optical density unit at 254 m/x ( x l O 3 ) . O, 5 min after injection; 15 min after injection.

CEREBRAL

RIBONUCLEIC T A B L E

ACID 20.4

I N F L U E N C E O F D E V E L O P M E N T ON T H E C O N V E R S I O N O F

3

H - L E U C I N E TO

A M I N O A C I D S I N C E R E B R A L C O R T I C A L G R A Y M A T T E R AND MEDULLARY WHITE

461

ALTERATIONS

M A T T E R OF THE INTACT

OTHER

HINDBRAIN-

RAT

Conversion* Age

Time after injection —

Gray matter

min 5 15 5 15 5 15

days newborn 14 42

White matter

%

%

trace 2.3 trace trace 20.3 41.7

— —

11.8 42.5 27.8 44.3

* Radioactive products formed from [4, 5- 3 H] leucine included phosphoserine, taurine, glutamic acid, and glutamine.

mals (Table 20.5). Moreover, the data suggested that the rate of protein synthesis in cerebral white matter was not lower than that in cerebral gray matter in either immature or adult animals. Radioactivity incorporated into total protein of postmitochondrial supernatants obtained from cerebral cortical tissue of the newborn rat increased during the 5- to 15-minute period following injection of the isotopic leucine (Table 20.5). In contrast, radioactivity incorporated into total protein may

T A B L E

20.5

D E V E L O P M E N T A L V A R I A T I O N S IN I N C O R P O R A T I O N OF

in vivo

Age

Time after injection

days

min

Cerebral cortical gray newborn 14 42 Cerebral white 14 42

L-[(4,5)-3H]LEUCINE

INTO P R O T E I N S OF C E R E B R A L POSTMITOCHONDRIAL

matter

matter 5 15 5 15 5 15 5 15 5 15

SUPERNATANTS

Radioactivity in protein Ribosomal

Soluble

Total

cpm/g tissue X10"3

cpm/g tissue xio-3

cpm/g tissue XIO" 3

30 .9 28. .9 11..2 9. .5 23 .4 14 .0

25. .5 36. 8 17..5 10..4 37 .2 26. .9

56. ,4 65. .7 28 .7 19.,9 60 .6 40 .9

52 .0 81 .5 113 107

88 .2 85 .2 143 206

140 167 256 313

Relative incorporation*

4 7 12 21 26 104 20 76 73 147

* Radioactivity incorporated into total protein (cpm/g tissueX 10~ 3 )-r radioactivity in free leucine (cpm/VmoleX10~ 6 ).

462

NEURAL

GROWTH

AND

DIFFERENTIATION

have declined after 5 minutes in analogous samples from mature animals. Approximately two-thirds of the protein synthesized on polyribosomes of cerebral cortical gray matter in the adult rat appeared as soluble protein during the 15-minute interval. Percent of soluble protein released was lowest in samples from cerebral cortical gray matter of the newborn rat and intermediate in value for the immature animal. These data suggested that the proportion of stable proteins synthesized on cerebral cortical polyribosomes decreased with age, even though overall synthesis of protein did not seem to be markedly diminished. A high proportion of the proteins synthesized by mature cerebral cortex were formed on large, unstable mRNA-ribosome complexes and turned over rapidly (compare Figures 20.8 and 20.11). Smaller ribosomes appeared to become progressively more active in the synthesis of relatively stable proteins in the developing brain. This possibility was supported by the finding that nuclear RNA preparations from adult rat brain contained a large fraction of messenger RNA species of relatively small molecular size (including species with sedimentation coefficients of 12 S or lower) which stimulated amino acid incorporation into proteins of brain ribosomes in vitro (12, 23) (Table 20.6). Investigations of the biosynthesis and metabolism of cerebral ribonucleic acids during development have generally produced inconclusive results. Advancement of knowledge in this area has been limited by difficulties in the fractionation and purification of various nucleic acids, instability of the isolated preparations, lack of knowledge of precursor pools and compartments, and unavailability of definitive methods for measurement of messenger RNA. Nevertheless, considerable evidence indicates that the synthesis and

T A B L E

20.6

S E D I M E N T A T I O N C H A R A C T E R I S T I C S AND S T I M U L A T O R Y OF N U C L E A R R N A

Addition

None Nuclear R N A >34 S '28 S' '18 S'