Progress in Brain Research Volume 15 Biology of Neuroglia

P R O G R E S S I N B R A I N RESEARCH VOLUME 1 5 BIOLOGY OF NEUROGLLA

PROGRESS IN BRAIN RESEARCH

ADVISORY BOARD W. Bargmann H. T. Chang

E. De Robertis J. C. Eccles

J. D. French

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

J. P. Schadt F. 0. Schmitt

Kiel Shanghai Buenos Aires Canberra Los Angeles

Goteborg Amsterdam Moscow Amsterdam B rookline (Mass.)

T. Tokizane

Tokyo

H . Wael sch

New York

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 15

BIOLOGY O F NEUROGLIA EDITED B Y

E. D. P. DE ROBERTIS Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires (Argentina) AND

R. C A R R E A Instituto Torcuato di Tella. Centre of Neurological Investigations, Buenos Aires (Argentina)

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

LONDON

1965

1 NEW

YORK

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

335 J A N VAN GALENSTRAAT, P.O. BOX 211, A M S T E R D A M

A M E R I C A N E L S E V I E R P U B L I S H I N G COMPANY, INC. 52 V A N D E R B I L T AVENUE, N E W Y O R K , N.Y. 10017

ELSEVIER PUBLISHING COMPANY LIMITED R I P P L E S I D E C O M M E R C I A L ESTATE R I P P L E ROAD, B A R K I N G , ESSEX

This volume contains the lectures delivered and the discussion that followed during a symposium on BIOLOGY O F NEUROGLIA

which was held as apart of the 10th Latin-AmericanCongress of Neurosurgery at the Academia Nacional de Medicina in Buenos Aires from 17-18 October, 1963. This Symposium was sponsored by: The Instituto Torcuato di Tella, Buenos Aires (Argentina); The International Brain Research Organization;The Centro de Cooperacidn Cientifca para America Latina de la UNESCO; and partially supported by grants from: The National Institutes of Health, U.S.Dept. of Health, Education and Welfare ( N B 0473541);The Consejo Nacional de Investigacione Cientifcas y Tecnicas, Argentina (No. 1423)

L I B R A R Y O F C O N G R E S S C A T A L O G C A R D N U M B E R 64-18525

W I T H 1 8 1 I L L U S T R A T I O N S A N D 2 3 TABLES

ALL RIGHTS RESERVED T H I S BOOK O R A N Y P A R T T H E R E O F M A Y N O T B E R E P R O D U C E D I N A N Y F O R I N C L U D I N G P H O T O S T A T I C O R M I C R O F I L M FORM, WITHOUT WRITTEN PERMISSION F R O M T H E PUBLISHERS

PRINTED IN THE NETHERLANDS

List of Contributors

L. BAKAY, The Division of Neurosurgery, State University of Jew Tork at Buffa Medical School, Buffalo General Hospital, Buffalo, N. Y. (U.S.A.).

0

M. BRADBURY, Medical Research Council, University College London, London. H. COLLEWIJN, Netherlands Central Institute for Brain Research, Amsterdam. H. DAVSON, Medical Research Council, University College London, London. Institute of General Anatomy and Embryology, Univei sity of E. D. P. DE ROBERTIS, Buenos Aires, Buenos Aires. B. D. DRUJAN, Department of Neurobiology, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas. R. FATEHCHAND, Department of Neurobiology, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas. R. L. FRIEDE, Mental Health Research Institute and Department of Pathology, The University of Michigan, Ann Arbor, Mich. (U.S.A.). R. GALAMBOS, Department of Psychology, Yale University, New Haven, Conn. (U. S.A.). C. A. GARCIAARGIZ,Department of Biochemistry, Faculty of Pharmacology and Biochemistry, University of Buenos Aires, Buenos Aires.

W. HAYMAKER, National Aeronautics and Space Administration, Ames Research Center, Moffett Field, Calif. (U.S.A.). I. KLATZO,Section of Neuropathology, Surgical Neurology Branch, National Institute of Neurological Diseases and Blindness, Bethesda 14, Md. (U.S.A.). A. LASANSKY, Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires.

E. LEVIN,Department of Biochemistry, Faculty of Pharmacology and Biochemistry, University of Buenos Aires, Buenos Aires. J. MIQUEL,National Aeronautics and Space Administration, Ames Research Center, Moffett Field, Calif. (U.S.A.). K. NEGISHI,Department of Neurobiology, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas. G. J. NOGUEIRA, Department of Biochemistry, Faculty of Pharmacology and Biochemistry, University of Buenos Aires, Buenos Aires.

VI

LIST OF CONTRIBUTORS

H. M. PAPPIUS,The Donner Laboratory of Experimental Neurochemistry, Montreal Neurological Institute and the Department of Neurology and Neurosurgery, McGill University, Montreal.

M. POLAK,Fundacion Roux-Ocefa, Laboratory of Histological and Histopathological Investigations, Buenos Aires.

J. P. SCHADB,Netherlands Central Institute for Brain Research, Amsterdam. DE TESTA,Department of Neurobiology, Instituto Venezolano de InvestigaA. SELV~N ciones Cientificas (IVIC), Caracas.

D. E. SMITH,Section of Neuropathology, Surgical Neurology Branch, National Institute of Neurological Diseases and Blindness, Bethesda 14, Md. (U.S.A.). Department of Neurobiology, Instituto Venezolano de Investigaciones G. SVAETICHIN, Cientificas (IVIC), Caracas. I. TASAKI,Laboratory of Neurobiology, National Institutes of Mental Health, National Institutes of Health, Bethesda, Md. (U.S.A.).

F. WALD,Department of Biophysics and Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires. H. WI~NIEWSKI, Section of Neuropathology, Surgical Neurology Branch, National Institute of Neurological Diseases and Blindness, Bethesda 14, Md. (U.S.A.). J. A. ZADUNAISKY, Department of Biophysics and Institute of General Anatomy and Embryology, University of Buenos Aires, Buenos Aires.

VII

Other volumes in this series:

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

Volume 2: Nerve, Brain and Memory Models Edited by Norbert Wiener? and J. P. SchadC Volume 3 : The Rhinencephalon and Related Structures Edited by W . Bargmann and J. P. Schade Volume 4: Growth and Mafurafionof the Brain Edited by D. P. Purpura and J. P. Schade

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

Volume 6: Topics in Basic Neurology Edited by W. Bargmann and J. P. Schade Volume 7: Slow Electrical Processes in the Brain by N . A. Aladjalova Volume 8 : Biogenic Afnines Edited by Harold E. Himwich and Williamina A. Himwich

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

Volume 10: The Strucfrrre and Function of the Epiphysis Cerebri Edited by J. Ariens Kappers and J. P. SchadC

Volume 1 1 : Organization of the Spinal Cord Edited by J. C. Eccles and J. P. SchadC Volume 12 : Physiology of Spinal Neurons Edited by J. C. Eccles and J. P. SchadC

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

VIlI

Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P. Schad6 Volume 16 : Horizons in Neuropsychopharmacology Edited by Williamina A. Hmwich and J. P. SchadC Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener? and J. P. Schade Volume 18: Sleep Mechanisms Edited by K. Akert, Ch. Bally and J. P. SchadC Volume 19: Experimental Epilepsy by A. Kreindler Volume 20: Pharmacology and Physiology of the Reticular Formation Edited by A, V. Valdman Volume 21 : Correlative Neurosciences Edited by T. Tokizane and J. P. SchadC Volume 22: Brain Reflexes Edited by E. A. Asratyan Volume 23 : Sensory Mechanisms Edited by Y . Zotterman Volume 24: Carbon Monoxide Poisoning Edited by H. Bour and I. McA. Ledingham Volume 25 : The cerebellum Edited by C . A. Fox and R. S. Snider Volume 26 : Developmenfa1 Neurology

IX

Preface

The Latin-American Congress of Neurosurgery has been regularly held every two years since 1943 and a Symposium on Neurological Research has been added to each Congress in recent years. The Executive Committee of the 10th Latin-American Congress of Neurosurgery, presided over by Professor Ricardo Morea, decided upon our suggestion that a suitable subject for the 1963 Buenos Aires meeting would be the Biology of Neuroglia and recommended that some related aspects of brain edema should be included. There were three justifications for the choice of this subject: an interdisciplinary presentation on the subject had not been held since the 1956 Conference on the Biology of Neuroglia, organized and edited by W. F. Windle; there was a group of local investigators who could contribute their own experience to the Symposium, and finally, this was a subject of obvious interest to the predominantly neurosurgical audience of the Congress. The responsibility of selecting the subjects and the speakers and of conducting the Symposium was given to us as Coordinator and Secretary of the Symposium, respectively. The Executive Committee of the Congress deserves our gratitude for giving us complete freedom to carry out this responsibility. In the planning of the Symposium, we formulated a program including the morphological, biochemical and neurophysiological aspects of the problem. A small group of distinguished investigators were included who could cover different aspects of these three major subdivisions of the subject. This volume presents enlarged versions of the lectures held, some of which are of a review type as encouraged by Progress in Brain Research. This provides the readers with complementary detailed information, therefore, which could not be made available to the audience of the Symposium. An unrestricted opportunity was given to the members of the Symposium and the invited discussants to express their ideas at the end of each of three sessions. A fairly accurate version of the recorded discussion is also included in this volume at the points where it actually took place, i.e. after each of the sessions and at the end of the Symposium. A considerable amount of additional information can be gathered from this material. The Symposium began with some introductory remarks by Antonio de Veciana from the UNESCO’s Centro de Cooperaci6n Cientifica para America Latina and by Guido Di Tella, Vice-president of the Instituto Torcuato di Tella, both representing the institutional interest in a meeting of this nature. Prof. Bernard0 A. Houssay, President of the Consejo Nacional de Investigaciones Cientificas y Tecnicas of Argentina gave the opening address outlining the history of knowledge about neuroglia. This Symposium was made possible through the joint efforts of the institutions represented

X

PREFACE

by these three speakers and by the U.S. Department of Health, Education and Welfare, Public Health Service (NB 04735-OI), the International Brain Research Organization and the Executive Committee of the 10th Latin-American Congress of Neurosurgery. It was agreed upon from the beginning that the Secretary of the Symposium would not participate in the discussion no matter how tempting it would be to do so. This decision was made not only because the research subject was not his own but also because we thought it more important that one of us should concentrate on keeping the meeting running smoothly. The entire material for this volume was, however, in his hands - which is why it became mandatory for him to be one of the Editors. Last but not least we wish to express our gratitude to our secretary, Mrs. Joan Firmat, who assisted in so many secretarial and other duties. E. DE ROBERTIS R. CARREA

XI

Contents

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

List of contributors Preface

v IX

Some new electron microscopical contribations to the biology of neuroglia (Introduction) 1 E.D.P. De Robertis (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . . . Morphological and functional characteristics of the central and peripheral neuroglia (light microscopical observations) M. Polak (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Enzyme histochemistry of neuroglia R. L. Friede (Ann Arbor, Mich.) . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Functional implications of structural findings in retinal glial cells A. Lasansky (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Observations on penetration of serum proteins into the central nervous system I. Klatzo, H. WiSniewski and D. E. Smith (Bethesda, Md.) . . . . . . . . . . . . . . . 73 Astroglial reactions to ionizing radiation : with emphasis on glycogen accumulation J. Miquel and W. Haymaker (Moffett Field, Calif.) . . . . . . . . . . . . . . . . . . 89 First discussion period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 The extracellular space of the brain H. Davson and M. Bradbury (London) . . . . . . . . . . . . . . . . . . . . . . . 124 The distribution of water in brain tissues swollen in vitro and in vivo H. M. Pappius (Montreal) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The movement of electrolytes and albumin in different types of cerebral edema L. Bakay (Buffalo, N.Y.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Changes in the size of astrocytes and oligodendrocytes during anoxia, hypothermia and spreading depression H. Collewijn and J. P. Schadk (Amsterdam) . . . . . . . . . . . . . . . . . . . . . Osmotic behaviour and glial changes in isolated frog brains J. A. Zadunaisky, F. Wald and E. D. P. De Robertis (Buenos Aires) . . . . . . . . . Some aspects of amino acid transport in the central nervous system E. Levin, G. J. Nogueira and C. A. Garcia Argiz (Buenos Aires) . . . . . . . . . . . Second discussion?period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excitability of neurons and glial cells I. Tasaki (Bethesda, Md.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nxvous function based on interactions between neuronal and non-neuronal elements G. Svaerichin, K. Negishi, R. Fatehchand, B. D. Drujan and A. Selvin de Testa (Caracas) Introductory discussion on glial function R. Galambos (New Haven, Conn.) . . . . . . . . . . . . . . . . . . . . . . . . . General discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions of the symposium E. D. P. De Robertis (Buenos Aires) . . . . . . . . . . . . . . . . . . . . . . . .

135 155

184

. 196

. 219 225 234

. 243 267 278 284

Author Index.

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287

Subject Index.

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1

Some New Electron Microscopical Contributions to the Biology of Neuroglia INTRODUCTION

E D U A R D O D. P. DE ROBERTIS

Institute of General Anatomy and Embryology, University of Biienos Aires, Buenos Aires (Argentina)

When I was asked by the Committee of the 10th Latin-American Congress of Neurosurgery to organize an International Symposium of Neurological Research, as has been done with great success at the previous Congress in Mexico, I thought that a discussion on the Biology of Neuroglia might be of interest to all participants in the Congress. In fact, glia cells contribute greatly to the pathology and clinical treatment of neurological diseases and certainly to most of the surgical treatment. Furthermore, one of the main problems that neurosurgery has to tackle as a complication of traumatisms, tumours or toxic lesions, is that of brain oedema and as will be shown at this Symposium, this pathological condition depends to a certain extent on the glia cells and on their relationship to the blood vessels of the brain. The different types of glial cells constitute quantitatively an important part of the central nervous system (CNS), but until recent years their special functions and relationships with the neurons and vascular elements were practically unknown. The classical work of the Spanish school (Cajal, del Rio Hortega) was fundamental in defining the different types of glial elements -astroglia, oligodendroglia, microglia -and their histogenic relationships, but unable to throw definite light on their physiological significance. Only in recent years, with the development of new cytochemical and ultrastructural techniques and the use of physiological methods for the study of movement of ions and water between the different compartments, of microphysiological techniques for recording potentials in the glia membranes, and the biochemical studies on isolated glial cells in different physiological conditions, has new light been thrown on the role of glial cells in myelination, brain permeability and metabolism. In addition, the possible participation of glial cells in more complex electrophysiological mechanisms of the brain has been suggested. In a review on the ‘Submicroscopic Morphology and Function of Glial Cells’ De Robertis and Gerschenfeld (1961) analyzed some of the possible functions of these cells. Interfascicular OIigodendroglia of the white matter is recognized as having a prevaReferences p . I 1

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E. D. P. DE R O B E R T I S

lent role in the formation, maintenance and disposal of myelin and thus to be the main point of attack of agents that may cause demyelinating diseases. The possible role of perineuronal oligodendroglia has been mainly studied by HydCn and coworkers (Hyden and Pigon, 1960; HydCn and Lange, 1962; Hamberger and Hyden, 1963), who have obtained data indicating a possible metabolic interaction with the neuronal elements. These studies suggest that neurons and perineuronal oligodendroglia cells are linked in an energetic system, which may react as a functional unit. Astroglia is thought to be involved in the transport of water, electrolytes and metabolites within the brain and to be the site of an active homeostatic mechanism that regulates the content of water and prevents swelling of this tissue. The cell appears to be involved also in the physiologic barriers of the brain including the blood-brain barrier (BBB) and possibly the liquor-brain barrier (LBB) with the cerebrospinal fluid. In the above-mentioned review, together with the data on the bioelectric activity of astrocytes, we also discussed astroglia as a possible pool of electrolytes probably containing a high sodium content. The impact that some of these new concepts have on the physiology of the brain, was reflected on the postulation by Galambos (1961) of a continuous physiological interaction between glial cells and neurons in the electrophysiological and more complex brain functions. The recent evidence supplied by Hydkn and Egyhiizi (1963) that the RNA of glial cells may change during learning experiments in rats, adds even more interest to this new field of research and I am only sorry that Prof. HydCn could not accept our invitation to participate in the Symposium. Since some of the recent experimental works from our laboratory referring to glial cells in the retina and to the ependymoglial cells of amphibian brain will be presented by Dr. Lasansky and Dr. Zadunaisky, I would like to mention here only some of the newer contributions that electron microscopy is making to this field of study. ( a ) A formalin perfusion Jixation method f o r the study of brain First of all I would like to mention to you briefly the method for perfusion fixation with formaldehyde that has been recently developed in our laboratory (Gonzalez Aguilar and De Robertis, 1963) and which permits an excellent preservation of the entire CNS for electron microscopy. For the development of this technique a study was made on the water changes of the tissue after fixation and the components of the solutions for washing of the blood and fixation were so adjusted as to leave the water Fig. 1 . Diagram showing some of the concepts discussed by De Robertis and Gerschenfeld (1961) on the relationshipof astroglia with other cellular components of the CNS and its possible function. In the centre an astroglial cell with the clear cytoplasm and processes that make contact with the basal membrane (bas. m.) of a blood capillary (Cap), with the pial membrane and a neuron. This astroglial cell is supposed to be involved in the blood-brain (BBB), the liquor-brain barrier (LBB) with the cerebrospinal fluid (CSF), and the synaptic barrier (SB). The possible movement of fluids within the astroglial cytoplasm is indicated with arrows. The neuron has surface relationship with astroglia, oligodendroglia (oligo), and the synaptic endings. A microelectrode (elect) recording extraneuronally is supposed to be implanted in a glial process (end = capillary endothelium). This diagram emphasizes the small extracellular space. (For further description see De Robertis and Gerschenfeld, 1961.)

ELECTRON MICROSCOPICAL CONTRIBUTIONS

Fig. 1. For legend see p. 2.

3

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E. D. P. D E R O BER TI S

content of the brain unchanged. The effect of the perfusion with the washing solution was followed by EEG recordings. Excellent preservation of the glial cells and nerve elements in gray and white matter was observed (Figs. 2 and 3). This method has permitted new observations on the disposition of the glial membranes, which will be discussed below. The advantage of this method resides in the use of a cheap, innocuous fixative and a very simple modus operandi. The>otal preservation obtained permits a systematic study of any neuro-anatomical region and the carrying out of histophysiological or neuropathological experiments in the CNS.

(b) The problem of the extracellular space in the CNS The second part I would present, refers to the problem of the extracellular space in the brain. Physiologically, the extracellular space of a tissue can be determined by calculating the volume distribution of a substance incapable of penetration through cell membranes (Robinson, 1960). In the case of the brain this operational approach has given figures that vary considerably. Using radioactive C1- spaces of 31.4 to 50% and with radioactive Na+, spaces of 26.6 to 40% were calculated. From the above figures down to 4-5 % with 35S04-or inulin in vivo, many intermediary ones have been obtained. With in vitro techniques the most accepted figures vary between 1 6 1 5 % for inulin or sucrose and 17 for ferrocyanide (see Table I in De Robertis and Gerschenfeld, 1961). From the very early observations with the electron microscope (lit. in De Robertis and Gerschenfeld, 1961), it was demonstrated that in the CNS there are no large extracellular spaces and that the membranes of all the cellular components of the nervous tissue are in intimate contact among themselves and with the basal membrane of capillaries. Only fine spaces in the order of 100 to 250 A were observed in between the adjacent membranes. This morphological extracellular space was calculated by Horstmann and Meves (1959) to be not larger than 5 %. This apparent contradiction with the physiological data became more marked when Gerschenfeld et ul. (1959) found that on incubation of brain slices in vitro, as is done for the determination of the extracellular space, there is a marked swelling of the cell body and processes of astrocytes, but absolutely no change of the extracellular spaces. These studies demonstrated also that, at least in the gray matter of the brain, there is not a true extracellular oedema, but an intracellular swelling of the astrocytes. The implications of these observations in the pathology and treatment of human cases of brain oedema need no further comment. As a consequence of these experimental findings and those which will be reported by Dr. Zadunaisky at this Symposium, the following hypotheses on the function of astroglia can be advanced. (I)Astroglia constitutes a cellular compartment that has a special osmoticbehaviour reacting with a swelling in different conditions in vitro or in vivo when the BBB mechanism is altered. (2) In addition to having important mechanical and homeostatic functions, astroglia constitutes a pool of water and electrolytes interposed between the blood plasma,

ELECTRON MICROSCOPICAL CONTRIBUTIONS

5

Fig. 2. Electron micrograph of the cerebral cortex of a rat showing a pericapillary region. cl = capillary lumen; e = endothelium; bm= basement membrane; As = astrocytic feet. With arrows are indicated two tight junctions at which the astrocytic processes are adherent. See therest of theneuropi1 with small intercellular spaces. Fixation by formaldehyde perfusion followed by osmium and uranyl acetate in all Figs. 2-5. x 68,000.

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E. D. P. DE R O B E R T I S

the neurons and other glial elements. Gerschenfeld et al. (1959) found that under in vivo conditions of water overload as marked as to produce a 40% increase in the total extracellular space of the body, no real brain oedema could be produced. This was explained as being due to the presence of a BBB mechanism preventing the entrance of excess water and ions and/or an actively pumping back of ions and water into capillaries. Either or both mechanisms would prevent the swelling of the brain in the living animal. The narrow gaps observed under the electron microscope led some authors to think that they could not adequately serve as diffusion channels and an extreme view that in the CNS there is not a true extracellular space, was sometimespropounded. The experimentsof Lasansky and Wald (1962) on the retina, in which with the electron microscope ferrocyanidewas observed to diffuse in between the intercellular gaps with great rapidity, indicated that these gaps are truly extracellular in their function and therefore that the neuronal membrane is exposed to an extracellularfluid (see Lasansky, this Symposium). These findings are not necessarily opposed to the concept that the access of solutes to the extracellular space can be regulated by the glial cells by way of the astrocytic feet (Fig. 1). Recently Farquhar and Palade (1963) have drawn attention to the fact that at the junction between certain epithelial cells there may be occluding zones in which the intercellular gap disappears. At these points the membranes of adjacent cells are completely adherent. The presence of these so-called tight junctions could explain the impermeability shown by the furface of some epithelia. Zones of adhesion between the plasma membrane of adjacent astrocytic feet were observed by Gray (1961) and have been observed by us in specimens perfused with formaldehyde in the best conditions for the preservation of the tissue (Figs. 2 and 3). Further studies should be carried out to determine if these occluding zones are constant in all asstrocytic feet and if they are permanent or transient phenomena in the astrocytes. The presence of series of tight junctions around the capillaries would have the effect of occluding the intercellular spaces. If this is the case, the water, electrolytes and other solutes that traverse the endothelium and the basement membrane, should find a barrier and be taken up by the glial cell before entering into the open extracellular spaces. ( c ) Morphological bases of the synaptic barrier Another interestingproblem is that of the relationship that glial elements may have with synaptic transmission. The idea that glial processes might be interposed in between endings and neurons and involved in synaptic activity, which was especially propounded by De Castro (1951), has not been substantiated by electron microscopy. Starting with the work of De Robertis and Bennett (1954, 1955), and Palade and Palay (1954), all subsequent studies have demonstrated that glial processes are not interposed at the synaptic junction and that at this level a direct contact of the two neuronal elements exists. Recently the finer organization of the synaptic junction has been determined in synapses of the CNS (De Robertis, 1962). The synaptic cleft of about 300 A is crossed by a series of fine (50 A) intersynapticfilaments that join the

ELECTRON MICROSCOPICAL CONTRIBUTIONS

7

Fig. 3. The same description as Fig. 2 with a very long tight junction between astrocytic feet (arrows). se = synaptic ending; is = intercellular space in the neuropil; mi = mitochondria. x 45,000. Referenccs p . 11

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E. D. P. D E R O B E R T I S

Fig. 4. Synaptic region of the hypothalamus of a rat, showing the synaptic vesicles (sv), the synaptic membranes (sm) and subsynaptic web (ssw). The synaptic complex is surrounded by glial processes (gp), which at certain points (marked with arrows) are adherent to the ending. This may be the basis of the synaptic barrier. mi = mitochondria. x 120,000.

ELECTRON M I C R O S C O P I C A L C O N T R I B U T I O N S

9

Fig. 5. The same as Fig. 4, showing between arrows several tight junctions between glial processes and nerve endings. x 105,000. References p . I 1

10

E. D. P. DE R O B E R T I S

two synaptic membranes and in the postsynaptic cytoplasm there is an irregular system of filaments, the subsynaptic web. Both synaptic membranes are so tightly bound that upon homogenization of the brain the isolated synaptic endings generally carry with them the postsynaptic component. Surrounding this synaptic complex (Fig. 4) glial processes may be frequently observed. Recent observations on brains fixed by formaldehyde perfusion (Gonzalez Aguilar and De Robertis, 1963), have led us to observe the rather frequent presence of tight junctions around the endings. At certain points the plasma membrane of the glial process adheres to the membrane of the ending (Figs. 4 and 5). This sealing, either total or partial, of the extracellular space around the synaptic complexes may physiologically act as a kind of synaptic glial barrier slowing down or preventing diffusion of transmitters released at the junction to penetrate into the intercellular gaps. Physiological evidence of barriers to the diffusion of transmitters has been observed in different synapses in which there is a prolonged or residual action after a presynaptic stimulus (Curtis and Eccles, 1959). In fact, repetitive discharges due to longer excitatory postsynaptic potentials have been observed at certain synapses in response to the firing of a single presynaptic volley. Studies with microelectrodes in which drugs were injected electrophoretically, have also indicated the possible existence of such a synaptic barrier (Curtis and Eccles, 1958a, b). The finding of tight junctions between glial processes and the synaptic complexes thus appears as the most conspicuous morphological basis for a synaptic barrier. By this mechanism glial cells may certainly influence in a subtle manner the physiology of synaptic transmission. I hope that these few examples, in which submicroscopic studies contributed to the Biology of Neuroglia, may serve as introductory remarks to the important papers that will follow and in which the chemical, physiological and pathological aspects of glial cells will be discussed by some of the best experts in the field. I would like to express my sincere gratitude to the eminent foreign scientists who have come from such long distances to take part in this Symposium, thus giving to all of us the light of their wisdom and their stimulus and inspiration to carry on our own humble share in the progress of science. SUMMARY

References are made to a previous review on the submicroscopic morphology and function of glial cells published in the International Review of Neurobiology, Vol. 3, p. 1, 1961, De Robertis, E.D.P., and Gerschenfeld, H.M. Some recent contributions are related to the problem of fixation of the CNS. A new method of formalin perfusion which gives the best preservation of glial and nerve elements at the electron microscope level, is presented. The problem of the extracellular space in the CNS and the discrepancy between physiological methods and electron microscopy is discussed. The bases for an agreement are mentioned.

ELECTRON MICROSCOPICAL CONTRIBUTIONS

11

Astroglia constitutes a cellular compartment with a special osmotic behavior that reacts with swelling in several ‘in vivo’ and ‘in vitro’ conditions. The presence of tight junctions between the glial processes surrounding the capillaries, may occlude the intercellular space and thus the astroglia may have a regulating effect on the composition of the extracellular fluid. The so-called synaptic barrier is discussed and findings on the presence of tight junctions between glial processes and nerve endings, which could be the bases for such a barrier, are described. REFERENCES CURTIS, D. R., AND ECCLES, R. M., (1958a); The excitation of Renshaw cells by pharmacological agents applied electrophoretically. J . Physiol. (Lond.), 141,435-445. CURTIS, D. R., AND ECCLES, R. M., (1958b);The effect of diffusional barriers upon the pharmacology of cells within the central nervous system. J. Physiol. (Lond.), 141,44&463. CURTIS, D. R., AND ECCLES, J. C., (1959); The time courses of excitatory and inhibitory synaptic actions. J. Physiol. (Lond.), 145,529-546. DE CASTRO, F., (1951); Aspects anatomiques de la transmission synaptique ganglionnaire chez les mammifckes. Arch. int. Physiol., 59, 479. DE ROBERTIS, E., (1962); Fine structure of synapses in the CNS. Proceedings of the International Congress of Neuropathology, Munich, Vol. 2 (p. 35). DE ROBERTIS, E., AND BENNETT, H. S., (1954); Some features of the submicroscopic morphology of synapses in frog and earthworm. Fed. Proc., 13,35. DE ROBERTIS, E., AND BENNETT, H. S., (1955); Some features of the submicroscopic morphology of synapses in frog and earthworm. J. biophys. biochern. Cytol., 1,47-58. DE ROBERTIS, E., AND GERSCHENFELD, H. M., (1961); Submicroscopic morphology and function of glial cell. Znt. Rev. Neurobiol., 3, 1. FARQUHAR, M. G., AND PALADE, G. E., (1963); Junctional complexes in various epithelia. J. Cell Biol., 17,375-412. GALAMBOS,R., (1961);Aglia-neuronal theory of brain function. Proc. nut. Acad. Sci. (Wash.),47,129. GERSCHENFELD, H. M., WALD,F., ZADUNAISKY, J. A., AND DEROBERTIS, E. D. P., (1959);Function of astroglia in the water ion metabolism of the central nervous system. Neurology, 9,412-425. GONZALEZ AGUILAR, F.,AND DE ROBERTIS, E., (1963); A formalin-perfusion fixation method for histophysiological study of the central nervous system with the electron microscope. Neurology, 13,758-771. GRAY,E. G., (1961); Ultrastructure of synapses of the cerebral cortex and certain specialization of neuroglial membranes. Electron Microscopy in Anatomy. London, E. Arnold, Publishers (p. 54). HAMBERGER, A., AND HYDEN,H., (1963); Inverse enzymatic changes in neurons and glia during increased function and hypoxia. J . Cell Biol., 16,521-525. HORSTMANN, E., AND MEVES,H., (1959); Die Feinstruktur des molekularen Rindengraues und ihre physiologische Bedeutung. Z. ZeNforsch., 49, 569404. HYDBN,H., AND EGYH~ZI, E., (1963); Glial RNA changes during a learning experiment in rats. Proc. nut. Acad. Sci. (Wash.),49,618-624. HYDEN,H., AND LANGE, P. W., (1962);A kinetic study of the neuron-glia relationship. J. Cell Biol., 13,233-237. HYDBN,H., AND PIGON, A., (1960);A cytophysiological study of the functional relationship between oligodendroglial cells and nerve cells of Deiters’ nucleus. J. Neurochem., 6, 57-72. LASANSKY, A., AND WALD,F., (1962); The extracellular space in the toad retina as defined by the distribution of ferrocyanide. A light and electron microscope study. J. Cell Biol., 15,463-479. PALADE, G. E., AND PALAY, S. L., (1954); Electron microscope observations of interneuronal and neuromuscular synapses. Anat. Rec., 118, 335-336. ROBINSON, J. R., (1960);Metabolism of intracellular water. Physiol. Rev., 40,112-149.

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Morphological and Functional Characteristics of the Central and Peripheral Neuroglia (Light Microscopical Observations) M. POLAK Fudacion Roux-Ocefa. Laboratory of Histological and Histopathological Investigations, Buenos Aires (Argentina)

The cells constituting the so-called central and peripheral neuroglia are at present intensively studied. They are being analyzed by numerous methods and techniques, in human and animal material, normal, pathological and experimental. Some investigators with a thorough knowledge of the classical publications of Golgi, Cajal, Hortega and others, have carefully analyzed the structure of neuroglial cells. Others, however, ignoring these studies, when applying histological techniques now out-dated by the silver and gold impregnations, have rediscovered various types of glial cells. Therefore we believe that a summary of the morphological characteristics of the neuroglial elements as seen with the light microscope may be useful. In particular, sections impregnated by the metallic techniques of Golgi, Cajal and Hortega will allow us to distinguish between the different cellular types in great detail. These techniques clearly show the relationship between the neuroglial cells on the one hand, and the nerve cells, their processes, the blood vessels and the meninges, on the other hand. An important advance in knowledge of the morphological and functional interpretation of neuroglial cells was made by Hortega (1919-1921)) when he proved that the ‘third element’ of Cajal was in fact made up by two types of cell: one authentically neuroglic, the other of mesenchymal origin, the oligodendrocyte and the microglial cell. This discovery was partly due to the use of a new histological technique, now called the Hortega method (ammoniacal silver-carbonate). The microglia or mesoglia has been considered by many authors as part of the neuroglia. Glees (1955) adds to this confusion when he says: ‘The origin of neuroglial cells is twofold : some differentiate from neuroectoderm and others from mesoderm. Those of mesodermal origin appear to be of negligible importance except in traumatic reactions in the brain and need not be considered here’. This statement contains two misinterpretations: (1) the consideration that the microglia is part of the neuroglia; and (2) the microglia is of little importance in normal conditions. Hortega clearly explains that ‘there exist two kinds of cells: the true neuroglia and the false neuroglia; the one which has an ectodermal origin, in the same way, the

CHARACTERISTICS OF CENTRAL A N D PERIPHERAL NEUROGLIA

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same place and the same time as the nerve cells, and the other which has a mesodermal origin. The latter corresponds to the elements which I first described under the title of microglia’. Therefore, we shall not include the microglia in this report, and in referring to neuroglia we shall divide it into two groups: the central neuroglia, made up of ependymoventricular glioepithelial cells, astrocytes and oligodendrocytes; and the peripheral neuroglia made up of gliocytes in the spinal and sympathetic ganglia, Schwann and Remak cells of the nervous fibers and the terminal gliocytes related with the free nerve endings and the capsulated endings. CENTRAL NEUROGLIA

Embryological data The modifications of the ectoderm that lead to the formation of the neural plate, neural canal and neural tube are well known to the embryologists. The epithelium, lining the primitive neural tube, consists of prisma-like cells which rapidly proliferate at the inner limiting membrane, thus resembling a stratified epithelium on account of the different heights at which the nuclei are placed. Among these elements appear spherical cells in mitosis, named germinative cells, from which neuroblasts originate exclusively. His (1889) gave the name of spongioblasts to the primitive epithelial cells from which the ependymal-ventricular epithelium and the remaining neuroglial elements originate. However, some authors deny that the differentiation into neuroblasts and neuroglial elements takes place so early. Schaper (1897) regards the prisma-like cells as undifferentiated neuroepithelial elements, and germinative cells as the same cells in mitosis, giving forth new neuroepithelial cells or undifferentiated apolar cells that will produce new generations of undifferentiated cells or cells differentiated in neuroblasts and spongioblasts through mitotic division. At first, Schaper’s concepts were accepted by many scientists, and Cajal himself, referring to the problem, remarks: ‘We consider it more likely, in agreement with Kolliker and Schaper, that the spherical elements on the way to mitosis may be undifferentiated forms, whose progeny is represented as much by primitive epithelial cells as by neuroblasts. Specificity would occur later in the spongioblast and neuroblast phases’. Later, however, studying the formation of the neuroglia in the early phases of the development of the medulla, Cajal disposed of Schaper’s undifferentiated elements : ‘As our investigations have definitely proved, the elements of the neuroglia are nothing but displaced and transformed epithelial cells’. The germinative cells are transformed into neuroblasts, which, in turn, move from their original site among the spongioblasts to settle between the marginal nuclear zone and the marginal velum going through the following phases of evolution: apolar-, bipolar-, monopolar and young neurocyte. In the final formation of the ependymal and ventricular epithelium, the spongioblast goes through the following stages : primitive spongioblast, primordial epithelium, ramified epithelium and final epithelium. References p. 33/34

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M. P O L A K

According to Cajal, the neuroglial cells appear in the chick embryo on the 13th day, and in the human embryo in the 3rd month, and their genetic mechanism would be as follows: the cell body leaves the neural duct at different heights, where it undergoes various changes: atrophy of the processes, transversal growth of the soma while short extensions appear that later ramify, and which leads to a greater development of its radial outgrowth. At the beginning the cells are anchored in the outer limiting membrane. This insertion disappears in mammals shortly before birth and the displaced epithelial cells reduce their peripheral expansion, reabsorb their processes and become stellate. Cajal describes two phases in the neuroglial differentiation : (1) displaced epithelial cells whose radial expansions terminate in the pia mater by terminal thickenings (primordial neuroglial cells or astroblasts, according to LenhossGk); and (2) young neuroglial cells, which retract their two radial processes. These two types of cell will develop, according to the zones in which they are present, to become protoplasmic or fibrous astrocytes respectively. As far as the development of the oligodendroglia is concerned, Hortega described two kinds of elements which differentiate from the primitive neuroepithelium : the glioblasts originated from the spongioblasts and the neuroblast originated from the germinative cell. As the former leave the neural tube, some will become astroblasts and others oligodendroblasts. The astroblasts immediately come into contact with mesodermal structures (vessels and meninges) and the oligodendroblasts become intimately related to nerve fibers. These two facts constitute the fundamental morphological principles of the concepts of ‘angiogliona’ and ‘neurogliona’,to which we shall refer later. The astroblasts develop into protoplasmic and fibrous elements. The oligodendroblasts remain associated with nerve fibers. Although the microglia does not belong to the neuroglia, we shall refer briefly to its genesis. The microglial cells, representing the reticulo-endothelial system in the nervous system, originate from the pia mater, particularly from the superior and inferior choroidal tissues. The cells also originate from the adventitial cells of the blood vessels that penetrate into the nervous parenchyma. When an adventitial cell loses its connection with the vascular wall, it changes its shape in order to adapt to the new environment and then it is impossible to differentiate it from other microgliozytee. When a microglial element moves to the wall of a blood vessel, to which it becomes attached, it becomes an adventitial cell. The above-mentioned data allow us to draw up the following scheme: Primitive neural epithelium

F

spongio? ast 4 ependymoventricular glioepithelium

>minative I

glioblast

/

astroblast

microgliocyte

1

microglioblast

astrocyte

t

+

Leptomeniigeal ‘midus’

cell

CHARACTERISTICS OF CENTRAL A N D P E R I P H E R A L NEUROGLIA

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Ependy mal-Yentr icular epithelium (Figs. 1-3) On the basis of cytogenetic, morphological and functional criteria, neurohistologists consider that the ventricular and ependymal epithelium belongs to the group of neuroglial elements. The ventricular cavities and the spinal central canal are lined with cubical or cylindrical cells. In some species the apical part of these cells show cilia, for which Hild (1954) demonstrated in tissue cultures the presence of a continuous and coordinated movement in onkdirection. This phenomenon was pointed out i:t situ in-1836.b~Purkinje and simultaneously by Valentin who related-it to the I

Fig. 1. Glial-epithelial cells of ependyma. (Golgi-Hortega staining method.)

Fig. 2. Glial-epithelial cells with ‘feet’ on a blood vessel. (Hortega triple impregnation.) I