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UBC Theses and Dissertations

Macro-glial specialization in the brain Thompson, Sharleen Grace 1986

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MACRO-GLIAL SPECIALIZATION IN THE BRAIN BY SHARLEEN GRACE THOMPSON B. SC. The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PSYCHIATRY, DIVISION OF NEUROSCIENCES We a c c e p t t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER 198 6 ( c ) S h a r l e e n G r a c e Thompson, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date THESIS ABSTRACT T h i s t h e s i s e x a m i n e s t h e e v i d e n c e f o r g l i a l c e l l s p e c i a l i z a t i o n . I t s t a r t s w i t h an h i s t o r i c a l d e s c r i p t i o n o f t h e d e v e l o p m e n t o f i d e a s a b o u t g l i a l c e l l s , d e m o n s t r a t i n g how e a c h t e c h n o l o g i c a l a d v a n c e a l l o w e d an i n c r e a s e i n u n d e r s t a n d i n g o f v a r i o u s m o r p h o l o g i c a l l y d i f f e r e n t t y p e s o f g l i a l c e l l s and how e a c h t e c h n i q u e p r o v i d e d more e v i d e n c e f o r g l i a l h e t e r o g e n e i t y . The most s p e c t a c u l a r r e c e n t d e v e l o p m e n t i s t h e i n c r e a s i n g e v i d e n c e f o r b i o c h e m i c a l h e t e r o g e n e i t y i n c e l l s i n v i v o , i n d i f f e r e n t c e l l l i n e s and i n p r i m a r y c u l t u r e s f r o m v a r i o u s r e g i o n s . T h e s e b i o c h e m i c a l d i f f e r e n c e s h a v e been f o u n d b o t h among c e l l s t h a t a r e m o r p h o l g i c a l l y s i m i l a r and b e t w e e n d i f f e r e n t c e l l t y p e s . The r e s u l t s o f t h r e e e x p e r i m e n t s w h i c h p r o v i d e d i r e c t o r i n d i r e c t e v i d e n c e f o r g l i a l c e l l h e t e r o g e n e i t y a r e p r e s e n t e d . The f i r s t e x p e r i m e n t i s an a n a t o m i c a l a n a l y s i s o f t h e c e l l u l a r l o c a l i z a t i o n o f h e m o s i d e r i n i n r a t b r a i n . The r e s u l t s show p r i m a r y l o c a l i z a t i o n t o o l i g o d e n d r o c y t e s b u t n o t a l l o l i g o d e n d r o c y t e s a s t h e r e a r e d i s t i n c t r e g i o n a l d i f f e r e n c e s i n b o t h d e n s i t y and numbers o f o l i g o d e n d r o c y t e s s t a i n i n g . I n t h e s e c o n d e x p e r i m e n t , an a l t e r n a t e r o u t e o f g l u t a m a t e f o r m a t i o n f r o m p r o l i n e o r o r n i t h i n e v i a 1 - p y r r o l i n e d e h y d r o g e n a s e i s d e m o n s t r a t e d and shown t o be p r e s e n t i n o n l y a s m a l l s u b s e t o f g l i a l c e l l s and n o t i n o t h e r c e l l t y p e s . I n t h e t h i r d e x p e r i m e n t t h e g l i a l h e t e r o g e n e i t y c o n c e p t i s u s e d t o p r o v i d e an a l t e r n a t e i n t e r p r e t a t i o n o f a l l d a t a on b i o c h e m i c a l e f f e c t s o f t h i a m i n e d e f i c i e n c y i n r a t b r a i n . i i The conclusion summarizes the contribution of the experiments to the already strong evidence for g l i a l heterogeneity and suggests ways that assumptions of g l i a l heterogeneity rather than homogeneity could a f f e c t research the neurosciences. TABLE OF CONTENTS T i t l e Page i Thesis Abstract i i Table of Contents i v L i s t of Tables v L i s t of Figures v i Table of Abbreviations v i i D e f i n i t i o n of G l i a C e l l Types v i i i - x i i Introduction 1 History of Development of Today's Ideas on Structure and Function 1 Function of G l i a 5 G l i a and Neurotransmitters 11 New Techniques Enabling Advances i n Understanding G l i a 21 (a) Tissue Culture 21 (b) Freeze Fracture Techniques 21 (c) Markers 25 i) Fibrous Protein 25 i i ) Glutamine Synthetase 29 i i i ) Carbonic Anhydrase 3 0 iv) Other Markers 31 G l i a Heterogeneity-Morphological 34 Developmental Differences - a source of Heterogeneity 44 Heterogeneity i n Tissue Cultures 49 (A) Developmental Changes i n Culture 49 (B) Culture Conditions, Development and Heterogeneity 53 (C) C e l l Development and Dif f e r e n t a t i o n i n Response to Injury 61 (D) Heterogeneity Between Different G l i a Not Explained By Development or Cultural Conditions 63 Heterogeneity Between and Within G l i a C e l l Lines: Different Areas Labelling In Vivo 65 Differences i n G l i a l C e l l s from Different Areas of the Brain 77 Summary of Evidence for Biochemical Differences i n G l i a 86 Experimental Rationale and Abstract 88 Experiment 1 91 Experiment 2 117 Experiment 3 127 Conclusion 143 Acknowledgements 144 References 145 - iv - L I S T OF TABLES T a b l e I M i n o r a s t r o c y t e c e l l m a r k e r Pg. 3 2-33 T a b l e I I O l i g o d e n d r o c y t e and m y e l i n m a r k e r s Pg. 35-36 T a b l e I I I E f f e c t s o f c u l t u r e c o n d i t i o n s on c e l l c h a r a c t e r i s t i c s Pg. 59-60 T a b l e I V C o m p a r a t i v e v a l u e s o f g l u t a m a t e u p t a k e Pg. 70 T a b l e V C o m p a r a t i v e v a l u e s o f g l u t a m i n e u p t a k e Pg. 72 T a b l e V I C o m p a r a t i v e v a l u e s o f h i g h a f f i n i t y GABA u p t a k e Pg. 73 T a b l e V I I I r o n s t a i n i n g i n r a t b r a i n b y a r e a Pg. 115-116 T a b l e V I I I Enzyme l e v e l s i n c o n t r o l , t h i a m i n e d e f i c i e n t and r e c o v e r e d r a t s Pg. 136 - v - L I S T OF FIGURES F i g . 1 Microscopic pictures of iron s t a i n i n g i n r a t brain. Pg. 104, 106 Fig . 2 Photographs of whole s a g i t t a l sections of iron staining i n rat brain. Pg. 108 Fig. 3 Schematic diagram of Fig . 2 showing c e l l u l a r types by area Pg. 110 Fig . 4 Half photographs and ha l f schematic drawings of coronal section of iron staining i n rat brain. Pg. 112, 114 Fi g . 5 Schematic representation of the conversion of proline and ornithine to glutamate and GABA. Pg. 124 Fig . 6 1-Pyrroline dehydrogenase st a i n i n g of Bergmann g l i a i n cerebellum Pg. 12 6 F i g . 7 1-Pyrroline dehydrogenase staining of astrocytes of dentate gyrus. Pg. 12 6 F i g . 8 Proline oxidase staining of Bergmann g l i a i n cerebellum Pg. 12 6 F i g . 9 GABA-T staining i n thiamine d e f i c i e n t r a t Pg. 142 - vi - TABLE OF ABBREVIATIONS FULL WORD ABBREVIATION 1ST PG. USED Acetylcholine ACh 15 Acetylcholinesterase AChE 15 Adenosine Triphosphate ATP 79 Adenosine-5-Triphosphatase ATPase 10 Calcium Ca++ 12 Catechol-O-Methyl Transferase COMT 15 Central Nervous System CNS 6 Choline Acetyltransferase CAT 89 C y c l i c Adenine Monophosphate CAMP 18 Diaminobenzaldehyde DAB 89 Dibutyryl C y c l i c Adenine Monophosphate dBcAMP 17 Dopamine DA 11 Electroencephalogram EEG 89 Gamma-Aminobutyric Acid GABA 9 Gamma-Aminobutyric Acid Transaminase GABA-T 15 G l i a l F i b r i l l a r y A c i d i c Protein GFAP 25 Glutamic Acid Decarboxylase GAD 89 Glutamine Synthetase GS 16 Histamine Type I Receptor HI 18 Histamine Type II Receptor H2 18 Magnesium Mg++ 76 Maximum V e l o c i t y of Reaction Vmax 12 Michaelis Constant (Concentration of Substate at 1/2 Vmax) KM 70 Monoamine Oxidase MAO 15 Niacine Adenine Dinucleotide NAD 117 Noradrenaline NA 11 Ornithine Oxo-Acid Aminotransferase OrnT 117 Potassium K+ 8 Pyrithiamine PT 127 1-Pyrroline-5-Carboxylate P5C 89 Pyrroline-5-Carboxylate Dehydrogenase Pro 89 1-Pyrroline Dehydrogenase PDH 89 Serotonin 5HT 11 Sodium Na+ 11 Thiamine Defi c i e n t TD 127 Thiamine Triphosphate TTP 128 T r i c a r b o x y l i c Acid Cycle TCA CYCLE 17 - v i i - DEFINITIONS OF GLIAL CELL TYPES OLIGODENDROCYTES A class of g l i a c e l l f i r s t stained and seen by Golgi a f t e r he invented his s i l v e r s t a ining technique. There i s considerable morphological heterogeneity within t h i s group. They are t r a d i t i o n a l l y c l a s s i f i e d by where they are located and how they associate with other c e l l s or by t h e i r nuclear and c y t o p l a s t i c d e n s i t i e s . ASTROCYTES The second major cl a s s of g l i a l c e l l s . They are larger than oligodendrocytes, have pale s t a i n i n g nuclei and electron l i g h t cytoplasm. Cajal, the f i r s t to describe them divided them into two subclasses: fibrous and protoplasmic, based on the presence or number of fi b e r s within the c e l l body. Those described by Cajal are now considered as OOastrocytes. £-ASTROCYTE May be intermediate type between an OC-astrocyte and l i g h t oligodendrocyte. - v i i i - MICROGLIA A class of g l i a o r i g i n a l l y defined by the s i l v e r carbonate method of Del Rio Hortega. Their o r i g i n and c l a s s i f i c a t i o n i s highly controversial. They are not discussed i n t h i s paper. DISTINCT SUB-CLASSES OF GLIA BERGMANN GLIA Also c a l l e d Golgi E p i t h e l i a l C e l l s ; they are g l i a l c e l l s with c e l l bodies located i n or j u s t below the Purkinje c e l l layer of the cerebellum and having r a d i a t i n g f i b e r s extending upward through to the outer surface of the molecular layer. They share many of the development and biochemical c h a r a c t e r i s t i c s of g l i a but also have many differences. MULLER GLIA CELLS A g l i a c e l l i n the r e t i n a of the eye. Though not the only g l i a c e l l i n the eye, they have been extensively studied and have considerable overlap of c h a r a c t e r i s t i c s with g l i a of the central nervous system. - ix - RADIAL GLIA CELLS PITUICYTES EPENDYMAL CELLS TANYCYTES A developmental stage of many g l i a c e l l s where the c e l l body has long arms extending perpendicularly to some outer surface. These rad i a t i n g f i b e r s may a s s i s t i n guiding other c e l l s to t h e i r correct place during development. In some areas the r a d i a l form may p e r s i s t u n t i l adulthood. The Bergmann g l i a may be an example of t h i s . P i t u i c y t e s are g l i a l c e l l s i n the neurohypophysis. They have many c h a r a c t e r i s t i c of central g l i a . These g l i a - l i k e c e l l s l i n e the ve n t r i c u l a r system within the brain and central canal of the spinal cord. They may have special function i n blood brain b a r r i e r , and production of cerebrospinal f l u i d . They have many ch a r a c t e r i s t i c s of g l i a c e l l s and may evolve from r a d i a l g l i a . Specialized g l i a with ra d i a t i n g processes that l i n e the v e n t r i c l e , p a r t i c u l a r l y the t h i r d v e n t r i c l e . They have many g l i a c h a r a c t e r i s t i c s . - x - ENTERIC GLIA G l i a - l i k e c e l l s of the enteric nervous system that are more l i k e the central g l i a than the peripheral Schwann c e l l s SCHWANN CELLS C e l l s of the peripheral nervous system that wrap the peripheral nerves with layers of t h e i r external membrane to insulate nerves from each other. In s p e c i a l cases, such as those i n the eye, they grow with the optic nerve into the brain and are located c e n t r a l l y . Occasionally, as i n the Schwann c e l l s of the o l f a c t o r y nerve, they have properties s i m i l a r to the central g l i a . RESEARCH CELL TYPES GLIA CELL LINES Permanent c e l l cultures maintained in laboratories and o r i g i n a l l y created by transformation by c e r t a i n viruses or chemicals. They are believed to be models of brain tumors and thus to c e r t a i n c h a r a c t e r i s t i c s of various types of brain tumors, and are extensively used i n research because the l i n e s are stable and can be purchased. They have well defined c h a r a c t e r i s t i c s which cannot be assumed to be l i k e those of untransformed g l i a c e l l s i n vivo but some normal c h a r a c t e r i s t i c s have been retained. Some l i n e s , such as C - l or C-6, may have c h a r a c t e r i s t i c s of gliomas, while others may resemble neural tumors or astrocytomas. PRIMARY CULTURES Cultures recently derived from f e t a l or neonatal brain and cultured for short periods of time. During t h i s time they develop through several changes of morphology and biochemical c h a r a c t e r i s t i c s that can be manipulated by culture conditions. They are thus useful i n t r y i n g to understand the c h a r a c t e r i s t i c s of g l i a . Since culture conditions are never i d e n t i c a l to those i n vivo, many in vivo c h a r a c t e r i s t i c s never develop. - xi i - I n t r o d u c t i o n This thesis examines g l i a l c e l l l i t e r a t u r e f o r evidence of g l i a l heterogeneity and then presents my r e s u l t s on s p e c i f i c g l i a l s t a i ning and the e f f e c t s of experimental manipulation on subsets of g l i a . The r e s u l t s show considerable evidence for macroglial heterogeneity, both on a regional and a c e l l u l a r basis. My research shows, i n two unrelated procedures, that only c e r t a i n subsets of g l i a l c e l l are stained, further supporting the evidence for biochemical differences between g l i a l c e l l s . The current r e s u l t s suggest that generalization from one g l i a l system to another i s no longer v a l i d . I f g l i a l c e l l heterogenity e x i s t s , why i s the evidence so l a t e i n coming, and why i s there tremendous resistance to the acceptance of t h i s idea? Much of our understanding of g l i a l c e l l function i s based on work done very early i n t h i s century. The early assumptions were so well accepted that more recent re s u l t s have l a r g e l y been ignored by neuroscientists. Basic neuroscience texts s t i l l do not devote more than a small amount of space to g l i a , giving l i t t l e more than a simple des c r i p t i o n of the basic types and perhaps an h i s t o r i c a l note on t h e i r function. H i s t o r y o f Development A look at the h i s t o r i c a l work done on g l i a w i l l serve to introduce the topic of g l i a l c e l l s t r u c t u r a l and functional - 1 - heterogeneity. The early researchers faced a number of problems which led to assumptions that formed biases which now prevent the acceptance of some of the findings of heterogeneity. Virchow i n 1846 was the f i r s t to mention the existence of neuroglia i n the brain. He thought that, since neurons did not appear to occupy a l l the space i n the brain, there must be something holding the neurons together; t h i s he c a l l e d the nerve glue, or neuroglia and the German word came to be adopted. He did not see that the "glue" was composed of c e l l s because the early c e l l preservation techniques were crude and neuroglia were the f i r s t c e l l s to swell and disintegrate, which made them d i f f i c u l t to see under l i g h t microscopes. H i s t o r i c a l l y the main reason for concentration on neurons was the d i f f i c u l t y i n studying g l i a . The fact that the spaces between the nerves were occupied by c e l l s now c a l l e d g l i a was f i r s t observed by Golgi (1879) a f t e r he invented the Golgi s i l v e r s t a i n i n g method for those g l i a l c e l l s now c a l l e d oligodendrocytes. In 1913 Cajal invented the gold sublimate method which he found stained another type of non-neural c e l l , the astrocyte, thus allowing the d i f f e r e n t i a t i n g of two types of c e l l s , the oligodendrodcytes, stained best by the Golgi technique, and the astrocyte. In 1919 del Rio Hortega invented the s i l v e r carbonate method which stained microglia, the t h i r d major type of g l i a l c e l l . Although there were advances i n the understanding of the development of these c e l l s , there were no major additions - 2 - t o t h e u n d e r s t a n d i n g o f c e l l t y p e s u n t i l c e l l s were f i r s t s e p a r a t e d , t h e e l e c t r o n m i c r o s c o p e was i n v e n t e d and c e l l s p e c i f i c m a r k e r s became a v a i l a b l e . I h a v e n o t i n c l u d e d f u r t h e r d i s c u s s i o n o f t h e m i c r o g l i a i n t h i s t h e s i s b e c a u s e I h a v e no r e s e a r c h t o p r e s e n t on m i c r o g l i a and t h e y a r e s u r r o u n d e d b y c o n s i d e r a b l e c o n t r o v e r s y . O l i g o d e n d r o c y t e s a r e now u n d e r s t o o d t o be s m a l l o v a l c e l l s t h a t c o m p r i s e a b o u t 20% o f t h e b r a i n mass ( V a r o n , 1 9 7 8 ) . O l i g o d e n d r o c y t e s come i n s e v e r a l d i f f e r e n t m o r p h o l o g i e s and h a v e b e e n c l a s s i f i e d by two methods. The f i r s t method i s by where t h e y a r e l o c a t e d and how t h e y a s s o c i a t e w i t h o t h e r c e l l s . O l i g o d e n d r o c y t e s o c c u r i n rows i n w h i t e m a t t e r , c a l l e d i n t r a f a s i c u l a r g l i a l , where t h e i r p r o c e s s e s a r e a s s o c i a t e d w i t h g r o u p s o f m y e l i n a t e d n e r v e f i b e r s . I n g r e y m a t t e r , t h e y may a p p e a r a s i n d e p e n d e n t o r a s s a t e l l i t e c e l l s i n c l o s e a s s o c i a t i o n w i t h n e u r o n s . O l i g o d e n d r o c y t e s a r e f o u n d t o have a w i d e r a n g e o f n u c l e a r and c y t o p l a s m i c d e n s i t i e s ( C a l e y and M a x w e l l , 1 9 6 8 ) . On t h e b a s i s o f t h e s e e l e c t r o n m i c r o s c o p i c d e n s i t i e s , M o r i and L e b l o n d (1970) c l a s s i f i e d them i n t o l i g h t , medium and d a r k o l i g o d e n d r o c y t e s , w h i c h seem t o be p r o g e s s i n g d e v e l o p m e n t a l s t a g e s ( M o r i and L e b l o n d , 1 9 7 0 ) f r o m l i g h t t o d a r k , w i t h c o n c u r r e n t r e d u c t i o n s i n t h e s i z e and i n c r e a s e i n t h e d e n s i t y o f t h e n u c l e u s , r e d u c t i o n s i n c y t o p l a s m i c volume, i n c r e a s i n g c o m p l e x i t y o f r o u g h e n d o p l a s m i c r e t i c u l a and G o l g i o r g a n e l l e s , and r e d u c t i o n i n t h e number o f p r o c e s s e s . T h e i r d e v e l o p m e n t p a r a l l e l s m y e l i n a t i o n . The f i n a l d e v e l o p m e n t a l p r o d u c t , t h e m a t u r e o l i g o d e n d r o c y t e s , a r e c h a r a c t e r i z e d by a - 3 - s m a l l e l e c t r o n - d e n s e n u c l e u s , o f t e n p o s i t i o n e d e c c e n t r i c a l l y , s c a n t and d e n s e c y t o p l a s m w i t h a h i g h l y d e v e l o p e d G o l g i a p p a r a t u s , s t a c k s o f r o u g h e n d o p l a s m i c r e t i c u l a c i s t e r n a e and l a m e l l a r b o d i e s f r e q u e n t l y a s s o c i a t e d w i t h i n t r a c e l l u l a r membranes, and a s m a l l g r o u p o f p r o c e s s e s c o n t a i n i n g m i c r o t u b u l e s b u t no g l i o f i l a m e n t s . A s t r o c y t e s a r e t h e s e c o n d o f t h e m a j o r c l a s s e s o f g l i a . T h e y c o m p r i s e 20 t o 25% ( V a r o n , 1978) o f b r a i n t i s s u e , a r e l a r g e r t h a n o l i g o d e n d r o c y t e s , h a v e p a l e s t a i n i n g n u c l e i and e l e c t r o n l i g h t c y t o p l a s m , h a v e numberous p r o c e s s e s w i t h g l i a l f i l a m e n t s and a c c u m u l a t e g l y c o g e n g r a n u l e s u n d e r a n o x i c c o n d i t i o n s . C a j a l o r i g i n a l l y d i v i d e d them i n t o f i b r o u s and p r o t o p l a s m i c t y p e s , b a s e d on l o c a t i o n , m o r p h o l o g y and f u n c t i o n . Now, w i t h t h e e l e c t r o n m i c r o s c o p e , m a t u r e f i b r o u s a s t r o c y t e s a r e n o t e d t o h a v e e x t e n s i v e , w e l l o r g a n i z e d c y t o p l a s m i c f i l a m e n t s ( P a l a y e t a l . 1962), and p r o t o p l a s m i c a s t r o c y t e s do n o t . T h e r e a r e a l s o a w i d e v a r i e t y o f o t h e r c e l l s w i t h a s t r o c y t e c h a r a c t e r i s t i c s . T h e s e w i l l be d i s c u s s e d more f u l l y when g l i a l h e t e r o g e n e i t y i s b e i n g e x a m i n e d i n a f o l l o w i n g s e c t i o n . I n t h e e a r l y y e a r s s e v e r a l t h e o r i e s were p u t f o r w a r d as t o t h e f u n c t i o n o f n e u r o g l i a . G o l g i (1894) t h o u g h t t h a t t h e y n o u r i s h e d n e u r o n s b e c a u s e he o b s e r v e d t h a t t h e y h a d end f e e t t h a t were o p p o s e d t o c a p i l l a r i e s . I n 1885 W e i g a r t (1895) s u g g e s t e d t h e i r f u n c t i o n was t o g i v e a s t r u c t u r a l s u p p o r t . I n 1896 M a r i n e s c o s u g g e s t e d t h a t t h e y h a d a h i s t o l y t i c r o l e i n t h e c l e a r i n g o f d y i n g n e u r o n s . H i s , i n 1887, was t h e f i r s t t o v i e w g l i a l c e l l s as p r o v i d i n g g u i d a n c e f o r t h e g r o w t h o f t h e - 4 - n e r v e f i b e r s d u r i n g e m b r y o l o g i c a l d e v e l o p m e n t . L u g a r o (19 07) was t h e f i r s t t o s p e c u l a t e on t h e i r f u n c t i o n a s a d e t o x i f i c a t i o n f i l t e r b e tween b l o o d and b r a i n a n d a l s o s u g g e s t e d t h a t t h e y s e r v e d t o remove and c h e m i c a l l y s p l i t compounds s e c r e t e d b y n e r v e e n d i n g s . L u g a r o r e j e c t e d G o l g i ' s n u t r i t i o n a l h y p o t h e s i s b e c a u s e he d i d n o t b e l i e v e t h a t d e n d r i t e s were l i k e r o o t s t o p l a n t s , and he a l s o r e j e c t e d t h e i r r o l e i n p r o v i d i n g b i o c h e m i c a l s u p p o r t f o r n e u r o n s s i n c e he s t i l l t h o u g h t t h a t t h e y were b a s i c a l l y t h e p a c k i n g m a t e r i a l f o r t h e more " n o b l e " n e u r o n s . C a j a l (1913) t h o u g h t t h a t t h e y s e r v e d t o i n s u l a t e n e r v e f i b e r s and f i b e r b u n d l e s . T h e s e c o n c e p t s o f g l i a l f u n c t i o n r e m a i n e d i n t a c t u n t i l K o r y e t a l . (1958) i s o l a t e d g l i a l c e l l s and t h e e l e c t r o n m i c r o s c o p e s t i m u l a t e d u l t r a s t r u c t u r a l r e s e a r c h . F u n c t i o n o f G l i a We now u n d e r s t a n d t h a t t h e f u n c t i o n s o f g l i a a r e complex, b u t t h e c u r r e n t l y u n d e r s t o o d f u n c t i o n s i n c l u d e some o f t h o s e a s s i g n e d t o g l i a b y many o f t h e e a r l i e r r e s e a r c h e r s . I n o r d e r t o u n d e r s t a n d g l i a h e t e r o g e n e i t y t h e i r b a s i c f u n c t i o n s must be u n d e r s t o o d . The c o n c e p t o f s t r u c t u r a l s u p p o r t , as p r o p o s e d by W e i g a r t ( 1 8 9 5 ) , i s no l o n g e r s e r i o u s l y t h o u g h t o f a s a f u n c t i o n even t h o u g h t h e h i s t o r i c a l f a c t i s f r e q u e n t l y m e n t i o n e d i n t e x t s w i t h o u t much e l a b o r a t i o n . I n f a c t , e x c e p t when e x t e n s i v e g l i o s i s h a s f o r m e d s c a r t i s s u e , g l i a a r e p e r h a p s s o f t e r t h a n n e u r o n s a s t h e y a r e more s u s p e c t i b l e t o i s c h e m i a and m e c h a n i c a l d i s r u p t i o n . T h ey do, however, p e r f o r m s e v e r a l - 5 - s t r u c t u r a l f u n c t i o n s . The o l i g o d e n d r o g l i a do wrap t h e n e r v e f i b e r s i n t h e b r a i n and s p i n a l c o r d w i t h many l a y e r s o f t h e i r c e l l , membranes, f o r m i n g t h e c e n t r a l n e r v o u s s y s t e m (CNS) m y e l i n . G l i a f i r s t a p p e a r j u s t p r i o r t o t h e t i m e o f m y e l i n a t i o n . The most r a p i d r a t e o f m y e l i n a t i o n i s s y n c h r o n o u s w i t h t h e most r a p i d p r o l i f e r a t i o n and d i f f e r e n t i a t i o n o f o l i g o d e n d r o c y t e s . I n humans, m y e l i n a t i o n s t a r t s a t a b o u t 4 months g e s t a t i o n and c o n t i n u e s t i l l a b o u t age 2; i t s t a r t s a t t h e n e u r o n a l c e l l body and grows d i s t a l l y . T h i s s e r v e s t o s p e e d t h e s a l t a t o r y c o n d u c t i o n o f e l e c t r i c a l i m p u l s e s a l o n g t h e n e r v e s and p r o v i d e s some s t r u c t u r a l s t r e n g t h e n i n g o f t h e s e d e l i c a t e f i b e r s . I n t h e w h i t e m a t t e r t h e o l i g o d e n d r o c y t e s do p r o v i d e a warp and woof l i k e m a t r i x w i t h t h e n e r v e f i b e r b u n d l e s . T h e i r end f e e t and d e n d r i t e s a l s o p r o v i d e s h e a t h s o v e r a l l o u t e r s u r f a c e s o f t h e CNS. They do h a v e many t y p e s o f c o n n e c t i o n s b e t w e e n t h e i r p l a s m a membranes and some t y p e s o f c o n n e c t i o n s may p r o v i d e s t r u c t u r a l s u p p o r t . M a r i e s c o ' s o r i g i n a l p r o p o s a l o f h i s t o l y t i c a c t i v i t y f o r t h e r e m o v a l o f d y i n g n e u r o n s may be c o r r e c t i n t h a t g l i a may a b s o r b t h e d e b r i s o f t h e d y i n g n e u r o n s , b u t most o f t h i s seems t o be done by m a c r o p h a g e s t h a t i n v a d e t h e a r e a o f damage. T h i s d o e s n o t mean t h a t g l i a a r e n o t i n v o l v e d i n t h e a c t i v i t y a t damaged s i t e s . When r e p a i r s a r e n e e d e d t h e y p r o l i f e r a t e , i n c r e a s e i n s i z e and c hange s t r u c t u r e s o t h a t t h e y a r e more f i b r o u s and t h u s f o r m a t o u g h s c a r t i s s u e a t t h e c o r e o f t h e damaged a r e a . T h ey a l s o w a l l o f f t h e damaged a r e a o f t h e b r a i n f r o m t h e o v e r l y i n g l e p t o m e n i n g e a l c e l l s . I n f a c t i t was - 6 - t h i s s c a r t i s s u e t h a t gave us t h e o r i g i n a l c o n c e p t o f a s t r u c t u r a l s u p p o r t r o l e f o r g l i a . C a j a l ' s i d e a t h a t g l i a s e r v e t o i n s u l a t e and i s o l a t e n e r v e f i b e r s and b u n d l e s i s s t i l l h i g h l y s u p p o r t e d t o d a y . N o t o n l y a r e t h e n e r v e s m y e l i n a t e d f o r i n c r e a s e d e f f i c i e n c y b u t t h e s y n a p t i c t e r m i n a l s a r e a l s o s e p a r a t e d f r o m e a c h o t h e r by g l i a l c e l l s . T h i n a s t r o c y t i c p r o c e s s e s b r e a k up t h e n e u r o p i l i n t o m o s a i c s o f s m a l l r e g i o n s e a c h c o n t a i n i n g a s y n a p t i c f i e l d . A s i m i l a r p a r c e l l i n g o c c u r s a r o u n d c l u s t e r s o f s y n a p t i c t e r m i n a l s . A s t r o c y t i c p r o c e s s e s o f t e n i n t e r v e n e between c e l l t y p e s o r n e u r o n a l g r o u p s , i s o l a t i n g n e u r o n a l s u r f a c e s i n s u c h a way a s t o p r e v e n t f l o w o f i m p u l s e s i n a h a p h a z a r d manner ( L a s a n s k y , 1 9 7 1 ) . T h e s e a r e a s h a v e many membrane s p e c i a l i z a t i o n s and seem t o h a v e c o n t i n u o u s d y n a m i c a l t e r a t i o n s ( W o l f f and G u l d n e r , 1978) a s i f a c t i v e l y i n v o l v e d i n t h e i s o l a t i o n p r o c e d u r e . L u g a r o ' s (1907) c o n c e p t o f a d e t o x i f i c a t i o n f i l t e r c a n be compared t o t h e m i n o r r o l e t h a t t h e a s t r o c y t i c e nd f e e t may p l a y i n t h e b l o o d b r a i n b a r r i e r . T h e y a r e no l o n g e r b e l i e v e d t o p r o v i d e a b a r r i e r a r o u n d c a p i l l a r i e s b u t , b e c a u s e most i n c o m i n g c h e m i c a l s must go t h r o u g h t h e i r n o n - o c c l u d i n g and n o n - c o n t i n u o u s j u n c t i o n s , t h e y may have t h e f i r s t o p p o r t u n i t y a t t h e s e l e c t i o n o f i n c o m i n g c h e m i c a l s . G o l g i (1894) o r i g i n a l l y t h o u g h t t h a t t h e g l i a p r o v i d e d b i o c h e m i c a l s u p p o r t and n u t r i t i o n f o r t h e n e u r o n s . T h i s i d e a h a s e v o l v e d t o i n c l u d e s e v e r a l d i f f e r e n t c o n c e p t s . S a t e l l i t e o l i g o d e n d r o c y t e s ( t h o s e c e l l b o d i e s l y i n g n e a r l o n g axons) may be i n v o l v e d i n n e u r o n a l n u t r i t i o n : F r e i d e , (1966) t h o u g h t o f - 7 - them a s a u x i l i a r y m e t a b o l i c u n i t s f o r t h e a x o n s o f n e u r o n s . A s t r o c y t i c end f e e t may be i n v o l v e d i n t h e t r a n s p o r t o f s u b s t a n c e s i n w a r d t o n e u r o n s and g l i a a t t h e c e n t r e o f t h e b r a i n mass. I n many c a s e s n e u r o n s and g l i a do h a v e c o m p l i m e n t a r y m e t a b o l i s m s . C u l t u r e d n e u r o n s o n l y s u r v i v e a few d a y s w i t h o u t g l i a u n l e s s n e r v e g r o w t h f a c t o r s o r b r a i n e x t r a c t s a r e a d d e d . Medium t h a t h a s s u r r o u n d e d g l i a w i l l s u p p o r t n e u r i t e g r o w t h ( E b e n d a l and J a c o b s o n , 1 9 7 5 ) . T h i s means some s o l u b l e f a c t o r ( s ) must be i n v o l v e d i n m a i n t a i n i n g t h e n e u r o n s b u t t h i s may n o t be a n u t r i t i o n a l s u b s t a n c e . C o n t r a r y t o t h e c l a s s i c a l a s s u m p t i o n s on g l i a f u n c t i o n , t h e m e t a b o l i c r a t e o f g l i a c e l l s i s now known t o be q u i t e h i g h . H e r t z (1978) showed t h a t t h e e a r l y work done on g l i a l c e l l l i n e s a n d g l i a l s c a r t i s s u e h a d g i v e n e r r o n e o u s l y low m e t a b o l i c r a t e s f o r g l i a . E n e r g y m e t a b o l i s m i n some t y p e s i s c o m p a r a b l e t o t h a t o f n e u r o n s ( H e r t z , 1 9 8 2 ) . G l i a h a v e t h e m a j o r i t y o f o x i d a t i v e enzymes, and a l s o h a v e r e d u c t i v e enzymes, a l t h o u g h a s t r o c y t e s a r e l o w e r i n o x i d o r e d u c t a s e enzymes t h a n a r e o l i g o d e n d r o c y t e s . The r a t e o f p r o t e i n s y n t h e s i s s u g g e s t s t h a t t h e a s t r o c y t e s a r e m a k i n g a c o n s i d e r a b l e p r o p o r t i o n o f t h e t o t a l b r a i n p r o t e i n (White and H e r t z , 1 9 8 1 ) . O l i g o d e n d r o c y t e s h a v e a h i g h e r o x y g e n u t i l i z a t i o n t h a n a s t r o c y t e s and consume much o f t h e o x y g e n i n w h i t e m a t t e r ( P e v s n e r , 1 9 7 9 ) . Cummins e t a l . (1979) showed t h a t t h e u p t a k e by g l i a o f 2 r a d i o a c t i v e m e t a b o l i s m m a r k e r s c a n be i n c r e a s e d b y p o t a s s i u m (K+) o r h i g h l e v e l s o f n e u r o t r a n s m i t t e r s , i n d i c a t i n g a m e t a b o l i c r e s p o n s i v e n e s s t o t h e i r e n v i r o n m e n t . - 8 - G l i a may n o t o n l y i n t e r a c t w i t h n e u r o n s b u t may have some o f t h e i o n c o n d u c t a n c e and r e c e p t o r p r o p e r t i e s t r a d i t i o n a l l y a s s o c i a t e d w i t h n e u r o n s . A s t r o c y t e s h a v e a r e s t i n g membrane p o t e n t i a l t h a t i s s l i g h t l y h i g h e r t h a n t h a t o f n e u r o n s , b e i n g 70-90 mV, and t h a t v a r i e s w i t h t h e e x t e r n a l K+ l e v e l s a c c o r d i n g t o t h e N e r n s t e q u a t i o n ( P e v z n e r , 1 9 7 9 ) . Thus t h e y may h a v e some r o l e i n t h e p r o d u c t i o n o f e x t r a c e l l u l a r c u r r e n t . Bowman and K i m e l b e r g (1984) showed t h a t a s t r o c y t e s c a n be d e p o l a r i z e d i n p r i m a r y c u l t u r e , a p r o p e r t y p r e v i o u s l y t h o u g h t t o be e x c l u s i v e t o n e u r o n s . They d e p o l a r i z e i n t h e p r e s e n c e o f 0 - a m i n o b u t y r i c a c i d (GABA), L - g l u t a m a t e , D- and L - a s p a r t a t e and k a i n i c a c i d i n c u l t u r e . I n v i v o t h e amino a c i d s h a v e been r e p o r t e d t o d e p o l a r i z e a l l a s t r o c y t e s l y i n g i n t h e v i c i n i t y o f n e u r o n s . B u t s u b s t a n c e s t h a t r e v e r s i b l y b l o c k K+ c o n d u c t a n c e a b o l i s h e d t h e d e p o l a r i z a t i o n o f g l i a l c e l l s (Bowman and K i m e l b e r g , 1 9 8 4 ) . T h e r e f o r e t h e g l i a l c e l l s may n o t h a v e r e c e p t o r s f o r t h e s e amino a c i d s , b u t d e p o l a r i z e d b e c a u s e o f t h e e f f l u x o f K+ f r o m t h e n e u r o n s . T h i s s u b j e c t i s s t i l l c o n t r o v e r s i a l . L u g a r o (1907) was t h e f i r s t t o p o s t u l a t e t h a t g l i a remove and c a t a b o l i z e s u b s t a n c e s r e l e a s e d f r o m n e r v e s . Now we know o f many s u b s t a n c e s t h a t a r e removed f r o m t h e s y n a p t i c c l e f t by g l i a a n d u n d e r s t a n d s u c h a c t i o n s t o be o f m a j o r i m p o r t a n c e i n t h e f u n c t i o n i n g b r a i n . G l i a a l s o seem t o c o n t r o l e x t r a c e l l u l a r K+ l e v e l s . They f u n c t i o n a s a f i n e t u n i n g mechanism a f t e r t h e n e u r o n s do most o f t h e u p t a k e . I t c a n be shown t h a t g l i a c o u l d t a k e up enough K+ t o c l e a r t h e e x c e s s t h a t l e a k s o u t o f n e u r o n s b u t w h e t h e r - 9 - t h e y a c t u a l l y do t h i s i s s t i l l i n q u e s t i o n . K+ r e l e a s e d f r o m n e u r o n s c a u s e s an i n c r e a s e i n e x t r a c e l l u l a r K+ w h i c h e v e n t u a l l y c a u s e s t h e n e u r o n s t o f i r e ( P r i n c e , 1 9 7 8 ) . The g l i a l s h e e t s a c t as dams r e s t r i c t i n g t h e d i f f u s i o n o f K+. G l i a l c e l l s remove K+ and m i n i m i z e t h e s p r e a d o f K+ t o o t h e r r e g i o n s , t h u s a c t i n g a s a b u f f e r zone ( T r a c h t e n b e r g and P o l l e n , 1 9 7 0 ) . G l i a a r e i d e a l l y s u i t e d f o r t h i s r o l e b e c a u s e t h e y h a v e a h i g h r e s t i n g membrane p o t e n t i a l , a r e s e l e c t i v e l y p e r m e a b l e t o K+, a r e e l e c t r i c a l l y e x c i t a b l e , and h a v e i r r e g u l a r b o d i e s w i t h many p r o c e s s e s . T h i s u p t a k e p r o c e s s may be b y a c t i v e t r a n s p o r t b e c a u s e g l i a h a v e an a d e n o s i n e - 5 t r i p h o s p h a t a s e (ATPase) t h a t i s s p e c i f i c a l l y a c t i v a t e d b y K+ ( F r a n c k e t a l . , 1978, G r i s a r and S c h o f f e n i e l s , 1978, Grossman, 1978, and P r i n c e e t a l . , 1 9 7 8 ) . T h i s A T P a s e i s a s s e n s i t i v e a s n e u r o n a l A T P a s e t o o u a b a i n (Walz and H e r t z , 1 9 8 2 ) . The c o n c e p t o f an a c t i v e r o l e f o r g l i a i n K+ h o m o s t a s i s h a s t h r e e p r e r e q u i s i t e s . F i r s t , t h a t t h e K+ r e l e a s e d f r o m n e u r o n s l e a d s t o a b u i l d - u p o f e x t r a c e l l u l a r K+; t h i s i s u n a n i m o u s l y a c c e p t e d . S e c o n d , t h a t t h e e x c e s s K+ i s removed by s u r r o u n d i n g c e l l s , n o t t h r o u g h d i f f u s i o n ; t h i s h a s now b e en d e m o n s t r a t e d and p r o b a b l y r e q u i r e s e n e r g y a s i t h a s b e e n shown t h a t e x c e s s K+ l e a d s t o a t r a n s i e n t i n c r e a s e i n r e s p i r a t i o n i n m i c r o d i s s e c t e d g l i a l c e l l s , b u l k - p r e p a r e d a s t r o g l i a , o r c u l t u r e d a s t r o c y t e s i f t h e c u l t u r e d c e l l s a r e 3-4 weeks o l d ( H e r t z , 1982) . T h i r d , t h a t K+ u p t a k e i n t o a s t r o c y t e s i s more i n t e n s e t h a n i n t o n e u r o n s and i s f u r t h e r i n c r e a s e d b y an i n c r e a s e i n t h e e x t r a c e l l u l a r K+ l e v e l b e y o n d t h e r e s t i n g l e v e l ; t h e l a r g e K+ c o n t e n t i n a s t r o c y t e s and h i g h membrane - 10 - p o t e n t i a l s u n e q u i v o c a l l y show t h a t t h e s e c e l l s a r e a b l e t o a c c u m u l a t e l a r g e amount o f K+. H e r t z and Chaban (1982) showed t h a t a s t r o c y t e s h a v e u p t a k e r a t e s h i g h e r t h a n n e u r o n s i n c u l t u r e s . T h i s t h u s s a t i s f i e s t h e t h i r d c r i t e r i a o f a c t i v e g l i a l t r a n s p o r t . T h ey a l s o showed t h i s u p t a k e i s i n h i b i t e d by o u a b a i n i n b o t h C-6 c e l l s (a much s t u d i e d g l i a l c e l l l i n e ) and p r i m a r y c u l t u r e s o f a s t r o c y t e s , i n d i c a t i n g t h a t a s o d i u m (Na+) -K+ e x c h a n g e c a t a l y z e d b y t h e Na+,K+-ATPase e x i s t s . A l t h o u g h u p t a k e i s r e d u c e d by o u b a i n , i t i s n o t c o m p l e t e l y a b o l i s h e d . T h e r e f o r e a n o t h e r mechanism must e x i s t w h i c h o u b a i n d o e s n o t i n h i b i t ; t h i s mechanism i s p r o b a b l y d e p e n d a n t on c a r b o n i c a n h y d r a s e b e c a u s e a c e t a z o l a m i d e , an i n h i b i t o r o f c a r b o n i c a n h y d r a s e , i n h i b i t s some p o t a s s i u m u p t a k e i n t o c e l l s ( H e r t z and Chaban, 1 9 8 2 ) . G l i a and N e u r o t r a n s m i t t e r s G l i a l c e l l s a l s o seem t o be i n v o l v e d i n many a s p e c t s o f n e u r o t r a n s m i t t e r f u n c t i o n . As L u g a r o (1907) s u g g e s t e d , g l i a may t a k e up s u b s t a n c e s r e l e a s e d b y n e u r o n s . T hey may a c t i v e l y t a k e up n e u r o t r a n s m i t t e r s b y mechanisms t h a t a r e n o t a l w a y s i d e n t i c a l t o t h e u p t a k e i n t o n e u r o n s . T h e y a r e c a p a b l e o f c a t a b o l i z i n g some o f t h e s e n e u r o t r a n s m i t t e r s and t h e r e i s e v i d e n c e o f r e c e p t o r s on some t y p e s o f g l i a . A. A c t i v e u p t a k e o f s e v e r a l n e u r o t r a n s m i t t e r s h a s b e e n d e m o n s t r a t e d i n g l i a l c e l l p o p u l a t i o n s . U s i n g p r i m a r y c u l t u r e s o f a s t r o c y t e s , S c h o u s b o e (1978) showed t h a t g l i a c a n t a k e up n o r a d r e n a l i n e (NA), dopamine (DA), and s e r o t o n i n - 11 - (5HT) . H e r t z (1982) showed t h a t t h i s o c c u r r e d i n an energy- d e p e n d e n t manner r e q u i r i n g b o t h Na+ and K+. H a n s s o n e t a l . (1984a) d i d n o t c o n f i r m t h i s d a t a f o r DA. S e v e r a l o f t h e amino a c i d n e u r o t r a n s m i t t e r s a l s o seem t o be t a k e n up b y g l i a . L e v i e t a l . (1982) a n d W i l k i n e t a l . (1982) showed t h a t t h e u p t a k e o f amino a c i d s by s l i c e s o f c e r e b e l l u m was p r e d o m i n a n t l y i n t o a s t r o g l i a l c e l l s r a t h e r t h a n n e u r o n s . A h i g h a f f i n i t y u p t a k e o f g l y c i n e i n t o g l i a h a s been r e p e a t e d l y d e m o n s t r a t e d ( H o k f e l t and L u n g d a h l , 1971, Matus and D e n n i s o n , 1971, Henn, 1 9 7 6 ) . GABA h a s r e p e a t e d l y b e e n shown t o be t a k e n up by g l i a l c e l l s (Henn, 1976, C u r r i e and K e l l y , 1 9 8 1 ) . H a n s s o n e t a l . (1984b) and L a r s o n e t a l . (1980) showed t h a t t h i s u p t a k e was Na+ d e p e n d e n t . T h e r e i s c o n t r o v e r s y o v e r w h e t h e r g l i a l u p t a k e o f GABA i s g r e a t e r o r l e s s t h a n i n t o n e u r o n s . B a l c a r e t a l . (1982) f o u n d t h e u p t a k e i n t o g l i a l e s s i n t e n s e t h a n i n t o n e u r o n s , w h e r e a s S c h o u s b o e (1978) c a l c u l a t e d , b a s e d on t h e maximum v e l o c i t y (Vmax) i n a s t r o c y t e s i n p r i m a r y c u l t u r e , t h a t t h e r a t e c o u l d be 2 t o 6 t i m e s h i g h e r t h a n i n t o n e u r o n s and c o u l d b e i n c r e a s e d b y c a l c i u m (Ca++) o r low K+. S c h o u s b o e (1981) a l s o r e v i e w e d t h e work o f many o t h e r s and f o u n d t h a t c u l t u r e d a s t r o c y t e s e x h i b i t a Vmax c o m p a r a b l e t o t h a t f o u n d i n b r a i n s l i c e s . L - G l u t a m a t e and D- o r L - a s p a r t a t e a p p e a r t o s h a r e common t r a n s p o r t s y s t e m s . H i g h a f f i n i t y u p t a k e o f g l u t a m a t e o r D - a s p a r t a t e i n t o g l i a h a s b e e n r e p e a t e d l y d e m o n s t r a t e d a u t o r a d i o g r a p h i c a l l y ( H o k f e l d and L j u n g d a h l , 1972, Sc h o n and K e l l y , 1974, L a s h e r , 1975, McLennan, 1976, C u r r i e and K e l l y , - 12 - 1 9 8 1 ) . The g l i a l u p t a k e r e q u i r e s t h e p r e s e n c e o f b o t h Na+ and K+ and i s b o t h e n e r g y and t e m p e r a t u r e d e p e n d e n t ( S c h o u s b o e , 1978) . U p t a k e o f g l u t a m a t e h a s a l s o b e e n d e m o n s t r a t e d i n t o many c u l t u r e d c e l l l i n e s (Hamberger, 1971, Henn e t a l . , 1974, W e i l e r e t a l . , 1 9 7 9 ) . I t h a s a l s o b e e n d e m o n s t r a t e d i n a s t r o c y t e s p r e p a r e d b y g r a d i e n t c e n t r i f u g a t i o n ( F a i v r e - B a u m a n n e t a l . , 1974, Henn e t a l . , 1974, B a l c a r e t a l . , 1977, P f e i f f e r e t a l . , 1 9 7 0 ) , i n c l u d i n g a s t r o c y t o m a s ( S n o d g r a s s and I v e r s e n , 1 9 7 4 ) , r e t i n a l M u l l e r c e l l s ( B r u u n and E h i n g e r , 1974, W h i t e and N e a l , 1976) and a s t r o c y t e s i n p r i m a r y c u l t u r e ( S c h o u s b o e e t a l . , 1977b, H e r t z e t a l . , 1979, and B a l c a r a n d H o u s e r , 1 9 7 8 ) . S c h o u s b o e (1978) showed t h a t t h e g l u t a m a t e Vmax f o r a s t r o c y t e s i n p r i m a r y c u l t u r e was much h i g h e r t h a n t h a t f o r g l u t a m a t e u p t a k e i n t o s e n s o r y g a n g l i a o r g l i a l c e l l l i n e s . I n some a s t r o c y t e c e l l l i n e s i t may be h i g h enough t o keep p a c e w i t h r e l e a s e f r o m n e u r o n s and a l s o h i g h enough s o t h a t t h e g l u t a m a t e may be t h e i r o n l y f u e l s o u r c e ( H e r t z , 1 9 7 9 ) . I n some o f t h e o t h e r g l i a l c e l l l i n e s t h i s r a t e i s p r o b a b l y n o t enough t o p r o v i d e t h e s o l e f u e l s o u r c e . We know t h a t t r a n s m i t t e r u p t a k e i n g l i a i s n o t a g e n e r a l phenomenom b e c a u s e n e u t r a l amino a c i d s o t h e r t h a n GABA g l y c i n e m a t e r i a l s , and c l o s e l y r e l a t e d m a t e r i a l s , h a v e no h i g h a f f i n i t y u p t a k e i n t o g l i a . B. A l t h o u g h t h e u p t a k e o f n e u r o t r a n s m i t t e r s h a s b e e n d e m o n s t r a t e d , t h e r e i s e v i d e n c e t h a t some o f t h i s u p t a k e i s n o t i d e n t i c a l t o t h a t w h i c h o c c u r s i n n e u r o n s i n e i t h e r - 13 - c h a r a c t e r o r q u a n t i t y . The u p t a k e o f monoamines i n t o p r i m a r y c u l t u r e s , f o r i n s t a n c e , i s a t l o w e r r a t e s t h a n i n t o n e u r o n a l c u l t u r e s ( H e r t z , 1982) b u t t h e u p t a k e o f L - g l u t a m a t e i s h i g h e r ( S c h o u s b o e , 1 9 7 8 a ) . However a l a r g e number o f g l i a l c e l l l i n e s show h i g h a f f i n i t y u p t a k e s s i m i l a r i n r a t e t o t h o s e o f n e u r o n s (Edwards e t a l . , 1 9 7 9 ) . The mechanism c a n a l s o be q u i t e d i f f e r e n t . F o r example, W a n i e w s k i and M a r t i n (1983) f o u n d t h a t 4 - a c e t a m i d o - 4 1 - i s o t h i o c y a n o - 2 , 2 ' - d i s u l f o n i c a c i d s t i l b e n e , an i n h i b i t o r o f a n i o n e x c h a n g e , was a p o t e n t and s e l e c t i v e i n h i b i t o r o f L - g l u t a m i c a c i d u p t a k e by c u l t u r e d g l i o m a c e l l l i n e and r a t b r a i n a s t r o c y t e s b u t d i d n o t a f f e c t s y n a p t o s o m a l u p t a k e . T h e r e f o r e g l u t a m a t e t r a n p o r t s y s t e m s d i f f e r b e t w e e n n e u r o n s and g l i a . R a m a h a r o - B r a n d r o e t a l . (1982) r e p o r t t h a t n e u r o n a l and g l i a l g l u t a m a t e c a r r i e r s e x h i b i t d i f f e r e n c e s i n t e r m s o f b o t h s u b s t r a t e s p e c i f i c i t y a nd i n t e r m s o f d e p e n d e n c y on mono- o r d i - v a l e n t c a t i o n s . O n l y n e u r o n a l u p t a k e i s d e p e n d a n t on b o t h Na+ a n d Ca++, and i s t h e r e f o r e more s u s c e p t i b l e t o c h a n g e s i n e x t e r n a l i o n i c c o n c e n t r a t i o n s . F u r t h e r m o r e , a s t r o c y t e u p t a k e o f g l u t a m a t e was f o u n d t o be n o n - c o m p e t i t i v e l y i n h i b i t e d by D - a s p a r t a t e w h e r e a s u p t a k e by g r a n u l e c e l l s was c o m p e t i t i v e l y i n h i b i t e d . U p t a k e o f g l u t a m a t e i n a s t r o c y t e s f r o m p r e f r o n t a l c o r t e x was c o u p l e d t o 1 Na+ i o n i n c o n t r a s t t o 2 f o r t h e g r a n u l e c e l l . G l i a l c e l l s e x h i b i t e d no K+ i n d u c e d r e l e a s e o f g l u t a m a t e i n c o n t r a s t t o n e u r o n s ( D r e j e r e t a l . , 1 9 8 2 ) . A s i m i l a r d i f f e r e n c e between n e u r o n a l and g l i a l u p t a k e c a n - 14 - be shown f o r GABA u p t a k e . K e l l y and D i c k (1978) showed t h a t - a l a n i n e i s a s p e c i f i c b l o c k e r o f GABA u p t a k e i n g l i a b u t n o t n e u r o n s and a c y c l o h e x a n e a m i n e d e r i v a t i v e i s a b l o c k e r s p e c i f i c f o r GABA u p t a k e i n t o n e u r o n s . Thus t h e two GABA u p t a k e s y s t e m s a r e b i o c h e m i c a l l y d i f f e r e n t . G l i a l c e l l s c a n a l s o r e l e a s e some n e u r o t r a n s m i t t e r s and t h i s r e l e a s e c a n be d e m o n s t r a t e d t o be d i f f e r e n t i n some c a s e s f r o m t h a t o f n e u r o n s . C. G l i a c e l l s c a n a l s o p o s s e s s c a t a b o l i c enzymes. A l t h o u g h a c e t y l c h o l i n e (Ach) u p t a k e h a s n o t b e e n d e m o n s t r a t e d i n t o g l i a o r n e u r o n s , a c e t y l c h o l i n e s t e r a s e (AChE) a c t i v i t y c a n be f o u n d i n c e r t a i n c l o n a l l i n e s o f g l i a l c e l l s (C-6) ( V e r n a d a k i s and A r n o l d , 1 9 8 0 ) . G l i a a l s o p o s s e s s h i g h e r s p e c i f i c a c t i v i t i e s o f t h e monoamine c a t a b o l i z i n g enzymes, monoamine o x i d a s e (MAO) (Hazama e t a l . , 1976, H a n s s o n and S e l l s t r o m , 1983) and c a t c h o l - O - m e t h y l t r a n s f e r a s e (COMT) t h a n f o u n d i n w h o l e b r a i n . The p r e s e n c e o f COMT and MAO h a s b e e n shown i n s e v e r a l g l i a l c e l l l i n e s w h i c h s u g g e s t s t h e y h a v e t h e a b i l i t y t o i n a c t i v a t e c a t e c h o l a m i n e s ( S i l b e r s t e i n e t a l . , 1 9 7 2 ) . The d e g r a d a t i v e enzyme f o r GABA, GABA t r a n s a m i n a s e (GABA-T), h a s b e e n d e m o n s t r a t e d i n g l i a . B u l k p r e p a r e d g l i a a nd c u l t u r e d a s t r o c y t e s s t a i n f o r GABA-T ( S e l l s t r o m e t a l . , 1977, T a r d y e t a l . , 1 978), as do a s t r o c y t e s c u l t u r e d f r o m n e o n a t a l b r a i n ( S c h o u s b o e e t a l . , 1 9 7 2 ) . GABA-T a c t i v i t y i n g l i a l c u l t u r e s , however, i s l o w e r t h a n i n c e r e b r a l h e m i s p h e r e s ( H a n s s o n and S e l l s t r o m , 1983) and l o w e r t h a n i n n e u r o n s ( K e l l y - 15 - and D i c k , 1 9 7 8 ) . The g l u t a m a t e e v e n t u a l l y g e n e r a t e d by t h e breakdown o f GABA and f r o m o t h e r s o u r c e s c a n a l s o be c a t a b o l i z e d i n g l i a . I n f a c t g l u t a m i n e s y n t h e t a s e (GS), t h e d e g r a d a t i v e enzyme w h i c h p l a y s a m a j o r r o l e i n t h e c h e m i s t r y o f g l u t a m a t e , i s f o u n d o n l y i n a s t r o c y t e s . F o r a number o f y e a r s g l u t a m a t e has b e e n t h o u g h t t o e x i s t i n two p o o l s , one i n t h e n e u r o n s f r o m w h i c h g l u t a m a t e i s r e l e a s e d when t h e n e u r o n s f i r e , and a s e c o n d s m a l l e r p o o l i n t h e g l i a , where g l u t a m a t e i s c o n v e r t e d i n t o g l u t a m i n e w i t h t h e h e l p o f GS. T h i s g l u t a m i n e i s t h e n r e l e a s e d t o be t a k e n up b y n e u r o n s and r e c o n v e r t e d t o g l u t a m a t e . T h i s schema r e m a i n s h i g h l y c o n t r o v e r s i a l . T he r e s t r i c t i o n o f GS a c t i v i t y i n t h e b r a i n i n v i v o t o a s t r o c y t e s ( N o r e n b e r g and M a r t i n e z - H e r m a n d e z , 1979) and t h e h i g h a c t i v i t y o f t h i s enzyme i n p r i m a r y c u l t u r e s o f a s t r o c y t e s ( S c h o u s b o e e t a l . , 1980) a r e c o n s i s t e n t w i t h t h e c o n c e p t t h a t any g l u t a m a t e a c c u m u l a t e d i n a s t r o c y t e s , i s t o a l a r g e e x t e n t c o n v e r t e d t o g l u t a m i n e . Some r e s e a r c h e r s , however, have f o u n d GS a c t i v i t y low i n g l i a l c e l l s r e l a t i v e t o w h o l e b r a i n ( N i c k l a s and B r o w n i n g , 1978) b u t t h i s i s a f u n c t i o n o f t h e age o f t h e g l i a l c e l l s . H i g h GS a c t i v i t y m a t u r e s l a t e i n d e v e l o p m e n t . I n a c c o r d a n c e w i t h t h e l a t e m a t u r i n g o f GS, t h e r a t e o f g l u t a m i n e s y n t h e s i s i s f a s t e r i n 3 week c u l t u r e s t h a n 1 week o l d o n e s b u t i t d o e s n o t i n c r e a s e i n r e s p o n s e t o d i - b u t y l c y c l i c a d e n i n e mono-phosphate (dBcAMP) w h i c h i s g e n e r a l l y t h o u g h t t o c a u s e m a t u r a t i o n . Two o t h e r g l u t a m a t e - m e t a b o l i z i n g enzymes, g l u t a m a t e d e h y d r o g e n a s e and g l u t a m a t e o x a l o a c e t a t e t r a n s f e r a s e , w h i c h - 16 - c o n v e r t g l u t a m a t e t o °\ - k e t o g l u t a r a t e , a r e a l s o p r e s e n t i n a s t r o c y t i c c u l t u r e s a t h i g h a c t i v i t i e s ( S c h o u s b o e e t a l . , 1 9 8 0 a ) . T h i s s u g g e s t s t h a t g l u t a m a t e a c c u m u l a t e d i n a s t r o c y t e s may be c o n v e r t e d t o t r i c a r b o x y l i c a c i d (TCA) c y c l e c o n s t i t u e n t s and t h u s be a m e t a b o l i c s u b s t r a t e . T h i s w o u l d mean t h a t t h e g l u t a m a t e t o g l u t a m i n e t o g l u t a m a t e l o o p w o u l d n o t be c o m p l e t e d . S u p p o r t f o r s u c h an a l t e r n a t i v e r o u t e was s u p p l i e d b y s t u d i e s o f t h e f a t e o f r a d i o a c t i v e g l u t a m a t e i n d e v e l o p i n g c u l t u r e s o f mouse a s t r o c y t e s ( P o t t e r e t a l . , 1982); t h e r a d i o a c t i v i t y o f g l u t a m i n e n e v e r e x c e e d e d t h a t o f i t s p r e c u r s o r g l u t a m a t e i n d i c a t i n g t h e o t h e r m e t a b o l i c r o u t e s must e x i s t . P o s s i b l e r o l e s f o r g l i a i n h e l p i n g t o d i s p o s e o f p e p t i d e n e u r o t r a n s m i t t e r s h a v e n o t y e t b e e n w i d e l y i n v e s t i g a t e d . However, L e n t z e n and P a l e n d k e r (1983) showed, by u s i n g s p e c i f i c enzyme i n h i b i t o r s and e x a m i n i n g t h e p r o d u c t s , t h a t g l i a l c e l l s h a v e t h e a b i l i t y t o d e g r a d e e n k e p h a l i n , a p e p t i d e n e u r o t r a n s m i t t e r , b y b o t h a m i n o p e p t i d a s e and membrane bound e n k e p h a l i n a s e A. D. T h e r e i s c o n s i d e r a b l e e v i d e n c e t h a t some g l i a c a n p o s s e s s r e c e p t o r o r b i n d i n g s i t e s f o r v a r i o u s n e u r o c h e m i c a l s . F o r example, on s e l e c t i v e l y d e s t r o y i n g t h e M u l l e r g l i a c e l l s i n t h e r e t i n a , Memo e t a l . (1981) were a b l e t o show a s e l e c t i v e l o s s o f DA and 5HT b i n d i n g s i t e s , s u g g e s t i n g t h a t M u l l e r c e l l s c a r r y t h e s e r e c e p t o r s . Henn and Henn (1980) showed dopamine b i n d i n g s i t e s on a s t r o c y t e s t h a t a r e l i n k e d t o a d e n y l a t e c y c l a s e and s t i m u l a t e - 17 - cAMP f o r m a t i o n , w h i c h i s b l o c k e d by a n t i p s y c h o t i c d r u g s . A s t r o c y t e s p r e p a r e d f r o m a r e a s r i c h i n dopamine show dopamine b i n d i n g t h a t c a n be b l o c k e d b y a n t i p s y c h o t i c d r u g s (Hansson e t a l . , 1 9 8 4 ) , and a n t i p s y c h o t i c e f f e c t i v e n e s s i s c o r r e l a t e d w i t h t h e i r a b i l i t y t o d i s p l a c e dopamine ( H e r t z , 1981) i n b u l k p r e p a r e d c e l l s . The p o t e n c y o f t h e d r u g s i n b l o c k i n g t h e f o r m a t i o n o f cAMP i n a s t r o c y t i c c u l t u r e s i s a l s o s a i d t o be w e l l c o r r e l a t e d t o t h e i r e f f e c t i v e n e s s a s a n t i p s y c h o t i c s ; t h i s i s n o t t r u e w i t h n e u r o n a l p r e p a r a t i o n s where t h e a n t i p s y c h o t i c a c t i o n seems more c l o s e l y r e l a t e d t o t h e dopamine b i n d i n g s i t e s w h i c h a r e n o t c l o s e l y c o u p l e d w i t h 3 ' - 5 ' c y c l i c a d e n i n e m onophosphate (cAMP). H o s l i e t a l . (1984) showed t h a t a s t r o c y t e s c u l t u r e s f r o m r a t b r a i n s t e m and s p i n a l c o r d h a d h i s t a m i n e t y p e 1 (HI) and h i s t a m i n e t y p e 2 (H2) r e c e p t o r s . The HI a g o n i s t t h i a z o l e t h y l a m i n e p r o d u c e d m a i n l y d e p o l a r i z a t i o n s w h i l e i m p r o m i d i n e , a H2 a g o n i s t , c a u s e d h y p e r p o l a r i z a t i o n s . T h e r e a r e many o t h e r i n s t a n c e s o f g l i a c e l l s i n t e r a c t i o n s w i t h d r u g s w h i c h i n d i c a t e t h a t g l i a l c e l l s may h a v e r e c e p t o r s f o r t h e d r u g s and show r e c e p t o r m e d i a t e d r e s p o n s e s s i m i l a r t o t h o s e s e e n i n n e u r o n s . A r e c e n t a r t i c l e b y H e r t z and R i c h a r d s o n (1984) r e v i e w e d t h e d a t a on t h i s t o p i c . C-6 c e l l s , a g l i o m a c e l l l i n e , i n c r e a s e t h e i r l e v e l s o f cAMP i n r e s p o n s e t o NA o r i s o p r o t e r e n o l ( G i l m a n and N i r e n b e r g , 1 9 7 1 ) . A s i m i l a r i n c r e a s e was n o t e d i n t h e human g l i o m a l i n e number 1181 ( C l a r k and P e r k i n s , 1 9 7 1 ) . B o t h t h e s e c e l l t y p e s must t h e n p o s s e s s a l l t h e known components o f t h e cAMP r e g u l a t i n g s y s t e m ( P e r k i n s e t a l . , 1971) and h a v e r e c e p t o r s - 18 - f o r a d r e n e r g i c d r u g s . However, a d r e n e r g i c r e c e p t o r s i n a s t r o c y t e s and n e u r o n s may show d i f f e r e n t p h a r m a c o l o g i c a l p r o f i l e s ( B e n d e r and H e r t z , 1 9 8 4 ) . C h r o n i c e x p o s u r e t o a d r e n e r g i c d r u g s c a n c a u s e a down r e g u l a t i o n o f a d r e n e r g i c r e c e p t o r s on g l i a ( H e r t z a n d R i c h a r d s o n , 1984) a s on n e u r o n s . F o r example, c h r o n i c e x p o s u r e o f a s t r o c y t i c c e l l l i n e s t o i s o p r o t e r e n o l l e a d s t o a d e c r e a s e d a c c u m u l a t i o n o f cAMP and a d e c r e a s e d r e s p o n s e t o some d r u g s w i t h ^ - a g o n i s t p r o p e r t i e s ( H e r t z and R i c h a r d s o n , 1983) . V a r i o u s a n t i d e p r e s s a n t d r u g s , s u c h a s d o x e p i n ( H e r t z and R i c h a r d s o n , 1983) and i m i p r a m i n e ( W h i t a k e r e t a l . , 1 9 8 3 ) , a r e bound t o o r t a k e n up b y i n t a c t a s t r o c y t e s b u t t h i s m i g h t be b e c a u s e o f t h e l i p o p h i l i c n a t u r e o f t h e s e d r u g s . A n t i d e p r e s s a n t s m i g h t a l s o i n t e r a c t w i t h t h e Ot7- and ^ - a d r e n e r g i c r e c e p t o r s i t e s on a s t r o c y t e s . T h e s e s i t e s a r e known t o e x i s t and an i n t e r a c t i o n o f a n t i d e p r e s s a n t s w i t h . - a d r e n o r e c e p t o r s i s e v i d e n t s i n c e s u c h d r u g s i n h i b i t i s o p r o t e r e n o l - i n d u c e d s t i m u l a t i o n o f cAMP p r o d u c t i o n ( H e r t z and R i c h a r d s o n , 1983) . The b i n d i n g o f / 3 - a d r e n e r g i c l i g a n d s t o C-6 and a s t r o c y t o m a c e l l l i n e s i s a l s o i n h i b i t e d by a l l g r o u p s o f a n t i d e p r e s s a n t s b u t n o t by a n x i o l y t i c o r a n t i p s y c h o t i c d r u g s ( H e r t z e t a l . , 1 9 82b). Henn i n 1980 d e m o n s t r a t e d b i n d i n g o f t h e b e n z o d i a z e p i n e , d i a z e p a m , t o a s t r o c y t e s . T h i s s e l e c t i v e b i n d i n g c a n be b e t t e r d e m o n s t r a t e d w i t h a n o t h e r b e n z o d i a z e p i n e , R05-4864, b e c a u s e i t d i s s o c i a t e s l e s s r a p i d l y f r o m a s t r o c y t e s t h a n f r o m n e u r o n a l b i n d i n g s i t e s (Shoemaker e t a l . , 1 9 8 3 ) . - 19 - H e r t z and M r u e r j i (1980) showed a l a r g e amount o f s p e c i f i c d i a z e p a m b i n d i n g on p r i m a r y a s t o c y t e s i n c u l t u r e . Diazepam may be d i s p l a c e d by o t h e r b e n z o d i a z e p i n e s o r b y h i g h c o n c e n t r a t i o n s o f b a r b i t u a t e s . Thus t h e s e d r u g s may be a c t i n g t h r o u g h t h e same r e c e p t o r s . B a r b i t u a t e s s u p p r e s s p o t a s s i u m - i n d u c e d s t i m u l a t i o n o f t h e o x y g e n u p t a k e w h i c h o c c u r s i n b r a i n s l i c e s and i n a s t r o c y t e s b u t n o t i n n e u r o n s . T h i s m i g h t be t h e i n v i t r o m a n i f e s t a t i o n o f t h e b a r b i t u a t e - i n d u c e d r e d u c t i o n i n n o r m a l m e t a b o l i c r a t e . B a r b i t u a t e s a l s o i n h i b i t GABA u p t a k e i n t o a s t r o c y t e s and t h i s may be one b a s i s o f t h e i r a n t i - c o n v u l s a n t a c t i o n . I t was p r e d i c t e d t h a t n o n - b a r b i t u a t e s w h i c h i n h i b i t GABA u p t a k e i n t o g l i a m i g h t be e f f e c t i v e a n t i c o n v u l s a n t s , and t h i s was l a t e r f o u n d t o be t r u e f o r t h e d r u g THPO (Meldrum e t a l . , 1 9 8 2 ) . T h u s t h e e f f e c t s o f v a r i o u s d r u g s on a s t r o c y t e s may be s i m i l a r t o t h o s e on n e u r o n s , h a v e a d i f f e r e n t p r o f i l e , o r be s e l e c t i v e f o r a s t r o c y t e s . I n some c a s e s , t h e d r u g - g l i a i n t e r a c t i o n may be more c l i n i c a l l y r e l e v a n t t h a n t h e d r u g - n e u r o n i n t e r a c t i o n . New techniques enabling advances i n understanding g l i a Our u n d e r s t a n d i n g o f g l i a and how t h e y e x h i b i t h e t e r o g e n e i t y o n l y came a b o u t b e c a u s e o f new t e c h n i q u e s d e v e l o p e d o v e r t h e p a s t d e c a d e o r s o . A) Tissue Cultures The u n d e r s t a n d i n g o f g l i a h a s p r o g r e s s e d i n t h e l a s t d e c a d e b e c a u s e o f r e c e n t a d v a n c e s i n t e c h n i q u e s f o r s e p a r a t i n g p u r e , homogeneous s a m p l e s . T h e s e a r e now r o u t i n e l y p r e p a r e d , - 20 - u s i n g g r a d i e n t c e n t r i f u g a t i o n o f t i s s u e c u l t u r e . The a s t r o c y t e s p r e p a r e d by g r a d i e n t c e n t r i f u g a t i o n f r o m f r e s h t i s s u e a r e n o r m a l a s t r o c y t e s b u t may be c o n t a m i n a t e d by o t h e r c e l l t y p e s and d e b r i s and t h e i r f u n c t i o n a l i n t e g r i t y may be i m p a i r e d . A s t r o c y t e s i n c u l t u r e a r e o f two g e n e r a l t y p e s : e s t a b l i s h e d c e l l l i n e c u l t u r e s t h a t a r e t r a n s f o r m e d c e l l s w h i c h do n o t r e p r e s e n t t r u e g l i a t y p e s , and p r i m a r y c u l t u r e s , f r e s h t i s s u e c u l t u r e s t h a t a r e t r e a t e d b y p r o c e d u r e s w h i c h s e l e c t f o r c e r t a i n c e l l t y p e s . T h e y a r e u s u a l l y p r e p a r e d f r o m immature b r a i n s o t h e i r d i f f e r e n t i a t i o n must o c c u r d u r i n g c u l t u r e . P r i m a r y c u l t u r e s a r e q u i t e homogeneous, b e i n g l e s s t h a n 5% n o n - s p e c i f i c , and a r e b e l i e v e d t o be f u n c t i o n a l l y s i m i l a r t o i n v i v o g l i a o f t h e t y p e s e l e c t e d , most f r e q u e n t l y a s t r o c y t e s . The k n o w l e d g e on h e t e r o g e n e i t y t h a t t h e s e t e c h n i q u e s h a v e added a r e p a r t i c u l a r l y a b o u t r e g i o n a l h e t e r o g e n e i t y o r d i f f e r e n c e s b etween v a r i o u s c e l l t y p e s . B) F r e e z e F r a c t u r i n g T e c h n i q u e s T h e r e i s now a f r e e z e f r a c t u r e t e c h n i q u e w h i c h a l l o w s more d e t a i l e d e l e c t r o n m i c r o s c o p e v i e w s o f c e l l s u r f a c e s t h a n p r e v i o u s l y a v a i l a b l e . T h i s new d e v e l o p m e n t h a s l e d t o some e v i d e n c e o f g l i a l h e t e r o g e n e i t y . F r e e z e f r a c t u r e i s a t e c h n i q u e i n w h i c h c e l l s a r e f r o z e n a n d m e c h a n i c a l l y f r a c t u r e d ; t h e f r a c t u r e d s u r f a c e i s r e p l i c a t e d w i t h p l a t i n i u m and c a r b o n , w h i c h r e v e a l s t h e t e x t u r e o f t h e f r a c t u r e l i n e s upon e x a m i n a t i o n b y t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . T h i s e x a m i n a t i o n y i e l d s s e v e r a l t y p e s o f s t r u c t u r a l i n f o r m a t i o n . I t c a n show t h e e x i s t e n c e and - 21 - o r g a n i z a t i o n o f t h e f i l a m e n t s i n t h e c y t o p l a s m , c o n f i r m and show d i f f e r e n c e s i n j u n c t i o n b etween c e l l s , a n d r e v e a l t h e e x i s t e n c e o f r e p e a t i n g p a t t e r n s o f bumps o f unknown f u n c t i o n on c e l l membranes. T h i s t e c h n i q u e c a n show h e t e r o g e n e i t y w i t h i n c e l l u l a r p a r t s , among c e l l t y p e s , and among c e l l s o f t h e same t y p e f r o m d i f f e r e n t a r e a s . The a s t r o c y t i c c e l l p r o c e s s e s c a n be d i s t i n g u i s h e d f r o m t h o s e o f o t h e r g l i a l c e l l t y p e s on t h e b a s i s o f b o t h 10 nm. c y t o p l a s m i c f i l a m e n t s and t h e c h a r a c t e r i s t i c membrane s t r u c t u r e (Massa and M u g n a i n i , 1 9 8 2 ) . The membranes o f o l i g o d e n d r o c y t e s and a s t r o c y t e s h a v e d i f f e r i n g i n t r a - m e m b r a n o u s p a r t i c l e s . Waxman and B l a c k (1984) e x a m i n e d nodes o f R a n v i e r i n a d u l t r a t o p t i c n e r v e and f o u n d most h a d a s t r o c y t i c p r o c e s s e s s u r r o u n d i n g them. The c y t o p l a s m o f t h e s e a s t r o c y t e s c o n t a i n s 10 nm f i l a m e n t s . The e x t e r n a l f a c e s a r e c h a r a c t e r i z e d by o r t h o g o n a l a r r a y s o f p i t s w i t h a c e n t r e t o c e n t r e p e r i o d i c i t y o f 6 nm, w h i c h c o r r e s p o n d s t o p a r t i c l e s on t h e i r p r o t o p l a s m i c o r i n n e r membrane f a c e s . The d e n s i t y o f p a r t i c l e s i s s i m i l a r t o t h a t i n p e r i p a r e n c h y m a l a s t r o c y t i c membranes and l e s s t h a n i n p e r i c a p i l l a r y a s t r o c y t i c and s u b p i a l a s t r o g l i a l membranes. Waxman a n d B l a c k (1984) showed t h a t t h e o r t h o g o n a l a r r a y s and gap j u n c t i o n s p a t t e r n c a n be u s e d t o i d e n t i f y t h e s e a s t r o c y t i c p r o c e s s e s . A n d e r s and B r i g h t m a n , (1979) showed t h a t t h e s e o r t h o g o n a l a r r a y s o f p a r t i c l e s i n c r e a s e i n number f r o m e m b r y o n i c d a y 20 on i n r a t s . T h ey a l s o showed r e a c t i v e a s t r o c y t e s n o t o n l y had an i n c r e a s e d number o f p a r t i c l e s b u t t h a t t h e y were a l s o - 22 - r e a r r a n g e d t o a more h i g h l y o r d e r e d s t r u c t u r e compared t o t h a t s e e n i n n o r m a l a s t r o c y t e s . The number o f o r t h o g o n a l a r r a y s o f p a r t i c l e s on a s t r o c y t i c membranes i n c r e a s e where t h e y a r e i n c o n t a c t w i t h n o n - n e u r o n a l t i s s u e (Wujek and R e i e r , 1981, A n d e r s and B r i g h t m a n , 1979, L a n d i s a n d R e e s e , 1 9 8 1 ) . B u t L a n d i s and R e e s e (1981) d i d n o t f i n d o r t h o g o n a l a s s e m b l i e s i n t h e C-6 g l i o m a c e l l l i n e . I t i s n o t c l e a r w h e t h e r t h i s means t h a t n o t a l l g l i a h a v e them o r t h a t a g l i o m a l i n e , m o d i f i e d u n d e r t h e C-6 c u l t u r e c o n d i t i o n s , w i l l n o t h a v e them. Gotow (1984) f o u n d t h a t f i l i p i n , a c h e m i c a l t h a t p r o d u c e s a c h a r a c t e r i s t i c d i s r u p t i o n o f membranes by a c t i n g on t h e c h o l e s t e r o l i n t h e membranes, h a d l e s s e f f e c t on o r t h o g o n a l a r r a y - c r o w d e d a s t r o c y t i c membranes c o n t a c t i n g t h e b a s a l l a m i n a t h a n on o t h e r membrane a r e a s . T h i s means e i t h e r t h a t t h e s e membrane a r e a s c o n t a i n l e s s c h o l e s t e r o l o r t h a t t h e c h o l e s t e r o l i s somehow p r o t e c t e d f r o m t h e f i l i p i n . S u c h a r e a s a l s o c o n t a i n l e s s a l k a l i n e p h o s p h a t a s e and Na+,K+-ATPase, w h i c h a r e b o t h a s s o c i a t e d w i t h t h e membrane t r a n s p o r t n o r m a l l y f o u n d i n p e r i v a s c u l a r p r o c e s s e s . T h i s s u g g e s t s a r e g i o n a l s p e c i a l i z a t i o n o f t h e a s t r o c y t e s i n v i v o . Gotow s u g g e s t e d t h a t t h e o r t h o g o n a l a r r a y s may be s t r u c t u r a l i n f u n c t i o n , as t h e y o c c u r s p e c i f i c a l l y where a c t i v e t r a n s p o r t i s l e s s , o r t h a t t h e y a r e i n v o l v e d i n f o r m i n g a b a r r i e r t o c h o l e s t e r o l and p r o t e i n s . He f o u n d o r t h o g o n a l a r r a y s o n l y i n a s t r o c y t e s and e p e n d y m a l c e l l s . I n c u l t u r e s t h e y a p p e a r on a l l t h e s u r f a c e s and t h e r e f o r e may d e v e l o p on s u r f a c e s e x p o s e d t o l a r g e - 23 - e x t r a c e l l u l a r s p a c e s . The f r e e z e f r a c t u r e s t u d i e s c a n a l s o be u s e d t o d e f i n e v a r i o u s t y p e s o f j u n c t i o n s . Gap j u n c t i o n s o c c u r b e t w e e n a s t r o c y t e s and b etween o l i g o d e n d r o c y t e s and a s t r o c y t e s b u t n o t b e t w e e n o l i g o d e n d r o c y t e s , b u t a d j a c e n t o l i g o d e n d r o c y t e s do f o r m t i g h t j u n c t i o n s (Massa and Mugnami, 1 9 8 2 ) . S a i n t M a r i e and C a r l s o n (1983) u s e d f r e e z e f r a c t u r e t e c h n i q u e s t o d e s c r i b e g l i a h e t e r o g e n e i t y i n t h e r e t i n a o f t h e compound ey e o f t h e h o u s e f l y . C e l l s i n e a c h l a y e r o f t h e r e t i n a h a d a c h a r a c t e r i s t i c p a t t e r n o f t h r e e t y p e s o f j u n c t i o n s (gap j u n c t i o n s , t i g h t j u n c t i o n s , and s e p t a t e j u n c t i o n s ) and desmosomes w h i c h may be e q u i v a l e n t t o t h e o r t h o g o n a l a r r a y s . G l i a l c e l l s o f e a c h o f t h e l a y e r s h a d c h a r a c t e r i s t i c p a t t e r n s and d e n s i t i e s o f t h e s e f e a t u r e s as w e l l a s o f s h a p e s an d p h y s i c a l r e l a t i o n s h i p s t o t h e o t h e r c e l l s o f t h e l a y e r . The v a r i o u s t y p e s o f c o n t a c t s may h a v e d i f f e r e n t f u n c t i o n s : gap j u n c t i o n s - i n t r a c e l l u l a r c o m m u n i c a t i o n ; t i g h t j u n c t i o n s - o c c l u s i o n o f e x t r a c e l l u l a r m a t e r i a l ; s e p t a t e - f i r m b u t f l e x i b l e a d h e s i o n o r t i s s u e i m p e d a n c e ; desmosomes - i n t e r c e l l u l a r a d h e s i o n . The d i f f e r i n g p a t t e r n s t h u s i m p l y t h a t t h e c e l l s h a v e d i f f e r i n g f u n c t i o n s . T h i s t y p e o f work may w e l l be e x t e n d e d i n t h e f u t u r e t o g l i a o f t h e CNS. C) M a r k e r s A v a r i e t y o f m a r k e r s h a v e b e e n f o u n d t h a t a l l o w d i s t i n c t i o n s t o be made betw e e n and w i t h i n t h e v a r i o u s c l a s s i f i c a t i o n s o f g l i a c e l l s . T h e s e m a r k e r s h a v e p r o v i d e d a - 24 - w e a l t h o f i n f o r m a t i o n on g l i a l h e t e r o g e n e i t y . Some m a r k e r s c a n be u s e d t o i d e n t i f y a s g l i a , c e l l t y p e s t h a t were n o t p r e v i o u s l y s o c l a s s i f i e d , o t h e r m a r k e r s c a n be u s e d t o i d e n t i f y s u b s e t s and t o p r o v i d e c l u e s a s t o h e t e r o g e n e i t y o f g l i a l f u n c t i o n . T h e r e h a v e b e e n s e v e r a l g o od r e v i e w s o f g l i a l m a r k e r s ( R o o t s , 1981, S c h a c h n e r , 1982) b u t t h e y g e n e r a l l y do n o t e m p h a s i z e t h e g l i a l h e t e r o g e n e i t y r e v e a l e d b u t r a t h e r t h e u s e o f some m a r k e r s f o r d e f i n i n g p u r i t y o f c u l t u r e s o r s i m i l a r p u r p o s e s . 1) F i b r o u s p r o t e i n s o f a s t r o c y t e s a) G l i a l f i b r i l l a r y a c i d i c p r o t e i n A s t r o c y t e s a r e most r e l i a b l y i d e n t i f i e d b y t h e p r e s e n c e o f g l i a l f i l a m e n t s u n d e r t h e e l e c t r o n m i c r o s c o p e . The C a j a l g o l d method s p e c i f i c a l l y s t a i n s t h e s e f i b e r s ( C a j a l , 1 9 1 3 ) . They a r e composed o f t h e most s t u d i e d o f g l i a l s p e c i f i c m a r k e r s , g l i a l f i b r i l l a r y a c i d i c p r o t e i n (GFAP). GFAP was o r i g i n a l l y i s o l a t e d f r o m m u l t i p l e s c l e r o s i s p l a q u e s (Uyeda e t a l . , 1972) and CNS g l i o t i c a r e a s ( B i g n a m i e t a l . , 1 9 7 2 ) , and c a n be r e a d i l y s t a i n e d b y i m m u n o h i s t o c h e m i s t r y . I t i s t h e p r i n c i p l e c o n s t i t u e n t o f t h e f i l a m e n t s t h a t d e v e l o p i n a s t r o c y t e s t h a t may h a v e some f u n c t i o n i n m a i n t a i n i n g t h e s h a p e o f a s t r o c y t e s . D u f f y e t a l . (1982) l o o k e d a t GFAP i n human a s t r o c y t o m a c e l l s i n c u l t u r e and f o u n d a r e l a t i o n s h i p b e t w e e n t h e shape o f t h e a s t r o c y t e s and t h e l o c a t i o n o f t h e GFAP. S p i n d l e s h a p e d c e l l s h a d a b u n d a n t GFAP i n body and p r o c e s s e s , w h e r e a s i n r o u n d o r p o l y h e d r a l a s t r o c y t o m a s t h e GFAP was l a r g e l y p e r i n u c l e a r . As p r o c e s s e s d e v e l o p e d , GFAP e x t e n d e d i n d e n s e p a r a l l e l a r r a y s . - 25 - T h e r e may a l s o be a r e l a t i o n s h i p b e t w e e n m o t i l i t y and GFAP. S t e l l a t e c e l l s i n c u l t u r e , w i t h e x t e n s i v e p a r a l l e l a r r a y s o f GFAP f i b e r s , were more r i g i d w h i l e s p i n d l e c e l l s , w i t h o u t t h e s e p a r a l l e l a r r a y s were c o n s t a n t l y e x t e n d i n g and r e t r a c t i n g p r o c e s s e s . S a l m e t a l . (1982) showed t h a t r a t p i t u i c y t e s , w h i c h a r e v e r y GFAP p o s i t i v e , do n o t have g l i a l f i l a m e n t s , a s do o t h e r a s t r o c y t e s . T h i s means t h a t GFAP d o e s n o t h a v e t o be o r g a n i z e d i n t o f i l a m e n t s t o g i v e p o s i t i v e GFAP s t a i n i n g i n c e l l s . S u e s s and P l i s k a (1981) f o u n d t h a t t h e p i t u i c y t e s r e m a i n e d s t r o n g l y GFAP p o s i t i v e e v e n a f t e r t r a n s p l a n t o f t h e p i t u i t a r y t o a r e g i o n u n d e r t h e k i d n e y c a p s u l e where t h e r e a r e no n e u r a l i n f l u e n c e s . GFAP i s f o u n d m a i n l y i n m a t u r e a s t r o c y t e s . I t i s d e n s e r i n t h e f i b r o u s s u b t y p e b u t i s a l s o i n p r o t o p l a s m i c a s t r o c y t e s . I t i s a l s o f o u n d i n s e v e r a l c e l l t y p e s r e l a t e d t o a s t r o c y t e s , i n c l u d i n g r a d i a l g l i a l c e l l s ( B i g n a m i and D a h l , 1974), t h e Bergmann g l i a , e n t e r i c g l i a a nd a s m a l l p e r c e n t a g e o f g l i a i n t h e p e r i p h e r a l n e r v o u s s y s t e m ( J e s s e n e t a l . , 1 9 8 4 ) . Kennedy (1982) n o t e d t h a t t h e q u e s t i o n o f w h e t h e r a l l a s t r o c y t e s c o n t a i n GFAP h a s y e t t o be a n s w e r e d . A p e r m a n e n t i n c r e a s e i n GFAP c o n t e n t r a p i d l y f o l l o w s i n j u r y and p r e c e d e s a s t r o g l i o s i s ( B i g n a m i and D a h l , 1975). I n c u l t u r e d a s t r o c y t e s GFAP s t a i n i n g c a n be i n c r e a s e d b y g l i a m a t u r a t i o n f a c t o r ( L i m e t a l . , 1 977), b r a i n e x t r a c t s o r dBcAMP w h i c h c a u s e s m a t u r a t i o n . I n some c e l l t y p e s i t may o n l y be e x p r e s s e d t r a n s i e n t l y ; i n humans i t i s o n l y e x p r e s s e d i n ependymal c e l l s b e t w e e n week 13 and f u l l t e r m , and i s a l s o - 26 - t r a n s i e n t l y i n t a n y c y t e s ( R o o t s , 1 9 8 1 ) . H e t e r o g e n e i t y c a n be f o u n d i n s u b s e t s o f c e l l s . D a h l e t a l . (1982) n o t i c e d t h a t a n t i G F A P s t a i n e d o n l y a s u b s e t o f Schwann c e l l s i n r a t s c i a t i c n e r v e . T h e s e were m a i n l y s u r r o u n d i n g n o n - m y e l i n a t e d a x o n s , and i n c r e a s e d i n number d u r i n g W a l l e r i a n d e g e n e r a t i o n . T h e r e may a l s o be h e t e r o g e n e i t y o f t h e GFAP w i t h i n t h e CNS. GFAP h a s b e e n shown t o be composed o f v a r i o u s c h e m i c a l l y d i f f e r e n t p r o t e i n s a l t h o u g h t h e s u b t y p e s h a v e t h e same m o l e c u l a r w e i g h t i n p e r i p h e r a l and c e n t r a l g l i a . The p r o b l e m s w i t h GFAP s t a i n i n g a r e m a i n l y t h o s e o f p r o d u c i n g t h e a n t i b o d y i t s e l f and t h e c r o s s - r e a c t i v i t y t h a t i m p u r i t i e s c a u s e . b) V i m e n t i n T h e r e a r e two o t h e r p r o t e i n s , r e l a t e d t o b u t d i f f e r e n t f r o m GFAP, w h i c h a r e f o u n d i n some g l i a and a r e c o n s t i t u e n t s o f f i b e r s y s t e m s w i t h i n t h e c e l l . One o f t h e s e i s v i m e n t i n . A n t i b o d i e s t o v i m e n t i n and GFAP were u s e d i n a d o u b l e l a b e l i n g e x p e r i m e n t t o examine a s t r o c y t i c f i l a m e n t s i n d e v e l o p m e n t and w o u n d i n g ( P i x l e y and D e V e l l i s , 1 9 8 4 ) . F i l a m e n t s s t a i n e d f o r v i m e n t i n o n l y i n newborn r a t s and f o r GFAP i n 2 0 day and o l d e r r a t s , w i t h a g r a d u a l s w i t c h i n b e t w e e n . S t a b wounds were made t o c o r t i c a l a r e a s a t a t i m e when t h e r e were n o r m a l l y no v i m e n t i n - p o s i t i v e c e l l s i n t h e r e g i o n . V i m e n t i n o n l y o c c u r r e d a t t h e edge o f t h e wound. T h i s l e d t o t h e h y p o t h e s i s t h a t v i m e n t i n o c c u r s when t h e r e i s c o n t a c t w i t h w i d e open s p a c e s and i s l o s t when s u c h c o n t a c t w i t h t h e s e s p a c e s d i s a p p e a r . A l l v i m e n t i n - p o s i t i v e c e l l s seem - 27 - t o h a v e a t l e a s t one p o r t i o n o f t h e c e l l i n c o n t a c t w i t h CSF, e g . e pendymal c e l l s , t a n y c y t e s , Bergmann g l i a l f i b e r s , M u l l e r c e l l s , and r a d i a l g l i a l . The d i s a p p e a r a n c e o f v i m e n t i n p o s i t i v e c e l l s c o r r e l a t e s w i t h t h e l o s s i n e x t r a c e l l u l a r v o l u m e . C e l l s i n c u l t u r e , where t h e r e i s much e x t r a c e l l u l a r f l u i d , d e v e l o p v i m e n t i n r e g a r d l e s s o f o r i g i n . T h i s h y p o t h e s i s w o u l d e x p l a i n t h e a p p e a r a n c e o f v i m e n t i n o n l y i n c e l l s a t t h e e dge o f t h e wound, c l o s e t o t h e f l u i d t i s s u e b o u n d a r y . I n t h e a d u l t r a t , v i m e n t i n was o n l y o b s e r v e d i n f i b r o b l a s t s , c e l l s o f r e l a t i v e l y l a r g e b l o o d v e s s e l s , e p endymal c e l l s , and a s t r o c y t e s . A t e m b r y o n i c r a t day 11, t h e v i m e n t i n was o b s e r v e d o n l y i n r a d i a l f i b e r s , v e n t r i c u l a r c e l l s , and b l o o d v e s s e l s . I n c u l t u r e S c h n i t z e r e t a l . (1981) f o u n d GFAP and v i m e n t i n o c c u r r i n g t o g e t h e r i n t h e c e l l s w h i c h h a v e f i b r o b l a s t m o r p h o l o g y . c) Desmin D a h l and B i g n a m i (1982) showed t h a t d e s m i n f o r m s a t h i r d f i b e r s y s t e m i n g l i a c e l l s . I t was u n i f o r m l y d i s t r i b u t e d i n a s t r o c y t e s o f b r a i n and s p i n a l c o r d and i n M u l l e r c e l l s . A c o m p a r i s o n w i t h GFAP showed t h a t b o t h were s i m i l a r l y l o c a l i z e d i n b r a i n and s p i n a l c o r d b u t n o t i n t h e f i b e r s o f M u l l e r c e l l s . 2) G l u t a m i n e s y t h e t a s e GS i s a m a j o r a s t r o c y t e m a r k e r w h i c h c a t a l y s e s t h e r e a c t i o n : g l u t a m a t e + ammonia + ATP > G l u t a m i n e + ADP + P i I t may t h u s be i n v o l v e d i n g l u t a m a t e m e t a b o l i s m and ammonia - 28 - d e t o x i f i c a t i o n . M a r t i n e z - H e r n a n d e z e t a l . (1977) u s e d i m m u n o h i s t o c h e m i c a l t e c h n i q u e s t o show t h a t GS i s l o c a t e d e x c l u s i v e l y o v e r g l i a l c e l l s i n t h e b r a i n and M u l l e r c e l l s o f t h e r e t i n a . S a r t h y and Lam (1978) c o n f i r m e d i t s l o c a t i o n i n M u l l e r c e l l s . E l e c t r o n m i c r o s c o p e s t u d i e s o f r a t b r a i n h a v e r e v e a l e d a l o c a l i z a t i o n i n b o t h p r o t o p l a s m i c and f i b r o u s a s t r o c y t e s ( N o r e n b e r g and M a r i n e z - H e r n a n d e z , 1979, Kennedy, 1 9 8 2 ) . N o r e n b e r g (1983) a l s o d e s c r i b e d GS s t a i n i n g i n e pendymal c e l l s , Bergmann g l i a , p e r i k a r y o n , v a s c u l a r end f e e t i n t h e g l i a l i m i t a n s and a s t r o c y t i c p r o c e s s e s w h i c h ended j u s t b e n e a t h t h e ependymal s u r f a c e . T h i s d i s t r i b u t i o n s u p p o r t s a f u n c t i o n f o r g l i a o f p r o v i d i n g a b a r r i e r a g a i n s t ammonia. S c h o u s b o e e t a l . (1977b) f o u n d h i g h a c t i v i t y o f t h i s enzyme i n p r i m a r y a s t r o c y t e c u l t u r e s . B u t N i c k l a s and B r o w n i n g (1978) f o u n d t h a t t h e C-6 a s t r o c y t o m a c e l l l i n e h a d v e r y low GS a c t i v i t y . I t s l o c a l i z a t i o n t o g l i a i m p l i c a t e s g l i a as t h e l o c a t i o n o f t h e s m a l l g l u t a m a t e compartment ( N o r e n b e r g and M a r i n e x - H e r n a n d e z , 1979), as d i s c u s s e d p r e v i o u s l y . S i n c e p r i m i t i v e g l i a do n o t c o n t a i n GFAP, b u t do c o n t a i n GS, t h e l a t t e r may be a b e t t e r m a r k e r o f a s t r o c y t e s . G l i o t i c s c a r t i s s u e , however, d o e s n o t c o n t a i n GS; t h i s may l e a d t o b u i l d u p o f ammonia and g l u t a m a t e w h i c h c o u l d e x p l a i n t h e e p i l e p t o g e n i c n a t u r e o f s u c h t i s s u e ( N o r e n b e r g , 1 9 8 3 ) . T h e r e a r e s i g n i f i c a n t r e g i o n a l v a r i a t i o n s i n t h e i n t e n s i t y o f GS s t a i n i n g i n a s t r o c y t e s . The m o l e c u l a r l a y e r s o f t h e c e r e b e l l u m and h ippocumpus a r e p a r t i c u l a r l y h e a v i l y s t a i n e d ( N o r e n b e r g , 1 9 7 9 ) . - 29 - 3) C a r b o n i c a n h y d r a s e C a r b o n i c a n h y d r a s e c o m b i n e s r e v e r s i b l y CO2 and H2O t o form c a r b o n i c a c i d , h y d r a t e s a l d e h y d e g r o u p s t o a l c o h o l s , and a c t s a s an e s t e r a s e . I t t h u s may be i n v o l v e d i n t h e r e g u l a t i o n o f pH, s e c r e t o r y a c t i v i t i e s a nd movement o f i o n s . I t d e v e l o p s i n t h e b r a i n a t t h e same t i m e as g l i a l p r o l i f e r a t i o n . I t u s e d t o be c o n s i d e r e d an a s t r o c y t e s p e c i f i c m a r k e r b u t i s now known t o be a l s o on M u l l e r c e l l s ( S a r t h y and Lam, 1978) a n d o l i g o d e n d r o c y t e s a s w e l l (Ghandour e t a l . , 1979, 1980, R o u s e l l e t a l . , 1979, M a n d e l e t a l . , 1 9 7 8 ) . P r i m a r y c u l t u r e s o f a s t r o c y t e s h a v e c a r b o n i c a n h y d r a s e ( K i m e l b e r g e t a l . , 1978b), b u t C-6 c e l l s do n o t ( D e V e l l i s and B r o o k e r , 1 9 7 3 ) . K i m e l b e r g e t a l . (1982) f o u n d t h e h i s t o c h e m i c a l method f o r c a r b o n i c a n h y d r a s e s t a i n e d a s t r o c y t e s i n t h e m o n o l a y e r o f p r i m a r y c u l t u r e s f r o m r a t c e r e b r a l h e m i s p h e r e s , a s w e l l a s c e l l s b e l i e v e d be be o l i g o d e n d r o c y t e s above t h i s l a y e r . C a r b o n i c a n h y d r a s e e x i s t s i n s e v e r a l i s o e n z y m e s t h a t h a v e d i f f e r e n t amino a c i d s e q u e n c e s and a r e i m m u n o l o g i c a l l y d i s t i n c t . A n t i b o d i e s t o two o f t h e s e i s o e n z y m e s (Ca-1 and CA-2) may s t a i n d i s t i n c t s u b s e t s o f g l i a . I t was t h o u g h t t h a t CA-1 m i g h t be a s t r o c y t e s p e c i f i c and CA-2 o l i g o d e n d r o c y t e s p e c i f i c . However, i n c u l t u r e cDbAMP c a u s e d a s t r o c y t e s t o d i f f e r e n t i a t e and s t a i n f o r CA-2 a s i n t e n s e l y a s o l i g o d e n d r o c y t e s . - 30 - 4) O t h e r m a r k e r s T h e r e a r e a number o f m a r k e r s t h a t h a v e b e e n r e p o r t e d t o s t a i n a s t r o g l i a p r e d o m i n a n t l y . Many o f t h e s e a r e i s o l a t e d r e p o r t s w i t h v e r y l i t t l e c o n f i r m a t i o n . T a b l e I s u m m a r i z e s many o f t h e s e r e c e n t o r p o o r l y s u b s t a n t i a t e d f i n d i n g s . T h e r e a r e a number o f m a r k e r s t h a t u s e d t o be c o n s i d e r e d a s t r o c y t e s p e c i f i c and now a r e known t o be on o t h e r c e l l t y p e s a s w e l l o r whose s p e c i f i c i t y i s now h i g h l y c o n t r o v e r s i a l . - 31 - MARKER TABLE I : MINOR ASTROCYTE CELL MARKERS TYPE OF CELL AUTHORS Non-neuronal Astrocytes cerebellum including Langley & Ghandour enolase * Bergmann g l i a & cytoplasmic processes (1982) OL-2 -Glycoprotein Astrocytes; astrocytomas not glioblastomas Langley et a l . (1982) Tamm-horsefall glycoprotein Ependymal c e l l s & astrocyte processes Zalc et of Bergmann f i b e r s or astrocytic feet i n contact with blood vessels or meninges a l . (1984) Sulfogalactosyl ceramide (SGC) ** 11 Zalc et a l . (1984) Ml - antigen Distinguishes sub-cerebellar astrocytes Lagenaur some but not a l l GFAP+ c e l l s • et a l . (1980) C-l - antigen Only processes of Bergmann g l i a & Muller Sommer et a l . (1981) c e l l s , and ependymal c e l l s , but not other astrocytes except i n early postnatal astrocytes of white matter Purkinje c e l l layer and r a d i a l l y oriented structures of telencephalon, pons, p i t u i t a r y and retina IgG - RAN 2 # Astrocyte precursor c e l l s , ependymal Bartlett et a l . (1981) c e l l s , Muller c e l l s . leptomeningeal c e l l s * Catalyzes oxidation of phosphoglyceric acid to phosphopyruvate ** C l " possibly involved i n C l transport # IgC made by antibody secreting hybridomas and defined by antibody TABLE I ( c o n t i n u e d ) : MINOR ASTROCYTE CELL MARKERS MARKER TYPE OF CELL AUTHORS THY 1 O n l y s u b t y p e s o f a s t r o c y t e s t h a t a r e a l s o g l a c t o c e r e b r o s i d e + S c h n i t z e r and S c h a c h n e r (1981) THY 1 Two c e l l l i n e s Kemshead e t a l . (1982) A n t i g e n A2B5 Immature a s t r o c y t e s ; o l i g o d e n d r o c y t e s n e u r o n s ; h a s c o n s i d e r a b l e r e g i o n a l , d e v e l o p m e n t a l and s p e c i e s v a r i a t i o n S c h n i t z e r & S c h a c h n e r (1982) S 100 P r o t e i n * ( c o n t r o v e r s i a l ) s p e c i f i c a s t r o c y t e m a r k e r OR i n o l i g o d e n d r o c y t e s , e n d o t h e l i a l c e l l s and n e u r o n s a l s o Gandour e t a l . (1981a) Hyden e t a l . (1980) Hansson e t a l . (1976) SSEA-1 G l y c o l i p i d ** a n t i g e n S u b t y p e s o f a s t r o c y t e s . I n e a r l y mouse c e r e b e l l u m o n l y i n e x t e r n a l g r a n u l a r l a y e r a n d m o l e c u l a r l a y e r ; l a t e r o n l y s m a l l a r e a s i n m o l e c u l a r a r e a L a g e n a u r e t a l . (1982a) L a g e n a u r e t a l . (1982b) N11N1 M o n o c l o n a l # a n t i b o d y Human f e t a l b r a i n c u l t u r e s and p r i m a t e s p i n a l c o r d ; s u b t y p e s 80-90% GFAP+ i n some c e l l s o n l y D i c k s o n e t a l . (1983) 308 M o n o c l o n a l # a n t i b o d y D i f f e r e n c e s f o u n d between GFAP+ and GFAP- a s t r o c y t e s D i c k s o n e t a l . (1983) * May be i n v o l v e d i n b i n d i n g Ca++ & movement o f m o n o v a l e n t c a t i o n s o r GABA t r a n s p o r t ** O r i g i n a l l y f o u n d on s u r f a c e o f F9 t e t r a c a r c i n o m a c e l l # O r i g i n a l l y r a i s e d t o human n e u r o b l a s t o m a As c a n be s e e n f r o m T a b l e I , most o f t h e s e m i n o r m a r k e r s f o r a s t r o c y t e s a r e o n l y s p e c i f i c f o r s u b s e t s o f a s t r o c y t e s . Thus, w i t h t h e d e v e l o p m e n t o f m a r k e r t e c h n o l o g y came c o n s i d e r a b l e e v i d e n c e f o r g l i a l h e t e r o g e n e i t y . More r e s e a r c h on e a c h o f t h e s e m a r k e r s may be t h e b a s i s f o r f u t u r e c l a s s i f i c a t i o n s y s t e m s f o r a s t r o c y t e s . O l i g o d e n d r o c y t e s and CNS m y e l i n a l s o h a v e t h e i r m a r k e r s t h a t a r e more o r l e s s s p e c i f i c . T a b l e I I su m m a r i z e s d a t a a v a i l a b l e on m a r k e r s t h a t c o u l d o r h a v e b e e n c o n s i d e r e d o l i g o d e n d r o c y t e m a r k e r s . F u t u r e r e s e a r c h w i l l u s e t h e s e and o t h e r o l i g o d e n d r o c y t e m a r k e r s y e t t o be f o u n d t o s t a r t t o c l a s s i f y o l i g o d e n d r o c y t e s i n t o s u b t y p e s b a s e d on h e t e r o g e n e i t y s e e n b e t w e e n a r e a s , s p e c i e s , and p h y s i c a l c e l l t y p e s . S i n c e some m a r k e r s a r e n o t o n l y t o o l s b u t a r e a l s o d i r e c t i n d i c a t o r s o f g l i a l s t r u c t u r e and b i o c h e m i s t r y , t h e w i d e v a r i e t y o f m a r k e r s s t a i n a b l e i n v a r i o u s s u b s e t s o f g l i a i s i n d i c a t i v e o f g r e a t f u n c t i o n a l h e t e r o g e i t y o f g l i a l c e l l s . W i t h a d v a n c e s i n t e c h n i q u e s f o r s t a i n i n g , u s i n g m a r k e r s , f r e e z e f r a c t u r e , and e l e c t r o n m i c r o s c o p y , t h e r e came a new u n d e r s t a n d i n g o f t h e m o r p h o l o g i c a l d i v e r s i t y o f g l i a and how t h i s d i v e r s i t y c a n sometimes be c o r r e l a t e d t o t h e b i o c h e m i c a l d i v e r s i t y t h a t c a n be f o u n d . - 34 - TABLE I I : MARKERS FOR OLIGODENDROCYTES AND MYELIN MARKER LOCATION AUTHORS G a l a c t o c e r e b r o s i d e M y e l i n s p e c i f i c PNS + CNS o l i g o d e n d r o c y t e s e p i t h e l i a l c e l l o f v e n t r i c l e s and c h o r o i d p l e x u s R a f f e t a l . (1978) M y e l i n b a s i c p r o t e i n * E a r l y o l i g o d e n d r o c y t e s and m y e l i n s h e a t h s S t e r n b e r g e r e t a l . (1978) Hartman e t a l . (1979) R o u s s e l & Nussbaum(1981) M y e l i n b a s i c p r o t e i n C u l t u r e s o f g a l a c t o c e r e b r o s i d e + c e l l s B h a t e t a l . (1981) A n t i - p r o t e o l i p i d a n t i s e r u m M y e l i n s h e a t h s and a c t i v e l y m y e l i n a t i n g o l i g o d e n d r o c y t e s A g r a w a l & Hartman (1979) W o l f g r a m P r o t e i n WI & W2 C e n t r a l m y e l i n & o l i g o d e n d r o c y t e s L a b o u r d e t t e e t a l . (1979) 2 ' , 3 ' - C y c l i c n u c l e o t i d e 3 ' - p h o s p h o h y d r o l a s e ** I n n e r & o u t e r most s h e a t h m y e l i n S p e c i e s s p e c i f i c , n o t i n f i s h ; I t s f u n c t i o n may o n l y be c o i n c i d e n t a l l y r e l a t e d t o m y e l i n N o t i n o l d o l i g o d e n d r o c y t e c u l t u r e s S z u c h e t and S t e f a n s s o n (1980) 0 1 — > 0 4 # O l i g o d e n d r o c y t e s o f e a r l y p o s t n a t a l c e r e b e l l u m , c e r e b r u m , s p i n a l c o r d , o p t i c n e r v e & r e t i n a : 01 & 02, and 03 & 04 o c c u r i n d i f f e r e n t d e v e l o p m e n t a l t i m e s i n d i f f e r e n t a r e a s Sommer & S c h a c h n e r (1981) S c h a c h n e r (1982) * A n t i g e n d e f i n e d ** C a t a l y s e s h y d r o l y s i s o f 2 ' , 3 ' - c y c l i c n u c l e o s i d e s t o t h e 2 • - n u c l e o t i d e s ; m a j o r component W o l f g r a m p r o t e i n # F o u r m o n o c l o n a l a n t i b o d i e s f r o m mouse myeloma immunized w i t h w h i t e m a t t e r f r o m c o r p u s c a l l o s u m TABLE II (continued): MARKERS FOR OLIGODENDROCYTES AND MYELIN MARKER LOCATION AUTHORS MAG O l i g o d e n d r o c y t e s , Schwann c e l l s and c e r t a i n a r e a s o f p e r i a x o n a l r e g i o n o f t h e c e n t r a l and p e r i p h e r a l m y e l i n s h e a t h S t e r n b e r g e r e t a l . (1979) S u c c i n i c d e h y d r o g e n a s e O l i g o d e n d r o c y t e s and a s t r o c y t e s M o s s a k o w s k i ( 1 9 6 2 ) B u t y r y l C h o l i n e s t e r a s e * More a c t i v e i n o l i g o d e n d r o c y t e s t h a n i n o t h e r g l i a (1954) Cavangh & Thompson , Oehmichen (1980) A n t i m y e l i n a n t i s e r u m M y e l i n a t e d f i b e r s , medium and l i g h t o l i g o d e n d r o c y t e s ; n o t d a r k (mature) a s t r o c y t e s , G o l g i e p i t h e l i u m c e l l s , Bergmann f i b e r s and some subependymal c e l l s R o u s s e l & Nussbaum (1983) P-2 M y e l i n s p e c i f i c p r o t e i n R a b b i t CNS m y e l i n : more i n c a u d a l a r e a s H i g h e s t i n s p i n a l c o r d , l o w e s t i n f r o n t a l c o r t e x ; o n l y i n l a r g e r d i a m e t e r axons T r a p p e t a l . (1983) G l y c e r o l - 3 - p h o s p h a t e 0 1 i g o d e n d r o c y t e s D e V e l l i s e t a l . (1978) TU-01 ** C e r e b e l l a r g l i a c e l l s ; o n l y i n m i c r o t u b u l e s ; smooth e n d o p l a s m i c r e t i c u l u m o u t e r m i t o c h o n d r i a l membrane; r i b o s o m a l r o s e t t e s H a j o s & R o s t o m i a n (1984) * N o n - s p e c i f i c c h o l i n e s t e r a s e ** A n t i - t u b u l i n a n t i b o d y G l i a l Heterogeneity - Morphological G i v e n t h e d e v e l o p m e n t o f new t o o l s and r e c e n t i mprovements o f o l d t o o l s t h e h e t e r o g e n e i t y , as we now know i t , c a n be d i s c u s s e d . The most o b v i o u s f i r s t way t o c o n s i d e r h e t e r o g e n e i t y w o u l d be t o d i s c u s s t h e v i s u a l d i f f e r e n c e s b e t w e e n c e l l s . A b r i e f d i s c r i p t i o n o f g l i a l c e l l t y p e s h a s a l r e a d y b e e n g i v e n i n t h e i n t r o d u c t i o n b u t w i l l be d e v e l o p e d f u r t h e r h e r e a s t h e r e a p p e a r s t o be a v a r i a t i o n i n m o r p h o l o g y w i t h i n t h e c l a s s i c a l g l i a t y p e s . C a j a l o r i g i n a l l y d i v i d e d a s t r o c y t e s i n t o f i b r o u s and p r o t o p l a s m i c t y p e s , b a s e d on l o c a t i o n , m o r p h o l o g y and f u n c t i o n . S e v e r a l o t h e r d i s t i n c t a s t r o g l i a l c e l l t y p e s h a v e b e e n d i s c r i b e d b u t i t r e m a i n s t o be s e e n w h e t h e r t h e y a r e r e a l l y d i s t i n c t i v e c e l l t y p e s , t r a n s i t i o n a l f o r m s r e f l e c t i n g d e v e l o p m e n t a l s t a g e s , a d a p t i o n s t o l o c a l p h y s i c a l o r c h e m i c a l e n v i r o n m e n t s , o r a r e v e r s i b l e e x p r e s s i o n o f a n a t u r a l a s t r o c y t i c p l a s t i c i t y . The m o r p h o l o g i c a l t y p e s w h i c h h ave b e e n d e s c r i b e d h a v e n o t b e e n c a t a g o r i z e d i n t o u s a b l e s u b g r o u p s . M o s t a r e s t i l l c a t a g o r i z e d as m a t u r e a s t r o c y t e s by t h e a p p e a r a n c e o f c y t o p l a s m i c f i l a m e n t s i n t h e e l e c t r o n m i c r o s c o p e , a s o r i g i n a l l y d e s c r i b e d by P a l a y e t a l . ( 1 9 6 2 ) . The d i s t i n c t i o n b etween a s t r o c y t e s and o l i g o d e n d r o c y t e s may s o m e t i m e s be d i f f i c u l t . B o t h c a n be s t a i n e d b y t h e s i l v e r c a r b o n a t e method s o t h a t t h e c h e m i s t r y o f t h e i r c y t o p l a s m s must be s i m i l a r . R e y n e r s e t a l . (1982) d e s c r i b e d a h i g h l y r a d i o s e n s i t i v e c e l l t h a t h a s u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s i n t e r m e d i a t e b e t w e e n t h e n o r m a l p r o t o p l a s m i c a s t r o c y t e and t h e - 37 - l i g h t o l i g o d e n d r o c y t e . I t i s p r e s e n t i n s i g n i f i c a n t numbers and may be a m u l t i p o t e n t i a l r e s e r v e g l i a l c e l l , c a p a b l e o f r e p l a c i n g o l i g o d e n d r o c y t e s o r m i c r o g l i a l c e l l s . T h i s b e t a - a s t r o c y t e , as i t i s c a l l e d , c a n be d i s t i n g u i s h e d f r o m a l p h a - a s t r o c y t e s , as n o r m a l a s t r o c y t e s a r e c a l l e d , b y i t s i r r e g u l a r l y s h a p e d n u c l e u s , d e n s e r r i b o s o m a l c o v e r o f t h e e n d o p l a s m i c r e t i c u l u m , c o a r s e r l y s o s o m a l m o r p h o l o g y , l a c k o f c e l l u l a r p r o c e s s e s , and t o t a l a b s e n c e o f g l i o f i l a m e n t s . The ^ - a s t r o c y t e s a r e n e v e r f o u n d n e a r t h e o u t e r membranes o f t h e b l o o d v e s s e l s b u t , l i k e m i c r o g l i a , a r e f r e q u e n t l y f o u n d i n p e r i v a s c u l a r a r e a s , and a r e f r e q u e n t l y s a t e l l i t e s t o n e r v e c e l l s . K o e n i g and B a r r o n (1963) a l s o n o t e d t h a t t h e r e i s a c o n t i n u u m o f t r a n s i t i o n a l f o r m s between o l i g o d e n d r o c y t e s and r e a c t i v e a s t r o c y t e s . O l i g o d e n d r o c y t e m o r p h o l o g y h a s l o n g b e e n s u b - d i v i d e d i n t o s u b - t y p e s . R i o H o r t e g a (1928) was t h e f i r s t t o d e s c r i b e d i f f e r e n c e s i n o l i g o d e n d r o c y t e s . He c l a s s i f i e d o l i g o d e n d r o c y t e s i n t o p e r i n e u r o n a l and i n t e r f a s i c u l a r , t h e l a t t e r c e l l s b e i n g f r e q u e n t l y a l i g n e d i n rows w i t h soma c l o s e t o one a n o t h e r . B o t h o f t h e s e s u b c l a s s e s were h e t e r o g e n e o u s i n t e r m s o f s i z e and s h a p e o f t h e c e l l s b o d i e s , i n t h e number and f e a t u r e s o f t h e i r p r o c e s s e s and i n t h e number and s i z e o f t h e a x o ns w i t h w h i c h t h e c e l l s were a s s o c i a t e d . He t h u s s u b c l a s s i f i e d them i n t o 4 p r o t o t y p e s . M o s t p e r i n e u r o n a l and p e r i v a s u l a r o l i g o d e n d r o c y t e s a r e t y p e 1: t h e s e a r e s m a l l c e l l s 15-20 fjim i n d i a m e t e r w i t h many f i n e p r o c e s s e s t h a t show g r e a t v a r i a b i l i t y i n t h e manner i n w h i c h t h e y emanate f r o m t h e c e l l b u t e x h i b i t l i t t l e b r a n c h i n g . E a c h p r o c e s s a b u t s a n e r v e - 38 - f i b e r . T h e y a r e f o u n d i n t h e c e r e b r u m , c e r e b e l l u m , and m e d u l l a . M o s t i n t e r f a s i c u l a r o l i g o d e n d r o c y t e s a r e t y p e 2: t h e y h a v e f e w e r and t h i c k e r p r o c e s s e s t h a n t y p e 1, r a n g e f r o m 20 t o 4 0 ^ r a i n d i a m e t e r , d i f f e r i n t h e manner i n w h i c h t h e p r o c e s s e s come o u t f r o m t h e c e l l , and a r e o n l y i n w h i t e m a t t e r . T ype 3 o l i g o d e n d r o c y t e s a r e d i s t i n g u i s h e d by t h e i r l a r g e s i z e and few b u t l a r g e p r o c e s s e s . T h e y a r e most f r e q u e n t l y f o u n d a s mono- o r b i - p o l a r c e l l s a s s o c i a t e d w i t h l a r g e a x o n s . They a r e l e s s numerous t h a n t y p e s 1 o r 2. Type 4 a r e e i t h e r mono- o r b i - p o l a r and h a v e h i g h l y e l o n g a t e d b o d i e s and a r e a t t a c h e d d i r e c t l y t o a x o n s . T h e s e s u b - t y p e s show s i m i l a r d e p o s i t i o n o f s i l v e r c a r b o n a t e , b e i n g d e n s e t h r o u g h o u t t h e c e l l body. T h e s e s u b t y p e s t h e r e f o r e h a v e s i m i l a r b i o c h e m i s t r y f o r t h i s s t a i n . A n o t h e r c l a s s i f i c a t i o n scheme f o r o l i g o d e n d r o c y t e s i s b a s e d on t h e w i d e r a n g e o f n u c l e a r and c y t o p l a s m i c d e n s i t i e s ( C a l e y a n d M a x w e l l , 1968, M o r i and L e b l o n d 1970) . T h ey u s e d t h e s e d e n s i t y d i f f e r e n c e s t o c l a s s i f y them i n t o l i g h t , medium and d a r k o l i g o d e n d r o c y t e s . I t i s n o t known how much o f an o v e r l a p t h e r e i s between t h e two c l a s s i f i c a t i o n s y s t e m s . I t i s h i g h l y p r o b a b l e t h a t t h e i n c r e a s i n g d e n s i t y i s a f u n c t i o n o f d e v e l o p m e n t a l m a t u r i t y . Many r e c e n t d a t a s u g g e s t t h a t a w i d e v a r i e t y o f c e l l s n o t t r a d i t i o n a l l y t h o u g h t o f a s b e i n g g l i a s h a r e c h a r a c t e r i s t i c s o f t h e c l a s s i c a l g l i a and u s u a l l y r e s e m b l e a s t r o c y t e s . E p endymal c e l l s c o n t a i n v i m e n t i n and r e s e m b l e a s t r o c y t e s i n m o r p h o l o g y . P i t u i c y t e s o f t h e n e u r o h y p o p h y s i s a r e G F A P - p o s i t i v e and - 39 - r e t a i n t h i s c h a r a c t e r i s t i c e v e n when no l o n g e r u n d e r n e u r a l i n f l u e n c e . M u l l e r c e l l s o f t h e r e t i n a h a v e l o n g b e e n r e c o g n i z e d as b e i n g g l i a , t h o u g h t h e y a r e n o t t h e o n l y g l i a i n t h e r e t i n a . T h e y a r e G F A P - p o s i t i v e , and v i m e n t i n - p o s i t i v e , and c o n t a i n b o t h GS and c a r b o n i c a n h y d r a s e ( L i n s e r and Muscona, 1 9 8 1 ) . The two enzymes d i f f e r m a r k e d l y i n d e v e l o p m e n t a l p r o f i l e ( L i n s e r and Muscona, 1981) . I n e a r l y e m b r y o n i c c h i c k s , c a r b o n i c a n h y d r a s e i s i n a l l r e t i n a c e l l s , b u t d u r i n g d e v e l o p m e n t t h e s p e c i f i c i t y o f M u l l e r c e l l s i n c r e a s e s w i t h t h e f i n a l d i s a p p e a r a n c e f r o m a m a c r i n e c e l l s o n l y on t h e 1 6 t h day. Bussow (1980) showed t h a t M u l l e r c e l l s o f t h e r e t i n a a r e n o t l i k e g l i a o f o t h e r a r e a s as t h e y seem t o h a v e a s p e c i a l i z e d f u n c t i o n . T h ey a r e t h r o u g h o u t t h e e n t i r e t h i c k n e s s o f t h e r e t i n a and t h e i r b a s a l p r o c e s s e s a l i g n w i t h t h e n e r v e f i b e r t o f o r m s e p t i t h a t f a s i c u l a t e t h e a x o n s o f t h e g a n g l i o n i c c e l l s . I n t h e i n n e r and o u t e r p l e x i f o r m l a y e r o f t h e r e t i n a o f Macaque monkeys t h e r e a r e , b a s e d on m o r p h o l o g y , a t l e a s t two o t h e r g l i a c e l l t y p e s b e s i d e s M u l l e r c e l l s ( B o y c o t t and H o p k i n s , 1 9 8 1 ) . Bussow (1980) f o u n d a s t r o c y t e s t h a t a r e n o t h o m o g e n e o u s l y d i s t r i b u t e d t h r o u g h o u t t h e l a y e r s o f t h e r e t i n a , b u t h a v e p r o c e s s e s o n l y i n t h e g a n g l i o n and n e r v e f i b e r l a y e r s t h a t r u n p a r a l l e l t o t h e a x o n s . He a l s o saw i n t h e o p t i c n e r v e f i b r o u s a s t r o c y t e s w i t h p r o c e s s e s t h a t r u n p e r p e n d i c u l a r t o t h e a x on b u n d l e s , t h e s e m i g h t be c o n s i d e r e d s p e c i a l i z e d g l i a f o r t h e g a n g l i o n i c c e l l a x o n s . B a r b e r and L i n d s a y (1982) f o u n d t h a t t h e Schwann c e l l s o f t h e o l f a c t o r y and v o m e r o n a s a l n e r v e s a r e more c l o s e l y r e l a t e d - 40 - t o c e n t r a l a s t r o c y t e s and g l i a l c e l l s o f t h e m y e n t e r i c p l e x u s t h a n t o Schwann c e l l s o f o t h e r p a r t s o f t h e p e r i p h e r y b e c a u s e t h e y r e a c t t o a n t i b o d i e s t o b o t h GFAP and a 49k d a l t o n g l i a l f i l a m e n t p r o t e i n f r o m human b r a i n . T h e s e g l i a , w h i c h grow f r o m t h e p e r i p h e r y i n t o t h e CNS a l o n g t h e n e r v e a s i t d e v e l o p s i n t o b r a i n , a r e t r a d i t i o n a l l y c a l l e d Schwann c e l l s b e c a u s e o f t h e i r p e r i p h e r a l o r i g i n . T h e y a r e , however, a l s o m o r p h o l o g i c a l l y d i f f e r e n t f r o m t r u e p e r i p h e r a l Schwann c e l l s i n t h a t t h e y do n o t e n s h e a t h a x o ns i n d i v i d u a l l y b u t e x t e n d t o n g u e s o f c y t o p l a s m w h i c h b r a n c h between t h e a x o n s and s e p a r a t e them i n t o b u n d l e s . T h e y a l s o h a v e no basement membrane s u r r o u n d i n g i n d i v i d u a l c e l l s , and c o n t a i n no c o l l a g e n . T a n y c y t e s a r e s p e c i a l i z e d g l i a - l i k e c e l l s w i t h r a d i a l l y o r i e n t e d p r o c e s s e s t h a t l i n e p a r t s o f t h e v e n t r i c l e s , e s p e c i a l l y t h e t h i r d v e n t r i c l e . They a r e G F A P - p o s i t i v e a t an e a r l i e r s t a g e t h a n a r e g l i a and c o n t i n u e s o t h r o u g h o u t d e v e l o p m e n t ( B a s c o e t a l . , 1981, D e V i e t r y e t a l . , 1 9 8 1 ) . They r e m a i n GFAP+ i n t o a d u l t h o o d , i n w h i c h r e g a r d t h e y a r e l i k e Bergmann g l i a o f t h e c e r e b e l l u m , some s i m i l a r c e l l s o f t h e c o r t e x , h i p p o c a m p a l g l i a o f t h e d e n t a t e g y r u s , and n o r m a l a s t r o c y t e s . They, l i k e o t h e r g l i a i n c o n t a c t w i t h c e r b r o s p i n a l f l u i d , a r e v i m e n t i n - p o s i t i v e . Bergmann g l i a , o r G o l g i e p i t h e l i a l c e l l s a s t h e y a r e s o m e t i m e s c a l l e d , a r e s p e c i a l i z e d g l i a o f t h e c e r e b e l l u m w i t h c e l l b o d i e s a r o u n d t h e P u r k i n j e c e l l s and r a d i a l l y o r i e n t e d p r o c e s s e s e x t e n d i n g t h o u g h o u t t h e m o l e c u l a r l a y e r . T h ey s t a i n f o r most a s t r o c y t i c m a r k e r s . They e v o l v e f r o m f i b e r s i n t h e - 41 - m o l e c u l a r l a y e r o f t h e e a r l y c e r e b e l l u m ( F u l o p e t a l . , 1979) t h a t t r a n s f o r m i n t o Bergmann g l i a l c e l l s a f t e r g u i d i n g g r a n u l e c e l l s t o t h e i r f i n a l p o s i t i o n ( R a k i c , 1 9 7 1 ) . C o n t e s t a b i l e and A n d e r s e n (1978) s t u d i e d Bergmann g l i a l c e l l s a n d f o u n d a d i f f e r e n t p r o f i l e o f enzyme a c t i v i t i e s t h a n i n r e g u l a r a s t r o c y t e s . They f o u n d h i g h a c t i v i t i e s o f n o n s p e c i f i c L - g l u t a m a t e d e h y d r o g e n a s e , g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e and T P N H - t e t r a z o l i u m r e d u c t a s e , b u t low a c t i v i t y o f l a c t a t e d e h y d r o g e n a s e , and no s u c c i n a t e d e h y d r o g e n a s e . T h e r e f o r e , t h e Bergmann g l i a a r e low i n c i t r i c a c i d c y c l e enzymes and h i g h i n t h o s e o f t h e p e n t o s e p h o s p h a t e s h u n t . Bergmann g l i a a r e n o t t h e o n l y g l i a i n t h e c e r e b e l l u m . H a t t e n e t a l . (1984) s t u d i e d g l i a l c e l l s o f d i f f e r e n t t y p e s i n e a r l y p o s t n a t a l mouse c e r e b e l l u m . T h e r e were two t y p e s t h a t were GFAP p o s i t i v e : one t y p e h a d two t o t h r e e n e u r o n s a s s o c i a t e d w i t h i t and r e s e m b l e d Bergmann g l i a , and t h e o t h e r was l a r g e r , h a d s h o r t e r arms i n w h i c h i t c l u s t e r e d a d o z e n o r more n e u r o n s and r e s e m b l e d a s t r o c y t e s o f t h e g r a n u l a r l a y e r . T h e r e were a l s o g a l a c t o c e r e b r o s i d e - p o s i t i v e g l i a w h i c h d i d n o t a s s o c i a t e w i t h c e r e b e l l a r n e u r o n s d u r i n g t h e t i m e s t u d i e d . Time l a p s e p h o t o g r a p h y r e v e a l e d e x t e n s i v e m i g r a t i o n a l o n g t h e arms o f t h e B e r g m a n n - l i k e a s t r o c y t e s b u t n o t t h e s t e l l a t e a s t r o c y t e s . R a d i a l g l i a l c e l l s h a v e b e e n d e s c r i b e d b y a number o f r e s e a r c h e r s . T h ey t r a n s f o r m i n t o d i s t i n g u i s h a b l e t y p e s o f g l i a , a r e r e l a t e d t o a s t r o c y t e s , Bergmann g l i a , M u l l e r c e l l s and t a n y c y t e s w h i c h a l l c o n t a i n GFAP a t some p o i n t i n t h e i r d e v e l o p m e n t and h a v e s i m i l a r m o r p h o l o g y . The immature r a d i a l - 42 - g l i a a r e c l a s s i f i e d as a s t r o c y t i c b e c a u s e o f t h e b u n d l e s o f f i l a m e n t s and a c c u m u l a t i o n o f g l y c o g e n i n t h e i r c y t o p l a s m ( R a k i c , 1 9 7 2 ) , b u t t h e y may a l s o d e v e l o p i n t o o l i g o d e n d r o c y t e s . A k e r s (1977) and C a j a l (1929) b o t h d e s c r i b e t h e d e v e l o p m e n t a l c h a n g e s t h a t o c c u r i n r a d i a l g l i a i n t h e c o r t e x . T r a n s i t i o n a l f o r m s h a v e b e e n n o t e d b e t w e e n r a d i a l g l i a a n d a s t r o c y t e s ( S c h m e c h e l and R a k i c , 1979) and between r a d i a l g l i a and o l i g o d e n d r o c y t e s ( C h o i e t a l . , 1 9 8 3 ) . R a d i a l g l i a h a v e b e e n shown t o t r a n s f o r m i n t o m a t u r e a s t r o c y t e s ( R a k i c , 1972, C a j a l , 1929, S c h m e c h e l and R a k i c , 1 9 7 9 ) , and most a s t r o c y t e s p a s s t h r o u g h a r a d i a l g l i a l p h a s e . They may a l s o d e v e l o p f r o m ependymal and s u b e p e n d y m a l l a y e r s by way o f g l i o b l a s t s a n d a s t r o b l a s t s , a s w i l l be d i s c u s s e d i n t h e n e x t s e c t i o n . U s i n g GFA t o f o l l o w t h e d e v e l o p i n g b r a i n i n monkey, L e v i t t and R a k i c (1980) showed t h e e v o l u t i o n o f r a d i a l g l i a l c e l l s as t h e y f a n n e d o u t f r o m r e t i c u l a r and s u b r e t i c u l a r z o n e s t o t h e p i a l s u r f a c e where t h e y h a d end f e e t t h a t s t a y e d u n t i l t h e t r a n s i t i o n t o a s t r o c y t e s . R a d i a l g l i a a r e t r a d i t i o n a l l y s u p p o s e d t o be t h e g u i d e l i n e s t h a t n e u r o n s u s e t o grow a l o n g d u r i n g d e v e l o p m e n t . However, i n t h e d e v e l o p i n g mouse c o r t e x and h i p p o c a m p u s , Woodhams e t a l . (1981) n o t i c e d t h a t c o r t i c a l p l a t e f o r m a t i o n and t i m e o f t h e f i r s t a p p e a r a n c e o f GFAP+ r a d i a l g l i a d i d n o t c o r r e l a t e v e r y w e l l . A c l e a r a s s o c i a t i o n i s e v i d e n t i n t h e l a t e s t a g e s b u t n o t i n t h e e a r l y s t a g e s . T h i s a r g u e s a g a i n s t a p r i m a r y r o l e i n c o r t i c a l p l a t e f o r m a t i o n . C h o i and Lapham (1980) f o u n d two t y p e s o f r a d i a l g l i a l - 43 - c e l l s i n t h e d e v e l o p i n g c e r e b e l l u m o f t h e human f e t u s a t 9 weeks, w h i c h i s e a r l i e r t h a n p r e v i o u s l y t h o u g h t . The l o w e r o n e s e x t e n d e d f r o m t h e v e n t r i c u l a r a r e a t o t h e v a s c u l a r w a l l s o f t h e i n t e r m e d i a t e l a y e r , t h e u p p e r ones f r o m b e l o w t h e P u r k i n j e c e l l l a y e r t o t h e p i a . The l a t t e r , a t 20 weeks, c l o s e l y r e s e m b l e d Bergmann g l i a . S e r e s s (1980) e x a m i n e d t h e r a d i a l g l i a l c e l l s o f p o s t n a t a l r a t b r a i n . U n t i l d a y 10 r a d i a l g l i a l c e l l s were s e e n i n t h e w a l l s o f t h e t h i r d and f o r t h v e n t r i c l e s , a n d h a d v e r y v a r i a b l e m o r p h o l o g y i n d i f f e r e n t r e g i o n s . A f t e r d a y 10, o n l y t a n y c y t e s were s e e n i n t h e w a l l o f t h i r d v e n t i c l e , s h o w i n g t h e p o s t n a t a l d e v e l o p m e n t o f t a n y c y t e s . ¥ Developmental Differences - A Source Of Heterogeneity G l i a c e l l s c a n h a v e v e r y d i f f e r e n t d e v e l o p m e n t a l h i s t o r i e s . U n d e r s t a n d i n g t h e d e v e l o p m e n t i s s t i l l n o t c o m p l e t e and h a s u n d e r g o n e many c h a n g e s . I n 1888 G o l g i , u s i n g t h e G o l g i s t a i n i n g t e c h n i q u e , p r o p o s e d t h a t t h e p r e c u r s o r s o f a l l n o n - n e u r o n a l c e l l s , t h e s p o n g i o b l a s t s , a r o s e f r o m c o l u m n a r e p i t h e l i a l c e l l s i n t h e w a l l s o f t h e n e u r a l t u b e . H i s (1889) f i r s t p o s t u l a t e d t h e t h e o r y o f 2 t y p e s o f g e r m i n a l c e l l s i n t h e n e u r a l t u b e , g e r m i n a l c e l l s w h i c h a r e n e u r o n p r o d u c i n g c e l l s a nd s p o n g i o b l a s t s w h i c h a r e g l i a l p r o d u c i n g . L e n h o s s e c k (1891) was t h e f i r s t t o p r o v e t h a t g l i a l c e l l s were e p i t h e l i a l r a t h e r t h a n mesenchymal i n o r i g i n and d i f f e r e n t i a t e f r o m t h e p r i m a t i v e ependyma j u s t a s n e u r o n s do. S c h a p e r (1897) a r g u e d t h a t g e r m i n a l c e l l s p r o d u c e a b i p o t e n t i a l c e l l t h a t m i g r a t e d away f r o m t h e l a y e r t o d i f f e r e n t i a t e i n t o g l i a l o r n e u r o n a l - 44 - c e l l s . I t was n o t u n t i l C a j a l (1909-1911), i n h i s c l a s s i c a l s t u d i e s o f t h e s p i n a l c o r d o f t h e c h i c k embryo, f o u n d t r a n s i t i o n a l c e l l s f r o m n e u r o e c t o d e r m t o m a t u r e n e u r o g l i a t h a t t h e o r i g i n o f t h e n e u r o e c t o d e r m was f i r m l y e s t a b l i s h e d . C a j a l n o t e d t h r e e c e l l t y p e s : n e u r o n s a p p e a r e d f i r s t , a s t r o c y t e s and an u n i d e n t i f i e d t h i r d t y p e a p p e a r e d l a t e r . T h i s t h i r d t y p e was l a t e r e l u c i d a t e d b y d e l R i o H o r t e g a (1919) who u s e d t h e s i l v e r c a r b o n a t e method t o i d e n t i f y them a s m i c r o g l i a . The p r o b l e m w i t h t h i s e a r l y work i s t h a t t h e s t a i n i n g was n o t r e a l l y a d e q u a t e f o r d e v e l o p m e n t a l work. The g o l d s u b l i m a t e method o f C a j a l s e l e c t i v e l y s t a i n e d a s t r o c y t e s w e l l b u t t h e s i l v e r s t a i n s were n o t c o m p l e t e l y s e l e c t i v e f o r o l i g o d e n d r o c y t e s and n e i t h e r o f t h e s e two methods s t a i n e d u n d i f f e r e n t i a t e d c e l l p r e c u r s o r s . P e n f i e l d (1932) n o t e d t h a t some o f t h e ependymal c e l l s were d e r i v e d f r o m n e u r o e p i t h e l i a l c e l l s w h i c h h a d p r o c e s s e s e x t e n d i n g t o t h e e x t e r n a l l i m i t i n g membrane an d were known as s u p p o r t i v e s p o n g i o b l a s t s . T h e s e c e l l s l o s t t h e i r p r o c e s s e s and became s u b p i a l a s t r o c y t e s w h i c h t h e n f o r m e d a t t a c h m e n t s t o b l o o d v e s s e l s . T h i s was t h u s an a l t e r n a t e r o u t e t h a n f r o m b i p o t e n t i a l c e l l s f o r g l i a l d e v e l o p m e n t . U n t i l t h e d e v e l o p m e n t o f t h e e l e c t r o n m i c r o s c o p e , t h e ependymal c e l l was t h o u g h t t o be t h e p r e c u r s o r o f t h e m a c r o g l i a . The c u r r e n t t h e o r y i s t h a t g l i a d e v e l o p f r o m n e u r o e c t o d e r m a f t e r n e u r o b l a s t f o r m a t i o n d e c l i n e s . The g l i o b l a s t s g i v e r i s e t o b o t h a s t r o c y t e s and o l i g o d e n d r o c y t e s . U s i n g t h y m i d i n e i n j e c t e d a f t e r b i r t h i n r a t s f r o m day 1-21, and s a c r i f i c e d a t day 25, g l i a l g e n e s i s i n t h e s o m a t o s e n s o r y - 45 - c o r t e x was o b s e r v e d ( I c h i k a w a and H i r a t e , 1 9 8 2 ) . I t o c c u r e d f r o m an i n s i d e t o o u t s i d e manner i n t h e f i r s t two weeks. The g l i o b l a s t d e v e l o p m e n t , however, i s s t i l l i n d o u b t . H i s ' s o r i g i n a l t h e o r y o f two p r e c u r s o r c e l l s was c h a l l e n g e d a f t e r 90 y e a r s b y F u j i t a ( 1 9 8 0 ) , who showed t h a t t h e s e two c e l l t y p e s a r e n o t h i n g b u t t h e same c e l l i n d i f f e r e n t p h a s e s o f t h e m i t o t i c c y c l e . I n t h e e a r l y s t a g e o f d e v e l o p m e n t t h e n e u r a l t u b e i s composed o n l y o f m a t r i x c e l l s ( s t a g e I ) . Out o f t h e s e d e v e l o p p o s t - m i t o t i c c e l l s t h a t a r e t h e n e u r o b l a s t s ( s t a g e I I ) . When n e u r o b l a s t p r o d u c t i o n comes t o an end, s t a g e I I I b e g i n s w h i c h i s t h e p r o d u c t i o n o f g l i o b l a s t s and ependymal c e l l s , w h i c h m i g r a t e and m a t u r e i n t o o l i g o d e n d r o c y t e s o r a s t r o c y t e s and r e s t i n g m i c r o g l i a . F u j u i t a e t a l . (1981) c o n c l u d e d t h a t r e s t i n g m i c r o g l i a e v o l v e d f r o m n e u r o e c t o d e r m and c a n g i v e r i s e t o f i b r o u s a s t r o c y t e s upon i n j u r y . S t u r r o c k (1976) o b s e r v e d f o u r d i f f e r e n t t y p e s o f immature g l i a i n t h e c o r p u s c a l l o s u m o f m i c e . T h e y a r e t h e e a r l y g l i o b l a s t , s m a l l g l i o b l a s t , l a r g e g l i o b l a s t , and y o u n g a s t r o c y t e . He p r o p o s e d t h e f o l l o w i n g s e q u e n c e o f d e v e l o p m e n t : e a r l y g l i o b l a s t > l a r g e g l i o b l a s t > l i g h t o l i g o d e n d r o c y t e >medium o l i g o d e n d r o c y t e >dark o l i g o d e n d r o c y t e e a r l y g l i o b l a s t > s m a l l g l i o b a s t >young a s t r o c y t e >mature a s t r o c y t e S k o f f (1980) d i s p u t e d t h e c o n c e p t t h a t g l i o g e n e s i s o c c u r s o n l y a f t e r n e u r o g e n e s i s . He c i t e s t h e o b s e r v a t i o n t h a t r a d i a l g l i a e x i s t t o g u i d e t h e n e u r o n s t o t h e i r p l a c e , t h o u g h t h i s i s now i n q u e s t i o n i n t h e e a r l i e r s t a g e s (Woodhams e t a l . , 1981). - 46 - He p r o p o s e d t h a t a s t r o c y t e s and o l i g o d e n d r o c y t e s o r i g i n a t e f r o m a s t r o b l a s t s and o l i g o d e n d r o b l a s t s , n o t g l i o b l a s t s . A s t r o c y t e s c a n d i v i d e d u r i n g d e v e l o p m e n t ( H a j o s e t a l . , 1981) and, o n c e f o r m e d , c a n d i v i d e b u t o l i g o d e n d r o c y t e s do n o t . K e e n i k o v a e t a l . (1979) s t a i n e d b o t h b a s i c a n d a c i d i c p r o t e i n s d u r i n g d e v e l o p m e n t and n o t i c e d s u c c e s s i v e c h a n g e s i n t h e r a t i o o f b a s i c t o a c i d i c , s u g g e s t i n g s u c c e s s i v e p o p u l a t i o n s o f g l i a t y p e s . P o l y c l o n a l a n t i b o d i e s t o v i m e n t i n h a v e shown t h e e x i s t a n c e o f s u b p o p u l a t i o n s o f a s t r o c y t e s d u r i n g d e v l e o p m e n t ( D a h l e t a l . , 1981, Shaw e t a l . , 1981, Yen and F i e l d , 1 9 8 1 ) . O t h e r d e v e l o p m e n t a l p r o f i l e s seem t o e x i s t i n some s y s t e m s . R a f f e t a l . (1984) d e s c r i b e t h r e e t y p e s o f g l i a l c e l l s i n t h e o p t i c n e r v e t h a t a p p e a r a t d i f f e r e n t t i m e s . Type 1 a s t r o c y t e s a p p e a r a t e m b r y o n i c day 16, o l i g o d e n d r o c y t e s a t p o s t n a t a l d a y 2 and t y p e 2 a s t r o c y t e s on p o s t n a t a l d a y 10. I n c u l t u r e i t was shown t h a t t h e o l i g o d e n d r o c y t e s and t y p e 2 a s t r o c y t e s came f r o m a d i f f e r e n t c e l l t y p e t h a n t y p e 1. O t h e r s showed t h a t t h e o p t i c n e r v e a s t r o b l a s t s c a n be t r a c e d b a c k t o v e n t r i c u l a r c e l l s w i t h o u t g o i n g t h r o u g h g l i o b l a s t s t a g e . T h i s i s e v i d e n c e f o r a l t e r n a t e g l i o g e n e s i s i n b r a i n . The e x p l a n a t i o n f o r t h e s e g r o s s l y d i f f e r e n t t h e o r i e s o f g l i o g e n e s i s may be t h a t d i f f e r e n t r e s e a r c h e r s o b s e r v e d i f f e r e n t p o p u l a t i o n s o f g l i a w h i c h a r e h e t e r o g e n e o u s i n t h e i r d e v e l o p m e n t . T h e s e d i f f e r e n t d e v e l o p m e n t a l p r o f i l e s may be a m a j o r s o u r c e o f c o n f u s i o n i n i n t e r p r e t i n g t h e l i t e r a t u r e on g l i a h e t e r o g e n e i t y . Not o n l y a r e t h e r e d i f f e r e n t p a t t e r n s o f - 47 - g l i o g e n e s i s b u t t h e r e a r e d i f f e r e n t p a t t e r n s o f d e v e l o p m e n t o f b i o c h e m i c a l a b i l i t i e s w i t h i n t h e s e c e l l s . Some b i o c h e m i c a l phenomena d e v e l o p e a r l y , s u c h a s v i m e n t i n , w h e r e a s some d e v e l o p l a t e . F o r example p o t a s s i u m i s n o t e d t o h a v e a s t i m u l a t o r y e f f e c t on t h e Na+,K+-ATPase o n l y i n v e r y l a t e o n t o g e n i c s t a g e s ( G r i s a r , 1979) o r i n o l d e r a s t r o c y t e c u l t u r e s (Moonen and F r a n c k , 1977) A n o t h e r example o f d i f f e r i n g b i o c h e m i c a l m a t u r a t i o n i s t h e i n c o r p o r a t i o n o f r a d i o a c t i v e g l u c o s e i n t o a s p a r t a t e , g l u t a m a t e , and g l u t a m i n e w h i c h d e v e l o p s l a t e , a s i t i s much l e s s p r o n o u n c e d i n b r a i n s f r o m immature a n i m a l s t h a n i n a d u l t b r a i n (Van den B e r g , 1 9 7 0 ) . The d e v e l o p m e n t o f m e t a b o l i c c o m p a r t m e n t a t i o n c o i n c i d e s w e l l w i t h t h e t i m e p e r i o d when c o n v e r s i o n o f g l u c o s e c a r b o n i n t o t h e s e amino a c i d s i n t e n s i f i e s ( P a t e l and B a l a z s , 1 9 7 4 ) . The i n c r e a s e d i n t e n s i t y o f g l u t a m a t e u p t a k e i n t o c u l t u r e d a s t r o c y t e s o c c u r s a t t h e same age ( S c h o u s b o e e t a l , 1976, H e r t z e t a l . , 1 9 7 9 ) . A l s o GS l e v e l s r i s e i n v i v o a t t h e same age and t h e same o c c u r s i n c u l t u r e d a s t r o c y t e s ( H e r t z e t a l . , 1 9 7 8 ) . T h e r e f o r e some g l i a l d i f f e r e n c e s may e v o l v e a l o n g w i t h t h e r e l a t i v e l a t e d e v e l o p m e n t o f m e t a b o l i c c o m p a r t m e n t a l i z a t i o n . O b s e r v a t i o n s l i k e t h i s means t h a t c a u t i o n must be u s e d when i n t e r p r e t i n g t h e b i o c h e m i c a l d i f f e r e n c e s r e p o r t e d f o r g l i a a s some h e t e r g e n e i t y may be due t o t h e d e v e l o p m e n t s t a g e o f t h e c e l l s u s e d i n t h e r e s e a r c h . G l y c o g e n s t o r a g e c h a n g e s w i t h i n r a d i a l g l i a l c e l l s o f d e v e l o p i n g r a t b r a i n i s a n o t h e r example o f d e v e l o p m e n t a l c h a n g e s t h a t o c c u r . S u c h s t o r a g e f i r s t a p p e a r s on e m b r y o n i c - 48 - day 14 i n t h e c h o r o i d p l e x u s and i n t h e r a d i a l g l i a l c e l l s o f m i d b r a i n and m e d u l l a r y r a p h e ( B r u c k n e r and B i e s o l d , 1 9 81). T h e s e c e l l s r e t a i n e d t h e h i g h e s t c a p a c i t y t h r o u g h o u t d e v e l o p m e n t b u t o t h e r r a d i a l g l i a a l s o showed some g l y c o g e n s t o r a g e a s t h e y d e v e l o p e d . G l y c o g e n s t o r a g e t h e n d e c r e a s e d t o a d u l t l e v e l s b y p o s t n a t a l d a y 21. T h i s m i g h t i n d i c a t e t h a t g l y c o g e n i s u s e d a s an e n e r g y s o u r c e i n p e r i n a t a l m e t a b o l i s m . I n o t h e r work, C o l m a n t (1965) n o t i c e d i n c r e a s e s i n a c i d p h o s p h a t a s e s , DPNH- and TPNH- t e t r a z o l i u m r e d u c t a s e s , s u c c i n a t e d e h y d r o g e n a s e , 5 ' n u c l e o t i d a s e , p h o s p h a m i d a s e , and ^ - n a p h t h o l e s t e r a s e i n o l i g o d e n d r o c y t e s d u r i n g p o s t n a t a l d e v e l o p m e n t . L a g e n a u r e t a l . (1980) u s e d t h e a n t i b o d y t h e y d e s i g n a t e d M l t o d i s t i n g u i s h s u b g r o u p s o f a s t r o c y t e s i n mouse c e r e b e l l u m . S t a i n i n g a p p e a r e d i n w h i t e m a t t e r a t d a y 7 and l a s t e d u n t i l a d u l t h o o d b u t i n Bergmann g l i a a nd i n t h e g r a n u l a r l a y e r on day 10 and l a s t e d o n l y a s h o r t t i m e . I n c u l t u r e i t i s i n some b u t n o t a l l GFAP+ c e l l s . N e u r o n s a n d g l i a a r e c o m m i t t e d t o c e l l l i n e s p r i o r t o c e s s a t i o n o f d i v i s i o n b u t i n most o f t h e s e schemes t h e f i n a l d i f f e r e n t i a t i o n o c c u r s t h r o u g h o u t t h e parenchyma, t h u s c l o s e t o t h e c e l l s a nd b l o o d v e s s e l s t h e y m i g h t e v e n t u a l l y i n t e r a c t w i t h , a l l o w i n g f o r t h e l o c a l b i o c h e m i c a l c l i m a t e t o i n d u c e v a r i a b i l i t y . - 49 - Heterogeneity i n tissue cultures A) D e v e l o p m e n t a l c h a n g e s i n c u l t u r e P r i m a r y c u l t u r e s o f g l i a p r o v i d e some p e r t i n a n t i n f o r m a t i o n on d e v e l o p m e n t a l q u e s t i o n s and h e t e r o g e n e i t y . P r i m a r y c u l t u r e s a r e t h o u g h t t o m i m i c c l o s e l y t h e i n v i v o s i t u a t i o n and d e v e l o p o r r e d e v e l o p many i n v i v o c h a r a c t e r i s t i c s . M assa e t a l . ( 1 9 8 3 ) , f o r i n s t a n c e , showed t h a t o l i g o d e n d r o c y t e s d e s i g n a t e d B 3 , f e v e n r e d e v e l o p membrane s p e c i a l i z a t i o n s s u c h a s t i g h t j u n c t i o n s . C u l t u r e s a l l o w e x p e r i m e n t a l m a n i p u l a t i o n s and d e v e l o p m e n t a l o b s e r v a t i o n s t o be c a r r i e d o u t . S e v e r a l r e s e a r c h e r s h a v e m o n i t o r e d p r i m a r y g l i a l c u l t u r e s o f v a r i o u s a g e s f o r c h a n g e s w i t h t i m e i n c u l t u r e . T h e s e a r e b e l i e v e d t o m i m i c d e v e l o p m e n t a l c h a n g e s i n v i v o . F e d o r o f f e t a l . (1984a) e x a m i n e d newborn mouse a s t r o c y t e s i n c u l t u r e s , o r i g i n a l l y p l a t e d a t low d e n s i t y , l o n g i t u d i n a l l y f r o m t h r e e d a y s t o f o u r weeks. The e a r l i e s t a s t r o c y t e p r e c u r s o r c e l l s o r g l i o b l a s t s a r e c l o s e l y a p p o s e d e p i t h e l i a l c e l l s t h a t r a r e l y h a v e j u n c t i o n s . T h e i r s c a n t y c y t o p l a s m c o n t a i n s many f r e e r i b o s o m e s b u t few m i c r o f i l a m e n t s . The c e l l s o f t h e n e x t s t a g e o f a s t r o c y t e l i n e a g e , p r o a s t r o b l a s t s , a r e f l a t a nd s e p a r a t e f r o m e a c h o t h e r t o a v a r i a b l e d e g r e e . T h ey h a v e i n t e r c e l l u l a r j u n c t i o n s a s s o c i a t e d w i t h m i c r o f i l a m e n t s and c o n t a i n s i n g l y d i s p e r s e d i n t e r m e d i a t e f i l a m e n t s . The p r o a s t r o b l a s t s g r a d u a l l y d i f f e r e n t i a t e i n t o a s t r o b l a s t s w h i c h h ave a s i m i l a r m o r p h o l o g y e x c e p t t h a t t h e y a l s o c o n t a i n b u n d l e s o f i n t e r m e d i a t e f i b e r s . When n e u r o b l a s t p r o d u c t i o n comes t o an end, t h e t h i r d s t a g e b e g i n s w h i c h i s - 50 - t h e p r o d u c t i o n o f a s t r o b l a s t s a nd ependymal c e l l s . T h e s e m i g r a t e a nd m a t u r e i n t o o l g o d e n d r o c y t e s o r a s t r o c y t e s a nd r e s t i n g m i c r o g l i a . The m a t u r e f i b r o u s a s t r o c y t e s h a v e w e l l d e f i n e d p r o c e s s e s a nd d i s t i n c t p e r i k a r y a . T h i s s t u d y showed t h a t t h e r o u t e f r o m r a d i a l g l i a t o f i b r o u s a s t r o c y t e s i s n o t t h e o n l y r o u t e . I t s u p p o r t s t h e g e n e r a l o b s e r v a t i o n o f t h e v e n t r i c u l a r o r s u b v e n t r i c u l a r o r i g i n o f a s t r o c y t e s . T h e s e o b s e r v a t i o n s a l s o i l l u s t r a t e s how l a c k o f d e f i n i t i o n a s t o c u l t u r e c o n d i t i o n s o r c e l l u l a r s t a g e c o u l d l e a d t o o b s e r v a t i o n s o f h e t e r o g e n e i t y w h i c h w o u l d i n r e a l i t y be d i f f e r e n t s t a g e s . M a r k e r c h a n g e s a l s o o c c u r a nd compound t h e d i f f i c u l t i e s i n r e s e a r c h . S c h o u s b o e e t a l . (1980) showed t h a t GFAP i n c r e a s e d w i t h t i m e i n a s t r o c y t e c u l t u r e s t o e x c e e d t h e l e v e l i n 4 week o l d w h o l e f o r e b r a i n . L a b o u r d e t t e a nd Mar k s (1975) showed t h a t S100 i s s y n t h e s i z e d m a i n l y a f t e r d i f f e r e n t i a t i o n i n t h e C-6 l i n e . C hanges i n enzymes a l s o o c c u r d u r i n g d e v e l o p m e n t . S c h o u s b o e e t a l . (1980) m o n i t o r e d v a r i o u s enzymes i n p r i m a r y c u l t u r e s o f a s t r o c y t e s f r o m t h e c o r t e x o f m i c e o r r a t s . Na+,KH—ATPase r e a c h e d i t s p e a k a t 2-3 weeks i n c u l t u r e b u t t h e s t i m u l a t o r y e f f e c t s o f K+ d i d n o t o c c u r u n t i l 4 weeks. T h i s p a r a l l e l s t h e i n v i v o c h a n g e s . L a c t a t e d e h y d r o g e n a s e p e a k s a t two weeks i n c u l t u r e t o a l e v e l a bove t h a t o f a d u l t b r a i n , t h e n d r o p s t o t h e a d u l t l e v e l . The i s o - e n z y m e p a t t e r n o f l a c t a t e d e h y d r o g e n a s e c h a n g e s f r o m immature t o m a t u r e f r o m one t o t h r e e weeks. GABA-T i n a s t r o c y t e c u l t u r e s d r o p s i n t h e f i r s t week b u t t h e n i n c r e a s e s b a c k t o l e v e l s c o m p a r a b l e t o - 51 - t h o s e i n n e o n a t a l mouse b r a i n . C a r b o n i c a n h y d r e a s e was low b u t f o u n d t o i n c r e a s e t o w a r d i n v i v o l e v e l s i n d i f f e r e n t i a t e d c u l t u r e s . COMT and MAO i n c r e a s e d w i t h t i m e i n p r i m a r y a s t r o c y t e c u l t u r e s ( Hansson and S e l l s t r o m , 1 9 8 3 ) . L e v i and C i o t t i (1983) showed GABA b u t n o t D - a s p a r t a t e u p t a k e was r e s t r i c t e d t o m a t u r e s t e l l a t e a s t r o c y t e s i n c u l t u r e . T h e r e f o r e GABA t r a n s p o r t i s a d i f f e r e n t i a t e d phenomenon o r i s due t o a s u b s e t i n c u l t u r e t h a t become p r o m i n e n t . M e l l e r and W a e l s c h (1984) s t u d i e d c u l t u r e s o f c e l l s f r o m e m b r y o n i c b r a i n f o r a y e a r . A n t i - G F A P and m y e l i n b a s i c p r o t e i n were u s e d t o i d e n t i f y g l i a l c e l l t y p e s . F o u r c e l l t y p e s were o b s e r v e d and m o n i t o r e d : f l a t e p i t h e l o i d c e l l s t h a t were GFAP- and e i t h e r m y e l i n b a s i c p r o t e i n + o r -, a s t r o g l i a l c e l l s w h i c h were 92% GFAP+, and o l i g o d e n d r o c y t e s w h i c h were m y e l i n b a s i c p r o t e i n + . The a s t r o c y t e s o r i g i n a t e c o n t i n u o u s l y where a s t h e o l i g o d e n d r o c y t e s d i f f e r e n t i a t e e v e r y 2 0 t o 3 0 d a y s . V e r n a d a k i s and Mangoura (1983) compared c u l t u r e s f r o m newborn and a d u l t m i c e and f o u n d t h a t t h o s e f r o m newborn m i c e h a d b o t h o l i g o d e n d r o c y t e s and a s t r o c y t e s , a s d e t e r m i n e d by m a r k e r s , and t h e s e b o t h i n c r e a s e d i n c u l t u r e . On t h e o t h e r hand, i n c u l t u r e s f r o m a d u l t m i c e o n l y t h e a s t r o c y t e s i n c r e a s e d a n d t h e s e f o r o n l y a few d a y s , w h e r e a s t h e o l i g o d e n d r o c y t e s d e c r e a s e d i n number. T h i s p a r a l l e l s t h e a s t r o g l i o s i s s e e n i n a g i n g b r a i n . O t h e r p e o p l e h a v e shown a v a r i e t y o f c e l l t y p e s i n p r i m a r y a s t r o c y t e c u l t u r e s . W i l k i n e t a l . (1983) e x a m i n e d p r i m a r y - 52 - c u l t u r e s made f r o m c e r e b e l l a r a s t r o c y t e s w h i c h were GFAP p o s i t i v e and were o f two d i s t i n c t m o r p h o l o g i c a l t y p e s . One c l a s s was s t e l l a t e i n sh a p e w i t h r a d i a l l y d i s t r i b u t e d f i n e p r o c e s s e s w h i l e t h e o t h e r was v a r i e d i n s h a p e , b e i n g e i t h e r p o l y g o n a l o r e l o n g a t e d . T h ey b o t h c o u l d i n c o r p o r a t e t h y m i d i n e and t h e r e f o r e were c a p a b l e o f d i v i s i o n . B o t h t o o k up a s p a r t a t e b u t o n l y t h e s t e l l a t e c e l l s t o o k up GABA. The s t e l l a t e c e l l s d i s a p p e a r e d o v e r t h e 12 d a y s o f c u l t u r e b u t l a s t e d l o n g e r i n l o w e r d e n s i t y c u l t u r e s , p o s s i b l y u n d e r g o i n g a ch a n g e i n s h a p e f o l l o w i n g c e l l t o c e l l i n t e r a c t i o n s . N o n - s t e l l a t e c e l l s t h a t d i d show a weak GABA u p t a k e a b i l i t y l o s t t h i s a t l a t e r s t a g e s . c-AMP, w h i c h i n c r e a s e s s t e l l a t e m o r p h o l o g y , d i d n o t i n c r e a s e GABA u p t a k e , s u g g e s t i n g c-AMP i s n o t a t r u e a g e n t o f d i f f e r e n t i a t i o n and t h a t m o r p h o l o g y i s n o t a t r u e i n d i c a t o r o f b i o c h e m i c a l f u n c t i o n . The f a c t t h a t n o n - s t e l l a t e c e l l s c o n t i n u e d t o d i v i d e b u t s t e l l a t e d i d n o t may i n d i c a t e t h a t t h e s e a r e two d i f f e r e n t t y p e s o f a s t r o c y t e s , b u t o t h e r f a c t o r s s u c h a s s t a t e o f c o m m i t t a l t o d i f f e r e n t i a t i o n a t t i m e o f p l a t i n g , o r p r e s e n c e o f p a r t i c u l a r t y p e s o f n e u r o n s may be f a c t o r s . B) E f f e c t o f c u l t u r e c o n d i t i o n s on c e l l d e v e l o p m e n t Some o f t h e v a r i a b i l i t y i n c e l l t y p e may be due t o s l i g h t d i f f e r e n c e s i n c u l t u r e mediums. M o r r i s o n and D e V e l l i s (1983) a t t e m p t e d t o s t u d y t h i s b y u s i n g a c h e m i c a l l y d e f i n e d medium. Th e y f o u n d t h a t a c h e m i c a l l y d e f i n e d medium p r o d u c e d p u r e r and more c o n t r o l l e d c u l t u r e s t h a t were 95% a s t r o c y t e s (GFAP+) and o n l y 1% + f o r f i b r o n e c t i n , a m a r k e r f o r m e n i n g e a l o r - 53 - e n d o t h e l i a l c e l l s . The c e l l s were m o r p h o l o g i c a l l y d i f f e r e n t i a t e d and p o s i t i v e f o r b o t h S-100P and GS, i n d i c a t i n g a t l e a s t some b i o c h e m i c a l d i f f e r e n t i a t i o n . N o t a l l a s t r o c y t e s r e s p o n d t o d i f f e r e n t i a t i n g f a c t o r s i n t h e same way. R a f f e t a l . (1983) d e s c r i b e two t y p e s o f a s t r o c y t e s i n c u l t u r e o f w h i t e m a t t e r , b o t h b e i n g GFAP+. T y p e 1 a r e f i b r o b l a s t - l i k e , d i d n o t b i n d t e t a n u s t o x i n o r t h e m o n o c l o n a l a n t i b o d y A2B5, were s t i m u l a t e d t o d i v i d e by b o v i n e p i t u i t a r y e x t r a c t o r e p i d e r m a l g r o w t h f a c t o r and a r e a l s o f o u n d i n g r e y m a t t e r . T ype 2 h a d a n e u r o n - l i k e m o r p h o l o g y , bound t e t a n u s t o x i n and A2B5, and were n o t s t i m u l a t e d by b o v i n e p i t u i t a r y e x t r a c t o r e p i d e r m a l g r o w t h f a c t o r . Type 1 c o u l d be c o n v e r t e d t o n e u r o n - l i k e m o r p h o l o g y i n t h e p r e s e n c e o f dBcAMP, p i t u i t a r y e x t r a c t o r b r a i n e x t r a c t s , e s p e c i a l l y i n s e r u m - f r e e medium, b u t d i d n o t g a i n t h e s p e c i f i c b i n d i n g p r o p e r t i e s o f t y p e 2. I n n e o n a t a l c u l t u r e s most o f t h e t y p e 2 c e l l s d e v e l o p e d f r o m GFAP- c e l l s w h i c h were i n d u c e d t o e x p r e s s GFAP b y c u l t u r e c o n d i t i o n s . C u l t u r e c o n d i t i o n s c a n t h u s i n d u c e c h a n g e s i n m o r p h o l o g y . I f c h a n g e s i n c u l t u r e c o n d i t i o n s c a n i n d u c e c h a n g e s i n m o r p h o l o g y , u n d e r s t a n d i n g t h e mechanism c o u l d g i v e u s an u n d e r s t a n d i n g o f i n v i v o c e l l d i f f e r e n c e s . Much work h a s b een done on how c u l t u r e c o n d i t i o n s i n f l u e n c e c u l t u r e s . T rimmer e t a l . (1984) e x p l o r e d t h e c u l t u r e c o n d i t i o n s w h i c h i n f l u e n c e t h e c e l l u l a r c o m p o s i t i o n o f c e r e b r a l c o r t i c a l c u l t u r e s . A d e c r e a s e o f p l a t i n g d e n s i t y , i n c r e a s e d age o f t h e a n i m a l s and s u p p l e m e n t a t i o n o f t h e c o r t i c a l c u l t u r e s w i t h m e n i n g e a l f i b r o b l a s t s a l l c a u s e d an i n c r e a s e i n f i b r o n e c t i n - 54 - s t a i n i n g , and a d e c r e a s e i n GFAP, an i n c r e a s e i n £ 2 a d r e n e r g i c r e c e p t o r s and a d e c r e a s e i n A s t r o g l i a l c e l l s n o r m a l l y e x p r e s s b o t h t y p e s o f b i n d i n g s i t e s , w i t h 60% fil and 40% jS2. Goldman and C h i u (1984) showed t h a t a s t r o c y t e s p l a t e d a t h i g h d e n s i t y r e a c h e d h i g h e r d e n s i t i e s q u i c k l y , h a d s m a l l p e r i k a r y a and s e v e r a l l o n g p r o c e s s e s t h a t were GFAP+ and c o n t a i n e d l e s s a c t i n and more i n t e r m e d i a t e f i l a m e n t s , whereas t h o s e t h a t were p l a t e d a t low i n i t i a l d e n s i t i e s d i d n o t i n c r e a s e i n c e l l number, were f l a t and p o l y g o n a l , s t a i n e d f o r GFAP and r e t a i n e d l a r g e amounts o f c y t o s k e l e t a l a c t i n r e l a t i v e t o i n t e r m e d i a t e f i l a m e n t s . T h e s e r e s u l t s were m i r r o r e d i n t h e r a t e s o f s y n t h e s i s o f t h e s e c y t o s k e l e t a l p r o t e i n s . B o t h o f t h e s e f o r m s t a k e t i m e t o d e v e l o p f r o m t h i n s p i n d l e - s h a p e d c e l l s w i t h a few n a r r o w p r o c e s s e s . L i n d s e y e t a l . ( 1 9 8 2 ) , u s i n g a s t r o g l i a l c e l l s f r o m t h e c o r p u s c a l l o s u m , showed t h a t a s t r o c y t e s c h a n g e s h a p e a s t h e c e l l s a p p r o a c h c o n f l u e n c y . D i f f e r e n c e s i n c u l t u r e c o n d i t i o n s c a n e x i s t w i t h i n t h e same c u l t u r e . F e d o r o f f e t a l . (1983) f o u n d a c e l l i n n o r m a l c u l t u r e s w i t h o u t dBcAMP w h i c h f o r m s on t o p o f t h e l a y e r o f p r e c u r s o r c e l l s ; t h i s c e l l i s s m a l l e r t h a n t h e l o w e r c e l l s b u t r e s e m b l e s t h e dBcAMP s t i m u l a t e d l a r g e a s t r o c y t e . B o t h c o n t a i n GFA and v i m e n t i n , w i t h v i m e n t i n d e v e l o p i n g f i r s t . S u c h c e l l s seem t o d e v e l o p s p o n t a n e o u s l y where t h e r e a r e s p e c i a l c o n d i t i o n s a t t h e i n t e r f a c e b etween t h e c e l l c o n f l u e n t l a y e r a n d t h e medium. Thus much o f t h e h e t e r o g e n e i t y t h a t e x i s t s i s r e a l l y t h e r e s u l t o f t h e same c e l l r e s p o n d i n g t o d i f f e r i n g c o n d i t i o n s . - 55 - C u l t u r e d i f f e r e n c e s c a n be u s e d t o s e l e c t f o r s u b s e t s o f c e l l s . A s u b s e t o f o l i g o d e n d r o c y t e s were s e l e c t e d b y t h e i r i n a b i l i t y t o p l a t e on p l a s t i c c u l t u r e p l a t e s , b u t o n l y on p o l y l y s i n e c o a t e d p l a t e s . T h e s e were o l i g o d e n d r o c y t e s as t h e y were 98% g a l a c t o c e r b r o s i d e +, a r e h i g h l y d i f f e r e n t i a t e d and r e m a i n s o i n c u l t u r e . T h i s i s shown by h i g h l e v e l s o f CNPase a c t i v i t y , h i g h i n c o r p o r a t i o n o f H2SO4 i n t o s u l f i d e s , and a l i p i d m e t a b o l i s m t h a t m i m i c s t h a t a s s o c i a t e d w i t h m y e l i n o g e n e s i s , i . e . , t h e p r e s e n c e o f m y e l i n a s s o c i a t e d g l y c o p r o t e i n s and m y e l i n b a s i c p r o t e i n . I f c u l t u r e c o n d i t i o n s c a n c h a n g e c e l l s , p e r h a p s we a r e l o o k i n g a t b i p o t e n t i a l o r m u l t i p o t e n t i a l c e l l s . T h e y h a v e b e e n p o s t u l a t e d t o e x i s t i n v i v o and h a v e b e e n d e m o n s t r a t e d i n c u l t u r e s . R a f f e t a l . (1984) d e s c r i b e a p r o g e n i t o r c e l l t h a t d i f f e r e n t i a t e s i n t o an o l i g o d e n d r o c y t e i f c u l t u r e d i n a serum f r e e medium and an a s t r o c y t e i f c u l t u r e d w i t h f e t a l c a l f serum. G a l a c t o c e r e b r o s i d a s e was u s e d a s a m a r k e r f o r o l i g o d e n d r o c y t e s and GFAP a s a m a r k e r f o r a s t r o c y t e s . The c e l l c o n t a i n s v i m e n t i n f i l a m e n t s w h i c h i t r e t a i n s i f i t v becomes an a s t r o c y t e and l o s e s i f i t becomes an o l i g o d e n d r o c y t e . The commitment i s r e v e r s i b l e f o r 1 t o 2 d a y s . N o b l e an d M u r r a y (1984) f o u n d t h e same o r v e r y s i m i l a r c e l l s i n o p t i c n e r v e s o f n e o n a t a l r a t s . T h e s e were s t i m u l a t e d t o d i v i d e b y t h e p r e s e n c e o f p u r i f i e d t y p e 1 a s t r o c y t e s o r s o l u b l e f a c t o r s f r o m s u c h a s t r o c y t e s , p r o d u c i n g a l a r g e number o f p r o g e n i t o r c e l l s and o l i g o d e n d r o c y t e s . N o b l e and M u r r a y - 56 - s p e c u l a t e d t h a t t h i s s u b p o p u l a t i o n may be t h e s o u r c e o f t h e o p t i c n e r v e o l i g o d e n d r o c y t e s and t y p e 2 a s t r o c y t e s b u t n o t t y p e 1 a s t r o c y t e s . T h ey h a d a p r o f i l e o f a n t i g e n s i d e n t i c a l t o t h e c e l l s r e p o r t e d b y R a f f e t a l . ( 1 9 8 3 ) . J u u r l i n k e t a l . (1981) f o u n d t h a t immature e p i t h e l i a l - l i k e c e l l s t h a t f o r m t y p e A c o l o n i e s i n c u l t u r e come m a i n l y f r o m t h e s u b v e n t r i c u l a r zone and d e v e l o p i n t o t y p e C c u l t u r e s w h i c h a r e m o r p h o l o g i c a l l y d i f f e r e n t . S i n c e t h e s e r e s p o n d t o dBcAMP i n t h e same ways a s a s t r o c y t e s do, t h e a u t h o r s p r o p o s e t h a t t y p e A c e l l s a r e a s t r o c y t e p r o g e n i t o r c e l l s , p r o b a b l y e q u i v a l e n t t o t h e p a l e c e l l s f r o m t h e s u b v e n t r i c u l a r z one. As p o s t n a t a l d e v l e o p m e n t o c c u r s , t h e number o f c o l o n y - f o r m i n g c e l l s d e c r e a s e . Thus t h e r e a r e a l s o i n t e r a c t i o n s b e t w e e n c e l l t y p e a n d c u l t u r e c o n d i t i o n s . A n o t h e r example o f i n t e r a c t i o n was shown b y Yu and H e r t z (1982) who f o u n d t h a t t h e p r o p o r t i o n o f MAO t y p e A t o t y p e B d e c r e a s e d i n mouse b r a i n p r i m a r y a s t r o c y t e c u l t u r e s a f t e r t r e a t m e n t w i t h dBcAMP. A t 31 d a y s , u n t r e a t e d c e l l s e x p r e s s m a i n l y t y p e A b u t dBcAMP t r e a t m e n t c a u s e s 30% e x p r e s s i o n o f t y p e B. T h i s i n c r e a s e i n t y p e B i s s i m i l a r t o t h a t s e e n w i t h i n c r e a s i n g age o f t h e c u l t u r e o r i n a d u l t r a t s . S i n c e most c e l l l i n e s e x p r e s s one o r t h e o t h e r o f t h e s e enzymes, b u t n o t b o t h , t h i s f i n d i n g may be an example o f t r u e i n d u c e d d i f f e r e n t i a t i o n t h a t p a r a l l e l s t h a t o c c u r r i n g i n v i v o . P r u s s e t a l . (1982) f o u n d t h a t a s t r o c y t e s i n c u l t u r e r e s p o n d t o f i b r o b l a s t g r o w t h f a c t o r and Schwann c e l l m i t o g e n w h i l e o l i g o d e n d r o c y t e s w i l l o n l y do s o i n s u s p e n d e d c u l t u r e s b u t n o t i n p r i m a r y c u l t u r e s . T h i s may be r e l a t e d t o t h e - 57 - a s t r o c y t e s a b i l i t y t o d i v i d e a f t e r i n j u r y . S i n c e t h e c o n t e n t o f serum change w i t h age o f t h e a n i m a l , i n v i v o serum c h a n g e s may w e l l be what c o n t r o l s g l i a l d i f f e r e n t i a t i o n , w i t h d i f f e r e n t c e l l s o r i g i n a t i n g f r o m t h e same p r o g e n i t o r c e l l s a t d i f f e r e n t t i m e s d i c t a t e d b y t h e c h a n g e s i n serum. T a b l e I I I l i s t s some o f t h e e f f e c t s i n p r i m a r y c u l t u r e s o f c h a n g e s o f v a r i o u s s u b s t a n c e s i n t h e c u l t u r e medium. - 58 - TABLE I I I : EFFECTS OF CULTURE CONDITIONS ON CELL CHARACTERISTICS MEDIUM CHANGES PROBABLE MECHANISM C E L L TYPE OBSERVED CHANGES AUTHORS dcAMP T h r o u g h P r i m a r i l y F l a t e p i t h e l o i d - l i k e c e l l s change cAMP A s t r o c y t e t o l a r g e r s t e l l a t e - t y p e t h a t C u l t u r e s r e s e m b l e a s t r o c y t e s I n c r e a s e GFAP and V i m e n t i n A c t i n i n c r e a s e s A c t i n l e s s o r g a n i z e d M i c r o t u b u l e s o r g a n i z e d i n t o b u n d l e s e x t e n d i n g i n t o p r o c e s s e s L e v e l o f most enzymes i n c r e a s e L e v e l s o f GS d e c r e a s e d Change i n p a t t e r n o f p r o t e i n s y n t h e s i s I n c r e a s e d e f f e c t o f p o t a s s i u m s t i m u l a t i o n on Na+,K+-ATPase MAO and COMT i n c r e a s e d P r o p o r t i o n o f MAO Type A t o Type B F e d o r o f f e t a l . (1984b); H a n s s o n and Ronnbeck (1983) C i e s i e l s k i - T r e s k a e t a l . (1984) C i e s i e l s k i - T r e s k a e t a l . (1982b) C i e s i e l s k i - T r e s k a e t a l . (1982a) Schousboe e t (1980a) a l . W hite Se H e r t z (1981) K i m e l b e r g e t a l . (1978a) Hansson & S e l l s t r o m (1983) Yu & H e r t z (1982) d e c r e a s e d a s i n o l d e r c e l l c u l t u r e s o r i n v i v o w i t h age. A s t r o c y t e s t o s t a i n f o r CA-II as i n t e n s e l y a s o l i g o d e n d r o c y t e s K i m e l b e r g e t a l , (1982) D e c r e a s e d GABA u p t a k e w i t h b o t h Vmax and K a f f e c t e d H a n sson e t a l . (1984b) TABLE I I I ( c o n t i n u e d ) : EFFECTS OF CULTURE CONDITIONS ON CELL CHARACTERISTICS MEDIUM PROBABLE CELL CHANGES MECHANISM TYPE OBSERVED CHANGES AUTHORS dcAMP P r o b a b l y t h r o u g h AMP H o r s e Serum F e t a l c a l f serum r e m o v a l H y d r o c o r t i s o n e P r o s t a g l a n d i n PGE1 C-6 P r i m a r y a s t r o g l i a l C-6 [S 100] p r o t e i n i n c r e a s e d I n c r e a s e d a s p a r t a t e a m i n o t r a n s f e r a s e B i n d i n g p a t t e r n o f c o n c a v a l i n - A becomes c o n f l u e n t A s t r o c y t e c u l t u r e s f r o m non-serum T a b u c h i e t a l . (1981) T a r d y e t a l (1981) T a b u c h i e t a l , (1981) F i s c h e r e t a l . c o n t a i n i n g medium b e g i n t o e x p r e s s GFAP (1982) P r i m a r y a s t r o c y t e c u l t u r e s R e t r a c t i o n o f c e l l soma; e x t e n s i o n Hansson & Ronnback o f c e l l p r o c e s s e s ; d e c r e a s e d 3H v a l i n e (1983) i n c o r p o r a t i o n i n t o p r o t e i n ; d e c r e a s e d t o t a l s o l u a b l e p r o t e i n ; d e c r e a s e d p r o t e i n s e c r e t i o n ( a l l r e t u r n e d b y s o l u b l e b r a i n e x t r a c t s ) I n c r e a s e d g l u t a m a t e d e h y d r o g e n a s e and GABA-T (as i n aged mice) Schousboe e t a l , (1980a) COMT and MAO i n c r e a s e d I n c r e a s e d Na+,K+,ATPase and GS T h r o u g h cAMP Hansson & S e l l s t r o m (1983) Schousboe e t a l . (1980) I n c r e a s e d GABA-T and a s p a r t a t e amino- T a r d y e t a l . t r a n s f e r a s e ; same m o r p h o l o g i c a l c h a n g es (1981) a s w i t h GFAP G l i a m a t u r a t i o n S u r f a c e G l i o b l a s t s f a c t o r r e c e p t o r A s t r o g l i a l m a t u r a t i o n Lim (1977) I t o e t a l . (1982) C y t o s i n e M i t o t i c C e r e b e l l a r A s t r o g l i a l m a t u r a t i o n a r a b i n o s i d e i n h i b i t o r n e u r o n a l c u l t u r e s Leu e t a l . (1983) C) C e l l d e v e l o p m e n t and d i f f e r e n t i a t i o n i n r e s p o n s e t o i n j u r y A n o t h e r k i n d o f c e l l d i f f e r e n t i a t i o n o c c u r s i n r e s p o n s e t o i n j u r y . The t y p e o f r e s p o n s e t o some e x t e n t i s v a r i a b l e , d e p e n d i n g on t h e t y p e o f i n j u r y , t h e age o f t h e a n i m a l and t h e l o c a t i o n o f t h e i n j u r y . I n g e n e r a l a s t r o c y t e s i n c r e a s e i n number, i n s i z e and i n number o f p r o c e s s e s i n r e s p o n s e t o i n j u r y t o become r e a c t i v e a s t r o c y t e s . A l l o x i d o r e d u c t i v e enzymes become more a c t i v e , as do most o t h e r enzymes (Oehmichen, 1 9 8 0 ) . The i n c r e a s e o c c u r s e a r l i e r f o r t h o s e enzymes i n v o l v e d i n g l y c o l y s i s o r t h e h e x o s e monophosphate s h u n t t h a n f o r t h o s e o f t h e c i t r i c a c i d c y c l e , s u c h a s s u c c i n i c d e h y d r o g e n a s e ( F r i e d e , 1966, Oehmichen, 1 9 8 0 ) . The enzyme i n c r e a s e s a r e p e r m a n e n t a s t h e y p e r s i s t e v e n i n o l d s c a r s . M o r p h o l o g i c a l c h a n g e s i n a s t r o c y t e s i n r e s p o n s e t o i s c h e m i c i n j u r y were e x a m i n e d ( P e t i t o and B a b i a k , 1982) and f o u n d t o o c c u r w i t h i n 4 0 m i n s . a f t e r i n j u r y . T h e s e c h a n g e s c o n s i s t e d o f e x p a n s i o n and i n c r e a s e d number o f m i t o c h o n d r i a , c y t o p l a s m and r o u g h ER, s u g g e s t i n g i n c r e a s e d m e t a b o l i c a c t i v i t y . The number o f a s t r o c y t i c n u c l e i a l s o i n c r e a s e d v e r y e a r l y . Murabe e t a l . (1981) f o u n d t h a t o n l y a s t r o c y t e s c h a n g e d m o r p h o l o g y i n r e s p o n s e t o k a i n i c a c i d - i n d u c e d damage i n t h e h i p p o c a m p u s . They f i r s t s w e l l e d , t h e n f i l a m e n t s d e v e l o p e d . P o l y n u c l e a r a s t r o c y t e s e x t e n d e d p r o c e s s e s i n a r e a s v a c a t e d by t h e n e u r o n s . A s t r o c y t e s a p p e a r e d t o h a v e p h a g o c y t i c a c t i v i t y . - 61 - Primary cultures derived from ka i n i c acid lesioned r at striatum lead to 2 morphologically distinguishable c e l l types (Van Alstyne et a l . , 1983). They were mainly (95%) composed of large f l a t c e l l s with i l l defined junctions and no c e l l u l a r processes but 5% of the c e l l s were small with processes. Upon treatment with dBcAMP, the large immature c e l l s transform to the smaller type. These newly derived smaller c e l l s exhibit c e l l - s p e c i f i c markers (galactocerbroside on 10% and GFAP on 80%), plus some f e t a l c h a r a c t e r i s t i c s . Therefore the larger c e l l s were g l i o b l a s t s that were i n the k a i n i c acid damaged ti s s u e . Freide (1966) showed that the oxidoreductive enzymes i n oligodendrocytes are more active than i n r e s t i n g astrocytes but they increase to above the oligodendrocytic l e v e l i n reactive astrocytes. Although oligodendrocytes do not p r o l i f e r a t e , they do grow and t h e i r oxidoreductase enzymes do become more active i n response to trauma (Ibrahim et a l . , 1974). Colmant (1965) noticed increases i n acid phosphatases, DPNH- and TPNH- tetrazolium reductases, succinate dehydrogenase, 5 'nucleotidase, phosphamidase, and /5-naphthol esterase. I f too much damage occurs, the oligodendrocytes w i l l die. Even with knowing that there are several sources of v a r i a t i o n between c e l l s that can explain much observed heterogeneity, there i s s t i l l heterogeneity that does not seen to be due to these variables. C e l l types i n vivo and i n culture are found to have a number of biochemical differences - 62 - that remain unexplained. I f there are biochemical differences i n g l i a they must subserve some differences i n function. D) Heterogeneity between d i f f e r e n t g l i a not explained by development or culture conditions Different g l i a l systems even have d i f f e r e n t c e l l u l a r d e n s i t i e s depending on t h e i r o r i g i n (Henn, 1980), and u l t r a s t r u c t u r a l heterogeneity has been described (Mori and Lebond, 1970). Schachner et a l . (1977) found that GFAP was located i n var i a b l e places i n astrocytes of the mouse cerebellum. The la b e l was found i n c e l l s around the glomerular complexes i n the granular layer, i n r a d i a l f i b e r s i n the molecular layer, i n the sheath surrounding Purkinje c e l l s , and i n a s t r o c y t i c end feet impinging on meninges and blood vessels; i n white matter c e l l bodies there was d i f f u s e cytoplasmic l a b e l and elongated strings of l a b e l . Mize et a l . (1981) observed t r i t i a t e d GABA l a b e l l i n g i n the superior c o l l i c u l u s of cats and noted that dark oligodendrocytes and astrocytes accumulated GABA moderately, while l i g h t oligodendrocytes and microglia did not. The dark oligodendrocytes wrap around presynaptic terminals and are therefore l i k e l y candidates for the removal of GABA. Hosli and H o s l i (1978) observed t h i s b a r r i e r function working i n the cultured g l i a l c e l l s of dorsal root ganglia. In mixed cultures the g l i a , not the neurons, would take up GABA, but, i f the neurons were i s o l a t e d they took GABA up better than the g l i a l c e l l s . This was interpreted as meaning that the g l i a l - 63 - c e l l s n o r m a l l y a c t as a b u f f e r zone, f o r m i n g a b a r r i e r w h i c h p r e v e n t s t h e u p t a k e i n t o n e u r o n s . L e v i e t a l . (1983) c o r r e l a t e d t h e m o r p h o l o g y e x p r e s s e d by a s t r o g l i a l c e l l s i n p o s t - n a t a l c e r e b e l l a r , i n t e r n e u r o n - e n r i c h e d p r i m a r y c u l t u r e s w i t h t h e a b i l i t y o f t h e s e c e l l s t o a c c u m u l a t e p u t a t i v e amino a c i d n e u r o t r a n s m i t t e r s . The c u l t u r e s were o r i g i n a l l y composed m o s t l y o f u n d i f f e r e n t i a t e d G F A P - c o n t a i n i n g c e l l s , b u t , d u r i n g t h e n e x t 12 d a y s , t h e number o f s t e l l a t e a s t r o c y t e s i n c r e a s e d t o be 70-80% o f t h e a s t r o c y t e s p r e s e n t and t h e y were l a r g e r . L e v i e t a l . n o t e d t h a t a s p a r t a t e a c c u m u l a t e d , a s shown b y 3 H - D - a s p a r t a t e a u t o r a d i o g r a p h y , i n t o t h e u n d i f f e r e n t i a t e d G F A P - c o n t a i n i n g c e l l s b u t t h a t 3H-GABA was a c c u m u l a t e d i n s u b s t a n t i a l amounts by t h e s t e l l a t e a s t r o c y t e s . E a r l y a s t r o c y t e s o f o t h e r s h a p e s s t a i n e d o n l y l i g h t l y f o r GABA, and, e v e n w i t h i n t h e s t e l l a t e p o p u l a t i o n t h e e x t e n t o f GABA l a b e l i n g was v a r i a b l e f r o m one c e l l t o a n o t h e r . A u t o r a d i o g r a p h i c e x a m i n a t i o n s and d e t e r m i n a t i o n s o f t h e I C 50s f o r GABA u p t a k e i n h i b i t o r s c o n s i s t e n t l y i n d i c a t e d t h a t t h e GABA t r a n s p o r t s y s t e m s p r e s e n t i n s t e l l a t e a s t r o c y t e s d i d n o t h a v e t h e f e a t u r e s g e n e r a l l y a t t r i b u t e d t o a g l i a l t r a n s p o r t s y s t e m b u t i n s t e a d m a t c h e d t h a t o f t h e i n h i b i t o r y i n t e r n e u r o n s p r e s e n t i n t h e c u l t u r e . T h e y n o t i c e d i n n e u r o n - e n r i c h e d c u l t u r e s t h a t a s t r o c y t e s may l o s e t h e i r a b i l i t y t o t a k e up GABA as c u l t u r e s grow o l d e r , e v e n t h o u g h t h e s t e l l a t e m o r p h o l o g y i s m a i n t a i n e d . F r e i d e (1966) n o t e d t h a t n o t a l l o l i g o d e n d r o c y t e s h a v e t h e same enzyme a c t i v i t y . S a t e l l i t e c e l l s , f o r example, have a marked c y t o c h r o m e o x i d a s e a c t i v i t y w h i c h i s n o t o b s e r v e d i n - 64 - o t h e r o l i g o d e n d r o c y t e s . S z u c h e t and Yim (1984) f o u n d an o l i g o d e n d r o c y t e l i n e t h e y d e s i g n a t e d B 3 , f , w h i c h was m o r p h o l o g i c a l l y homogeneous i n c u l t u r e , b u t h a d a n t i - m y e l i n a s s o c i a t e d g l y c o p r o t e i n g a l a c t o c e r e b r o s i a l s t a i n i n g t h a t v a r i e d f r o m weak t o s t r o n g b e t w e e n c e l l s . Some o f t h e d i f f e r e n c e s r e p o r t e d i n v a r i o u s a s t r o c y t e c u l t u r e s may be due t o i n t e r s p e c i e s d i f f e r e n c e s . T h e s e have b e e n shown i n a few s y s t e m s . Low r a t e s o f p o t a s s i u m u p t a k e were o b s e r v e d i n y o u n g r a t a s t r o c y t e s ( K i m e l b e r g , 1 9 7 9 ) . The r a t e was h i g h e r i n p r i m a r y c u l t u r e s o f c h i c k a s t r o c y t e s , where i t was a l m o s t t o t a l l y i n h i b i t e d b y o u a b a i n ( L a t z o v i t s , 1 9 7 8 ) . Mouse b r a i n a s t r o c y t e s h a d a much h i g h e r r a t e ( H e r t z , 1978d). T h i s l a s t o b s e r v a t i o n seems t o be a t r u e s p e c i e s v a r i a t i o n s i n c e t h e e x p e r i m e n t s were done i n t h e same l a b o r a t o r y . H e t e r o g e n e i t y b e t w e e n and w i t h i n g l i a l c e l l l i n e s T h e r e i s a v e r y e x t e n s i v e body o f r e s e a r c h on d i f f e r e n c e s b e t w e e n d i f f e r e n t g l i a l c e l l l i n e s , b u t t h e r e s e a r c h i s n o t w i t h o u t i t s p r o b l e m s . The g l i a l c e l l l i n e f i r s t p r o d u c e d was C-6 (Benda, 1968). Now t h e r e a r e many t y p e s o f g l i a l c e l l l i n e s f r e q u e n t l y s t u d i e d . T h e y show c h a r a c t e r i s t i c s t h a t a r e b e l i e v e d t o some e x t e n t t o r e s e m b l e n o r m a l g l i a . T h e s e e s t a b l i s h e d c e l l l i n e s h a v e s e v e r a l a d v a n t a g e s : t h e y a r e r e a d i l y a v a i l a b l e , r e l a t i v e l y e a s y t o m a i n t a i n f o r l o n g p e r i o d s o f t i m e and, b e c a u s e t h e i r c h a r a c t e r i s t i c s a r e r e l a t i v e l y s t a b l e , t h e y c a n be compared between l a b o r a t o r i e s . However, b e c a u s e t h e y were - 65 - o r i g i n a l l y t r a n s f o r m e d b y c h e m i c a l s o r v i r u s e s , t h e i r c h a r a c t e r i s t i c s a r e n o t e n t i r e l y l i k e g l i a f o u n d i n n o r m a l b r a i n . T h e y may a c t u a l l y be q u i t e d i f f e r e n t . F o r example, t h e m e t a b o l i c r a t e o f g l i a l c e l l s was o r i g i n a l l y b e l i e v e d t o be q u i t e low, b a s e d on e a r l y work done on e a r l y g l i a l c e l l l i n e s and g l i a l s c a r t i s s u e t h a t g a v e e r r o n o u s l y low m e t a b o l i c r a t e s f o r g l i a ( H e r t z , 1 978b). They may h a v e c h a r a c t e r i s t i c s o f two o r more t y p e s o f g l i a c e l l o r may e v e n h a v e n e u r o n a l c h a r a c t e r i s t i c s . F o r example, g l u t a m a t e i s t r a n s p o r t e d i n t o a l a r g e number o f g l i a l c e l l l i n e s w h i c h h a v e a h i g h a f f i n i t y u p t a k e s i m i l a r t o t h a t s e e n i n n e u r o n s (Edwards e t a l . , 1979) b u t n o t i n p r i m a r y c u l t u r e s o f g l i a . S u c h f i n d i n g s mean t h a t e x t r a p o l a t i o n c a n n o t be made f r o m g l i a l c e l l l i n e s t o n o r m a l g l i a w i t h o u t c o r r o b o r a t i v e e v i d e n c e . T h e y r e m a i n , however, u s e f u l t o o l s f o r p r e l i m i n a r y r e s e a r c h b e c a u s e o f t h e i r e a s e o f u s e . G l i a l c e l l l i n e s a r e d e f i n e d a s g l i a b e c a u s e o f m a r k e r s o r o t h e r c h a r c t e r i s t i c s t h e y s h a r e w i t h g l i a . T h e y c a n sometimes be e a s i l y d i s t i n g u i s h e d f r o m n e u r o n a l c e l l l i n e s b u t t h e s e d i s t i n c t i o n s a r e n o t a l w a y s c l e a r . S p e c i f i c a n t i g e n s s u c h as NS-1 ( S c h a c h n e r , 1974), GI and G2 ( S t a l l c u p and Cohn, 1976) a r e c o n s i d e r e d g l i a l s u r f a c e m a r k e r s b e c a u s e t h e y a r e on t h e s u r f a c e o f g l i a l tumor l i n e s b u t n o t n e u r o n a l tumor l i n e s and t h u s a r e u s e d i n d e f i n i n g new g l i a l l i n e s . S h i n e e t a l . (1981) f o u n d more ^ - g l u t a m y l t r a n s p e p t i d a s e i n g l i a l c e l l l i n e s t h a n i n n e u r a l o n e s . W i l s o n e t a l . (1981) worked e x t e n s i v e l y t o d e f i n e g l i a l a nd n e u r o n a l c e l l l i n e s . He u s e d a n t i s e r a a g a i n s t - 66 - p s e u d o n e u r o n a l and p s e u d o g l i a l c e l l l i n e s t o d e f i n e t h e r e l a t i o n s h i p b etween t h e c l a s s i c c e l l l i n e s and b e t w e e n e a c h o t h e r . F o r example, t h e N4 a n t i g e n was e x p r e s s e d b y t h e p s e u d o n e u r o n a l c e l l l i n e s and b y 7/10 n e u r o n a l l i n e s . P s e u d o n e u r o n a l and n e u r o n a l l i n e s were f u r t h e r r e l a t e d b y t h e f i n d i n g o f s i m i l a r Na+ and K+ c h a n n e l s . On t h e o t h e r hand, p s e u d o n e u r o n a l c e l l l i n e s and p s e u d o g l i a l c e l l l i n e s were f o u n d t o be r e l a t e d b e c a u s e b o t h p o s s e s s a n t i g e n s c a l l e d NG1 a n d NG2. W i l s o n e t a l . c o n c l u d e d t h a t t h e r e must be d e v e l o p m e n t a l l i n k a g e s b e t w e e n n e u r o n a l and g l i a l c e l l l i n e s . O s b o r n e t a l . (1981) f o u n d t h a t g l i a l l i n e s d i f f e r e d f r o m e a c h o t h e r and f r o m p r i m a r y a s t r o c y t e c u l t u r e s i n e x p r e s s i o n o f GFAP. C u l t u r e s o f n o r m a l b i o p s i e d human g l i a l m a t e r i a l showed no GFAP+ a f t e r s e v e n d o u b l i n g s b u t t h e g l i a l l i n e U251 MG showed 3% a n d U333CG/343 MG 98% GFAP+ c e l l s . O s b o r n e t a l . f o u n d t h e d i f f e r e n c e b etween p r i m a r y c u l t u r e s and g l i a l c e l l l i n e s seemed p e r m a n e n t a s i t d i d n o t r e v e r s e i n r e s p o n s e t o dBcAMP. T h i s d i f f e r e n c e may be b e c a u s e o f c h a n g e s i n t h e g e n e t i c m a r k e r f o r GFAP i n t r a n s f o r m e d c e l l s o r b e c a u s e t h e r e was a s u b p o p u l a t i o n o f c e l l s c o n t a i n i n g d i f f e r e n t g e n e t i c m a t e r i a l t h a t t h r i v e d i n c u l t u r e . The b a s i c b i o c h e m i c a l l e v e l o f f u n c t i o n i n g i n most g l i o m a c e l l l i n e s i s l o w e r t h a n i n p r i m a r y c u l t u r e s o f g l i a . The C-6 l i n e ( K i m e l b e r g , 1974) and o t h e r g l i o m a c e l l l i n e s ( H e r t z , 1977) , f o r example, h a v e b e e n f o u n d t o h a v e a l o w e r Na+,KH—ATPase a c t i v i t y and a l o w e r t h a n N e r n s t i a n s l o p e f o r p o t a s s i u m u p t a k e t h a n p r i m a r y c u l t u r e s o f g l i a l c e l l s f r o m t h e c e r e b e l l u m w h i c h show a c l a s s i c N e r n s t i a n s l o p e (Sugaya e t - 67 - a l . , 1 9 7 9 ) . NN c e l l s a r e f o u n d t o be l e s s r e s p o n s i v e t o K+ s t i m u l a t i o n ( C i e s i e l s k i - T r e s k a , 1976) t h a n p r i m a r y c u l t u r e s . G l i o m a c e l l l i n e s a l s o h a v e a much l o w e r l i p i d c o n t e n t t h a n b u l k - s e p a r a t e d a s t r o g l i a ( N o r t o n e t a l . , 1 9 7 5 ) . N o t a l l s y s t e m s f u n c t i o n a t a l o w e r l e v e l , however; f o r example C-6 c e l l s h a v e a h i g h e r g l y c o l y t i c r a t e t h a n p r i m a r y a s t r o c y t e c u l t u r e s a s shown by t h e h i g h e r i n c o r p o r a t i o n o f g l u c o s e i n t o l a c t a t e . T h e i r a b i l i t y t o m a i n t a i n a h i g h e r amount o f ATP i n t h e a b s e n c e o f o x y g e n may be r e l a t e d t o t h e i r h i g h e r g l y c o l y t i c r a t e ( P a s s o n n e a u e t a l . , 1 9 7 8 ) . C a r b o n i c a n h y d r a s e seems t o be e n r i c h e d i n a s t r o g l i a ( R o u s s e l e t a l . , 1979, K i m e l b e r g e t a l . , 1 9 7 8 b ) , b u t i t i s n o t i n C-6 c e l l s ( D e V e l l i s and B r o o k e r , 1 9 7 3 ) . V a r i o u s c e l l l i n e s a r e f o u n d t o h a v e d i f f e r e n t l e v e l s o f MAO and p r o p o r t i o n s o f t y p e s . A s t r o c y t e s p o s s e s s h i g h e r MAO a c t i v i t i e s t h a n b r a i n , and a c t i v i t i e s i n C-6 c e l l s (Murphy e t a l . , 1976) a r e e v e n h i g h e r . T h i s enzyme e x i s t s i n two f o r m s : C-6 a n d most o t h e r g l i a l c e l l l i n e s c o n t a i n o n l y f o r m A (Haber and H u t c h i s o n , 1976) b u t a s t r o c y t e s i n p r i m a r y c u l t u r e c o n t a i n b o t h , e s p e c i a l l y a f t e r e x p o s u r e t o dBucAMP ( H e r t z , 1 982). E v i d e n c e f o r c o m p a r t m e n t a t i o n o f g l u t a m a t e m e t a b o l i s m i s n o t a s s t r o n g i n g l i a l c e l l l i n e s as i n p r i m a r y c u l t u r e s . C-6 d i d n o t seem t o show e v i d e n c e o f c o m p a r t m e n t a l i z a t i o n i n p r o p e r t i e s s u c h a s GS a c t i v i t y b u t b u l k i s o l a t e d g l i a l c e l l s d i d . H i g h a f f i n i t y u p t a k e o f g l u t a m a t e h a s b e e n d e m o n s t r a t e d a u t o r a d i o g r a p h i c a l l y i n g l i a l c e l l l i n e s ( F a i v r e - B a u m a n n e t a l . , 1974, Henn e t a l . , 1974, B a l c a r e t a l . , 1977, P f e i f f e r e t - 68 - a l . , 1 9 7 6 ) , i n c l u d i n g a s t r o c y t o m a ( S n o d g r a s s and I v e r s e n , 1 9 7 4 ) . T h i s g l u t a m a t e u p t a k e h a s b e e n shown t o be s e n s i t i v e t o Na+ s t i m u l a t i o n i n C-6 c e l l s (Henn, 1 9 7 5 ) . The u p t a k e s y s t e m may be d i f f e r e n t f r o m t h a t i n p r i m a r y c u l t u r e s . C a l c u i m i s n o t r e q u i r e d f o r g l u t a m a t e u p t a k e i n t o p r i m a r y a s t r o c y t e s c u l t u r e s ( S c h o u s b o e e t a l . , 1977b) o r t h e NN g l i a l c e l l l i n e ( B a l c a r e t a l . , 1977) b u t i s f o r u p t a k e i n t o C-6 g l i o m a c e l l s ( F a i v e - B a u m a n e t a l . , 1 9 7 4 ) . T a b l e I V i n d i c a t e s t h a t t h e Vmax v a l u e s f o r g l u t a m a t e u p t a k e a r e g e n e r a l l y h i g h e r f o r p r i m a r y c u l t u r e s o f a s t r o c y t e s t h a n f o r u p t a k e i n t o a s t r o c y t e s p r e p a r e d b y g r a d i e n t c e n t r i f u g a t i o n , o r g l i a l c e l l l i n e s . - 69 - T a b l e IV: C o m p a r a t i v e V a l u e s o f G l u t a m a t e U p t a k e ( f r o m H e r t z , 1979) C e l l D e s c r i p t i o n Km(^M) Vmax* R e f e r e n c e A s t r o c y t e s i n p r i m a r y c u l t u r e 22 0 0.8 A " II II II 50 5.9 B " " " 11 30-90 3-7.5 C " " 11 11 10-20 0.4-0.6 D C-6 g l i o m a 15 0.4 E " " 66 F NN g l i o m a c e l l s 14 0.07 G " 11 " 12-19 0.02-0.03 H MGM-LM g l i o m a c e l l s 2 0 0.3 I 138 MG g l i o m a c e l l s 65 0.14 J B u l k p r e p a r e d a s t r o c y t e s 12 F " 11 » 12 K " " " 10 0.06 L B u l k p r e p a r e d c e r e b e l l a r a s t r o c y t e s 15 0.2 M R e t i n a 21 3.5 N * ( pi m o l /min p e r g p r o t e i n ) 2 R e f e r e n c e c o d e s A = S c h o u s b o e e t a l . , 1977b, B = H e r t z e t a l . , 1978b, C = H e r t z e t a l . , 1979b, D = B a l c a r and H a u s e r , 1978, E = F a i v r e - B a u m a n e t a l . , 1974, F = Henn e t a l . , 1974, G = B a l c a r e t a l . , 1977, H = B a l c a r e t a l . , 1978, I = S t e w a r t e t a l . , 1976, J = Walum and W e i l e r , 1978, K = Henn, 1976, L = W e i l e r e t a l . , 1979, M = L e C a m p e l l and Shank, 1978, N = W h i t e and N e a l , 1976, 0 = S c h o u s b o e e t a l . , 1979, P = Henn and Hamberger, 1971, Q = L a s h e r , 1975, R = S c h r i e r and Thompson, 1974, S = H u t c h i s o n e t a l . , 1974, T = S c h o u s b o e e t a l . , 1977a. - 70 - B a e t g e e t a l . (1979) a l s o r e v i e w e d t h e r e s e a r c h on a w i d e v a r i e t y o f g l i a l c e l l l i n e s and f o u n d d i f f e r e n t u p t a k e r a t e s f o r g l u t a m a t e . Two g l i a l c e l l l i n e s , B28 and BE11, h a d v e r y h i g h g l u t a m a t e u p t a k e r a t e s and a n o t h e r two, B15 and B i l l , h ad o n l y m o d e r a t e l y h i g h u p t a k e ; some g l u t a m a t e u p t a k e , however, o c c u r r e d i n most o f t h e o t h e r c e l l l i n e s . E v e n t h o u g h t h e i r u p t a k e r a t e s v a r i e d , t h e b a s i c mechanism d i d n o t seem t o v a r y . T h ey h a d t h e same s p e c i f i c i t y and were c o u p l e d t o Na+ i n i d e n t i c a l ways and t h e Km was t h e same. S c h o u s b o e (1978b) f o u n d d i f f e r i n g g l u t a m a t e Km's between c e l l l i n e s a n d p r i m a r y c u l t u r e s (12 0 mM f o r NN c e l l s and 18 mM i n p r i m a r y c u l t u r e s ) and t h e i n f l u e n c e o f Na+ on t h e u p t a k e o f g l u t a m a t e d i f f e r e d f r o m c e l l l i n e t o c e l l l i n e . He a l s o f o u n d t h a t t h e u p t a k e was Ca++-dependent i n C-6 and some o t h e r g l i o m a l i n e s , b u t n o t i n NN, p r i m a r y c u l t u r e s o f a s t r o c y t e s o r b u l k p r e p a r e d g l i a l c e l l s . H e r t z (1979) a l s o e x a m i n e d g l u t a m i n e u p t a k e i n t o v a r i o u s p r e p a r a t i o n s . A g a i n t h e k i n e t i c c o n s t a n t s v a r i e d f r o m p r e p a r a t i o n t o p r e p a r a t i o n b u t t h e g l i o m a l i n e was a b o u t t h e a v e r a g e o f t h e n o r m a l l i n e s as i n d i c a t e d i n T a b l e V. - 71 - T a b l e V: C o m p a r a t i v e v a l u e s o f g l u t a m i n e u p t a k e i n t o d i f f e r e n t g l i a l p r e p a r a t i o n s C e l l T y p e Km(^M) Vmax* R e f e r e n c e B u l k - p r e p a r e d a s t r o c y t e s 63 0 0.16 L A s t r o c y t e s i n p r i m a r y c u l t u r e 3 3 00 5.0 0 " " " " 150 0.2 D D138 MG g l i o m a c e l l l i n e 490 2.9 J *(amol/min p e r 100 mg p r o t e i n ( f o r r e f e r e n c e s s e e T a b l e IV) GABA u p t a k e a l s o v a r i e s b e t w e e n n o r m a l g l i a a nd t r a n f o r m e d g l i a l c e l l l i n e s . Henn (1975) f o u n d C-6 c e l l s t o h a v e a h i g h a f f i n i t y u p t a k e f o r GABA w h i c h was Na+ s e n s i t i v e . S c h o u s b o e (1981) compared t h e work o f many o t h e r s and f o u n d t h a t c u l t u r e d a s t r o c y t e s e x h i b i t a Vmax c o m p a r a b l e t o t h a t f o u n d i n b r a i n s l i c e s and i n n e u r o n s d e r i v e d f r o m t h e c e r e b e l l u m b u t t h a t C-6 c e l l s h a d a much l o w e r c a p a c i t y t h o u g h i t was s t i l l h i g h a f f i n i t y u p t a k e . H e r t z (1979) r e v i e w e d much o f t h e l i t e r a t u r e ( T a b l e VI) and s i m i l a r l y c o n c l u d e d t h e c a p a c i t y o f C-6 was l o w e r t h a n t h a t o f a s t r o c y t e s i n c u l t u r e . - 72 - T a b l e V I : C o m p a r a t i v e V a l u e s o f h i g h a f f i n i t y u p t a k e o f GABA i n t o v a r i o u s g l i a l p r e p a r a t i o n s C e l l T y p e Km(>JM) Vmax* R e f e r e n c e s B u l k p r e p a r e d a s t r o c y t e s 0.27 P " " " 0.6 K C u l t u r e d c e r e b e l l a r g l i a 0.29 0.0001-0.0002 Q C-6 g l i o m a 32 0.002 R " " 0.22 0.0001 S " " 50 K C u l t u r e d c e r e b r a l a s t r o c y t e s 40 0.035 T " " 11 45 0.040 B * |Jmol/min p e r g wet w e i g h t ( f o r r e f e r e n c e s s e e T a b l e IV) C-6 a l s o h a v e a v e r y low a c t i v i t y o f GABA-T compared t o c u l t u r e d a s t r o c y t e s and b u l k p r e p a r e d g l i a ( N i c k l a s and B r o w n i n g , 1977) and much l o w e r a c t i v i t y t h a n f o u n d i n t h e b r a i n o f m i c e o f s i m i l a r age ( N i c k l a s and B r o w n i n g , 1 9 7 8 ) . D i f f e r e n t c e l l l i n e s c a n a l s o show s t r u c t u r a l d i f f e r e n c e s . P i l k i n g t o n e t a l . (1982) showed t h a t 3 c e l l l i n e s d e r i v e d f r o m a s p o n t a n e o u s m u r i n e a s t r o c y t o m a d i f f e r e d i n t h e number and r a t i o o f 10 nm f i l a m e n t s and 234 nm m i c r o t u b u l e s and t h a t t h e s e d i f f e r e n c e s were r e l a t e d t o t h e d e g r e e o f d i f f e r e n t i a t i o n o f t h e c e l l l i n e . However, e v e n a g i v e n c e l l l i n e c a n v a r y m o r p h o l o g i c a l l y w i t h i n and b e t w e e n l o t s . Benda (1978) e v e n n o t e d t h a t C-6 c e l l s i n a s i n g l e p l a t e d i s p l a y d i f f e r e n c e s i n m o r p h o l o g y , p a t t e r n s o f c o l o n y f o r m a t i o n , and p a t t e r n s o f b i o c h e m i s t r y s u c h as a c c u m u l a t i o n o f S-lOOp. T h e s e c h a r a c t e r i s t i c s c a n be m a n i p u l a t e d b y serums, p l a t i n g - 73 - d e n s i t y and o t h e r f a c t o r s . F o r example, i n s e r u m - l e s s medium t h e a b i l i t y t o a c c u m u l a t e S-lOOp i s l o s t ( P f e i f f e r e t a l . , 1 9 7 0 ) . H i g h l e v e l s o f f e t a l c a l f serum b r i n g o u t a s e l e c t i v e i n c r e a s e i n amino a c i d u p t a k e and m o r p h o l o g i c a l c h a n g e s ( L o g a n , 1 9 7 6 ) . G l i a m a t u r a t i o n f a c t o r ( b o v i n e ) h a s more e f f e c t on n o r m a l c e l l s t h a n on C-6 tumor c e l l s and i t h a s t o be p r e s e n t w i t h i n a c r i t i c a l t i m e f a c t o r t h a t m a t c h e s t h e p e r i o d o f p o s t n a t a l g l i o g e n e s i s ( K a t o e t a l . , 1 9 8 1 ) . T h e r e a r e s i t u a t i o n s where t h e r e a p p e a r s t o be s p o n t a n e o u s d i f f e r e n t i a t i o n u n d e r e x a c t l y t h e same c u l t u r e c o n d i t i o n s o f c e l l l i n e s w h i c h d i f f e r b i o c h e m i c a l l y and m o r p h o l o g i c i a l l y . F o r example, t h r e e d i s t i n c t t y p e s o f a s t r o c y t i c c e l l c l o n e s came o u t o f e s t a b l i s h e d c u l t u r e s o f 8-day p o s t n a t a l mouse c e r e b e l l a ( A l l i o t and P r e s s a c , 1 9 8 4 ) . T h e y were a l l GFAP+ b u t d i f f e r e d m o r p h o l o g i c a l l y . T ype 1 h a d s m a l l somata, s e v e r a l s h o r t p r o c e s s e s , were p s e u d o d i p l o i d and were t h o u g h t t o r e s e m b l e f i b r o u s a s t r o c y t e s . T ype 2 bound m o n o c l o n a l a n t i b o d i e s BSP-3, M2 and M3, and h a d s m a l l somata, w i t h two p r o c e s e s , one o f w h i c h was l o n g and t h i n . T h e y were t h o u g h t t o r e s e m b l e G o l g i e p i t h e l i a l c e l l s . T y p e 3 h a d l a r g e f l a t s omata, no p r o c e s s e s , were h e t e r o d i p l o i d , and were t h o u g h t t o r e s e m b l e f i l a m e n t o u s a s t r o c y t e s . T h e s e c h a r a c t e r i s t i c s were a l l s t a b l e i n c u l t u r e o v e r t i m e and t h u s r e p r e s e n t t r u e d i f f e r e n t i a t i o n . C e l l l i n e s c a n d i f f e r f r o m e a c h o t h e r i n b a s i c b i o c h e m i s t r y . F o r example, C-6 h a s h i g h e r l e v e l s o f S-lOOp t h a n do n e u r o n s , and h a s an Na+,K+ pumping a c t i o n s i m i l a r t o many n e u r o n s b u t h i g h e r t h a n o t h e r g l i a l c e l l l i n e s . A n o t h e r - 74 - example was f o u n d by S h i n e e t a l . ( 1 9 8 1 ) . T h e y showed t h a t y - g l u t a m y l t r a n s p e p t i d a s e , w h i c h i s t h o u g h t t o be i n v o l v e d i n t h e t r a n s p o r t o f amino a c i d s a c r o s s membranes, i n t h e a c t i v a t i o n o f b i o p e p t i d e s and i n t h e d e t o x i f i c a t i o n o f v a r i o u s s u b s t a n c e s , h a s a t r e m e n d o u s v a r i a t i o n b e t w e e n g l i a l c e l l l i n e s . I t i s h i g h e s t i n C-6 and l o w e s t i n human A1B1. C e l l l i n e s d i f f e r i n v a r i o u s t r a n s m i t t e r s y s t e m s as w e l l . AChE a c t i v i t y c a n be f o u n d i n o n l y c e r t a i n c l o n a l l i n e s o f g l i a l c e l l s s u c h a s C-6 ( V e r n a d a k i s and A r n o l d , 1 9 8 0 ) . C e r t a i n c e l l c l o n e s e x i s t t h a t a r e p a r t i c u l a r l y h i g h i n one o r a n o t h e r p u t a t i v e amino a c i d t r a n s m i t t e r . C a m b i e r e t a l . (1983) c r e a t e d a g l y c i n e - e n r i c h e d a s t r o c y t e s c l o n e , K55, d e r i v e d f r o m mouse c e r e b e l l a r a s t r o c y t e c u l t u r e s t r a n s f o r m e d by s i m i a n v i r u s - 4 0 . T h ey f o u n d t h a t a h i g h p e r c e n t a g e o f t h e a s t r o c y t i c c e l l c l o n e s , d e r i v e d f r o m mouse c e r e b e l l a r c u l t u r e b y s i m i a n v i r u s - 4 0 o r b y s p o n t a n e o u s t r a n s f o r m a t i o n , c o n t a i n h i g h amounts o f g l y c i n e ( C a m b i e r and P e s s a c , 1 9 8 3 ) , w h i l e t h e o l i g o d e n d r o c y t e - l i k e c l o n e s were h i g h i n a l a n i n e . T h ey a l s o n o t e d t h a t a s t r o c y t i c c e l l c l o n e s u s e d g l u t a m i n e d i f f e r e n t l y t h a n d i d t h e o t h e r c e l l t y p e s ( C a m b i e r and P e s s a c , 1 9 8 3 ) . S c h o u s b o e (1978a) f o u n d t h a t C-6 a s t r o c y t o m a and p r i m a r y c u l t u r e s o f a s t r o c y t e s h a v e a h i g h c a p a c i t y f o r t a u r i n e u p t a k e w h i l e t h a t i n t h e NN l i n e was l o w e r . Drummond and P h i l l i p s (1977) f o u n d d i f f e r e n c e s i n amino a c i d l e v e l s i n d i f f e r e n t c e l l l i n e s w h i c h were n o t w e l l c o r r e l a t e d w i t h t h e c e l l c l a s s . The amino a c i d l e v e l s were d e p e n d e n t on t i s s u e c u l t u r e c o n d i t i o n s b u t , i f t h e s e c o n d i t i o n s were c a r e f u l l y c o n t r o l l e d , some s t a t i s t i c a l l y - 75 - s i g n i f i c a n t differences were s t i l l found. GABA l e v e l s were found to be p a r t i c u l a r l y high i n both C-6 and B92 g l i a l l i n e s . Glutamate l e v e l s i n various c e l l l i n e s varied between 50.8 to 158 nmol/mg protein and glutamine l e v e l s from 0.8 to 107 nmol/mg protein. S t a t i s t i c a l l y s i g n i f i c a n t differences were also observed f o r aspartate, proline, glycine, alanine, v a l i n e , cystathionine, isoleucine, and leucine. The uptake of amino acids by these d i f f e r e n t clones does not necessarily vary i n the same way as the l e v e l s . Schier and Thompson (1974) examined uptake of putative neurotransmitters by three cultured g l i a l c e l l l i n e s . The c e l l l i n e s exhibited s i m i l a r rapid uptake of glutamate and Na+ dependent uptake of GABA, as well as pyridoxal-dependent GABA synthesis and excretion. Taurine uptake occurred i n a l l three, with each showing a fas t saturable component and a slow non-saturable component which varied i n magnitude between the c e l l l i n e s . There was one c e l l l i n e which could maintain a high concentration gradient of taurine. Synthesis of taurine from cysteine was only found i n one of these l i n e s . G l i a l c e l l l i n e s may also respond d i f f e r e n t l y to drugs. Elkouby et a l . (1982) found that two g l i a l c e l l l i n e s , NN astrocytoma and C-6 glioma, responded d i f f e r e n t l y to the hormones hydrocortisone and thyroxine. The a c t i v i t y of Ca++,-Mg++ ATPase increased i n the NN l i n e but decreased i n C-6 i n response to these hormones. Another drug, dexametiasone, can be used to induce GS i n only a subset of C-6 c e l l s (Holbrook et a l . , 1981). Bigner et a l . (1981) examined various c h a r a c t e r i s t i c s of - 76 - f i f t e e n l i n e s of human c e l l s t r a d i t i o n a l l y thought of as being gliomas. They showed a wide v a r i e t y of human leukocyte antigen phenotypes. A l l but two, which were from a black patient, had type B glucose-6-phosphate dehydrogenase isoenzymes. Only four could be transplanted into athymic mice, two of which grew and then regressed. Only two were GFAP+. Thus each l i n e had a unique p r o f i l e . C e l l l i n e research therefore shows a v a r i e t y of types of heterogeneity. The s i g n i f i c a n c e i s unknown but there are a number of sources of variance that may explain some of the differences. F i r s t , being transformed c e l l s , they may be expressing some new genotype. Second, the transformed c e l l may be expressing d i f f e r e n t parts of the genotype than i s normally expressed by the parent c e l l r e s u l t i n g i n a mixing of c h a r a c t e r i s t i c s . Third, these various g l i a l types may be derived from d i f f e r e n t types of parent c e l l s and r e t a i n the differences. Different subtypes of the progenitor c e l l s may be r e l a t e d to variables we have already discussed or perhaps to the areas of the brain from which the c e l l came. Differences i n g l i a l c e l l s from d i f f e r e n t areas of the brain There has been a wide v a r i e t y of research that has shown regional heterogeneity i n g l i a l c e l l s . Much of the data were generated by people who did not set out to show differences between regions or are minor observations i n a paper on another t o p i c . There are probably many more examples buried i n the l i t e r a t u r e . Such differences have not been emphasized i n indices to the l i t e r a t u r e because i t was not u n t i l recently - 77 - that an i n t e r e s t i n t h i s subject developed . It has been known for a long time that the morphology of g l i a l c e l l s varies between d i f f e r e n t areas of the brain. Examples include g l i a l c e l l s that are s p e c i a l i z e d enough to have s p e c i f i c names, such as Bergmann g l i a , whose variance i n morphology has been previously discribed. In addition, there are areas of brain where g l i a l c e l l s appear morphologically d i f f e r e n t but have not been given s p e c i f i c names. Astrocytes of the hippocampus, for example, have a c h a r a c t e r i s t i c shape that i s d i f f e r e n t from that seen i n other areas. Astrocytes are known to have several d i f f e r e n t types of GFAP of d i f f e r e n t molecular weights and d i f f e r e n t s o l u b i l i t i e s i n water, with those of high molecular weights being the least water soluble (Eng, 1982). These forms are unevenly d i s t r i b u t e d i n the brain even though they are a l l c a r r i e d on the same gene (Gheuens et a l . , 1984). Since GFAP i s known to influence the shape of astrocytes, t h i s might be part of an explanation f o r some of the shape differences. GFAP varies not only i n structure but i n i t s schedule of appearance during development. Weir et a l . (1984) measured GFAP i n o l f a c t o r y bulbs, forebrain and cerebellum of rats during development, using a double antibody radioimmunoassay. Each brain region showed a d i f f e r e n t pattern of development for GFAP. At b i r t h , GFAP protein i n the o l f a c t o r y bulb was 85 times that i n forebrain, and 485 times that i n cerebellum. The increase i n GFAP corresponded with maturation more than p r o l i f e r a t i o n . The pattern of increase i n GS a c t i v i t y was s i m i l a r to that of GFAP i n the forebrain and o l f a c t o r y bulbs - 78 - but d i f f e r e d markedly i n the cerebellum. In the cerebellum the maximum increase i n GFAP occurred a f t e r the peak of a s t r o g l i a l p r o l i f e r a t i o n and 1 week before maximum acquis i t i o n of GS and S-100 protein. The d i s t r i b u t i o n of a s t r o g l i a l contacts on the surface of neurons varies greatly among brain areas as well as among d i f f e r e n t types of neurons (Guldner and Wolff, 1973, Peters and Palay, 1965, and Wolff, 1965). Neurons and synapses may even be wrapped d i f f e r e n t l y by several layers of g l i a l lamellae (Guldner and Wolff, 1973, Palay, 1966, Specek, 1968, and Szentagothai, 1970). Palay and Chan-Palay (1974) showed, for example, that Purkinje c e l l s are l a r g e l y covered by Bergmann g l i a i n contrast to cerebell a r interneurons which are not wrapped. Wolff and Guldner (1978) found that e l e c t r i c a l stimulation produced swelling of a s t r o c y t i c processes i n the neocortex. Since t h i s experimentally produced feature of c o r t i c a l astrocytes e x i s t s normally i n ce r t a i n other a s t r o g l i a l c e l l s i t i s suggested that v a r i a t i o n s of the structure and arrangement of a s t r o g l i a l processes between d i f f e r e n t brain regions may r e f l e c t neuronal a c t i v i t i e s . There i s , however, some evidence (discussed below) that these c h a r a c t e r i s t i c s are not j u s t responses to neuronal influences but are stable c h a r a c t e r i s t i c s of the g l i a i n various regions. There have been numerous observations of differences i n number of g l i a l c e l l s i n various brain areas. Szeligo and Leglond (1977) not only found differences i n numbers but also showed that handling or enriched environments caused increases - 79 - i n t h e numbers o f a s t r o c y t e s and o l i g o d e n d r o c y t e s i n o n l y c e r t a i n l a y e r s o f t h e c o r t e x and n o t i n o t h e r a r e a s , s u c h as t h e c o r p u s c a l l o s u m . Oehmichen (1980) r e p o r t e d t h a t a s t r o c y t e s a r e o b s e r v e d i n v a r y i n g d e n s i t i e s i n t h e CNS a n d t h a t t h e f u n c t i o n a l a c t i v i t y i n t h e r e s t i n g s t a t e i s q u a n t i t a t i v e l y d i f f e r e n t d e p e n d i n g on l o c a t i o n . F o r example, s t r o n g p h o s p h o r y l a s e a c t i v i t y h a s been f o u n d i n t h o s e a r e a s t h a t h a v e a t e n d e n c y t o a c c u m u l a t e g l y c o g e n ( M o s s a k o w s k i and P e n a r , 1972, Oehmichen, 1 9 8 0 ) . O t h e r s h a v e c o n f i r m e d t h e v a r i a b i l i t y f r o m a r e a t o a r e a o f g l y c o g e n s t o r a g e i n r a d i a l g l i a l c e l l s o f d e v e l o p i n g r a t b r a i n ( B r u c k n e r and B i e s o l d , 1 9 8 1 ) . N o t o n l y c a n g l y c o g e n s t o r a g e be s e e n i n d i f f e r e n t c o n c e n t r a t i o n i n v a r i o u s a r e a s b u t t h e r a t i o o f g l i a v s . n e u r o n a l i n c o r p o r a t i o n o f p r e c u r s o r s i n t o g l y c o c o n j u g a t e s v a r i e s f r o m a r e a t o a r e a . H i g h e r i n c o r p o r a t i o n l e v e l s were f o u n d i n t h e s u p r a o p t i c and a r c u a t e n u c l e u s a n d l o w e s t i n c e r e b e l l u m . O t h e r i n d i c e s o f g l i a l m e t a b o l i s m c a n a l s o v a r y . G l u c o s e u p t a k e v a r i e s w i d e l y f r o m a r e a t o a r e a . Thompson e t a l . (1980) showed t h a t c r e a t i n e k i n a s e BB i s o e n z y m e was l o c a l i z e d o n l y t o a s t r o c y t e s o f t h e w h i t e m a t t e r o f human c e r e b r u m . T h i s enzyme i s n o r m a l l y a s s o c i a t e d w i t h c e l l s t h a t h a v e h i g h a d e n o s i n e t r i p h o s p h a t e (ATP) r e g e n e r a t i n g c a p a b i l i t i e s s u c h as c e l l s i n v o l v e d i n t r a n s p o r t o r c o n t r a c t i l e s y s t e m s . T h e r e f o r e a s t r o c y t e s o f t h e w h i t e m a t t e r w o u l d a p p e a r t o h a v e s p e c i a l i z e d f u n c t i o n s . D e V e l l i s e t a l . (1967) f o u n d t h a t t h e r e i s a r e g i o n a l - 80 - d i f f e r e n c e i n t h e i n d u c a b i l i t y o f g l u c o s e p h o s p h a t e d e h y d r o g e n a s e w i t h t h e c e r e b e l l u m and b r a i n s t e m s h o w i n g h i g h e r l e v e l s o f i n d u c t i o n t h e n t h e c e r e b r a l h e m i s p h e r e s . T h i s c a n n o t be e x p l a i n e d by d e v e l o p m e n t a l t i m e t a b l e s f o r g l i a . K r e u t z b e r g and H u s s a i n (1982) showed t h a t M u l l e r c e l l s o f t h e e x t e r n a l r e t i n a l l a y e r s b u t n o t t h e i n t e r n a l l a y e r s h a v e 5 1 - n u c l e o t i d a s e on t h e i r membranes. T h i s enzyme f u n c t i o n s t o h y d r o l y s e m o n ophosphates s u c h a s AMP. The r e a s o n f o r t h i s d i f f e r e n c e i s unknown b u t t h e enzyme h a s n o t b e e n f o u n d on a s t r o c y t e s o f o t h e r a r e a s . T h e r e i s a l a r g e l i t e r a t u r e on d i f f e r e n c e s i n v a r i o u s t r a n s m i t t e r - r e l a t e d i n d i c e s b e t w e e n g l i a i s o l a t e d f r o m v a r i o u s r e g i o n s o f t h e b r a i n . D i f f e r e n c e s b etween p r e f r o n t a l c o r t e x a n d v i s u a l c o r t e x h a v e b e e n f o u n d i n c o n t e n t o f c a t c h o l a m i n e s ( B j o r k l u n d e t a l . , 1 978), and i n membrane b i n d i n g f o r n a l o x o n e , d i a z e p a m and a m u s c a r i n i c l i g a n d q u i n u c l i d i n y l b e n z i l a t e ( D i v a c and B r a e s t r u p , 1 9 7 8 ) . H a n s s o n e t a l . (1984a) showed t h a t a s t r o g l i a l c u l t u r e s f r o m v a r i o u s r e g i o n s o f t h e b r a i n showed i n c r e a s e d cAMP a f t e r i n c u b a t i o n w i t h dopamine o r a p o m o r p h i n e ; t h e i n c r e a s e c o u l d be b l o c k e d b y a dopamine a n t a g o n i s t . S u c h i n c r e a s e was most p r o n o u n c e d i n a s u b p o p u l a t i o n o f c e l l s f r o m t h e s t r i a t u m and l e a s t i n c e l l s f r o m t h e b r a i n stem. A s t r o c y t e s p r e p a r e d from a r e a s r i c h i n dopamine show dopamine b i n d i n g t h a t c a n be b l o c k e d b y t h e dopamine a n t a g o n i s t s c h l o r o p r o m a z i n e , h a l o p e r i d o l , and o t h e r a n t i p s y c h o t i c d r u g s , b u t a s t r o c y t e s f r o m non-dopamine c o n t a i n i n g p a r t s o f t h e b r a i n do n o t have t h i s a b i l i t y ( H ansson e t a l , 1 9 84b). T h i s means t h a t t h e r e - 81 - must be spe c i a l i z e d c e l l s i n dopamine-rich areas and that these c h a r a c t e r i s t i c s of such s p e c i a l i z e d g l i a are stable i n culture where they are not under neuronal influence. Hansson (1984) measured the a c t i v i t i e s of both MAO and COMT i n primary a s t r o g l i a l cultures from newborn r a t brain c u l t i v a t e d from s i x d i f f e r e n t regions and i n brain homogenates from these same regions. The areas compared were the cerebral cortex, striatum, hippocampus, brain stem, and cerebellum. MAO a c t i v i t y was higher i n the cultures from the striatum than i n those from the other brain regions. S t r i a t a l homogenates showed the same trend which c o n f l i c t s with the r e s u l t s of Hazama et a l . (1976) who found no differences i n the homogenates. COMT a c t i v i t y was the same i n neonatal cultures and adult brain homogenates and also showed regional differences. The lowest a c t i v i t y was found i n the brain stem, with higher l e v e l s i n the cortex, striatum and cerebellum and the highest i n the hippocampus. Henn and Henn (1980) found that g l i a from the caudate had a much higher number of haloperidol binding s i t e s and more dopamine s e n s i t i v e adenylate cyclase than those from other brain regions. Even so, the binding s i t e s are located on only a f r a c t i o n of the a s t r o g l i a l c e l l s of the caudate. A very s u r p r i s i n g finding was that by Denis-Donini et a l . (1984) who showed that d i f f e r e n t g l i a l populations a f f e c t the morphology of mouse mesencephalic dopaminergic neurons. G l i a l monolayers cultured from the s t r i a t a l or the mesencephalic region of the embryonic brain were used to grow dopaminergic neurons from the mesencephalon. On mesencephalic g l i a l c e l l s - 82 - the majority of the dopamine neurons developed a great number of highly branched and varicose neurites, whereas on s t r i a t a l g l i a they only exhibited one long, t h i n , l i n e a r neurite. The morphology of the underlying g l i a was not very d i f f e r e n t but they were not equally stained with GFAP, thus showing some heterogeneity i n the l e v e l of expression of g l i a l filament. Thus the c l a s s i c assumption of g l i a only responding to t h e i r neuronal environment i s ac t u a l l y found to be reversed. G o l d l e f t e r (1976) found that the p e r i v e n t r i c u l a r g l i a of the hypothalamus were p o s i t i v e with gonadotrophin or to Gomori's s t a i n and such staining increased on treatment with dopamine. Thus the g l i a of t h i s area respond to neurotransmitter and to a hormone produced by surrounding c e l l s . Schousboe (1978b) found that high a f f i n i t y uptake of GABA occurred i n peripheral ganglia, r a t re t i n a , glioma c e l l l i n e s , s p i n a l cord explant cultures, and primary cultures of g l i a l c e l l s from the cerebellum and cerebrum but not from other areas of the brain. I t was only i n g l i a l c e l l cultures from the cerebellum and cerebrum that the l e v e l of uptake was comparable to that i n brain s l i c e s . This a s t r o c y t i c uptake was d i f f e r e n t from the neuronal system since i t was s e l e c t i v e l y i n h i b i t e d by |3-proline but not by two selec t i v e i n h i b i t o r s of neuronal GABA uptake. G l i a l c e l l s may also vary i n t h e i r response to neurotransmitters. Krnjevic and Schwartz (1967) f i r s t showed that GABA applied iontophoretically caused depolarization of some, but not a l l , g l i a l c e l l s i n the cortex. They could not, - 83 - however, ru l e out the p o s s i b i l i t y that the s e l e c t i v i t y depended on proximity to GABA-depolarized neurons which released K+ that, i n turn, depolarized the nearby g l i a . GABA-T, the degradative enzyme for GABA, showed no regional differences (Hansson, 1984) i n primary cultures from the cerebral cortex, striatum, hippocampus, brain stem, and cerebellum of newborn rat brain. Glutamate indices have also been measured and found to vary r e g i o n a l l y . Autoradiographical studies of glutamate or D-aspartate high a f f i n i t y uptake (Currie and Kelly, 1981) showed extensive uptake into ce r e b e l l a r g l i a , e s p e c i a l l y Bergmann g l i a , and that t h i s decreased a f t e r transection of c e r t a i n projections. This implied that these differences are a r e s u l t of influences of neurons on g l i a , not the stable g l i a c h a r a c t e r i s t i c s that other research was indicated. They also noted other differences i n glutamate uptake from d i f f e r e n t regions. Hansson (1983) also used autoradiography to show regional differences i n uptake. She found that glutamate, and to a l e s s e r extent, aspartate, was taken up r e a d i l y i n cultures from the cerebral cortex, hippocampus, and striatum and, to a l e s s e r extent, i n cultures from the brainstem and cerebellum. This i s evidence for stable g l i a l differences i n glutamate uptake. Valine, an amino acid which i s incorporated mostly into protein, was used as an i n t e r n a l control and was found to be accumulated to the same extent i n the various primary cultures. Schousboe (1978a) showed a range i n values i n glutamate uptake by astrocytes cultured from d i f f e r e n t brain regions. The Vmax ranged from 8 nmol./min/mg c e l l - 84 - protein i n c e l l s from whole cerebrum to 60 nmol./min/mg i n c e l l s cultured from cerebral cortex, with Km varying from 22 0^ M to 50 J A M . Schousboe and Divac (1979) further showed that the glutamate uptake i n primary astrocyte cultures from neonatal mice a f t e r three weeks i n culture was greater i n c e l l s o r i g i n a t i n g from the prefrontal cortex and neostriatum than i n those o r i g i n a t i n g from the o c c i p i t a l cortex or cerebellum. These r e s u l t s generally correlate with the synaptosomal uptake of glutamate i n these regions and indicate that t h i s g l i a l c h a r a c t e r i s t i c was stable for at l e a s t three weeks i n culture without neuronal influences. Drejer et a l . (1982) did a s i m i l a r experiment and found the following Vmax values for astrocytes: p r e f r o n t a l cortex - 13.9, o c c i p i t a l cortex - 11.4, neostriatum - 27.3, and cerebellum - 5.8 nmol/min/mg c e l l protein. There were only minor differences i n Km between regions except i n the neostriatum where i t was s l i g h t l y higher. Differences i n Vmax and not Km mean that there are differences i n the number but not i n the properties of the transport s i t e s . Again the authors noted the apparent r e l a t i o n s h i p between the regional a b i l i t y of g l i a to accumulate glutamate and number of glutaminergic terminals. Glycine i s an important i n h i b i t o r y transmitter at the spi n a l l e v e l but not i n the forebrain. I t has been found that gradient-separated astrocytes from spinal cord, but not those from f r o n t a l cortex, show a high a f f i n i t y uptake of glycine (Henn, 1980). Others have confirmed that the d i s t r i b u t i o n of g l i a l transport systems for glycine follows the same - 85 - d i s t r i b u t i o n as glycine (Hokfelt and Lungdahl, 1971, Matus and Dennison, 1971) . The conclusions of these observations on GABA, glutamate, and glycine i s that there are probably differences i n the numbers of uptake s i t e s i n g l i a l c e l l s i n various brain regions and that these are stable i n culture. Moreover, the g l i a l uptake seems to correlate to some extent with the regional density of the amino acid boutons. Schousboe et a l . (1980b) suggested that t h i s g l i a l heterogeneity must be taken into account i n the int e r p r e t a t i o n of neurochemical changes r e s u l t i n g from s p e c i f i c neuronal degrenerations. For example, the e f f e c t s of g l i o s i s a f t e r k a i n i c acid lesions could s e r i o u s l y a f f e c t i n t e r p r e t a t i o n of biochemical changes. Summary of evidence for biochemical d i f f e r e n t i a t i o n i n g l i a The evidences for regional biochemical differences i n g l i a or cultured g l i a i s thus quite strong. Some differences may be because of d i r e c t e f f e c t s of surrounding neurons but some are stable i n culture a f t e r the e f f e c t of the neurons i s no longer there. These stable differences may be i n t e g r a l parts of the genetic makeup of these g l i a or may be i n i t i a t e d at some c r i t i c a l developmental point by i t s environment. Questions of t h i s nature have not yet been answered. There i s evidence that reverse e f f e c t s may be operative. Paterson et a l . (1977) showed that g l i a l c e l l s release some factor that influences the amount of neurotransmitter synthesized by sympathetically derived neurons eith e r by co-cultured or conditioned medium. They also found that C-6 and sympathetic - 86 - s a t e l l i t e c e l l s both influence growth and development of cho l i n e r g i c synapses and ACh synthesis. There i s also some evidence of species v a r i a t i o n s . There are, f o r example, considerable differences i n the rate of potassium uptake i n astrocytes cultured from chick, r a t or mouse brain. Thus future research must be extremely careful i n t r a n s f e r i n g experiments from one species to another. I f these biochemical differences between g l i a of d i f f e r e n t areas and species stand the t e s t of time, then the difference must be explored further and considered i n much of the on-going neurochemical research. In experimental conditions causing damage leading to g l i o s i s , some of the biochemical changes w i l l undoubtedly be found to be due to g l i a l changes. Research on many diseases may have to consider g l i a as being possibly involved i n the etiology. There are already research findings i n some diseases that point to t h i s . For example, Carter (1981) observed that GS a c t i v i t y was reduced i n Huntington's disease i n some areas where i t could not be accounted for by c e l l l o s s . I t has also been observed that thiamine d e f i c i e n t models of Wernicke-Korsakoff's syndrome produce damage f i r s t i n g l i a l c e l l s of c e r t a i n areas of the brain ( C o l l i n s , 1968; C o l l i n s and Converse, 1970). Research aimed at i d e n t i f y i n g differences i n g l i a has yielded much. But there i s also i n the vast l i t e r a t u r e on sta i n i n g of brain c e l l s many coincidental reports of staining of subsets of g l i a ; such reports tend to be buried i n the generalized l i t e r a t u r e because i n t e r e s t i n g l i a has been so l i t t l e compared to the in t e r e s t i n neurons. - 87 - EXPERIMENTAL RATIONALE AND ABSTRACT I have used two unrelated staining procedures that s t a i n predominantly g l i a l c e l l s , but not a l l g l i a l c e l l s , only subsets of them. I have also looked at a model of Wernicke-Korsakoff's syndrome that demonstrates that the disease may damage the g l i a l c e l l s of only some areas and before neuronal damage occurs i n these areas. In Experiment 1, hemosiderin, a form of iron, was examined i n the brains of rats using a Prussian Blue followed by diaminobenzidine (DAB) procedure. The areas of the brain containing the various types of c e l l u l a r and non-cellular s t a i n i n g were mapped. Iron was found to be predominantly located i n or on oligodendrocytes, but not i n a l l areas as there was a d i s t i n c t regional pattern of staining. There was also some sta i n i n g i n neurons, ependymal c e l l s and astrocytes of s p e c i f i c and r e s t r i c t e d areas, and various l e v e l s of background staining. The background s t a i n i n g i s probably terminal boutons on unstained c e l l s or neuronal or g l i a l processes. The r e s u l t s are compared to the known anatomy of several neurotransmitter systems. S i g n i f i c a n t overlap of the loc a t i o n of ir o n staining was noted with GABA, dopamine, endorphins and enkephalins. In Experiment 2, a modification of the method of Van Gelder (1965) for histochemical staining of GABA-T containing c e l l s was used to s t a i n c e l l s containing some enzymes catalyzing a possible a l t e r n a t i v e route for glutamate production i n brain: from proline or ornithine which i s - 88 - oxidized to glutamate v i a l-pyrroline-5-carboxylate (P5C) by 1-pyrroline dehydrogenase (EC 1.5.1.12;PDH). PDH has been demonstrated i n several bacteria and mammalian systems (Fig. 5) and, i n our experiment, was found to be exclusively i n g l i a l c e l l s such as the Bergmann g l i a of the cerebellum and astrocytes of the hippocampus. P5C can be formed from proline by the action of p r o l i n e oxidase (pyrroline-5-carboxylate reductase, EC 1.5.1.2,PrO). This enzyme was also l o c a l i z e d e x c l u s i v e l y i n g l i a l c e l l s but the staining was much less d i s t i n c t . Both of these experiments provide a d d i t i o n a l evidence of g l i a l c e l l s p e c i a l i z a t i o n . Experiment 3 only postulates g l i a l involvement i n thiamine deficiency as the technique does not allow f o r c e l l u l a r histochemistry. Pyrithiamine, a thiamine phosphokinase i n h i b i t o r , was fed to rats on a thiamine-deficient d i e t to create an animal model of Wernicke's encephalopathy. Symptoms of weight loss, ataxia, and loss of r i g h t i n g r e f l e x were produced i n rats i n ten days. At t h i s time some rats were s a c r i f i c e d and the rest of the rats were returned to a normal d i e t , to be s a c r i f i c e d only when t h e i r weight had returned to t h e i r pre-experimental l e v e l . Rats used for biochemical measurements were s a c r i f i c e d by c e r v i c a l fracture,the brains dissected into eight regions, and glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) a c t i v i t y measured on the brain homogenates. Other rats were perfused f o r h i s t o l o g i c a l observation of GABA-T, by a modification of a - 89 - method of Van Gelder (1965). GAD a c t i v i t y was found to be s i g n i f i c a n t l y reduced i n symptomatic rats i n the thalamus > cerebellum > pons/medulla > and mid-brain. GABA-T staining was found to be s i m i l a r l y reduced, with greatest losses i n the thalamus > i n f e r i o r c o l l i c u l u s > pons > and medulla. CAT a c t i v i t y was not s i g n i f i c a n t l y altered i n any brain areas. Upon return to a normal d i e t , recovery of GAD was s i g n i f i c a n t only i n the thalamus, while GABA-T staining recovered at least p a r t i a l l y i n a l l areas affected. These r e s u l t s are discussed i n terms of g l i a l s p e c i f i c i t y and e f f e c t s these new assumptions might have on the in t e r p r e t a t i o n of the r e s u l t s . - 90 - EXPERIMENT 1 There have been few studies examining the c e l l u l a r d i s t r i b u t i o n of iron i n brain but iron may have an important r o l e i n the brain, and may be involved i n several disease processes. The ro l e of iron i n the CNS i s not yet understood but low dietary iron i s known to have a number of e f f e c t s on brain function, including e f f e c t s on the electroencephalogram (EEG) (Tucker, 1982), and disturbances i n circadian rhythm, thermoregulation, motor a c t i v i t y (Youdim et a l . , 1981) and decreased attentiveness (Youdim et a l . , 1980). The mechanism of production of symptoms i n iron d e f i c i e n t y i s thought to be at l e a s t p a r t l y through neurotransmitters although the reduced capacity of the blood to carry oxygen may have an i n d i r e c t e f f e c t on the brain. Chronic, s l i g h t l y elevated l e v e l s of iro n have been shown to be tox i c to both ACh and GABA neurons (Swainman, 1984). Iron deposits can also occur i n c e r t a i n diseases such as i n Hallervorden-Spatz (Bronson, 1980), Huntington's disease or Parkinson's disease (Swainman, 1981). Non-heme iro n e x i s t s i n two forms i n the brain: f e r r i t i n i s i ron held i n storage by a protein forming globules i n lysosomes i n some parts of the brain, and hemosiderin, which i s f e r r i c hydroxide granules deposited more evenly i n c e l l bodies and processes. Hemosiderin i s probably the form active i n the brain. Hemosiderin releases f e r r i c iron on exposure to hydrogen chloride and potassium ferrocynanide can react with the f e r r i c iron producing f e r r i c ferrocyanide (Prussian Blue). This c l a s s i c Perl's reaction can be i n t e n s i f i e d using a - 91 - procedure of Nguyen-Legros et a l (1980). Diaminobenzidine i s added to the Prussian Blue, allowing the Prussian Blue to act as a c a t a l y s t for the oxidation of DAB by hydrogen peroxide forming an intense brown deposit where the iron i s . This i s the procedure we used to s t a i n for iro n . We did a det a i l e d map and analysis of both c e l l u l a r and non-cellular iron which allowed c o r r e l a t i o n of iron with known neurotransmitter anatomy. METHOD A method s i m i l a r to that of Nguyen-Legros et a l . (1980) was used. Two solutions were made up ju s t before experimental procedures were started. Solution A: 4% hydrogen chloride. Solution B: 4% ferrocyanide. Brains that had been perfused with phosphate buffered s a l i n e followed by 4% formaldehyde/4% gluteraldehyde, and stored for at le a s t three days i n the same f i x a t i v e s , were used. S l i c e s were cut on a cryostat at 50 and reacted for twenty minutes i n a 50% mixture of solutions A and B. I f the reaction proceeded c o r r e c t l y , the solution should be yellow not blue or green. The s l i c e s were then washed i n 0.1 M phosphate buffer, pH 7 . 4 , f o r 3 to 5 min. While the sections were washing, the DAB reagent was made up. 2 0 mg DAB was mixed into 100 ml t r i s buffer pH. 7.6, and 2 drops of hydrogen peroxide (30%) are added. The DAB (3.3'-diaminobenzidine tetrahydrochloride monohydrate 97%) was obtained from A l d r i c h . - 92 - The sections were placed i n t h i s reaction mixture for 1 0 min. i n the dark, then taken out and washed, mounted, dehydrated and coverslipped. (The darkness of the s t a i n depends on the amount of H2O2 and the time i n t h i s reaction mixture). RESULTS Several types of staining were seen. There were various l e v e l s of background staining without c l e a r c e l l u l a r morphology present (Fig. l a & b), areas of high background with d e f i n i t e c e l l u l a r s t a ining (Fig. Ic & d), and areas with very low background staining but with d e f i n i t e c e l l u l a r s t a i n i n g (Fig. Ie & f) as well as gradations i n between. There were also areas of neuronal staining (Fig. lg) and of a s t r o c y t i c s t a i n i n g (Fig. l h ) . There were gradations i n the background staining of various areas such that i t was a matter of judgement to decide which areas were to be c a l l e d high, medium, low or no background staining. I f e e l that the background staining i s probably a combination of staining of c e l l u l a r processes and nerve endings. F i g . 2 shows photographs of s a g i t t a l sections of iron stained sections showing the density of s t a i n . F i g . 3 gives maps corresponding to the photographs showing where there was i n d i v i d u a l c e l l u l a r staining ( c i r c l e s ) , or high or medium l e v e l s of background staining (dots) or both. F i g . 4 presents coronal sections and the corresponding maps. Because of the judgemental nature of the mapping, the photographs may be useful i n providing more detailed information as to the - 93 - density of the background staining than the maps and table can provide, but the photographs must be used cautiously i n t h i s regard since some dark areas may ju s t r e f l e c t a high density of the c e l l u l a r staining. A l l areas containing c e l l u l a r s t a i n i n g are marked on the schematic maps and are judged to be nonambiguous. Most of the c e l l u l a r staining i s thought to be of oligodendrocytes but there are i s o l a t e d i n d i v i d u a l c e l l s that are probably neuronal (Fig. l g ) . There were also l i m i t e d areas i n the ol f a c t o r y bulb and olfa c t o r y t r a c t that had what appeared to be staining of fibrous astrocytes on a low background area (Fig. Ih) and other areas i n the ol f a c t o r y bulb that had what appeared to be a mixture of stained fibrous astrocytes and neurons or oligodendrocytes i n a high background area. There were also regions i n the area postrema and around the v e n t r i c l e s where the staining appeared to be predominantly i n e p i t h e l i a l c e l l s . Table VIII summarizes the areas showing various types of stai n i n g . D I S C U S S I O N The most i n t e r e s t i n g observation that can be made from our re s u l t s i s that g l i a l c e l l s t a i ning i s not the same i n a l l areas of the brain. This uneven d i s t r i b u t i o n of stained g l i a l c e l l s tends to support further other observations of g l i a l c e l l s p e c i a l i z t i o n . This must indicate that g l i a l c e l l s are biochemically d i f f e r e n t i n t h e i r iron metabolism and i n iron - r e l a t e d functions, whatever they may be. The function of - 94 - i r o n i s not understood i n the brain, but our observations of regional heterogeneity i n iron density and c e l l u l a r location may be correlated with other information i n an attempt to assess i n which transmitter systems i r o n - r i c h g l i a l c e l l s may be involved. There are numerous theories proposed as to how i r o n i s involved i n the brain. Early iron l o c a l i z a t i o n studies using Turnbull blue (Spatz, 1922, Diezel, 1954) l o c a l i z e d iron to the g l i a l c e l l s of the globus p a l l i d u s , and the substantia nigra, and, to a lesser extent, the red nucleus, s t r i a t e body and Luys 1 body, a l l structures of the extrapyramidal system. Spatz noted that iron deposits occurred i n diseases involving the extrapyramidal system such as Parkinson's, Hallervorden Spatz's and Huntington's diseases. Based on these observations i t was proposed that iron may be involved i n dopamine metabolism because of the known importance of dopamine i n the extrapyramidal system. Supporting evidence included observations that low iron caused a reduced hypothermic e f f e c t of D-amphetamine and increased apomorphine induced stereotypic behaviour (Youdim et a l . , 1981). Both e f f e c t s are mediated by dopamine systems. I t was postulated that iron may function as a cofactor for tyrosine and tryptophan hydroxylases (Youdim et a l . , 1984) or may be involved i n dopamine receptor functions (Youdim et a l . , 1980). My findings are s i m i l a r to those i n Spatz's early work except there i s no staining i n the subthalamus (Luys body). My findings do show some c o r r e l a t i o n with dopamine d i s t r i b u t i o n but there are areas high i n dopamine that do not - 95 - have s p e c i f i c iron s taining and areas of iron s t a i n i n g where there are no known dopamine t r a c t s , projections or c e l l bodies. One of the major dopamine pathways i s that from the zona compacta of the substantia nigra and c e l l s j u s t medial to i t to the caudate, putamen, globus p a l l i d u s , o l f a c t o r y tubercle, nucleus accumbens, and l a t e r a l amygdala nucleus and f r o n t a l cortex. In my findings the substantia nigra has stained g l i a l c e l l s , as do the caudate-putamen, globus p a l l i d u s , amygdala and ol f a c t o r y tubercle and a l l these areas have high or medium background staining as well. But I fin d no i r o n s t a i n i n g i n c e l l s medial to the substantia nigra, and only medium background staining i n the nucleus accumbens. Small branches of t h i s dopamine system are supposed to ascend to the f r o n t a l cortex, anterior cortex, and septum. I f i n d no stai n i n g i n any part of the cortex although there i s a uniform low background l e v e l . There i s , however, medium background st a i n i n g i n some septal areas as well as neuronal staining i n the l a t e r a l septum. There are other dopamine pathways such as the one from the arcuate nucleus of the hypothalamus to the median eminence. My findings show the arcuate nucleus has oligodendrocyte s t a i n i n g on a medium background. I did not s t a i n sections containing the median eminence but H i l l and Switzer (1984) found a high concentration of iron stained ependymal c e l l s i n that region. There are c e l l s i n the medial dorsal nucleus of the hypothalamus that are thought to be dopaminergic that project to the thalamus and zona incerta. I f i n d that the medial - 96 - dorsal nucleus of the hypothalamus has stained oligodendrocytes with a high background, the thalamus has medium sta i n i n g and some areas with p o s i t i v e oligodendrocytes, and the zona incerta has stained oligodendrocytes but no background staining. There are also dopamine interneurons i n the hypothalamus, brain stem and ol f a c t o r y bulb. These are a l l areas that contain some background staining with stained c e l l s i n the ol f a c t o r y bulb. Thus a l l areas of dopamine c e l l bodies except the area medial to the substantia nigra also contain stained oligodendrocytes but i n the dopamine terminal areas there i s everything from p o s i t i v e staining of various c e l l types to no c e l l s t aining, and a range from low to high i n background sta i n i n g . I t may be relevant that the dopaminergic areas which show the lea s t iron s taining are generally those of the A10 system i n which dopamine and cholecystokinin are co l o c a l i z e d . My evidence i s somewhat supportive of iron involvement i n dopamine metabolism, p a r t i c u l a r l y around non-peptidergic dopamine c e l l bodies, but the lack of a t o t a l match means that iron does not ex i s t exclusively i n association with dopamine. Other researchers have t r i e d to correlate iron d i s t r i b u t i o n with GABA neuroanatomy. Glutamate-binding protein i s known to contain iron and i s required f o r GABA regeneration (Michaelis et a l . , 1982). Francois et a l . (1981) observed that the GABA s t r i a t o - or p a l l i d o - n i g r a l and cer e b e l l a r c o r t i c a l pathways overlap s i g n i f i c a n t l y with iron - 97 - d i s t r i b u t i o n . They also noted that areas high i n GAD such as i n the superior c o l l i c u l u s and the nucleus interpeduncularis, were also high i n iron (Francois et a l . , 1981.). My observations confirm t h e i r findings of iron i n a l l these areas except the cortex, where we f i n d only low to medium background and no c e l l u l a r staining, and the cerebellar cortex, where there i s only medium background staining. H i l l and Switzer (1984) d i d a study s i m i l a r to mine using the same technique and found s i m i l a r but not i d e n t i c a l r e s u l t s and concluded that high i r o n concentrations i n g l i a overlapped most s i g n i f i c a n t l y with areas high i n GAD and GABA; these areas included the ventral pallidum, globus p a l l i d u s , substantia nigra pars r e t i c u l a t a , and cerebellar n u c l e i . They pointed out that i n j e c t i o n s of GABA into the globus p a l l i d u s led to reductions i n i r o n i n the i p s i l a t e r a l ventral pallidum, globus p a l l i d u s and substantia nigra ( H i l l , 1984). They thought, however, that the d i s t r i b u t i o n of iron indicated i t was not exclusively r e l a t e d to GABA but might be involved i n other neurotransmitter systems such as enkephalins. My r e s u l t s do not support the involvement of iron i n GABA as strongly as do those of H i l l and Switzer. GABA i s thought to be the transmitter of the Purkinje c e l l s of the cerebellum which project to the cerebellar nuclei and of the cerebellar basket c e l l s , Golgi c e l l s and the s t e l l a t e c e l l s , which are a l l wholly contained i n the cerebellar grey matter. However, my r e s u l t s do not show any c e l l u l a r s t a ining i n the cerebellar cortex and only a medium amount of background staining. Although I do f i n d s i g n i f i c a n t oligodendrocyte and possibly - 98 - neuronal s t a i n i n g i n the cerebellar n u c l e i . Overall, t h i s does not provide strong evidence for the involvement of iron i n c e r e b e l l a r GABA systems. The pattern of hippocampal s t a i n i n g i s consistent with iron's involvement i n GABA processes i n that nucleus as the only area of sta i n i n g i s a narrow band of medium background staining around the middle of hippocampal layers where there are basket c e l l s which are GABAergic. The high l e v e l s of stained c e l l s and background i n the globus p a l l i d u s and the pars r e t i c u l a t a of the substantia nigra are consistent with the well established GABAergic projections between these two structures. GABA i s also the neurotransmitter of interneurons of the o l f a c t o r y bulb which might be consistent with the observations of c e l l u l a r staining f o r i r o n i n that region. However, GABA i s so ubiquitous i n brain that i f a l l GABA systems were associated with i r o n - r i c h g l i a or other structure, one would expect a f a r more even d i s t r i b u t i o n of iron than found i n t h i s or previous studies. There i s l i t t l e c o r r e l a t i o n between areas of high [3H]-GABA uptake (Iversen and Schon, 1973), and high iron s taining areas, except that the substantia nigra i s high i n both. Thus my r e s u l t s only give l i m i t e d support to ir o n involvement i n GABA metabolism i n some areas of brain with these areas including those i n which H i l l showed reductions i n iron a f t e r p a l l i d a l i n j e c t i o n of GABA. Several researchers have suggested a connection between 5HT and iron . I t was observed that i r o n - d e f i c i e n t synaptosomes take up less 5HT than normal synaptosomes and, when iro n i s returned to the di e t , uptake increased (Kaladhar - 99 - and Rao, 1982). This phenomena extended to o f f s p r i n g of iron d e f i c i e n t mothers (Kaladher and Rao, 1983). These authors suggest an ir o n dependent serotonin binding protein or some other involvement of iron i n v e s i c u l a r storage of 5HT. Tamir et a l . (1976) noted that the binding of serotonin by serotonin binding protein was enhanced by Fe2+. Most 5HT neurons are located i n the raphe or r e t i c u l a r system and project to the neostriatum, cortex, thalamus, hippocampus, cerebellum, preoptic nucleus, septal n u c l e i or pons. Our r e s u l t s show p o s i t i v e c e l l s or, at lea s t , medium background st a i n i n g i n a l l the above areas except the cortex, but again there i s no consistent s t a i n i n g pattern d i f f e r e n t i a t i n g areas of projection and c e l l bodies. The ependymal c e l l s l i n i n g the t h i r d v e n t r i c l e are serotonergic and s t a i n heavily for iron which might be interpreted as some support f o r the involvement of i r o n i n 5HT systems. There i s a s t r i k i n g overlap of iron d i s t r i b u t i o n with some aspects of enkephalin neuroanatomy. There are both iron s t a i n i n g and enkephalin c e l l s i n the l a t e r a l septum, bed nucleus of the s t r i a terminalis, striatum, hypothalamus, amygdala, substantia nigra, medial v e s t i b u l a r nucleus, nucleus of the spinal t r a c t of the trigeminal, and the periaquaductal gray, although i n the bed nucleus of the s t r i a terminalis and the spinal t r a c t of the trigeminal the sta i n i n g i s only a medium background staining. P-Endorphins and related substances also have a sim i l a r and extensive overlapping pattern with iron d i s t r i b u t i o n . Our evidence indicates that, i f iron i s involved i n any - 100 - s p e c i f i c neurotransmitter system, i t i s not involved i n a simple way. I t may be involved i n two or more transmitter systems, or be involved i n some other, as yet unhypothesized, processes. Our evidence does not eliminate any of the theories previously advanced but neither does i t wholly support any one of them either. A recent study (Y. Noda unpublished) examined the effects of a 20 month normal, iron d e f i c i e n t , or i r o n abundant di e t on three enzymes: CAT, GAD, and tyrosine hydroxylase (TH). The r e s u l t s showed that GAD and, to some extent, TH a c t i v i t y i s inversely r e l a t e d to the amount of iron i n the d i e t i n a l l brain regions examined. CAT a c t i v i t i e s were unaffected. Noda thought that iron must be e s s e n t i a l for both GABAergic and catcholaminergic systems but concluded that excessive iron might r e s u l t i n degeneration of the neurons. Iron deposits can be harmful and do occur i n some diseases as mentioned e a r l i e r , and i n the same structures that are normally high i n i r o n . High l e v e l s of iron may be harmful because i t can lead to the generation of oxygen free r a d i c a l s , OH1 due to the iron mediated coupling of O2 and H2O2/ the so c a l l e d Haber-Weiss reaction. The presence of iron i n subsets of g l i a l c e l l s might suggest that: (1) the iron i s necessary fo r some function of these p a r t i c u l a r g l i a l c e l l s ; or (2) the iron i s e s s e n t i a l for c e r t a i n types of neurons, and the g l i a l c e l l s surrounding them are e i t h e r supplying iron to these neurons or scavenging i t from the e x t r a c e l l u l a r space around them. The most s i g n i f i c a n t finding of t h i s research i s the - 101 - extensive l o c a l i z a t i o n of hemosiderin to g l i a and the fact that t h i s l o c a l i z a t i o n i s d i f f e r e n t i n various brain regions. - 102 - Figure 1: Various Types of Staining for Iron i n Rat Brain F i g . IA Midbrain areas showing several densities of background st a i n i n g without any i n d i v i d u a l l y stained c e l l s . C a l i b r a t i o n bar = 1000 /um. Fi g . IB Band of medium staining with no c e l l s above the pyramidal c e l l layer of the hippocampus. Ca l i b r a t i o n bar = 300 jam. Fi g . IC Area i n the globus p a l l i d u s with moderately heavy background st a i n i n g and c l e a r l y stained c e l l s , probably oligodendrocytes. C a l i b r a t i o n bar = 300 fKm. F i g . ID Strands of l i g h t and dark background staining with heavily stained oligodendrocytes among dark strands i n the striatum. C a l i b r a t i o n bar = 300 pirn. - 103 - - 104 - F i g . IE Several stained oligodendrocytes i n a l i g h t l y stained area of the striatum. C a l i b r a t i o n bar = 300^on. Fi g . IF I n t e r f a s i c u l a r oligodendrocytes against l i g h t background staining of corpus callosum. C a l i b r a t i o n bar = 300 /̂m. F i g . 1G L i g h t l y stained neurons on a l i g h t background i n l a t e r a l septum. C a l i b r a t i o n bar = 300 /im. F i g . IH Area i n o l f a c t o r y bulb with l i g h t background staining showing probable a s t r o c y t i c staining. C a l i b r a t i o n bar = 300 ijim. - 105 - - 106 - F i g . 2 Photographs of s a g i t t a l sections of whole rat brain. F i g . 2a 0.5mm of the midline. F i g . 2b, 1.2 mm of the midline. F i g . 2c, 2.9 mm o f f the midline. C a l i b r a t i o n bar = 1 mm. - 107 - - 1 0 8 - F i g . 3 Schematic diagrams of figure 2. C i r c l e s indicate area of c e l l u l a r staining, and dots indicate high background st a i n i n g . C a l i b r a t i o n bar = 1 mm. (See Table VII for abbreviations.) - 109 - - 110 - F i g . 4 Half photographs and h a l f schematic drawing of coronal sections of rat brain. F i g . 4a, 3.2 mm anterior to bregma. F i g . 4b, 1.4 mm anterior to bregma. F i g . 4c, 0.6 mm anterior to bregma. F i g . 4d, 2.0 mm poster i o r to bregma. C a l i b r a t i o n bar = 1mm. (See Table VII for abbreviation explanations) - I l l - - He! F i g . 4 (Continued) Half photographs and h a l f schematic drawing of coronal sections of rat brain. F i g . 4 e, 4.0 mm poster i o r to bregma. F i g . 4f, 6.0 mm poster i o r to bregma. F i g . 4g, 8.8 mm poster i o r to bregma. F i g . 4h, 11.4 mm posterior to bregma. C i r c l e s indicate areas of c e l l u l a r s t a ining and dots of high background staining. C a l i b r a t i o n bar = 1 mm. (See Table VII for abbreviation explanations) - 113 - - 114 TABLE VII: IRON STAINING IN VARIOUS AREAS OP THE BRAIN Table VII summarizes the type of staining i n various structures. A l l structures not mentioned have no c e l l s and low or no background staining. H, M, L - high, medium, low background staining, 0 - oligodendrocytes, N - neurons, A - astrocytes,E - e p i t h e l i a l c e l l s . Brain Structure Symbol Background C e l l Types Nucleus Accumbens Septi ACB M none Central Amygdala ACE M 0 Anterior Hypothalamic Area AHA M 0, N? Lateral Amygdaloid Nucleus AL M none Accessory Olfactory Bulb AOB M 0, N? Area Postrema AP H E? Arcuate Nucleus of Hypothalamus ARH M 0 Bed Nucleus of the Anterior BCA M 0 Commissure Bed Nucleus of S t r i a Terminalis BST M none Anterior Commissure CA none Strings Corpus Callosum CC none Strings Cerebellar Grey CG M none I n f e r i o r C o l l i c u l u s CIF M 0 Caudate Putamen CPUH in Strings 0 Superior C o l l i c u l u s CS M 0 Commissure of the Superior CSC none 0 C o l l i c u l u s L ateral Cuneate Nucleus CUL M none Decussations of Medial Lemniscus DLM M 0 Dorsal Medial Nucleus of DMH H 0 the Hypothalamus Dorsal Raphe DR M none Endopeduncular Nucleus EP L 0 External Plexiform Layer EPL L 0, A of Olfactory Bulb Fornix FX M i n strands non< Geniculate Body G M none Globus P a l l i d u s GP H 0, N? Nucleus G r a c i l i s GR L 0? Hypothalamus ( a l l other areas) H M 0 Habenular Nucleus HN H none Hippocampus CA 3 HP M none Islands of C a l l e j a IC H none I n f e r i o r Olfactory Bulb IGL M 0?, A Interpeduncular Nucleus IP H 0 Locus Coeruleus LC M 0 Lateral Hypothalmic Area LHA M none Lateral Lemniscus LL H 0 Dorsal Nucleus of the LLD M 0 Lateral Lemniscus Medial Lemniscus LM L 0 Lateral Septal Nucleus LS M N Lateral Nucleus of Thalamus LT M 0 Medial Forebrain Bundle MFB M 0 - 115 TABLE VII (continued) B r a i n S t r u c t u r e Symbol Background C e l l L a t e r a l Mammillary Nucleus ML H none M e d i a l Mammillary Nucleus MM M 0 M e d i a l S e p t a l Nucleus MS M none Nucleus Accumbens NA M none C o c h l e a r Nucleus NC M none Dentate Nucleus ND M 0 F a s t i g i a l Nucleus NF M 0,N? I n t e r p o s i t u s Nucleus of NI M 0 Cerebellum P r e p o s i t u s Nucleus NPH M 0 P o s t e r i o r Nucleus of Thalamus NPT M 0 Red Nucleus NR L 0 Nucleus o f S p i n a l T r a c t of NTST M none the T r i g e m i n a l Nerve L a t e r a l V e s t i b u l a r Nucleus NVL M 0 M e d i a l V e s t i b u l a r Nucleus NVM M 0 S u p e r i o r V e s t i b u l a r Nucleus S p i n a l V e s t i b u l a r Nucleus I n f e r i o r O l i v a r y Nucleus NVS M none NVSP M none OL M 0 O p t i c T r a c t OT none 0 Pons P H none P o s t e r i o r Hypothalamus PH L none P r e t e c t a l Area PRT M 0 L a t e r a l P r e o p t i c Area POA H 0 P e r i v e n t r i c u l a r Grey PVG L 0 P a r a v e n t r i c u l a r Hypothalamus PVH M none R e t i c u l a r Formation RF none 0 Rhomboid Nucleus of Thalamus RH M N R e t i c u l a r Nucleus o f Thalamus RT M 0 , N? Suprachiasmic Nucleus SC M N? S t r i a M e d u l l a r i s Thalami SM H none S u b s t a n t i a N i g r a SN H 0 S u p r a o p t i c Nucleus of the SO M N? Hypothalamus S o l i t a r y Nucleus SOL M none S u p e r i o r O l i v a r y Complex SOC M 0 Thalamus ( a l l o t h e r areas) T M none Intermediate O l f a c t o r y T r a c t TO I none A? Nucleus T r i a n g u l a r i s S e p t i TS H none O l f a c t o r y T u b e r c l e TUO M 0 V e n t r a l Nucleus of Thalamus VE M 0 V e r t r o m e d i a l Hypothalamus VMH M none V e n t r a l Tegmental Nucleus VTN M none Zona I n c e r t a AI none 0 O l f a c t o r y Nerve I H 0 , N?, A? Vermian Lobule C l M none Nucleus of the T h i r d Nerve I I I L 0? F a c i a l Nerve VII M 0 H y p o g l o s s a l Nucleus XII M none - 116 - EXPERIMENT 2 1-Pyrroline dehydrogenase (EC 1.5.1.12; PDH) has been shown i n several b a c t e r i a l and mammalian systems to be a key enzyme i n the pathways from ornithine and pr o l i n e to glutamate (Figure 5). Ornithine i s converted to glutamic acid semialdehyde by ornithine ^-transaminase (Ornithine - oxo-acid aminotransferase, EC 2.6.1.13, OrnT) and the semialdehyde i s i n equilibrium with P5C which can also be formed from proline by the action of PrO. The P5C i s oxidized by PDH to glutamate (Roberts, 1982). Glutamate i s an important putative neurotransmitter i n i t s own r i g h t and i s also an immediate precursor of GABA. Most brain glutamate i s formed from glucose through the t r i c a r b o x y l i c acid cycle but the route from ornithine or proline o f f e r s a possible a l t e r n a t i v e for a small glutamate pool. Proline i s also a possible neurotransmitter which has been shown, when injected, to end up as GABA i n g l i a l c e l l s (Van den Berg, 1970). One of the enzymes, PDH, has been p u r i f i e d from beef l i v e r and i s a mitochondrial enzyme that requires nicotinamide adenine dinucleotide (NAD) (Strecker, 1971). The other, PrO, has moderate a c t i v i t y i n brain (Kawabata et a l . , 1980); although not f u l l y characterized, i t appears to be a membrane bound enzyme which also uses NAD (Boggess et a l . , 1978). Since both enzymes function i n the presence of NAD, we thought they might be histochemically l o c a l i z e d by va r i a t i o n s of the technique developed f o r GABA-T by Van Gelder (1965). Van Gelder used the NADH produced during the metabolism of GABA to - 117 - reduce n i t r o blue tetrazolium to the dye formazan which stayed i n the c e l l s containing the GABA-T. Modifications of t h i s technique with P5C or L-proline as a substrate were t r i e d as a means of demonstrating the histochemical l o c a l i z a t i o n of PDH and PrO i n brain. METHOD P5C was prepared from i t s precursor (supplied by Calbiochem of La J o l l a , C a l i f o r n i a ) according to the manufacturer's d i r e c t i o n s : 1 gm of the precursor i s dissolved i n 33 ml of 6N HC1 and brought to 100 C f o r 45 min. The P5C was p u r i f i e d on a 150 x 3 0ml column of Dowex 50, 8% crosslinked, mesh 50-100 H+, using the procedures of Strecker (1960). The P5C was eluted and a portion of each f r a c t i o n was analyzed f o r P5C by reaction with some o-aminobenzaldehyde and measurement of the absorbance at 440 nm. The samples showing presence of P5C were combined and l y o p h i l i z e d . Male Wistar rats weighing 250-350 gm obtained from Canadian Breeding Laboratories were perfused i n t r a c a r d i a l l y with 150 ml of i c e cold 0.1M phosphate buffered s a l i n e (pH 7.4) followed by 2% gluteraldehyde/2% paraformaldehyde. Sections were cut on a vibratome and c o l l e c t e d i n 0.1M phosphate buffer. The free f l o a t i n g sections were stained for PDH by preincubating them for 2 0 min. at 37°C i n the dark i n 5 ml t r i s hydrogen-chloride 0.1M (pH 8.6), plus 0.5 ml NAD+ (10 mg/ml), plus 1.5 ml of solution containing 144 mg/ml NaCl, 2 0 mg/ml MgCl and 1 mg/ml KCN. After the preincubation, 10 mg of n i t r o blue tetrazolium were mixed i n 0.25 ml dimethyl - 118 - sulfoxide which was added with 0.25 ml d i s t i l l e d water, followed by 0.2 ml of phenazine methosulfate (2 mg/ml) and 0.1-0.5 ml of 150 mg/ml of P5C. The incubation was continued at 37°C i n the dark for 45 min. The reaction was stopped by tran s f e r to a phosphate buffer. The sections were mounted on g e l a t i n coated s l i d e s , dried at le a s t overnight, dehydrated i n xylene and coverslipped i n Permamount. A l l solutions were i n d i s t i l l e d water unless otherwise s p e c i f i e d . The procedure for the L-proline oxidase staining was i d e n t i c a l except that 0.1-0.5 ml of 250 mg/ml of commercially av a i l a b l e L-proline was substituted from the P5C. Controls were done without L-proline or P5C. RESULTS A l l concentrations of P5C gave some background staining, but there was a much darker s p e c i f i c s t a i n i n g of c e r t a i n types of c e l l s . This was most evident i n the cerebellum where a high proportion of Bergmann type astrocytes were darkly and d i s t i n c t l y stained. Although there was a high background l e v e l i n the granular layer of the cerebellum, no c e l l u l a r morphology was evident i n that layer and the staining density never approached h a l f of that i n the Bergmann g l i a l c e l l s . Figure 6a shows the stained Bergmann g l i a l c e l l bodies i n the Purkinje c e l l layer and t h e i r f i b e r - l i k e projections into the molecular layer. Figure 5b shows t h i s s t a i n i n g i s consistent throughout the cerebel l a r sections. The next most consistent and c l e a r f i n d i n g was i n the pyramidal c e l l layer of the dentate gyrus of the hippocampus. Here the st a i n i n g was l i g h t - 119 - but a d i s t i n c t band of stained hippocampal astrocytes could be distinguished (Fig. 7). The only other c l e a r l y stained c e l l s were occasional a s t r o c y t e - l i k e c e l l s of the corpus callosum and other prominent white t r a c t s . PrO s t a i n i n g was much less d i s t i n c t and was li m i t e d to the Bergmann g l i a l c e l l s (Fig. 8). Sections stained without either L-proline or P5C showed no c e l l u l a r s t a i n i n g and only a f a i n t pink background staining. D I S C U S S I O N Our modifications of the Van Gelder technique f o r the histochemistry of GABA transaminase gave some i n d i c a t i o n of the probable l o c a l i z a t i o n of PDH and PrO. In both cases there was some non-specific background staining, but i n neither case was i t high enough to i n t e r f e r e with microscopic i n t e r p r e t a t i o n of s p e c i f i c c e l l s taining. The technique could probably be used s i m i l a r l y for the histochemical l o c a l i z a t i o n of other NAD requiring enzymes. I t may not, however, reveal a l l l o c i of such enzymes and i s probably not suitable f o r quantitative analysis. Thus, for example, no c e l l u l a r s t a i n i n g for PDH was seen i n the cortex although appreciable, i f r e l a t i v e l y low, a c t i v i t y was found i n that region on biochemical assay (Thompson et a l . , 1985). I t i s possible that the technique only gives c l e a r s t a i n i n g of c e l l s containing PDH at a c t i v i t i e s that approach the l e v e l s i n the cere b e l l a r Bergmann g l i a l c e l l s . Increasing the substrate concentration did not r e s u l t i n s p e c i f i c s t a i n i n g of more c e l l types. - 120 - The s t a i n i n g for prol i n e was les s d i s t i n c t than that for PDH and may, i n fact, be due to PDH since the product of PrO i s P5C which could be acted on by PDH to produce glutamate and further NADH (Fig. 5). This p o s s i b i l i t y gains some support from the fac t that the only d e f i n i t e c e l l s t a i n i n g for PrO was seen i n the cerebellum although regional d i s t r i b u t i o n data for PrO suggest the highest a c t i v i t i e s are i n the midbrain and brain stem (Thompson et a l . , 1985). The most consistent histochemical finding i s that only c e r t a i n g l i a l c e l l populations were stained. I t can be argued that Bergmann g l i a l c e l l s are a spe c i a l type of g l i a l c e l l but the astrocytes of the hippocampus, although morphologically d i f f e r e n t to astrocytes i n the res t of the brain, are not recognized as a s p e c i f i c subtype. This f i n d i n g thus supports a growing body of evidence of g l i a l c e l l s p e c i a l i z a t i o n . I t i s tempting to speculate that the chemical s p e c i a l i z a t i o n of g l i a i s to provide materials important to the neurons i n the v i c i n i t y . I t i s true that the densest s t a i n i n g (of Bergmann c e l l s and of astrocytes i n the hippocampal dentate gyrus) i s i n regions where important glutamate t r a c t s as well as GABA interneurons are expected. On the other hand, there i s considerable evidence that many co r t i c o f u g a l and c o r t i c a l commissural t r a c t s also use glutamate as a transmitter and that many c o r t i c a l interneurons are GABAergic, but astrocytes s t a i n i n g f o r PDH or PrO were not seen i n the c o r t i c a l grey matter. Our research may be pertinent to the study of a number of f a m i l i a l conditions i s which the peripheral metabolism of - 121 - p r o l i n e or ornithine i s known to be affected. In the hyperprolinemias there i s a deficiency of PrO i n type 1 (Haysaka et a l . , 1982) and of PDH i n type II (Valle et a l . , 1974). A v a r i e t y of neurological symptoms, including EEG abnormalities, convulsions, and mental deficiency, have been reported i n such cases but the fact that many are asymptomatic suggests there i s not a causal r e l a t i o n s h i p (Molicca and Pavone, 1976). Nevertheless, i t would be of i n t e r e s t to study brain region l e v e l s of these enzymes i n post mortem tissue from such cases as compared with controls. The same might be true of cases of hyperornithinemia which have been reported to show atrophy of ocular t i s s u e (Haysaka et a l . , 1982); since OrnT l e v e l s are normally much higher i n the r e t i n a than i n brain (Rao and C o t l i e r , 1984), the former may show the most d r a s t i c changes i f t h i s enzyme i s d e f i c i e n t . - 122 - F i g . 5 S c h e m a t i c r e p r e s e n t a t i o n o f t h e c o n v e r s i o n s o f p r o l i n e a n d o r n i t h i n e t o g l u t a m a t e a n d G A B A . - 123 - H 2 C H 2 C C H - C H - C O O H N H 2 H 2 N ~ C H 2 C H 2 _ C H 2 C H ^QQQ|_J O R N I T H I N E H P R O L I N / N A D P R O L I N E OX IDASE H , C - H C N A D H - C H 2 C H - C O O H ORNITHINE ^ -TRANSAMINASE ~ H C - C H 2 - C H 2 - C H - C O O H NH- P Y R R O L ! N E - 5- C A R B O X Y L A T E G L U T A M I C A C I D S E M I A L D E H Y D E / N A ° I - P Y R R O L I N E D E H Y D R O G E N A S E N A D H H O O C - C H . - C H , - C H - C O O H NH- G A D G L U T A M I C A C I D H O O C - C H 2 - C H 2 - C H 2 N H 2 G A B A - 124 - F i g . 6 PDH staining i n cerebellum (A) Bergmann g l i a l c e l l s showing f i b e r s projecting up to the cerebellar molecular layer. C e l l bodies are loosely arranged around the Purkinje (P) c e l l layer. C a l i b r a t i o n bar = 100 M m . (B) Same at lower magnification. C a l i b r a t i o n bar = 300 jum. F i g . 7 PDH stained astrocytes i n layer of dentate gyrus of hippocampus. C a l i b r a t i o n bar = 300 Min. Fig . 8 PrO staining of Bergmann g l i a l c e l l s of cerebellum. C a l i b r a t i o n bar = 100 pirn. - 125 -  EXPERIMENT 3 Thiamine deficiency leading to Wernicke-Korsakoff's syndrome occurs among several populations of Western people, most commonly among al c o h o l i c s , but also i n people on d i a l y s i s , people with i n t e s t i n a l absorption diseases (Sassaris et a l . , 1983), and the e l d e r l y (Iber et a l . , 1982). Thiamine deficiency (TD) can also lead to b e r i b e r i , a p o l y n e u r i t i s which can occur with congestive heart f a i l u r e . Werniche's encephalopathy i s a neurological disorder with symptoms of confusion, disturbances i n ocular m o t i l i t y , p u p i l l a r y a l t e r a t i o n s , nystagmus, and ataxia with tremors. I t s symptoms are believed to be the d i r e c t r e s u l t of a biochemical l e s i o n which can l a r g e l y be reversed by thiamine administration. Korsakoff's syndrome i s characterized by impaired memory for recent events and p o l y n e u r i t i s . I t occurs frequently with Wernicke's but does not reverse with thiamine therapy. Its thiamine r e s i s t a n t symptoms may be the r e s u l t of s t r u c t u r a l damage because of repeated or long term thiamine deficiency. In humans the st r u c t u r a l damage of Korsakoff's syndrome occurs as hemorrhagic lesions i n the mammillary bodies, p e r i v e n t r i c u l a r regions of the thalamus and hypothalamus, periaquaductal regions of the midbrain and f l o o r of the fourth v e r t r i c l e and i n parts of the cerebellum. Wernicke's pathology i s i n s i m i l a r structures i f i t i s present. We used the pyrithiamine animal model i n which rats are put on a thiamine d e f i c i e n t d i e t and given pyrithiamine (PT), - 127 - an antagonist of thiamine phosphokinase, the enzyme which converts thiamine to thiamine pyrophosphate. Using t h i s model, symptoms of weight l o s t , ataxia, and loss of r i g h t i n g r e f l e x occur i n about 10 days and death i n 14 days. PT produces lesions i n the l a t e r a l v e s t i b u l a r nucleus, f l o o r of the fourth v e n t r i c l e , mammillary bodies, thalamus, i n f e r i o r o l i v e , and cerebellum; these are thus s i m i l a r but not i d e n t i c a l to the human patterns seen i n Wernicke-Korsakoff's syndrome. Understanding the nature of the early biochemical lesions has been the goal of much research since 1936, when Peters proposed the biochemical l e s i o n theory to explain the neurological e f f e c t s of thiamine deficiency. Peters' o r i g i n a l theory was that the biochemical l e s i o n when found must explain two observations, the s e l e c t i v e v u l n e r a b i l i t y of c e r t a i n structures i n the brain, and the r e v e r s i b i l i t y upon treatment with thiamine. Explaining these observations remains important i n current research. The enzymes for which thiamine triphosphate (TTP) i s a co-enzyme, as well as several enzymes asociated with various neurotransmitters, have been examined by previous authors but the r e s u l t s do not explain f u l l y the nature of the i n i t i a l biochemical l e s i o n . In t h i s experiment we examined the synthetic enzymes for GABA and ACh and the degradative enzyme for GABA f o r t h e i r possible r o l e i n the i n i t i a l biochemical l e s i o n . ACh i s one of the neurotransmitters previously studied i n thiamine deficiency. A decrease i n the TTP-dependent enzyme, - 128 - pyruvate dehydrogenase, which i s e s s e n t i a l for the production of acetyl-CoA and therefore of ACh, would t h e o r e t i c a l l y lead to reduced synthesis of ACh and therefore reduced concentrations of ACh. Decreased synthesis of ACh has i n fact been observed, but, although there were e a r l i e r reports (Hamel et a l . , 1980) of decreased ACh concentrations, most recent reports do not confirm t h i s (Reynolds and Blass, 1975, Vorhees et a l . , 1977). The difference may l i e i n the speed at which the brain was fixed and the resultant extent to which the metabolically active pools of ACh are measured (Barclay et a l . , 1981). Since some of the pools are of l i t t l e functional value, turnover i s thought to be a better index of functional change (Cheney et a l . , 1977). Decreased turnover of ACh has been observed even i n the presence of adequate l e v e l s of choline and CAT (Thornber et a l . , 1980). I t i s postulated that the decrease i n pyruvate dehydrogenase i n vivo i n thiamine d e f i c i e n t animals i s not enough to explain a l l the reduction i n ACh synthesis. The l e v e l s of CAT (Bhatgat and Lockett, 1962, Heinrich et a l . , 1973, Reddy, 1982, Sacchi et a l . , 1978) and the a c t i v i t i e s of cholinesterase are reportedly not decreased (Takats et a l . , 1981). We examined the regional a c t i v i t i e s of CAT i n controls, a f t e r the appearance of symptoms of thiamine deficiency, and a f t e r recovery to o r i g i n a l weight. GABA has been found to be decreased i n the whole brain, pons/medulla, midbrain, cortex and cerebellum p r i o r to neurological symptoms i n rats on pyrithiamine (Butterworth et a l . , 1979, Butterworth, 1982a). These findings have not been - 129 - confirmed by other researchers ( P l a i t a k i s et a l . , 1979). GABA high a f f i n i t y uptake i s not affected i n any brain areas ( P l a i t a k i s , 1982). We examined both GAD and GABA-T i n s p e c i f i c regions of the brain at the peak of the pyrithiamine induced symptoms and a f t e r return to o r i g i n a l weight on a normal d i e t . I f the l e s i o n i s fundamentally biochemical i n nature, then there should be at l e a s t some recovery when thiamine i s returned to the d i e t . There have been no studies done on ei t h e r GABA or ACh enzymes to see i f these change s e l e c t i v e l y and i f they recover upon reintroduction of thiamine to the d i e t . I f there i s a biochemical l e s i o n involving a p a r t i c u l a r enzyme, and the thiamine deficiency i s stopped j u s t before the onset of symptoms, there should be t o t a l recovery of enzymatic function; but i f the c r i t i c a l time to stop i s past, there may be residual damage due to prolonged biochemical disruption of the c e l l with some consequent c e l l death. Recovery of biochemical function might be explained i n yet another way. I f lesions are i n i t i a t e d i n the g l i a , the r e c o v e r a b i l i t y of the early lesions may be because g l i a l c e l l s have the capacity to p r o l i f e r a t e . The i n i t i a l anatomical l e s i o n appears to occur f i r s t i n g l i a l c e l l s i n the areas known to be most affected by thiamine deficiency such as the l a t e r a l v e s t i b u l a r nucleus ( C o l l i n s , 1967). These early lesions consist of swelling of both g l i a l c e l l s and the myelin sheath (Robertson et a l . , 1968) and may involve astrocytes more than other c e l l types (Watanabe and Kanabe, 1978). C o l l i n s and Converse (1970) noted also that - 130 - the Bergmann g l i a l f i b e r s associated with degenerating neurons of the cerebell a r molecular layer were the f i r s t to accumulate glycogen i n thiamine deficiency. Since g l i a appear to be the f i r s t structures to change and since they do not change equally i n a l l areas there may be fundamental differences i n thiamine dependence of various g l i a . This experiment exemplifies a type of research where concepts of g l i a l heterogeneity may be relevant to the int e r p r e t a t i o n of the data. M E T H O D Male Wister rats from Canadian Breeding Farms, weighing 300+12 gms, were given free access to water and commercially a v a i l a b l e thiamine d e f i c i e n t d i e t from N u t r i t i o n a l Biochemicals and were injected i n t r a p e r i t o n e a l l y with 0.5 mg/kg of pyrithiamine d a i l y . The rats were housed i n d i v i d u a l l y i n rooms with other rodents on a 12 hour on, 12 hour o f f l i g h t schedule. A l l rats were weighed d a i l y and gross behavioural changes were noted. When rats exhibited signs of ataxia and loss of r i g h t i n g r e f l e x , usually on day 10 or 11, they were either s a c r i f i c e d f or immediate use or were put on to a normal d i e t and given a few shots of thiamine hydrochloride (0.5 mg/kg i n t r a p e r i t o n e a l l y ) . These rats were kept u n t i l they had reattained t h e i r o r i g i n a l weights, upon which time they had regained t h e i r r i g h t i n g r e f l e x and had l o s t most of t h e i r ataxia. This was usually within two weeks. Rats f o r biochemical studies were s a c r i f i c e d by c e r v i c a l fracture. The brains were immediately removed and dissected - 131 - into eight regions: cerebellum, pons/medulla, neostriatum, midbrain, hypothalamus, thalamus, hippocampus, and cortex. Each t i s s u e sample was homogenized i n 0.3 ml or 10 volumes (whichever was less) of cold 0.25 M sucrose. Portions of the homogenate were used for determination of eit h e r CAT or GAD by methods described below. CAT was measured by a modification of the method of F. Fonnum (1969). 60-180 mg of tissue was activated by treatment with T r i t o n X-100 and then incubated with acetyl-coenzyme A l a b e l l e d with [C14]. The [14C] acetylcholine was absorbed onto an ion exchange column (IG50) and eluted with 3 ml of 4N ac e t i c acid. Radioactivity i n the eluant was counted. GAD a c t i v i t y was determined by a modification of the method of Lupien et a l . (1968). L-(1-[14C]}-Glutamic acid i s incubated with t i s s u e homogenates i n the presence of pyrrdoxal phosphase, and the [14C02] produced i s trapped on hyamine hydroxide soaked paper, and the r a d i o a c t i v i t y counted. Separate rats, s a c r i f i c e d by perfusion under deep barbituate anesthesia, were used for the GABA-T histochemistry which was done by a method a Van Gelder (1965) modified as follows. Rats anaethesized with sodium pentabarbital and perfused i n t r a c a r d i a l l y with 150 ml ice cold 0.1M phosphate buffer, pH 7.4, had t h e i r brains removed, kept i n 0.1M phosphate buffer, sectioned at 50 /Um on an Oxford Vibratome and stained for GABA-T by preincubating free f l o a t i n g sections i n the dark f o r 2 0 min. i n a reaction mixture containing 5.0 ml t r i s HCl 0.1M, 0.2 ml of 250 mg/ml alpha-ketoglutarate, 1.5 ml of a soluti o n containing 144 mg/ml NaCl, 2 0 mg/ml MgCl2, - 132 - and 1 mg/ml KCN, and 0.5ml 10 mg/ml NAD at pH 8.6. After the preincubation, 10 mg of n i t r o blue tetrazolium dissolved i n 2.5 ml dimethyl sulfoxide and 2.5 ml water, 9.5 ml of lmg/5ml phenazine methosulfate and 0.2 ml of 250 mg/ml GABA are added to the pre-incubation medium. The sections are incubated for 45 min. at 37°C. The reaction i s stopped with the transfer of these sections to 0.1M phosphate buffer. The sections are mounted on g e l a t i n coated s l i d e s , a i r dried, dehydrated i n xylene, and coverslipped with Permamount. RESULTS In symptomatic thiamine d e f i c i e n t (TD), pyrithiamine .treated r a t s GAD a c t i v i t i e s were found to be s i g n i f i c a n t l y decreased i n four areas of the brain: the thalamus > cerebellum > pons/medulla > midbrain (see Table VIIIA). After body weight had returned to pre-experimental l e v e l s , there was s i g n i f i c a n t recovery of GAD a c t i v i t y except i n the thalamus. GABA-T sta i n i n g was most dramatically reduced i n the thalamus (see F i g . 9a, 9b), and next i n the i n f e r i o r c o l l i c u l u s . There was some loss i n the pons and medulla, but no change i n other areas of the brain including the cerebellum. A f t e r return to a normal d i e t , there i s at least p a r t i a l recovery of staining i n a l l areas affected (see F i g . 9b, 9c). There was no s i g n i f i c a n t change i n CAT a c t i v i t y i n any brain area (see Table VIIIB). - 133 - DISCUSSION Our f i n d i n g of a s p e c i f i c loss of the two GABA related enzymes, GAD and GABA-T, i n several brain areas i s compatible with the findings of several other workers. I t i s compatible with the reduction of GABA i n whole brain (Gaitonde, 1975, Gubler et a l . , 1974) i n both PT and TD rats, and with the findings of reduced GABA concentrations i n PT rats i n the cerebellum (Butterworth et a l . , 1978, Butterworth, 1982a, Butterworth, 1982b), medulla/pons (Butterworth et a l . , 1978), and midbrain (Butterworth, 1982b). We d i d not, however, observe a decrease i n GABA-T or GAD i n cerebral cortex as Butterworth (1982b) did. Our findings are not compatible with those of P l a i t a k i s et a l . (1979) who found no change i n cerebellum or pons/medulla i n pyrithiamine treated r a t s . The thalamic changes i n GABA we observed had not been reported elsewhere, but the p e r i v e n t r i c u l a r region of the thalamus, where there i s a high density of presumptive GABAergic neurons (Nagai et a l . , 1983), i s an area which, l i k e the cerebellum and midbrain, have notable histopathology i n Korsakoff's syndrome. The fact that GAD remained reduced i n the thalamus of rats put on a normal d i e t with thiamine supplementation suggests some s t r u c t u r a l damage to GABAergic systems i n t h i s area. I t i s in t e r e s t i n g that the thalamus, which has the largest e f f e c t s of thiamine deficiency on GAD and GABA-T, i s also the region showing the largest drops i n GAD and GABA on aging (McGeer and McGeer, 1982). The intracytoplasmic inclusions - 134 - found i n the thalamus i n thiamine-deficient mice have been said to be morphologically indistinguishable from those i n aged mice (Aikawa et a l . , 1983). TABLE VIII: Enzyme Levels i n Control, Thiamine-Deficient and Recovered Rats ( moles/hr-100 mg protein; Mean+S.D.; number rats i n parentheses). Brain Area Controls (7) Thiamine Def.(7) Recovered (5) A. Glutamic Acid Decarboxylase Cerebellum 19. 39 + 1. 78 12. 14 + 1. 65# 17. 64 + 3. 25 Pons/Medulla 13. 24 + 1. 21 11. 38 + 0. 60** 12 . 92 + 1. 83 Neostriatum 15. 11 + 1. 06 12. 12 + 5. 60 14. 98 + 1. 24 Midbrain 19. 46 + 0. 95 13. 11 + 5. 21* 17. 74 + 3. 02 Hypothalamus 16. 28 + 1. 52 12. 13 + 3. 33 14. 26 + 2. 98 Thalamus 21. 34 + 1. 88 10. 02 + 2. 30# 14. 91 + 2. 99* Hippocampus 13 . 56 + 2. 15 13. 95 + 0. 97 13. 47 + 0. 85 Cortex 15. 56 + 2. 94 12 . 67 + 4. 29 13. 92 + 2. 84 Choline Acetyltransferase Cerebellum 1. 77 + 0. 26 1. 50 + 0. 22 1. 63 + 0. 24 Pons/Medulla 17. 90 + 2. 13 20. 32 + 2. 15 19. 74 + 1. 93 Neostriatum 33 . 06 + 5. 49 26. 93 + 2. 76 34. 32 + 3. 27 Midbrain 12. 08 + 1. 21 11. 54 + 1. 40 12. 09 + 1. 02 Hypothalamus 6. 19 + 0. 48 6. 93 + 0. 64 6. 37 + 0. 35 Thalamus 10. 54 + 1. 59 11. 08 + 1. 69 10. 91 + 0. 99 Hippocampus 11. 21 + 1. 34 10. 12 + 1. 35 11. 37 + 0. 85 Cortex 12. 76 + 3. 65 10. 90 + 2. 05 12 . 93 + 2. 64 #p<0.001, **p<0.001, *p<0.02 for comparison with controls. - 135 - The lack of change i n CAT during thiamine deficiency i s consistent with previous reports (Bhatgat and Lockett, 1962, Heinrich et a l . , 1973, Reddy, 1982, Sacchi et a l . , 1978, Thornber et a l . , 1980). This, combined with the f a c t that AChE i s also unaffected i n thiamine deficiency (Gibson et a l . , 1982, Takata et a l . , 1981) and that a decreased turnover of acetylcholine i s observed even i n animals showing normal l e v e l s of CAT (Sacchi et a l . , 1978, Thornber et a l . , 1980), i s consistent with the b e l i e f that the amount of enzyme i s not normally rate c o n t r o l l i n g and that factors such as decreased a v a i l a b i l i t y of acetyl coenzyme A (Vorhees et a l . , 1978) or an i n h i b i t o r y e f f e c t of thiamine deficiency on acetylcholine release (Dunant and Eder, 1983, Eder et a l . , 1976) may play important r o l e s . A hypothesis as to the mechanism of the losses i n GAD and GABA-T must take into account the regional s p e c i f i c i t y observed. A s p e c i f i c loss of GAD i s assumed to be because of destruction of GABAergic synaptosomes (Butterworth, 1982a) and perhaps of the GABA neurons themselves. I f i t i s assumed that only neurons are involved, i t i s hard to see why GABA neurons are not destroyed equally i n a l l areas. Why for instance i s GAD not s i g n i f i c a n t l y reduced i n the neostriatum or hippocampus where there are high concentrations of GABA neurons or interneurons? I t has been suggested that the most affected areas are those with high turnover rates of thiamine and high oxidative metabolism which i s dependent, for at least one step, on thiamine as a cofactor (Dreyfus, 1976). The - 136 - cerebellum i s one such area (Ritchie et a l . , 1980, 1984). Decreased a c t i v i t y of pyruvate dehydrogenase, which i s dependent on thiamine triphosphate as a coenzyme, would, for example, lead to decreased incorporation of glucose into amino acids and keto acids of both the TCA cycle and GABA shunt (Butterworth et a l . , 1978, Butterworth, 1982a). GAD a c t i v i t y , however, i s not known to be affected by precursor a v a i l a b i l i t y . Another hypothesis involving only neurons i s some interneuronal reaction. For example, the changes i n cer e b e l l a r GAD might be secondary to changes i n 5HT system which may innervate GABA neurons. Such a s i t u a t i o n has been suggested (Chan-Palay et a l . , 1977) i n the loss of serotonergic mossy f i b e r s i n contact with the cerebell a r Purkinje c e l l s which are GABAergic (Chan-Palay et a l . , 1977, Onodera et a l . , 1981, P l a i t a k i s et a l . , 1978a, P l a i t a k i s et a l . , 1978b, P l a i t a k i s et a l . , 1979). Thus serotonergic changes might lead to GABA changes and GAD changes and these serotonergic neurons are known to be susceptible to thiamine deficiency ( P l a i t a k i s et a l . , 1978a). This hypothesis could presumably be tested by examining GAD l e v e l s i n rats where lesions of the serotonergic neurons have been produced by other means such as 5,7-dihydroxytryttamine. The recovery of GAD i n our re s u l t s may indicate: biochemical reversal of changes which reduced the a c t i v i t y of GAD, regrowth of GABAergic synaptosomes containing GAD, or, i f GAD i s a c t u a l l y s e n s i t i v e to precursor a v a i l a b i l i t y , the recovery of precursors. GABA-T loss and recovery could be - 137 - i n d i c a t i v e of loss and recovery of synaptosomes which contain GABA-T to regulate GABA l e v e l s presynaptically. The regional s p e c i f i c i t y and p a r t i a l r e v e r s i b i l i t y of the changes i n GABAergic systems analyzed under t h i s scenario would indicate a possible r o l e of thiamine i n ei t h e r GABA neurons or afferents to such neurons; there i s no good explanation why a l l GABA neurons are not affected. I f g l i a l c e l l heterogeneity i s taken into account and combined with the observations that the f i r s t changes that occur i n the thiamine deficiency models are i n g l i a l c e l l s , then the in t e r p r e t a t i o n of our observations could be quite d i f f e r e n t . The recovery upon return of thiamine, a part of the d e f i n i t i o n of a biochemical l e s i o n as defined by Peters, could depend upon p r o l i f e r a t i o n of the remaining g l i a l c e l l s i n the damaged areas, or of g l i a l c e l l s from surrounding areas to restore normal g l i a l factors needed to support the GABAergic neurons. Since GABA-T i s i n g l i a as well as neurons, g l i a l p r o l i f e r a t i o n might help to explain the recoveries i n GABA-T. The second part of Peters' d e f i n i t i o n of the biochemical nature of thiamine deficiency, the s e l e c t i v e v u l n e r a b i l i t y of ce r t a i n regions, may not be due to regional differences i n neurons but be due to g l i a l heterogeneity. I t has been shown that only g l i a l c e l l s of c e r t a i n areas show early thiamine deficiency changes. A l l the areas where GABA-T loss and recovery were noted are areas where g l i a l c e l l damage occurs early; an anomaly i s the cerebellum where there are early g l i a l changes but no GABA-T losses. This area i s also the - 138 - only one i n which GAD losses and recovery do not seem to p a r a l l e l GABA-T changes. This may be because any loss of neuronal GABA-T i s concealed by GABA-T a c t i v i t y i n the Bergmann g l i a which seem to contain unusually high concentrations of t h i s enzyme which i s found i n both GABAergic neurons and g l i a (Nagai et a l . , 1983). In the cerebellum i t i s the g l i a l c e l l s of the molecular layer that are the f i r s t to change. Therefore the i n i t i a l biochemical l e s i o n may be i n sub-types of g l i a l c e l l s leading to regional g l i a l c e l l loss which i n turn causes changes i n neurons of the surrounding area. As reviewed i n the main body of t h i s thesis c e r t a i n types of g l i a appear to have the a b i l i t y to take up and metabolize glutamate and to form the glutamine required as a GABA precursor by GABAergic neurons. Changes i n these symbiotic g l i a might lead to changes i n the a c t i v i t y of GABA neurons. Butterworth (1982a) suggests that the types of g l i a l c e l l s may be important i n determining the s e l e c t i v e v u l n e r a b i l i t y of ce r t a i n areas and notes that g l i a l c e l l l i n e s are more susceptible to thiamine deficiency than are neuronal l i n e s . Thiamine pyrophosphatase a c t i v i t y was found to be very high i n the plasma membrane of microglia, and oligodendrocytes and astrocytes also had s i g n i f i c a n t s t aining i n the Golgi apparatus (Murabe and Sano, 1981) so an association between thiamine and g l i a has been made. Other people have also suggested key roles f o r g l i a i n thiamine deficiency. Butterworth (1982a) suggested that - 139 - s e l e c t i v e changes i n g l i a l c e l l i n t e g r i t y may explain GABA changes i n the l a t e r a l v e s t i b u l a r nucleus. He also postulated that the observed enhanced glutamate uptake i n early thiamine deficiency may be explained by the p r o l i f e r a t i o n of g l i a l c e l l s that occurs i n damaged areas. In conclusion, i f g l i a l c e l l heterogeneity i s assumed, the GABA enzyme changes we have observed may be the d i r e c t or i n d i r e c t r e s u l t of the early changes i n a subtype of g l i a l c e l l s . This then serves to i l l u s t r a t e an example of the types or research where concepts of g l i a l heterogeneity may be relevant to the int e r p r e t a t i o n of the r e s u l t s . - 140 - Figure 9. S a g i t t a l sections of rat brains (at 2.5 mm from midline) stained for GABA-T. A, Control; B, Thiamine-deficient; C, Recovered, th, thalamus; p, pons; i c , i n f e r i o r c o l l i c u l u s . - 141 - - 142 - C O N C L U S I O N I have i n t h i s thesis reviewed the data on many morphologically defined types of g l i a or g l i a l - l i k e c e l l s i n the brain. These c e l l types have variable marker staining, vary biochemically, have d i f f e r e n t develpment p r o f i l e s , and respond d i f f e r e n t l y to d i f f e r e n t culture condition and to injury. Culture work shows even more v a r i a b i l i t y . There are differences not only between c e l l l i n e s but between primary cultures from d i f f e r e n t areas of the brain i n c e l l s that are morphologically s i m i l a r . My experiments have added to t h i s picture. Experiment 1 showed that g l i a can s t a i n for iron with a d i s t i n c t regional pattern of density and types of c e l l s t a i ning. This i s just one more example of regional heterogeneity. Experiment 2 showed PDH, an enzyme only recently known to e x i s t , can be stained f o r i n a selected few g l i a l c e l l s . This would t h e o r e t i c a l l y indicate that an alternate route of glutamate synthesis e x i s t s i n these few selected g l i a l c e l l s . Experiment 3 i l l u s t r a t e s how assumptions on the existence of g l i a l heterogeneity may shed a d i f f e r e n t l i g h t on the int e r p r e t a t i o n of research data. There remains much research to be done on g l i a l heterogeneity. I foresee that i t i s highly probable that a complimentary map of s p e c i f i c g l i a functions w i l l be created with a complexity that may approach that now emerging for neurons. - 143 - ACKNOWLEDGEMENTS I would l i k e to thank a l l the members of the U.B.C. Di v i s i o n of Neurological Research who were a l l very h e l p f u l , e s p e c i a l l y my advisor Dr. Edie McGeer whose warmth and generosity meant a tremendous amount to me. I would also l i k e to thank the Huntington's Disease Society and the graduate student summer fund which supported me f i n a n c i a l l y , and my husband who typed the document. The work on the iron experiment was supported by the Medical Research Council of Canada. Dr. Y. Noda, a s c i e n t i s t from the Chugai Research Laboratories, Tokyo, Japan, greatly a s s i s t e d me with the iron research, and was the co-author of a paper submitted to J.Neurochem. that came out of t h i s work. I would l i k e to thank Dr. T.W. McBride for the use of h i s microscope. The p y r r o l i n e dehydrogenase experiments would not have been done without the o r i g i n a l suggestion from Peter Wong, who also collaborated with a paper published i n J.Neurochem. I was supported by the Garfield-Western Foundation and M.R.C. of Canada i n t h i s work. The thiamine experiment was supported by the M.R.C. of Canada and required the technical assistance of Mrs. Kim Singh. The data contained i n the portion of my thesis formed the basis for an abstract for the International Association of Neurochemists meeting i n Vancouver i n 1983 which was published by the Journal of Neurochemistry Vol. 41 supplement, and a paper published i n The Neurochemical Research. - 144 - REFERENCES Adams E. and F r a n k L. (1980) M e t a b o l i s m o f p r o l i n e and h y d r o x y p r o l i n e s . Ann. Rev. Biochem. 49, 1005-1061. Agr a w a l H.C. and Hartman B.K. 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