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Glutathione as a neurotransmitter in primary visual cortex : binding sites and neuronal uptake Bowlsby, Stephen 1991

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GLUTATHIONE AS A NEUROTRANSMITTER IN PRIMARY VISUAL CORTEX: BINDING SITES AND NEURONAL UPTAKE by STEPHEN BOWLSBY B . S c , The U n i v e r s i t y o f B r i t i s h C o lumbia, 1977 .F.A., The U n i v e r s i t y o f Southern C a l i f o r n i a , 1980 A THESIS SUBMITTED IN PARTIAL FULLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f P h y s i o l o g y We a c c e p t t h i s 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 September 1991 (c) Stephen A r t h u r Bowlsby, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia/ I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives, it is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^U*^S7 ft/tfCji^ The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT U n d e r s t a n d i n g t h e response p r o p e r t i e s and p l a s t i c i t y o f p r i m a r y v i s u a l ( s t r i a t e ) c o r t e x depends on t h e d e t e r m i n a t i o n o f i t s " c h e m i c a l c i r c u i t r y " , y e t t h e n e u r o t r a n s m i t t e r s t h a t m e d i a t e s e n s o r y i n p u t t o s t r i a t e c o r t e x a r e n o t known, and such e v i d e n c e as e x i s t s i s c o n t r a d i c t o r y . The g e n i c u l p s t r i a t e i n p u t b e a r s a s i m i l a r i t y t o g l u t a m a t e r g i c n e u r o t r a n s m i s s i o n , but g l u t a m a t e i s not a good c a n d i d a t e i n t h i s pathway. I n c o n t r a s t , g l u t a t h i o n e (GSH) has been s u g g e s t e d t o be one o f t h e e x c i t a t o r y - a m i n o - a c i d n e u r o t r a n s m i t t e r s i n c o r t e x ( O g i t a and Yoneda, 1987). C r i t e r i a f o r t h e i d e n t i f i c a t i o n o f a n e u r o t r a n s m i t t e r i n c l u d e t h e d e m o n s t r a t i o n o f t h e p r e s e n c e o f r e c e p t o r s f o r t h e s u b s t a n c e ( n e c e s s a r y ) and t h e d e m o n s t r a t i o n o f r e t r o g r a d e u p t a k e and t r a n s p o r t o f t h e s u b s t a n c e ( s u p p o r t i v e ) . The p r e s e n t s t u d y attempted t o c h a r a c t e r i z e GSH b i n d i n g s i t e s , examine t h e i r d i s t r i b u t i o n , and examine p o s s i b l e GSH i n t e r a c t i o n w i t h e x c i t a t o r y amino a c i d r e c e p t o r s i n r a t and c a t p r i m a r y v i s u a l c o r t e x u s i n g i n - v i t r o r a d i o l i g a n d methods on 20-/xm-thick c o r t e x s e c t i o n s . I n a d d i t i o n , t h e p r e s e n t s t u d y used i n - v i v o uptake and t r a n s p o r t o f r a d i o l a b e l e d t r a c e r s t o attempt t o s u p p o r t t h e l o c a l i z a t i o n o f GSH n e u r o t r a n s m i s s i o n t o pathways w i t h i n t h e v i s u a l system. S a t u r a t i o n b i n d i n g e x p e r i m e n t s u s i n g r a d i o l a b e l e d GSH i n r a t p r i m a r y o c c i p i t a l c o r t e x s e c t i o n s r e v e a l e d a h i g h -a f f i n i t y s i t e (K(j = 5.4 nM; B m a x = 235 fmol/mg p r o t e i n ) and a i i i d e n s e r l o w - a f f i n i t y s i t e (K^ = 1.3 /iM; B m a x = 1.3 pmol/mg p r o t e i n ) , t h e KQ< o f which i s t y p i c a l o f e x c i t a t o r y amino a c i d r e c e p t o r s . K i n e t i c and c o m p e t i t i o n e x p e r i m e n t s y i e l d e d s i m i l a r K<j v a l u e s . C o m p e t i t i o n s t u d i e s o f t h e l o w - a f f i n i t y GSH b i n d i n g s i t e showed a s e p a r a t e s i t e as w e l l as b i n d i n g w i t h a f f i n i t y f o r t h e n e u r o t r a n s m i t t e r c a n d i d a t e s c y s t e i n e , a s p a r t a t e , and g l u t a m a t e . E x c i t a t o r y - a m i n o - a c i d r e c e p t o r s u b t y p e a f f i n i t y was shown f o r AMPA a t pH 7.4 and f o r NMDA a t pH 6.9.. R a d i o l a b e l e d GSH b i n d i n g i n a d u l t r a t v i s u a l c o r t e x showed a r e l a t i v e l y u n i f o r m d i s t r i b u t i o n a c r o s s a l l c o r t i c a l l a y e r s . B i n d i n g d i s t r i b u t i o n s t u d i e s i n c a t s t r i a t e c o r t e x showed d e n s e s t [ 3 5 S ] G S H b i n d i n g i n l a y e r 4, t h e g e n i c u l o s t r i a t e i n p u t l a y e r , from 13 days p o s t n a t a l t o a d u l t . D i s t r i b u t i o n o f [ 3 5 S ] G S H b i n d i n g s i t e s a l s o showed a d i s t i n c t p r e f e r e n c e f o r lower l a y e r 4 i n monkey s t r i a t e c o r t e x . M i c r o i n j e c t i o n o f [ 3 5S]GSH, [ 3H]GSH, and i t s c o n s t i t u e n t amino a c i d s , [ 3 H ] g l u t a m a t e , [ 3 5 S ] c y s t e i n e , and [ 3 H ] g l y c i n e , i n t o p r i m a r y and secondary v i s u a l c o r t e x i n t h e r a t produced u p t a k e t o v i s u a l - s y s t e m t h a l a m i c n u c l e i and s u p e r i o r c o l l i c u l u s . P o s s i b l e r e t r o g r a d e uptake t o c e l l b o d i e s was d e t e r m i n e d f o r [ 3H]GSH and [ 3 5 S ] c y s t e i n e i n t h e d o r s a l l a t e r a l g e n i c u l a t e and l a t e r a l p o s t e r i o r n u c l e i i n t h e r a t . M i c r o i n j e c t i o n o f [ 3H]GSH i n c a t c o r t i c a l a r e a 17 produc e d u p t a k e t o t h e d o r s a l l a t e r a l g e n i c u l a t e n u c l e u s , b u t p o s s i b l e r e t r o g r a d e uptake t o c e l l b o d i e s c o u l d n o t be d e t e r m i n e d . These r e s u l t s s u p p o r t t h e p r o p o s i t i o n t h a t GSH p l a y s a r o l e as a n e u r o t r a n s m i t t e r i n p r i m a r y v i s u a l c o r t e x , i n i v p a r t i c u l a r as a g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r , and may t a k e p a r t i n n e u r o t r a n s m i s s i o n by i n t e r a c t i n g w i t h e x c i t a t o r y amino a c i d r e c e p t o r s i n a d d i t i o n t o GSH r e c e p t o r s . V TABLE OF CONTENTS page ABSTRACT i i TABLE OF CONTENTS V LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ANATOMICAL ABBREVIATIONS X ACKNOWLEDGEMENTS x i INTRODUCTION 1 Chemical C i r c u i t r y and V i s u a l Cortex 1 The G e n i c u l o s t r i a t e Neurotransmitter 8 An I n s t r u c t i v e Case — Neurotransmission w i t h NAAG 17 G l u t a t h i o n e i n B r a i n 21 E x c i t a t o r y Amino A c i d Receptors and T r a n s m i t t e r s 25 Ne u r o t r a n s m i t t e r C r i t e r i a 29 T h e s i s R a t i o n a l e 31 MATERIALS AND METHODS 36 Ligands and T r a c e r s 3 6 In V i t r o Receptor B i n d i n g Procedures 3 6 B i n d i n g S i t e A n a l y s i s 41 In V i v o Uptake Procedures 49 RESULTS 52 Para m e t r i c S t u d i e s 52 B i n d i n g S i t e C h a r a c t e r i z a t i o n 70 E x c i t a t o r y Amino A c i d Receptor I n t e r a c t i o n s 84 B i n d i n g S i t e D i s t r i b u t i o n s 89 Uptake Experiments In Viv o v 98 v i page DISCUSSION 107 B i n d i n g S i t e C h a r a c t e r i z a t i o n 107 Laminar D i s t r i b u t i o n 112 Pathways 115 A s t r o c y t e s 120 E x c i t a t o r y Amino A c i d R e c e p t o r I n t e r a c t i o n s 122 S p e c u l a t i o n — A N o v e l Form o f N e u r o t r a n s m i s s i o n ? 125 SUMMARY AND CONCLUSIONS 128 REFERENCES 130 v i i LIST OF TABLES page N u c l e i showing p r e s e n c e o f r a d i o l a b e l from u p t a k e . 101 V l l l LIST OF FIGURES page 1. A s c h e m a t i c d i s t r i b u t i o n o f t h e l a m i n a r b i n d i n g p a t t e r n s i n n e o n a t a l k i t t e n s and a d u l t c a t s . 6 2. Diagram showing t h e t e r m i n a t i o n o f a f f e r e n t f i b r e s and t h e o r i g i n o f e f f e r e n t f i b r e s i n g e n e r a l i z e d n e o c o r t e x . 10 3. T e r m i n a t i o n s o f X - c e l l and Y - c e l l e f f e r e n t s i n a r e a 17 o f t h e c a t . 11 4. Diagram o f t h e l a m i n a r d i s t r i b u t i o n o f t h a l a m i c a f f e r e n t s t o c a t and monkey a r e a 17. 12 5. The c h e m i c a l s t r u c t u r e o f reduced g l u t a t h i o n e and o x i d i z e d g l u t a t h i o n e d i s u l f i d e . 20 6. An o u t l i n e o f t h e metabolism and f u n c t i o n o f g l u t a t h i o n e . 24 7. B i n d i n g o f [ H]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n o f c o n c e n t r a t i o n o f b u f f e r . 54 8. B i n d i n g o f [ H]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n o f pH. 54 9. B i n d i n g o f [ 3H]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n b u f f e r t y p e . 56 10. B i n d i n g o f [ 3H]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n o f i o n c o n c e n t r a t i o n . 57 11. B i n d i n g o f [ H]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n o f f i x a t i o n . 61 12. B i n d i n g o f [ 3H]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f v a r i o u s b i o c h e m i c a l a g e n t s . 62 13. S e p a r a t i o n o f d i s u l f i d e bonds by t h e r e d u c i n g a c t i o n o f d i t h i o t h r e i t o l , d u r i n g w h i c h t h e d i t h i o t h r e i t o l i s i n a c t i v a t e d . 68 14. B i n d i n g o f L - [ 3 H ] g l u t a m a t e and [ 3H]CPP i n r a t b r a i n membranes as a f u n c t i o n o f pH. 69 15. A s s o c i a t i o n and d i s s o c i a t i o n r a t e s o f r a d i o l a b e l e d GSH b i n d i n g i n r a t v i s u a l c o r t e x . 71 16. C o m p e t i t i o n c u r v e s f o r [ 3H]GSH b i n d i n g i n a d u l t r a t and c a t v i s u a l c o r t e x . 76 i x 17. Competition of GSH with 18 nM [ 3 5S]GSH, and i t s d e r i v e d "B versus B*I" p l o t t o r e v e a l the sepa r a t e v a l u e s . 78 18. S a t u r a t i o n b i n d i n g curves and Eadie-Hofstee p l o t s o f [ 3H]GSH and [ 3 5S]GSH b i n d i n g s i t e s i n r a t v i s u a l c o r t e x . 82 19. Competition by GSH with [ 3H]GSH a t 20 minutes i n c u b a t i o n (pH 7.4) t o reduce b i n d i n g t o the dense, nonreceptor, LAHC s i t e , the presence of which would obscure displacements. 86 20. Percent c o n t r o l b i n d i n g of L-[ 3H]glutamate i n r a t v i s u a l c o r t e x a t pH 8.0 compared t o b i n d i n g a t pH 6.9 and a t pH 6.9 wit h v a r i o u s 1 mM co m p e t i t o r s . 87 21. Percent c o n t r o l b i n d i n g of [ 3H]GSH i n r a t v i s u a l c o r t e x with s i g n i f i c a n t displacement by v a r i o u s l mM competitors a t pH 7.4 and a t pH 6.9. 88 22. A u t o r a d i o g r a p h i c d i s t r i b u t i o n s of [ 3 5S]GSH b i n d i n g i n r a t , c a t , and monkey v i s u a l c o r t e x . 91 23. Q u a n t i t a t i v e a n a l y s i s of laminar b i n d i n g d e n s i t y on a u t o r a d i o g r a p h i c f i l m a c r o s s c a t area 17 c o r t e x a t v a r i o u s ages. 96 24. A u t o r a d i o g r a p h i c d i s t r i b u t i o n of r a d i o l a b e l e d t r a c e r uptake t o the thalamus from i n j e c t i o n i n v i s u a l c o r t e x i n r a t s . 102 25. Photomicrographs of combined p y r o n i n Y s t a i n i n g f o r c e l l bodies and a u t o r a d i o g r a p h i c d i s t r i b u t i o n of s i l v e r g r a i n s f o l l o w i n g uptake of [ 3H]GSH from r a t v i s u a l c o r t e x t o the DLG. 106 X LIST OF ANATOMICAL ABBREVIATIONS ATPD a n t e r i o r p r e t e c t a l nucleus, d o r s a l d i v i s i o n CP c e r e b r a l peduncle DLG d o r s a l l a t e r a l g e n i c u l a t e nucleus IGL i n t e r g e n i c u l a t e l e a f IMA i n t r a m e d u l l a r y t h a l a m i c area LP l a t e r a l p o s t e r i o r nucleus LPLR l a t e r a l p o s t e r i o r nucleus, l a t e r a l r o s t r a l d i v i s i o n LPMC l a t e r a l p o s t e r i o r nucleus, medial c a u d a l d i v i s i o n LPMR l a t e r a l p o s t e r i o r nucleus, medial r o s t r a l d i v i s i o n O c l primary o c c i p i t a l c o r t e x Oc2 secondary o c c i p i t a l c o r t e x Oc2L secondary o c c i p i t a l c o r t e x , l a t e r a l OC2ML secondary o c c i p i t a l c o r t e x , m e d i o l a t e r a l OC2MM secondary o c c i p i t a l c o r t e x , mediomedial P a r i p a r i e t a l c o r t e x , area 1 RSA r e t r o s p l e n i a l agranular c o r t e x RSG r e t r o s p l e n i a l g r a n u l a r c o r t e x Rt r e t i c u l a r t h a l a m i c nucleus sc s u p e r i o r c o l l i c u l u s , s u p e r i o r and i n f e r i o r grey SubG s u b g e n i c u l a t e nucleus VLG v e n t r a l l a t e r a l g e n i c u l a t e nucleus VLGMC v e n t r a l l a t e r a l g e n i c u l a t e nucleus, m a g n o c e l l u l a r VLGPC v e n t r a l l a t e r a l g e n i c u l a t e nucleus, p a r v o c e l l u l a r ZI zona i n c e r t a ZID zona i n c e r t a , d o r s a l ZIV zona i n c e r t a , v e n t r a l x i ACKNOWLEDGEMENTS I would l i k e t o e x t e n d my thanks and a p p r e c i a t i o n t o : Dr. C h r i s Shaw f o r p r o v i d i n g t h e o p p o r t u n i t y o f w o r k i n g i n h i s l a b and f i n d i n g out what r e s e a r c h i s a l l about; f o r h i s a s s i s t a n c e and s u p p o r t i n c o m p l e t i n g t h i s t h e s i s ; and f o r p r o v i d i n g t h e c o s t l y r a d i o l a b e l e d c h e m i c a l s . Dr. S t e v e n K e h l , D e r r i c k March, Mala G l e n w r i g h t , and John Sanker f o r t e c h n i c a l a s s i s t a n c e . Dr. Ken B a i m b r i d g e and Dr. W i l l i a m Ovale f o r use o f t h e i r c r y o s t a t microtomes. Dr. S t e v e n V i n c e n t f o r use of h i s computer image a n a l y s i s system. Dr. Ken C u r r y f o r h e l p f u l comments. Dr. Ray Pederson f o r h i s s u p p o r t as g r a d u a t e a d v i s o r . My w i f e J o y c e f o r f i n a n c i a l s u p p o r t when t h e s t u d e n t l o a n s u n e x p e c t e d l y d r i e d up. 1 INTRODUCTION CHEMICAL CIRCUITRY AND VISUAL CORTEX To u n d e r s t a n d t h e b r a i n , and t o be a b l e t o i n t e r v e n e on i t s b e h a l f , i t i s n e c e s s a r y t o model t h e b r a i n ' s a c t i v i t y (McNaughton and M o r r i s , 1987; Z i p s e r and Andersen, 1988; Ambros-Ingerson and Lynch, 1990; S e r v a n - S c h r e i b e r e t a l . , 1990). Knowledge o f a n a t o m i c a l c o n n e c t i v i t y i s i n s u f f i c i e n t t o do t h i s , s i n c e neurons a r e not s i m p l e t h r e s h o l d d e v i c e s . A major r e a s o n f o r t h i s i s t h e c o m p l e x i t y o f c h e m i c a l n e u r o t r a n s m i s s i o n . To u n d e r s t a n d t h e c o m p u t a t i o n s p e r f o r m e d by any p a r t o f t h e b r a i n i t i s n e c e s s a r y t o know t h e i d e n t i t i e s o f t h e n e u r o t r a n s m i t t e r s and r e c e p t o r s u b t y p e s employed a t each synapse. C h e m i c a l n e u r o t r a n s m i s s i o n produces a wide range o f e f f e c t s . V a r i o u s i o n conductances and second-messenger systems can be a c t i v a t e d by d i f f e r e n t r e c e p t o r s u b t y p e s f o r t h e same n e u r o t r a n s m i t t e r , depending on t h e pathway s t u d i e d ( f o r r e v i e w see N i c o l l , 1988). R e c e p t o r a c t i v a t i o n may produce a f a s t s y n a p t i c p o t e n t i a l change, o r i t may a l t e r , o v e r a l o n g e r t i m e span, t h e p r o p e r t i e s o f v o l t a g e - d e p e n d e n t i o n i c c o n d u c t a n c e s t h a t a r e i n v o l v e d i n c o n t r o l o f c e l l e x c i t a b i l i t y , r e s u l t i n g i n such changes as i n c r e a s e d a c t i o n p o t e n t i a l d u r a t i o n , changes i n f i r i n g f r e q u e n c y and f i r i n g p a t t e r n , and i n c r e a s e d C a 2 + e n t r y d u r i n g an a c t i o n p o t e n t i a l ( f o r r e v i e w see Kaczmarek and L e v i t a n , 1987). R e c e p t o r 2 p o p u l a t i o n s may be u p - r e g u l a t e d o r d o w n - r e g u l a t e d by t h e i r own a c t i v a t i o n o r by t h e a c t i v a t i o n o f o t h e r r e c e p t o r s , and r a t e s o f r e g u l a t i o n may be modulated by o t h e r n e u r o t r a n s m i t t e r s ( f o r r e v i e w see H o l l e n b e r g , 1985). A c t i v a t i o n o f one r e c e p t o r s i t e may d i r e c t l y modulate o r c o -a c t i v a t e t h e e f f e c t s o f a r e c e p t o r s i t e f o r a d i f f e r e n t n e u r o t r a n s m i t t e r ( f o r example, see Thompson e t a l . , 1989). The second-messengers from a c t i v a t i o n o f d i f f e r e n t r e c e p t o r s by d i f f e r e n t n e u r o t r a n s m i t t e r s may converge on t h e same i o n i c c o n d u c t a n c e , even as each d i v e r g e s t o a f f e c t s t i l l o t h e r c o n d u c t a n c e s (see r e v i e w by N i c o l l , 1988). Long t e r m s y n a p t i c s t r e n g t h s may be a l t e r e d , f o r example by t h e a c t i o n i n i t i a t e d by a v o l t a g e - d e p e n d e n t r e c e p t o r ( A r t o l a and S i n g e r , 1987) . N e u r o t r a n s m i s s i o n may a f f e c t gene e x p r e s s i o n ( f o r r e v i e w see Morgan and C u r r a n , 1989), and n e u r o t r a n s m i t t e r s may a c t as i n d u c e r s d u r i n g development t o a f f e c t b i o c h e m i c a l (Moran and P a t e l , 1989) and s t r u c t u r a l ( r e v i e w e d by L i p t o n and K a t e r , 1989) d i f f e r e n t i a t i o n . Widespread p a r a c r i n e e f f e c t s may even be i m p o r t a n t i n t h e c o m p u t a t i o n s p e r f o r m e d by t h e b r a i n (Herkenham, 1987). F i n a l l y , d i f f e r e n t n e u r o t r a n s m i t t e r s can be c o - r e l e a s e d ( f o r example see Lundberg and H o k f e l t , 1983) and r e c e p t o r s may a c t a t axo-a x o n i c and d e n d r o s o m a t i c synapses (Cheramy and G l o w i n s k i , 1981) and as p r e s y n a p t i c a u t o r e c e p t o r s (see McGeer e t a l . , 1987) . The v i s u a l c o r t e x t r a n s f o r m s t h e p r o p e r t i e s o f i t s t h a l a m i c i n p u t t o g e n e r a t e a wide v a r i e t y o f f u n c t i o n a l 3 properties required for v i s i o n . Each c e l l i n the v i s u a l cortex has a receptive f i e l d where s t i m u l i of the appropriate orientation, shape, and d i r e c t i o n e l i c i t a discharge. Recently i t has become possible to make a d i r e c t association between i n d i v i d u a l c o r t i c a l connections and p a r t i c u l a r receptive f i e l d properties by the pharmacological "microdissection" of c o r t i c a l c i r c u i t s using neurotransmitter receptor antagonists. For example, l o c a l iontophoresis of b i c u c u l l i n e , an antagonist of the i n h i b i t o r y neurotransmitter gamma-aminobutyric acid (GABA) at GABA^ receptors, abolishes o r i e n t a t i o n and d i r e c t i o n a l s e l e c t i v i t y (reviewed i n Bolz, et a l . , 1988). Here the "chemical c i r c u i t r y " i s beginning to be understood. Experience-dependent synaptic p l a s t i c i t y i s c r u c i a l to the development of the capacities of the v i s u a l system, and receptor a c t i v a t i o n plays a dominant r o l e (for review see Cynader et a l . , 1990). For example, competetive binocular interactions occur within a c r i t i c a l period that peaks i n kit t e n s at approximately 4 weeks of age (Hubel and Weisel, 1970). At t h i s time, ey e l i d closure for as b r i e f a period as a few hours produces profound and prolonged e f f e c t s on both v i s u a l capacities and c o r t i c a l neuronal responses. Axon terminals from dorsal l a t e r a l geniculate nucleus r e l a y c e l l s connected to the deprived eye are found to occupy 20 % of the area of the cortex while the undeprived eye has an expanded representation covering 80 % of the thalamic r e c i p i e n t zone. Blockade of neurotransmission by the cholinergic and 4 n o r a d r e n e r g i c n o n - s p e c i f i c a f f e r e n t s t o v i s u a l c o r t e x s u b s t a n t i a l l y r e d u c e s t h i s o c c u l a r dominance s h i f t (Bear and S i n g e r , 1986). S i m i l a r l y , b l o c k a d e o f N - m e t h y l - D - a s p a r t a t e (NMDA) r e c e p t o r s on l a t e r a l g e n i c u l a t e c e l l s w i t h s p e c i f i c a n t a g o n i s t s d u r i n g t h e p e r i o d o f su b l a m i n a f o r m a t i o n p r e v e n t s r e t i n a l a f f e r e n t s from s e g r e g a t i n g i n t o "On" and " O f f " s u b l a m i n a e (Hahm e t a l . , 1991). I n f u s i o n o f t h e GABA^ r e c e p t o r a g o n i s t muscimol i n v i s u a l c o r t e x d u r i n g monocular e x p o s u r e produces an i n c r e a s e i n e f f e c t i v e n e s s o f t h e d e p r i v e d eye a t t h e expense o f t h e n o r m a l l y v i e w i n g eye ( R e i t e r and S t r y k e r , 1988). O c c u l a r dominance s h i f t s due t o monocular d e p r i v a t i o n a r e a s s o c i a t e d w i t h an i n c r e a s e i n GABA^ r e c e p t o r s i n v i s u a l c o r t e x (Shaw and Cynader, 1988) . Most n e u r o t r a n s m i t t e r r e c e p t o r s a r e found t o have an i d i o m o r p h i c l a m i n a r d i s t r i b u t i o n i n v i s u a l c o r t e x , c o r r e s p o n d i n g t o s p e c i f i c i n p u t / o u t p u t zones (see b e l o w ) , and changes i n t h e s e d i s t r i b u t i o n s a r e a s s o c i a t e d w i t h t h e c r i t i c a l p e r i o d f o r s y n a p t i c p l a s t i c i t y ( f o r r e v i e w see Shaw e t a l . , 1986; Cynader e t a l , 1990). F i g u r e 1 shows t h e s e d i s t r i b u t i o n s f o r s e l e c t e d r e c e p t o r s , and t h e i r change i n d i s t r i b u t i o n o v e r t h e c r i t i c a l p e r i o d . Some o f t h e s e r e c e p t o r s have been shown t o be r e - e x p r e s s e d on d i f f e r e n t neurons (Van H u i z e n e t a l . , 1988), and from p o s t - s y n a p t i c t o p r e s y n a p t i c elements (Shaw e t a l . , 1989b). The s i g n i f i c a n c e o f t h e s e changes i n r e c e p t o r d i s t r i b u t i o n i s n o t known, b u t t h e y emphasize t h e importance o f d e t e r m i n i n g and u n d e r s t a n d i n g t h e c h e m i c a l c i r c u i t r y o f t h e v i s u a l c o r t e x . 5 To u n d e r s t a n d c o m p u t a t i o n , p l a s t i c i t y , and development i n b r a i n , and i n p a r t i c u l a r i n p r i m a r y v i s u a l c o r t e x , a c o m p l e t e mapping of t h e n e u r o t r a n s m i t t e r s i n each pathway and t h e i r c o r r e s p o n d i n g s y n a p t i c e f f e c t s must be made. The g e n i c u l s t r i a t e n e u r o t r a n s m i t t e r i s a major one o f t h e s e . 6 F i g u r e 1. A s c h e m a t i c d i s t r i b u t i o n o f t h e l a m i n a r b i n d i n g p a t t e r n s s t u d i e d i n n e o n a t a l k i t t e n s and i n a d u l t c a t s . The t o p row o f t h e f i g u r e l i s t s t h e c l a s s e s o f n e u r o t r a n s m i t t e r s whose r e c e p t o r s have been examined. These i n c l u d e a c e t y l c h o l i n e (ACh), a d r e n a l i n e (R), o p i a t e s (OP), p e p t i d e s (PEP) and a d e n o s i n e (AD) w i t h i n t h e b road c l a s s o f n e u r o m o d u l a t o r y t r a n s m i t t e r s . The b road c l a s s o f amino a c i d t r a n s m i t t e r s i n c l u d e s t h e e x c i t a t o r y amino a c i d s , r e p r e s e n t e d by g l u t a m a t e (GLUT), and t h e i n h i b i t o r y amino a c i d s , r e p r e s e n t e d by gamma-aminobutyric a c i d (GABA). The v a r i o u s r e c e p t o r s u b t y p e s a r e l i s t e d i n t h e row below. The t r i t i a t e d l i g a n d employed i s l i s t e d i n t h e subsequent row. The c o r t e x i s d i v i d e d i n t o s i x l a y e r s and t h e d e n s i t y o f t h e b i n d i n g i s i l l u s t r a t e d u s i n g t h e s h a d i n g s c a l e g i v e n a t t h e bottom o f t h e f i g u r e . I n n e o n a t a l k i t t e n s t h e most common p a t t e r n s o f b i n d i n g o b s e r v e d a r e e i t h e r l i t t l e o r no d i s c e r n a b l e b i n d i n g ( n i c o t i n e , o x y t r e m o r i n e , b a c l o f e n ) , o r a pronounced peak i n l a y e r 4. I n a d u l t a n i m a l s , t h e p a t t e r n i s v e r y d i f f e r e n t , w i t h r e l a t i v e l y few b i n d i n g s i t e s ( n i c o t i n e , m u s c i m o l , and f l u n i t r a z e p a m ) c o n c e n t r a t e d i n l a y e r 4. Most r e c e p t o r s show a d i s t r i b u t i o n e m p h a s i z i n g l a y e r s 1 t h r o u g h 3 and 6. (From Cynader e t a l . , 1990, a f t e r an e a r l i e r v e r s i o n i n Shaw e t a l . , 1986) 1 - 3 DAY OLD Neuromodulators AA Transmitters NT/NM Subtype ACh 0 OP. PEP. AO. GLUT. GABA Ca4* N M 2 M, Al Q K A BZ B in < o o co m < >< o < • 0. c' (0 N OJ 6 x ^ z I 10 O I Z ID z. Z O a. 2 O Q Z o o o < 2 LL m a A D U L T Relative Binding Densities Very high High m Moderate No Light 8 THE GENICULOSTRIATE NEUROTRANSMITTER A r e a 17, t h e c o r t i c a l r e t i n o t o p i c map t h a t r e c e i v e s t h e m a j o r i t y o f t h e v i s u a l s e n s o r y i n p u t , i s commonly r e f e r r e d t o as t h e s t r i a t e c o r t e x because i n p r i m a t e s a w h i t e band i n upper l a y e r 4, t h e l i n e o f G e n n a r i , g i v e s a r e a 17 a s t r i a t e d a ppearance (Lund, 1973). The main t h a l a m i c i n p u t t o a r e a 17, w h i c h o r i g i n a t e s from t h e d o r s a l l a t e r a l g e n i c u l a t e n u c l e u s (DLG), i s t h u s r e f e r r e d t o as t h e g e n i c u l o s t r i a t e p r o j e c t i o n o r pathway ( f o r r e v i e w see G i l b e r t , 1983; Stone, 1983). The l i n e o f G e n n a r i b e a r s l i t t l e r e l a t i o n s h i p t o t h e a r e a o f t e r m i n a t i o n o f g e n i c u l a t e a f f e r e n t s and most p r o b a b l y r e p r e s e n t s m y e l i n a t e d i n t r i n s i c axons (Lund, 1973) . A l t h o u g h c a t a r e a 17, w h i c h has no l i n e o f G e n n a r i (Stone, 1983) i s commonly r e f e r r e d t o as s t r i a t e c o r t e x , t h e p r i m a r y v i s u a l c o r t e x o f t h e r a t (Ocl) i s n o t . For s i m p l i c i t y , however, i n t h i s t h e s i s t h e g e n i c u l a t e i n p u t t o p r i m a r y v i s u a l c o r t e x w i l l be r e f e r r e d t o as t h e g e n i c u l o s t r i a t e p r o j e c t i o n o r pathway i n t h e r a t as w e l l . A r e a 17 i s h i g h l y o r g a n i z e d ( f o r r e v i e w see Lund, 1973; G i l b e r t , 1983; Stone, 1983; S e f t o n and D r e y e r , 1986). Neurons a r e a r r a n g e d i n columns e x t e n d i n g from p i a t o w h i t e m a t t e r h a v i n g common r e c e p t i v e f i e l d p r o p e r t i e s w i t h i n each column. A h o r i z o n t a l o r g a n i z a t i o n i s a l s o e v i d e n t c o r r e s p o n d i n g t o c o r t i c a l l a y e r i n g s e p a r a t i n g s i m p l e r monocular r e s p o n s e s i n l a y e r 4 from more complex b i n o c u l a r r e s p o n s e s i n l a y e r s 2, 3, 5 and 6. The l a y e r s a l s o 9 c o r r e s p o n d t o r e s t r i c t e d i n p u t and o u t p u t zones. F i g u r e 2 i l l u s t r a t e s t h i s w i t h a s c h e m a t i c diagram t h a t g e n e r a l l y h o l d s t r u e f o r a l l n e o c o r t e x . L a y e r 4 i s t h e p r i m a r y i n p u t l a y e r , where a f f e r e n t s synapse m a i n l y on s p i n y s t e l l a t e c e l l s c o n f i n e d t o t h i s l a y e r , and on a p i c a l d e n d r i t e s o f p y r a m i d a l c e l l s o f l o w e r l a y e r s . L a y e r 2 and 3 p y r a m i d a l c e l l s make c o r t i c a l a s s o c i a t i o n a l and c o m m i s s u r a l p r o j e c t i o n s . L a y e r 5 p y r a m i d a l c e l l s i n a r e a 17 p r o j e c t t o t h e s u p e r i o r c o l l i c u l u s (SC) and t h e p u l v i n a r o r i t s homolog t h e l a t e r a l p o s t e r i o r n u c l e u s (LP) i n t h e r a t , and L P / p u l v i n a r complex i n t h e c a t . L a y e r 6 neurons i n a r e a 17 p r o j e c t back t o t h e DLG and t o t h e r e t i c u l a r t h a l a m i c n u c l e u s ( R t ) . Other i n p u t s i n c l u d e c o r t i c o c o r t i c a l a f f e r e n t s , w h i c h g e n e r a l l y r a m i f y i n t h e s u p e r f i c i a l l a y e r s ; c a l l o s a l a f f e r e n t s c o n f i n e d t o t h e a r e a 17/18 b o r d e r ; and b r a i n s t e m m o d u l a t o r y a f f e r e n t s such as t h o s e m e d i a t e d by s e r o t o n i n o r n o r a d r e n a l i n e w h i c h have t h e i r own d i s t i n c t i v e l a m i n a r d i s t r i b u t i o n s i n a r e a 17. O t h e r p r o j e c t i o n s e x i s t t o v a r i o u s s u b c o r t i c a l n u c l e i from b o t h a r e a 17 and a r e a 18 (secondary v i s u a l c o r t e x ) . The predominant zone f o r s e n s o r y i n p u t n e u r o t r a n s m i s s i o n i n v i s u a l c o r t e x i s l a y e r 4 i n a r e a 17 ( O c l ) , because t h i s i s where most of t h e t h a l a m i c t e r m i n a l s synapse, but t h e r e a r e m i n o r e x c e p t i o n s . F i g u r e 3 shows examples o f t h e dense t e r m i n a l a r b o r a t i o n s f o r t h e two main p h y s i o l o g i c a l t y p e s o f g e n i c u l a t e i n p u t s i n t h e c a t . F i g u r e 4 diagrams t h e l a y e r s o f t e r m i n a t i o n s o f a l l t h e v a r i o u s t h a l a m i c i n p u t s t o a r e a 17 i n t h e c a t and monkey. The X and Y g e n i c u l a t e r e l a y 10 Layers NEOCORTEX Afferents Efferents 1 Molecular 2 External granular External pyramidal 4 Internal granular 5 Internal pyramidal V Projection Association and commissural F i g u r e 2. Diagram showing the t e r m i n a t i o n of a f f e r e n t f i b r e s and the o r i g i n of e f f e r e n t f i b r e s i n g e n e r a l i z e d neocortex. (From Changeux, 1985) 11 F i g u r e 3. T e r m i n a t i o n s o f X - c e l l and Y - c e l l e f f e r e n t s i n a r e a 17 o f t h e c a t . The upper diagram shows t h e mode o f t e r m i n a t i o n i n a r e a 17 o f an axon o f a Y - c l a s s g e n i c u l a t e r e l a y c e l l . The axons spr e a d p r i n c i p a l l y i n l a y e r IVab, f o r m i n g two c l u s t e r s t h a t may c o r r e s p o n d t o o c c u l a r dominance columns. A few s m a l l branches e x t e n d i n t o l a y e r I I I , and t h e axon a l s o g i v e s c o l l a t e r a l s t o t h e upper p a r t o f l a y e r V I . The l o w e r diagram shows t h e mode o f t e r m i n a t i o n i n a r e a 17 o f an axon o f an X - c l a s s g e n i c u l a t e r e l a y c e l l . The axon t e r m i n a t e s p r i n c i p a l l y i n l a y e r I V c , a l t h o u g h a few s m a l l b r a n c h e s e x t e n d i n t o l a y e r IVab. T h i s axon a l s o g i v e s c o l l a t e r a l s t o t h e upper p a r t o f l a y e r V I . (From F e r s t e r and LeVay, 1978) 12 F i g u r e 4. Diagram o f t h e l a m i n a r d i s t r i b u t i o n o f t h a l a m i c a f f e r e n t s i n c a t and monkey ar e a 17. The C l a m i n a e i n c a t l a t e r a l g e n i c u l a t e n u c l e u s c o n t a i n Y- and W - c e l l s . P a r v o c e l l u l a r and m a g n o c e l l u l a r l a y e r s o f t h e monkey l a t e r a l g e n i c u l a t e c o n t a i n Y- and X - c e l l s , r e s p e c t i v e l y . LP i n t h e d i a g r a m i n c a t i s t h e L P / p u l v i n a r complex, homologous t o t h e monkey p u l v i n a r . (From G i l b e r t , 1983) 13 p r o j e c t i o n s i n t h e c a t and t h e p a r v o c e l l u l a r and m a g n o c e l l u l a r g e n i c u l a t e r e l a y p r o j e c t i o n s i n t h e monkey c o r r e s p o n d t o t h e p h y s i o l o g i c a l l y d e f i n e d r e t i n a l g a n g l i o n X c e l l ( t o n i c , s m a l l r e c e p t i v e f i e l d s , slow) and Y c e l l ( p h a s i c , l a r g e f i e l d s , f a s t ) a f f e r e n t s . The L P / p u l v i n a r a c t s as a r e l a y f o r r e t i n a l i n p u t but p r o j e c t s t o a r e a 17 o n l y s p a r c e l y . The DLG and and LP a l s o p r o j e c t t o a r e a 18 i n r a t , c a t , and monkey, a l t h o u g h much l e s s d e n s e l y t h a n t h e g e n i c u l o s t r i a t e p r o j e c t i o n . D i f f e r e n c e s e x i s t between s p e c i e s ( F i g u r e 4 ) . The p r i m a t e p r i m a r y v i s u a l c o r t e x i s more s h a r p l y d i v i d e d i n t o a s e r i e s o f c l e a r l y l i m i t e d laminae t h a n i t i s i n t h e c a t o r r a t . W-type g e n i c u l a t e r e l a y c e l l s e x i s t i n c a t and r a t . The DLG shows p r o j e c t i o n s t o a l l l a y e r s i n t h e c a t , b u t n o t so i n t h e monkey. I n t h e r a t t h e r e a r e p r a c t i c a l l y no X - l i k e c e l l s , g e n i c u l a t e r e l a y c e l l s a r e not a g g r e g a t e d i n t o l a m i n a e , t h e DLG does not p r o j e c t t o l a y e r 5 as i t does i n c a t , and t h e LP p r o j e c t s t o l a y e r s 5 and 6. The n e u r o t r a n s m i t t e r s m e d i a t i n g s e n s o r y i n p u t t o p r i m a r y v i s u a l c o r t e x , whether i n t h e r a t , c a t , o r monkey, i n c l u d i n g t h e g e n i c u l o s t r i a t e i n p u t , a r e unknown, and what e v i d e n c e does e x i s t i s c o n t r a d i c t o r y . Glutamate i s p r o p o s e d as a n e u r o t r a n s m i t t e r i n v i s u a l c o r t e x ( r e v i e w e d by Tsumoto, 1990). Glutamate a c t i v a t e s s e v e r a l p h a r m a c o l o g i c a l l y - d i s t i n c t r e c e p t o r s u b t y p e s known as e x c i t a t o r y amino a c i d (EAA) r e c e p t o r s , but so do s e v e r a l o t h e r endogenous s u b s t a n c e s , and t h e a c t u a l n e u r o t r a n s m i t t e r s f o r t h e s e r e c e p t o r s u b t y p e s a r e 14 n o t d e t e r m i n e d (see below; f o r r e v i e w see F o s t e r and Fagg, 1984; C o y l e e t a l . , 1986; Mayer and Westbrook, 1987; Monaghan e t a l . , 1989). Glutamate can e x c i t e c e l l s i n a l l l a y e r s o f v i s u a l c o r t e x , i n c l u d i n g l a y e r 4, t h e predominant s e n s o r y i n p u t l a y e r ( K r n j e v i c and P h i l l i s , 1962), and t h e b r o a d -s p e c t r u m EAA r e c e p t o r a n t a g o n i s t k y n u r e n i c a c i d can b l o c k evoked r e s p o n s e s i n l a y e r 4 o f v i s u a l c o r t e x (Tsumoto e t a l . , 1986; H a g i h a r a e t a l . , 1988). However, b o t h r e c e p t o r b i n d i n g and u p t a k e s t u d i e s suggest t h a t g l u t a m a t e may n o t be t h e p r i m a r y g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r . R e c e p t o r b i n d i n g s t u d i e s i n a d u l t v i s u a l c o r t e x show r e l a t i v e l y l i g h t b i n d i n g d e n s i t y i n l a y e r 4, compared t o o t h e r l a y e r s , f o r g l u t a m a t e o r f o r s e v e r a l EAA r e c e p t o r subtype l i g a n d s i n t h e r a t (Monaghan and Cotman, 1982, 1985; Monaghan e t a l . , 1984) i n t h e c a t (Shaw e t a l . , 1986, 1990; see F i g u r e 1 ) , and i n t h e monkey (Shaw and Cynader, 1986; Shaw e t a l . , 1989, 1990, 1991) ( f o r r e v i e w see Tsumoto, 1990). S i n c e t h e dense t e r m i n a t i o n s o f g e n i c u l o s t r i a t e axons c o n t r i b u t e a l a r g e p r o p o r t i o n o f t h e synapses i n l a y e r 4, t h i s r e l a t i v e l y l i g h t d e n s i t y o f g l u t a m a t e b i n d i n g would be u n e x p e c t e d i f g l u t a m a t e i s t h e p r i m a r y g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r , u n l e s s t h e r e i s a l o w e r r e c e p t o r a f f i n i t y o r lower number o f r e c e p t o r s p e r synapse i n t h i s l a y e r . The o n l y r e c e p t o r s found t o d a t e i n r e l a t i v e l y h i g h d e n s i t y i n a d u l t v i s u a l c o r t e x l a y e r 4 b e s i d e s GABA^ and b e n z o d i a z a p i n e r e c e p t o r s i n t h e c a t (Shaw e t a l . , 1986; see F i g u r e 1) and p r e s y n a p t i c n i c o t i n i c a c e t y l c h o l i n e r e c e p t o r s on g e n i c u l a t e t e r m i n a l s i n t h e c a t 15 ( P r u s k y e t a l . , 1987; see F i g u r e 1) a r e f o r t a c h y k i n i n p e p t i d e s i n t h e r a t and c a t (Danks e t a l . , 1986; Mantyh e t a l . , 1984, 1989; March and Shaw, 1990), w h i c h a r e p o s t s y n a p t i c i n t h e c a t (March and Shaw, 1990). T a c h y k i n i n s , however, a r e so f a r known o n l y as n e u r o m o d u l a t o r s o r as p r o d u c i n g s l o w e x c i t a t o r y s y n a p t i c p o t e n t i a l s ( r e v i e w e d i n J e s s e l and Womack, 1985), which i s not c o n s i s t e n t w i t h a r o l e as a p r i m a r y g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r . P r e l i m i n a r y d a t a s u g g e s t t h a t t h e NMDA subtype o f EAA r e c e p t o r i s c o n c e n t r a t e d i n s u b - l a y e r 4C6 (see F i g u r e 4) o f monkey s t r i a t e c o r t e x (Shaw e t a l , 1991), and c o u l d t h e r e f o r e c o n c e i v a b l y p l a y a r o l e i n g e n i c u l o s t r i a t e s y n a p t i c t r a n s m i s s i o n , a l t h o u g h t h e s e r e c e p t o r s do n o t appear t o p l a y s u c h a r o l e i n t h e r a t o r c a t ( f o r r e v i e w see Tsumoto, 1990). A second form o f s u p p o r t i n g e v i d e n c e f o r t h e i d e n t i t y o f a n e u r o t r a n s m i t t e r i n a g i v e n pathway i s t h e p r e s e n c e o f r e t r o g r a d e u p t a k e and t r a n s p o r t o f t h e c a n d i d a t e s u b s t a n c e from t e r m i n a l s t o c e l l b o d i e s . Uptake s t u d i e s w i t h [ 3 H ] g l u t a m a t e i n c a t have c o n s i s t e n t l y i m p l i c a t e d as g l u t a m a t e r g i c o n l y t h e r e c u r r e n t o u t p u t p r o j e c t i o n from c o r t i c a l l a y e r 6 t o t h e DLG and not t h e g e n i c u l o s t r i a t e p r o j e c t i o n (Baughman and G i l b e r t , 1981), w i t h c o n t r o v e r s y e x i s t i n g r e g a r d i n g t h e p r o j e c t i o n from l a y e r 5 t o t h e p u l v i n a r and SC (Baughman and G i l b e r t , 1981; Fosse e t a l . , 1984). I n t h e r a t , s t u d i e s employing c o r t i c a l a b l a t i o n and measures o f endogenous g l u t a m a t e have i m p l i c a t e d as g l u t a m a t e r g i c o n l y t h e c o r t i c a l p r o j e c t i o n s t o t h e DLG, LP, and SC (Lund-Karlsen and Fonum, 1978; Fosse and Fonnum, 1987). For review see Tsumoto (1990). 17 AN INSTRUCTIVE CASE: NEUROTRANSMISSION WITH NAAG The m o d i f i e d d i p e p t i d e N - a c e t y l - a s p a r t y l - g l u t a m a t e (NAAG) has been r e c e n t l y i m p l i c a t e d as a n e u r o t r a n s m i t t e r i n s e v e r a l b r a i n pathways. NAAG i s found i n h i g h l e v e l s i n b r a i n , r e g i o n a l l y d i s t r i b u t e d i n neurons ( C o y l e e t a l , 1986). I t i s e p i l e p t o g e n i c i n hippocampus, w i t h a h i g h a f f i n i t y f o r t h e 2-amino-4-phosphonobutyrate (AP4) s e l e c t i v e s u b t y p e o f EAA r e c e p t o r s (Zaczeck, 1983). The d e t e r m i n a t i o n o f a n e u r o t r a n s m i t t e r r o l e f o r NAAG i n p a r t i c u l a r pathways i s i n s t r u c t i v e , n o t o n l y f o r t h e importance o f m u l t i p l e c r i t e r i a i n i d e n t i f y i n g any su b s t a n c e as a n e u r o t r a n s m i t t e r (see b e l o w ) , b u t because i t shows how an endogenous s m a l l p e p t i d e c a n t a k e t h e s t a g e as t h e p r i m a r y c a n d i d a t e f o r n e u r o t r a n s m i t t e r s t a t u s i n pathways p r e v i o u s l y t h o u g h t t o be m e d i a t e d by t h e u b i q u i t o u s EAA n e u r o t r a n s m i t t e r c a n d i d a t e g l u t a m a t e . E v i d e n c e such as r e l e a s e and e x c i t a t i o n i n p y r i f o r m c o r t e x had su g g e s t e d t h a t g l u t a m a t e o r a s p a r t a t e were t h e n e u r o t r a n s m i t t e r s o f t h e l a t e r a l o l f a c t o r y t r a c t (LOT) ( B r a d f o r d and R i c h a r d s , 1976; C o l l i n s , 1978). More r e c e n t l y , t h e EAA a n t a g o n i s t AP4 was shown t o b l o c k t h e e l e c t r o p h y s i o l o g i c a l r e s ponse o f p y r i f o r m c o r t e x p y r a m i d a l c e l l s t o b o t h t h e monosynaptic e x c i t a t i o n e l i c i t e d by s t i m u l a t i o n o f t h e LOT and t o i o n t o p h o r e t i c a l l y - a p p l i e d NAAG, w h i l e l e a v i n g u n a f f e c t e d t h e re s p o n s e s t o a p p l i e d g l u t a m a t e and a s p a r t a t e ( f f r e n c h - M u l l e n e t a l . , 1985). Then 18 immunocytochemical s t a i n i n g l o c a l i z e d NAAG, b u t n o t g l u t a m a t e o r a s p a r t a t e , t o t h e o l f a c t o r y b u l b m i t r a l c e l l s ( which p r o j e c t as t h e LOT) ( B l a k e l y e t a l . , 1987). F u r t h e r n e u r o t r a n s m i t t e r c r i t e r i a a r e f u l f i l l e d by t h e d e m o n s t r a t i o n o f c a l c i u m - d e p e n d e n t r e l e a s e i n r a t b r a i n s l i c e s ( Z o l l i n g e r e t a l . , 1988), and a method o f r a p i d i n a c t i v a t o n by a s e l e c t i v e d i p e p t i d a s e , f o l l o w e d by r e u p t a k e o f t h e degraded g l u t a m a t e i n synaptosomes ( B l a k e l y e t a l , 1986; R o b i n s o n e t a l . , 1987). R e l e a s e o f g l u t a m a t e and a s p a r t a t e i n t h e LOT i s t h e r e f o r e most l i k e l y due t o t h e f a c t t h a t t h e y a r e d e g r a d a t i o n p r o d u c t s o f r e l e a s e d NAAG. Most r e c e n t l y NAAG has been i m p l i c a t e d as a n e u r o t r a n s m i t t e r o f t h e s e p t o -h i p p o c a m p a l pathway (Senut e t a l . , 1990) and a t a l l r e t i n a l t a r g e t zones, i n c l u d i n g t h e DLG and SC ( M o f f e t t e t a l . , 1991). An i n t e r e s t i n g p o s s i b i l i t y i s s u g g e s t e d by C o y l e e t a l . (1986). I n a n a l o g y t o t h e c h o l i n e r g i c system, c h o l i n e i t s e l f i s known t o have a g o n i s t a c t i o n s a t b o t h m u s c a r i n i c and n i c o t i n i c c h o l i n e r g i c r e c e p t o r s . Glutamate has b r o a d and u n i f o r m e x c i t a t o r y e f f e c t s on neurons, a c t i n g on d i s t i n c t EAA r e c e p t o r s u b t y p e s . A p o s s i b l e e x p l a n a t i o n f o r t h i s b r o a d a c t i o n i s t h a t g l u t a m a t e o r a s p a r t a t e a r e common components o f a group o f s m a l l p e p t i d e s l i k e NAAG w h i c h i n t e r a c t w i t h d i s c r e t e E/AA r e c e p t o r s . Glutamate o r a s p a r t a t e i n s u f f i c i e n t c o n c e n t r a t i o n s may produce a g o n i s t e f f e c t s by b i n d i n g t o t h a t domain o f t h e EAA r e c e p t o r t h a t r e c o g n i z e s t h e g l u t a m a t e o r a s p a r t a t e p r e s e n t w i t h i n t h e endogenous n e u r o t r a n s m i t t e r . 19 Such an i n t e r p r e t a t i o n i s not i n c o n s i s t e n t w i t h t h e o b s e r v a t i o n t h a t gamma-D-glutamyl-glycine i s a p o t e n t a n t a g o n i s t o f EAA r e c e p t o r s . I t i s f u r t h e r p o s s i b l e t h a t t h e d e g r a d a t i o n p r o d u c t s o f t h e s e s m a l l p e p t i d e s may have a d i r e c t p h y s i o l o g i c a l r o l e t o p l a y i n i n t e r a c t i o n w i t h EAA r e c e p t o r s , as "secondary" n e u r o t r a n s m i t t e r s . S m a l l d i - and t r i - p e p t i d e s may t a k e p a r t i n f a s t s y n a p t i c p o t e n t i a l EAA-type n e u r o t r a n s m i s s i o n a t d i s t i n c t , known EAA r e c e p t o r t y p e s , as w e l l as a t p o s s i b l y n o v e l r e c e p t o r s u b t y p e s s p e c i f i c t o t h a t s m a l l p e p t i d e . The r e l a t i v e p a u c i t y o f known EAA r e c e p t o r s i n l a y e r 4 i n p r i m a r y v i s u a l c o r t e x c o u p l e d w i t h t h e e x c i t a t i o n o f l a y e r 4 by g l u t a m a t e s u g g e s t t h e p o s s i b i l i t y o f a n o v e l r e c e p t o r t y p e f o r a n o v e l n e u r o t r a n s m i t t e r , w i t h some a f f i n i t y f o r g l u t a m a t e and antagonism by k y n u r e n i c a c i d (see a b o v e ) . NAAG i t s e l f i s p r e s e n t i n l o w e s t amounts i n n e o c o r t e x , w i t h i t s h i g h e s t b i n d i n g i n upper l a y e r s ( C o y l e , e t a l . , 1986). G l u t a t h i o n e , o r g a m m a - g l u t a m y l - c y s t e i n y l - g l y c i n e (GSH), an endogenous t r i p e p t i d e ( s t r u c t u r e shown i n F i g u r e 5) i s a p o s s i b l e a l t e r n a t i v e c a n d i d a t e f o r n e u r o t r a n s m i s s i o n i n v i s u a l c o r t e x , and f o r t h e g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r i n p a r t i c u l a r . 20 0. Gly H—C—H N—H 0 = C Cys H—C—CH 2 — SH y-Glu—C s—Gly N—H S 0 = C CH 2 CH y-Glu—Cys—Gly Oxidized glutathione Y-Glu +H,N—C— H Reduced glutathione (y-Glutamylcysteinylglycine) F i g u r e 5. The c h e m i c a l s t r u c t u r e o f r e d u c e d g l u t a t h i o n e (GSH) and o x i d i z e d g l u t a t h i o n e d i s u l f i d e (GSSG). (From S t r y e r , 1988) 21 GLUTATHIONE IN BRAIN . GSH p l a y s many b i o c h e m i c a l r o l e s t h r o u g h o u t t h e body, i n c l u d i n g t h e b r a i n ( f o r r e v i e w see O r l o w s k i and Karkowsky, 1976; M e i s t e r , 1989). GSH a c t s t o p r o t e c t c e l l u l a r t h i o l g roups a g a i n s t o x i d a t i v e damage by c y c l i n g between t h e predominant r e d u c e d form and an o x i d i z e d , d i s u l f i d e form (GSSG, see F i g u r e 5 ) , o f t e n i n c o n j u n c t i o n w i t h enzymes such as g l u t a t h i o n e p e r o x i d a s e . GSH a c t s as an S - c o n j u g a t e o f t o x i c s u b s t a n c e s , i n c o n j u n c t i o n w i t h v a r i o u s g l u t a t h i o n e S-t r a n s f e r a s e s o r n o n - e n z y m a t i c a l l y , t o a i d e x c r e t i o n . GSH a c t s as a coenzyme i n h y d r a t i o n , d e h y d r o g e n a t i o n , i s o m e r i z a t i o n , and d e h y d r o c h l o r i n a t i o n r e a c t i o n s . G l u t a t h i o n e r e d u c t a s e m a i n t a i n s GSH i n i t s p r e dominant r e d u c e d form (GSH:GSSG = 500:1). A membrane-bound, e x t r a c e l l u l a r - a c t i n g enzyme, g a m m a - 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 c o n c e n t r a t e d i n c a p i l l a r i e s , c h o r o i d p l e x u s , and ependymal c e l l s , degrades GSH i n t o c y s t e i n y l - g l y c i n e and i n t o g l u t a m a t e t h a t i s c o n j u g a t e d w i t h f r e e amino a c i d s (gamma-g l u t a m y l - A A ) . The b i o c h e m i s t r y o f GSH i s i l l u s t r a t e d i n F i g u r e 6. GSH may p l a y a- r o l e i n n e u r o t r a n s m i s s i o n i n a d d i t i o n t o i t s o t h e r f u n c t i o n s . A l t h o u g h GSH i s found i n h i g h c o n c e n t r a t i o n s i n b r a i n ( R e i c h e l t and Fonnum, 1969; O r l o w s k i and Karkowsky, 1976), and i s shown t o be n e c e s s a r y f o r maintenance o f m i t o c h o n d r i a l f u n c t i o n i n b r a i n ( J a i n e t a l . , 1991), t h e r e i s l i t t l e d e t o x i f i c a t i o n by GSH c o n j u g a t i o n i n 22 b r a i n ( O r l o w s k i and Karkowsky, 1976) . GSH i s c o n c e n t r a t e d i n t h e n e u r o p i l and w h i t e m a t t e r t h r o u g h o u t r o d e n t and p r i m a t e b r a i n , w i t h t h e e x c e p t i o n o f c e l l b o d i e s o f c e r e b e l l a r P u r k i n j e c e l l s ( S l i v k a e t a l . , 1987; P h i l b e r t e t a l . , 1991). I n r a t b r a i n , GSH p e r o x i d a s e i s c o n f i n e d l a r g e l y t o t h e n u c l e i o f o n l y some neurons ( U s h i j i m a e t a l . , 1986) and t h e GSH S - t r a n s f e r a s e s a r e found o n l y i n a s t r o c y t e s ( S e n j o e t a l . , 1986). GSH b i n d i n g s i t e s a r e seen i n c r u d e s y n a p t i c membrane p r e p a r a t i o n s from r a t b r a i n , i n c l u d i n g c o r t e x , showing r e g i o n a l v a r i a t i o n ( O g i t a e t a l . 198 6b; O g i t a and Yoneda 1987, 1988). These s i t e s i n c l u d e a p o s s i b l e GSH r e c e p t o r w i t h a K^ v a l u e t y p i c a l o f EAA r e c e p t o r s , as w e l l as a p u t a t i v e Na-independent, t e m p e r a t u r e dependent u p t a k e s i t e . GSH a l s o shows a f f i n i t y f o r g l u t a m a t e b i n d i n g s i t e s , w i t h s e l e c t i v i t y f o r t h e AMPA and AP4 r e c e p t o r s ( O g i t a and Yoneda, 1987; O j a , 1988) and f o r t h e NMDA r e c e p t o r a n t a g o n i s t -p r e f e r r i n g s i t e ( O g i t a and Yoneda, 1990) (see b e l o w ) . C y s t e i n e i s r e l e a s e d i n a calcium-dependent manner from d e p o l a r i z e d r a t c o r t i c a l s l i c e s , and may t h u s be a d e g r a d a t i o n p r o d u c t o f a l a r g e r r e l e a s e d p r e c u r s o r s u c h as GSH ( K e l l e r e t a l . , 1989). The above d a t a s uggest t h a t GSH c o u l d be a n e u r o t r a n s m i t t e r o f t h e t y p e h y p o t h e s i s e d i n t h e p r e v i o u s s e c t i o n , and i t has now been shown t o be e x c i t a t o r y i n p r i m a r y v i s u a l c o r t e x . R e c e n t l y Timothy J . T e y l e r a t N.E. Ohio U n i v e r s i t y C o l l e g e o f M e d i c i n e ( p e r s o n a l communication) d e m o n s t r a t e d a s t r o n g f i e l d - p o t e n t i a l s o u r c e i n s l i c e s o f r a t 23 v i s u a l c o r t e x i n l a y e r 6 i n r e s ponse t o i o n t o p h o r e t i c a p p l i c a t i o n o f GSH t o l a y e r 4, a p p a r e n t l y by a c t i v a t i n g a p i c a l d e n d r i t e s o f l a y e r 6 c e l l s i n l a y e r 4. T h i s i s c o n s i s t e n t w i t h t h e p o s s i b i l i t y t h a t GSH i s a g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r . 2 F i g u r e 6. An o u t l i n e o f t h e metabolism and f u n c t i o n o f g l u t a t h i o n e . Enzymes: (1) g a m m a - g l u t a m y l t r a n s p e p t i d a s e ; (2) g a m m a - g l u t a m y l c y c l o t r a n s f e r a s e ; (3) 5 - o x o p r o l i n a s e ; (4) gamma-glutamylcysteine s y n t h e t a s e ; (5) g l u t a t h i o n e s y n t h e t a s e ; (6) d i p e p t i d a s e ; (7) g l u t a t h i o n e S - t r a n s f e r a s e ; (8) g l u t a t h i o n e p e r o x i d a s e ; (;9) g l u t a t h i o n e r e d u c t a s e ; (10) t r a n s h y d r o g e n a s e s . AA = amino a c i d s ; X = compounds t h a t r e a c t w i t h g l u t a t h i o n e t o y i e l d c o n j u g a t e s . (From M e i s t e r , 1983) 25 EXCITATORY AMINO ACID RECEPTORS AND TRANSMITTERS As demonstrated by o t h e r i n v e s t i g a t o r s , GSH may i n t e r a c t w i t h a range o f EAA r e c e p t o r s u b t y p e s , i n a d d i t i o n t o a p o s s i b l e GSH r e c e p t o r . I t may a l s o be e n z y m a t i c a l l y degraded i n t o a s e r i e s o f amino a c i d s and d i p e p t i d e s t h a t can t h e m s e l v e s i n t e r a c t w i t h EAA r e c e p t o r s . GSH i s degraded i n t o c y s t e i n y l - g l y c i n e and gamma-glutamyl-amino a c i d s by gamma-g l u t a m y l t r a n s p e p t i d a s e ; c y s t e i n y l - g l y c i n e i s degraded i n t o c y s t e i n e and g l y c i n e by c y s t e i n y l g l y c i n a s e ; c y s t e i n e i s m e t a b o l i z e d t o c y s t e i n e s u l f i n i c a c i d (CSA) by c y s t e i n e d i o x e g e n a s e ; and CSA i s m e t a b o l i z e d t o c y s t e i c a c i d by c y s t e i n e s u l f i n i c a c i d d e c a r b o x y l a s e ( M e i s t e r , 1983; R a s s i n and G a l l , 1987; H i l l e t a l . , 1985). The f o l l o w i n g s e c t i o n b r i e f l y d e s c r i b e s some of t h e c h a r a c t e r i s t i c s o f t h e known EAA r e c e p t o r s u b t y p e s and t h e p r o c e s s e s i n w h i c h t h e y a r e i n v o l v e d , and l i s t s t h e i r proposed endogenous n e u r o t r a n s m i t t e r s . F o r r e v i e w s see F o s t e r and Fagg (1984), Mayer and Westbrook (1987), Monaghan e t a l . (1989), and G r i f f i t h s (1990). The EAA r e c e p t o r s u btypes a r e d i s t i n g u i s h e d by t h e s p e c i f i c i t y o f r a d i o l i g a n d b i n d i n g and t h e p h y s i o l o g i c a l e f f e c t s o f a g o n i s t s and a n t a g o n i s t s , each t y p e named a f t e r i t s most p r e f e r e n t i a l o r p o t e n t a g o n i s t ( n o m e n c l a t u r e t a k e n from Monaghan e t a l [ 1 9 8 9 ] ) . The i o n o t r o p i c NMDA, KAIN ( k a i n a t e ) , and AMPA ( a l p h a - a m i n o - 3 - h y d r o x y - 5 - m e t h y l i s o x a z o l e -4 - p r o p i o n a t e ) r e c e p t o r subtypes a l l i n c r e a s e N a + and K + 26 c o n d u c t a n c e s , p r e f e r e n t i a l l y a c t i v a t i n g d i f f e r e n t c o n d u c t a n c e s t a t e s o f t h e same i o n c h a n n e l t y p e ( J a r and S t e v e n s , 1987). The NMDA r e c e p t o r i s more complex. I t i s v o l t a g e dependent, permeable t o t h e second messenger C a 2 + , c o n t r i b u t e s b o t h a f a s t and a second, slow s y n a p t i c p o t e n t i a l component n o t seen w i t h t h e o t h e r two c h a n n e l r e c e p t o r s (Angelo e t a l . , 1990), and c o n t r i b u t e s t o t o n i c background v o l t a g e - d e p e n d e n c e and f a c i l i t a t i o n o f a c t i o n p o t e n t i a l s (Sah e t a l . , 1989). The NMDA r e c e p t o r i s c o - a c t i v a t e d by t h e n e u r o t r a n s m i t t e r g l y c i n e , modulated by Z n 2 + , modulated by a n o n - c o m p e t i t i v e a n t a g o n i s t b i n d i n g s i t e i n s i d e t h e c h a n n e l , and modulated by an a n t a g o n i s t - p r e f e r r i n g r e c o g n i t i o n s i t e t h a t may be e i t h e r a s e p a r a t e s i t e from t h e a g o n i s t s i t e o r an a l t e r n a t e s t a t e . A c t i v a t i o n o f NMDA, AMPA, and KAIN r e c e p t o r s can a l s o d e c r e a s e i n o s i t o l - t r i s - p h o s p h a t e (IP3) p r o d u c t i o n i n d u c e d by m u s c a r i n i c , s e r o t o n i n , o r h i s t a m i n e r e c e p t o r a c t i v a t i o n (Nobel e t a l . , 1989). A second KAIN r e c e p t o r s u b t y p e i s p e r m i a b l e t o C a 2 + ( l i n o e t a l . , 1990; H o l o p a i n e n e t a l . , , 1990). Recent e v i d e n c e i n d i c a t e s t h e p r e s e n c e o f a common AMPA/KAIN r e c e p t o r i n a d d i t i o n t o s e p a r a t e AMPA and KAIN r e c e p t o r s u b t y p e s ( r e v i e w e d i n B a r n a r d and Henl e y , 1990). The m e t a b o t r o p i c ACPD ( t r a n s - l - a m i n o - 1 , 3 -c y c l o p e n t a n e d i c a r b o x y l a t e ) r e c e p t o r i s G - p r o t e i n l i n k e d t o an i n c r e a s e i n IP3 p r o d u c t i o n , l e a d i n g t o a d e c r e a s e i n C a 2 + -dependent v o l t a g e - g a t e d K + conductance i n hippocampus, i n c r e a s i n g r e s p o n s i v i t y o f t h e c e l l (Charpak e t a l . , 1990). The AP4 (L-2-amino-4-phosphonobutyrate) r e c e p t o r i s n o t y e t 27 w e l l c h a r a c t e r i z e d ; i t appears t o p l a y a dominant r o l e as a p r e s y n a p t i c r e c e p t o r a t t e n u a t i n g r e l e a s e a t g l u t a m a t e r g i c t e r m i n a l s , as w e l l as o c c u r i n g as a p o s t s y n a p t i c r e c e p t o r ( B r i d g e s e t a l . , 1986). F i n a l l y , t h e r e appears t o be a CSA ( c y s t e i n e s u l f i n a t e ) r e c e p t o r ( P u l l a n e t a l . , 1987; P i n e t a l . , 1987), w h i c h may s t i m u l a t e c y c l i c AMP p r o d u c t i o n i n t h e hippocampus (Baba e t a l . , 1988) and p o s s i b l y a N a + c o n d u c t a n c e ( P u l l a n e t a l . , 1987). D e s e n s i t i z a t i o n o f EAA r e c e p t o r s o c c u r s i n two forms: f a s t (10 t o 100 ms) f o r NMDA r e c e p t o r s , and slow (seconds) f o r non-NMDA r e c e p t o r s ( T r u s s e l e t a l . , 1987). EAA r e c e p t o r s a r e i m p l i c a t e d i n a range o f p l a s t i c i t y and d e v e l o p m e n t a l p r o c e s s e s . NMDA r e c e p t o r s a r e i n s t r u m e n t a l i n a s s o c i a t i v e l o n g - t e r m p o t e n t i a t i o n (LTP) i n s t r i a t e c o r t e x ( A r t o l a and S i n g e r , 1987). ACPD r e c e p t o r s a r e a s s o c i a t e d w i t h LTP i n s t r i a t u m (Dumuis e t a l . , 1990), and w i t h t h e development o f t h e c r i t i c a l p e r i o d i n s t r i a t e c o r t e x (Dudek and S i n g e r , 1989). Q u i s q u a l a t e (QUIS) s e n s i t i v e r e c e p t o r s ( i . e . , AMPA r e c e p t o r s , ACPD r e c e p t o r s , o r both) a r e i m p l i c a t e d i n a s s o c i a t i v e l o n g - t e r m d e p r e s s i o n i n c e r e b e l l u m (Kano and K a t o , 1987). D i f f e r e n t EAA r e c e p t o r s can have d i f f e r e n t e f f e c t s on n e u r o n a l development depending on b r a i n r e g i o n and age, but i n g e n e r a l t h e r e i s a continuum o f a c t i o n s f o r EAA n e u r o t r a n s m i t t e r s , where a t low l e v e l s t h e y s t i m u l a t e s p r o u t i n g , a t h i g h e r l e v e l s h a l t o u t g r o w t h , and a t s t i l l h i g h e r l e v e l s prune d e n d r i t i c morphology ( f o r r e v i e w see L i p t o n and K a t e r , 1989). 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 can 28 a l s o be i n d u c e d by a c t i v a t i o n o f NMDA r e c e p t o r s : s u c h a c t i v a t i o n i n d u c e s e x p r e s s i o n o f t h e d o p a m i n e r g i c phenotype i n c u l t u r e d c e r e b e l l a r g r a n u l e c e l l s (Moran and P a t e l , 1989). V a r i o u s endogenous s u b s t a n c e s have been p r o p o s e d as t h e n e u r o t r a n s m i t t e r s f o r t h e EAA r e c e p t o r s . Glutamate and a s p a r t a t e a r e proposed f o r a l l s u b t y p e s . Q u i n o l i n i c a c i d and h o m o c y s t e i c a c i d a r e proposed f o r t h e NMDA r e c e p t o r i n p a r t i c u l a r . K y n u r e n i c a c i d may be a p h y s i o l o g i c a l a n t a g o n i s t o f NMDA r e c e p t o r s , a c t i n g a t t h e g l y c i n e s i t e (Swartz e t a l . , 1990). C y s t e i n e i s r e l e a s e d upon d e p o l a r i z a t i o n o f r a t c o r t e x s l i c e s ( K e l l e r e t a l . , 1989), r e d u c e s p r o d u c t i o n o f IP3 i n d u c e d by n o r a d r e n a l i n e ( X i and Jope, 1989), and has t h e same a f f i n i t y f o r t h e AP4 r e c e p t o r as does NAAG (about 300 nM K(j) ( P u l l a n e t a l . , 1987; Zaczek e t a l . , 1983), so t h a t e i t h e r o f t h e s e s u b s t a n c e s may be endogenous l i g a n d s f o r t h e AP4 r e c e p t o r . C y s t e i n e s u l f i n a t e may be t h e n e u r o t r a n s m i t t e r f o r t h e CSA r e c e p t o r (Iwata e t a l . , 1982a,b; f o r r e v i e w see G r i f f i t h , 1990). C y s t e i c a c i d (CA) has a f f i n i t y f o r KAIN and AP4 r e c e p t o r s ( P u l l a n e t a l . , 1987). Gamma-g l u t a m y l g l u t a m a t e i s proposed f o r TAMPA, NMDA, and KAIN r e c e p t o r s , and gamma-glutamylaspartate f o r KAIN r e c e p t o r s (Varga e t a l . , 1989). G l u t a t h i o n e i n t h e form o f GSH and GSSG has been proposed as a modulator o f AMPA r e c e p t o r s (Oja e t a l . , 1988; Varga e t a l . , 1989; O g i t a e t a l . , 1987), and GSH has been proposed as an endogenous l i g a n d f o r t h e c o m p e t i t i v e a n t a g o n i s t - p r e f e r r i n g s i t e o f NMDA r e c e p t o r s ( O g i t a and Yoneda, 1990). 29 NEUROTRANSMITTER CRITERIA I n 1966, Werman r e v i e w e d t h e c r i t e r i a f o r i d e n t i f i c a t i o n o f a n e u r o t r a n s m i t t e r i n t h e c e n t r a l nervous system. He p o i n t e d o u t many problems o f i n t e r p r e t a t i o n o f d a t a and pr o p o s e d many t h e o r e t i c a l o b j e c t i o n s t o t h e n e c e s s i t y o f many o f t h e s o - c a l l e d " c r i t e r i a " a p p l i e d i n t h e c a s e o f a new t r a n s m i t t e r c a n d i d a t e . The o n l y r e c o u r s e i s t o c o n d u c t m u l t i p l e t e s t s and d e v e l o p as l a r g e a body o f s u p p o r t i v e e v i d e n c e as p o s s i b l e , c o n s i s t e n t w i t h t h e p r o p o s i t i o n t h a t a g i v e n s u b s t a n c e i s r e l e a s e d as a n e u r o t r a n s m i t t e r from t e r m i n a l s o f a g i v e n neuron. The f o l l o w i n g o u t l i n e o f t e s t s t h a t s h o u l d be conducted i s adapted from McGeer e t a l . (1987). The c a n d i d a t e s u b s t a n c e s h o u l d p r o b a b l y d e m o n s t r a t e many o f t h e f o l l o w i n g : 1. P r e s e n c e i n adequate amounts. 2. Non-homogeneous d i s t r i b u t i o n i n b r a i n . 3. Show a drop i n c o n c e n t r a t i o n f o l l o w i n g l e s i o n s o f s u s p e c t e d l ong-axon pathways. 4. I n c r e a s e i n c o n c e n t r a t i o n p o s t n a t a l l y , c o n c u r r e n t w i t h synapse p r o l i f e r a t i o n , i n b r a i n r e g i o n s where i t i s f u n c t i o n a l l y i n v o l v e d . 5. L o c a l i z a t i o n by h i s t o c h e m i c a l o r i m m u n o h i s t o c h e m i c a l s t u d i e s o f t h e su b s t a n c e o r i t s s y n t h e s i z i n g enzyme a t t h e c e l l u l a r l e v e l . 6. R e l e a s e (a) from s u i t a b l e t i s s u e p r e p a r a t i o n s i n v i t r o by a p r o c e s s t h a t i s C a 2 + - d e p e n d e n t and s t i m u l a t e d by 30 K o r o t h e r d e p o l a r i z a t i o n ; and (b) upon n e r v e s t i m u l a t i o n i n v i v o ( r e c o v e r e d by, f o r example, b r a i n d i a l y s i s ) . 7. H i g h - a f f i n i t y uptake of t h e s u b s t a n c e o r i t s m e t a b o l i t e s i n t o n erve t e r m i n a l s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t (not demonstrated f o r p e p t i d e s ) . As d i s c u s s e d i n t h e T h e s i s R a t i o n a l e , r e t r o g r a d e t r a n s p o r t c o r r e l a t e s w i t h s e l e c t i v e u p t a k e of n e u r o t r a n s m i t t e r s o r t r a n s m i t t e r m e t a b o l i t e s and can t h u s d e l i n e a t e , u s i n g exogenous r a d i o l a b e l e d s u b s t a n c e s , t h e n e u r o n a l pathway e m p l o y i n g t h e t r a n s m i t t e r . 8. P r e s e n c e o f d e g r a d i n g enzymes, p a r t i c u l a r l y f o r non-p e p t i d e t r a n s m i t t e r s . These may be i n d e n d r i t e s o r g l i a l c e l l s o r on c e l l membranes. 9. B i n d i n g w i t h h i g h a f f i n i t y t o p o s t s y n a p t i c r e c e p t o r s . C r i t e r i a f o r r e c e p t o r b i n d i n g a r e d i s c u s s e d i n t h e T h e s i s R a t i o n a l e . R e c e p t o r d i s t i b u t i o n s s h o u l d c o r r e l a t e w i t h t e r m i n a l f i e l d s o f t h e proposed pathways u t i l i z i n g t h e t r a n s m i t t e r . 10. I d e n t i t y o f a c t i o n : exogenous a p p l i c a t i o n o f t h e presumed n e u r o t r a n s m i t t e r s h o u l d mimic t o a l a r g e degree t h e e f f e c t s o f s t i m u l a t i o n o f t h e n e u r o n a l pathway f o r w h i c h i t i s b e l i e v e d t o be t h e t r a n m s i t t e r . 11. P h a r m a c o l o g i c a l a g e n t s : i t s h o u l d be p o s s i b l e t o f i n d d r u gs t h a t i n t e r f e r e w i t h o r enhance t h e a c t i o n o f t h e n e u r o t r a n s m i t t e r a t any o f t h e s t a g e s o f s y n t h e s i s , s t o r a g e , r e l e a s e , o r r e c e p t o r a c t i v a t i o n . A d r ug t h a t b l o c k s r e c e p t o r a c t i v a t i o n s h o u l d show i d e n t i t y o f a c t i o n as i n #10. 31 THESIS RATIONALE U n d e r s t a n d i n g t h e response p r o p e r t i e s , development, and p l a s t i c i t y o f p r i m a r y v i s u a l c o r t e x depends on t h e d e t e r m i n a t i o n o f i t s c h e m i c a l c i r c u i t r y . The major n e u r o t r a n s m i t t e r s m e d i a t i n g f a s t t r a n s m i s s i o n a r e l i k e l y t o be s i m i l a r t o t h o s e proposed as EAA n e u r o t r a n s m i t t e r c a n d i d a t e s , w h i c h may i n c l u d e d i - and t r i - p e p t i d e s . G e n i c u l o s t r i a t e i n p u t b e a r s a s i m i l a r i t y t o g l u t a m a t e r g i c n e u r o t r a n s m i s s i o n , y e t t h e r e i s a r e l a t i v e p a u c i t y o f EAA b i n d i n g i n l a y e r 4, t h e major i n p u t l a y e r , and t h e g e n i c u l o s t r i a t e axons do not t a k e up and r e t r o g r a d e l y t r a n s p o r t g l u t a m a t e (see b e l o w ) . I t was h y p o t h e s i z e d t h a t t h e a c i d i c g l u t a m y l t r i p e p t i d e GSH, wh i c h shows r e c e p t o r b i n d i n g i n r a t c o r t i c a l s y n a p t i c membranes, i s a n e u r o t r a n s m i t t e r i n v i s u a l c o r t e x , and may be, more s p e c i f i c a l l y , a g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r . The g o a l o f t h i s s t u d y was t o use r a d i o l a b e l e d GSH t o (1) c h a r a c t e r i z e p o s s i b l e GSH r e c e p t o r b i n d i n g i n r a t O c l ; (2) v i s u a l i z e GSH b i n d i n g s i t e d i s t r i b u t i o n i n r a t , c a t , and monkey p r i m a r y v i s u a l c o r t e x ; (3) d e t e r m i n e t h e change i n d i s t r i b u t i o n o f GSH b i n d i n g s i t e s o v e r t h e p o s t n a t a l " c r i t i c a l p e r i o d " i n c a t a r e a 17; (4) d i s c e r n p o s s i b l e i n t e r a c t i o n s o f GSH w i t h o t h e r known EAA r e c e p t o r s u btypes i n r a t O c l ; and 32 (5) l o c a l i z e p u t a t i v e GSH n e u r o t r a n s m i s s i o n t o s p e c i f i c v i s u a l system pathways i n r a t and c a t b r a i n by d e m o n s t r a t i n g i n v i v o t h e p r e s e n c e o f r e t r o g r a d e u p t a k e and t r a n s p o r t t o c e l l b o d i e s i n s p e c i f i c n u c l e i . B i n d i n g e x p e r i m e n t s i n v i t r o P r e v i o u s b i n d i n g s t u d i e s w i t h r a d i o l a b e l e d GSH were c o n d u c t e d w i t h s u s p e n s i o n s o f s y n a p t i c membrane f r a c t i o n s o f b r a i n homogenates ( O g i t a e t a l , 1986; O g i t a and Yoneda, 1987, 1988, 1989). The s t u d y r e p o r t e d here used i n - v i t r o b i n d i n g o f r a d i o l a b e l e d GSH i n t h i n s e c t i o n s o f r a t and c a t p r i m a r y v i s u a l c o r t e x . Young and Kuhar (1979) showed f o r o p i o i d r e c e p t o r s t h a t b i n d i n g i n v i t r o t o postmortem, p r e v i o u s l y f r o z e n , mounted t i s s u e s e c t i o n s d i d not change t h e r e c e p t o r c h a r a c t e r i s t i c s as d e t e r m i n e d i n a homogenate p r e p a r a t i o n , o r t h e d i s t r i b u t i o n s as d e t e r m i n e d by i n - v i v o b i n d i n g . T h i s method a l l o w s b o t h a u t o r a d i o g r a p h y and q u a n t i t a t i v e c h a r a c t e r i z a t i o n t o be performed. The pharmacology o f t h e r e c e p t o r s can be d e t e r m i n e d f i r s t , and endogenous l i g a n d r e d u c e d , p r i o r t o a u t o r a d i o g r a p h y . W h i l e no s i n g l e p r o p e r t y u n i q u e l y s e r v e s t o i d e n t i f y a r e c e p t o r i n t e r a c t i o n , s e v e r a l c r i t e r i a t a k e n t o g e t h e r can s e r v e t o d i s t i n g u i s h r e c e p t o r from n o n r e c e p t o r i n t e r a c t i o n s ( C a u t r e c a s a s and H o l l e n b e r g , 1976; B u r t , 1978; L i m b r i d , 1986). To demonstrate t h e p o s s i b l e p r e s e n c e of r e c e p t o r s f o r GSH, t h e b i n d i n g o f r a d i o l a b e l e d GSH must show s a t u r a t i o n a t 33 p h y s i o l o g i c a l c o n c e n t r a t i o n s , r e v e r s i b i l i t y , r e l a t i v e l y h i g h a f f i n i t y (<10 /iM K<j) , s p e c i f i c i t y i n t h e p r o f i l e o f i n h i b i t i o n o f b i n d i n g by d i f f e r e n t l i g a n d s , and a non-homogeneous d i s t r i b u t i o n . Other c r i t e r i a , s u ch as c o r r e l a t i o n o f b i n d i n g w i t h dose-response c u r v e s , o r b i n d i n g o f GSH a n t a g o n i s t s , c o u l d not be t e s t e d w i t h i n t h e scope o f t h i s s t u d y . The e x p e r i m e n t s d e m o n s t r a t i n g r e c e p t o r i n t e r a c t i o n s a l s o p r o v i d e d measures o f s t a n d a r d p a r a m e t e r s f o r c o m p a r i s o n t o t h e l i t e r a t u r e and between s p e c i e s ( f o r p r o c e d u r e s and a n a l y s i s o f b i n d i n g see Methods). Time c o u r s e , c o m p e t i t i o n , and s a t u r a t i o n b i n d i n g e x p e r i m e n t s were conducted under c o n d i t i o n s t h a t m i n i m i z e d b i n d i n g t o EAA r e c e p t o r s and a l l o w e d c h a r a c t e r i z a t i o n o f a s e p a r a t e b i n d i n g s i t e f o r GSH. F u r t h e r c o m p e t i t i o n e x p e r i m e n t s w i t h EAA r e c e p t o r a g o n i s t s , a n t a g o n i s t s , and p u t a t i v e n e u r o t r a n s m i t t e r s were conducted under more p h y s i o l o g i c a l c o n d i t i o n s t o i n d i c a t e p o s s i b l e i n t e r a c t i o n s w i t h EAA r e c e p t o r s . I t was p r e d i c t e d t h a t b i n d i n g would i n c r e a s e i n c a t c o r t e x w i t h i n c r e a s i n g p o s t n a t a l age, o v e r t h e c r i t i c a l p e r i o d , and t h a t , i f GSH i s t h e g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r , t h e d e n s e s t b i n d i n g would be found i n l a y e r 4, c o n t i n u i n g i n t h e a d u l t , i n r a t , c a t , and monkey p r i m a r y v i s u a l c o r t e x . 34 Uptake e x p e r i m e n t s i n v i v o A n o t h e r form o f s u p p o r t i n g e v i d e n c e f o r n e u r o t r a n s m i t t e r s t a t u s i s t h e d e m o n s t r a t i o n o f a h i g h - a f f i n i t y u p t a k e system t o pump t h e m a t e r i a l o r i t s m e t a b o l i t e s back i n t o n e r v e e n d i n g s ( I v e r s e n , 1971). The f e a s i b i l i t y o f s i m u l t a n e o u s l y d e t e r m i n i n g c o n n e c t i v i t y and t r a n s m i t t e r s p e c i f i c i t y by t h e i n - v i v o u p t a k e and r e t r o g r a d e a x o n a l t r a n s p o r t o f r a d i o l a b e l e d n e u r o t r a n s m i t t e r s has a l s o been d e m o n s t r a t e d ( M a l t h e - S o r e n s s e n e t a l . , 1979; S t r e i t , 1980). T e r m i n a l u p t a k e and r e t r o g r a d e t r a n s p o r t back t o c e l l b o d i e s i s s e l e c t i v e f o r t h e n e u r o t r a n s m i t t e r o f t h a t pathway, b u t n o t f o r i t s p r e c u r s o r s , and does not o c c u r f o r n o n - t r a n s m i t t e r amino a c i d s ( S t r e i t , 1980). H i g h a f f i n i t y u p t a k e o f non-t r a n s m i t t e r amino a c i d s i s found i n most neurons (Hannuniemi and O j a , 1981), but i s c o n f i n e d t o t h e c e l l body, where t h e amino a c i d s a r e i n c o r p o r a t e d i n t o p r o t e i n s and a n t e r o g r a d e l y t r a n s p o r t e d e x c l u s i v e l y i n t h i s form by a c t i v e a x o p l a s m i c t r a n s p o r t (Octis e t a l . , 1967), t h u s d e m o n s t r a t i n g a x o n a l c o n n e c t i o n s i f t h e amino a c i d s a r e r a d i o l a b e l e d (Cowan e t a l . , 1972). A n t e r o g r a d e t r a n s p o r t o f n e u r o t r a n s m i t t e r s i s much p o o r e r ( S t r e i t , 1980). R e t r o g r a d e t r a n s p o r t o f t h e t r a n s m i t t e r - c a n d i d a t e amino a c i d g l u t a m a t e i n c a t v i s u a l s ystem pathways o c c u r s by f r e e d i f f u s i o n and i s c o n f i n e d t o s p e c i f i c pathways (Baughman and G i l b e r t , 1981). A pathway t h a t shows r e t r o g r a d e uptake o f g l u t a m a t e from d i s t a n t t e r m i n a l s i n t o c e l l b o d i e s a l s o shows a n t e r o g r a d e t r a n s p o r t from c e l l b o d i e s t o t e r m i n a l s , presumably b e i n g f i r s t t a k e n 35 up r e t r o g r a d e l y t o the c e l l body by l o c a l l y r a m i f y i n g axon t e r m i n a l s , whereas pathways t h a t showed no r e t r o g r a d e t r a n s p o r t of glutamate a l s o showed no anterograde t r a n s p o r t (Baughman and G i l b e r t , 1981). Thus r e t r o g r a d e t r a n s p o r t of an amino a c i d i n d i c a t e s a r o l e as a n e u r o t r a n s m i t t e r or n e u r o t r a n s m i t t e r m e t a b o l i t e , while anterograde t r a n s p o r t i s e q u a l l y c o n s i s t e n t w i t h a metabolic or a n e u r o t r a n s m i s s i o n f u n c t i o n . Terminal uptake may be assumed t o be s e l e c t i v e , but not a b s o l u t e l y s p e c i f i c , s i n c e , f o r example, the uptake s i t e f o r L-glutamate a l s o takes up L - a s p a r t a t e , D-aspartate, c y s t e i n e s u l f i n a t e , and c y s t e i c a c i d ( B a l c a r and Johnson, 1972; Wilson and Pastusko, 1986). In the uptake experiments, r a d i o l a b e l e d GSH and i t s c o n s t i t u e n t amino a c i d s c y s t e i n e , g l y c i n e , and glutamate, as w e l l as the amino a c i d r e c e p t o r a g o n i s t s muscimol and k a i n a t e were i n d i v i d u a l l y i n j e c t e d i n t o area O c l of e l e v e n r a t s . In one c a t area 17 was i n j e c t e d with [ 3H]GSH. A f t e r s u i t a b l e s u r v i v a l , uptake t o other b r a i n r e g i o n s was v i s u a l i z e d by autoradiography. I t was p r e d i c t e d t h a t , i f GSH i s a g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r , t h e r e might be uptake of l a b e l i n c e l l bodies of the DLG from the i n j e c t i o n i n t o primary v i s u a l c o r t e x of r a d i o l a b e l e d GSH, or of any of i t s c o n s t i t u e n t amino a c i d s , or both. Such a f i n d i n g would be s u p p o r t i v e of a n e u r o t r a n s m i t t e r r o l e , but i s not an e s s e n t i a l c r i t e r i o n . 36 MATERIALS AND METHODS LIGANDS AND TRACERS F o r i n v i t r o b i n d i n g e x p e r i m e n t s [ 3 5 S ] G S H ( 3 1 . 4 — 1 1 2 . 5 Ci/mmol), l a b e l e d on t h e c y s t e i n e r e s i d u e , [ 3H]GSH ( 1 — 0 . 8 5 3 Ci/mmol), l a b e l e d on t h e g l y c i n e r e s i d u e , and [ 3 H ] g l u t a m a t e ( 3 6 — 4 9 Ci/mmol) were o b t a i n e d from New E n g l a n d N u c l e a r (NEN, B o s t o n , MA). A l l o t h e r c h e m i c a l s were o b t a i n e d from Sigma ( S t . L o u i s , MO). R a d i o l a b e l e d t r a c e r s f o r i n v i v o u p t a k e e x p e r i m e n t s were a l l o b t a i n e d from New England N u c l e a r (NEN, B o s t o n , MA) and i n c l u d e d [ 3 5 S ] G S H ( l a b e l e d on t h e c y s t e i n e r e s i d u e , 57.5 Ci/mmol), [ 3H]GSH ( l a b e l e d on t h e g l y c i n e r e s i d u e , 1-0.85 Ci/mmol), [ 3 5 S ] c y s t e i n e (1,113 Ci/mmol), [ 3 H ] g l y c i n e (53 Ci/mmol), [ 3 H ] g l u t a m a t e (36 Ci/mmol), [ 3 H ] k a i n a t e (58 Ci/mmol), and [ 3H]muscimol (20 Ci/mmol). IN VITRO RECEPTOR BINDING PROCEDURES S t a n d a r d methods of r a d i o l i g a n d b i n d i n g and c o r r e s p o n d i n g d a t a a n a l y s i s (see f o l l o w i n g s e c t i o n ) a r e w e l l e s t a b l i s h e d ( r e v i e w e d by B e n n e t t , 1978; B y l u n d , 1980; L i m b r i d , 1986; McGonigle and M o l i n o f f , 1989; Yamamura e t a l . , 1 990), and a r e d e s c r i b e d as f o l l o w s . Time c o u r s e e x p e r i m e n t s 37 a r e c o n d u c t e d i n wh i c h t h e amount bound i s d e t e r m i n e d as a f u n c t i o n o f t i m e , w i t h f r e e r a d i o l i g a n d c o n c e n t r a t i o n h e l d c o n s t a n t ( f o r a s s o c i a t i o n ) o r brought t o z e r o ( f o r d i s s o c i a t i o n ) . The t i m e r e q u i r e d t o r e a c h e q u i l i b r i u m ( i . e . , where t h e r a t e o f a s s o c i a t i o n e q u a l s t h e r a t e o f d i s s o c i a t i o n , so t h a t t h e b i n d i n g has r e a c h e d s t e a d y s t a t e ) i s t h e n used f o r t h e o t h e r e x p e r i m e n t s . The d i s s o c i a t i o n h a l f - t i m e i s used t o det e r m i n e o p t i m a l r i n s e t i m e s ( i . e . , r e m o v a l o f f r e e r a d i o l i g a n d w i t h m i n i m a l r e d u c t i o n o f s p e c i f i c b i n d i n g ) . S p e c i f i c i t y i s shown by c o m p e t i t i o n e x p e r i m e n t s . I n t h e s e e x p e r i m e n t s b i n d i n g i s d e t e r m i n e d as a f u n c t i o n o f t h e i n c r e a s e i n t h e c o n c e n t r a t i o n o f an u n l a b e l e d l i g a n d w h i l e t h e c o n c e n t r a t i o n o f r a d i o l i g a n d i s h e l d c o n s t a n t . I f t h e r e i s a f f i n i t y f o r t h e b i n d i n g s i t e by t h e u n l a b e l e d l i g a n d , t h e b i n d i n g o f l a b e l e d l i g a n d w i l l d e c r e a s e o v e r a c o n c e n t r a t i o n range o f u n l a b e l e d l i g a n d c o r r e l a t e d w i t h i t s a f f i n i t y f o r t h e s i t e . S a t u r a t i o n b i n d i n g e x p e r i m e n t s a r e t h e n performed i n which t h e c o n c e n t r a t i o n o f f r e e l i g a n d i s i n c r e a s e d and b i n d i n g i s d e t e r m i n e d a t e q u i l i b r i u m . A v a l u e f o r t h e d e n s i t y o f maximum b i n d i n g a t s a t u r a t i n g c o n c e n t r a t i o n s i s c a l c u l a t e d from t h e s a t u r a t i o n b i n d i n g d a t a . A v a l u e f o r t h e a f f i n i t y o f b i n d i n g can be c a l c u l a t e d from each o f t h e t h r e e t y p e s o f e x p e r i m e n t s as a check on t h e r e l i a b i l i t y o f an a f f i n i t y v a l u e . A d i f f e r e n c e o f a f a c t o r o f two between v a l u e s from d i f f e r e n t a s s a y s f o r t h e same parameter i s c o n s i d e r e d t o be good agreement i n c h a r a c t e r i z a t i o n s t u d i e s ( L i m b r i d , 1986). 38 S a t u r a t i o n and t i m e c o u r s e e x p e r i m e n t s measure " s p e c i f i c b i n d i n g " . The b a s i c assumption i n a b i n d i n g a s s a y i s t h a t t h e t i s s u e c o n t a i n s a f i n i t e amount o f s p e c i f i c r e c e p t o r ( r e c o g n i t i o n ) s i t e s w hich become s a t u r a t e d when t h e p r e p a r a t i o n i s exposed t o i n c r e a s i n g c o n c e n t r a t i o n s o f f r e e r a d i o l i g a n d . The b i n d i n g o f t h e l i g a n d can be e n v i s a g e d as t h e sum o f a t l e a s t two p r o c e s s e s — " s p e c i f i c " b i n d i n g t o s a t u r a b l e s i t e s and " n o n s p e c i f i c " b i n d i n g t h a t i s n o n s a t u r a b l e , i n c r e a s e s l i n e a r l y w i t h f r e e r a d i o l i g a n d c o n c e n t r a t i o n , and i n c l u d e s b i n d i n g t o o t h e r membrane components and o t h e r m a t e r i a l . A d d i t i o n o f an e x c e s s amount o f n o n r a d i o a c t i v e m o l e c u l e s of t h e same o r r e l a t e d compound w i l l , by c o m p e t i t i o n , d i s p l a c e a p o r t i o n o f bound r a d i o a c t i v i t y . N o n s p e c i f i c b i n d i n g i s g e n e r a l l y d e f i n e d as t h a t r a d i o l a b e l e d b i n d i n g t h a t i s not d i s p l a c e d by a c o n c e n t r a t i o n o f n o n r a d i o l a b e l e d c o m p e t i t o r a t l e a s t 100 t i m e s t h e (see below) of t h e s p e c i f i c b i n d i n g s i t e , o r a c o n c e n t r a t i o n t a k e n from t h e p l a t e a u a t t h e bottom o f t h e c u r v e g e n e r a t e d by t h e c o m p e t i t i o n e x p e r i m e n t s . The s p e c i f i c b i n d i n g a t each c o n c e n t r a t i o n o r t i m e i s t h e n c a l c u l a t e d as t h e d i f f e r e n c e between t o t a l and n o n s p e c i f i c b i n d i n g . A l l e x p e r i m e n t s were performed on r a t p r i m a r y o c c i p i t a l c o r t e x (Ocl) ( P a x i n o s and Watson, 1986) and c a t a r e a 17 (Tusa e t a l . , 1981; S n i d e r , 1961). A d u l t Sprague-Dawley r a t s w e i g h i n g 200 t o 600 g were a n e s t h e t i z e d w i t h h a l o t h a n e and k i l l e d by d e c a p i t a t i o n . C a t s from 6 t o 230 days p o s t n a t a l age were s a c r i f i c e d w i t h an overdose o f sodium p e n t o b a r b i t o l 39 and r a p i d l y p e r f u s e d through the h e a r t w i t h c o l d phosphate b u f f e r s o l u t i o n (PBS) f o l l o w e d i n most cases by a PBS/0.2% formaldehyde s o l u t i o n . The b r a i n s were d i s s e c t e d out and f r o z e n i n l i q u i d Freon or isopentane f o r s t o r a g e a t -2 0 t o -60° C. S p e c i f i c b i n d i n g of [ 3H]GSH i n r a t c o r t e x showed no decrease from storage f o r up t o one year. Coronal s e c t i o n s (20 /xm) were c u t on a c r y o s t a t (Hacker-Bright) and thaw-mounted onto subbed g l a s s s l i d e s ( i . e . , s l i d e s c oated w i t h g e l a t i n and chromium potassium s u l f a t e ) . S e c t i o n s were p r e - i n c u b a t e d i n c o p l i n j a r s i n c o l d (4° C) b u f f e r , f o l l o w e d by two f i v e - m i n u t e r i n s e s i n the same b u f f e r , w i t h the i n t e n t t o remove as much endogenous l i g a n d as p o s s i b l e . The s l i d e s were then p l a c e d f a c e up on a p l a s t i c t r a y , d r i e d i n a stream of c o o l a i r , and then 500 /ul of i n c u b a t i o n s o l u t i o n c o n t a i n i n g r a d i o l a b l e d l i g a n d was d r i p p e d onto each s e c t i o n . Incubations f o r autoradiography i n c l u d e d 0.2% bovine serum albumin i n order t o reduce b i n d i n g t o n o n s p e c i f i c s i t e s . Some time course and autoradiography i n c u b a t i o n s were performed a t 4° C t o e l i m i n a t e b i n d i n g t o p o s s i b l e Na-independent uptake s i t e s (Ogita and Yoneda, 1989). I n c u b a t i o n was terminated by a s i n g l e 6-second wash i n c o l d (4° C) b u f f e r and r a p i d d r y i n g i n a stream of c o o l a i r , except f o r autoradiography, i n which t h r e e 5-second washes were used t o attempt t o f u r t h e r reduce LAHC b i n d i n g (see B i n d i n g S i t e A n a l y s i s — s p e c i f i c b i n d i n g t h a t i s not a r e c e p t o r ) . 40 A s s a y s o f t o t a l b i n d i n g were r u n i n a t l e a s t t r i p l i c a t e ; n o n - s p e c i f i c b i n d i n g was de t e r m i n e d i n a t l e a s t t r i p l i c a t e by a d d i t i o n o f 50 ul o f 1 0 " 1 M GSH and 1 0 _ 1 M GSSG t o g i v e a f i n a l c o n c e n t r a t i o n o f 2 x 1 0 ~ 2 M t o t a l d i s p l a c e r as i n d i c a t e d from c o m p e t i t i o n e x p e r i m e n t s (see R e s u l t s ) . Twenty jul samples o f i n c u b a t i o n medium were removed t o d e t e r m i n e f r e e l i g a n d c o n c e n t r a t i o n . The washed and d r i e d s e c t i o n s were s c r a p e d onto a s m a l l c i r c l e o f g l a s s m i c r o f i b r e f i l t e r p a per (Whatman FG/B), and p l a c e d d i r e c t l y i n c o u n t i n g v i a l s i n 4 ml o f Formula NEF-963 (NEN). V i a l s were capped, shaken, l e f t o v e r n i g h t , and t h e amount o f bound r e c e p t o r was d e t e r m i n e d i n an LS 6000 IC Beckman S c i n t i l l a t i o n C o u n t e r ( e f f i c i e n c y 55% f o r t r i t i u m , 95% f o r [ 3 5 S ] ) . I n t h e s a t u r a t i o n b i n d i n g e x p e r i m e n t s a l t e r n a t e s e c t i o n s were used f o r d e t e r m i n a t i o n o f p r o t e i n c o n t e n t by t h e methods o f Lowry e t a l . (1951). S e c t i o n s were p r o c e s s e d f o r a u t o r a d i o g r a p h y by a p p o s i t i o n f o r a p p r o x i m a t e l y 24 hours t o Amersham H y p e r f i l m -3H, w h i c h was t h e n c o n v e n t i o n a l l y d e v e l o p e d and f i x e d . O r i g i n a l s e c t i o n s were t h e n s t a i n e d f o r cytochrome o x i d a s e l e v e l s by t h e method o f Wong-Riley (1979), w h i c h c l e a r l y and s p e c i f i c a l l y d e l i n e a t e s l a y e r 4 o f a r e a 17 i n c a t (Wong-R i l e y , 1979) and i n monkey ( C a r r o l and Wong-Riley, 1984), and i s s u g g e s t e d t o c o r r e l a t e w i t h t h e d e n s i t y o f t h a l a m i c t e r m i n a t i o n s (Wong-Riley, 1979). Q u a n t i t a t i v e a n a l y s i s o f l a m i n a r b i n d i n g d e n s i t y o f [ 3 5 S ] on a u t o r a d i o g r a p h i c f i l m s was done u s i n g an Imaging R e s e a r c h I n c . ( S t . C a t h e r i n e ' s , ONT 41 Canada) image a n a l y s i s system, "MCID", c a l i b r a t e d w i t h [ C] s t a n d a r d s (ARC, S t . L o u i s , MD; .002 - 35.0 /^Ci/g) . A d e n s i t y p r o f i l e was c o n s t r u c t e d a c r o s s a l l c o r t i c a l l a y e r s i n one r e p r e s e n t a t i v e s e c t i o n o f s t r i a t e c o r t e x from each a n i m a l . BINDING SITE ANALYSIS M a t h e m a t i c a l a n a l y s i s o f t h e b i n d i n g d a t a , as d e s c r i b e d by B y l u n d (1980) and o t h e r a u t h o r s as n o t e d , i s as f o l l o w s . The a s s u m p t i o n made i s t h a t l i g a n d - r e c e p t o r b i n d i n g i s a r e v e r s i b l e b i m o l e c u l a r r e a c t i o n t h a t a t e q u i l i b r i u m obeys t h e law o f mass a c t i o n and can be d e s c r i b e d as f o l l o w s : [L] + [R] <====> [LR] [1] k - i where [L] i s t h e c o n c e n t r a t i o n o f f r e e l i g a n d , [R] t h e c o n c e n t r a t i o n o f t h e f r e e r e c e p t o r , [RL] i s t h e c o n c e n t r a t i o n o f t h e l i g a n d / r e c e p t o r complex, and k + i and k _ i a r e t h e a s s o c i a t i o n and d i s s o c i a t i o n c o n s t a n t s , r e s p e c t i v e l y . The e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t (K^) f o r t h e l i g a n d -r e c e p t o r i n t e r a c t i o n i s used as a measure of t h e a f f i n i t y o f t h e l i g a n d f o r t h e r e c e p t o r and i s d e f i n e d by t h e law o f mass a c t i o n : K d = [ L ] [ R ] / [ L R ] = k _ ! / k + 1 [2] 42 A s s o c i a t i o n and d i s s o c i a t i o n k i n e t i c s : The can be d e t e r m i n e d from k + i and k_! ( e q u a t i o n 2 ) . F o r t h e d e t e r m i n a t i o n o f k + i , t h e p s e u d o - f i r s t o r d e r method was used, where i t i s assumed t h a t [L] >> [ R ] , and t h u s i s can be assumed t h a t t h e c o n c e n t r a t i o n o f r a d i o l i g a n d added a p p r o x i m a t e s a c o n s t a n t [ L ] . From e q u a t i o n 1 t h e f o l l o w i n g i n t e g r a t e d r a t e e q u a t i o n i s t h e n d e r i v e d : l n ( B e / ( B e - B t ) ) = ( k + 1 [ L ] + k _ ! ) t = k o b t . [3] where B-t i s t h e amount bound a t any t i m e and B e i s t h e amount bound a t e q u i l i b r i u m . A p l o t o f I n ( B e / ( B e - B t ) ) v e r s u s t w i l l t h u s have a s l o p e o f k ^ . O b t a i n i n g k_! from i n d e p e n d e n t e x p e r i m e n t s a l l o w s c a l c u l a t i o n o f k + i from k 0j-, and [L] ( e q u a t i o n 3 ) . To det e r m i n e k_x , t h e b i n d i n g r e a c t i o n i s a l l o w e d t o p r o c e e d t o e q u i l i b r i u m and t h e n , a t t = 0, r e b i n d i n g o f l i g a n d i s p r e v e n t e d by an e f f e c t i v e l y i n f i n i t e d i l u t i o n , and t h e amount bound i s d e t e r m i n e d a t v a r i o u s t i m e s t h e r e a f t e r . I f B = Bg a t t = 0, t h e n t h e i n t e g r a t e d d i s s o c i a t i o n r a t e e q u a t i o n i s d e r i v e d from e q u a t i o n 1: I n (B/B 0) = - k _ ! t [4] su c h t h a t a p l o t o f I n ( B / B Q ) v e r s u s t has a s l o p e o f -k _ ] _ . 43 S a t u r a t i o n b i n d i n g e x p e r i m e n t s : F o r r e c e p t o r b i n d i n g , a p l o t o f [RL] v e r s u s [L] y i e l d s a r e c t a n g u l a r h y p e r b o l e , t h e e q u a t i o n o f w h i c h i s d e r i v e d from e q u a t i o n 2: [RL] = [Rip] [ L ] / ( K d + [L]) [5] where t h e t o t a l r e c e p t o r c o n c e n t r a t i o n [RT] = [R] + [ R L ] . I t can be seen t h a t f o r t h e p o i n t on t h e c u r v e where [L] = K d, [RL] = 1/2 R T ( e q u a t i o n 7) , t h u s t h e K d i s e q u a l t o t h e c o n c e n t r a t i o n o f L which o c c u p i e s one h a l f t h e t o t a l b i n d i n g s i t e s . E q u a t i o n 7 can be t r a n s f o r m e d i n t o t h e l i n e a r E a d i e -H o f s t e e r e l a t i o n s h i p : [RL] = [RT] - K d ( [ R L ] / [ L ] ) [6] These p a r a m e t e r s a r e more commonly r e p r e s e n t e d by t h e f o l l o w i n g symbols: B = B m a x - K d(B/F) [7] I n a p l o t o f B v e r s u s (B/F) t h e B m a x i s t h u s t h e i n t e r c e p t on t h e Y - a x i s and t h e K d i s t h e n e g a t i v e s l o p e . A n a l y s i s o f E a d i e - H o f s t e e p l o t s i s somewhat more c o m p l i c a t e d . F o r s i n g l e s i t e E a d i e - H o f s t e e p l o t s , Z i v i n and Waud (1982) show t h a t t h e v a l u e s f o r B m a x and K d a r e s y s t e m a t i c a l l y u n d e r e s t i m a t e d , and t h e y p r o v i d e a means o f c o r r e c t i n g f o r t h i s b i a s , based on an e s t i m a t e o f background 44 e r r o r , E r a d . The q u a l i t y o f s a t u r a t i o n - b i n d i n g d a t a can be a s s e s s e d by i n s p e c t i o n o f t h e s t a n d a r d d e v i a t i o n o f t h e background e r r o r , t h e S D ( E r a d ) . I f t h e S D ( E r a d ) i s more t h a n 0.2, t h e d a t a w i l l be o f l i t t l e v a l u e . F o r E a d i e - H o f s t e e p l o t s o f d u a l b i n d i n g s i t e s t h e p l o t i s a c u r v e w i t h two s l o p e components t h a t must be s e p a r a t e d . Hunston (1975) p r o v i d e s a method o f d i r e c t l y c a l c u l a t i n g t h e f o u r p a r a m e t e r s (two each o f B m a x and K d) from an i n i t i a l e s t i m a t e o f t h e l i m i t i n g s l o p e s a t each end o f t h e c u r v e . A n a l y s i s o f s a t u r a t i o n b i n d i n g w i t h r a d i o l a b e l e d GSH was pe r f o r m e d u s i n g a computer program f o r t h e s i n g l e - s i t e E a d i e - H o f s t e e method d e s c r i b e d by Z i v i n and Waud (1982) w i t h t h e f o l l o w i n g m o d i f i c a t i o n . F o r d u a l b i n d i n g s i t e s , t h e c o r r e c t e d B m a x and K d v a l u e s c a l c u l a t e d from t h e two i s o l a t e d components o f t h e c u r v e were used t o dete r m i n e i n t e r c e p t v a l u e s f o r r e c a l c u l a t i o n o f B m a x and K d v a l u e s by t h e d u a l - s i t e method of Hunston (1975). A n o t h e r parameter d e r i v e d from s a t u r a t i o n b i n d i n g d a t a i s t h e H i l l c o e f f i c i e n t . The H i l l c o e f f i c i e n t i s an i n d i c a t i o n o f c o o p e r a t i v i t y : a v a l u e o f 1.0 i n d i c a t e s t h a t l i g a n d - r e c e p t o r i n t e r a c t i o n o c c u r s v i a a r e a c t i o n o b e y i n g a s i m p l e mass a c t i o n law. H i g h e r o r lower v a l u e s s u g g e s t p o s i t i v e o r n e g a t i v e c o o p e r a t i v i t y , r e s p e c t i v e l y . The H i l l c o e f f i c i e n t i s t a k e n from t h e g e n e r a l i z e d form o f e q u a t i o n 5, where t h e b i n d i n g o f each l i g a n d m o l e c u l e f a c i l i t a t e s t h e b i n d i n g o f t h e ne x t one: 4 5 B = B m a x - F n / ( K d + F n) [8] which can be l o g a r i t h m i c a l l y transformed thus: log(B/(B 'max - B)) = n l o g F - l o g [9] so t h a t a p l o t of log(B/(B-'max - B)) versus l o g F y i e l d s a s t r a i g h t l i n e w i t h a s l o p e n, the H i l l c o e f f i c i e n t . C o r r e c t e d H i l l c o e f f i c i e n t s were c a l c u l a t e d f o r each component by the program based on the method of Z i v i n and Waud (1982), and f o r the whole curve by t a k i n g the s l o p e of the p l o t of equation 9. Competition experiments: The a f f i n i t y of a compound f o r the GSH b i n d i n g s i t e or s i t e s can be determined by i t s I C 5 0 / the c o n c e n t r a t i o n of the u n l a b e l e d compound t h a t d i s p l a c e s h a l f of the b i n d i n g of r a d i o l a b e l e d GSH, which depends on the c o n c e n t r a t i o n of r a d i o l a b e l e d GSH added, [ L ] . "K^" i s the term g i v e n t o r e f e r t o the e q u i l i b r i u m c o n s t a n t of the competing compound (or " i n h i b i t o r " ) , which i s d e r i v e d from e q u a t i o n 1: K i = I C 5 0 / ( 1 + [L]/K d) [10] A c o m p e t i t i o n curve f o r a competing l i g a n d w i t h s i g n i f i c a n t l y d i f f e r e n t a f f i n i t i e s f o r two or more b i n d i n g - s i t e subtypes shows a c l e a r l y b i p h a s i c shape and d e v i a t e s from the s i n g l e -4 6 s i t e r u l e . The s i n g l e s i t e r u l e s t a t e s t h a t when t h e c o n c e n t r a t i o n o f c o m p e t i t o r i s i n c r e a s e d from 0 . 1 t o 1 0 t i m e s t h e I C 5 0 , t h e amount o f b i n d i n g d e c r e a s e s from 9 1 % t o 9 % . When GSH i t s e l f i s used as t h e competing l i g a n d , t h e K d can be c a l c u l a t e d . F o r m u l t i p l e s i t e s t h e most a c c u r a t e method o f d e t e r m i n i n g t h e s e p a r a t e K d v a l u e s was pr o p o s e d by B y l u n d ( 1 9 8 6 ) , as f o l l o w s . The e q u a t i o n d e s c r i b i n g c o m p e t i t i v e i n h i b i t i o n i s where I i s t h e c o n c e n t r a t i o n o f f r e e " i n h i b i t o r " ( c o m p e t i t o r ) and L i s t h e f r e e r a d i o l i g a n d c o n c e n t r a t i o n . When t h e r a d i o l i g a n d and c o m p e t i t o r have t h e same a f f i n i t y f o r t h e r e c e p t o r ( K d = K ^ ) , t h e n B = B m a x ' L / ( K d ( l + I / K i ) + L [ 1 1 ] B = B m a x ' L / ( K d + I + L) [ 1 2 ] w h i c h can be r e a r r a n g e d t o produce B = - B ' I - 1 / ( K d + L) + B-'max •L/(Kd + L) [ 1 3 ] I f we d e f i n e B Q as t h e v a l u e o f B when 1 = 0 , t h e n B = - B ' I - 1 / ( K d + L) + B 0 [ 1 4 ] Thus a p l o t o f B v e r s u s B*I has a s l o p e e q u a l t o - l / ( K d + L ) . 47 A d u a l s i t e c o m p e t i t i o n p l o t y i e l d s a c u r v e d B v e r s u s B ' l p l o t a n a l o g o u s t o an E a d i e - H o f s t e e p l o t composed o f two components from w h i c h t h e two independent s l o p e s can be e x t r a c t e d . S p e c i f i c b i n d i n g t h a t i s not a r e c e p t o r : U n f o r t u n a t e l y , r e c e p t o r b i n d i n g c r i t e r i a can be "mimicked" by o t h e r phenomena ( C u a t r e c a s a s and H o l l e n b e r g , 1976). F o r example, s p e c i f i c b i n d i n g o f [ 1 2 5 I ] g l u c a g o n t o m i l l i p o r e f i l t e r s i n t h e absence o f t i s s u e shows h i g h a f f i n i t y , s a t u r a b i l i t y , r e v e r s i b i l i t y , and s p e c i f i c i t y i n i t s d i s p l a c e m e n t by n a t i v e g l u c a g o n , growth hormone, and v a s o p r e s s i n . The i m p o r t a n c e o f s u c h phenomena f o r d a t a a n a l y s i s i s emphasized by t h i s quote from C u a t r e c a s a s and H o l l e n b e r g (1976): " S i n c e b i o l o g i c a l t i s s u e s w i l l s u r e l y a l s o e x h i b i t some of t h e " s p e c i f i c " t y p e s o f i n t e r a c t i o n s d e s c r i b e d here f o r s i m p l e n o n r e c e p t o r s ystems, awareness o f such i n t e r a c t i o n s s h o u l d encourage g r e a t e r s c r u t i n y , p a r t i c u l a r l y f o r t h o s e i n t e r a c t i o n s o b s e r v e d w i t h h i g h c o n c e n t r a t i o n s o f hormone, where t h e r e s u l t s a r e f r e q u e n t l y i n t e r p r e t e d t o i n d i c a t e "second" o r " m u l t i p l e " c l a s s e s o f r e c e p t o r s . I n v i e w o f t h e u b i q u i t y w i t h w h i c h n o n r e c e p t o r , but " s p e c i f i c " , b i n d i n g o f moderate — ft o a f f i n i t y ( a p p a r e n t Kg-, 10 ° t o 10 M) can be d e m o n s t r a t e d i n a v a r i e t y o f s i m p l e systems t h a t do not c o n t a i n t i s s u e , i t w ould i n d e e d be s u r p r i s i n g i f complex b i o l o g i c a l t i s s u e s d i d n o t a l s o e x h i b i t such p r o p e r t i e s . I n t h i s r e s p e c t , i t i s p e r t i n e n t t h a t v i r t u a l l y a l l p e p t i d e and o t h e r hormones 48 ( e . g . , c a t e c h o l a m i n e s , a c e t y l c h o l i n e , s t e r o i d ) can be de m o n s t r a t e d , under p r o p e r c o n d i t i o n s , t o e x h i b i t a "second" l o w - a f f i n i t y and h i g h - c a p a c i t y b i n d i n g component when d a t a a r e a n a l y z e d by S c a t c h a r d [a v a r i a n t o f E a d i e - H o f s t e e ] p l o t s . U n f o r t u n a t e l y , i t i s most d i f f i c u l t t o s t u d y t h e s e low-a f f i n i t y s i t e s p r o p e r l y i n complex t i s s u e s . However, s p e c i a l c a r e s h o u l d be e x e r c i s e d i n i n t e r p r e t i n g t h e "second" s i t e as a s i n g l e , u n i q u e b i n d i n g s t r u c t u r e o r c l a s s o f r e c e p t o r s r a t h e r t h a n as t h e sum o f a complex m i x t u r e o f components u n r e l a t e d t o r e c e p t o r s . " Such low a f f i n i t y , h i g h c a p a c i t y (LAHC) n o n r e c e p t o r s p e c i f i c b i n d i n g can produce anomalous b e h a v i o u r i n k i n e t i c a n a l y s e s . Mass a c t i o n assumptions may no l o n g e r h o l d . There may a l s o be a v i o l a t i o n o f t h e assumption o f c o n s t a n t [L] i n p s e u d o - f i r s t o r d e r a n a l y s i s o f a s s o c i a t i o n b i n d i n g c o n d u c t e d a t low [ L ] , due t o t h e l a r g e B m a x o f LAHC b i n d i n g . (See R e s u l t s . ) An a l t e r n a t i v e i n t e r p r e t a t i o n o f LAHC b i n d i n g i s t h a t i t r e p r e s e n t s b i n d i n g t o an uptake s i t e (see D i s c u s s i o n ) . P l o t t i n g and c u r v e s t r i p p i n g : P l o t s o f t h e d a t a were g e n e r a t e d as f o l l o w s . S t a n d a r d e r r o r s were c a l c u l a t e d f o r each p o i n t and used t o a i d drawing o f c u r v e s f o r u n t r a n s f o r m e d d a t a and t o c a l c u l a t e s i g n i f i c a n c e by S t u d e n t ' s t - t e s t . L i n e a r p l o t s f o r t r a n s f o r m e d d a t a were d e t e r m i n e d by a l i n e a r r e g r e s s i o n program (SIGMAPLOT). Each l i n e drawn f o r a s i n g l e component o f a d u a l component p l o t was d e t e r m i n e d by 49 l i n e a r r e g r e s s i o n on t h e s u b s e t o f p o i n t s a t t r i b u t a b l e t o t h a t component o n l y , w i t h t h e f o l l o w i n g e x c e p t i o n s . I n t h e c a s e o f t h e dual-component k i n e t i c a s s o c i a t i o n c u r v e ( F i g u r e 15A), new p o i n t s were f i r s t d e r i v e d f o r t h e e a r l y component by m a t h e m a t i c a l s t r i p p i n g o f t h e s l o w e r e x p o n e n t i a l component by a m o d i f i c a t i o n o f t h e method o f Lawson (1986). The f i t t i n g o f t h e l i n e s f o r t h e d u a l - s i t e B v e r s u s B*I p l o t ( F i g u r e 17B) was performed by t h e g r a p h i c method o f R o s e n t h a l (1967). The f i t t i n g o f t h e l i n e f o r t h e d u a l - s i t e E a d i e -H o f s t e e p l o t was de t e r m i n e d from t h e v a l u e s d e r i v e d from t h e Hunston (1975) method as d e s c r i b e d above. IN VIVO UPTAKE PROCEDURES Male Sprague-Dawley r a t s w e i g h i n g from 250 t o 650 grams were used f o r each o f t h e r a d i o a c t i v e t r a c e r s l i s t e d , and one a d u l t f e m a l e c a t (100 days o l d ) was used t o t e s t [ 3H]GSH u p t a k e . F o r s u r g e r y , r a t s were d e e p l y a n e s t h e t i z e d w i t h S o m n i t o l (sodium p e n t o b a r b i t o l ) (0.1 ml/100 g, i . p . ) . A t r o p i n e s u l f a t e (10 mg/kg, i.m.) was g i v e n t o p r e v e n t s u f f o c a t i o n . A n i m a l s were h e l d i n a s t e r e o t a x i c a p p a r a t u s , where h o l e s were made i n t h e s k u l l o v e r l y i n g a r e a O c l on one s i d e u s i n g a "Dremmel" hand d r i l l . A 50 ixl H a m i l t o n s y r i n g e w i t h a 33-gauge, b l u n t - e n d e d n e e d l e was p l a c e d i n t h e s t e r e o t a x i c a p p a r a t u s f o r i n j e c t i o n s . I n j e c t i o n s were made i n t o p r i m a r y v i s u a l c o r t e x o f e l e v e n r a t s (Ocl) ( P a x i n o s and 50 Watson, 1986), and one c a t (area 17) (Tusa e t a l . , 1981). Most i n j e c t i o n s i t e s c o n s i s t e d of a s e r i e s of i n j e c t i o n s a t one minute i n t e r v a l s spanning the depth of the c o r t e x from approximately 750 t o 2 00 fxra. from the s u r f a c e . In s e v e r a l cases the t r i t i a t e d t r a c e r s were used as s u p p l i e d (1 juCi/jul) , so t h a t a t two i n j e c t i o n s i t e s 2 ill of t r a c e r was p r e s s u r e -i n j e c t e d a t s e v e r a l c o r t i c a l l e v e l s f o r a t o t a l of 10 fil (10 /LtCi) . In other cases 20 jul of t r i t i a t e d t r a c e r was f i r s t evaporation-condensed t o approximately 4 jul and i n j e c t e d i n one s i t e a t two l e v e l s f o r a t o t a l of 20 fxCi i n a s m a l l e r volume. For the higher-energy beta emmitter, [ S ] , a 2 ul i n j e c t i o n i n one s i t e was s u f f i c i e n t . Most r a t s were allowed t o s u r v i v e f o r two days t o maximize r e t r o g r a d e t r a n s p o r t by p a s s i v e d i f f u s i o n (Baughman and G i l b e r t , 1981) w h i l e r e s t r i c t i n g i t t o the completed f a s t phase of axoplasmic t r a n s p o r t i n order t o minimize l a b e l i n g of f i b r e s of passage h. (Cowan e t a l . , *72; Hendrickson, 1972; Swanson e t a l . 1974). The c a t and one r a t ([ 3H]GSH) were s a c r i f i c e d a f t e r one day t o minimize t r a n s s y n a p t i c t r a n s p o r t and l o s s of t e r m i n a l l a b e l (Hendrickson, 1972). Rats were s a c r i f i c e d w i t h an overdose of sodium p e n t o b a r b i t o l and r a p i d l y p e r f u s e d w i t h c o l d phosphate b u f f e r s o l u t i o n (PBS) f o l l o w e d by e i t h e r a PBS/2% paraformaldehyde/ 1% g l u t a r a l d e h y d e s o l u t i o n , or a PBS/4% paraformaldehyde s o l u t i o n . The c a t was f i r s t a n e s t h e t i z e d w i t h halothane, then s a c r i f i c e d w i t h an overdose of sodium p e n t o b a r b i t o l and r a p i d l y p e r f u s e d through the h e a r t w i t h c o l d phosphate b u f f e r s o l u t i o n (PBS) f o l l o w e d by a 51 PBS/4% p a r a f o r m a l d e h y d e s o l u t i o n . The b r a i n s were d i s s e c t e d o u t and f r o z e n i n l i q u i d i s o p e n t a n e a t -60 °C. The c a t b r a i n was f i r s t p l a c e d i n a s a t u r a t e d 3 0% s u c r o s e s o l u t i o n . C o r o n a l s e c t i o n s (20 /im) were c u t on a c r y o s t a t , thaw-mounted onto subbed g l a s s s l i d e s ( i . e . , s l i d e s c o a t e d w i t h g e l a t i n and chromium p o t a s s i u m s u l f a t e ) , and p r o c e s s e d f o r a u t o r a d i o g r a p h y by a p p o s i t i o n t o Amersham H y p e r f i l m - [ H] f o r from one t o 90 days. F o l l o w i n g a u t o r a d i o g r a p h y on f i l m , some o f t h e s e c t i o n e d t e s t b r a i n s were s t a i n e d f o r N i s s l s u b s t a n c e w i t h c r e s y l v i o l e t t o i d e n t i f y t h a l a m i c n u c l e i and c o r t i c a l l a m i n a e . Other s e c t i o n e d t e s t b r a i n s f o r a l l r a d i o l i g a n d s e x c e p t [ 3 5 S ] G S H were t h e n used t o determine<a c e l l u l a r r e s o l u t i o n o f t h e r a d i o l a b e l uptake t o c e l l b o d i e s ( i . e . , r e t r o g r a d e t r a n s p o r t ) by t h e d i r e c t l i q u i d - e m u l s i o n d i p p i n g method d e s c r i b e d by Rogers (1979) u s i n g I l f o r d K2 n u c l e a r e m u l s i o n . The e m u l s i o n c o a t e d s l i d e s were were exposed a t 4 °C f o r 37 days f o r [ 3 5 S ] and f o r 4 months f o r t r i t i u m . F o l l o w i n g development o f t h e exposed e m u l s i o n c o a t e d s l i d e s , t h e s e s e c t i o n s were s t a i n e d f o r c e l l b o d i e s w i t h p y r o n i n Y. T h a l a m i c n u c l e i were i d e n t i f i e d from t h e c r e s y l - v i o l e t and p y r o n i n Y s t a i n e d s e c t i o n s i n r e f e r e n c e t o T a k a h a s h i (1985) and P a x i n o s and Watson (1986) f o r t h e r a t s and S n i d e r (1969) f o r t h e c a t . 52 RESULTS PARAMETRIC STUDIES Experiments were performed with r a t v i s u a l c o r t e x t o d e f i n e f i x e d v a l u e s f o r i n c u b a t i o n parameters a c r o s s a l l c h a r a c t e r i z a t i o n experiments, i n which the dependent v a r i a b l e , b i n d i n g , would be determined as a f u n c t i o n of v a r i o u s f a c t o r s (see Methods), and f o r the b i n d i n g d i s t r i b u t i o n experiments. The c r i t e r i a f o r d e t e r m i n i n g these i n c u b a t i o n parameters was taken t o be a maximization of s p e c i f i c b i n d i n g of [ 3H]GSH. B i n d i n g of [ 3H]glutamate and i n t e r a c t i o n s between glutamate and GSH b i n d i n g were a l s o t e s t e d under the c o n d i t i o n s so d e f i n e d , t o determine whether GSH was b i n d i n g t o a s i t e e x c l u s i v e of glutamate b i n d i n g s i t e s . The p a r a m e t r i c experiments were performed w i t h 500 nM [ 3H]GSH because, due t o the low s p e c i f i c a c t i v i t y s u p p l i e d by the manufacturer, t h i s was the minimum c o n c e n t r a t i o n t h a t would a l l o w dpm v a l u e s s i g n i f i c a n t l y above background i n low b i n d i n g c o n d i t i o n s . T h i s c o n c e n t r a t i o n of [ H]GSH sho u l d have r e s u l t e d i n roughly equal p r o p o r t i o n s of b i n d i n g t o the t h r e e independent b i n d i n g s i t e s found, as e s t i m a t e d from the K d and B m a x v a l u e s obtained i n the subsequent b i n d i n g s i t e c h a r a c t e r i z a t i o n experiments. The i n c u b a t i o n time r e q u i r e d t o r e a c h e q u i l i b r i u m (steady s t a t e b i n d i n g , see Methods) was determined i n p r e l i m i n a r y time course experiments w i t h 5 nM 53 [ J 3S]GSH and 355 nM [ JH]GSH i n 50 mM T r i s - a c e t a t e b u f f e r , pH 7.4, a t room temperature. Incubations were run f o r 12 0 minutes, the time r e q u i r e d t o reach e q u i l i b r i u m a t the lowest c o n c e n t r a t i o n , e n s u r i n g t h a t a l l c o n d i t i o n s were assayed a t e q u i l i b r i u m . The d i s s o c i a t i o n h a l f - t i m e from the aforementioned 355 nM time course experiment a l l o w e d c a l c u l a t i o n of optimal r i n s e times ( f o r removal of f r e e r a d i o l i g a n d ) t o maximize l o s s of n o n s p e c i f i c b i n d i n g and minimize l o s s of s p e c i f i c b i n d i n g (Bennett, 1978). Non-s p e c i f i c b i n d i n g d e f i n e d by the a d d i t i o n of 10~ 4 M GSH was up t o 50 p e r c e n t of t o t a l b i n d i n g f o r the p a r a m e t r i c experiments. For the subsequent b i n d i n g s i t e c h a r a c t e r i z a t i o n experiments, the n o n - s p e c i f i c b i n d i n g was reduced by the a d d i t i o n of 10" 2 M GSH p l u s 10" 2 M GSSG. The need f o r t h i s r e l a t i v e l y l a r g e amount of d i s p l a c e r was determined by com p e t i t i o n experiments (see R e s u l t s - B i n d i n g S i t e C h a r a c t e r i z a t i o n ; F i g u r e 16), due t o the presence of LAHC b i n d i n g (see Methods). V a r i o u s c o n d i t i o n s were t e s t e d f o r maximum s p e c i f i c b i n d i n g . F i g u r e 7 i n d i c a t e s optimal b u f f e r o s m o l a r i t y t e s t e d i n T r i s - a c e t a t e . The subsequent i n c u b a t i o n s were performed a t 100 mM b u f f e r . S p e c i f i c b i n d i n g showed a marked peak a t pH 8.0 ( F i g u r e 8), a t which the subsequent i n c u b a t i o n s were performed. ( V a r i a t i o n s i n b i n d i n g between assays a t p o i n t s r e p r e s e n t i n g the same c o n d i t i o n s are due t o v a r i a t i o n i n sample s i z e from one assay t o another.) S p e c i f i c b i n d i n g was s i g n i f i c a n t l y reduced by i n c l u s i o n of ions and b u f f e r s 54 F i g u r e 7. B i n d i n g of [ JH]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n o f c o n c e n t r a t i o n of b u f f e r . C r y o s t a t s e c t i o n s of a d u l t r a t O c l were incubated i n v a r i o u s c o n c e n t r a t i o n s of T r i s - a c e t a t e a t pH 7.4 f o r 120 minutes a t room temperature w i t h 500 nM [ HIGSH. N o n s p e c i f i c b i n d i n g was d e f i n e d by the a d d i t i o n of 10 M GSH. The graph r e p r e s e n t s one experiment conducted i n q u a d r u p l i c a t e . F i g u r e 8. B i n d i n g of [ JH]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n of pH. C r y o s t a t s e c t i o n s of a d u l t r a t O c l were in c u b a t e d i n 100 mM T r i s - a c e t a t e f o r 120 minutes a t room temperature w i t h 500 nM [ H]GSH a t v a r i o u s pH v a l u e s as i n d i c a t e d . N o n s p e c i f i c b i n d i n g was d e f i n e d by the a d d i t i o n of 10~ 4 M GSH. The graph r e p r e s e n t s combined data from two se p a r a t e experiments conducted i n q u a d r u p l i c a t e . 600 5.900 6.900 7.900 8.900 PH O—Ototal A — A nonspecific •—•specific 56 700.0 Tris-acetate Tris—citrate Tris—HCI F i g u r e 9. B i n d i n g o f [ H]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n o f b u f f e r t y p e . C r y o s t a t s e c t i o n s o f a d u l t r a t O c l were i n c u b a t e d i n 100 mM T r i s - a c e t a t e , T r i s - c i t r a t e , o r T r i s -HC1 a t pH 8.0 f o r 120 minutes a t room t e m p e r a t u r e w i t h 500 nM [ H I G S H . N o n s p e c i f i c b i n d i n g was d e f i n e d by t h e a d d i t i o n o f 10 M GSH. Data i s from one experiment c o n d u c t e d i n q u a d r u p l i c a t e . 57 F i g u r e 10. B i n d i n g of [ JH]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n of i o n c o n c e n t r a t i o n . C r y o s t a t s e c t i o n s o f a d u l t r a t O c l were incubated i n 100 mM T r i s - a c e t a t e a t pH 8.0 f o r 12 0 minutes a t room temperature with 500 nM [ 3H]GSH and v a r i o u s c o n c e n t r a t i o n s of ions as i n d i c a t e d . N o n s p e c i f i c b i n d i n g was d e f i n e d by the a d d i t i o n of 10~ 4 M GSH. The graph r e p r e s e n t s combined data from two separate experiments conducted i n q u a d r u p l i c a t e . (A) NaCl. (B) C a C l 2 . (C) M g C l 2 . (D) MgS0 4. (E) KC1. (F) KH 2P0 4. 58 O O total A A nonspecific • • specific OH 1 1 1 1 0 0.1 1 10 100 mM CaCI o O — O total A — A nonspecific • — •specific 59 0-| 1 1 1 1 0 0.01 0.1 1 10 mM MgCI 2 O-—Ototal A — - A nonspecific • — • specific 60 E .0 0.01 O O total A-0.1 mM KCI •A nonspecific specific 4 0 0 $ F 0 0.01 0.1 mM KHJ^0 4 O — O total A — A nonspecific >— # specific 61 1000.0 % formalin Figure 11. Binding of [JH]GSH i n rat vi s u a l cortex as a function of f i x a t i o n . Cryostat sections of adult r a t Ocl were preincubated i n cold (4°C) Tris-acetate (pH 8, 100 mM) with 0.6%, 0.2%, or no formalin for ten minutes and then incubated i n 100 mM Tris-acetate at pH 8.0 for 120 minutes at room temperature with 500 nM :[3H]GSH. Data i s from one experiment conducted i n quadruplicate. Nonspecific binding was defined by the addition of 10~ 4 M GSH. 62 F i g u r e 12. B i n d i n g of [ JH]GSH i n r a t v i s u a l c o r t e x as a f u n c t i o n of the c o n c e n t r a t i o n of v a r i o u s b i o c h e m i c a l agents. C r y o s t a t s e c t i o n s of a d u l t r a t O c l were incubated i n 100 mM T r i s - a c e t a t e a t pH 8.0 f o r 120 minutes a t room temperature w i t h 500 nM [ 3H]GSH and v a r i o u s c o n c e n t r a t i o n s of thes e agents as i n d i c a t e d . The graph r e p r e s e n t s combined data from two s e p a r a t e experiments conducted i n q u a d r u p l i c a t e . N o n s p e c i f i c b i n d i n g was d e f i n e d by the a d d i t i o n of 10 M GSH. (A) B a c i t r a c i n . (B) A c i v i c i n . (C) D i t h i o t h e i t o l . 63 400-F A O-0.1 1 mM bacitracin O total A A nonspecific 100 specific 400-F B 0 0.1 1 1 0 mM acivicin O Ototal A A nonspecific #—-< 100 specific 64 O O total A A nonspecific • •specific 65 commonly used i n s t u d i e s of EAA or p e p t i d e r e c e p t o r s ( B l a k e l y e t a l . , 1986; L u i n i e t a l . , 1984; P i n e t a l . , 1987; Mantyh e t a l . , 1989; P u l l a n e t a l . , 1987). These i n c l u d e d NaCl, C a C l 2 , MgCl, MgS0 4, or KCl, KH 2P0 4, T r i s - h y d r o c h l o r i d e , and T r i s -c i t r a t e ( F i g u r e s 9 and 10). F i x a t i o n with 0.2% f o r m a l i n i n c l u d e d i n the f i r s t s tep of the pr e i n c u b a t o n showed no s i g n i f i c a n t r e d u c t i o n of s p e c i f i c b i n d i n g ( F i g u r e 11), and was i n c l u d e d i n the f o l l o w i n g i n c u b a t i o n s . The phosphatase i n h i b i t o r b a c i t r a c i n , commonly i n c l u d e d i n r e c e p t o r b i n d i n g i n c u b a t i o n s , produced a s l i g h t r e d u c t i o n of s p e c i f i c b i n d i n g ( F i g u r e 12A). The i n c l u s i o n of a c i v i c i n t o b l o c k the p o s s i b l e degrading a c t i o n of endogenous plasma-membrane-bound gamma-glutamyl t r a n s p e p t i d a s e (GGTP) ( H i l l e t a l . , 1985) was om i t t e d from the subsequent c h a r a c t e r i z a t i o n experiments because i t was determinined t h a t up t o 10 mM a c i v i c i n showed no e f f e c t on s p e c i f i c b i n d i n g under these i n - v i t r o c o n d i t i o n s ( F i g u r e 12B). The r e d u c i n g agent d i t h i o t h r e i t o l (DTT) (see F i g u r e 13) had a n e g a t i v e impact on GSH b i n d i n g . A p a r a m e t r i c assay ( F i g u r e 12C) showed a smal l r e d u c t i o n of s p e c i f i c b i n d i n g and an anomalous e f f e c t on n o n s p e c i f i c b i n d i n g w i t h c o n c e n t r a t i o n . However, p r e l i m i n a r y c o m p e t i t i o n s t u d i e s of r a d i o l a b e l e d GSH b i n d i n g by nonlabeled GSH showed a marked r e d u c t i o n i n a f f i n i t y due t o the presence of the DTT i n c l u d e d by the manufacturer i n the r a d i o l i g a n d s t o c k s o l u t i o n (10 mM), a phenomemon t h a t was determined as f o l l o w s . C ompetition of 2 jiiM [ 3H]GSH with GSH showed an I C 5 0 v a l u e two 66 o r d e r s of magnitude hig h e r than c o m p e t i t i o n under the same c o n d i t i o n s w i t h 18 nM [ 3 5S]GSH. The s h i f t i n I C 5 0 due t o the h i g h e r r a d i o l i g a n d c o n c e n t r a t i o n should have been o n l y a f a c t o r of two (see equation 10, Methods). The c o n c e n t r a t i o n of DTT i n the i n c u b a t i o n medium a t 2 JUM [ 3H]GSH was c a l c u l a t e d t o be 10 /uM. T h i s c o n c e n t r a t i o n of DTT has been shown by Oja e t a l . (1988) t o reduce glutamate b i n d i n g and t o s h i f t the c o m p e t i t i o n curve of glutamate by GSH t o the r i g h t , s u g g e s t i n g t h a t DTT a f f e c t s a s u l f h y d r y l group on the r e c e p t o r b i n d i n g s i t e . Because of t h i s , 100 mM d i e t h y l d i s u l f i d e was added t o the i n c u b a t i o n b u f f e r i n a l l experiments a t hig h c o n c e n t r a t i o n s of r a d i o l a b e l e d GSH, i n order t o n e u t r a l i z e the DTT t h a t was i n c l u d e d i n the st o c k s o l u t i o n by the manufacturer (as shown i n F i g u r e 13). T h i s proved t o r e t u r n the GSH I C 5 0 v a l u e s t o t h e i r expected range. Under these c o n d i t i o n s (100 mM T r i s - a c e t a t e , pH 8, 120 minutes, room temperature) t h e r e was no displacement of [ H]GSH b i n d i n g by glutamate, and no displacement o f Na-independent [ 3H]glutamate b i n d i n g by GSH, as shown i n s i n g l e — 7 — T c o m p e t i t i o n experiments ranging from 10 t o 10 M competitor, a t f r e e r a d i o l i g a n d c o n c e n t r a t i o n s of 40 nM and 2 JUM f o r [ 3H]glutamate and i n two experiments a t 2 /iM f o r T . . . . [ H]GSH (data not shown). T h i s i s c o n s i s t e n t w i t h the known r e d u c t i o n i n glutamate b i n d i n g a t pH 8, as shown i n F i g u r e 14 (Fagg e t a l . , 1988). Under these c o n d i t i o n s , [ 3H]glutamate b i n d i n g was 14 percent of [ 3H]GSH b i n d i n g , and t h e r e was no displacement of [ H]glutamate by glutamate i t s e l f , or by 67 a s p a r t a t e , homocysteic a c i d , or q u i n o l i n i c a c i d a t 10" M (two experiments), i n d i c a t i n g lowered b i n d i n g a f f i n i t y f o r EAA r e c e p t o r s . These c o n d i t i o n s t h e r e f o r e a l l o w c h a r a c t e r i z a t i o n of b i n d i n g s i t e s separate from ot h e r EAA r e c e p t o r s . R — C H 2 — S — S — C H 2 — R Disulfide-linked chains H OH I I HS—CH,—C—C—CH,—SH I I OH H Dithiothreitol (Added in excess) R—CH 2—SH + H S — C H 2 — R ' Separated reduced chains F i g u r e 13. S e p a r a t i o n o f d i s u l f i d e bonds by t h e r e d u c i n g a c t i o n o f d i t h i o t h r e i t o l , d u r i n g which t h e d i t h i o t h r e i t o l i n a c t i v a t e d . (From S t r y e r , 1988) 69 F i g u r e 14. B i n d i n g o f L-f-^H] glutamate and [ J H ] 3 - ( 2 -c a r b o x y p i p e r a z i n - 4 - y l ) p r o p y l - l - p h o s p h o n a t e (CPP) i n r a t b r a i n membranes as a f u n c t i o n o f pH ( d i f f e r e n t y - a x i s s c a l e s f o r each l i g a n d ) . The c u r v e f o r CPP i s s i m i l a r t o t h a t f o r t h e ACPD r e c e p t o r a n t a g o n i s t 2-amino-3-phosphonopropionic a c i d (AP3), f o r AP4, and f o r t h e c o m p e t i t i v e NMDA r e c e p t o r a n t a g o n i s t AP5 (2-amino-5-phosphonovalerate). (From Fagg e t a l . , 1988) 70 BINDING SITE CHARACTERIZATION Inc u b a t i o n s were c a r r i e d out a t room temperature i n 100 mM T r i s - a c e t a t e b u f f e r a t pH 8.0 f o r 120 minutes (see above), except where noted below. N o n - s p e c i f i c b i n d i n g d e f i n e d by displacement u s i n g 10~ 2 M GSH and 10~ 2 M GSSG ranged from 10 t o 25% of t o t a l , i n c r e a s i n g l i n e a r l y with c o n c e n t r a t i o n of r a d i o l i g a n d . In r a t v i s u a l c o r t e x , h i g h - and l o w - c o n c e n t r a t i o n time course and s a t u r a t i o n b i n d i n g experiments were conducted i n or d e r t o r e v e a l h i g h - and l o w - a f f i n i t y r e c e p t o r subtypes, r e s p e c t i v e l y . The low c o n c e n t r a t i o n experiments used [ 3 5S]GSH, which had a h i g h s p e c i f i c a c t i v i t y s u f f i c i e n t t o r e s o l v e low amounts of b i n d i n g . Competition experiments were conducted i n both r a t and c a t v i s u a l c o r t e x . Two b i n d i n g s i t e s i n d i c a t i v e of r e c e p t o r subtypes were r e v e a l e d , as w e l l as an LAHC s i t e (see D i s c u s s i o n ) . Such " s p e c i f i c " but non-r e c e p t o r LAHC b i n d i n g i s l i k e l y t o appear as a second b i n d i n g - s i t e component i n data from b i n d i n g experiments conducted a t h i g h c o n c e n t r a t i o n s (see Methods - B i n d i n g S i t e A n a l y s i s ) . A c o m p e t i t i o n assay f o r c r o s s o v e r w i t h o t h e r EAA r e c e p t o r s or n e u r o t r a n s m i t t e r s i n r a t O c l was conducted a t 20-minutes i n c u b a t i o n a t pH 7.4, which minimized the LAHC b i n d i n g . F i g u r e 15 shows data from time course experiments i n r a t O c l t h a t r e v e a l e d a l o w - a f f i n i t y p o s s i b l e GSH-receptor 71 F i g u r e 15. A s s o c i a t i o n and d i s s o c i a t i o n r a t e s of r a d i o l a b e l e d GSH b i n d i n g i n c r y o s t a t s e c t i o n s of r a t v i s u a l c o r t e x . Data i s transformed t o c a l c u l a t e K 0 k s from the a s s o c i a t i o n s l o p e and K_^ from the d i s s o c i a t i o n s l o p e . On the o r d i n a t e , B e i s b i n d i n g a t e q u i l i b r i u m , w h i l e B i s b i n d i n g a t the time i n d i c a t e d . S e c t i o n s were i n c u b a t e d i n r a d i o l a b l e d GSH f o r v a r i o u s times up t o 120 minutes. Remaining s e c t i o n s were then i n t r o d u c e d i n t o an 11 i n f i n i t e d i l u t i o n " of b u f f e r and incubated f o r f u r t h e r times up t o 40 minutes. N o n s p e c i f i c b i n d i n g was d e f i n e d by the a d d i t i o n of 10~ 2 M GSH and 10" 2 M GSSG. (A) Data from s p e c i f i c b i n d i n g a t a f r e e l i g a n d c o n c e n t r a t i o n of 5 /uM [ 3H]GSH (room temperature) t o r e v e a l l o w - a f f i n i t y r e c e p t o r k i n e t i c s . The curves r e p r e s e n t normalized data from two s e p a r a t e experiments conducted i n q u a d r u p l i c a t e . R e g r e s s i o n l i n e s are drawn o n l y f o r the p o s s i b l e r e c e p t o r components. The l a t e component of the a s s o c i a t i o n curve and the e a r l y component of the d i s s o c i a t i o n curve show b i n d i n g t o the LAHC non-receptor s i t e (see R e s u l t s ) . For the f i r s t component of the a s s o c i a t i o n p l o t , l i n e a r r e g r e s s i o n was performed on p o i n t s d e r i v e d from mathematically s t r i p p i n g o f f the slower component by a m o d i f i c a t i o n of the method of Lawson (1986). L i n e a r r e g r e s s i o n was performed on the l a s t 8 p o i n t s of the d i s s o c i a t i o n . (B) Data from s p e c i f i c b i n d i n g a t f r e e l i g a n d c o n c e n t r a t i o n of 5 nM [ 3 5S]GSH (4° C) t o r e v e a l l o w - a f f i n i t y r e c e p t o r k i n e t i c s . Regression l i n e s are drawn o n l y f o r the p o s s i b l e r e c e p t o r components. The l a t e component of the a s s o c i a t i o n curve shows non-mass-action k i n e t i c s , w h i l e the e a r l y component of the d i s s o c i a t i o n curve r e f l e c t s non-r e c e p t o r (LAHC) k i n e t i c s (see R e s u l t s ) . The curves r e p r e s e n t n o r m a l i z e d data from t h r e e separate experiments conducted i n t r i p l i c a t e . L i n e a r r e g r e s s i o n was performed on the f i r s t 5 p o i n t s of the a s s o c i a t i o n and l a s t 4 p o i n t s of the d i s s o c i a t i o n . 72 Time (mins.) Time (mins.) 73 b i n d i n g s i t e ( F i g u r e 15A) and a hi g h a f f i n i t y p o s s i b l e GSH-r e c e p t o r b i n d i n g s i t e ( F i gure 15B). In the h i g h -c o n c e n t r a t i o n time course experiments (5 /xM) s p e c i f i c b i n d i n g reached e q u i l i b r i u m by 90 minutes; d i s s o c i a t i o n h a l f - t i m e was 1.5 minutes. F i g u r e 15A shows a s s o c i a t i o n and d i s s o c i a t i o n experiments a t h i g h c o n c e n t r a t i o n (see equations 3 and 4 ) . There were two components of each of the two curves shown, r e p r e s e n t i n g p o s s i b l e r e c e p t o r and non-receptor b i n d i n g (see s a t u r a t i o n experiments, below). The f i r s t component of the observed r a t e constant ( k 0 k s ) was used w i t h the second component of the d i s s o c i a t i o n r a t e constant ( k _ i ) t o c a l c u l a t e the a s s o c i a t i o n r a t e constant (k+i), w h i l e the remaining components, which d i d not r e p r e s e n t b i n d i n g t o a " r e a l " r e c e p t o r s i t e , c o u l d not be used t o c a l c u l a t e k i n e t i c c o n s t a n t s (see s a t u r a t i o n and co m p e t i t i o n experiments; Methods - B i n d i n g S i t e A n a l y s i s ; D i s c u s s i o n ; and below). The v a l u e f o r koj-, ( a f t e r e x p o n e n t i a l s t r i p p i n g , see F i g u r e 15) was 0.067 +/- 0.012 m i n - 1 , and the v a l u e f o r k _ i was 0.010 +/- 0.003 m i n - 1 (mean and S.E. from two sep a r a t e experiments each). The k + 1 v a l u e was c a l c u l a t e d t o be 0.0114 /LtM - 1min - 1 (equation 3, Methods). Since K d = k _ i / k + i , these r a t e c o n s t a n t s r e s u l t e d i n a K d of 0.88 /xM. The c h o i c e of the second component of the d i s s o c i a t i o n p l o t f o r k _ i i s i n d i c a t e d by an a l t e r n a t i v e method of independently d e r i v i n g k+i and k _ i from the a s s o c i a t i o n data alone, as f o l l o w s . Because k ^ = k + ^ [ L ] + k _ i (from e q u a t i o n 3) , a graph of k 0j-, versus [L] w i l l y i e l d the k+i as the s l o p e 74 and the k_! as the y - a x i s i n t e r c e p t . Using the k O D from the p r e l i m i n a r y time course experiment performed a t 355 nM [ 3H]GSH, two p o i n t s were a v a i l a b l e t o d e f i n e a l i n e which had a s l o p e of 0.01 /LiM - 1min - 1 (K+i) and a y - a x i s i n t e r c e p t of 0.017 m i n " 1 ( k _ i ) . T h i s value f o r k _ i i s very c l o s e t o the v a l u e of 0.01 m i n - 1 obtained from the second component of the d i s s o c i a t i o n p l o t , and very f a r from the v a l u e of 0.15 m i n - 1 f o r the f i r s t component. In order f o r the y - a x i s i n t e r c e p t t o y i e l d a k_^ v a l u e of 0.15, the s l o p e ( k + i ) would have t o , be n e g a t i v e , an i m p o s s i b i l i t y . The behaviour of the f i r s t component presumably r e s u l t s from non-receptor LAHC b i n d i n g . In the low-concentration, time course experiments a t 5 nM a t 4° C, s p e c i f i c b i n d i n g reached e q u i l i b r i u m by 120 minutes; d i s s o c i a t i o n h a l f - t i m e was 1 minute ( F i g u r e 15B). Values f o r k O D and k _ i were determined as d e s c r i b e d above: 0.014 +/-0.002 m i n - 1 and 0.009 +/- 0.001 m i n - 1 r e s p e c t i v e l y (mean and S.E. from t h r e e separate experiments each), y i e l d i n g a K + 1 of .001 nM'-'-min-1 and a K d of 9 nM. The remaining components of each of the two curves c o u l d not be used t o c a l c u l a t e k i n e t i c c o n s t a n t s (as above). The second component of the a s s o c i a t i o n curve v i o l a t e s the p s e u d o - f i r s t order assumptions of a c o n s t a n t f r e e l i g a n d c o n c e n t r a t i o n [ L ] , which r e q u i r e s t h a t [L] be much l a r g e r than the r e c e p t o r c o n c e n t r a t i o n i n t h a t i n c u b a t i o n volume [R] (see Methods - B i n d i n g S i t e A n a l y s i s ) . S i n c e t h i s component r e p r e s e n t e d the more s l o w l y -a s s o c i a t i n g l o w - a f f i n i t y b i n d i n g s i t e and LAHC s i t e (see s a t u r a t i o n and c o m p e t i t i o n experiments), the c o r r e s p o n d i n g 75 r a t i o s of [ L ] / [ R ] , as c a l c u l a t e d from B m a x v a l u e s o b t a i n e d i n the s a t u r a t i o n experiments ( d e s c r i b e d below), were 2.2 f o r the l o w - a f f i n i t y s i t e and 0.1 f o r the LAHC s i t e (while the h i g h - a f f i n i t y s i t e r a t i o was 12). The two low r a t i o s would have produced l a r g e d e v i a t i o n s i n [ L ] . A c o n s t a n t [L] i s assumed i n order t o c a l c u l a t e k i n e t i c v a l u e s (Bylund, 1980), and thus t h i s component of the a s s o c i a t i o n curve would y i e l d anomalous r e s u l t s i f used t o determine k i n e t i c c o n s t a n t s . Competition w i t h 2 juM [ 3H]GSH by n o n - r a d i o l a b l e d GSH i n r a t ( F i g u r e 16A) showed a marked d e v i a t i o n from a s i n g l e - s i t e p r o f i l e (91-9% r u l e ) with the e x t e n s i o n of the curve t o the r i g h t showing the LAHC s i t e . Because of the h i g h amount of b i n d i n g t o the LAHC s i t e a t 2 MM [ 3H]GSH, a t w o - s i t e a n a l y s i s of the K d v a l u e s was performed on data from c o m p e t i t i o n o f 18 nM [ 3 5S]GSH wit h GSH, as shown i n F i g u r e 17. The K d was determined t o be 1.3 +/- 0.5 JUM f o r the p o s s i b l e r e c e p t o r component and 92 +/- 2 /xM f o r the LAHC s i t e (mean and S.E. of t h r e e s e p a r a t e experiments). S p e c i f i c i t y was shown by other c o m p e t i t o r s . For GSSG the p o s s i b l e r e c e p t o r b i n d i n g s i t e IC 5o was the same as f o r GSH, w i t h a s l i g h t l y h i g h e r a f f i n i t y f o r the LAHC b i n d i n g ( F i g u r e 16A). Three other substances showed a f f i n i t y f o r [ 3H]GSH b i n d i n g a t 1 mM (Figure 16A), i n the f o l l o w i n g o r d e r : c y s t e i n e , S-methyl-GSH, and CSA. Under these c o n d i t i o n s (pH 8, 120 minutes) many substances f a i l e d t o show displacement of [ 3H]GSH b i n d i n g from 1 /xM t o 1 mM. These were L-glutamate, L - a s p a r t a t e , D/L-homocysteic a c i d , q u i n o l i n i c a c i d , D/L-2-76 F i g u r e 16. Competition curves f o r [ JH]GSH b i n d i n g i n a d u l t r a t (A) and c a t (B) c r y o s t a t s e c t i o n s of v i s u a l c o r t e x . S e c t i o n s were incubated f o r 12 0 minutes a t room temperature (pH 8) i n 2 jiM [ 3H]GSH and v a r i o u s c o n c e n t r a t i o n s o f co m p e t i t o r s as i n d i c a t e d . The curves i n (A) r e p r e s e n t n o r m a l i z e d data from two separate experiments conducted i n a t l e a s t t r i p l i c a t e . The curves i n (B) are from one experiment conducted i n a t l e a s t t r i p l i c a t e . Note t h a t the extended s e c t i o n o f the curves a t hig h competitor c o n c e n t r a t i o n s r e p r e s e n t s the LAHC non-receptor b i n d i n g s i t e . 77 120.0-% 100.0* o i—i n . 80.o-c C 60.0-£ 40.0 o 20.0-A 0.0 120.0 • GSH • GSSG A Cysteine oS-methyl-GSH •H-100.0 o i—i ^ 80.0-C7> C c lo "5 -M o •+-» 60.0-40.0-20.0-0.0 I 1 1 1 1 ' 1 1 — | r--8 - 7 - 6 - 5 - 4 1 A - 3 - 2 •GSH • GSSG ^ Cysteine B i • i — • — i — i — i — i — i — i — | — 8 - 7 - 6 - 5 - 4 - 3 - 2 Log M 78 F i g u r e 17. Competition of GSH with 18 nM [ J DS]GSH and i t s d e r i v e d "B versus B ' l " p l o t t o r e v e a l the sep a r a t e K d v a l u e s f o r the p o s s i b l e r e c e p t o r b i n d i n g s i t e and nonreceptor LAHC s i t e . C r y o s t a t s e c t i o n s of a d u l t r a t v i s u a l c o r t e x were i n c u b a t e d f o r 120 minutes a t room temperature (pH 8) i n 18 nM [ 3 5S]GSH and v a r i o u s c o n c e n t r a t i o n s of n o n l a b e l e d GSH as i n d i c a t e d . The graphs r e p r e s e n t normalized data from t h r e e s e p a r a t e experiments conducted i n t r i p l i c a t e . (A) Competition curve. (B) "B versus B*I" p l o t , where the n e g a t i v e i n v e r s e of the slo p e s of the f i r s t and second components y i e l d K d v a l u e s of 1.3 /zM and 92 /uM, r e s p e c t i v e l y . E x t r a c t i o n o f the l i n e s f o r the two separate components was performed by the method of Rosenthal (1967). 80 amino-3-phosphonopropionic a c i d (AP3), D/L-AP4, D/L-2-amino-5-phosphonopentanoic a c i d (AP5), gamma-D-glutamylglycine (gamma-DGG), AMPA, and QUIS. NMDA showed no s i g n i f i c a n t d i splacement a t 1 mM. A pH of 8 i s optimal f o r b i n d i n g of AP3, AP4, and AP5 t o EAA r e c e p t o r s (see F i g u r e 14). F i g u r e 18 shows data from s a t u r a t i o n experiments t h a t r e v e a l t he l o w - a f f i n i t y , p o s s i b l e GSH r e c e p t o r b i n d i n g s i t e ( F i g u r e 18A) and the h i g h - a f f i n i t y p o s s i b l e GSH r e c e p t o r b i n d i n g s i t e ( F i g u r e 18B). Eadie-Hofstee a n a l y s i s of the h i g h - c o n c e n t r a t i o n s a t u r a t i o n b i n d i n g experiment ( F i g u r e 18A; see Methods) y i e l d e d a K d of 1.3 +/-0.3 iiM and a B T O a x of 1.3 +/-0.2 pmoles/mg p r o t e i n (mean and S.E. from two se p a r a t e experiments). The second component shown i n the E a d i e -H o f s t e e p l o t i n F i g u r e 18A i s the non-receptor LAHC b i n d i n g s i t e i n v a r i a b l y found a t hig h c o n c e n t r a t i o n s (see Methods) wi t h a ve r y h i g h K d of 17.2 +/-7.8 /nM and a very l a r g e B m a x of 31.4 +/-11.4 pmoles/mg p r o t e i n (see D i s c u s s i o n ) . The low-c o n c e n t r a t i o n s a t u r a t i o n experiment ( F i g u r e 18B) was performed a t 45 minutes i n c u b a t i o n t o minimize b i n d i n g t o the l o w - a f f i n i t y b i n d i n g s i t e and the LAHC s i t e , i n or d e r t o ma i n t a i n mass-action assumptions, f o r the reason d e s c r i b e d above i n the time course experiments. E a d i e - H o f s t e e a n a l y s i s ( F i g u r e 18B) y i e l d e d a s i n g l e component wit h a low K d of 5.4 +/-0.8 nM and a s m a l l e r B m a x of 0.24 +/-0.02 pmoles/mg p r o t e i n (mean and S.E. of th r e e separate experiments). The q u a l i t y of s a t u r a t i o n - b i n d i n g data can be asses s e d by i n s p e c t i o n of the H i l l c o e f f i c i e n t s and S D ( E r a c j ) v a l u e s . 81 An SD(Erad) v a l u e more than 0.2 means the data i s of l i t t l e v a l u e . A H i l l c o e f f i c i e n t h i g h e r or lower than one suggests p o s i t i v e or n e g a t i v e c o o p e r a t i v i t y , r e s p e c t i v e l y (see Methods - B i n d i n g S i t e A n a l y s i s ) . H i l l c o e f f i c i e n t and S D ( E r a d ) v a l u e s f o r each separate component of the r a d i o l a b e l e d GSH s a t u r a t i o n b i n d i n g d e s c r i b e d above are as f o l l o w s : 0.99 +/-0.05 and 0.07, r e s p e c t i v e l y , f o r the h i g h - a f f i n i t y r e c e p t o r ; 0.98 +/- 0.09 and 0.09 f o r the l o w - a f f i n i t y r e c e p t o r ; 0.98 +/- 0.17 and 0.11 f o r the LAHC s i t e ; and a H i l l c o e f f i c i e n t of 0.92 +/- 0.11 f o r the h i g h - c o n c e n t r a t i o n s a t u r a t i o n experiment as a whole. A s i n g l e c o m p e t i t i o n experiment was c a r r i e d out f o r c a t v i s u a l c o r t e x i n order t o a s c e r t a i n t h a t a f f i n i t y and s p e c i f i c i t y under these i n c u b a t i o n c o n d i t i o n s were not r a d i c a l l y d i f f e r e n t from those i n r a t b r a i n , s i n c e a u t o r a d i o g r a p h i c d i s t r i b u t i o n s t u d i e s i n c a t c o r t e x were expected t o be more r e v e a l i n g of laminar b i n d i n g (based on p r e v i o u s r e c e p t o r b i n d i n g s t u d i e s [see I n t r o d u c t i o n ] and p r e l i m i n a r y a u t o r a d i o g r a p h y ) . In s e c t i o n s from a d u l t c a t area 17, c o m p e t i t i o n with 2 /iM [ 3H]GSH (pH 8, 120 min) showed s i m i l a r a f f i n i t y , s p e c i f i c i t y , and LAHC b i n d i n g as t h a t i n r a t O c l ( F i g u r e 16B). The c a t data i s not s u f f i c i e n t f o r d e t e r m i n i n g b i n d i n g parameters. A p o s s i b l e d i f f e r e n c e from r a t was displacement i n c a t area 17 by 1 mM glutamate a t pH 8 (p < 0.02). 82 F i g . 18. S a t u r a t i o n b i n d i n g curves and E a d i e - H o f s t e e p l o t s of [ 3H]GSH and [ 3 5S]GSH b i n d i n g s i t e s on c r y o s t a t s e c t i o n s of a d u l t r a t v i s u a l c o r t e x . N o n s p e c i f i c b i n d i n g was d e f i n e d by the a d d i t i o n of 10~ 2 M GSH and 10~ 2 M GSSG. (A) High f r e e -l i g a n d c o n c e n t r a t i o n range ([ 3H]GSH) t o r e v e a l parameters f o r l o w - a f f i n i t y r e c e p t o r s . The graphs r e p r e s e n t combined data from two separate experiments conducted i n q u a d r u p l i c a t e a t room temperature, 12 0 minutes i n c u b a t i o n , pH 8. In the E a d i e -Hof s t e e p l o t the component wit h steeper s l o p e (no l i n e drawn) r e p r e s e n t s the LAHC non-receptor s i t e . The l i n e f o r the p o s s i b l e r e c e p t o r component was determined by the method of Hunston (1975). (B) Low f r e e - l i g a n d c o n c e n t r a t i o n range ( [ 3 5 S ] G S H ) , t o r e v e a l parameters f o r h i g h - a f f i n i t y r e c e p t o r s , i n c u b a t e d f o r 45 minutes (pH 8, room temp.) t o minimize b i n d i n g t o the much denser LAHC s i t e s t o r e t a i n mass a c t i o n assumptions (see R e s u l t s ) . The graphs r e p r e s e n t combined data from t h r e e separate experiments conducted i n t r i p l i c a t e . The Ea d i e - H o f s t e e l i n e i s drawn by l i n e a r r e g r e s s i o n . Bound (pM) 84 EXCITATORY AMINO ACID RECEPTOR INTERACTIONS To f u r t h e r c h a r a c t e r i z e p o s s i b l e i n t e r a c t i o n s between GSH and EAA neu r o t r a n s m i s s i o n under p h y s i o l o g i c a l c o n d i t i o n s , c o m p e t i t i o n experiments were conducted a t 20 minutes i n c u b a t i o n a t pH 7.4, i n which t h e r e was a r e d u c t i o n o f the LAHC b i n d i n g as shown by the com p e t i t i o n curve i n F i g u r e 19, and a t pH 6.9 ( a l s o 20 min), which i s op t i m a l f o r glutamate r e c e p t o r b i n d i n g (Fagg e t a l . , 1988). C o n t r o l s p e c i f i c b i n d i n g of [ 3H]GSH with 2 0-minute i n c u b a t i o n s , r e d u c i n g the LAHC component, showed r a t i o s r e l a t i v e t o b i n d i n g a t pH 7.4 of 3.6 a t pH 6.9 and 2.5 a t pH 8.0, which d i f f e r s from the b i n d i n g w i t h pH seen a t 12 0 minutes i n c u b a t i o n ( F i g u r e 8 ) . In a d d i t i o n , b i n d i n g and com p e t i t i o n experiments w i t h [ 3H]glutamate were conducted a t pH 6.9, the optimum pH f o r glutamate b i n d i n g (Figure 14) . As shown i n F i g u r e 20, [ 3H]glutamate b i n d i n g was s i g n i f i c a n t l y (p < 0.05) g r e a t e r a t pH 6.9. At pH 6.9, as opposed t o a t pH 8, the r e was s i g n i f i c a n t displacement (p < 0.05) of [ 3H]glutamate b i n d i n g by both glutamate and GSH a t 1 mM GSH. There was, however, no displacement by 1 mM AMPA a t t h i s pH. At pH 7.4 t h e r e were s i g n i f i c a n t displacements o f [ 3H]GSH by substances a t 1 mM as shown i n F i g u r e 21. There was no s i g n i f i c a n t displacement by D/L-AP3, D/L-AP4, NMDA, KAIN, or CSA. At pH 6.9 t h e r e was s i g n i f i c a n t displacement of [ JH]GSH as shown i n F i g u r e 21, with no displacement by glutamate, L - a s p a r t a t e , D/L-AP3, D/L-AP4, or KAIN. 86 120.0-0.0-1 • 1 • , , . , . , . , . r-0 -8 -7 -6 - 5 -4 - 3 -2 log M F i g u r e 19. C o m p e t i t i o n by GSH w i t h [ JH]GSH a t 20 m i n u t e s i n c u b a t i o n (pH 7.4) t o reduce b i n d i n g t o t h e dense, n o n r e c e p t o r , LAHC s i t e , t h e presence of which would o b s c u r e d i s p l a c e m e n t s . The c u r v e i s compared t o t h e p r e v i o u s d a t a f r om c o m p e t i t i o n a t 120 minutes a t pH 8.0 ( d o t t e d l i n e ) t o show t h a t t h e r e i s a r e d u c t i o n i n t h e r i g h t h a n d , low a f f i n i t y component. These c o n d i t i o n s a r e o p t i m a l f o r e x a m i n i n g i n t e r a c t i o n s w i t h EAAs. C r y o s t a t s e c t i o n s o f a d u l t r a t v i s u a l c o r t e x were i n c u b a t e d a t room t e m p e r a t u r e (pH 8) i n 2.6 p,K [ H]GSH and v a r i o u s c o n c e n t r a t i o n s o f n o n l a b e l e d GSH as i n d i c a t e d . Data i s from one experiment where s i x s e c t i o n s were used a t each d a t a p o i n t . 87 200.0 TTI 150.0 CD -t-> o E D -t-> cn<+-o cn c TD ~5 100.0-~ 50.0-c o o 0.0 pH 8 K8pH6.9 contro l contro l GSH AMPA F i g u r e 20. Percent c o n t r o l b i n d i n g of L-[ H]glutamate i n r a t v i s u a l c o r t e x a t pH 8.0 compared t o b i n d i n g a t pH 6.9 and a t pH 6.9 w i t h v a r i o u s 1 mM competitors. C r y o s t a t s e c t i o n s of a d u l t r a t O c l were incubated i n T r i s - a c e t a t e f o r 45 minutes a t room temperature with 3 /nM [ 3H]glutamate and v a r i o u s c o m p e t i t o r s as i n d i c a t e d . Data i s combined from two separate experiments conducted i n t r i p l i c a t e . The d i f f e r e n c e between b i n d i n g without competitors a t pH 8.0 and pH 6.9 i s s i g n i f i c a n t (p <0.05). 88 F i g u r e . 21. P e r c e n t c o n t r o l b i n d i n g o f [ JH]GSH i n r a t v i s u a l c o r t e x w i t h s i g n i f i c a n t d i s p l a c e m e n t (p <.05) by v a r i o u s 1 mM c o m p e t i t o r s a t pH 7.4 and a t pH 6.9. C o n t r o l b i n d i n g i s r e l a t i v e t o b i n d i n g a t pH 7.4 w i t h o u t c o m p e t i t o r s . C r y o s t a t s e c t i o n s o f a d u l t r a t O c l were i n c u b a t e d i n T r i s - a c e t a t e w i t h 2 /xM [ 3H]GSH and c o m p e t i t o r s f o r 2 0 m i n utes (room temp.) t o m i n i m i z e LAHC b i n d i n g . The graph r e p r e s e n t s n o r m a l i z e d d a t a f r om t h r e e s e p a r a t e experiments conducted i n t r i p l i c a t e . O n l y s i g n i f i c a n t d i s p l a c e m e n t i s shown. 89 BINDING SITE DISTRIBUTIONS Q u a n t i t a t i o n of s p e c i f i c b i n d i n g of [ 3H]GSH i n r a t b r a i n showed r e g i o n a l v a r i a t i o n with s i g n i f i c a n t l y l e s s b i n d i n g i n more l a t e r a l , n o n - v i s u a l c o r t e x , brainstem, and white matter (p < .01). In c a t b r a i n , n o n - s p e c i f i c b i n d i n g of [ 3 5S]GSH , determined by the a d d i t i o n of 10~ 2 M n o n - r a d i o l a b e l e d GSH, showed densest b i n d i n g i n white matter and brainstem. The laminar d i s t r i b u t i o n of GSH t o t a l - b i n d i n g s i t e s i n v i s u a l c o r t e x was determined by autoradiography f o r a d u l t r a t s , f o r c a t s a t p o s t n a t a l ages of 6, 13, 30, 46, 61, 90, and 2 30 days, and f o r one a d u l t monkey. These i n c u b a t i o n s were performed with 10 nM [ 3 5S]GSH a t pH 7.4 f o r 3 0 minutes t o reduce L/AHC b i n d i n g . Rat c o r t e x showed r e l a t i v e l y u n iform b i n d i n g a c r o s s a l l l a y e r s (Figure 22A). Cat v i s u a l c o r t e x showed the h i g h e s t d e n s i t y of b i n d i n g i n l a y e r 4, c o n f i n e d t o area 17, from a f t e r 6 days p o s t n a t a l age. F i g u r e 22 shows r e p r e s e n t a t i v e autoradiographs and F i g u r e 23 shows q u a n t i t a t i v e densitometry of l a y e r s a t each age s t u d i e d . In 6-day o l d c a t area 17 no d i s t i n c t laminar p a t t e r n of [ J JS]GSH b i n d i n g was seen (Figure 23A). At a l l l a t e r ages, densest b i n d i n g was shown i n middle l a y e r s of area 17. Cytochrome oxi d a s e s t a i n i n g f o r l a y e r 4 was obtained i n the 46, 61, and 90 day o l d c a t s , which shows t h a t the band of densest [ 3 5S]GSH b i n d i n g corresponded c o n s i s t e n t l y w i t h l a y e r 4 al o n g i t s e n t i r e l e n g t h i n area 17 (see f o r example F i g u r e 22 D and E, F and G). Q u a n t i t a t i v e a n a l y s i s of b i n d i n g d e n s i t y , shown 90 i n F i g u r e 23, showed a peak i n middle l a y e r s a f t e r 6 days of age, w i t h b i n d i n g showing an i n c r e a s e from 6 t o 61 days of age and then a r e d u c t i o n a t 90 and 230 days of age, w i t h l a y e r 4 b i n d i n g d e n s i t y becoming more d i s c r e t e and s l i g h t l y l e s s prominant a t the l a t t e r two ages. The r e g i o n o f maximum b i n d i n g d e n s i t y f o r the 46, 61, and 90 day o l d s e c t i o n s corresponded w i t h the cytochrome oxidase band. A d u l t monkey area 17 showed a d i s t i n c t [ 3 5S]GSH laminar b i n d i n g p r e f e r e n c e f o r l a y e r 4C, as shown by the corresponding cytochrome oxi d a s e s t a i n e d s e c t i o n (Figure 221,J). 91 F i g . 22. A u t o r a d i o g r a p h i c d i s t r i b u t i o n of [ J DS]GSH b i n d i n g i n r a t , c a t , and monkey v i s u a l c o r t e x . Slide-mounted c o r o n a l s e c t i o n s (20 izm) from f r o z e n t i s s u e were in c u b a t e d f o r 3 0 minutes a t 8-10 nM [ 3 5S]GSH at room temperature, pH 7.4. Bar = 1 mm. In c a t and monkey s e c t i o n s s u c c e s s f u l l y s t a i n e d f o r cytochrome oxidase l e v e l s , as shown, the p o s i t i o n of the band of densest s t a i n i n g ( l a y e r 4, c i r c l e ) corresponds t o the p o s i t i o n of densest b i n d i n g of [ 3 5S]GSH as shown i n the a u t o r a d i o g r a p h s . (A) A d u l t r a t b r a i n autoradiograph from a c o r o n a l s e c t i o n a t the l e v e l of the SC and medial g e n i c u l a t e , l i n e s i n d i c a t i n g approximate borders of O c l . (B) 13-day o l d c a t v i s u a l c o r t e x autoradiograph, w i t h area 17 d e l i n e a t e d by the band of denser b i n d i n g a t the top r i g h t h a n d s i d e . (C) 3 0-day o l d c a t v i s u a l c o r t e x autoradiograph, w i t h area 17 d e l i n e a t e d by the band of densest b i n d i n g a l o n g the l e n g t h of the l e f t h a n d s i d e of the s e c t i o n , c o n t i n u i n g v e n t r a l l y i n t o the l a r g e s u l c u s . (D,E) 46-day o l d c a t v i s u a l c o r t e x a u t o r a d i o g r a p h (D) and c o r r e s p o n d i n g cytochrome oxidase s t a i n e d s e c t i o n ( E ) . (F,G) 61-day o l d c a t v i s u a l c o r t e x a u t o r a d i o g r a p h (F) and c o r r e s p o n d i n g cytochrome oxidase s t a i n e d s e c t i o n (G). (H) A d u l t (230 day old) c a t v i s u a l c o r t e x a u t o r a d i o g r a p h w i t h area 17 comprising the two top l e f t h a n d g y r i . (I/J) A d u l t monkey area 17 autoradiograph (I) and c o r r e s p o n d i n g cytochrome oxidase s t a i n e d s e c t i o n (J) w i t h arrows i n d i c a t i n g , from top t o bottom, l a y e r s 4A, 4Cot, and 4CB. 92 93 95 I J 96 F i g . 23. Q u a n t i t a t i v e a n a l y s i s of laminar b i n d i n g d e n s i t y on a u t o r a d i o g r a p h i c f i l m a c r o s s c a t area 17 c o r t e x r a d i a l l y from white matter t o p i a a t v a r i o u s p o s t n a t a l ages. O p t i c a l d e n s i t y was c a l i b r a t e d u s i n g ARC [ C] standards (0.002 - 35.0 ixCi/g) . The graph i s a histogram w i t h each p o i n t showing the mean value f o r a b i n dimension of 0.145 mm r a d i a l l y and approximately 0.460 mm t a n g e n t a l l y . The histograms f o r a l l ages are a l i g n e d w i t h the c e n t e r o f l a y e r 4, as determined by cytochrome oxidase and N i s s l s t a i n i n g . The approximate width of l a y e r 4 i s i n d i c a t e d by the h o r i z o n t a l bar. The p i a ( r i g h t side) and the boundary of the white matter ( l e f t side) are i n d i c a t e d by the v e r t i c a l bars c r o s s i n g the histogram f o r each age. Width i n mm on the o r d i n a t e i s p r o v i d e d f o r s c a l i n g , with i t s o r i g i n a t an a r b i t r a r y p o i n t i n the white matter. V a r i a t i o n s i n the t h i c k n e s s of c o r t e x and p o s i t i o n i n g of anatomical l a y e r s , depends on the p a r t i c u l a r sample taken a t any g i v e n age. A l l samples are taken from the medial bank, wi t h ages 6, 30, 46, and 61 days taken from the v e n t r a l gyrus, age 13 days from the d o r s a l gyrus, and ages 9 0 and 230 days from the s u l c u s . A peak i n b i n d i n g d e n s i t y i s seen i n l a y e r 4 a t a l l ages except day 6. (A) Densi t y p r o f i l e a t p o s t n a t a l ages of 6, 13, and 30 days. (B) Density p r o f i l e a t p o s t n a t a l ages 46, 61, 90, and 230 days. 97 10--b Z3 A 4 2 0 O — O 6 day old A A 13 day old • • 30 day old • i i i A A • i i i i 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 mm en b B 10 8 6 4 + 2 0 r A 1 A A • « 4 6 day old O — 0 6 1 day old A — A 90 day old A — A 230 day old 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 mm 98 UPTAKE EXPERIMENTS IN VIVO Usin g i n j e c t i o n s of r a d i o a c t i v e n e u r o t r a n s m i t t e r s or n e u r o t r a n s m i t t e r m e t a b o l i t e s , one can l a b e l , i n p r i n c i p l e , d i s t a n t c e l l bodies having p r o j e c t i o n s t o c o r t e x by making i n j e c t i o n s i n t h e i r e f f e r e n t t a r g e t area. T h i s r e t r o g r a d e uptake c o r r e l a t e s w i t h a n e u r o t r a n s m i t t e r r o l e i n the d e l i n e a t e d pathway ( S t r e i t , 1980; Baughman and G i l b e r t , 1981). One can a t the same time r e t r o g r a d e l y l a b e l l o c a l c e l l s v i a l o c a l l y r a m i f y i n g axons, which can then show uptake and anterograde t r a n s p o r t t o p r o j e c t e d t e r m i n a l s , thus a l s o i n d i c a t i n g a n e u r o t r a n s m i t t e r f u n c t i o n (Baughman and G i l b e r t , 1981). For amino a c i d s , however, t h e r e may a l s o be uptake by c e l l b o d i es f o r metabolic purposes, w i t h anterograde t r a n s p o r t of the l a b e l i n p r o t e i n s t o e f f e r e n t t a r g e t area t e r m i n a l s (Cowan e t a l . , 1972). Although our i n j e c t i o n s were made e x c l u s i v e l y i n primary v i s u a l c o r t e x i n both r a t and c a t (Ocl and area 17, r e s p e c t i v e l y ) , i n r a t c o r t e x t h e r e was spread of the t r a c e r s i n t o secondary v i s u a l c o r t e x (0c2) and sometimes i n t o adjacent n o n - v i s u a l c o r t e x and a c r o s s the corpus callosum; i n the c a t the i n j e c t e d t r a c e r remained c o n f i n e d w i t h i n a p o r t i o n of area 17. Thalamic and brainstem n u c l e i showing the presence of r a d i o l a b e l a f t e r i n j e c t i o n i n t o v i s u a l c o r t e x are summarized i n Table I. Examples of a u t o r a d i o g r a p h s are shown i n F i g u r e 24. A l l l a b e l e d t r a c e r s except [ H]muscimol and [ HJkainate showed uptake and t r a n s p o r t . A l l l a b e l e d n u c l e i (Table I) had d i r e c t 99 c o n n e c t i o n w i t h v i s u a l c o r t e x as f o l l o w s ( f o r a b b r e v i a t i o n s see L i s t of Anatomical A b b r e v i a t i o n s ) . In r a t b r a i n , the f o l l o w i n g n u c l e i are not known t o p r o j e c t t o v i s u a l c o r t e x but they do r e c e i v e p r o j e c t i o n s from both O c l and 0c2 : the VLG, APTD, Rt, and SC (Sefton and Dreyer, 1985; Swanson e t al.,1974; Takahashi, 1985), except f o r the i n f e r i o r grey of the SC, the p a r v o c e l l u l a r d i v i s i o n of the VLG, and the IMA, which r e c e i v e p r o j e c t i o n s only from 0c2. The DLG and ZI ( L i n e t a l . , 1990) have r e c i p r o c a l connections w i t h O c l and area 17 i n the r a t and c a t (Sefton and Dreyer, 1985; Takahashi, 1985). The SubG i s an a c e t y l c h o l i n e e s t e r a s e - s t a i n i n g area of the ZI, as determined by Paxinos and Watson (1985). The LP p r o j e c t i o n t o O c l i n the r a t ( i n c l u d i n g LPMC) i s not r e c i p r o c a l except t o the l a t e r a l r o s t r a l d i v i s i o n (LPLR) (S e f t o n and Dreyer, 1985; Takahashi, 1985). The p r o j e c t i o n s and f u n c t i o n s of the IGL have not been e l u c i d a t e d s e p a r a t e l y from the DLG and VLG (Sefton and Dreyer, 1985). [ 3 5 S ] C y s t e i n e t r a n s p o r t i n the d o r s a l CP i n one r a t presumably r e s u l t e d from d i f f u s i o n i n t o motor c o r t e x , and c a l l o s a l t r a n s p o r t seen i n many cases i n the r a t s may be due t o p e n e t r a t i o n of the white matter. Cat [ 3H]GSH uptake, r e s t r i c t e d t o area 17 only, was t r a n s p o r t e d e x c l u s i v e l y t o the DLG. R e g i s t r a t i o n of s i l v e r g r a i n s f o r c e l l u l a r r e s o l u t i o n was performed by the emulsion-dipping method f o r a l l t e s t animals except the [ 3 5 S ] G S H - i n j e c t e d r a t s . Images were o b t a i n e d i n one of the [ 3 5 S ] c y s t e i n e - i n j e c t e d and t h r e e of 100 the [^HJGSH-injected r a t s . The higher-energy beta e m i s s i o n i n s e c t i o n s from the [ 3 5 S ] c y s t e i n e - i n j e c t e d r a t produced a dense s i l v e r g r a i n d i s t r i b u t i o n over c e l l b odies and n e u r o p i l i n t h e n u c l e i l i s t e d (Table I ) . In emulsion-coated s e c t i o n s from the [ 3H]GSH-injected r a t s s i l v e r g r a i n s were seen over c e l l b o d i es and n e u r o p i l i n the DLG and LPLR, i n d i c a t i v e of r e t r o g r a d e t r a n s p o r t , as shown i n F i g u r e 25, but the same d i s t r i b u t i o n was seen i n the VLG and SC (see D i s c u s s i o n ) . No s i l v e r g r a i n s c o u l d be det e c t e d a f t e r f o u r months exposure time i n the emulsion-dipped [ 3H]GSH-injected c a t s e c t i o n s or i n the [ 3 H ] g l y c i n e - and [ 3 H ] g l u t a m a t e - i n j e c t e d r a t s e c t i o n s . Due t o time l i m i t s f o r the present study, i t was not p o s s i b l e t o wait up t o a year or more to o b t a i n s i l v e r g r a i n d i s t r i b u t i o n s i n these s e c t i o n s . I t was t h e r e f o r e i m p o s s i b l e t o determine whether uptake and t r a n s p o r t i n the s e b r a i n s was r e t r o g r a d e . 101 TABLE I. Nuclei showing presence of radiolabel Animal/tracer N nuclei Rat: [:?H] GSH ["S]GSH it S]cysteine 3 DLG VLG LPLR ZI/SubG SC IMA IGL 2 DLG LPLR 2 DLG VLG LPLR APTD ZI/SubG SC IGL Rt CP [?H]glutamate 1 DLG VLG LPMC ZI/SubG IMA [ 3H]glycine 1 DLG VLG LPLR APTD ZI/SubG SC IMA • [^HJmuscimol 1 [ 3H]kainate 1 Cat: [3H]GSH 1 DLG 102 F i g . 24. A u t o r a d i o g r a p h i c d i s t r i b u t i o n s of r a d i o l a b e l e d t r a c e r uptake t o thalamus from i n v i v o i n j e c t i o n i n v i s u a l c o r t e x i n r a t s . Bar = 1 mm. (A) A t l a s of r e l e v a n t s t r u c t u r e s a t the same l e v e l as the au t o r a d i o g r a p h i n (B) (Bregma minus 3.8 mm, from Paxinos and Watson, 1986). (B) [ 3H]GSH uptake t o DLG, IGL, and VLG. (C) A t l a s of r e l e v a n t s t r u c t u r e s a t the same l e v e l as the aut o r a d i o g r a p h s i n (D), (E), and (F) (Bregma minus 4.3 mm, from Paxinos and Watson, 1986). (D) [ 3 5 S ] C y s t e i n e uptake t o LPLR, DLG, ILG, VLG, and ZI/SubG. (E) [ 3H]Glutamate uptake t o LPLR, DLG, VLG, and ZI/SubG. (F) [ 3 H ] G l y c i n e uptake t o APTD, LPLR, IMA, DLG, VLG, and ZI/SubG. 103 104 105 106 F i g . 25. B r i g h t f i e l d p hotomicrograph of combined p y r o n i n Y s t a i n i n g f o r c e l l b o d i e s and a u t o r a d i o g r a p h i c d i s t r i b u t i o n o f s i l v e r g r a i n s i n t h e r a t DLG f o l l o w i n g uptake o f [ 3H]GSH from v i s u a l c o r t e x . I n t h e s e 20 /urn t h i c k s e c t i o n s , s i l v e r g r a i n s s h o u l d not be seen over most c e l l b o d i e s u n l e s s r e t r o g r a d e t r a n s p o r t i n t o t h e c e l l b o d i e s had t a k e n p l a c e . 107 DISCUSSION BINDING SITE CHARACTERIZATION The p r e s e n t r e s u l t s g i v e the f i r s t demonstration of g l u t a t h i o n e b i n d i n g s i t e s i n whole b r a i n . Three s i t e s were r e v e a l e d , w i t h K d v a l u e s of 5.4 t o 9 nM, 0.9 t o 1.3 /^ M, and 17 t o 92 /iM. The h i g h e s t v a l u e s r e p o r t e d i n the l i t e r a t u r e f o r r e c e p t o r b i n d i n g parameters are 8.3 /uM f o r K d and 6.9 pmol/mg p r o t e i n f o r B m a x , and these are e x c e p t i o n a l l y h i g h (reviewed i n F o s t e r and Fagg, 1984). The LAHC b i n d i n g s i t e v a l u e s a re s i g n i f i c a n t l y h i g h e r than these, p a r t i c u l a r l y the Bmax ( 3 1 pmol/mg p r o t e i n ) , i n d i c a t i n g nonreceptor b i n d i n g . T h i s i n t e r p r e t a t i o n i s supported by the d e v i a t i o n s from mass a c t i o n and p s e u d o - f i r s t order assumption seen i n k i n e t i c a n a l y s i s . For the low a f f i n i t y , p o s s i b l e GSH r e c e p t o r b i n d i n g s i t e the K d v a l u e s and B m a x (1.3 pmol/mg p r o t e i n ) are t y p i c a l of EAA r e c e p t o r s (Foster and Fagg, 1984). The K d v a l u e s and B m a x (0.24 pmol/mg p r o t e i n ) of the h i g h - a f f i n i t y GSH b i n d i n g s i t e are t y p i c a l of pe p t i d e and neuromodulator r e c e p t o r s (Shaw e t a l . , 1986). An e x p l a n a t i o n f o r the marked decrease i n [ H]GSH s p e c i f i c b i n d i n g seen a t p h y s i o l o g i c a l c o n c e n t r a t i o n s of N a + and M g 2 + i s suggested as f o l l o w s . For o p i o i d r e c e p t o r b i n d i n g i n b r a i n , Na + decreases a g o n i s t b i n d i n g t o 37% a t 50 mM and a b o l i s h e s b i n d i n g by 2 00 mM, due t o a decrease i n a f f i n i t y (with a corresponding i n c r e a s e i n a n t a g o n i s t 108 a f f i n i t y ) , and t h e r e i s a l s o a g e n e r a l r e d u c t i o n i n b i n d i n g caused by c a t i o n s (Simon e t a l . , 1975; Young and Kuhar, 1979). For t a c h y k i n i n p e p t i d e s i n r a t c o r t e x , r e c e p t o r b i n d i n g i s reduced t o 60% a t 150 mM NaCl, K C l , and NH 4C1 ( C a s c i e r i e t a l . , 1985). Even b i n d i n g of glutamate t o the Na-dependent h i g h a f f i n i t y glutamate uptake s i t e i n r a t c o r t e x synaptosomes i s s t e a d i l y decreased t o 50% over the Na + c o n c e n t r a t i o n range of 20 t o 200 mM (Bennett e t a l . , 1973). Thus Na + and other c a t i o n s can reduce r e c e p t o r and uptake s i t e b i n d i n g a t p h y s i o l o g i c a l c o n c e n t r a t i o n s and pH. I t i s c o n c e i v a b l e t h a t e f f e c t s such as these may be d r a m a t i c a l l y enhanced a t n o n - p h y s i o l o g i c a l pH ( f o r example, pH a f f e c t s b i n d i n g t o glutamate uptake s i t e s [Bennett e t a l . , 1973]). Such a s i t u a t i o n i s suggested f o r GSH b i n d i n g a t pH 8.0 i n the p r e s e n t study. The experiments of O g i t a and Yoneda (1987) support the i d e a t h a t GSH r e c e p t o r s are not N a + s e n s i t i v e a t pH 7.4, s i n c e [ H]GSH b i n d i n g was not decreased by 100 mM CU^COONa i n s y n a p t i c membranes, a p r e p a r a t i o n which may exclude m e t a b o l i c uptake s i t e s and LAHC b i n d i n g . The a l t e r n a t i v e p o s s i b i l i t y t h a t r e d u c t i o n by NaCl i s due t o C l ~ i s a l s o suggested by the r e d u c t i o n of [ 3H]GSH b i n d i n g c o n s i s t e n t l y found with C l _ s a l t s (NaCl, C a C l 2 , M gCl 2, and KCl) and w i t h T r i s - H C l . I t i s u n l i k e l y t h a t the p o s s i b l e GSH r e c e p t o r b i n d i n g s i t e s d e s c r i b e d here r e f l e c t b i n d i n g of GSH t o i t s m e t a b o l i z i n g enzymes or uptake s i t e s , f o r the f o l l o w i n g reasons. (1) The K d v a l u e s f o r GSH b i n d i n g were i n the low 109 nM and low /xM range, while K m v a l u e s f o r the v a r i o u s GSH S-t r a n s f e r a s e s are 0.2 t o 2 mM i n r a t l i v e r (Habig e t a l . , 1974) and t h e i r a c t i v i t y i s low i n b r a i n (Orlowski and Karkowsky, 197 6) ; the K m f o r GSH peroxidase i s 18 izM i n r a t l i v e r (Flohe e t a l . , 1972) and i s l a r g e l y c o n f i n e d t o neuronal n u c l e i (Ushijima, 1986); the K m f o r the m i t o c h o n d r i a l h i g h - a f f i n i t y GSH t r a n s p o r t e r i s 60 ;uM i n l i v e r (Martensson e t a l . , 1990); and the on l y e x t r a c e l l u l a r enzyme, gamma-glutamyl t r a n s p e p t i d a s e , which may a l s o be the b l o o d -b r a i n and blood-CSF t r a n s p o r t s i t e , may have a K m of about 6 mM i n r a t b r a i n (Kannan e t a l . , 1990; J a i n e t a l . , 1991), and a c i v i c i n had no a f f e c t on b i n d i n g . (2) GSSG had a s i m i l a r a f f i n i t y f o r the l o w - a f f i n i t y GSH b i n d i n g s i t e , as i t does f o r the h i g h - a f f i n i t y s i t e i n a s t r o c y t e s (Guo e t a l . , p e r s o n a l communicaton, see below). (3) There was no n e c e s s i t y f o r the -SH moiety i n the b i n d i n g , nor i s t h e r e n e c e s s i t y f o r the gamma-glutamyl s t r u c t u r e i n s y n a p t i c membrane b i n d i n g (Ogita and Yoneda, 1987). L i k e w i s e the p o s s i b l e r e c e p t o r b i n d i n g s i t e s are u n l i k e l y t o r e p r e s e n t degradation of [ S]GSH i n t o [ 3 5 S ] c y s t e i n e or of [ 3H]GSH i n t o [ 3 H ] g l y c i n e and subsequent b i n d i n g t o c y s t e i n e or g l y c i n e uptake s i t e s because (1) the b r a i n c y s t e i n e uptake s i t e i s Na-dependent (Hwang and Segal, 1979), and (2) the K m f o r the l o w - a f f i n i t y Na-independent c o r t i c a l g l y c i n e uptake s i t e was o r i g i n a l l y found t o be 300 /xM (Johnston and Iversen, 1971) , and i s found t o be 8 /xM i n i s o l a t e d c o r t i c a l a s t r o c y t e s and 10 /xM i n i s o l a t e d c o r t i c a l 110 neurons (Hannuniemi and Oja, 1981). The b i n d i n g i s a l s o u n l i k e l y t o r e p r e s e n t [ 3 H ] g l y c i n e b i n d i n g t o the s t r y c h n i n e -i n s e n s i t i v e NMDA r e c e p t o r g l y c i n e b i n d i n g s i t e because the K d f o r t h i s s i t e i s 80 nM i n r a t c o r t e x s e c t i o n s (Miyoshi e t a l . , 1990). In a d d i t i o n , GSH i s not degraded i n s y n a p t i c membrane p r e p a r a t i o n s i n T r i s - a c e t a t e b u f f e r (Ogita e t a l . , 1986a), and o p i o i d p e p t i d e s are not degraded i n l i g h t l y f i x e d , whole b r a i n s e c t i o n i n c u b a t i o n s i n T r i s - H C l (Young and Kuhar, 1979). A caveat i s expressed, however, by L i m b r i d (1986): "Before a l i g a n d b i n d i n g s i t e can be demonstrated t o be a r e c e p t o r of p h y s i o l o g i c a l i n t e r e s t , t h e r e must be a b i o l o g i c a l e f f e c t e l i c i t e d by t h i s l i g a n d t o which the p r o p e r t i e s of r a d i o l i g a n d b i n d i n g can be compared", such t h a t "the potency of u n l a b e l e d agents i n competing f o r b i n d i n g t o the r e c e p t o r should p a r a l l e l the order of potency of these agents i n promoting (agonists) or b l o c k i n g ( a n t a g o n i s t s ) the p h y s i o l o g i c a l e f f e c t ( s ) mediated v i a the p u t a t i v e r e c e p t o r . " T h i s b i n d i n g data by i t s e l f , t h e r e f o r e , cannot be taken t o unequivocably demonstrate the presence of GSH r e c e p t o r s . The l o w - a f f i n i t y p o s s i b l e r e c e p t o r b i n d i n g s i t e may correspond t o the h i g h e r - a f f i n i t y s i t e of the two GSH b i n d i n g s i t e s p r e v i o u s l y r e p o r t e d f o r r a t s y n a p t i c membranes, w i t h p u b l i s h e d v a l u e s (from the same l a b o r a t o r y ) of 1.9, 0.6, and 0.8 /xM f o r the K d and 9.6, 2.5, and 4.0 pmoles/mg p r o t e i n f o r the B m a x (Ogita e t a l . , 1986b; O g i t a and Yoneda, 1987, 1988, r e s p e c t i v e l y ) , and which i s s i m i l a r l y d i s p l a c e d by c y s t e i n e . I l l The LAHC s i t e may or may not correspond t o the p r e v i o u s l y r e p o r t e d l o w e r - a f f i n i t y , temperature-dependent, Na-independent p u t a t i v e uptake s i t e because, although the K d and Bmax v a l u e s are s i m i l a r t o the GSH LAHC s i t e (K d: 5.9, 12.6, 11.0 JUM; B m a x : 21.4, 28.5, 27.6 pmoles/mg p r o t e i n ; O g i t a e t a l . , 1986b; O g i t a and Yoneda 1987, 1988), the LAHC b i n d i n g was not p o t e n t i a t e d by c y s t e i n e as i s t h i s r e p o r t e d p u t a t i v e uptake s i t e (Ogita and Yoneda, 1989). From the p r e s e n t experiments i t cannot be d i s c e r n e d whether the LAHC b i n d i n g or the l o w - a f f i n i t y p o s s i b l e - r e c e p t o r b i n d i n g i s temperature dependent because the non-mass-action component of the 4° C time courses c o u l d be due t o e i t h e r or both of these s i t e s . I t i s suggested t h a t s y n a p t i c membrane p r e p a r a t i o n s may e l i m i n a t e the LAHC b i n d i n g . Such a p r e p a r a t i o n would a l s o e l i m i n a t e the h i g h - a f f i n i t y GSH b i n d i n g s i t e i f t h i s s i t e i s a hormone r e c e p t o r or i s c o n f i n e d t o a s t r o c y t e s (see below), but i n any case the hig h c o n c e n t r a t i o n range covered by the s a t u r a t i o n b i n d i n g a n a l y s i s i n these p r e v i o u s r e p o r t s would miss t h i s h i g h - a f f i n i t y b i n d i n g s i t e , as demonstrated i n the p r e s e n t study. I t i s p o s s i b l e t h a t the LAHC b i n d i n g r e v e a l e d i n the p r e s e n t study i n c l u d e s b i n d i n g t o one or more of the f o l l o w i n g : (1) GSH peroxidase i n neuronal n u c l e i ( K m = 18 /xM i n l i v e r [Flohe e t a l . , 1972]); (2) a m i t o c h o n d r i a l uptake s i t e ( K m = 60 /xM f o r the h i g h - a f f i n i t y component i n l i v e r [Martensson e t a l . , 1990]); (3) the p o s s i b l e neuronal plasma membrane uptake s i t e i m p l i c a t e d i n the pr e s e n t uptake s t u d i e s 112 i n v i v o , s i n c e uptake s i t e s g e n e r a l l y have h i g h K d and B m a x v a l u e s , except t h a t the high e r a f f i n i t y t e r m i n a l uptake systems appear t o be u n i v e r s a l l y Na-dependent, i n c l u d i n g those f o r n e u r o t r a n s m i t t e r amino a c i d s (Iversen, 1971; Bennett e t a l . , 1973; L a j t h a and Shershen, 1975), w h i l e the g e n e r a l m e t a b o l i c uptake systems have much h i g h e r K m v a l u e s (mM) than the K d f o r the LAHC s i t e shown i n the p r e s e n t study (Johnston and Iversen, 1971; Bennett e t a l . , 1973); and (4) d e g r a d a t i o n i n t o [ 3 H ] g l y c i n e (by r e a c t i o n s not i n v o l v i n g gamma-glutamyl t r a n s p e p t i d a s e ) and subsequent b i n d i n g t o the low a f f i n i t y g l y c i n e uptake s i t e , although t h i s a l s o i s u n l i k e l y (see above). LAMINAR DISTRIBUTION Most s t u d i e s of the d i s t r i b u t i o n s of r e c e p t o r t y p e s , i n c l u d i n g EAA subtypes, i n more h i g h l y - d e v e l o p e d v i s u a l c o r t e x such as i n the c a t (Shaw e t a l . , 1986) and p a r t i c u l a r l y the monkey (Shaw and Cynader, 1986; Shaw e t a l . , 1991) show g r e a t e r d e n s i t i e s i n s p e c i f i c l a y e r s . Rat v i s u a l c o r t e x shows l e s s d i s t i n c t (Monaghan e t a l . , 1984; Monaghan and Cotman, 1982, 1985; Shaw, unpublished) or un i f o r m (Miyoshi e t a l . , 1990) l a m i n a t i o n f o r many r a d i o l i g a n d s , i n c l u d i n g those f o r EAA r e c e p t o r s . The [ 3 5S]GSH a u t o r a d i o g r a p h i c d i s t r i b u t i o n s i n v i s u a l c o r t e x s e c t i o n s are c o n s i s t e n t w i t h these t r e n d s . 113 In a d u l t monkey area 17, cytochrome oxidase s t a i n i n g i s h i g h e s t i n the t h i n s u b l a y e r 4A, i n s u b l a y e r 4C.ct, and i n the lower p a r t of s u b l a y e r 4CB ( C a r r o l and Wong-Riley, 1984) (as i n d i c a t e d i n F i g u r e 22J; see a l s o F i g u r e 4). The [ 3 5S]GSH b i n d i n g i n comparison, then, showed a p r e f e r e n c e f o r s u b l a y e r 4C ( F i g u r e 221), which i s e x c l u s i v e l y s t e l l a t e c e l l s r e c e i v i n g g e n i c u l a t e t e r m i n a t i o n s , and t h i s b i n d i n g i s not found i n s u b l a y e r 4B, a l a y e r t h a t r e c e i v e s no DLG t e r m i n a t i o n s (see F i g u r e 4) and c o n t a i n s the l i n e of Gennari (Lund, 1973; G i l b e r t , 1983). T h i s p a t t e r n i s c o n s i s t e n t w i t h a r o l e as a g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r . Cat area 17 l a y e r 4 [ 3 5S]GSH b i n d i n g was somewhat more d i f f u s e than i n the monkey, which i s c o n s i s t e n t w i t h the presence i n the c a t of DLG a f f e r e n t s t o l a y e r s immediately above and below l a y e r 4, u n l i k e i n the monkey (see F i g u r e 4), and the l a c k of a l i n e of Gennari ( G i l b e r t , 1983). The g e n e r a l i n c r e a s e i n b i n d i n g from 6 t o 61 days of age i s c o i n c i d e n t w i t h the major synaptogenesis t h a t o c c u r s d u r i n g t h i s p e r i o d ( W i n f i e l d , 1981), while the r e d u c t i o n i n b i n d i n g a t 90 and 230 days of age i s c o i n c i d e n t w i t h the succeeding synapse e l i m i n a t i o n ( f o r review see Payne e t a l . , 1988). The l a c k of a b i n d i n g peak i n 6-day o l d c a t area 17 o c c u r s a t a time when g e n i c u l a t e i n n e r v a t i o n i s much more uniform, and synapses are fewer (Kato e t a l . , 1983; Payne e t a l . , 1988). At 32 days of age the most d i s t i n c t i v e [ 3 5S]GSH b i n d i n g d e n s i t y i n l a y e r 4 was seen, a t which time i n the c a t the a d u l t p a t t e r n of g e n i c u l a t e i n n e r v a t i o n has been reached 114 (Kato e t a l . , 1983; Payne e t a l . , 1988), and the c r i t i c a l p e r i o d f o r p l a s t i c i t y i s peaking (Hubel and W e i s e l , 1970; f o r review see Cynader e t a l . , 1990). I f GSH does a c t as a n e u r o t r a n s m i t t e r a t the sensory-i n p u t synapses t o l a y e r 4 i n area 17, then the uni f o r m d i s t r i b u t i o n i n the r a t must be e x p l a i n e d , as w e l l as the l e s s d i s t i n c t l a y e r 4 b i n d i n g seen i n area 17 f o l l o w i n g the peak of the c r i t i c a l p e r i o d i n the c a t . Perhaps the most obvious e x p l a n a t i o n i s t h a t GSH r e c e p t o r b i n d i n g i s "overshadowed" by b i n d i n g t o the much denser LAHC s i t e , s i n c e b i n d i n g t o the l a t t e r i s s t i l l p r esent i n 20 minute i n c u b a t i o n s a t h i g h r a d i o l i g a n d c o n c e n t r a t i o n ( F i g u r e 19), and may be pr e s e n t a t low c o n c e n t r a t i o n as w e l l . An a l t e r n a t i v e e x p l a n a t i o n i s the f o l l o w i n g . Layer 4 i s the predominant i n p u t zone f o r s p e c i f i c a f f e r e n t s from the DLG, but i n the r a t t h e r e i s a l s o g e n i c u l a t e i n p u t t o a t l e a s t l a y e r s 1, 3, and 6 ( f o r review see Seft o n and Dreher, 1985), w h i l e i n the c a t a l l other l a y e r s a l s o r e c e i v e d i r e c t , monosynaptic g e n i c u l a t e input ( f o r review see Stone, 1983), and such t e r m i n a l s may a l s o have GSH r e c e p t o r s . There are a l s o LP p r o j e c t i o n s t o a t l e a s t l a y e r s 5 and 6 i n the r a t (S e f t o n and Dreyer, 1985), and p o s s i b l y p r o j e c t i o n s f o r the L P / p u l v i n a r t o l a y e r s 1 and 5 i n the c a t ( G i l b e r t , 1983). I t has been suggested t h a t e x t r a g e n i c u l a t e v i s u a l i n p u t may r e p r e s e n t a p h y l o g e n e t i c a l l y o l d e r and l e s s s p e c i f i c a l l y o r g a n i z e d pathway (Diamond and H a l l , 1969). In the r a t , then, e x t r a g e n i c u l a t e v i s u a l i nput from the LP may r i v a l t he 115 g e n i c u l a t e i n p u t i n terms of number of r e c e p t o r s . S i n c e the i n v i v o uptake experiments i n r a t s showed p o s s i b l e r e t r o g r a d e uptake of r a d i o l a b e l e d GSH from O c l t o the LP, t h e r e i s support f o r the n o t i o n t h a t such e x t r a g e n i c u l a t e c o r t i c a l synapses a l s o have GSH r e c e p t o r s . In the case of c a t s t r i a t e c o r t e x , a f t e r 32 days of age t h e r e may occur an i n c r e a s e i n the r a t i o of the number, a f f i n i t y , or subtype of GSH r e c e p t o r s , r e l a t i v e t o l a y e r 4, a t e x t r i n s i c or i n t r i n s i c i n p u t s ( i n c l u d i n g other g e n i c u l a t e inputs) t o o t h e r l a y e r s , c o i n c i d e n t w i t h ongoing synaptogenesis up t o about day 60 ( W i n f i e l d , 1981). Conversely, or a d d i t i o n a l l y , t h e r e c o u l d be a down-regulation, a f f i n i t y decrease, or subtype change of GSH r e c e p t o r s a t l a y e r 4 g e n i c u l a t e - t e r m i n a l synapses. I t t h e r e f o r e seems p o s s i b l e t o s p e c u l a t e t h a t g l u t a t h i o n e may be a g e n i c u l o s t r i a t e n e u r o t r a n s m i t t e r i n the a d u l t monkey, c a t , and r a t . PATHWAYS Uptake showed s e l e c t i v i t y s i n c e [ H]muscimol and [ 3 H ] k a i n a t e were not taken up. P o s s i b l e e x c i t o t o x i c l e s i o n i n g by k a i n a t e or glutamate would not a f f e c t t e r m i n a l uptake (Coyle, 1987), so r e t r o g r a d e t r a n s p o r t would not be a f f e c t e d , and s i n c e t r a n s p o r t takes p l a c e s o l e l y by p a s s i v e d i f f u s i o n (Baughman and G i l b e r t , 1981), and the somal d e g e n e r a t i o n phase of e x c i t o t o x i c i t y i s dela y e d up t o a day 116 (Choi, 1988; Coyle, 1987), t h e r e f o r e anterograde uptake and t r a n s p o r t should not have been d i s r u p t e d e i t h e r . The p o s s i b l e presence of r e t r o g r a d e t r a n s p o r t suggested by c e l l u l a r r e s o l u t i o n i n d i c a t e s p o s s i b l e n e u r o t r a n s m i s s i o n r o l e s f o r GSH and c y s t e i n e i n the g e n i c u l o s t r i a t e and l a t e r a l p o s t e r i o r - c o r t i c a l p r o j e c t i o n s i n r a t , where c y s t e i n e may be a m e t a b o l i t e of r e l e a s e d GSH. The l a c k of L P / p u l v i n a r and o t h e r uptake i n the c a t i s c o n s i s t e n t with the known anatomy i n t h a t e x t r a g e n i c u l a t e p r o j e c t i o n s t o area 17 i n the p o s t n a t a l c a t are very sparce ( f o r review see G i l b e r t , 1983; Stone, 1983; Payne, 1988; Swanson e t a l . , 1974). Probable anterograde t r a n s p o r t shown by s i l v e r g r a i n s i n the n e u r o p i l i n a l l r a t n u c l e i t h a t showed uptake may r e p r e s e n t uptake f o r m e t a b o l i c purposes (Ochs e t a l . , 1967; Hannuniemi and Oja, 1981), e s p e c i a l l y i f GSH had been metabolized i n v i v o i n t o amino a c i d s (see below). C e l l u l a r r e s o l u t i o n f o r [3H]glutamate and [3H]glycine uptake i n r a t was not o b t a i n e d , so i t was not p o s s i b l e t o determine whether th e s e are i m p l i c a t e d i n n e u r o t r a n s m i s s i o n ( r e t r o g r a d e ) , p o s s i b l y as m e t a b o l i t e s of r e l e a s e d GSH, or may simply be p a r t i c i p a t i n g i n p r o t e i n s y n t h e s i s (anterograde). For the VLG, APTD, Rt, and SC t h e i r known anatomical c o n n e c t i v i t y p r e c l u d e s r e t r o g r a d e t r a n s p o r t , so presence i n these n u c l e i of [3H]glutamate or [3H]glycine does not i n d i c a t e n e u r o t r a n s m i t t e r - r e l a t e d r o l e s . Presence of [3H]glutamate or [ 3 H ] g l y c i n e i n the DLG and LP i s not i n c o n s i s t e n t w i t h these 117 amino a c i d s being m e t a b o l i t e s of GSH r e l e a s e d by c o r t i c a l t e r m i n a l s of p r o j e c t i o n s from these n u c l e i . A p o s s i b l e confounding f a c t o r f o r i n t e r p r e t a t i o n was observed i n the r a t s . Retrograde uptake was determined by the presence of s i l v e r g r a i n s over c e l l b o dies. In 2 0 /xm t h i c k s e c t i o n s , most c e l l bodies are s e c t i o n e d . I t t h e r e f o r e f o l l o w s t h a t i f uptake of r a d i o l a b e l were s o l e l y anterograde and c o n f i n e d t o t e r m i n a l s i n the n e u r o p i l , an area f r e e of s i l v e r g r a i n s should correspond t o many of the s t a i n e d c e l l b o d i e s , as demonstrated by Baughman and G i l b e r t (1981). In c o n f l i c t w i t h t h i s assumption was the o b s e r v a t i o n i n r a t of s i l v e r g r a i n s over many c e l l bodies i n the VLG and SC, which have no p r o j e c t i o n s t o v i s u a l c o r t e x . To address t h i s i s s u e , the f o l l o w i n g p o s s i b i l i t i e s are suggested. The one-day s u r v i v a l time f o r the c a t was chosen t o minimize t r a n s -s y n a p t i c t r a n s p o r t (Hendrickson 1972), and i t i s t h e r e f o r e p o s s i b l e t h a t some of the l a b e l i n g i n t h a l a m i c n u c l e i from r a t s w i t h two-day s u r v i v a l times r e p r e s e n t s t r a n s s y n a p t i c t r a n s p o r t . Since the p o s s i b i l i t y of t r a n s - s y n a p t i c t r a n s p o r t cannot be completely r u l e d out i n these r a t s , i t sho u l d be noted t h a t many of the uptake n u c l e i i n t e r c o n n e c t : SC <-> DLG/VLG; Rt <-> ZI/SubG,LP,DLG; ZI/SubG<->VLG; VLG -> APTD; APTD ->Rt. Retrograde uptake i n the VLG may t h e r e f o r e be v i a i t s p r o j e c t i o n s t o the SC, ZI/SubG, and APTD, a l l of which showed uptake. Another p o s s i b i l i t y i s noted by Young and Kuhar (1979). The t i s s u e s e c t i o n was coated w i t h wet emulsion t o o b t a i n a c l o s e r e g i s t e r of exposed s i l v e r g r a i n s . 118 T h i s m oisture can sometimes cause a d i s p e r s a l of the r a d i o l a b e l from i t s o r i g i n a l p o s i t i o n . Thus the d i s t i n c t i o n between r e t r o g r a d e and anterograde uptake may have been obscured i n a l l n u c l e i i n the r a t s . I t i s a l s o p o s s i b l e t h a t exogenous, r a d i o l a b e l e d GSH i s c a t a b o l i z e d i n v i v o t o c y s t e i n y l - g l y c i n e and gamma-glutamy1-amino a c i d s by gamma-glutamyl-transpeptidase, f o l l o w e d by the a c t i o n of c y s t e i n y l - g l y c i n a s e t o produce c y s t e i n e and g l y c i n e (reviewed by M e i s t e r , 1983, 1989). The [ 3 5 S ] c y s t e i n e from the breakdown of [ 3 5S]GSH, as w e l l as the [ 3 5 S ] c y s t e i n e a p p l i e d d i r e c t l y , may be taken up by the h i g h - a f f i n i t y Na-dependent or low a f f i n i t y c y s t e i n e uptake s i t e s found i n r a t c o r t i c a l synaptosomes (Hwang and Segal, 1979). A l t e r n a t i v e l y , [ 3 5 S ] c y s t e i n e may be o x i d i z e d t o c y s t e i n e s u l f i n a t e (Olney, e t a l . , 1971; M i s r a , 1983) and then be taken up by the s i n g l e axon-terminal t r a n s p o r t e r t h a t t a k e s up glutamate, a s p a r t a t e , c y s t e i n e s u l f i n a t e , and c y s t e a t e ( B a l c a r and Johnson, 1972; Wilson and Pastuszko, 1986). The breakdown of [ 3H]GSH ( l a b e l e d on the g l y c i n e r e s i d u e ) would l i k e w i s e produce [ H ] g l y c i n e , f o r which has been found a low l e v e l of both l o w - a f f i n i t y , n o n - s p e c i f i c ( n e u t r a l amino a c i d s ) c o r t i c a l synaptosomal uptake (Johnston and I v e r s e n , 1971) and h i g h - a f f i n i t y c o r t i c a l neuronal uptake (Hannuniemi, 1981). There are s e v e r a l p o s s i b l e i n t e r p r e t a t i o n s of the r a d i o l a b e l uptake r e p o r t e d here. Uptake of [ 3H]glutamate c o u l d r e p r e s e n t g l u t a m a t e r g i c pathways, e s p e c i a l l y s i n c e i t 119 i s not necessary f o r each m e t a b o l i t e of a s m a l l p e p t i d e n e u r o t r a n s m i t t e r t o be taken up by the t e r m i n a l , as shown f o r NAAG r e l e a s e ( B l a k e l y e t a l . , 1986; Robinson e t a l . , 1897; Coyle e t a l . , 1986), and s i n c e the known GSH-degrading enzyme, gamma-glutamyl-transpeptidase, would produce mostly gamma-glutamyl-amino a c i d s r a t h e r than f r e e glutamate, alt h o u g h t h i s enzyme may be r e s t r i c t e d t o c a p i l l a r i e s and c h o r o i d p l e x u s (Orlowski and Karkowsky, 1976). L i k e w i s e [ 3 5 S ] uptake from [ 3 5 S ] c y s t e i n e i n the form of c y s t e i n e s u l f i n a t e , as d i s c u s s e d above, c o u l d a l s o r e p r e s e n t g l u t a m a t e r g i c pathways or c y s t e i n e s u l f i n a t e - m e d i a t e d pathways (Pin e t a l . , 1987; Iwata e t a l . , 1982a,b). [ 3 5 S ] C y s t e i n e uptake c o u l d a l s o i n d i c a t e c y s t e i n e r g i c pathways ( K e l l e r e t a l . , 1989; L i and Jope, 1989). The uptake of [ 3H]GSH, l a b e l e d on the g l y c i n e r e s i d u e , i s perhaps the b e s t i n d i c a t o r t h a t we may be o b s e r v i n g uptake of n e u r o t r a n s m i t t e r m e t a b o l i t e s of GSH or of GSH i t s e l f , s i n c e g l y c i n e n e u r o t r a n s m i s s i o n i s r a r e i n f o r e b r a i n (reviewed i n McGeer e t a l . , 1987), and s i n c e g l y c i n e i s not l i k e l y t o be the g e n i c u l o c o r t i c a l n e u r o t r a n s m i t t e r , having no independent e x c i t a t o r y e f f e c t (Thompson e t a l . , 1989; Bonhaus e t a l . , 1989) . F i n a l l y , i n the mammalian v i s u a l system i n p a r t i c u l a r , l a b e l i n g may sometimes r e p r e s e n t f i b r e s of passage, even a t s h o r t s u r v i v a l times (Swanson e t a l . , 1974; Hendrickson e t a l . , 1972) . 120 A p o s s i b l e s i m i l a r i t y t o the LOT NAAG-transmission s t o r y (see I n t r o d u c t i o n ) occurs here. There i s no uptake and t r a n s p o r t of glutamate i n e i t h e r d i r e c t i o n between the SC and area 17 i n c a t (Baughman and G i l b e r t , 1981), but c o r t i c a l a b l a t i o n i n r a t decreases endogenous glutamate r e l e a s e i n the SC (Lund-Karlsen and Fonnum, 1978). One might c o n j e c t u r e t h a t i f r a t and c a t share the same n e u r o t r a n s m i t t e r i n these pathways the t r u e n e u r o t r a n s m i t t e r c o u l d c o n c e i v a b l y be GSH, r e l e a s i n g glutamate as a m e t a b o l i t e , s i m i l a r t o NAAG ,neurotransmission. ASTROCYTES Guo e t a l . (1991) have demonstrated GSH b i n d i n g s i t e s on c u l t u r e d a s t r o c y t e s and i n white matter i n r a t b r a i n . Using l o w - c o n c e n t r a t i o n [ 3 5S]GSH s a t u r a t i o n b i n d i n g they f i n d a h i g h - a f f i n i t y b i n d i n g s i t e on both f i b r o u s and p r o t o p l a s m i c c o r t i c a l a s t r o c y t e s i n c u l t u r e (K d = 9 nM). The a d d i t i o n a l presence of the low a f f i n i t y b i n d i n g s i t e and LAHC b i n d i n g s i t e on a s t r o c y t e s was supported by these a u t h o r s ' c o m p e t i t i o n data (Guo e t a l . , p e r s o n a l communication), which showed an apparent IC50 of 10 /uM, w i t h t w o - s i t e a n a l y s i s (Bylund and Yamamura, 1990) y i e l d i n g s eparate v a l u e s of 2.3 JUM and 46 /xM. C e l l u l a r r e s o l u t i o n o b t a i n e d w i t h c o l l o i d a l g o l d d e c o r a t i o n of b i o t i n y l - G S H b i n d i n g (Guo and Shaw, 1991) showed very h i g h d e n s i t y b i n d i n g on a s t r o c y t e s . 121 T h i s h i g h d e n s i t y i s i n keeping with the B m a x v a l u e s r e p o r t e d here, where the l o w - a f f i n i t y s i t e and LAHC s i t e c o n s t i t u t e 99% o f the t o t a l b i n d i n g (11% and 88%, r e s p e c t i v e l y ) . More r e c e n t r a d i o a c t i v e b i n d i n g experiments i n c o r t i c a l a s t r o c y t e s by Guo and Shaw (personal communication) d i f f e r e n t i a t e d the h i g h a f f i n i t y GSH b i n d i n g s i t e i n t o two sepa r a t e b i n d i n g s i t e s ( K d = 2 and 12 nM), so i t may be p o s s i b l e t h a t the h i g h a f f i n i t y b i n d i n g s i t e r e p o r t e d here f o r whole b r a i n i s r e s o l v a b l e i n t o two subtypes as w e l l . The presence of a low-a f f i n i t y b i n d i n g s i t e i n s y n a p t i c membranes (Og i t a e t a l . , 1986) makes i t u n l i k e l y t h a t the l o w - a f f i n i t y r e c e p t o r i s c o n f i n e d t o a s t o c y t e s , but the p o s s i b i l i t y t h a t the h i g h -a f f i n i t y r e c e p t o r i s c o n f i n e d t o a s t r o c y t e s cannot be r u l e d out. S i n c e GSH i s found i n hig h c o n c e n t r a t i o n s i n a s t r o c y t e s but not i n neurons, both i n c u l t u r e and i n v i v o (Raps e t a l . , 1989; S l i v k a e t a l . , 1987) and s i n c e GSH S - t r a n s f e r a s e i s c o n f i n e d t o a s t r o c y t e s as w e l l (Senjo e t a l . , 1986), one i n t e r p r e t a t i o n might be t h a t the h i g h - a f f i n i t y GSH r e c e p t o r subtype (or types) i s a g l i a l uptake s i t e . Another i n t e r p r e t a t i o n i s t h a t GSH i s i n v o l v e d i n a n o v e l form of n e u r o n - g l i a l s i g n a l l i n g (Marrero e t a l . , 1989; Usowicz e t a l . , 1989; Lieberman e t a l . , 1989; f o r reviews see Ba r r e s , 1989, and Kimelberg, 1988) i n a d d i t i o n t o neuronal s i g n a l l i n g . Guo e t a l . (1991) a l s o o b t a i n whole r a t b r a i n s e c t i o n d i s t r i b u t i o n s of s p e c i f i c b i n d i n g of b i o t i n y l a t e d GSH c o n f i n e d l a r g e l y t o white matter, and a l e s s e r amount i n brainstem. T h i s c o n t r a s t s w i t h autoradiographs from t h i s 122 study, which show densest s p e c i f i c [ DS]GSH b i n d i n g i n c o r t e x and LGN and densest n o n - s p e c i f i c b i n d i n g i n white matter and brainstem. Why b i o t i n y l a t e d GSH has a p r e f e r e n c e f o r white matter i s not understood. EXCITATORY AMINO ACID RECEPTOR INTERACTIONS None of the n e u r o t r a n s m i t t e r s f o r any of the EAA r e c e p t o r subtypes have been determined w i t h c e r t a i n t y , and displacement of [ 3H]GSH b i n d i n g by s u b t y p e - s p e c i f i c l i g a n d s or by p u t a t i v e t r a n s m i t t e r s may i n d i c a t e a p h y s i o l o g i c a l r o l e f o r GSH through i n t e r a c t i o n with EAA r e c e p t o r s (Coyle e t a l . , 1986). For EAA reviews see F o s t e r and Fagg (1984) , Mayer and Westbrook (1987), Monaghan e t a l . (1989), G r i f f i t h (1990), and Barnard and Henley (1990). At pH 7.4, the n e u r o t r a n s m i t t e r candidates glutamate, a s p a r t a t e , and c y s t e i n e ( K e l l e r e t a l . , 1989; L i and Jope, 1989) p a r t i a l l y d i s p l a c e d [ 3H]GSH b i n d i n g . Although c y s t e i n e d i s p l a c e d GSH, t h e r e was no need f o r the -SH moiety s i n c e S-methyl-GSH, GSSG, and CSA a l s o show a f f i n i t y f o r the GSH b i n d i n g s i t e r e v e a l e d a t pH 8.0, where t h e r e was no c r o s s o v e r w i t h glutamate b i n d i n g . Of the s i x d i f f e r e n t EAA s u b t y p e - s p e c i f i c l i g a n d s t e s t e d , o n l y AMPA p a r t i a l l y d i s p l a c e d [ 3H]GSH a t pH 7.4, s u g g e s t i n g a p o s s i b l e AMPA r e c e p t o r a f f i n i t y . T h i s s u g g e s t i o n i s supported by the o b s e r v a t i o n t h a t AMPA was a more e f f e c t i v e competitor than glutamate, because the 123 a f f i n i t y of glutamate f o r AMPA r e c e p t o r s i s f o u r times lower than the a f f i n i t y of AMPA i t s e l f ( I C 5 0 s = 0.3 /xM and 1.3 /xM, r e s p e c t i v e l y ) ( F o s t e r and Fagg, 1984). C y s t e i n e i s a can d i d a t e n e u r o t r a n s m i t t e r f o r the AP4 r e c e p t o r ( P u l l a n e t a l . , 1987), and although L-AP4 i t s e l f d i d not show c o m p e t i t i o n w i t h [ 3H]GSH, f r e e z i n g of the t i s s u e and c h l o r i n e - f r e e i n c u b a t i o n would have a b o l i s h e d the predominant AP4 uptake s i t e and l e v e l s of p o s t s y n a p t i c AP4 r e c e p t o r would be t oo low t o d e t e c t (Fagg e t a l . , 1983; Br i d g e s e t a l . , 1986), thus GSH a f f i n i t y f o r AP4 r e c e p t o r s i s not r u l e d out. F a i l u r e of L-AP3 t o d i s p l a c e [ 3H]GSH i n d i c a t e s a l a c k of a f f i n i t y f o r the ACPD r e c e p t o r (Schoepp and Johnson, 1989) . F a i l u r e of CSA i n d i c a t e s l a c k of a f f i n i t y f o r the CSA r e c e p t o r (Pin e t a l . , 1987; Iwata e t a l . , 1982a,b). To compare the r e s u l t s of t h i s study t o the l i t e r a t u r e , Oja e t a l . (1988) and Varga e t a l . (1989) found displacement of [ 3H]AMPA by GSH, and O g i t a and Yoneda (1987) found displacement of [ 3H]GSH by AMPA and by AP4 (1987) and a f f i n i t y of GSH f o r the NMDA r e c e p t o r a n t a g o n i s t - p r e f e r r i n g s i t e (Fagg e t a l . , 1988; Monaghan e t a l . , 1988) by i t s displacement of [ 3 H ] 3 - ( 2 - c a r b o x y p i p e r a z i n - 4 - y l ) p r o p y l - 1 -phosphonic a c i d (CPP) b i n d i n g (1990). At pH 6.9 the i n c r e a s e d b i n d i n g of [ 3H]GSH may have been due t o an i n c r e a s e of GSH r e c e p t o r b i n d i n g , of LAHC b i n d i n g , or of EAA r e c e p t o r b i n d i n g , although glutamate and a s p a r t a t e d i d not d i s p l a c e t h i s b i n d i n g . At t h i s pH [ 3H]GSH b i n d i n g i s d i s p l a c e d by NMDA as w e l l as by two other l i g a n d s w i t h 124 a f f i n i t y f o r the NMDA r e c e p t o r a g o n i s t - p r e f e r r i n g s i t e : QUIS and CSA (F o s t e r and Fagg, 1984; P u l l a n e t a l i ( 1987). These displa c e m e n t s may i n d i c a t e an a f f i n i t y f o r the NMDA a g o n i s t -p r e f e r r i n g s i t e . More l i k e l y , these displacements may i n d i c a t e a s t r u c t u r a l s i m i l a r i t y of the r e c o g n i t i o n s i t e of the GSH molecule t o c y s t e i n e (which d i s p l a c e d [ 3H]GSH a t pH 6.9, 7.4, and 8), and t h e r e f o r e AP4 r e c e p t o r a f f i n i t y , as w e l l as a f f i n i t y f o r AMPA r e c e p t o r s and f o r the NMDA c o m p e t i t i v e a n t a g o n i s t - p r e f e r r i n g s i t e . T h i s l a t t e r p o s s i b i l i t y i s suggested because (1) NMDA showed no displacement of [ 3H]GSH a t pH 7.4; (2) NMDA has a lower a f f i n i t y f o r NMDA r e c e p t o r s than does glutamate ( F o s t e r and Fagg, 1987), which d i d not d i s p l a c e the GSH b i n d i n g a t pH 6.9; (3) O g i t a and Yoneda (1990) show GSH a f f i n i t y f o r o n l y the c o m p e t i t i v e a n t a g o n i s t - p r e f e r r i n g s i t e of the NMDA r e c e p t o r ; (4) QUIS has g r e a t e r a f f i n i t y f o r the NMDA r e c e p t o r a n t a g o n i s t - p r e f e r r i n g s i t e (Ferkany and Coyle, 1983), f o r the AMPA r e c e p t o r ( f o r which GSH a f f i n i t y a t pH 7.4 was shown), and f o r AP4 r e c e p t o r s (Ogita and Yoneda, 1986); and (5) CSA a l s o has a f f i n i t y f o r the AP4 r e c e p t o r ( P u l l a n e t a l . , 1987). In r e f e r e n c e t o the r e p o r t e d abolishment o f v i s u a l l y -and e l e c t r i c a l l y - e v o k e d responses of neurons of l a y e r 4 i n c a t s t r i a t e c o r t e x by the e x c i t a t o r y amino a c i d r e c e p t o r a n t a g o n i s t k y n u r e n i c a c i d (Tsumoto, 1986; Hagihara e t a l . , 1988), i t i s p o s s i b l e t h a t t h i s broad-spectrum a n t a g o n i s t (Monaghan e t a l . , 1989) i s a c t u a l l y a c t i n g i n t h i s pathway as an a n t a g o n i s t of g l u t a t h i o n e r e c e p t o r s , b l o c k i n g t he e f f e c t 125 of endogenous r e l e a s e of GSH from g e n i c u l o s t r i a t e t e r m i n a l s v i a GSH r e c e p t o r s . T h i s idea i s supported by the r e l a t i v e p a u c i t y of EAA r e c e p t o r s found i n l a y e r 4 (see I n t r o d u c t i o n ) , a l t h o u g h ky n u r e n i c a c i d c o u l d have blocked the e f f e c t of GSH on AMPA r e c e p t o r s . Kynurenic a c i d should be t e s t e d as a p o s s i b l e a n t a g o n i s t of GSH r e c e p t o r s . SPECULATION - A NOVEL FORM OF NEUROTRANSMISSION? Bi n d i n g , uptake, and e l e c t r o p h y s i o l o g y ( T e y l e r , p e r s o n a l communication) of GSH i n v i s u a l c o r t e x suggest t h a t GSH may be a n e u r o t r a n s m i t t e r i n t h i s system, p a r t i c u l a r l y f o r the g e n i c u l o s t r i a t e p r o j e c t i o n . I t i s a l s o p o s s i b l e t h a t GSH may be r a p i d l y m e tabolized by known b r a i n enzymes i n t o a s e r i e s of seven substances, a l l of them neu r o t r a n s m i t t e r / m o d u l a t o r c a n d i d a t e s — gamma-glutamyl-amino a c i d s and glutamate; c y s t e i n e and g l y c i n e ; c y s t e i n e s u l f i n a t e ; c y s t e i c a c i d ; and t a u r i n e , i n t h a t order (Meister, 1983; R a s s i n and G a l l , 1987; H i l l e t a l . , 1985) — a l l p o s s i b l y w i t h i n the p e r i o d of a s i n g l e p o s t s y n a p t i c p o t e n t i a l ( J a i n e t a l . , 1991; Kozak and Tate, 1982; Legay e t a l . , 1987; Mis r a , 1983). I t i s t h e r e f o r e i n t e r e s t i n g t o s p e c u l a t e t h a t GSH n e u r o t r a n s m i s s i o n c o u l d be of a novel and h i g h l y complex form. GSH r e c e p t o r a c t i v a t i o n i t s e l f may, i n some systems, be metabotropic, s i n c e no sodium f l u x was found upon a p p l i c a t i o n t o r a t s t r i a t u m ( L u i n i , e t a l . , 1984). GSH may a l s o a c t i v a t e AMPA 126 and AP4 r e c e p t o r s , w h i l e a n t a g o n i z i n g NMDA r e c e p t o r s ( O g i t a and Yoneda, 1987, 1990; Varga e t a l . , 1989). NMDA r e c e p t o r antagonism would produce a decrease i n t o n i c background v o l t a g e dependence and r e d u c t i o n of f a c i l i t a t i o n o f a c t i o n p o t e n t i a l s (Sah e t a l . , 1989), and a decrease i n g l y c i n e - s i t e a f f i n i t y (Hood e t a l . , 1990). NMDA r e c e p t o r antagonism has a l s o been suggested t o reduce i n d u c t i o n of long-term p o t e n t i a t i o n and developmental p l a s t i c i t y (reviewed i n Monaghan e t a l . , 1989; Bear, 1988). Next, gamma-glutamyl amino a c i d s and some f r e e glutamate c o u l d a c t i v a t e the f u l l range of glutamate r e c e p t o r s (Varga e t a l . , 1989), and r e v e r s e a l l a n t a g o n i s t e f f e c t s on NMDA r e c e p t o r s (Hood e t a l . , 1990). C y s t e i n e may then a c t i v a t e AP4 r e c e p t o r s , which may i n c l u d e p r e s y n a p t i c a u t o r e c e p t o r s t h a t reduce r e l e a s e of glutamate (Monaghan e t a l . , 1989). G l y c i n e may then i n c r e a s e NMDA-receptor mediated responses (Thompson e t a l . , 1989; Bonhaus e t a l . , 1989), i n c l u d i n g s w i t c h i n g the r e c e p t o r toward the a g o n i s t - p r e f e r r i n g s t a t e (Monaghan e t a l . , 1988), f a c i l i t a t i n g a c t i o n p o t e n t i a l s (Sah e t a l . , 1989), and low e r i n g the t h r e s h o l d f o r i n d u c t i o n of long-term p o t e n t i a t i o n ( O l i v e r e t a l . , 1990) and of developmental s y n a p t i c p l a s t i c i t y (reviewed by Monaghan, 1989). Then CSA may a c t i v a t e the CSA r e c e p t o r (Pin e t a l . , 1987; Iwata e t a l . , 1982a,b; f o r review see G r i f f i t h , 1990), which may s t i m u l a t e c y c l i c AMP p r o d u c t i o n (Baba e t a l . , 1988). E x c i t a t i o n may be produced by CA, p o s s i b l y v i a KA and AP4 r e c e p t o r s ( P u l l a n e t a l . , 1987). F i n a l l y t a u r i n e , produced 127 l a s t , may have an i n h i b i t o r y e f f e c t (reviewed i n McGeer e t a l . , 1987). G l u t a t h i o n e n e u r o t r a n s m i s s i o n may even be modulated i n a novel manner: the i n i t i a l enzyme i n the m e t a b o l i c cascade, gamma-glutamyl-transpeptidase, i s modulated by s o m a t o s t a t i n and LHRH i n r a t b r a i n ( V a l i and V i j a y a n , 1990). Such a hypothesized n e u r o t r a n s m i s s i o n cascade c o u l d a l l o w a wide range of modulation and of r e g i o n a l v a r i a t i o n by the modulation of the enzymes i n v o l v e d and by the d i f f e r e n t i a l e x p r e s s i o n of s e t s of enzymes and r e c e p t o r s by a g i v e n neuron or synapse. I f t r u e , some n e u r o t r a n s m i t t e r candidates, i n some pathways, may t u r n out t o be secondary t o g l u t a t h i o n e r e l e a s e . 128 SUMMARY AND CONCLUSIONS 1. G l u t a t h i o n e b i n d i n g s i t e s i n r a t primary v i s u a l c o r t e x i n v i t r o s a t i s f i e d c r i t e r i a f o r r e c e p t o r b i n d i n g , i n c l u d i n g r e v e r s i b i l i t y , s a t u r a b i l i t y , s p e c i f i c i t y , h i g h a f f i n i t y , and heterogeneous d i s t r i b u t i o n . 2. G l u t a t h i o n e b i n d i n g s i t e s c h a r a c t e r i z e d i n t h i s study (pH 8) were sep a r a t e from EAA r e c e p t o r s , s i n c e t h e r e was no displacement of GSH by EAA t r a n s m i t t e r c a n d i d a t e s o r EAA s u b t y p e - s p e c i f i c l i g a n d s , and no displacement of glutamate b i n d i n g by GSH. 3. Two p o s s i b l e r e c e p t o r b i n d i n g s i t e s were p r e s e n t , one w i t h a low K d t y p i c a l of neuropeptide and neuromodulator r e c e p t o r s , and one with a h i g h e r K d t y p i c a l of EAA r e c e p t o r s . A low a f f i n i t y , h i g h c a p a c i t y b i n d i n g s i t e was a l s o p r e s e n t , t y p i c a l of nonreceptor b i n d i n g found commonly i n experiments a t h i g h c o n c e n t r a t i o n of l i g a n d . 4. G l u t a t h i o n e b i n d i n g s i t e s are prese n t i n c a t area 17 i n h i g h e s t d e n s i t y i n l a y e r 4, and i n monkey area 17 i n lower l a y e r 4, c o n s i t e n t with a r o l e as r e c e p t o r s f o r n e u r o t r a n s m i s s i o n by the g e n i c u l o s t r i a t e p r o j e c t i o n . Rat O c l b i n d i n g s i t e s are more u n i f o r m l y d i s t r i b u t e d , but n e v e r t h e l e s s do e x i s t i n l a y e r 4, which i s not i n c o n s i t e n t w i t h a r o l e i n the g e n i c u l o s t r i a t e p r o j e c t i o n . 129 5. R e s u l t s o f uptake e x p e r i m e n t s i n v i v o i n r a t and c a t p r i m a r y v i s u a l c o r t e x a r e c o n s i s t e n t w i t h t h e p r e s e n c e o f s e l e c t i v e t e r m i n a l uptake of GSH and/or i t s c o n s t i t u e n t amino a c i d s , w h i c h i n d i c a t e s t h a t GSH may be a n e u r o t r a n s m i t t e r a t s u c h t e r m i n a l s . The pathways i n v o l v e d a r e t h e g e n i c u l o s t r i a t e p r o j e c t i o n i n c a t and t h e g e n i c u l o s t r i a t e and l a t e r a l - p o s t e r i o r - o c c i p i t a l p r o j e c t i o n s i n r a t . , 6. C o m p e t i t i o n e x p e r i m e n t s a t pH 7.4 i n d i c a t e t h a t GSH c o u l d p l a y a r o l e i n EAA n e u r o t r a n s m i s s i o n by i n t e r a c t i n g w i t h EAA r e c e p t o r s such as t h e AMPA r e c e p t o r . 7. These r e s u l t s a r e i m p o r t a n t because, i n a d d i t i o n t o h e l p i n g d e t e r m i n e t h e c h e m i c a l c i r c u i t r y o f i m p o r t a n t pathways i n t h e v i s u a l system, t h e y s u g g e s t t h a t major n e u r o t r a n s m i t t e r s i n t h e b r a i n have y e t t o be i d e n t i f i e d , and t h a t t h e n e u r o t r a n s m i t t e r c a n d i d a t e s a s p a r t a t e and g l u t a m a t e may n o t p l a y as e x t e n s i v e a r o l e as p r e v i o u s l y t h o u g h t . 8 . To more d e f i n i t i v e l y s u p p o r t t h e p r o p o s i t i o n t h a t GSH b i n d i n g s i t e s a r e r e c e p t o r s , p h y s i o l o g i c a l r e s p o n s e s t o GSH and GSH a n t a g o n i s t s must be c o r r e l l a t e d w i t h r e c e p t o r b i n d i n g c h a r a c t e r i s t i c s . I n p a r t i c u l a r , k y n u r e n i c a c i d s h o u l d be t e s t e d as an a n t a g o n i s t o f p u t a t i v e GSH r e c e p t o r s , because of i t s a c t i o n i n b l o c k i n g g e n i c u l o s t r i a t e n e u r o t r a n s m i s s i o n . 130 REFERENCES Ambros-Ingerson, J . , Granger, R., and Lynch, G. 1990. S i m u l a t i o n of p a l e o c o r t e x performs h i e r a r c h i c a l c l u s t e r i n g . Science 247, 1344-1348. Angelo, E.D., R o s s i , P., and Garthwaite, J . 1990. Dual-component NMDA r e c e p t o r c u r r e n t s a t a s i n g l e c e n t r a l synapse. Nature 346, 467-469. A r t o l a , A. and Singer, W. 1987. Long-term p o t e n t i a t i o n and NMDA r e c e p t o r s i n r a t v i s u a l c o r t e x . Nature 330, 649-651. Baba, A., N i s h i u c h i , Y., Uemura, A., Tatsuno, T., Iwata, H. 1988. I n h i b i t i o n by f o r s k o l i n of e x c i t a t o r y amino a c i d -induced accumulation of c y c l i c AMP i n guinea p i g hippocampal s l i c e s . J . Neurochem. 51, 237-242. B a l c a r , V.L., and Johnston, G.A.R. 1972. The s t r u c t u r a l s p e c i f i c i t y of the hi g h a f f i n i t y uptake of L-glutamate and L - a s p a r t a t e by r a t b r a i n s l i c e s . J . Neurochem. 19, 2657-2666. Barnard, E. A. and Henley, J.M. 1990. The non-NMDA r e c e p t o r s : t y p e s , p r o t e i n s t r u c t u r e and molecular b i o l o g y . Trends Pharmacol. S c i . 11, 500-507. Ba r r e s , B.S. 1989. A new form of t r a n s m i s s i o n ? Nature 339, 343-344. Baughman, R.W., and G i l b e r t , C D . 1981. A s p a r t a t e and glutamate as p o s s i b l e n e u r o t r a n s m i t t e r s i n the v i s u a l c o r t e x . J . Ne u r o s c i . 1, 427-439. Bear, M.F. 1988. Involvement of e x c i t a t o r y amino a c i d r e c e p t o r mechanisms i n the experience-dependent development of v i s u a l c o r t e x . In: F r o n t i e r s i n E x c i t a t o r y Amino A c i d Research: Proceedings of an I n t e r n a t i o n a l Symposium, " E x c i t a t o r y A c i d s 1988", h e l d i n Manaus, Amazonas, B r a z i l , March 28 - A p r i l 2, 1988. Eds: C a v a l h i e r o , E.A., Lehmann, J . , T u r s k i , L. Manaus, B r a z i l . Bear, M.F. and Singer, w. 1986. Modulation of v i s u a l c o r t i c a l p l a s t i c i t y by a c e t y l c h o l i n e and n o r a d r e n a l i n e . Nature 320, 172-174. Bennett, J.P. 1978. Methods i n b i n d i n g s t u d i e s . In: Neu r o t r a n s m i t t e r Receptor B i n d i n g . Eds: Yamamura, H.I., Enna, S.J., and Kuhar, M.J. Raven Press, N.Y. 131 Bennett, J.P., Logan, W.J., and Snyder, S.H. 1973. Amino a c i d s as c e n t r a l nervous t r a n s m i t t e r s : the i n f l u e n c e of i o n s , amino a c i d analogues, and ontogeny on t r a n s p o r t systems f o r L-glutamic and L - a s p a r t i c a c i d s and g l y c i n e i n t o c e n t r a l nervous synaptosomes of the r a t . J . Neurochem. 21, 1533-1550. B l a k e l y , R.D., Ory-Lavolee, L., Grzanna, R., R o l l e r , K.J., and Coyle, J.T. 1987. S e l e c t i v e immunocytochemical s t a i n i n g of m i t r a l c e l l s i n r a t o l f a c t o r y bulb w i t h a f f i n i t y p u r i f i e d a n t i b o d i e s a g a i n s t N - a c e t y l - a s p a r t y l - g l u t a m a t e . B r a i n Res. 402, 373-378. B l a k e l y , R.D., Ory-Lavolee, Thompson, R.C., and Coyle, J.T. 1986. Synaptosomal t r a n s p o r t of r a d i o l a b e l from N - a c e t y l -a s p a r t y l - [ 3 H ] g l u t a m a t e suggests a mechanism of i n a c t i v a t i o n of an e x c i t a t o r y neuropeptide. J . Neurochem. 47, 1013-1019. B o l t z , J . , G i l b e r t , CD., and Wiesel, T.N. 1988. P h a r m a c o l o g i c a l a n a l y s i s of c o r t i c a l c i r c u i t r y . Trends N e u r o s c i . 11, 432-435. Bonhaus, D.W., Yeh, G.C., Skaryak, L., and McNamara, J.O. 1989. G l y c i n e r e g u l a t i o n of the N-methyl-D-aspartate r e c e p t o r - g a t e d i o n channel i n hippocampal membranes. M o l e c u l a r Pharmacology 36, 273-279. B r a d f o r d , H.F., and Richards, C D . 1976. S p e c i f i c r e l e a s e of endogenous glutamate from p y r i f o r m c o r t e x s t i m u l a t e d i n v i t r o . B r a i n Res. 105, 168. B r i d g e s , R.J., Hearn, T.J., Monaghan, D.T., and Cotman, CW. 1986. A comparison of 2-amino-4-phosphonobutyric a c i d (AP4) r e c e p t o r s and [ 3H]AP4 b i n d i n g s i t e s i n r a t b r a i n . B r a i n Res. 375, 204-209. Burt, D.R. 1978. C r i t e r i a f o r r e c e p t o r i d e n t i f i c a t i o n . In: N e u r o t r a n s m i t t e r Receptor B i n d i n g . Ed: Yamamura, H.I., Enna, S.J., and Kuhar, M.J. Raven Press, N.Y. Bylund, D.B. 1980. A n a l y s i s of r e c e p t o r b i n d i n g d a t a . In Receptor B i n d i n g Techniques, Short Course S y l l a b u s , Soc. f o r Neuroscience, pp. 70-99. Bylund, D.B. 1986. Graphic p r e s e n t a t i o n and a n a l y s i s of i n h i b i t i o n data from l i g a n d - b i n d i n g experiments. A n a l . Biochem. 159, 50-57. C a r r o l l , E.W., and Wong-Riley, M.T.T. 1984. Q u a n t i t a t i v e l i g h t and e l e c t r o n m i c r o s c o p i c a n a l y s i s of cytochrome o x i d a s e -r i c h zones i n the s t r i a t e c o r t e x of the s q u i r r e l monkey. J . Comp. Neurol. 222, 1-17. 132 C a s c i e r i , M.A., C h i c c h i , G.G., and Liang, T. 1985. Demonstration of two d i s t i n c t t a c h y k i n i n r e c e p t o r s i n r a t b r a i n c o r t e x . J . B i o l . Chem. 260, 1501-1507. Charpak, S., Gahwiler, B.H., Do, K.Q., and K n o p f e l , T. 1990. Potassium conductances i n hippocampal neurons b l o c k e d by e x c i t a t o r y amino-acid t r a n s m i t t e r s . Nature 347, 765-767. Cheramy, A., L e v i e l , V., and G l o w i n s k i , J . 1981. D e n d r i t i c r e l e a s e of dopamine i n the s u b s t a n t i a n i g r a . Nature, 289, 537-542. Choi, D. 1988. Calcium-mediated n e u r o t o x i c i t y : r e l a t i o n s h i p t o s p e c i f i c channel types and r o l e i n ischemic damage. Trends N e u r o s c i . 11, 465-469. C o l l i n s C.G.S. 1978. Evidence of n e u r o t r a n s m i t t e r r o l e f o r a s p a r t a t e and gamma-aminobutyric a c i d i n the r a t o l f a c t o r y c o r t e x . J . Ph y s i o l o g y 291, 51-54. Cowan, W.M., G o t t l i e b , D.I., Hendrickson, A.E., P r i c e , J.L., and Woolsey, T.A. 1972. The a u t o r a d i o g r a p h i c demonstration of axonal connections i n the c e n t r a l nervous system. B r a i n Res. 37, 21-51. Coyle, J.T. E x c i t o t o x i n s . 1987. In: Psychopharmacology: the T h i r d Generation of Progress. Ed: M e l t z e r , H.Y. Raven P r e s s , N.Y. Coyle, J.T., B l a k e l y , R., Zacseck, R., K o l l e r , K.J., Abreu, M., O r y - L a v o l l e e , L., F i s c h e r , R., f f r e n c h - M u l l e n , J.M.H., and Carpenter, D.O. 1986. A c i d i c P e p t i d e s i n B r a i n : Do they a c t a t p u t a t i v e g l u t a m a t e r g i c synapses? Adv. Exper. Med. B i o l . 203, 375-385. Cuatrecasas, P., and Hollenberg, M.D. 1976. Membrane r e c e p t o r s and hormone a c t i o n . Adv. P r o t e i n Chem. 30, 251-451. Cynader, M., Shaw, C., Prusky, G., and Van Huisen, F. 1990. Ne u r a l mechanisms u n d e r l y i n g m o d i f y a b i l i t y of response p r o p e r t i e s i n de v e l o p i n g c a t v i s u a l c o r t e x . In: V i s i o n and the B r a i n . Ed: Cohen, B., and Bodis-Wollner, I. Raven Press, New York. Danks, J.A., Rothman, R.B., C a s c i e r i , M.A., C h i c c h i , G.G., L i a n g , T., and Herkenham, M. 1986. A comparative a u t o r a d i o g r a p h i c study of the d i s t r i b u t i o n s of substance P and e l e d o i s i n b i n d i n g s i t e s i n r a t b r a i n . B r a i n Res. 385, 273-281. Diamond, I.T., and H a l l , W.C. 1969. E v o l u t i o n of neocortex. S c i e n c e 164, 251-262. 133 Dudek, S.M., and Bear, M.F. 1989. A b i o c h e m i c a l c o r r e l a t e of the c r i t i c a l p e r i o d f o r s y n a p t i c m o d i f i c a t i o n i n the v i s u l c o r t e x . Science 246, 673-675. Dumuis, A., P i n , J.P., Oomagari, K., Sebben, M., Bockaert, J . 1990. Aracadonic a c i d r e l e a s e d from s t r i a t a l neurons by j o i n t s t i m u l a t i o n of i o n o t r o p i c and metabotropic q u i s q u a l a t e r e c e p t o r s . Nature, 347, 182-184. Fagg, G.E., Baud, J , H a l l , R., and Dingwall, J . 1988. Do a g o n i s t s and c o m p e t i t i v e a n t a g o n i s t s b i n d t o d i s t i n c t s i t e s on the NMDA re c e p t o r ? In: F r o n t i e r s i n E x c i t a t o r y Amino A c i d Research: Proceedings of an I n t e r n a t i o n a l Symposium, " E x c i t a t o r y A c i d s 1988", h e l d i n Manaus, Amazonas, B r a z i l , March 28 - A p r i l 2, 1988. Eds: C a v a l h i e r o , E.A., Lehmann, J . , T u r s k i , L. Manaus, B r a z i l . Fagg, G.E., Mena, E.E.,Monaghan, D.T., and Cotman, C.W. 1983. F r e e z i n g e l i m i n a t e s a s p e c i f i c p o p u l a t i o n of L-glutamate r e c e p t o r s i n s y n a p t i c membranes. N e u r o s c i . L e t t . 38, 157-162. Ferkany, J.W. and Coyle, J.T. 1983. S p e c i f i c b i n d i n g of [ 3H]2-amino-7-phosophononeptanoic a c i d t o r a t b r a i n membranes i n v i t r o . L i f e S c i . 33, 1295-1305. f f r e n c h - M u l l e n , J.M.H., K o l l e r , K., Zaczek, R., Coyle, J.T., H o r i , N., and Carpenter, D.O. 1985. N - A c e t y l a s p a r t y l -glutamate: P o s s i b l e r o l e as the n e u r o t r a n s m i t t e r of the l a t e r a l o l f a c t o r y t r a c t . Proc. N a t l . Acad. S c i . USA 82, 3897-3900. Flohe, L., Loschen, G., Gunzler, W.A., and E i c h e l e , E. 1972. G l u t a t h i o n e peroxidase. V. The k i n e t i c mechanism. Z. P h y s i o l . Chem. 353, 987-999. Fosse, V. M., and Fonnum, F. 1987. B i o c h e m i c a l evidence f o r glutamate and/or a s p a r t a t e as n e u r o t r a n s m i t t e r s i n f i b r e s from the v i s u a l c o r t e x t o the l a t e r a l p o s t e r i o r nucleus ( p u l v i n a r ) i n r a t s . B r a i n Res. 400, 219-24. Fosse, V. M. , Heggelund, P., Iversen, E., and Fonnum, F. 1984. E f f e c t s of area 17 a b l a t i o n on n e u r o t r a n s m i t t e r parameters i n e f f e r e n t s t o area 18, l a t e r a l g e n i c u l a t e nucleus, p u l v i n a r , and s u p e r i o r c o l l i c u l u s i n the c a t . N e u r o s c i . L e t t . 52, 323-328. F o s t e r , A.C., and Fagg, G.E. 1984. A c i d i c amino a c i d b i n d i n g s i t e s i n mammalian neuronal membranes: t h e i r c h a r a c t e r i s t i c s and r e l a t i o n s h i p t o s y n a p t i c r e c e p t o r s . B r a i n Res. Rev. 7, 103-164. 134 F o s t e r . A . C , and Fagg, G.E. 1987. Comparison of L-[ 3H]glutamate, D - [ 3 H ] a s p a r t a t e , DL-[ 3H]AP5, and [ H]NMDA as l i g a n d s f o r NMDA r e c e p t o r s i n crude p o s t s y n a p t i c d e n s i t i e s from r a t b r a i n . Euro. J . Pharmacol. 133, 291-300. Galper, J.B., K l e i n , W., and C a t t e r a l l , W.A. 1977. M u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r s i n de v e l o p i n g c h i c k h e a r t . J . B i o l . Chem. 252, 8692-8699. G i l b e r t , C D . 1983. M i c r o c i r c u i t r y of the v i s u a l c o r t e x . Ann. Rev. N e u r o s c i . 6, 217-247. G r i f f i t h s , R. 1990. Cy s t e i n e s u l p h i n a t e (CSA) as an e x c i t a t o r y amino a c i d t r a n m s i t t e r c a n d i d a t e i n the mammalian c e n t r a l nervous system. Prog. N e u o r o b i o l . 35, 313-323. Guo, N., Mcintosh, C , and Shaw, C. 1991. GSH: new ca n d i d a t e neurohormone or n e u r o t r a n s m i t t e r i n the CNS. IBRO World Congress of Neuroscience, i n p r e s s . Guo, N., and Shaw, C. 1991. The c o l l o i d a l g o l d t e c h n i q u e f o r the c e l l u l a r l o c a l i z a t i o n of GSH r e c e p t o r s . S o c i e t y N e u r o s c i . A b s t r a c t s 17, i n p r e s s . Habig, W.H., Pabst, M.J., and Jakoby, W.B. 1974. G l u t a t h i o n e S - t r a n s f e r a s e s : the f i r s t enzymatic s t e p i n m e r c a p t u r i c a c i d f o r m a t i o n . J . B i o l . Chem. 249, 7130-7139. Hagihara, K., Tsumoto, T., Sato, H., and Hata, Y. 1988. A c t i o n s of e x c i t a t o r y amino a c i d a n t a g o n i s t s on g e n i c u l o c o r t i c a l t r a n s m i s s i o n i n the c a t l 9 s v i s u a l c o r t e x . Exp. B r a i n Res. 69, 407-416. Hahm, J.-O., Langdon, R.B., and Sur, M. 1991. D i s r u p t i o n of r e t i n o g e n i c u l a t e a f f e r e n t s e g r e g a t i o n by a n t a g o n i s t s t o NMDA r e c e p t o r s . Nature 351, 568-570. Hannuniemi, R, and Oja, S.S. 1981. Uptake of l e u c i n e , l y s i n e , a s p a r t i c a c i d , and g l y c i n e i n t o i s o l a t e d neurons and a s t o c y t e s . Neurochem. Res. 6, 873-884. Hendrickson, A.E. 1972. E l e c t r o n m i c r o s c o p i c d i s t r i b u t i o n of axoplasmic t r a n s p o r t . J . Comp. Neur. 144, 381-398. Hendrickson, A.E., Wagoner, N., and Cowan, W.M. 1972. An a u t o r a d i o g r a p h i c and e l e c t r o n m i c r o s c o p i c study o f re t i n o - h y p o t h a l a m i c connections. Z. Z e l l f o r s c h 13 5, 1-26. Herkenham, M. 1987. Mismatches between n e u r o t r a n s m i t t e r and r e c e p t o r l o c a l i z a t i o n s i n b r a i n : o b s e r v a t i o n s and ' i m p l i c a t i o n s . Neuroscience 23, 1-38. 135 H i l l , K.E., Von Hoff, D.D., and Burk, R.F. 1985. The e f f e c t of i n h i b i t i o n of gamma-glutamyl t r a n s p e p t i d a s e by AT-125 ( a c i v i c i n ) on g l u t a t h i o n e and c y s t e i n e l e v e l s i n r a t b r a i n and plasma. I n v e s t i g a t i o n a l New Drugs 3, 31-34. H o l l e n b e r g , M.D. 1985. Examples of ho m o s p e c i f i c and h e t e r o s p e c i f i c r e c e p t o r r e g u l a t i o n . Trends Pharmacol. S c i . 7, 242-245. Holopainen, I., Louve, M., E n k v i s t , M.O., and Akerman, K.E. 1990. C o u p l i n g of g l u t a m a t e r g i c r e c e p t o r s t o changes i n i n t r a c e l l u l a r C a 2 + i n r a t c e r e b e l l a r g r a n u l e c e l l s i n primary c u l t u r e . J . N e u r o s c i . Res. 25, 187-193. Hood, W.F., Compton, R.P., and Monahan, J.B. 1990. N-methyl-D-a s p a r t a t e r e c o g n i t i o n s i t e l i g a n d s modulate a c t i v i t y a t the coupled g l y c i n e r e c o g n i t i o n s i t e . J . Neurochem. 54, 1040-1046. Hubel, D.H., and Weisel, T.N. 1970. The p e r i o d of s u s c e p t i b i l i t y t o the p h y s i o l o g i c a l e f f e c t s of u n i l a t e r a l e y e l i d c l o s u r e i n k i t t e n s . J . P h y s i o l (Lond) 206, 419-436. Hunston, D.L. 1975. Two techniques f o r e v a l u a t i n g s m a l l molecule-macromolecule b i n d i n g i n complex systems. A n a l y t i c a l Biochem. 63, 99-109. Hwang, S-M., and Segal, S. 1979. Development and o t h e r a s p e c t s of [ 3 S ] c y s t e i n e t r a n s p o r t by r a t b r a i n synaptosomes. J . Neurochem. 33, 1303-1308. l i n o , M., Ozawa, S., and Tsusuki, K. 1990. Permeation of c a l c i u m through e x c i t a t o r y amino a c i d r e c e p t o r channels i n c u l t u r e d r a t hippocampal neurones. J . P h y s i o l . 424, 151-165. Ive r s e n , L.L. 1971. Role of t r a n s m i t t e r uptake mechanisms i n s y n a p t i c n e u r o t r a n s m i s s i o n . Br. J . Pharmac. 41, 571-591. Iwata, H., Yamagami, S., and Baba, A. 1982a. C y s t e i n e s u l f i n i c a c i d , i n the c e n t r a l nervous system: s p e c i f i c b i n d i n g of [ S ] c y s t e i c a c i d t o c o r t i c a l s y n a p t i c membranes — an i n v e s t i g a t o n of p o s s i b l e b i n d i n g s i t e s f o r c y s t e i n e s u l f i n i c a c i d . J . Neurochem. 38, 1275-1279. Iwata, H., Yamagami, S., Mizuo, H., and Baba, A. 1982b. C y s t e i n e s u l f i n i c a c i d i n the c e n t r a l nervous system: uptake and r e l e a s e of c y s t e i n e s u l f i n i c a c i d by a r a t b r a i n p r e p a r a t i o n . J . Neurochem. 38, 1268-1274. Jahr, C.E., and Stevens, C F . 1987. Glutamate a c t i v a t e s m u l t i p l e s i n g l e channel conductances i n hippocampal neurons. Nature 325, 522-525. 136 J a i n , A. , Martensson, J . , S t o l e , E. , Aula., P.A.M. , and M e i s t e r , A. 1991. G l u t a t h i o n e d e f i c i e n c y l e a d s t o m i t o c h o n d r i a l damage i n b r a i n . Proc. N a t l . Acad. S c i . 88, 1913-1917. J e s s e l , T.M., and Womack, M.D. 1985. Substance P and the n o v e l mammalian t a c h y k i n i n s : a d i v e r s i t y of r e c e p t o r s and c e l l u l a r a c t i o n s . Trends N e u r o s c i . 8, 43-45. Johnston, G.A.R., and Iversen, L.L. 1971. G l y c i n e uptake i n r a t c e n t r a l nervous system s l i c e s and homogenates: evidence f o r d i f f e r e n t uptake systems i n s p i n a l c o r d and c e r e b r a l c o r t e x . J . Neurochem. 18, 1951-1961. Kaczmarek, J.K., and L e v i t a n , I.B. 1987. Neuromodulaton: The b i o c h e m i c a l c o n t r o l of neuronal e x c i t a b i l i t y . Oxford U n i v e r s i t y Press, New York. Kannan, R., Kuhlenkamp, J.F., J e a n d i d i e r , E., T r i n h , H., Ookhtens, M., and Kaplowitz, N. 1990. Evidence f o r c a r r i e r - m e d i a t e d t r a n s p o r t of g l u t a t h i o n e a c r o s s the b l o o d - b r a i n b a r r i e r i n the r a t . J . C l i n . I n v e s t . 85, 2009-2013. Kano, M. and Kato, M. 1987. Quisq u a l a t e r e c e p t o r s are s p e c i f i c a l l y i n v o l v e d i n c e r e b e l l a r s y n a p t i c p l a s t i c i t y . Nature 325, 276-279. Kato, N., Kawaguchi, S., Yamamoto, T., Samejima, A., Miyata, H. 1983. P o s t n a t a l development of the g e n i c u l o c o r t i c a l p r o j e c t i o n i n the c a t : e l e c t r o p h y s i o l o g i c a l and m o r p h o l o g i c a l s t u d i e s . Exp. B r a i n Res. 51, 65-72. K e l l e r , H.J., Quan Do, Q., Z o l l i n g e r , M., W i n t e r h a l t e r , K.H., and Cuenod, M. 1989. C y s t e i n e : D e p o l a r i z a t i o n - i n d u c e d r e l e a s e from r a t b r a i n i n v i t r o . J . Neurochem. 52, 1801-1806. Kimelberg, H.K., ed. 1988. G l i a l C e l l Receptors. Raven Press, New York. Kozak, E.M. and Tate, S.S. 1982. G l u t a t h i o n e - d e g r a d i n g enzymes of m i c r o v i l l u s membranes. J . B i o l . Chem. 257, 6322-6327. K r n j e v i c , K., and P h i l l i s , J.W. 1963. I o n t o p h o r e t i c s t u d i e s of neurones i n the mammalian c e r e b r a l c o r t e x . J . P h y s i o l . 165, 274-304. K r n j e v i c , K, and Reinhardt, W. 1979. C h o l i n e e x c i t e s c o r t i c a l neurons. Science 296, 1321-1323. 137 L a j t h a , A., and Sershen, H. 1975. I n h i b i t i o n of amino a c i d uptake by the absence of Na + i n s l i c e s of b r a i n . J . Neurochem. 24, 667-672. Lawson, R. 1986. T r a c e r techniques and n u c l e a r medicine. In: Mathematical Methods i n Medicine I: S t a t i s t i c a l and A n a l y t i c a l Techniques. Eds: Ingram, D., and Block, R.F. W i l e y - I n t e r s c i e n c e , N.Y. Legay, F., Weise, V.K., O e r t e l , W.H., Tappaz, M.L. 1987. T a u r i n e b i o s y n t h e s i s i n b r a i n : a new s p e c i f i c and s e n s i t i v e microassay of c y s t e i n e s u l f i n a t e d e c a r b o x y l a s e (CSDI) a c t i v i t y through s e l e c t i v e immunotapping and i t s use f o r d i s t r i b u t i o n s t u d i e s . J . Neurochem. 48, 345-351. L i , X.H. and Jope, R.S. 1989. I n h i b i t i o n of r e c e p t o r - c o u p l e d p h o s p h o i n o s i t i d e h y d r o l y s i s by s u l f u r - c o n t a i n i n g amino a c i d s i n r a t b r a i n s l i c e s . Biochem. Pharmacol. 38, 2781-2787. Lieberman, E.M., Abbott, N.J., and Hassan, S. 1989. Evidence t h a t glutamate mediates axon t o Schwan c e l l s i g n a l l i n g i n the s q u i d . G l i a 2, 94-102. L i m b r i d , L.E. 1986. I d e n t i f i c a t i o n of n e u r o t r a n s m i t t e r r e c e p t o r s u s i n g r a d i o l i g a n d b i n d i n g t e c h n i q u e s . In: Chemical and F u n c t i o n a l Assays of Receptor B i n d i n g : Short Course 1 S y l l a b u s . Soc. f o r N e u r o s c i . Wash, D.C. p 3-31. L i n , C-S, N i c o l e l i s , M.A., Schneider, J.S., and Chapin, J.K. 1990. A major d i r e c t GABAergic pathway from zona i n c e r t a t o neocortex. Science 248, 1553-1556. L i p t o n , S.A., and Kater, S.B. 1989. N e u r o t r a n s m i t t e r r e g u l a t i o n of neuronal outgrowth, p l a s t i c i t y , and s u r v i v a l . Trends N e u r o s c i . 12, 265-270. Lowry, O.H., Rosebrough, N.J., F a r r , A.L., and R a n d a l l , R.J. 1951. P r o t e i n measurement with the F o l i n phenol reagent. J . B i o l . Chem. 193, 265-275. L u i n i , A., T a l , N., Goldberg, 0., Teic h b e r g , V . I . 1984. An e v a l u a t i o n of s e l e c t e d b r a i n c o n s t i t u e n t s as p u t a t i v e e x c i t a t o r y n e u r o t r a n s m i t t e r s . B r a i n Res. 324, 271-277. Lund, J.S. 1973. Organizaton of neurons i n the v i s u a l c o r t e x , area 17, of the monkey (Macaca m u l a t t a ) . J . Comp. Neu r o l . 147, 455-496. Lund-Karlsen, R., and Fonnum, F. 1978. Evidence f o r glutamate as a n e u r o t r a n s m i t t e r i n the c o r t i c o f u g a l f i b r e s t o the d o r s a l l a t e r a l g e n i c u l a t e body and the s u p e r i o r . c o l l i c u l u s i n r a t s . B r a i n Res. 151, 457-467. 138 Lundberg, J.M., and H o k f e l t . 1983. Coexistence of p e p t i d e s and c l a s s i c a l n e u r o t r a n s m i t t e r s . Trends N e u r o s c i . 6, 325-333. Malthe-Sorenssen, D., Skrede, K.K., and Fonnum, F. 1979. Calcium-dependent r e l e a s e of D - [ 3 H ] a s p a r t a t e evoked by s e l e c t i v e e l e c t r i c a l s t i m u l a t i o n of e x c i t a t o r y a f f e r e n t f i b r e s t o hippocampal pyramidal c e l l s i n v i t r o . N e u r o s c i . 4, 1255-1263 Mantyh, P.W., Gates, T., Mantyh, L.R., Maggio, J.E. 1989. A u t o r a d i o g r a p h i c l o c a l i z a t i o n and c h a r a c t e r i z a t i o n of t a c h y k i n i n r e c e p t o r b i n d i n g s i t e s i n the r a t b r a i n and p e r i p h e r a l t i s s u e s . J . N e u r o s c i . 9, 258-279. Mantyh, P.W., Hunt, S.P., and Maggio, J.E. 1984. Substance P r e c e p t o r s : l o c a l i z a t i o n by l i g h t m i c r o s c o p i c autoradiography i n r a t b r a i n u s i n g [ 3H]SP as the r a d i o l i g a n d . B r a i n Res. 307, 147-165. March, D., and Shaw, C. 1990. Is the g e n i c u l o s t r i a t e t r a n s m i t t e r a t a c h y k i n i n ? The Anatomical Record 1990: A b s t r a c t s of the American Assn. of Anatomists 266, 65A. Martensson, J . , L a i , J.C.K., and M e i s t e r , A. 1990. High-a f f i n i t y t r a n s p o r t of g l u t a t h i o n e i s p a r t of a multicomponent system e s s e n t i a l f o r m i t o c h o n d r i a l f u n c t i o n . Proc. N a t l . Acad. S c i . USA 87, 7185-7189. Mayer, M.L., and Westbrook, G.L. 1987. The p h y s i o l o g y of e x c i t a t o r y amino a c i d s i n the v e r t e b r a t e c e n t r a l nervous system. Progress N e u r o b i o l . 28, 197-276. Marrero, H., A s t i o n , M.L., Coles, J.A., and Orkand, R.K. 1989. Nature 339, 378-380. McGeer, P.L., E c c l e s , J.C, and McGeer, E.G. 1987. M o l e c u l a r Neurobiology of the Mamalian Brain,, Second E d i t i o n . Plenum Press, N.Y. McGonigle, P., and M o l i n o f f , P.B. 1989. Q u a n t i t a t i v e a s p e c t s of d r u g - r e c e p t o r i n t e r a c t i o n s . In: B a s i c Neurochemistry: M o l e c u l a r , C e l l u l a r , and M e d i c a l Aspects, 4th Ed. Ed: S i e g e l , G.J.. RAven Press, N.Y. McNaughton, B.L., and M o r r i s , R.G.M. 1987. Hippocampal s y n a p t i c enhancement and i n f o r m a t i o n s t o r a g e w i t h i n a d i s t r i b u t e d memory system. Trends N e u r o s c i . 10, 408-415 M e i s t e r , A. 1983. Metabolism and t r a n s p o r t of g l u t a t h i o n e and o t h e r gamma-glutamyl compounds. In: F u n c t i o n s of G l u t a t h i o n e : Biochemical, P h y s i o l o g i c a l , T o x i c o l o g i c a l , and C l i n i c a l Aspects. Ed: Larsson, A. Raven P r e s s , N.Y. 139 M e i s t e r , A. 1989. A b r i e f h i s t o r y of g l u t a t h i o n e and a survey of i t s metabolism and f u n c t i o n s . In: G l u t a t h i o n e : Chemical, B i o c h e m i c a l , and Med i c a l Aspects, P a r t A. Ed: Dolph i n , D., Avramovic, 0., and Poulson, R. W i l e y -I n t e r s c i e n c e , N.Y. M i s r a , C.H. 1983. In v i t r o study of c y s t e i n e o xidase i n r a t b r a i n . Neurochem. Res. 8, 1497-1508 M i y o s h i , R., K i t o , S., Doudou, N., Nomoto, T. 1990. Age-r e l a t e d changes of s t r y c h n i n e - i n s e n s i t i v e g l y c i n c e r e c e p t o r s i n r a t b r a i n as s t u d i e d by i n v i t r o a utoradiography. Synapse 6, 338-343. M o f f e t t , J.R., Williamson, L.C., Neale, J.H., P a l k o v i t s , M., and Namboodiri, M.A.A. 1991. E f f e c t of o p t i c nerve t r a n s e c t i o n on N - a c e t y l a s p a r t y l g l u t a m a t e immunoreactivity i n t he primary and accessory o p t i c p r o j e c t i o n systems i n the r a t . B r a i n Res. 538, 89-94. Monaghan, D.T., Bridges, R.J., and Cotman, CW. 1989. The e x c i t a t o r y amino a c i d r e c e p t o r s : t h e i r c l a s s e s , pharmacology, and d i s t i n c t p r o p e r t i e s i n the f u n c t i o n o f the c e n t r a l nervous system. Annu. Rev. Pharmacol. T o x i c o l . 29, 365-402. Monaghan, D.T., and Cotman, CW. 1982. D i s t r i b u t i o n of 3H-k a i n i c a c i d b i n d i n g s i t e s i n r a t CNS as determined by autoradiography. B r a i n Res. 252, 91-100. Monaghan, D.T., and Cotman, CW. 1985. D i s t r i b u t i o n of NMDA-s e n s i t i v e L- 3H-glutamate b i n d i n g s i t e s i n r a t b r a i n as determined by q u a n t i t a t i v e autoradiography. J . N e u r o s c i . 5, 2909-2919. Monagham, D.T., Olverman, H.J., Nguyen, L., Watkins, J . C , and Cotman, CW. 1988. Two c l a s s e s of N-methyl-D-aspartate r e c o g n i t i o n s i t e s : d i f f e r e n t i a l d i s t r i b u t i o n and d i f f e r e n t i a l r e g u l a t i o n by g l y c i n e . Proc. N a t l . Acad. S c i . U.S.A. 85, 9836-9840. Monaghan. D.T., Yao, D., and Cotman, CW. 1984. D i s t r i b u t i o n of H-AMPA b i n d i n g s i t e s i n r a t b r a i n as determined by q u a n t i t a t i v e autoradiography. B r a i n Res. 324, 160-164. Moran, J . , and P a t e l , A.J. 1989. S t i m u l a t i o n of the NMDA r e c e p t o r promotes 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 of c e r e b e l l a r granule neurons and not a s t r o c y t e s . B r a i n Res. 46, 97-105. Morgan, J . I . , and Curran, T. 1989. S t i m u l u s - t r a n s c r i p t i o n c o u p l i n g i n neurons: r o l e of c e l l u l a r immediate-early genes. Trends N e u r o s c i . 12,459-462. 140 N i c o l l , R.A. 1988. Coupling of n e u r o t r a n s m i t t e r r e c e p t o r s t o i o n channels i n the b r a i n . Science 241, 545-551. Nobel, E.P., S i n c i n i , E., Bergman, D., and Bruggencate, G. 1989. E x c i t a t o r y amino a c i d s i n h i b i t s t i m u l a t e d p h o s p h o i n o s i t i d e h y d r o l y s i s i n the r a t p r e f r o n t a l c o r t e x . L i f e S c i ence 44, 19-26. Ochs, S., Johnson, J . , and Ng, M.-H. 1967. P r o t e i n s i n c o r p o r a t i o n and axoplasmic flow i n motoneuron f i b r e s f o l l o w i n g i n t r a - c o r d i n j e c t i o n s of l a b e l l e d l e u c i n e . J . Neurochem. 14, 317-331. O g i t a , K. , K i t a g o , T., Nakamuta, H., Fukuda, Y., Koida, M., Ogawa, Y., and Yoneda, Y. 1986a. G l u t a t h i o n e - i n d u c e d i n h i b i t i o n of Na-independent and -dependent b i n d i n g s of L - [ 3 H ] - g l u t a m a t e i n r a t b r a i n . L i f e S c i . 39, 2411-2418. O g i t a , K., K i t a g o , T., and Yoneda, Y. 1986b. P o s s i b l e involvement of g l u t a t h i o n e i n c e n t r a l g l u t a m i n e r g i c n e u r o t r a n s m i s s i o n . In Contemporary Themes i n B i o c h e m i s t r y , Proceedings of the Fourth FAOB Congress, Singapore, 580-581. O g i t a , K., and Yoneda, Y. 1986. D i f f e r e n t i a t i o n of the C a 2 + -s t i m u l a t e d b i n d i n g from the Cl~-dependent b i n d i n g of [ H]glutamate i n s y n a p t i c membranes from r a t b r a i n . N e u r o s c i . Res. 4, 129-142. O g i t a , K., and Yoneda, Y. 1987. P o s s i b l e presence of [ 3 H ] g l u t a t h i o n e (GSH) b i n d i n g s i t e s i n s y n a p t i c membranes from r a t b r a i n . N e u r o s c i . Res. 4, 486-496. O g i t a , K., and Yoneda, Y. 1988. Temperature-dependent and -independent apparent b i n d i n g a c t i v i t i e s of [ 3 H ] g l u t a t h i o n e i n b r a i n s y n a p t i c membranes. B r a i n Res. 463, 37-46. O g i t a , K., and Yoneda, Y. 1989. S e l e c t i v e p o t e n t i a t i o n by L-c y s t e i n e of apparent b i n d i n g a c t i v i t y of [ H ] g l u t a t h i o n e i n s y n a p t i c membranes of r a t b r a i n . Biochem. Pharmacol. 38, 1499-1505. O g i t a , K., and Yoneda, Y. 1990. Temperature-independent b i n d i n g of [ 3 H ] - 3 - ( 2 - c a r b o x y p i p e r a z i n - 4 - y l ) p r o p y l - 1 -phosphonic a c i d i n b r a i n s y n a p t i c membranes t r e a t e d w i t h T r i t o n X-100. B r a i n Res. 515, 51-56. 141 Oja, S.S., Varga, V., Janaky, R., Kontro, P., A a r n i o , T., and Marnela, K.-M. 1988. G l u t a t h i o n e and g l u t a m a t e r g i c n e u r o t r a n s m i s s i o n i n the b r a i n . F r o n t i e r s i n E x c i t a t o r y Amino A c i d Research: Proceedings of an I n t e r n a t i o n a l Symposium, " E x c i t a t o r y A c i d s 1988", h e l d i n Manaus, Amazonas, B r a z i l , March 28 - A p r i l 2, 1988. Eds: C a v a l h i e r o , E.A., Lehmann, J . , T u r s k i , L. Manaus, B r a z i l . O l i v e r , M.W., K e s s l e r , M., Larson, J . , S c h o t t l e r , F., and Lynch, G. 1990. G l y c i n e s i t e a s s o c i a t e d w i t h the NMDA r e c e p t o r modulates long-term p o t e n t i a t i o n . Synapse 5, 265-270. Olney, J.W., Ho, O.L., and Rhee, V. 1971. C y t o t o x i c e f f e c t s of a c i d i c and sulphur c o n t a i n i n g amino a c i d s on the i n f a n t mouse c e n t r a l nervous system. Exp. B r a i n Res. 14, 61-76. Or l o w s k i , M. and Karkowsky, A. 1976. G l u t a t h i o n e metabolism and some p o s s i b l e f u n c t i o n s of g l u t a t h i o n e i n the nervous system. I n t . Rev. N e u r o b i o l . 19, 75-121. Paxinos, G., and Watson, C. 1986. The Rat B r a i n i n S t e r e o t a x i c C o o r d i n a t e s , 2nd E d i t i o n . Acadamic Press, Sydney, Orlando. Payne, B., Pearson, H., Cornwell, P. 1988. Development of v i s u a l and a u d i t o r y c o r t i c a l c onnections i n c a t . In: C e r e b r a l Cortex Volume 7: Development and M a t u r a t i o n of C e r e b r a l Cortex. Ed: P e t e r s , A., and Jones, E.G. Plenum Pre s s , N.Y. P h i l b e r t , M.A., Beiswanger, CM., Waters, D.K., Reuhl, K.R., and Lowndes, H.E. 1991. T o x i c o l . Appl. Pharmacol. 107, 215-227 P i n , J.-P., Rumigny, J.-F., Bockaert, J . , and Recasens, M. 1987. M u l t i p l e Cl-independent b i n d i n g s i t e s f o r the e x c i t a t o r y amino a c i d s : glutamate, a s p a r t a t e and c y s t e i n e s u l f i n a t e i n r a t b r a i n membranes. B r a i n Res. 402, 11-20. Prusky, G.T., Shaw, C , and Cynader, M.S. 1987. N i c o t i n e r e c e p t o r s are l o c a t e d on l a t e r a l g e n i c u l a t e n u c l e u s t e r m i n a l s i n c a t v i s u a l c o r t e x . B r a i n Res. 412, 131-138. P u l l a n , L.M., Olney, J.W., P r i c e , M.T., Compton, R.P., Hood, W.F., M i c h e l , J . , and Monahan, J.B. 1987. E x c i t a t o r y amino a c i d r e c e p t o r potency and s u b c l a s s s p e c i f i c i t y of s u l f u r - c o n t a i n i n g amino a c i d s . J . Neurochem. 49, 1301-1307. 142 Raps, S.P., L a i , J.C.K., Hertz, L., Cooper, A.J.L. 1989. G l u t a t h i o n e i s p r e s e n t i n h i g h c o n c e n t r a t i o n s i n c u l t u r e d a s t r o c y t e s but not i n c u l t u r e d neurons. B r a i n Res. 493, 398-401. R a s s i n , D.K., and G a u l l , G.E. 1975. S u b c e l l u l a r d i s t r i b u t i o n of enzymes of t r a n s m e t h y l a t i o n and t r a n s u l p h u r a t i o n i n r a t b r a i n . J . Neurochem. 24, 969-978. R e i c h e l t , K.L., and Fonnum, F. 1969. S u b c e l l u l a r l o c a l i z a t i o n o f N - a c e t y l - a s p a r t y l - g l u t a m a t e , N-acetyl-glutamate and g l u t a t h i o n e i n b r a i n . J . Neurochem. 16, 1409-1416. R e i t e r , H.O. and S t r y k e r , M.P. 1988. Neural p l a s t i c i t y without p o s t s y n a p t i c a c t i o n p o t e n t i a l s : Less a c t i v e i n p u t s become dominant when k i t t e n v i s u a l c o r t i c a l c e l l s are p h a r m a c o l o g i c a l l y i n h i b i t e d . Proc. N a t l . Acad. S c i . USA 85, 36-23-3627. Robinson, M., B l a k e l y , R.D., Couto, R., and Coyle, J.T. 1987. H y d r o l y s i s of the b r a i n d i p e p t i d e N - a c e t y l - L - a s p a r t y l - L -glutamate. J . B i o l . Chem. 262, 14498-14506. Robinson, M.B., B l a k e l y , R.D., Cuoto, R., and Coyle, J.T. 1987. H y d r o l y s i s of the b r a i n d i p e p t i d e N - a c e t y l - L -a s p a r t y l - L - g l u t a m a t e . J . B i o l . Chem. 262, 14498-14506. Rogers, A.W. 1979. Techniques of Autoradiography, T h i r d E d i t i o n , E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l P r e s s , N.Y. Rosenthal, H.E. 1967. A g r a p h i c method f o r the d e t e r m i n a t i o n and p r e s e n t a t i o n of b i n d i n g parameters i n a complex system. ' A n a l . Biochem. 20, 525-532. Sah, P., H e s t r i n , S., and N i c o l l , R.A. 1989. T o n i c a c t i v a t i o n of NMDA r e c e p t o r s by ambient glutamate enhances e x c i t a b i l i t y of neurons. Science 246, 815-818. Schoepp, D.D., and Johnson, B.G. 1989. I n h i b i t i o n of e x c i t a t o r y amino a c i d - s t i m u l a t e d p h o s p h o i n o s i t o l h y d r o l y s i s i n the neonatal r a t hippocampus by 2-amino-3-phosphonopropionate. J . Neurochem. 53, 1865-1870.. Schonbach, J . , Schonback, CH., and Cuenod, M. 1971. Rapic phase of axoplasmic flow and s y n a p t i c p r o t e i n s : an e l e c t r o n m i c r o s c o p i c a l a u t o r a d i o g r a p h i c study. J . Comp. Neur. 141, 485-498. S e f t o n , A.J., and Dreher, B. 1985. 6. V i s u a l System. In: The Rat Nervous System Volume 1 F p r e b r a i n and m i d b r a i n . Ed: Paxinos, G. Academic Press, A u s t r a l i a . 143 Sejno, M., I s h i b a s h i , T., Terashima, T., and Inoue, Y. 1986. S u c c e s s i v e appearance of g l u t a t h i o n e S - t r a n s f e r a s e -p o s i t i v e c e l l s i n dev e l o p i n g r a t b r a i n : c h o r o i d p l e x u s , p i a mater, v e n t r i c u l a r zone, and a s t r o c y t e s . Neuroscience L e t t e r s 66, 131-134. Senut, M.C., DeBilbao, F., and Lamour, Y. 1990. M e d i a l s e p t a l neurons c o n t a i n i n g N - a c e t y l a s p a r t y l g l u t a m a t e - l i k e immunoreactivity p r o j e c t t o the hippocampal f o r m a t i o n i n the r a t . N e u r o s c i . L e t t . 113, 12-16. Ser v a n - S c h r e i b e r , D., P r i n t z , H., and Cohen, J.D. 1990. A network .model of catecholamine e f f e c t s : g a i n , s i g n a l - t o -n o i s e r a t i o , and behavior. Science 249, 892-895. Shaw, C., Cameron, L., Prusky, G., Dyck, R., Cynader, M., and Hendrickson, A. 1989a. Pre- and p o s t n a t a l development of n e u r o t r a n s m i t t e r r e c e p t o r s i n monkey v i s u a l c o r t e x . S o c i e t y N e u r o s c i . A b s t r a c t s 15, 13 36. Shaw, C , and Cynader, M. 1986. Laminar d i s t r i b u t i o n of r e c e p t o r s i n monkey (Macaca f a s c i c u l a r i s ) g e n i c u l o s t r i a t e system. J . Comp. Neurol. 248, 301-312. Shaw, C., and Cynader, M. 1988. U n i l a t e r a l e y e l i d s u t u r e i n c r e a s e s GABA^ r e c e p t o r s i n c a t v i s u a c o r t e x . Dev. B r a i n Res. 40, 148-153. Shaw, C., Hendrickson, A., and E r i c k s o n , A. 1991. P r e - and p o s t n a t a l development of glutamate e x c i t a t o r y amino a c i d r e c e p t o r s i n monkey s t r i a t e c o r t e x . S o c i e t y N e u r o s c i . A b s t r a c t s 17, i n p r e s s . Shaw, C , Korn, J . , Hendrickson, A. 1990. L o c a l i z a t i o n of q u i s q u a l a t e r e c e p t o r s i n mammalian v i s u a l c o r t e x u s i n g NEN [ 3H]CNQX. Du Pont B i o t e c h Update 5, 95. Shaw, C., Prusky, G., Van Huizen, F., and Cynader, M. 1989b. D i f f e r e n t i a l e f f e c t s of q u i n o l i n i c a c i d l e s i o n s on m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r s i n c a t v i s u a l c o r t e x d u r i n g p o s t n a t a l development. B r a i n Res. B u l l . 22, 771-776. Shaw, C., W i l k i n s o n , M., Cynader, M., Needier, M., A o k i , C., and H a l l , S. 1986. The laminar d i s t r i b u t i o n s and p o s t n a t a l development of n e u r o t r a n s m i t t e r and neuromodulator r e c e p t o r s i n c a t v i s u a l c o r t e x . B r a i n Res. B u l l e t i n 22, 771-776. Simon, E . J . , H i l l e r , J.M., Groth, J . , and Edelman, I. 1975. F u r t h e r p r o p e r t i e s of s t e r e o s p e c i f i c o p i a t e b i n d i n g s i t e s i n r a t b r a i n : on the nature of the sodium e f f e c t . J . Pharmacol. Exp. Ther. 192, 531-537. /' 144 S l i v k a , A., M y t i l i n e o u , C , and Cohen, G. 1987. H i s t o c h e m i c a l e v a l u a t i o n of g l u t a t h i o n e i n b r a i n . B r a i n Res. 409, 275-284. Sn i d e r , R.S., and Niemer, W.T. 1961. A S t e r e o t a x i c A t l a s o f the Cat B r a i n . U. Chicago Press, Chicago and London. Stone, J . 1983. P a r a l l e l p r o c e s s i n g i n the v i s u a l system: the c l a s s i f i c a t i o n of r e t i n a l g a n g l i o n c e l l s and i t s impact on the neurobiology of v i s i o n . Plenum Pr e s s , N.Y. S t r e i t , P. 1980. S e l e c t i v e r e t r o g r a d e l a b e l i n g i n d i c a t i n g the t r a n s m i t t e r of neuronal pathways. J . Comp. Ne u r o l . 191, 429-463. Swanson, L.W., Cowan, W.M., and Jones, E.G. 1974. An a u t o r a d i o g r a p h i c study of the e f f e r e n t c o n n e c t i o n s of the v e n t r a l l a t e r a l g e n i c u l a t e nucleus i n the a l b i n o r a t and c a t . J . Comp. Neur. 156, 143-164. Swartz, K.J., During, M.J., Freese, A., and B e a l , M.F. 1990. C e r e b r a l s y n t h e s i s and r e l e a s e of kyn u r e n i c a c i d : an endogenous a n t a g o n i s t of e x c i t a t o r y amino a c i d r e c e p t o r s . J . N e u r o s c i . 10, 2965-2673. Takahashi, T. 1985. The o r g a n i z a t i o n of the thalamus of the hooded r a t . J . Comp. Neurol. 231, 281-3 09. Thompson, A.M., Walker, V.E., Flynn, D.M. 1989. G l y c i n e enhances NMDA-receptor mediated s y n a p t i c p o t e n t i a l s i n n e o c o r t i c a l s l i c e s . Nature 338, 422-424. T r u s s e l , L., Th i o , L., Zorumski, C., and F i s c b a c h , G. 1988. Rapid d e s e n s i t i z a t i o n of glutamate r e c e p t o r s i n v e r t e b r a t e c e n t r a l neurons. Proc. N a t l . Acad. S c i . USA 85, 2835-2838. Tsumoto, T. 1990. E x c i t a t o r y amino a c i d t r a n s m i t t e r s and t h e i r r e c e p t o r s i n n e u r a l c i r c u i t s of the c e r e b r a l neocortex. N e u r o s c i . Res. 9, 79-102. Tsumoto, T., Masui, H., Sato, H. 1986. E x c i t a t o r y amino a c i d t r a n s m i t t e r s i n neuronal c i r c u i t s of the c a t v i s u a l c o r t e x . J . Neurophysiol. 55, 469-483. Tusa, R., Palmer, L., and Rosenquist, A. 1981. M u l t i p l e c o r t i c a l v i s u a l areas: v i s u a l f i e l d topography i n the c a t . In C o r t i c a l Sensory O r g a n i z a t i o n , V o l 2, 1-31. U s h i j i m a , K., M i y a z a k i , H., and Morioka, T. 1986. Immunohistochemical l o c a l i z a t i o n of g l u t a t h i o n e p e r o x i d a s e i n the b r a i n of the r a t . R e s u s c i t a t i o n 13, 97-105. 145 Usowicz, M.M., G a l l o , V., and Cull-Candy, S.G. 1989. M u l t i p l e conductance channels i n type-2 c e r e b e l l a r a s t r o c y t e s a c t i v a t e d by e x c i t a t o r y amino a c i d s . Nature, 339, 380-383. V a l i , P.K., and V i j a y a n , E. 1990. G l u t a t h i o n e and gamma-glu t a m y l t r a n s p e p t i d a s e i n the a d u l t female r a t b r a i n a f t e r i n t r a v e n t r i c u l a r i n j e c t i o n of LHRH and so m a t o s t a t i n . Biochem. I n t . 21, 209-217. Van Huizen, F., Str o s b e r g , A.D., and Cynader, M.S. 1988. C e l l u l a r and s u b c e l l u l a r l o c a l i z a t i o n of m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r s d u r i n g p o s t n a t a l development of c a t v i s u a l c o r t e x u s i n g immunocytochemical procedures. Dev. B r a i n Res. 44, 296-301. Varga, V., Janaky, R., Marnela, K.-M., Gulyas, J . , Kontro, P. and Oja, S.S. 1989. Displacement of e x c i t a t o r y amino a c i d r e c e p t o r l i g a n d s by a c i d i c o l i g o p e p t i d e s . Neurochem. Res. 14, 1223-1227. Werman, R. 1966. C r i t e r i a f o r i d e n t i f i c a t i o n of a c e n t r a l nervous system t r a n s m i t t e r . Comp. Biochem. P h y s i o l . 18, 745-766. Wilson, D.F., and Pastuszko, A. 1986. T r a n s p o r t o f c y s t e a t e by synaptosomes i s o l a t e d from r a t b r a i n : evidence t h a t i t u t i l i z e s the same t r a n s p o r t e r as a s p a r t a t e , glutamate, and c y s t e i n e s u l f i n a t e . J . Neurochem. 47, 1091-1097. W i n f i e l d , D.A. 1981. The p o s t n a t a l development of synapses i n the v i s u a l c o r t e x of the c a t and the e f f e c t s of e y e l i d c l o s u r e . B r a i n Res. 206-, 166-171. Wong-Riley, M. 1979. Changes i n the v i s u a l system of monocularly sutured or enucleated c a t s demonstrable w i t h cytochrome oxidase h i s t o c h e m i s t r y . B r a i n Res. 171, 11-28. Yammamura, H.I., Enna, S.J., and Kuhar, M.J. (Eds.) 1990. Methods i n Neuro t r a n s m i t t e r Receptor A n a l y s i s . Raven P r e s s , N.Y. Young. W. S., and Kuhar, M.J. 1979. A new method f o r r e c e p t o r autoradiography: [ H ] o p i o i d r e c e p t o r s i n r a t b r a i n . B r a i n Res. 179, 255-270. Zaczek, R., K o l l e r , K.J., C o t t e r , R., H e l l e r , D., and Coyle, J.T. 1983. N - a c e t y l - a s p a r t y l - g l u t a m a t e : an endogenous p e p t i d e w i t h h i g h a f f i n i t y f o r a b r a i n "glutamate" r e c e p t o r . Proc. N a t l . Acad. S c i . USA, 80, 1116-1118. 146 Z i p s e r , D. and Andersen, R.A. 1988. A back-propogation programmed network t h a t s i m u l a t e s response p r o p e r t i e s of a subset of p a r i e t a l neurons. Nature 331, 679-684. Z i v i n , J.A., and Waud, D.R. 1982. How t o ana l y s e b i n d i n g , enzyme and uptake data: the s i m p l e s t case, a s i n g l e phase. L i f e S c i . 30, 1407-1422. Z o l l i n g e r , M., Amsler, U., Do, Q. K., S t r e i t , P., and Cuenod, M. 1988. Release of N - a c e t y l a s p a r t y l g l u t a m a t e on d e p o l a r i z a t i o n of r a t b r a i n s l i c e s . J . Neurochem. 51, 1919-1923. 

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