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The effects of excitatory amino acids and their antagonists on hippocampal electrophysiology Kehl, Steven James 1984

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THE EFFECTS OF EXCITATORY AMINO ACIDS AND THEIR ANTAGONISTS ON HIPPOCAMPAL ELECTROPHYSIOLOGY By S t e v e n James K e h l B . S c , The U n i v e r s i t y o f W a t e r l o o , 1976 M . S c , The U n i v e r s i t y o f W a t e r l o o , 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t 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 a s 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 B R I T I S H COLUMBIA May 1984 © S t e v e n James K e h l , 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Physiology  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 30, 1984 i i A b s t r a c t The e f f e c t s o f a c i d i c amino a c i d s and t h e i r a n t a g o n i s t s on h i p p o c a m p a l e l e c t r o p h y s i o l o g y The r o l e o f e x c i t a t o r y a c i d i c amino a c i d s i n s y n a p t i c t r a n s m i s s i o n and t h e m o d u l a t i o n o f e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s were examined i n t h e h i p p o c a m p a l s l i c e in v i t r o u s i n g c o n v e n t i o n a l e x t r a c e l l u l a r and i n t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s . I o n t o p h o r e s i s o f L - g l u t a m a t e , L - a s p a r t a t e and t h e more p o t e n t and s p e c i f i c a g o n i s t s N - m e t h y 1 - D L - a s p a r t a t e (NMA), k a i n a t e and q u i s q u a l a t e e x c i t e d C A l n e u r o n s ; NMA was u n i q u e i n c a u s i n g c e l l s t o f i r e i n b u r s t s o f r e p e t i t i v e d i s c h a r g e s i n c o n t r a s t t o t h e s u s t a i n e d f i r i n g o b s e r v e d w i t h t h e o t h e r compounds. I o n t o p h o r e t i c a l l y - a p p l i e d DL-2-amino-5-phos-p h o n o v a l e r a t e (APV) s e l e c t i v e l y b l o c k e d NMA e x c i t a t i o n s , an e f f e c t w h i c h was a t t r i b u t e d t o t h e D - i s o m e r ; ^ - D - g l u t a m y 1 g l y c i n e (DGG) i n h i b i t e d c e l l f i r i n g e l i c i t e d by NMA, k a i n a t e and L - a s p a r t a t e ; a t a h i g h e r dose i t a l s o i n h i b i t e d r e s p o n s e s t o q u i s q u a l a t e and L - g l u t a m a t e t o some e x t e n t . L - g l u t a m a t e d i e t h y l e s t e r , ( + ) - c i s - 2 , 3 - p i p e r i d i n e d i c a r b o x y l a t e and D L - 2 - a m i n o - 4 - p h o s p h o n o b u t y r a t e (APB) v a r i o u s l y a f f e c t e d amino a c i d - m e d i a t e d e x c i t a t i o n s b ut a l s o had c o m p l i c a t i n g s e c o n d a r y a c t i o n s . The e f f e c t s o f i o n t o p h o r e s i s t o t h e s t r a t u m r a d i a t u m i n i i i CA1 o f e x c i t a t o r y a m i n o a c i d s a n d some a n t a g o n i s t s w e r e e v a l u a t e d on t h e f i e l d E P S P e v o k e d by s t i m u l a t i o n o f t h e S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l (SC) p a t h w a y s . E a c h o f t h e s e l e c t i v e a g o n i s t s a s w e l l a s L - g l u t a m a t e a n d L - a s p a r t a t e d e p r e s s e d E P S P s . K a i n a t e - i n d u c e d d e p r e s s i o n s c o n s i d e r a b l y o u t l a s t e d t h e p e r i o d o f e j e c t i o n w h e r e a s NMA d e p r e s s i o n s w e re r a p i d l y r e v e r s i b l e and o f t e n f o l l o w e d by an i n c r e a s e d EPSP a m p l i t u d e . APV a b o l i s h e d r e s p o n s e s t o NMA, d i d n o t c h a n g e t h e EPSP b u t c o u l d b l o c k l o n g t e r m p o t e n t i a t i o n (LTP) o f s y n a p t i c t r a n s m i s s i o n . The o p t i c a l i s o m e r s o f APB had l i t t l e o r no e f f e c t o f E P S P s . DGG was an e f f e c t i v e s y n a p t i c a n t a g o n i s t w h i l s t h a v i n g no d e t e c t a b l e e f f e c t on t h e p r e s y n a p t i c f i b r e v o l l e y . I t i s c o n c l u d e d t h a t a k a i n a t e o r q u i s q u a l a t e t y p e r e c e p t o r may be i n v o l v e d i n S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l -- e v o k e d s y n a p t i c t r a n s m i s s i o n ; NMA r e c e p t o r s may p l a y a r o l e i n t h e n e u r o n a l p r o c e s s e s u n d e r l y i n g LTP. The i n t e r a c t i o n o f L - g l u t a m a t e and L - a s p a r t a t e w i t h t h e s e r e c e p t o r s i s d i s c u s s e d . To d e t e r m i n e t h e b a s i s f o r i t s c o n v u l s a n t and n e u r o -t o x i c p r o p e r t i e s t h e a c t i o n s o f k a i n a t e on s y n a p t i c p r o c e s s e s w e r e i n v e s t i g a t e d . I n C A l a n d t h e d e n t a t e g y r u s k a i n a t e p r o d u c e d a l a r g e and l o n g - l a s t i n g i n c r e a s e o f t h e o r t h o d r o m i c p o p u l a t i o n s p i k e (PS) and an a s s o c i a t e d d e c l i n e o f t h e EPSP; t h e a m p l i t u d e s o f p r e s y n a p t i c f i b r e v o l l e y s and a n t i d r o m i c PSs w e r e u n c h a n g e d . I n t h e CA3 r e g i o n l o w e r d o s e s o f k a i n a t e p o t e n t i a t e d s y n a p t i c e x c i t a t i o n s and e p i l e p t i f o r m f i r i n g a p p e a r e d s p o n t a n e o u s l y and f o l l o w i n g e l e c t r i c a l s t i m u l a t i o n . K a i n a t e a l s o r e d u c e d G A B A - m e d i a t e d i n h i b i t i o n s a nd i v IPSPs i n C A l and a b o l i s h e d i n CA3 an i n h i b i t i o n w h i c h , on t h e b a s i s o f s t u d i e s r e p o r t e d h e r e a p p e a r s t o be n o n - G A B A e r g i c , but t h i s d i s i n h i b i t o r y a c t i o n d i d n o t a p p e a r t o u n d e r l i e t h e i n c r e a s e d e x c i t a b i l i t y . O t h e r e x c i t a t o r y amino a c i d s c o u l d m imic o n l y t h e d e p r e s s a n t a c t i o n s o f h i g h e r d o s e s o f k a i n a t e . An i n c r e a s e d c o u p l i n g between d e n d r i t e s and somata r e l a t e d p e r h a p s t o a d e n d r i t i c d e p o l a r i z a t i o n i s s u g g e s t e d a s t h e b a s i s f o r t h e p o t e n t i a t i n g a c t i o n . The p o t e n t i a t i n g a n d d i s i n h i b i t o r y p r o p e r t i e s o f k a i n a t e a r e d i s c u s s e d i n t h e c o n t e x t o f i t s e p i l e p t o g e n i c i t y and n e u r o t o x i c i t y . I n C A l and t h e d e n t a t e g y r u s p e r f u s i o n o f f o l i c a c i d , a p o s s i b l e endogenous a c t i v a t o r o f t h e k a i n i c r e c e p t o r , c a u s e d a p o t e n t i a t i o n o f o r t h o d r o m i c PSs w h i l s t t h e EPSP was e i t h e r u n a f f e c t e d o r d e p r e s s e d ; a n t i d r o m i c r e s p o n s e s were unch a n g e d . In CA3 f o l a t e p o t e n t i a t e d PSs and c a u s e d b o t h s p o n t a n e o u s and 2 + e v o k e d e p i l e p t i f o r m d i s c h a r g e s . B u r s t f i r i n g was Ca d e p e n d e n t and was t r i g g e r e d by p a r o x y s m a l d e p o l a r i z a t i o n s h i f t s i n d i c a t i n g t h e i r s y n a p t i c o r i g i n . U n l i k e k a i n a t e , f o l a t e c a u s e d no m e a s u r a b l e change o f r e c u r r e n t i n h i b i t i o n i n C A l and n e v e r d i r e c t l y d e p r e s s e d e v o k e d a c t i v i t y . The i m p l i c a t i o n s o f t h e s e d a t a f o r f o l a t e n e u r o t o x i c i t y and t h e p o s s i b l e h o m o l o g i e s o f r e c e p t o r s f o r k a i n a t e and f o l a t e a r e d i s c u s s e d . V TABLE OF CONTENTS L I S T OF TABLES v i i L I S T OF FIGURES . v i i i - x i i i ACKNOWLEDGEMENTS x i v C h a p t e r 1. GENERAL INTRODUCTION 1 2. PREPARATION AND MAINTENANCE OF THE HIPPOCAMPAL S L I C E IN VITRO AND CHARACTERIZATION OF EVOKED RESPONSES 8 I n t r o d u c t i o n . 8 G e n e r a l M e t h o d s 9 I s o l a t i o n , c u t t i n g and m a i n t e n a n c e o f h i p p o c a m p a l t i s s u e 9 P r e p a r a t i o n o f i o n t o p h o r e t i c e l e c t r o d e s . . . . 14 S t i m u l a t i o n and r e c o r d i n g o f f i e l d r e s p o n s e s . . 14 I n t r a c e l l u l a r r e c o r d i n g 17 R e s u l t s 18 C h a r a c t e r i z a t i o n o f f i e l d r e s p o n s e s 19 C A l 19 D e n t a t e g y r u s 22 CA3 24 P a i r e d p u l s e s t i m u l a t i o n 27 H i g h f r e q u e n c y s t i m u l a t i o n 29 E f f e c t s o f e x t r a c e l l u l a r i o n c o n c e n t r a t i o n s on e v o k e d a c t i v i t y i n CA3 . . ^ . . . . . . . . . 31 Low [ C a _ ] , i n c r e a s e d [Mg ] 31 Low [CI ] ° . + ? 34 I n c r e a s e d [K ] 41 I n t r a c e l l u l a r r e c o r d i n g o f CA3 r e s p o n s e s . . . . 41 D i s c u s s i o n 46 3. POSTACTIVATION I N H I B I T I O N IN THE CA3 REGION . . . . 56 I n t r o d u c t i o n . 56 M e t h o d s 57 R e s u l t s 58 D i s c u s s i o n 68 v i 4. THE EFFECTS OF ANTAGONISTS ON AMINO ACID- AND SCHAFFER-COLLATERAL MEDIATED EXCITATION OF C A l NEURONS 75 I n t r o d u c t i o n 75 L - g l u t a m a t e and L - 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 : n e u r o c h e m i c a l e v i d e n c e 76 L - g l u t a m a t e and L - 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 t e l e c t r o p h y s i o l o g i c a l e v i d e n c e . . 80 Met h o d s 86 R e s u l t s 88 E f f e c t s o f e x c i t a t o r y a m i n o a c i d a g o n i s t s and a n t a g o n i s t s on s i n g l e u n i t a c t i v i t y . . . 88 E f f e c t s o f e x c i t a t o r y a m ino a c i d a g o n i s t s and a n t a g o n i s t s on s y n a p t i c a c t i v i t y 100 D i s c u s s i o n I l l 5. ACTIONS OF K A I N I C ACID ON SYNAPTIC PROCESSES IN THE HIPPOCAMPAL S L I C E 125 I n t r o d u c t i o n 125 M e t h o d s 133 R e s u l t s 134 E x t r a c e l l u l a r l y r e c o r d e d e f f e c t s o f k a i n a t e on e v o k e d and s p o n t a n e o u s a c t i v i t y 134 C A l 134 CA3 141 D e n t a t e g y r u s 147 E f f e c t s o f o t h e r a mino a c i d s i n C A l 149 K a i n a t e ' s m e c h a n i s m ( s ) o f a c t i o n 151 C A l - E x t r a c e l l u l a r a n a l y s i s o f k a i n a t e -i n d u c e d c h a n g e s o f r e c u r r e n t i n h i b i t i o n . . 153 C A l - I n t r a c e l l u l a r a n a l y s i s o f t h e a c t i o n s o f k a i n a t e on C A l p y r a m i d a l c e l l s 155 CA3 160 D e n t a t e g y r u s 167 D i s c u s s i o n 170 C o r r e l a t i o n b e t w e e n e l e c t r o p h y s i o l o g i c a l and n e u r o t o x i c a c t i o n s o f k a i n a t e 175 M e c h a n i s m ( s ) o f k a i n a t e ' s e p i l e p t o g e n i c i t y . . . 177 6. FOLIC ACID-INDUCED CHANGES OF NEURONAL A C T I V I T Y . . 180 I n t r o d u c t i o n 180 M e t h o d s 183 R e s u l t s 183 E f f e c t s o f f o l a t e i n C A l and t h e d e n t a t e g y r u s . 183 E f f e c t s i n CA3 187 A c t i o n s o f f o l a t e on r e c u r r e n t i n h i b i t i o n . . . 197 D i s c u s s i o n 200 M e c h a n i s m o f a c t i o n 202 REFERENCES 208 / APPENDIX I , . 235 v i i L I S T OF TABLES T a b l e I A c t i o n s o f a m i n o a c i d a n t a g o n i s t s o n t h e e x c i t a t o r y r e s p o n s e s t o NMA, k a i n a t e , q u i s q u a l a t e , L - g l u t a m a t e , L - a s p a r t a t e a nd ACh , 9 2 T a b l e I I Changes o f t h e S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l -e v o k e d EPSP f o l l o w i n g i o n t o p h o r e t i c a p p l i c a t i o n s o f e x c i t a t o r y a m i n o a c i d s 101 T a b l e I I I E f f e c t s o f a n t a g o n i s t s on S c h a f f e r c o l l a t e r a l -c o m m i s s u r a l - e v o k e d EPSPs 106 v i i i L I S T OF FIGURES F i g u r e 2.1 A s c h e m a t i c o u t l i n e o f t h e a p p a r a t u s f o r t h e c o n s t a n t f l o w p e r f u s i o n o f h i p p o c a m p a l s l i c e s w i t h a r t i f i c i a l c e r e b r o s p i n a l f l u i d 13 F i g u r e 2.2 A r r a n g e m e n t o f i n p u t and o u t p u t d e v i c e s f o r t h e r e c o r d i n g o f e x t r a c e l l u l a r and i n t r a c e l l -u l a r r e s p o n s e s 16 F i g u r e 2.3 P o s i t i o n s o f s t i m u l a t i n g and r e c o r d i n g e l e c -t r o d e s i n t h e C A l r e g i o n and t y p i c a l e x t r a -c e l l u l a r e v o k e d r e s p o n s e s 20 F i g u r e 2.4 P o s i t i o n s o f s t i m u l a t i n g and r e c o r d i n g e l e c -t r o d e s i n t h e d e n t a t e g y r u s and t y p i c a l e x -t r a c e l l u l a r e v o k e d r e s p o n s e s 23 F i g u r e 2.5 P o s i t i o n s o f s t i m u l a t i n g and r e c o r d i n g e l e c -t r o d e s i n t h e CA3 r e g i o n and t y p i c a l e x t r a -c e l l u l a r e v o k e d r e s p o n s e s 25 F i g u r e 2.6 Some c h a r a c t e r i s t i c s o f t h e mossy f i b r e -e v o k e d r e s p o n s e i n CA3 28 • F i g u r e 2.7 C h a n g e s i n f i m b r i a l a n d mossy f i b r e - e v o k e d r e s p o n s e s f o l l o w i n g t e t a n i c s t i m u l a t i o n 30 2+ 2 + F i g u r e 2.8 The a c t i o n s o f l o w [Ca ] / i n c r e a s e d [Mg ] on e v o k e d a c t i v i t y i n CA3 33 F i g u r e 2.9 E f f e c t s o f l o w [ C I ] o n s p o n t a n e o u s a n d e v o k e d a c t i v i t y i n CA3 36 i x F i g u r e 2.10 I n t h e CA3 r e g i o n c h a n g e s o f p o p u l a t i o n s p i k e a m p l i t u d e s i n ACSF c o n t a i n i n g l o w [ C l ] and 2+ 2 + [Ca ] and i n c r e a s e d [Mg ] 38 F i g u r e 2.11 S p o n t a n e o u s p o p u l a t i o n s p i k e s i n f i e l d CA3 i n t h e a b s e n c e o f s y n a p t i c t r a n s m i s s i o n 40 F i g u r e 2.12 E p i l e p t i f o r m a c t i v i t y i n CA3 f o l l o w i n g e x p o -s u r e t o i n c r e a s e d [ K + ] 42 o F i g u r e 2.13 C o r r e l a t i o n b e t w e e n f i m b r i a l and mossy f i b r e -e v o k e d e x t r a c e l l u l a r f i e l d s and i n t r a c e l l u l a r p o t e n t i a l s 45 F i g u r e 3.1 Homo- an d h e t e r o s y n a p t i c a l l y - e v o k e d i n h i b i -t i o n o f f i m b r i a l o r t h o d r o m i c p o p u l a t i o n s p i k e s 59 F i g u r e 3.2 The a c t i o n s o f mossy f i b r e c o n d i t i o n i n g s t i m -u l i on mossy f i b r e and a n t i d r o m i c t e s t p o p u -l a t i o n s p i k e s 61 F i g u r e 3.3 C h a r t r e c o r d i n g s o f i n t r a c e l l u l a r r e s p o n s e s t o s u b t h r e s h o l d mossy f i b r e and f i m b r i a l s t i m u l i 63 F i g u r e 3.4 T h e a c t i o n s o f b i c u c u l l i n e on e a r l y and l a t e i n h i b i t i o n s i n t h e CA3 r e g i o n 65 F i g u r e 3.5 C h a n g e s i n t h e p r o f i l e o f i n h i b i t i o n a f t e r b i c u c u l l i n e t r e a t m e n t 66 X F i g u r e 4.1 R a t e m e t e r r e c o r d s o f t h e e f f e c t s o f PDA on C A l e x c i t a t i o n s i n d u c e d by NMA, k a i n a t e and q u i s q u a l a t e 93 F i g u r e 4.2 B l o c k a d e o f a m i n o - a c i d - i n d u c e d r e s p o n s e s by APV and DGG 95 F i g u r e 4.3 E f f e c t s o f DL-APV and L-APV on r e s p o n s e s o f C A l n e u r o n s 96 F i g u r e 4.4 A l t e r a t i o n s o f amino a c i d - i n d u c e d c e l l f i r i n g by t h e o p t i c a l i s o m e r s o f APB 98 F i g u r e 4.5 D i f f e r e n t i a l a n t a g o n i s m o f NMA, L - a s p a r t a t e and L - g l u t a m a t e r e s p o n s e s by DGG 99 F i g u r e 4.6 D e p r e s s i o n o f S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l e v o k e d EPSPs by e x c i t a t o r y amino a c i d s 104 F i g u r e 4.7 The a c t i o n s o f NMA and APV on EPSPs 108 F i g u r e 4.8 B l o c k a d e by a n t a g o n i s t s o f e x c i t a n t - i n d u c e d d e p r e s s i o n s o f EPSPs 110 F i g u r e 4.9 E f f e c t s o f a n t a g o n i s t s on p o p u l a t i o n s p i k e s and l o n g t e r m p o t e n t i a t i o n 112 F i g u r e 4.10 The a c t i o n s o f b a t h - a p p l i e d D-APV on EPSPs and s y n a p t i c p l a s t i c i t y 113 F i g u r e 5.1 Enhancement o f s y n a p t i c e x c i t a t i o n i n C A l by k a i n a t e a p p l i e d e i t h e r i o n t o p h o r e t i c a l l y o r i n t h e p e r f u s a t e 135 x i F i g u r e 5.2 K a i n a t e - i n d u c e d c h a n g e s o f o r t h o d r o m i c a nd a n t i d r o m i c f i e l d p o t e n t i a l s i n C A l 138 F i g u r e 5.3 Changes o f i n p u t / o u t p u t r e l a t i o n s a f t e r p e r -f u s i o n o f k a i n a t e i n t h e C A l r e g i o n 139 F i g u r e 5.4 E f f e c t s o f k a i n a t e on EPSPs and o r t h o d r o m i c p o p u l a t i o n s p i k e s 140 F i g u r e 5.5 P o t e n t i a t i o n by k a i n a t e o f mossy f i b r e - e v o k e d a c t i v i t y i n t h e CA3 r e g i o n 143 F i g u r e 5.6 I n CA3 t h e a c t i o n s o f k a i n a t e on s p o n t a n e o u s and e v o k e d f i e l d p o t e n t i a l s 145 F i g u r e 5.7 S h i f t s o f i n p u t / o u t p u t c u r v e s i n t h e k a i n a t e -t r e a t e d d e n t a t e g y r u s 148 F i g u r e 5.8 D e p r e s s i o n o f EPSPs and enhancement o f o r t h o -d r o m i c e x c i t a t i o n i n t h e d e n t a t e a f t e r p e r -f u s i o n o f k a i n a t e 150 F i g u r e 5.9 The e f f e c t s o f o t h e r e x c i t a t o r y a m i n o a c i d s on p o p u l a t i o n s p i k e s i n C A l 152 F i g u r e 5.10 K a i n a t e - i n d u c e d r e d u c t i o n o f p a i r e d p u l s e i n h i b i t i o n i n C A l 154 F i g u r e 5.11 S i m u l t a n e o u s i n t r a c e l l u l a r and e x t r a c e l l u l a r a n a l y s e s o f k a i n a t e ' s a c t i o n s o n s y n a p t i c p r o c e s s e s 158 F i g u r e 5.12 A b o l i t i o n o f o r t h o d r o m i c a l l y and a n t i d r o m -i c a l l y - e v o k e d I P S P s d u r i n g k a i n a t e t r e a t m e n t i n C A l 159 x i i F i g u r e 5.13 C h a nges o f h e t e r o s y n a p t i c p a i r e d p u l s e e a r l y a n d l a t e i n h i b i t i o n s a f t e r i o n t o p h o r e s i s o f k a i n a t e i n s t r a t u m p y r a m i d a l e i n CA3 163 F i g u r e 5.14 S e l e c t i v e b l o c k a d e by k a i n a t e o f t h e l a t e i n h i b i t i o n i n CA3 165 F i g u r e 5.15 Summary o f t h e e f f e c t s o f b i c u c u l l i n e and k a i n a t e on h e t e r o s y n a p t i c i n h i b i t i o n s i n CA3....166 F i g u r e 5.16 A c t i o n s o f k a i n a t e on s y n a p t i c e x c i t a t i o n and i n h i b i t i o n i n t h e d e n t a t e g y r u s 169 F i g u r e 6.1 P o t e n t i a t i o n o f p o p u l a t i o n s p i k e s i n C A l f o l l o w i n g p e r f u s i o n o f f o l a t e 184 F i g u r e 6.2 F o l a t e - i n d u c e d a l t e r a t i o n s o f s y n a p t i c a c t i v -i t y i n t h e d e n t a t e g y r u s 185 F i g u r e 6.3 I n p u t / o u t p u t c u r v e s r e l a t i n g c h a n g e s o f p o p u -l a t i o n s p i k e s and EPSPs i n C A l and t h e d e n -t a t e g y r u s a f t e r t r e a t m e n t w i t h f o l a t e 186 F i g u r e 6.4 M ossy f i b r e - e v o k e d e p i l e p t i f o r m d i s c h a r g e s i n t h e f o l a t e - t r e a t e d CA3 r e g i o n 190 F i g u r e 6.5 E f f e c t s o f f o l a t e on o r t h o d r o m i c and a n t i -d r o m i c f i e l d p o t e n t i a l s i n CA3 191 F i g u r e 6.6 C o r r e l a t i o n b e t ween s p o n t a n e o u s b u r s t f i r i n g a n d t h e d e l a y e d o n s e t o f e v o k e d a c t i v i t y i n f o l a t e and b i c u c u l l i n e - t r e a t e d s l i c e s 192 x i i i F i g u r e 6.7 The e f f e c t s o f t h e t i m i n g a n d s t r e n g t h o f f i m b r i a l s t i m u l i on d e l a y e d b u r s t f i r i n g 194 F i g u r e 6.8 E x t r a c e l l u l a r and i n t r a c e l l u l a r c h a r a c t e r -i s t i c s o f f o l a t e - i n d u c e d s p o n t a n e o u s e p i l e p -t i f o r m a c t i v i t y i n CA3 196 F i g u r e 6.9 The a c t i o n s o f f o l a t e a n d k a i n a t e o n t h e r e c u r r e n t i n h i b i t i o n o f C A l n e u r o n s 199 x i v ACKNOWLEDGEMENTS F o r h i s g e n e r o u s s u p p o r t and c o u n s e l o v e r t h e c o u r s e o f t h i s s t u d y I am i n d e b t e d t o D r . Hugh McLennan. To D r . Graham C o l l i n g r i d g e , whose c o l l a b o r a t i v e e f f o r t s made p o s s i b l e t h e i n v e s t i g a t i o n s d e s c r i b e d i n C h a p t e r s 4 and 5, I e x p r e s s my s i n c e r e t h a n k s . G i f t s o f compounds f r o m D r . J . C . W a t k i n s ( B r i s t o l ) a r e g r a t e f u l l y a c k n o w l e d g e d . E x p e r t t e c h n i c a l a s s i s t a n c e i n t h e p r e p a r a t i o n o f t h e f i g u r e s was p r o v i d e d by K u r t H e nze. I w o u l d a l s o l i k e t o t h a n k D r . P. A n d e r s e n , t h e e x t e r n a l e x a m i n e r , and t h e members o f t h e a d v i s o r y c o m m i t t e e f o r r e v i e w i n g t h i s t h e s i s . F i n a l l y , I w i s h t o a c k n o w l e d g e t h e a w a r d o f a S t u d e n t s h i p by t h e M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a . 1 CHAPTER 1 GENERAL INTRODUCTION I n t h e r a t t h e h i p p o c a m p a l f o r m a t i o n o c c u p i e s t h e p o s t e r o - m e d i a l p o r t i o n o f e a c h c e r e b r a l h e m i s p h e r e and c o m p r i s e s more t h a n o n e - t h i r d o f t h e c o r t i c a l s u r f a c e a r e a . I t c o n s i s t s o f 4 m a j o r d i v i s i o n s : t h e e n t o r h i n a l c o r t e x , t h e d e n t a t e g y r u s , t h e h i p p o c a m p u s p r o p e r o r Amnion's h o r n ( c o r n u A m m o n i s ; f i e l d s C A 1 - C A 3 ) a n d t h e s u b i c u l a r c o m p l e x . The e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n s d e s c r i b e d i n t h i s t h e s i s f o c u s e d on t h e d e n t a t e g y r u s and t h e h i p p o c a m p u s p r o p e r , e a c h o f w h i c h i s c o m p o s e d o f a r c h i c o r t e x i n w h i c h c e l l b o d i e s , t h e i r d e n d r i t e s and a f f e r e n t and e f f e r e n t p a t h w a y s a r e a r r a n g e d i n a c l e a r l y d i s c e r n a b l e l a m i n a r f a s h i o n . T h e t r a n s f e r o f i n f o r m a t i o n w i t h i n t h e h i p p o c a m p u s o c c u r s v i a t h e s o - c a l l e d t r i - s y n a p t i c c i r c u i t w h i c h c o n s i s t s o f t h e p e r f o r a n t p a t h w a y f r o m t h e e n t o r h i n a l c o r t e x t o t h e d e n t a t e g y r u s , t h e mossy f i b r e p r o j e c t i o n f r o m t h e d e n t a t e t o f i e l d CA3 and f i n a l l y t h e S c h a f f e r c o l l a t e r a l f i b r e s y s t e m a r i s i n g f r o m CA3 a n d t e r m i n a t i n g i n f i e l d C A l ( F i g . 2 . 3 A ) . T h e 3 l a m i n a e o f t h e d e n t a t e g y r u s a r e : a c e n t r a l l a y e r , t h e s t r a t u m g r a n u l o s u m c o n t a i n i n g t h e s o m a t a o f ' d e n t a t e g r a n u l e c e l l s ; a n o u t e r m o l e c u l a r l a y e r c o n s i s t i n g o f t h e 2 d e n d r i t e s o f t h e s e c e l l s ; and t h e h i l u s o f t h e f a s c i a d e n t a t a ( f i e l d . CA4 o f L o r e n t e de No, 1 9 3 4 ) . E n t o r h i n a l f i b r e s a r e d i s t r i b u t e d a l o n g t h e d e n d r i t e s o f g r a n u l e c e l l s w i t h a f f e r e n t s f r o m t h e m e d i a l and l a t e r a l e n t o r h i n a l a r e a s t e r m i n a t i n g a p p r o x i m a t e l y i n t h e m i d d l e and o u t e r o n e - t h i r d o f t h e s t r a t u m m o l e c u l a r e , r e s p e c t i v e l y ( H j o r t h - S i m o n s e n and J e u n e , 1 9 7 2 ; S t e w a r t , 1 9 7 6 ) . C e l l s i n t h e d e n t a t e h i l u s r e c e i v e s y n a p t i c i n p u t f r o m b o t h t h e mossy f i b r e p a t h w a y ( B l a c k s t a d and K j a e r h e i m , 1961) and f i e l d CA3 ( G o t t l e i b and Cowan, 1 9 7 3 ; S c h w e r d t f e g e r and S a r v e y , 1 9 8 3 ) . The m a j o r o u t p u t s o f t h e s e n e u r o n s t e r m i n a t e o n t h e i n n e r t h i r d o f t h e d e n t a t e m o l e c u l a r l a y e r ( H j o r t h -S i m o n s e n and L a u r b e r g , 1 9 7 7 ) . F i e l d CA3 ( r e g i o i n f e r i o r ) i s a l s o a r r a n g e d i n 3 l a m e l l a e w i t h t h e s o m a t a o f p y r a m i d a l n e u r o n s a l i g n e d i n t h e s t r a t u m p y r a m i d a l e f r o m w h i c h b a s a l d e n d r i t e s e x t e n d i n t o t h e s t r a t u m o r i e n s and a p i c a l d e n d r i t e s p r o j e c t i n t o t h e m o l e c u l a r l a y e r . T h e m o s s y f i b r e i n p u t f r o m ' d e n t a t e g r a n u l e c e l l s t e r m i n a t e s p r i m a r i l y i n t h e s t r a t u m l u c i d u m , a n a r r o w zone i m m e d i a t e l y above t h e c e l l body l a y e r ( B l a c k s t a d and K j a e r -h e i m , 1 9 6 1 ; G a a r s k j a e r , 1 9 8 1 ) . A p r o j e c t i o n f r o m t h e e n t o r h i n a l c o r t e x t o r e g i o i n f e r i o r f o r m s s y n a p t i c c o n t a c t s en p a s s a g e w i t h d i s t a l a p i c a l d e n d r i t e s i n t h e s t r a t u m l a c u n o s u m -m o l e c u l a r e ( H j o r t h - S i m o n s e n and J e u n e , 1972; S t e w a r t , 1 9 7 6 ) . F i e l d CA3 a l s o r e c e i v e s c o m m i s s u r a l a f f e r e n t s f r o m t h e c o n t r a -l a t e r a l h o m o t o p i c f i e l d as w e l l a s i n p u t s f r o m i p s i l a t e r a l CA3 3 n e u r o n s ( G o t t l i e b a nd Cowan, 19 7 3 ; Swanson et. a _ l . , 1 9 8 1 ) . The f i b r e s o f t h e s e p a t h w a y s t e r m i n a t e i n t h e s t r a t a o r i e n s and r a d i a t u m ( G o t t l i e b and Cowan, 1 9 7 3 ) . The m a j o r e x c i t a t o r y i n p u t s t o C A l ( r e g i o s u p e r i o r ) p y r a m i d a l c e l l s a r e t h e c o m m i s s u r a l and S c h a f f e r c o l l a t e r a l f i b r e s w h i c h o r i g i n a t e i n CA3 c e l l s (Swanson et. a l . , 1980) and t e r m i n a t e m a i n l y i n t h e s t r a t a o r i e n s a nd r a d i a t u m r e s p e c -t i v e l y (Swanson e t a l . , 1978; V o n e i d a e t a l . , 1 9 8 1 ) . The m a j o r e f f e r e n t p r o j e c t i o n s o f t h e h i p p o c a m p a l f o r m a t i o n o f t h e r a t a r e t o t h e a n t e r i o r n u c l e a r c o m p l e x o f t h e t h a l a m u s , m a m m i l l a r y b o d y , l a t e r a l s e p t a l n u c l e i , n u c l e u s accumbens and c o r t i c a l a s s o c i a t i o n a r e a s (Swanson and Cowan, 1 9 7 7 ) . D e s p i t e i t s a n a t o m i c a l p r o m i n e n c e a p r e c i s e d e f i n i t i o n o f h i p p o c a m p a l f u n c t i o n has been e l u s i v e . I n d e e d Swanson et_ a l . ( 1 9 8 2 ) commented t h a t " t h e number o f g e n e r a l t h e o r i e s o f ' t h e f u n c t i o n 1 o f t h e h i p p o c a m p u s a p p r o a c h e s t h e number o f i n v e s t i g a t o r s who have s t u d i e d i t " . I n t h i s c o n t e x t i t i s w o r t h n o t i n g t h a t t h e a f f e r e n t i n p u t o f t h e h i p p o c a m p u s c a n n o t be c o n f i n e d t o a s i n g l e s e n s o r y m o d a l i t y and i t m i g h t r e a s o n a b l y be i n f e r r e d t h a t i t s e f f e c t o r o u t p u t i s r e l a t e d t o many c o r t i c a l f u n c t i o n s . I t i s b e y o n d t h e s c o p e o f t h i s t h e s i s t o c o n s i d e r t h e c o m p l e x i t i e s o f c u r r e n t h y p o t h e s e s r e g a r d i n g t h e r o l e o f t h e h i p p o c a m p u s i n c o r t i c a l a c t i v i t y . F o r t h a t p u r p o s e t h e r e c e n t r e v i e w s o f O ' K e e f e a n d N a d e l ( 1 9 7 9 ) a n d O l t o n a n d h i s c o -4 w o r k e r s (1980) a r e recommended. S u f f i c e i t t o s a y t h e r e i s a g e n e r a l c o n c e n s u s t h a t t h e h i p p o c a m p a l f o r m a t i o n p l a y s a v i t a l r o l e i n t h e a b s t r u s e c o r t i c a l a c t i v i t i e s o f t h e m a m m a l i a n b r a i n w h i c h s u b s e r v e c o g n i t i v e f u n c t i o n , p a r t i c u l a r l y l e a r n i n g and memory. T h a t i t a l s o r e c e i v e s i n p u t f r o m d i v e r s e r e g i o n s o f t h e b r a i n s u c h as t h e h y p o t h a l a m u s , r a p h e n u c l e i and l o c u s c o e r u l e u s s u g g e s t s t h e a d d i t i o n a l i n v o l v e m e n t o f t h e h i p p o -campus i n t h e m o d u l a t i o n o f b e h a v i o r a l s t a t e . F i n a l l y , w i t h r e g a r d t o c o r t i c a l d y s f u n c t i o n , t h e r e i s e v i d e n c e t h a t t h e h i p p o c a m p u s may a s s u m e some p r o m i n e n c e i n , t h e e t i o l o g y o f t e m p o r a l l o b e e p i l e p s y i n humans ( D e l g a d o - E s c u e t a , 1 9 7 9 ) . A f a c t o r w h i c h has no d o u b t c o n t r i b u t e d t o t h e s l o w d e v e l o p m e n t o f i n f o r m a t i o n r e g a r d i n g h i p p o c a m p a l f u n c t i o n has b e en t h e d i f f i c u l t y i n h e r e n t t o t h e a n a l y s i s i_n v i v o o f t h e p h y s i o l o g i c a l p r o p e r t i e s and i n t e r a c t i o n s o f v a r i o u s h i p p o -c a m p a l a f f e r e n t s . H o w e v e r , w i t h i n t h e l a s t 10 y e a r s t h e m a i n t e n a n c e o f s l i c e s o f b r a i n t i s s u e ^ r . v i t r o , a t e c h n i q u e p i o n e e r e d by Yamamoto and M c l l w a i n ( 1 9 6 6 ) , has p r o v i d e d n e u r o p h y s i o l o g i s t s w i t h a r e l a t i v e l y s i m p l e y e t p o w e r f u l m e t h o d f o r t h e e v a l u a t i o n o f n e u r o n a l a c t i v i t y u s i n g b o t h e x t r a c e l l u l a r and i n t r a c e l l u l a r r e c o r d i n g ( K e r k u t and W h e a l , 1 9 8 1 ) . I n t h i s c o n n e c t i o n , t h e h i p p o c a m p a l s l i c e has been p e r h a p s t h e most e x t e n s i v e l y s t u d i e d . P a r t i c u l a r l y i m p o r t a n t f e a t u r e s o f t h e i n c u b a t i o n ijn v i t r o o f s l i c e s o f h i p p o c a m p u s a r e t h e e l i m i n a t i o n o f t h e b l o o d b r a i n b a r r i e r and t h e a b i l i t y t o a l t e r t h e i o n i c c o m p o s i t i o n o f o r add d r u g s t o t h e medium 5 b a t h i n g t h e t i s s u e . F u r t h e r m o r e , t h e l a m e l l a r o r g a n i z a t i o n o f c e l l b o d i e s , t h e i r d e n d r i t e s and a f f e r e n t p a t h w a y s ( A n d e r s e n e t a l . , 1971a) a l l o w s s t i m u l a t i n g , r e c o r d i n g and i o n t o p h o r e t i c e l e c t r o d e s t o be p r e c i s e l y p o s i t i o n e d u n d e r v i s u a l c o n t r o l . I n t h e i n v e s t i g a t i o n s d e s c r i b e d i n t h i s t h e s i s t h e a d v a n t a g e s o f t h e s l i c e p r e p a r a t i o n h a v e been e x p l o i t e d t o d e t e r m i n e t h e r o l e o f e x c i t a t o r y a m i n o a c i d s i n s y n a p t i c p r o c e s s e s and t h e m o d u l a t i o n o f n e u r o n a l e x c i t a b i l i t y . T h e s e s t u d i e s a r e p r e f a c e d by a c h a r a c t e r i z a t i o n i n C h a p t e r 2 o f t h e a n t i d r o m i c a l l y and o r t h o d r o m i c a l l y - e v o k e d f i e l d p o t e n t i a l s i n e a c h o f t h e m a j o r h i p p o c a m p a l r e g i o n s : C A l , d e n t a t e g y r u s and e s p e c i a l l y t h o s e i n CA3 w h i c h h a v e n o t t o t h e a u t h o r ' s k n o w l e d g e been w e l l - d e s c r i b e d p r e v i o u s l y . E x p e r i m e n t s a s s e s s i n g h e t e r o s y n a p t i c i n t e r a c t i o n s i n r e g i o i n f e r i o r a l s o r e v e a l e d a p o t e n t and l o n g - l a s t i n g i n h i b i t i o n w h i c h , somewhat u n e x p e c t e d l y , was s h o w n t o be d u e t o i n h i b i t o r y p r o c e s s e s o t h e r t h a n t h o s e a t t r i b u t a b l e t o ^ - a m i n o b u t y r i c a c i d (GABA) t h e " c l a s s i c a l " i n h i b i t o r y t r a n s m i t t e r i n t h e h i g h e r r e g i o n s o f t h e m a m m a l i a n c e n t r a l n e r v o u s s y s t e m . B e c a u s e o f t h e p o s s i b l e f u n c t i o n a l s i g n i f i c a n c e o f t h i s a p p a r e n t l y n o v e l i n h i b i t i o n i t s p h y s i o l o g i c a l and p h a r m a c o l o g i c a l c h a r a c t e r -i s t i c s a r e d i s c u s s e d s e p a r a t e l y i n C h a p t e r 3. I t i s w e l l e s t a b l i s h e d t h a t t h e m a j o r i n t r i n s i c p a t h w a y s i n t h e t r i s y n a p t i c c i r c u i t a r e e x c i t a t o r y and t h e r e s u l t s o f n e u r o c h e m i c a l s t u d i e s have i m p l i c a t e d t h e a c i d i c a m i n o a c i d s L - g l u t a m a t e a n d L - a s p a r t a t e a s t h e i r p u t a t i v e 6 t r a n s m i t t e r s . T h e r e f o r e t h e e f f e c t s o f a number o f r e c e n t l y d e v e l o p e d compounds w h i c h have been d e s c r i b e d as a n t a g o n i s t s o f a m i n o a c i d - i n d u c e d e x c i t a t i o n s i n t h e s p i n a l c o r d w e r e e x a m i n e d on t h e e x c i t a t i o n o f C A l n e u r o n s m e d i a t e d by t h e s t i m u l a t i o n o f t h e S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l i n p u t . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e d i s c u s s e d w i t h r e g a r d t o t h e n a t u r e o f t h 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 ) m e d i a t i n g s y n a p t i c e x c i t a t i o n . The f i f t h c h a p t e r a d d r e s s e s t h e e l e c t r o p h y s i o l o g i c a l s e q u e l l a e o f t h e a c t i v a t i o n o f one c l a s s o f e x c i t a t o r y a m ino a c i d r e c e p t o r , t h a t w i t h w h i c h k a i n i c a c i d r e a c t s . K a i n i c a c i d , a compound w h i c h d oes n o t o c c u r n a t u r a l l y i n t h e c e n t r a l n e r v o u s s y s t e m , h a s p r o f o u n d e p i l e p t o g e n i c a n d n e u r o t o x i c e f f e c t s when i n j e c t e d i n t o t h e h i p p o c a m p u s r n v i v o , h o w e v e r t h e m e c h a n i s m s u n d e r l y i n g t h e s e a c t i o n s a r e n o t w e l l u n d e r -s t o o d . W i t h t h e a i m o f p r o v i d i n g a n i n s i g h t i n t o t h e s e p r o p e r t i e s o f k a i n a t e , t h e e l e c t r o p h y s i o l o g i c a l c h a n g e s p r o d u c e d by t h e a m i n o a c i d w e r e e x a m i n e d i n e a c h m a j o r h i p p o c a m p a l r e g i o n . S i n c e i n t r a c e r e b r a l i n j e c t i o n s o f k a i n a t e c a n r e p l i c a t e many o f t h e b e h a v i o r a l a n d n e u r o p a t h o l o g i c a l s y m p t o m s o f n e u r o d e g e n e r a t i v e d i s e a s e s s u c h as H u n t i n g t o n ' s c h o r e a and t e m p o r a l l o b e e p i l e p s y t h e r e i s b o t h s c i e n t i f i c and c l i n i c a l i n t e r e s t i n t h e i d e n t i f i c a t i o n o f t h e n a t u r a l l y - o c c u r r i n g l i g a n d f o r t h e k a i n i c a c i d r e c e p t o r . A compound w h i c h has r e c e n t l y r e c e i v e d a t t e n t i o n i n t h i s c a p a c i t y i s t h e v i t a m i n f o l i c a c i d w h i c h has been shown t o c a u s e k a i n a t e - l i k e s e i z u r e s 7 and t o x i c i t y . T h e r e f o r e i n t h e f i n a l c h a p t e r t h e q u e s t i o n o f a p h y s i o l o g i c a l " i d e n t i t y o f a c t i o n " b e t w e e n k a i n a t e a n d f o l a t e i s a d d r e s s e d . 8 CHAPTER 2 PREPARATION AND MAINTENANCE OF THE HIPPOCAMPAL S L I C E IN VITRO AND CHARACTERIZATION OF EVOKED RESPONSES I n t r o d u c t i o n S i n c e i t was f i r s t r e p o r t e d by Yamamoto and M c l l w a i n ( 1966 ) t h e r e c o r d i n g o f e l e c t r o p h y s i o l o g i c a l r e s p o n s e s i n  v i t r o h a s been s u c c e s s f u l l y a p p l i e d t o t i s s u e s o b t a i n e d f r o m v a r i o u s r e g i o n s o f t h e CNS. T h e s e i n c l u d e t h e s p i n a l c o r d , s u b s t a n t i a n i g r a , s e p t u m , o l f a c t o r y - a n d n e o c o r t e x , t h a l a m u s , c e r e b e l l u m and o p t i c t e c t u m ( f o r r e v i e w s e e K e r k u t and W h e a l , 1 9 8 1 ) . P e r h a p s t h e most e x t e n s i v e l y s t u d i e d o f t h e s e , t h e h i p p o c a m p a l s l i c e , has been t h e f o c u s o f t h e i n v e s t i g a t i o n s d e s c r i b e d i n t h i s t h e s i s . I n t h e f i r s t p a r t o f t h i s c h a p t e r t h e m e t h o d s f o r t h e p r e p a r a t i o n and m a i n t e n a n c e o f t h e r a t h i p p o c a m p a l s l i c e i n  v i t r o a r e d e s c r i b e d . S e v e r a l e x c e l l e n t r e v i e w s ( K e r k u t and W h e a l , 1 9 8 1 ; D i n g l e d i n e e_t a l . , 1980; L y n c h , 1980; T e y l e r , 1980) o f t h e s e p r o c e d u r e s have b e e n p u b l i s h e d r e c e n t l y and a r e recommended t o t h e r e a d e r . I n t h e m a i n t h e r e a r e o n l y m i n o r d i f f e r e n c e s b e t w e e n t h e t e c h n i q u e s r e p o r t e d h e r e a nd t h o s e d e s c r i b e d by o t h e r a u t h o r s . The m a j o r a i m o f t h i s c h a p t e r i s t o c h a r a c t e r i z e t h e e v o k e d p o t e n t i a l s o c c u r r i n g i n C A l , d e n t a t e g y r u s a n d CA3 9 p r e p a r a t o r y t o i n v e s t i g a t i o n s o f e l e c t r o p h y s i o l o g i c a l c h a n g e s e l i c i t e d by e x c i t a t o r y amino a c i d a g o n i s t s and a n t a g o n i s t s . B e c a u s e t h e y have been w e l l d e s c r i b e d by o t h e r s , t h e e v o k e d a c t i v i t y b o t h i n C A l and t h e d e n t a t e g y r u s i s c o n s i d e r e d o n l y b r i e f l y . S i n c e s u b s t a n t i a l l y l e s s i n f o r m a t i o n i s a v a i l a b l e r e g a r d i n g t h e n a t u r e o f and t h e b a s i s f o r t h e i r g e n e r a t i o n , a more d e t a i l e d c o n s i d e r a t i o n o f t h e CA3 e l e c t r o p h y s i o l o g y has b e en u n d e r t a k e n . G e n e r a l M e t h o d s  I s o l a t i o n , c u t t i n g and m a i n t e n a n c e o f h i p p o c a m p a l t i s s u e U n a n a e s t h e t i z e d m a l e a l b i n o r a t s ( W i s t a r , 150-250 gm) w e r e d e c a p i t a t e d a n d t h e s c a l p was r e f l e c t e d f o l l o w i n g a m i d l i n e i n c i s i o n . The p a r i e t a l , i n t e r p a r i e t a l a n d o c c i p i t a l b o n es were t h e n c a r e f u l l y removed u s i n g h e a v y f o r c e p s and t h e d u r a c o v e r i n g t h e b r a i n was r e s e c t e d . C o r o n a l c u t s a n t e r i o r -l y , a t t h e l e v e l o f t h e f r o n t o - p a r i e t a l s u t u r e , and p o s t e r i o r -l y a t t h e c e r e b e l l o - p o n t i n e a n g l e w e re made t o f r e e t h e b r a i n . A f t e r r e m o v a l f r o m t h e s k u l l , t h e b r a i n was p l a c e d i n a b e a k e r c o n t a i n i n g c o l d (8-12 °C) g a s s e d ( 0 2 / C 0 2 : 95/5%) a r t i f i c i a l c e r e b r o s p i n a l f l u i d ( A C S F ) . T h i s l a t t e r t r e a t m e n t washed away any p o o l e d b l o o d a n d , by c a u s i n g i t t o become f i r m e r , f a c i l i t a t e d s u b s e q u e n t h a n d l i n g o f t h e t i s s u e . A f t e r 30-60 s e c t h e b r a i n was removed f r o m t h e b e a k e r , p l a c e d on ACSF-m o i s t e n e d f i l t e r p a p e r and b i s e c t e d s a g i t a l l y u s i n g a s c a l p e l . M i d b r a i n and d i e n c e p h a l i c s t r u c t u r e s were t h e n d i s s e c t e d f r o m t h e r i g h t h e m i s p h e r e t o e x p o s e t h e v e n t r o - m e d i a l a s p e c t o f t h e 10 hippocampus and i t s s e p t a l - a n t e r i o r and t e m p o r a l - p o s t e r i o r c o n n e c t i o n s were c u t . A t e f l o n - c o a t e d s p a t u l a i n s e r t e d b e n e a t h t h e v e n t r o - m e d i a l m a r g i n i n t o t h e c a v i t y o f t h e v e n t r i c l e was u s e d t o r o l l o v e r t h e hippocampus so t h a t t h e v e n t r o - m e d i a l s u r f a c e now f a c e d t h e f i l t e r p a p e r a n d t h e a l v e a r s u r f a c e was u p p e r m o s t . Any r e m a i n i n g c o n n e c t i o n s were c u t and t h e i s o l a t e d hippocampus was l i f t e d w i t h a s p a t u l a and p l a c e d on i t s v e n t r o - m e d i a l s i d e on t h e c u t t i n g s t a g e o f a M c l l w a i n t i s s u e c h o p p e r . T o p r e s e r v e t h e i n t e g r i t y o f t h e t r i - s y n a p t i c l o o p w i t h i n e a c h s l i c e , t h e l o n g a x i s o f t h e hippocampus was p l a c e d a t an a n g l e o f 7 0 ° t o t h e c u t t i n g b l a d e ( c f . F i g . 8 o f T e y l e r , 1 9 8 0 ) . S l i c e s o f a 400^A,m t h i c k n e s s ( n o m i n a l ) t a k e n f r o m t h e m i d d l e t h i r d o f t h e r i g h t hippocampus were t r a n s f e r r e d f r o m t h e c u t t i n g b l a d e ( G i l l e t t e S u p e r B l u e ) t o c o l d , g a s s e d ACSF u s i n g a f i n e - t i p p e d s o f t - b r i s t l e d b r u s h . I n t h i s manner 6-8 s l i c e s were o b t a i n e d and t r a n s f e r r e d , u s i n g a wide-mouthed d r o p p e r , t o t h e n y l o n mesh s u p p o r t o f t h e s l i c e b a t h i n t h e i n c u b a t i o n c h a m b e r . T h e l e v e l o f t h e p e r f u s i n g ACSF was a d j u s t e d t o m a i n t a i n a t h i n l a y e r o v e r t h e s u r f a c e o f t h e s l i c e s . The e n t i r e p r o c e d u r e n o r m a l l y r e q u i r e d 7-9 m i n u t e s . To o b t a i n h e a l t h y s l i c e s i t was i m p o r t a n t t o m i n i m i z e m e c h a n i c a l trauma t o t h e hippocampus d u r i n g r e m o v a l f r o m t h e b r a i n and s l i c i n g . A p e r i o d o f 1-1.5 h was a l l o w e d f o r e q u i l i b r a t i o n b e f o r e e x p e r i m e n t s began. A d i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e p e r f u s i o n 11 a p p a r a t u s and t h e i n c u b a t i o n chamber i s g i v e n i n F i g . 2.1. D i s t i l l e d w a t e r i n t h e o u t e r p l e x i g l a s s w a t e r j a c k e t was m a i n t a i n e d a t 34 °C by DC c u r r e n t a p p l i e d t o a s u b m e r g e d n i c h r o m e c o i l v i a a t h e r m i s t o r - c o n t r o l l e d h e a t i n g c i r c u i t . By b u b b l i n g i t t h r o u g h t w o a i r s t o n e s i m m e r s e d i n t h i s h e a t e d w a t e r t h e g a s ( 0 2 / C 0 2 : 95/5%) f o r m i n g t h e a t m o s p h e r e o f t h e c hamber was warmed and h u m i d i f i e d . G a s s e d ACSF prewarmed t o 34 °C ( t o p r e v e n t d e g a s s i n g i n t h e f e e d l i n e s ) was d r a w n t h r o u g h a p e r i s t a l t i c pump a n d d r i p p e d a t a r a t e o f c a . 1.0 m l / m i n i n t o a r e s e r v o i r . By a d j u s t i n g t h e h e i g h t o f t h e r e s e r v o i r t h e o u t f l o w t o t h e b a t h was m a t c h e d t o t h e i n f l o w and as l o n g as t h i s c o n d i t i o n was m a i n t a i n e d , a c o n s t a n t p r e s s u r e h e a d e x i s t e d f o r p e r f u s i o n o f t h e s l i c e s w i t h ACSF. The o u t f l o w f r o m t h e r e s e r v o i r e n t e r e d t h e w a t e r j a c k e t t h r o u g h p o l y e t h y l e n e t u b i n g , was warmed a g a i n t o 34 °C and e n t e r e d t h e s l i c e b a t h . H e r e t h e ACSF c o u r s e d b e l o w a n d a r o u n d t h e s l i c e s t o t h e o t h e r s i d e o f t h e b a t h where i t was removed by a s u c t i o n n e e d l e . T h e s y s t e m ' s d e s i g n c a u s e d a l a g t i m e o f c a . 3.5 m i n b e t w e e n a c h a n g e o v e r t o d r u g - c o n t a i n i n g p e r f u s a t e and i t s a r r i v a l a t t h e e n v i r o n s o f t h e s l i c e . F u r t h e r m o r e , t h e s l i c e b a t h v o l u m e r e q u i r e d t h a t a p p r o x i m a t e l y 10 ml be p e r f u s e d t o p r o d u c e a c o m p l e t e r e p l a c e m e n t o f m e d i u m s u r r o u n d i n g t h e s l i c e . The i o n i c c o m p o s i t i o n o f t h e ACSF was (mM): N a C l ( 1 2 4 ) ; N a H 2 P 0 4 ( 1 . 2 5 ) , NaHC0 3 ( 2 6 ) , KC1 ( 5 ) , C a C l 2 ( 2 ) , M g S 0 4 (2) and D - g l u c o s e ( 1 0 ) . The pH was m a i n t a i n e d a t 7.3-7.4 by c o n s t a n t 12 F i g . 2.1 A s c h e m a t i c o u t l i n e o f t h e a p p a r a t u s f o r t h e c o n s t a n t f l o w ( c a . 1 m l / m i n ) p e r f u s i o n o f h i p p o c a m p a l s l i c e s i n v i t r o w i t h a r t i f i c i a l c e r e b r o s p i n a l f l u i d ( A C S F ) . The p l a n e s o f t h e c r o s s - s e c t i o n a l v i e w s o f t h e s l i c e b a t h i n B and C a r e i n d i c a t e d i n A; t h e b a t h i s drawn a p p r o x i m a t e l y t o s c a l e i n A. I n t h e e x p e r i m e n t s , s l i c e s on t h e n y l o n s u p p o r t n e t were i l l u m i n a t e d f r o m b e l o w and w i t h t h e a i d o f t h e o v e r h e a d m i c r o s c o p e (1.6X-4X) r e c o r d i n g e l e c t r o d e s were p o s i t i o n e d i n t h e c e l l body l a y e r , w h i c h was d i s t i n g u i s h a b l e a s a band o f more t r a n s l u c e n t t i s s u e , a n d / o r i n t h e d e n d r i t i c l a y e r o f t h e h i p p o c a m p a l f i e l d t o be e x a m i n e d . I n a s i m i l a r f a s h i o n , s t i m u l a t i n g e l e c t r o d e s w e r e p l a c e d t o a c t i v a t e t h e a p p r o p r i a t e a f f e r e n t and e f f e r e n t p a t h w a y s . The numbers i n t h e f i g u r e r e f e r t o : 1. t h e r m i s t o r , 2. w a t e r j a c k e t , 3. h e a t i n g c o i l , 4. ACSF o u t l e t p o r t , 5. A g / A g C l r e f e r e n c e e l e c t r o d e , 6. n y l o n n e t s u p p o r t r i n g , 7. s l i c e b a t h , 8. ACSF i n l e t p o r t , 9. p o l y e t h y l e n e t u b i n g , 10. a i r s t o n e s p r o v i d i n g 0^/Q.O^'.§b/b %, 1 1 . p e r i s t a l t i c pump, 12. i n l e t f o r Q^/dO^'.^b/b %, 13. pre- w a r m e d ACSF, 14. Haake w a t e r b a t h ( c a . 34°C), 15. o v e r h e a d m i c r o s c o p e , 16. n y l o n n e t , 17. s u c t i o n n e e d l e , 18. d i s t i l l e d w a t e r ( c a . 34°C), 19. f i b r e o p t i c s f o r i l l u m i n a t i o n o f s l i c e s , 20. ACSF r e s e r v o i r . 13 14 b u b b l i n g w i t h 0 2 / C 0 2 : 9 5 / 5 % . . I n s t u d i e s where t h e e f f e c t o f c h a n g e s o f i o n i c c o m p o s i t i o n on n e u r o n a l a c t i v i t y was e x a m i n e d , o s m o l a r i t y was m a i n t a i n e d by a d j u s t i n g t h e c o n c e n -2 + t r a t i o n o f N a C l e x c e p t : a) i n l o w Ca - c o n t a i n i n g medium (0.2 mM) t h e c o n c e n t r a t i o n o f MgSO^ was i n c r e a s e d t o 4.7 mM and b) i n C I r e p l a c e m e n t e x p e r i m e n t s where s o d i u m p r o p i o n a t e was s u b s t i t u t e d f o r N a C l . P r e p a r a t i o n o f i o n t o p h o r e t i c e l e c t r o d e s M u l t i b a r r e l e d e l e c t r o d e s f o r i o n t o p h o r e s i s were drawn on a v e r t i c a l p u l l e r and t h e t i p b r o k e n back t o a d i a m e t e r o f 5 - 1 0 ^ m u n d e r v i s u a l c o n t r o l w i t h a L e i t z m i c r o - m a n i p u l a t o r . B a r r e l s were t h e n w a t e r - f i l l e d by s u b m e r s i o n i n b o i l i n g d i s t i l l e d w a t e r , a l l o w e d t o c o o l and t h e w a t e r t o w i t h i n 1 cm o f t h e t i p s was removed by s u c t i o n . Once f i l l e d w i t h t h e t e s t d r u g s o l u t i o n s a t l e a s t 18 h was a l l o w e d f o r d i f f u s i o n t o t h e b a r r e l s ' t i p s . When i t was ne e d e d f o r r e c o r d i n g , t h e c e n t r a l b a r r e l o f t h e e l e c t r o d e was f i l l e d w i t h 1 o r 4 M N a C l ( 0 . 5 - 2 . 0 megohms). S t i m u l a t i o n and r e c o r d i n g o f f i e l d r e s p o n s e s M o n o p h a s i c s q u a r e wave p u l s e s ( 0 . 1 msec d u r a t i o n , up t o 80 V, <100 y * A ) f r o m a c o n s t a n t - v o l t a g e i s o l a t e d s t i m u l a t o r w e r e d e l i v e r e d i n some e x p e r i m e n t s t h r o u g h a g l a s s m i c r o -e l e c t r o d e ( 1 M N a C l - f i l l e d ; t i p d i a m e t e r 1-2_/*m) b u t m o r e commonly v i a a b i p o l a r e l e c t r o d e c o m p r i s e d o f 62y*m d i a m e t e r s t a i n l e s s s t e e l w i r e s t w i s t e d t o g e t h e r and i n s u l a t e d t o t h e i r 15 t i p s . S t i m u l u s f r e q u e n c y was 0.05-0.1 Hz e x c e p t where n o t e d . The p o s i t i o n s o f s t i m u l a t i n g e l e c t r o d e s a r e i l l u s t r a t e d i n F i g . 2.3-2.5. F i g . 2.2 i l l u s t r a t e s t h e a r r a n g e m e n t o f t h e a p p a r a t u s f o r r e c o r d i n g e x t r a - and i n t r a c e l l u l a r r e s p o n s e s ; i n p u t a n d o u t p u t d e v i c e s a r e i d e n t i f i e d i n t h e l e g e n d t o t h a t f i g u r e . T o r e c o r d e x t r a c e l l u l a r s o m a t i c o r d e n d r i t i c f i e l d r e s p o n s e s , a s i n g l e g l a s s e l e c t r o d e o r t h e c e n t r a l b a r r e l o f a m u l t i - b a r r e l e d i o n t o p h o r e t i c e l e c t r o d e was u s e d . S i n g l e -b a r r e l e d r e c o r d i n g e l e c t r o d e s p u l l e d f r o m 1.5 mm m i c r o -c a p i l l a r y t u b i n g w e r e f i l l e d w i t h 1 o r 4 M N a C l a n d h a d r e s i s t a n c e s o f 1-5 megohms. A c h l o r i d e d s i l v e r w i r e c o n n e c t e d t h e N a C l s o l u t i o n o f t h e e l e c t r o d e b a r r e l t o t h e p r e a m p l i f i e r i n p u t w h i c h f e d t o t h e i n v e r t i n g o r n o n - i n v e r t i n g i n p u t o f a d i f f e r e n t i a l a m p l i f i e r on a T e k t r o n i x 5111 d u a l beam s t o r a g e o s c i l l o s c o p e . The s i g n a l was r e c o r d e d r e l a t i v e t o a g r o u n d e d s i l v e r / s i l v e r c h l o r i d e w i r e l o c a t e d i n t h e b o t t o m o f t h e s l i c e b a t h ( F i g . 2 . 2 ) . F o r t h e e x p e r i m e n t s a N a r i s h i g e m i c r o m a n i p u l a t o r a d v a n c e d a r e c o r d i n g e l e c r o d e u n d e r v i s u a l c o n t r o l t h r o u g h t h e a p p r o p r i a t e r e g i o n o f t h e s l i c e t o o b t a i n t h e maximum f i e l d p o t e n t i a l e v o k e d by a l o w f r e q u e n c y (<0.2 Hz) a c t i v a t i o n o f t h e t e s t p a t h w a y . O n c e t h e o p t i m u m r e c o r d i n g d e p t h was e s t a b l i s h e d , t h e s t i m u l a t i n g e l e c t r o d e was s l o w l y a d v a n c e d u n t i l f u r t h e r i n c r e a s e s o f t h e r e s p o n s e were n o t o b t a i n a b l e ; most o f t e n a p o s i t i o n o f t h e s t i m u l a t i n g e l e c t r o d e t i p w i t h i n 10-20^am o f t h e s l i c e s u r f a c e was u s e d . 16 A B Current Pump Main Oscillo-scope Stimulus Timer Micromanipulators Ag/AgCI Reference Electrode Computer Chart R a t e . Recorder meter Record Oscillo-scope Piezoelectric Microdrive Amplifier Heodstage \ - A g / A g C l half-cell —<<* Audio Discriminator /Ratemeter Monitor Balance • Probe Output to main oscilloscope D C Amplifier Probe Driver (to stim. timer) Cycle Timer Microdrive Control F i g . 2.2 A r r a n g e m e n t o f i n p u t and o u t p u t d e v i c e s f o r t h e r e c o r d i n g o f (A) e x t r a c e l l u l a r a n d ( B ) i n t r a c e l l u l a r r e s p o n s e s . M a n u a l l y o p e r a t e d m i c r o m a n i p u l a t o r s were u s e d t o p o s i t i o n (a) m u I t i b a r r e l e d i o n t o p h o r e t i c - , (b) s i n g l e b a r r e l e d - and ( c ) b i p o l a r m e t a l s t i m u l a t i n g e l e c t r o d e s i n t h e s l i c e ( l e f t o f u p p e r f i g u r e ) . In a d d i t i o n to. t h e s e , i n t r a -c e l l u l a r e x p e r i m e n t s a l s o i n c o r p o r a t e d a p i e z o e l e c t r i c m i c r o d r i v e t o advance an e l e c t r o d e ( l o w e r f i g u r e ) t o i m p a l e h i p p o c a m p a l n e u r o n s . The d e v i c e s ( n u m b e r e d ) u s e d f o r t h e r e c o r d i n g and a n a l y z i n g r e s p o n s e s were: 1. T e k t r o n i x 5111 d u a l beam s t o r a g e o s c i l l o s c o p e , 2. D i g i t i m e r D100 a n d i s o l a t e d s t i m u l a t o r DS 2, 3. W i n s t o n E l e c t r o n i c s RAD-II-A window d i s c r i m i n a t o r / r a t e m e t e r , 4. T e k t r o n i x 502 d u a l beam o s c i l l o s c o p e , 5. E s t e r l i n e Angus 601R r e c t i l i n e a r r e c o r d e r , 6. G o u l d 2200 b r u s h r e c o r d e r , 7. A p p l e I I computer, 8. W-P I n s t r u m e n t s M-701 m i c r o p r o b e DC a m p l i f i e r , 9. O r t e c p r o g r a m and d e l a y c o n t r o l , 10. B u r l e i g h Inchworm c o n t r o l l e r PZ-500. 17 A m p l i t u d e s o f e v o k e d r e s p o n s e s were m e a s u r e d d i r e c t l y f r o m t h e o s c i l l o s c o p e , f r o m f i l m e d r e c o r d s o r on l i n e w i t h an A p p l e c o m p u t e r . E P S P s w e r e m e a s u r e d f r o m t h e p r e - s t i m u l u s b a s e l i n e t o t h e peak a m p l i t u d e e x c e p t where n o t e d ; a m p l i t u d e s o f P S s were c a l c u l a t e d f r o m t h e b a s e l i n e o r t h e p r e c e d i n g peak p o s i t i v i t y t o t h e peak n e g a t i v i t y . I n t r a c e l l u l a r r e c o r d i n g I n t r a c e l l u l a r r e c o r d s f r o m C A l and CA3 n e u r o n s were made u s i n g c o n v e n t i o n a l t e c h n i q u e s . M i c r o e l e c t r o d e s ( F r e d e r -i c k Haer-Omega D o t ; 1.5 mm O.D.) were p u l l e d j u s t b e f o r e u s e on a N a r i s h i g e PE-2 v e r t i c a l p u l l e r , b a c k - f i l l e d w i t h 1 M p o t a s s i u m a c e t a t e and c o n n e c t e d t o t h e s i l v e r / s i l v e r c h l o r i d e h a l f c e l l o f t h e e l e c t r o m e t e r (WP I n s t r u m e n t s , M-701) v i a a 1 M N a C l s o l u t i o n . S i g n a l s were r e c o r d e d w i t h r e s p e c t t o t h e g r o u n d e d s i l v e r / s i l v e r c h l o r i d e w i r e i n t h e b a t h ( c f . F i g . 2.2) . The m i c r o e l e c t r o d e , t i s s u e a n d r e c o r d i n g chamber f o r m e d •one arm o f a W h e a t s t o n e b r i d g e . W i t h t h e e l e c t r o d e t i p i n t h e e x t r a c e l l u l a r f l u i d , c u r r e n t was i n j e c t e d t h r o u g h t h e e l e c t r o d e and i t s r e s i s t a n c e (40-60 megohms) was n u l l e d by a d j u s t i n g t h e b r i d g e b a l a n c e . U s i n g a p i e z o e l e c t r i c m i c r o -d r i v e , t h e e l e c t r o d e was s l o w l y a d v a n c e d and i m p a l e m e n t o f c e l l s was a i d e d by o v e r c o m p e n s a t i n g t h e n e g a t i v e c a p a c i t y a d j u s t m e n t . I n p u t r e s i s t a n c e o f n e u r o n s was e s t i m a t e d by t h e s t e a d y s t a t e v o l t a g e d e f l e c t i o n c a u s e d by h y p e r p o l a r i z i n g c u r r e n t s o f up t o -1 nA and c a . 200 msec i n d u r a t i o n . The 18 a m p l i f i e d o u t p u t s i g n a l was d i s p l a y e d on an o s c i l l o s c o p e and p l o t t e d on a G o u l d DC c h a r t r e c o r d e r . The a m p l i t u d e s o f a c t i o n p o t e n t i a l s , EPSPs and I P S P s w e r e m e a s u r e d e i t h e r d i r e c t l y f r o m t h e o s c i l l o s c o p e , f r o m f i l m e d r e c o r d s o r f r o m t h e c h a r t r e c o r d s . The r e s t i n g membrane p o t e n t i a l was d e t e r m i n e d a f t e r w i t h d r a w a l f r o m t h e c e l l . R e s u l t s The v a l u e o f d a t a o b t a i n e d f r o m t h e h i p p o c a m p a l s l i c e i n v i t r o i s d e p e n d e n t o n t h e p r e s e r v a t i o n o f e l e c t r o p h y s -i o l o g i c a l a c t i v i t i e s r e c o r d e d i_n s i t u , a n d t h e r e s u l t s o f o t h e r s have i n d i c a t e d a c o n s i d e r a b l e p a r a l l e l i s m i n n e u r o n a l f u n c t i o n b e t w e e n t h e two p r e p a r a t i o n s ( A l g e r and T e y l e r , 1976; S c h w a r t z k r o i n , 1 9 7 5, 1 9 7 7 ) . The p r e s e r v a t i o n o f t h i s a p p a r e n t l y n o r m a l a c t i v i t y in v i t r o i s a l s o c o r r e l a t e d w i t h t h e p e r s i s t e n c e o f n o r m a l h i s t o l o g i c a l f e a t u r e s ( F r o t s c h e r e_t a l . , 1 9 8 1 ; C o b b e t t a nd C o t t r e l l , 1 9 8 1 ) . The f o l l o w i n g c r i t e r i a were e s t a b l i s h e d t o v e r i f y t h e e l e c t r o p h y s i o l o g i c a l i n t e g r i t y o f t h e s l i c e s a n d e n s u r e c o n s i s t e n c y b e t w e e n e x p e r i m e n t s . G e n e r a l l y , s l i c e s were j u d g e d t o be u n a c c e p t a b l e i f t h e i r r e s p o n s e s i n n o r m a l ACSF were c l e a r l y d i f f e r e n t f r o m t h o s e r e p o r t e d as o c c u r r i n g i n  v i t r o o r ir\ v i v o . T h e r e f o r e , s l i c e s were d i s c a r d e d f o r one o r m o r e o f t h e f o l l o w i n g r e a s o n s . F i r s t , t h e a p p e a r a n c e i n e i t h e r C A l o r t h e d e n t a t e g y r u s o f a s e c o n d a r y (2°) p o p u l a t i o n s p i k e (PS) i n t h e o r t h o d r o m i c a l l y - e v o k e d r e s p o n s e . T h i s was 19 i n t e r p r e t e d as a f a i l u r e o f s y n a p t i c i n h i b i t i o n . S e c o n d , i f t h e r a t i o o f t h e f i b r e v o l l e y : E P S P a m p l i t u d e s r e c o r d e d i n t h e d e n d r i t i c l a y e r was g r e a t e r t h a n c a . 1:2. M o s t o f t e n t h i s was c o r r e l a t e d w i t h a r e q u i r e m e n t f o r h i g h i n t e n s i t i e s o f s t i m u l a t i o n t o p r o d u c e a d e n d r i t i c EPSP. T h i r d , i f i n C A l o r t h e d e n t a t e g y r u s t h e maximum a n t i d r o m i c PS w h i c h c o u l d be e l i c i t e d was l e s s t h a n 15 mV ( b a s e l i n e t o peak n e g a t i v i t y ) ; i n CA3 l e s s t h a n 10 mV. F o u r t h , t h e a p p e a r a n c e i n CA3 o f s p o n t a n e o u s o r e v o k e d a c t i v i t y c o m p r i s e d o f b u r s t s o f P S s , an e f f e c t w h i c h was a t t r i b u t e d t o a l o s s o f i n h i b i t i o n . A b e r r a n t e l e c t r o p h y s i o l o g i c a l a c t i v i t y was o f t e n c o r r e l a t e d w i t h an a t y p i c a l a p p e a r a n c e o f t h e t i s s u e . F o r e x a m p l e , t h o s e s l i c e s w h i c h w e r e g r o s s l y e d e m a t o u s a t a n y s t a g e o f t h e i r i n c u b a t i o n o r had i n d i s t i n c t c e l l l i n e s o f t e n showed p o o r e l e c t r i c a l a c t i v i t y . T h i s was a l s o t h e c a s e i n s l i c e s w h i c h h a d a g r a n u l a r , m o t t l e d a p p e a r a n c e t o t h e i r s u r f a c e s - a c o n d i t i o n u s u a l l y a t t r i b u t a b l e t o p o o r h u m i d i f -i c a t i o n o r i m p r o p e r a d j u s t m e n t o f t h e g a s f l o w r a t e o r t h e ACSF l e v e l i n t h e b a t h . C h a r a c t e r i z a t i o n o f f i e l d r e s p o n s e s C A l T h e p o s i t i o n s o f s t i m u l a t i n g ( S ) a n d r e c o r d i n g (R) e l e c t r o d e s a r e i l l u s t r a t e d i n F i g . 2.3A. O r t h o d r o m i c a c t i v a t i o n o f S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l (SC) f i b r e s i n t h e s t r a t u m r a d i a t u m (S ) w i t h low i n t e n s i t y ( F i g . 2.3C: 5V) e l i c i t e d a d e n d r i t i c r e s p o n s e ( R 2 ' 100-200j^m f r o m t h e soma) 20 C. Orthodromic Antidromic E P S P Population Spike S C ( S A ) S C ( S A ) F i g . 2.3 A. P o s i t i o n s o f e l e c t r o d e s f o r s t i m u l a t i n g and r e c o r d i n g e x t r a c e l l u l a r r e s p o n s e s i n "the C A l r e g i o n . A s t i m u l a t i n g e l e c t r o d e ( S A ) i n t h e s t r a t u m r a d i a t u m a c t i v a t e d t h e S e h a f f e r c o l l a t e r a l - c o m m i s s u r a l (SC) i n p u t ; s t i m u l a t i o n o f e f f e r e n t f i b r e s i n t h e a l v e u s ( S D ) e x c i t e d C A l p y r a m i d s a n t i d r o m i c a l l y . D e n d r i t i c and s o m a t i c f i e l d p o t e n t i a l s were r e c o r d e d w i t h e l e c t r o d e s i n t h e s t r a t u m r a d i a t u m (R^-'^n beam" w i t h S^) and t h e s t r a t u m p y r a m i d a l e ( R ^ ) , r e s p e c t i v e l y . B. S i m p l i f i e d s c h e m a t i c o f C A l n e u r o n a l c i r c u i t r y . The p y r a m i d a l c e l l r e c e i v e s SC e x c i t a t o r y i n p u t ( i i ) t o t h e a p i c a l d e n d r i t e s a n d f e e d b a c k i n h i b i t i o n r e s u l t s f r o m t h e a c t i v a t i o n o f a G A B A e r g i c i n t e r n e u r o n ( i i i ) v i a a c o l l a t e r a l axon ( i v ) . E f f e r e n t s ( i ) o f C A l p y r a m i d a l c e l l s c o u r s e t h r o u g h t h e a l v e u s . C. T y p i c a l C A l f i e l d EPSPs ( R 2 ? p r e s y n a p t i c f i b r e v o l l e y i n d i c a t e d by f i l l e d s q u a r e below r e c o r d s ) and s o m a t i c f i e l d p o t e n t i a l s (R,) e v o k e d by a n t i d r o m i c (S_J and v a r i o u s i n t e n s i t i e s o f o r t h o d r o m i c (S.) s t i m u l i . See t e x t f o r e x p l a n a t i o n . In t h i s and s u b s e q u e n t r e c o r d s the time o f t h e s t i m u l u s i s i n d i c a t e d by an a r r o w . 21 c o n s i s t i n g o f two p r e d o m i n a n t l y n e g a t i v e w a v e s . The f i r s t o f t h e s e r e p r e s e n t e d t h e d e p o l a r i z a t i o n o f p r e s y n a p t i c f i b r e s a n d t e r m i n a l s ( p r e s y n a p t i c f i b r e v o l l e y ; A n d e r s e n e t a _ l . , 1 9 7 8 ) . T h e s e c o n d a n d l a r g e r wave ( f i e l d e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l , EPSP; A n d e r s e n , 1960) r e f l e c t e d t h e c u r r e n t f l o w a r i s i n g f r o m t h e d e n d r i t i c c o n d u c t a n c e change p r o d u c e d p o s t s y n a p t i c a l l y . S i n c e t h e c e l l body b e h a v e s as a c u r r e n t s o u r c e f o r t h e d e n d r i t i c E P S P , t h i s was r e c o r d e d i n t h e s t r a t u m p y r a m i d a l e (R-^) as a p o s i t i v e - g o i n g wave ( r e v e r s e d E P S P ) . As t h e s t i m u l u s i n t e n s i t y was i n c r e a s e d ( F i g . 2.3C: 7V) t h e t h r e s h o l d f o r f i r i n g i n some c e l l s was r e a c h e d and a n e g a t i v e - g o i n g p o p u l a t i o n s p i k e (PS) r e p r e s e n t i n g t h e s y n c h r o n o u s m o n o s y n a p t i c d i s c h a r g e o f s o m a t i c a c t i o n p o t e n -t i a l s ( A n d e r s e n et. a _ l . , 1966) was s u p e r i m p o s e d on t h e r e v e r s e d EPSP. B e c a u s e i n t h i s s i t u a t i o n t h e d e n d r i t e s a c t as a s o u r c e f o r t h e c u r r e n t s i n k o c c u r r i n g i n t h e s t r a t u m p y r a m i d a l e a p o s i t i v e - g o i n g wave ( r e v e r s e d PS) was s o m e t i m e s a p p a r e n t i n t h e f i e l d p o t e n t i a l r e c o r d e d i n t h e d e n d r i t e s . A n t i d r o m i c (AD) a c t i v a t i o n o f C A l c e l l s was p r o d u c e d by e x c i t i n g e f f e r e n t a x o n s c o u r s i n g t h r o u g h t h e a l v e u s (S ) . I n s t r a t u m p y r a m i d a l e t h e AD - e v o k e d r e s p o n s e c o n s i s t e d o f a s h o r t l a t e n c y PS f o l l o w e d by a s m a l l a m p l i t u d e p o s i t i v e wave ( F i g . 2.3C, S _ ) . As w o u l d be e x p e c t e d , t h e p o t e n t i a l r e c o r d e d i n t h e d e n d r i t e s i n t h i s s i t u a t i o n was c o m p r i s e d o f a r e v e r s e d PS o n l y ( n o t s h o w n ) . A l v e a r s t i m u l a t i o n , i n a d d i t i o n t o c a u s i n g AD i n v a s i o n o f C A l p y r a m i d a l c e l l s , p r o d u c e d a m o n o s y n a p t i c a c t i v a t i o n , 22 v i a a r e c u r r e n t c o l l a t e r a l p a t h w a y ( F i g . 2 . 3 B ) , o f i n h i b i t o r y i n t e r n e u r o n s r e l e a s i n g ^ - a m i n o - b u t y r i c a c i d ( G A B A ) . M e a s u r e -m e n t o f t h i s r e c u r r e n t i n h i b i t i o n c o u l d be made i n t h e f o l l o w i n g manner. An o r t h o d r o m i c s t i m u l u s e v o k i n g a PS was on a l t e r n a t e o c c a s i o n s p r e c e d e d (15-30 msec) by c o n d i t i o n i n g AD a c t i v a t i o n . The i n h i b i t i o n was t h e n c a l c u l a t e d a s t h e p e r c e n t d i f f e r e n c e b e t w e e n t h e mean a m p l i t u d e (3-6 d e t e r m i n a t i o n s ) o f t h e c o n d i t i o n e d ( C ) a n d u n c o n d i t i o n e d (U) o r t h o d r o m i c PS ( P S u - P S c / P S L J x l O O % ) . T h e same m e t h o d was a p p l i e d i n t h e a s s e s s m e n t o f i n h i b i t i o n i n t h e d e n t a t e g y r u s a n d t h e CA3 r e g i o n . D e n t a t e g y r u s F i g . 2.4A i n d i c a t e s t h e p o s i t i o n s o f s t i m u l a t i n g and r e c o r d i n g e l e c t r o d e s . Low i n t e n s i t y s t i m u l a t i o n o f e i t h e r t h e m e d i a l ( S c ) o r t h e l a t e r a l (Sp) p e r f o r a n t p a t h w a y (MPP and L P P , r e s p e c t i v e l y ) p r o d u c e d i n t h e m i d d l e and o u t e r m o l e c u l a r l a y e r s r e s p e c t i v e l y a p r e s y n a p t i c f i b r e v o l l e y and EPSP ( F i g . 2.4C, R 5:5 V ) . As i n t h e C A l r e g i o n , t h e EPSP was r e v e r s e d when r e c o r d e d i n t h e s t r a t u m g r a n u l o s u m ( F i g . 2.4C, : 5 V) . T h r e s h o l d o r t h o d r o m i c s t i m u l a t i o n c a u s e d a n e g a t i v e - g o i n g PS t o a p p e a r i n t h e s o m a t i c p o t e n t i a l a nd t h i s was e v i d e n t as a r e v e r s e d PS i n t h e d e n d r i t i c r e c o r d i n g s ( F i g . 2.4C, Rj.:6.2 V) . An AD r e s p o n s e c o u l d be e l i c i t e d by a s t i m u l u s b a c k f i r i n g mossy f i b r e s (MF) i n t h e s t r a t u m l u c i d u m o f CA3 (S ) o r t h e 23 ! i C. Orthodromic Antidromic E P S P Population Spike M P P ( S C ) M P P ( S C ) F i g . 2.4 A. P o s i t i o n s o f e l e c t r o d e s i n t h e d e n t a t e g y r u s . D e n d r i t i c f i e l d p o t e n t i a l s i n t h e m i d d l e (R^) a n d o u t e r (R,-) l a y e r s o f s t r a t u m m o l e c u l a r e were e v o k e d by s t i m u l a t i o n o f t h e m e d i a l ( S c ) a n d l a t e r a l ( S Q ) p e r f o r a n t pathways (LPP and MPP) r e s p e c t i v e l y . A s t i m u l a t i n g e l e c t r o d e i n t h e s t r a t u m l u c i d u m o f t h e CA3 r e g i o n (S„) o r t h e h i l u s (Sg) was u s e d t o e x c i t e mossy f i b r e s and evoke an a n t i d r o m i c r e s p o n s e . The s o m a t i c r e s p o n s e was r e c o r d e d i n the s t r a t u m g r a n u l o s u m ( R ^ ) . B. The g r a n u l e c e l l r e c e i v e s e x c i t a t o r y s y n a p t i c i n p u t v i a t h e LPP ( i ) and MPP ( i i ) . An e f f e r e n t axon a c t i v a t e s a G A B A e r g i c i n t e r n e u r o n ( i i i ) by a r e c u r r e n t c o l l a t e r a l a n d t e r m i n a t e s i n CA3 as t h e m o s s y f i b r e i n p u t ( i v ) . C. T y p i c a l d e n d r i t i c EPSPs (R^, ; p r e s y n a p t i c f i b r e v o l l e y i n d i c a t e d by a f i l l e d s q u a r e ) and the s o m a t i c f i e l d r e s p o n s e (R3) e v o k e d by s t i m u l a t i o n o f a f f e r e n t ( S ^ , S D ) and e f f e r e n t (Sg) p a t h w a y s . Changes o f f i e l d r e s p o n s e s between s u b t h r e s h o l d (5V) and s u p r a t h r e s h o l d (6.2V) MPP s t i m u l a t i o n a r e s i m i l a r t o t h o s e which o c c u r i n C A l ( c f . F i g . 2.3C). See t e x t f o r e x p l a n a t i o n . 24 h i l u s ( S E ) ( F i g . 2 . 4 C ) . As i n C A l t h i s AD s t i m u l a t i o n c o u l d a c t i v a t e G A B A e r g i c i n t e r n e u r o n s v i a a r e c u r r e n t c o l l a t e r a l a x o n ( F i g . 2 . 4 B ) . CA3 The p o s i t i o n s o f e l e c t r o d e s e v o k i n g a n t i - a nd o r t h o -d r o m i c r e s p o n s e s i n CA3 a r e i l l u s t r a t e d i n F i g . 2.5A. To a v o i d c o m p l i c a t i o n s a r i s i n g f r o m d i f f e r i n g r e s p o n s e c h a r a c t e r -i s t i c s i n CA3 s u b r e g i o n s (Masukawa et. a _ l . , 1982) t h e r e c o r d i n g e l e c t r o d e was p o s i t i o n e d f o r e a c h e x p e r i m e n t i n t h e s t r a t u m p y r a m i d a l e (Rg) i n t h e r e g i o n c o r r e s p o n d i n g a p p r o x i m a t e l y t o CA3a ( L o r e n t e de No, 1 9 3 4 ) . The d e n d r i t i c f i e l d p o t e n t i a l was n o t r e c o r d e d . Though n o t a l w a y s t h e c a s e ( s e e b e l o w ) , a l o w i n t e n s i t y s h o c k t o t h e f i m b r i a (FIM) c o u l d e v o k e a s m a l l r e v e r s e d EPSP upon w h i c h a n e g a t i v e - g o i n g PS was s u p e r i m p o s e d ( F i g . 2.5C, S ^ - t o p t r a c e ) . ' R a i s i n g t h e s t i m u l u s i n t e n s i t y i n c r e a s e d t h e a m p l i t u d e o f b o t h t h e r e v e r s e d EPSP and PS (up t o 15 mV) and t h e l a t e n c y o f t h e PS d e c r e a s e d b u t was n e v e r l e s s t h a n 2 m s e c . U s u a l l y a h i g h e r i n t e n s i t y s h o c k a l s o r e c r u i t e d a s h o r t e r l a t e n c y PS ( 0 . 2 -0.4 msec) r e p r e s e n t i n g t h e AD i n v a s i o n ( s e e b e l o w ) o f CA3 p y r a m i d s ( F i g . 2.5C, S ^ - l o w e r t r a c e ) . H o w ever, o c c a s i o n a l l y w i t h FIM s t i m u l a t i o n i t was p o s s i b l e t o e v o k e a p u r e AD r e s p o n s e a t a l o w s t i m u l u s i n t e n s i t y ( e . g . F i g . 3 . l E i v ) . F u r t h e r , i n some s l i c e s an o r t h o d r o m i c r e s p o n s e c o u l d be e l i c i t e d w h i c h e v e n a t h i g h s t i m u l u s i n t e n s i t i e s was n o t p r e c e d e d by an AD PS ( F i g . 5 . 1 4 A ) . 25 C -FIM(S G) F i g . 2.5 A. A s c h e m a t i c o f t h e h i p p o c a m p a l s l i c e s h o w ing t h e l o c a t i o n s o f e l e c t r o d e s f o r s t i m u l a t i n g and r e c o r d i n g r e s p o n s e s i n CA3. F i e l d p o t e n t i a l s i n t h e s t r a t u m py r a m i d a l e (R^,) 'were evo.k.e.d by . s t i m u l a t i o n o f mossy f i b r e s (MF) i n t h e h i l u s (S„) , a f f e r e n t and e f f e r e n t f i b r e s (S„) i n t h e f i m b r i a ( F I M ) , and by a n t i d r o m i c a c t i v a t i o n o f S c h a f f e r c o l l a t e r a l f i b r e s i n t h e s t r a t u m r a d i a t u m o f CA1/CA2 (S ). Pi D e n d r i t i c r e s p o n s e s w e r e n o t r e c o r d e d . B. A d i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e i n p u t , o u t p u t and l o c a l c i r c u i t r y i n t h e CA3 r e g i o n . Two m a j o r e x c i t a t o r y s y n a p t i c i n p u t s a r e t h e c o m m i s s u r a l ( i i ) and MF ( i i i ) pathways w h i c h t e r m i n a t e m a i n l y on t h e b a s a l and a p i c a l d e n d r i t e s r e s p e c t i v e l y . A r e c u r r e n t c o l l a t e r a l axon m e d i a t e s f e e d b a c k e x c i t a t i o n e i t h e r d i r e c t l y as shown ( v ) o r p o s s i b l y v i a an e x c i t a t o r y i n t e r n e u r o n ; p o s t a c t i v a t i o n i n h i b i t i o n i s p r o d u c e d a t l e a s t i n p a r t v i a t h e a c t i v a t i o n o f a G A B A e r g i c i n t e r n e u r o n ( i v ) . The CA3 e f f e r e n t axon b i f u r c a t e s t o p r o v i d e th e S c h a f f e r c o l l a t e r a l i n p u t t o i p s i l a t e r a l C A l c e l l s ( v i ) and t h e c o m m i s s u r a l i n p u t ( i ) t o t h e C A l r e g i o n o f t h e c o n t r a l a t e r a l hippocampus (Swanson et. a l . , 1 9 8 0). C. I l l u s t r a t e d a r e the f i e l d p o t e n t i a l s r e c o r d e d i n t h e c e l l body l a y e r (Rg) f o l l o w i n g t h e s t i m u l a t i o n o f a f f e r e n t and e f f e r e n t p a t h w a y s . See t e x t f o r e x p l a n a t i o n . 26 A s t i m u l a t i n g e l e c t r o d e i n t h e s t r a t u m r a d i a t u m o f C A l o r CA2 ( F i g . 2.5A, S A ) e v o k e d i n CA3a a s h o r t l a t e n c y AD PS r e f l e c t i n g t h e a c t i v a t i o n o f S c h a f f e r c o l l a t e r a l f i b r e s . T h i s AD PS was f o l l o w e d by a p o s i t i v e wave upon w h i c h a 2° PS was u s u a l l y s u p e r i m p o s e d ( F i g . 2.5C, S A ) • A l t h o u g h i t was s o m e t i m e s p o s s i b l e t o e v o k e o n l y t h e p o s i t i v e wave/PS a p u r e AD r e s p o n s e was n e v e r o b t a i n e d . CA3 n e u r o n s were a l s o d r i v e n by an e l e c t r o d e p o s i t i o n e d t o a c t i v a t e t h e MF p a t h w a y c o u r s i n g t h r o u g h t h e h i l u s . The f i e l d p o t e n t i a l e v o k e d by MF s t i m u l a t i o n was c o m p r i s e d o f a n e g a t i v e - g o i n g b i - o r t r i p h a s i c w a v e f o r m a t l e a s t a p o r t i o n o f w h i c h r e p r e s e n t s t h e s y n c h r o n o u s d i s c h a r g e o f CA3 c e l l s ( A n d e r s e n e_t a _ l . , 1 9 7 1 b , a n d s e e b e l o w ) . T h i s i n i t i a l r e s p o n s e i s s u b s e q u e n t l y r e f e r r e d t o a s t h e p r i m a r y (1°) PS, a l t h o u g h e v e n t s a d d i t i o n a l t o c e l l f i r i n g may c o n t r i b u t e t o i t s g e n e r a t i o n . The maximum a m p l i t u d e o f t h e 1° PS w i t h h i l a r s t i m u l a t i o n ( 50-70 V) was 2-4 mV a l t h o u g h a s t i m u l u s a p p l i e d c l o s e r t o t h e r e c o r d i n g s i t e c o u l d e l i c i t a PS o f up t o 10 mV ( n o t s h o w n ) . The l a t e n c y t o o n s e t o f t h e 1° PS e v o k e d by h i l a r s t i m u l a t i o n was 3.7 + 0.2 m s e c ( n = 10) f o r a 1-2 mV r e s p o n s e . The 1° PS was u s u a l l y f o l l o w e d by a p o s i t i v e wave upon w h i c h a 2° PS was s o m e t i m e s s u p e r i m p o s e d ( F i g . 2. 5C, S^,) . I t p r o v e d d i f f i c u l t t o e l i c i t a CA3 r e s p o n s e by d i r e c t l y a c t i v a t i n g a l a r g e PS i n t h e s t r a t u m g r a n u l o s u m o f t h e d e n t a t e g y r u s . However, i n t h r e e e x p e r i m e n t s where t h i s was s u c c e s s f u l , t h e CA3 r e s p o n s e was s i m i l a r t o t h a t o c c u r r i n g w i t h MF s t i m u l a t i o n i n t h e h i l u s . I n c r e a s i n g t h e s t i m u l u s 27 i n t e n s i t y i n b o t h c a s e s i n c r e a s e d t h e a m p l i t u d e o f t h e 1 PS and t h e p o s i t i v e wave w h i c h f o l l o w e d i t ( F i g . 2 . 6 A ) . P a i r e d P u l s e S t i m u l a t i o n A f i m b r i a l c o n d i t i o n i n g s t i m u l u s ( F I M C ) e v o k i n g a 2-4 mV s u b m a x i m a l o r t h o d r o m i c PS c o u l d have e i t h e r o f two e f f e c t s o n t h e t e s t ( F I M T ) r e s p o n s e e v o k e d 20-60 msec l a t e r . Thus e i t h e r p o t e n t i a t i o n (up t o 300%) o r v a r i o u s d e g r e e s o f i n h i b i t i o n ( F i g . 3.1A,B) o f t h e F I M ^ r e s p o n s e s c o u l d be o b s e r v e d . The u s u a l e f f e c t a t l o n g e r c o n d i t i o n i n g - t e s t (C-T) i n t e r v a l s was an i n h i b i t i o n o f t h e F I M T a c t i v i t y and i n some c a s e s t h i s a c t i o n p e r s i s t e d up t o 2 s e c a f t e r t h e c o n d i t i o n i n g s t i m u l u s . An e l e c t r o p h y s i o l o g i c a l and p h a r m a c o l o g i c a l a n a l y s i s o f t h i s i n h i b i t i o n i s p r e s e n t e d i n t h e n e x t c h a p t e r . P a i r e d s t i m u l a t i o n i n t h e h i l u s r e v e a l e d a b e h a v i o r o f t h e 2° PS o f MF T r e s p o n s e s w h i c h r e s e m b l e d t h a t o b s e r v e d w i t h F I M - e v o k e d o r t h o d r o m i c a c t i v i t y . T h u s a t 2 0 - 6 0 m s e c C-T i n t e r v a l s e i t h e r p o t e n t i a t i o n ( F i g . 2.6D) o r i n h i b i t i o n o f t h e 2° PS o c c u r r e d , w h e r e a s a t 60 t o 2000 msec an i n h i b i t i o n o f t h i s e v o k e d a c t i v i t y was e v i d e n t ( F i g . 3. 2B) . The 1° PS o f t h e M F -evoked r e s p o n s e e x h i b i t e d p a i r e d p u l s e p o t e n t i a t i o n a t C-T i n t e r v a l s up t o 60 msec ( F i g . 2.6B,C) i n c a . 20% o f t h e s l i c e s . H o wever, i n t h e m a j o r i t y o f t h e e x p e r i m e n t s , t h e 1° PS o f M F T r e s p o n s e s was n e i t h e r i n h i b i t e d n o r p o t e n t i a t e d t o any g r e a t d e g r e e d e s p i t e t h e s u b s t a n t i a l c h a n g e s w h i c h were o b s e r v e d i n t h e 2° PS o r F I M T o r t h o d r o m i c a c t i v i t y ( F i g . 2.6D,E; F i g . 3 . 2 A , B ) . 28 v \ , 1 ImV x ' I msec E. (i) (ii) 14 6 -Vi 10 _ J . L M V 5 msec 5 msec F i g . 2.6 Some c h a r a c t e r i s t i c s o f t h e e x t r a c e l l u l a r l y r e c o r d e d r e s p o n s e i n CA3 f o l l o w i n g a c t i v a t i o n o f t h e MF p a t h w a y i n t h e h i l u s . A. W i t h i n c r e a s i n g i n t e n s i t i e s o f s t i m u l a t i o n (4-16V) t h e r e was an i n c r e a s e b o t h o f t h e n e g a t i v e - g o i n g 1° r e s p o n s e and t h e p o s i t i v e wave w h i c h f o l l o w e d i t ; n o t e t h e d e c r e a s e o f l a t e n c y f o r the o n s e t o f a c t i v i t y . B. O v e r l a p p e d t r a c e s i n d i c a t e t h e c o n d i t i o n i n g ( u p p e r r e c o r d ) and t h e t e s t r e s p o n s e t o p a i r e d MF s t i m u l i s e p a r a t e d by 30 msec. P o t e n t i a t i o n o f t h e a m p l i t u d e o f t h e t e s t MF r e s p o n s e i s a l s o a p p a r e n t i n C. However, i n a n o t h e r s l i c e (D) a t a s i m i l a r C-T i n t e r v a l i t was t h e 2° PS o n l y w h i c h i n c r e a s e d . E. F i l m e d r e c o r d s i n ( i ) i l l u s t r a t e t h e r e f r a c t o r i n e s s t o i n h i b i t i o n of t h e MF-evoked 1° PS a t 6-14 msec i n t e r s t i m u l u s i n t e r v a l s , whereas w i t h p a i r e d FIM s h o c k s ( i i ) i n t h e same s l i c e , t h e t e s t o r t h o d r o m i c PS was s u b -s t a n t i a l l y i n h i b i t e d a t t h e 6 and 10 msec i n t e r v a l s . 29 H i g h F r e q u e n c y S t i m u l a t i o n F i g . 2.7 i l l u s t r a t e s t h e c h a n g e s i n t h e F I M - e v o k e d o r t h o d r o m i c r e s p o n s e f o l l o w i n g a 1 s e c t r a i n o f 100 Hz s t i m u l i . I n 6/8 s l i c e s t h e r e was w i t h i n 20 s e c ( t h e s h o r t e s t i n t e r v a l e x a m i n e d ) o f t h e t e r m i n a t i o n o f t h e h i g h f r e q u e n c y s t i m u l a t i o n a 6 3 - 4 3 2 % p o t e n t i a t i o n o f a PS h a v i n g a p r e -t r e a t m e n t a m p l i t u d e o f 0.5-1.5 mV ( F i g . 2 . 7 A ) . T h i s i n c r e a s e o f t h e PS a m p l i t u d e p e r s i s t e d t o t h e e n d o f t h e 15 m i n r e c o r d i n g p e r i o d a n d r e s e m b l e s t h e l o n g t e r m p o t e n t i a t i o n ( L T P ) r e p o r t e d a s o c c u r r i n g i n t h e d e n t a t e g y r u s ( B l i s s and Ljzfaio, 1973) and C A l ( S c h w a r t z k r o i n and W e s t e r , 1975; and s e e C h a p t e r 4 ) . I n 2 s l i c e s t h e e n h a n c e m e n t o f t h e e v o k e d a c t i v i t y a f t e r t e t a n u s l a s t e d f o r o n l y 1-2 m i n ( p o s t t e t a n i c p o t e n t i a t i o n ) b e f o r e a r e t u r n t o t h e p r e - t r e a t m e n t a m p l i t u d e . A l t h o u g h t h e t r a n s i e n t a p p e a r a n c e o f a 2° PS was an o c c a s i o n a l o b s e r v a t i o n f o l l o w i n g t e t a n i c s t i m u l a t i o n t h e r e was a t no t i m e t h e g e n e r a t i o n o f a f t e r d i s c h a r g e s i n c o n t r a s t t o t h e s i t u a t i o n i n v i v o ( K a n d e l and S p e n c e r , 1961a) and in v i t r o (Yamamoto, 1 9 7 2 a ; b u t s e e M a c V i c a r and Dudek, 1 9 7 9 ) . S t i m u l i p r o d u c i n g LTP o f t h e FIM o r t h o d r o m i c i n p u t had no a p p a r e n t e f f e c t on t h e f i e l d r e s p o n s e e v o k e d by t h e h i l a r s t i m u l a t i o n ( F i g . 2 . 7 B ) . The AD PS e v o k e d by FIM s h o c k d i d n o t e x h i b i t LTP. U s i n g the same s t i m u l u s paradigm, LTP of the MF-evoked 1° PS was o b s e r v e d i n o n l y 1 o f 8 s l i c e s w h i c h h a d p r e v i o u s l y o r s u b s e q u e n t l y d e m o n s t r a t e d LTP o f t h e FIM o r t h o d r o m i c i n p u t ( F i g . 2.7B) ( b u t s e e Y a m a moto, 1 9 7 2 b ; Yamamoto a n d C h u j o , 1 9 7 8 ) . 30 K> > E 2-0 J • F I M evoked -00 P S i M F e v o k e d - 1 ° P S F I M 1 0 0 H z . I s e c ( i ) • F I M ( 1 3 4 V ) 1 0 Time (min) t (13.4 V ) (Iii) (IIV) (IIV) ] * 2 m V 4 m s e c (v) M F ( 4 0 V ) (vi) ( 4 0 V ) (vi) ( 4 0 V ) (vii) ( 4 0 V ) FIM 00Hz. I sec MF 100 Hz;I sec F i g . 2.7 C h a n g e s i n e v o k e d a c t i v i t y f o l l o w i n g t e t a n i c s t i m u l a t i o n . A. H i g h f r e q u e n c y s t i m u l a t i o n o f t h e f i m b r i a a t t=0 m i n p r o d u c e d a l o n g - l a s t i n g i n c r e a s e o f t h e a m p l i t u d e o f t h e F I M - e v o k e d o r t h o d r o m i c (OD) P S , b u t t h e MF 1° PS r e m a i n e d u n a f f e c t e d o r d e c r e a s e d . B. F i l m e d r e c o r d s o f c o n t r o l r e s p o n s e s t o F I M ( i ) a n d MF ( v ) s t i m u l i (3 c o n s e c u t i v e s u p e r i m p o s e d s w e e p s ) . T r a c e s o b t a i n e d 15 m i n a f t e r h i g h f r e q u e n c y s t i m u l a t i o n o f t h e FIM show p o t e n t i a t i o n o f t h a t o r t h o d r o m i c PS ( i i ) w h e r e a s t h e MF-evoked f i e l d r e s p o n s e was u n a f f e c t e d ( v i ) . I n ( i i i ) t h e ' F I M s t i m u l u s i n t e n s i t y was d e c r e a s e d t o e l i c i t a PS o f an a m p l i t u d e c o m p a r a b l e t o t h e c o n t r o l r e s p o n s e ; MF was n o t c h a n g e d ( v i i ) . S u b s e q u e n t t e t a n i z a t i o n o f t h e h i l a r MF p a t h w a y h a d no e f f e c t on t h e FIM ( i v ) o r t h e MF r e s p o n s e ( v i i i ) e i t h e r i m m e d i a t e l y o r , a s shown, 15 m i n l a t e r . 31 E f f e c t s o f e x t r a c e l l u l a r i o n c o n c e n t r a t i o n s  on e v o k e d a c t i v i t y i n CA3 2+ 2 + Low [Ca ] q , i n c r e a s e d [Mg ] P e r f u s i o n o f s l i c e s w i t h ACSF i n w h i c h t h e c o n c e n t r a -t i o n s o f C a 2 + ( [ C a 2 + ] Q ) (0.2 mM) and M g 2 + ( [ M g 2 + ] Q ) (4.7 mM) w e r e m o d i f i e d t o b l o c k s y n a p t i c r e s p o n s e s a l l o w e d t h e c o n t r i b u t i o n o f a n t i - a n d o r t h o d r o m i c t r a n s m i s s i o n t o t h e v a r i o u s c o mponents o f CA3 f i e l d p o t e n t i a l s t o be a s s e s s e d . The c o m p l e t e a b o l i t i o n o f t h e F I M - e v o k e d o r t h o d r o m i c r e s p o n s e u n d e r t h e s e c o n d i t i o n s ( F i g . 2 . 8 A i - i i i ) d e m o n s t r a t e d t h e p o t e n t b l o c k a d e o f s y n a p t i c a c t i v i t y . I n t h e same s l i c e t h e s h o r t l a t e n c y PS e l i c i t e d by SC s t i m u l a t i o n was u n a f f e c t e d b u t t h e p o s i t i v e wave w h i c h f o l l o w e d i t was s u b s t a n t i a l l y r e d u c e d and t h e 2° PS e l i m i n a t e d ( F i g . 2 . 8 A i v - v i ) . A l t h o u g h i t b l o c k e d t h e p o s i t i v e wave and t h e 2° PS e v o k e d by a s h o c k t o MF i n t h e h i l u s ( 1 0 / 1 0 s l i c e s ) , t h e a c t i o n s o f t h i s m o d i f i e d ACSF on t h e 1° PS w e re i n c o n s i s t e n t . Thus i n 3 s l i c e s a maximum r e d u c t i o n o f 37+12% o f t h e r e s p o n s e was m e a s u r e d , w h e r e a s i n 7 o t h e r s t h e r e was no e f f e c t on t h e PS a m p l i t u d e . E v e n w i t h a p r o l o n g e d e x p o s u r e (40-80 m i n ) t o t h i s m o d i f i e d ACSF, t h e M F -evoked 1° PS p e r s i s t e d . F u r t h e r m o r e t h e l a t t e r t r e a t m e n t c a u s e d t h e g r a d u a l r e c r u i t m e n t o f a d d i t i o n a l P S s i n b o t h t h e MF- a n d t h e A D - e v o k e d r e s p o n s e ( F i g . 2 . 8 B ) . T h e r e was a l s o a t t h i s t i m e a n i n c r e a s e o f s i n g l e u n i t a c t i v i t y ; and w i t h e a c h i n p u t p a i r e d homonymous s t i m u l i s e p a r a t e d by 20-50 msec r e v e a l e d a s u b s t a n t i a l 32 F i g . 2 . 8 A. C o n t r o l r e s p o n s e s i n CA3 ( R & ) t o FIM ( i ) a n d S c h a f f e r c o l l a t e r a l ( i v ) s t i m u l a t i o n a r e s h o w n i n t h e f i r s t c o l u m n . A f t e r t h e a d m i n i s t r a t i o n o f ca. 2 0 m l o f a 2 + medium a l t e r e d t o c o n t a i n low [Ca ] ( 0 . 2 mM) and i n c r e a s e d 2 + [Mg ] (4.7 mM) t h e FIM r e s p o n s e was a b o l i s h e d ( i i ) . A t t h e same t i m e t h e S c h a f f e r c o l l a t e r a l - e v o k e d AD PS was u n a f f e c t e d w h e r e a s t h e p o s i t i v e wave f o l l o w i n g i t was r e d u c e d and t h e s e c o n d a r y PS was e l i m i n a t e d ( v ) . T h e s e e f f e c t s were r e v e r s e d ( t h i r d c o l u m n ) a f t e r a w a s h - o u t w i t h n o r m a l medium. B. The 2 + 2 + a c t i o n s o f l o w [Ca ] Q / h i g h [Mg ] o n p a i r e d MF a n d F I M r e s p o n s e s . ( i ) I n n o r m a l medium t h e f i r s t MF s h o c k e l i c i t e d a t y p i c a l f i e l d p o t e n t i a l and t h e t e s t MF a c t i v i t y e v o k e d 2 0 msec l a t e r showed no e f f e c t on t h e 1 ° and a s m a l l i n c r e a s e o f t h e 2 ° PS a m p l i t u d e . ( i i i ) As f o r ( i ) b u t w i t h p a i r e d FIM s t i m u l i p r o d u c i n g a s h o r t l a t e n c y AD PS f o l l o w e d by o r t h o -d r o m i c a c t i v i t y . I n l o w [ C a 2 + ] Q / h i g h [ M g 2 + ] Q t h e 1 ° PS o f t h e c o n d i t i o n i n g MF r e s p o n s e was d e c r e a s e d a n d a d d i t i o n a l P S s f o l l o w e d i t ; t h e t e s t 1 ° PS was i n h i b i t e d ( i i ) . C o n c u r r e n t l y , t h e c o n d i t i o n i n g AD PS i n ( i v ) was u n a f f e c t e d b u t a g a i n m u l t i p l e PSs o c c u r r e d ; i n c o n t r a s t t o t h e s i t u a t i o n i n n o r m a l medium a s u b s t a n t i a l r e d u c t i o n o f t h e t e s t AD PS was e v i d e n t . T h e s e c h a n g e s o f e v o k e d a c t i v i t y r e v e r s e d a f t e r r e t u r n i n g t o n o r m a l medium ( n o t s h o w n ) . 3 3 34 r e d u c t i o n o f a c t i v i t y i n t h e t e s t r e s p o n s e ( F i g . 2 . 8 B i i , i v ) w h i c h was n o t a p p a r e n t i n t h e p r e - t r e a t m e n t s i t u a t i o n ( F i g . 2 . 8 B i , i i ) ( s e e a l s o Yamamoto, 1 9 7 2 b ) . Low [ C l ~ ] I n a s t u d y o f t h e i o n s i n v o l v e d i n e p i l e p t i f o r m d i s c h a r g e s , O g a t a (1975) r e p o r t e d an i n c r e a s e i n t h e a m p l i t u d e o f t h e M F -evoked ( h i l a r s t i m u l a t i o n ) 1° PS i n ACSF i n w h i c h e x t r a c e l l u l a r c h l o r i d e ( [ C I ] ) was r e p l a c e d w i t h p r o p i o n a t e . S i n c e t h i s l a r g e r a n i o n d o e s n o t p a s s t h r o u g h t h e C I i o n o -p h o r e ( I t o e_t a _ l . , 1962 ) t h i s t r e a t m e n t h a s t h e a c t i o n o f r e d u c i n g G A B A - m e d i a t e d C I - d e p e n d e n t i n h i b i t i o n and t h i s was s u g g e s t e d by O g a t a as t h e b a s i s f o r t h e i n c r e a s e o f t h e 1° P S . To d e m o n s t r a t e a s y n a p t i c component t o t h i s l a t t e r r e s p o n s e , t h e e f f e c t s o f l o w [CI ] on CA3 a c t i v i t y were r e - e x a m i n e d h e r e . I n t h e p r e l i m i n a r y e x p e r i m e n t s [ C I ] was r e d u c e d t o a f i n a l c o n c e n t r a t i o n o f 7 mM by s u b s t i t u t i o n o f N a C l w i t h s o d i u m p r o p i o n a t e . P e r f u s i o n o f t h i s l o w C I A C S F h a d a b i p h a s i c e f f e c t on e v o k e d r e s p o n s e s . W i t h i n 2-4 m i n o f i t s e n t r y i n t o t h e b a t h t h e r e was a l a r g e r e d u c t i o n o f t h e F I M - e v o k e d o r t h o d r o m i c PS ( F i g . 2 . 9 A i i ) and t h e 2° PS o f t h e M F - evoked r e s p o n s e , b u t t h e 1° MF PS r e m a i n e d u n a f f e c t e d ( F i g . 2 . 9 A v ) . Some 3-7 rain l a t e r t h e r e s p o n s e s w h i c h h a d b e e n s u p p r e s s e d r e c o v e r e d t o c o n t r o l l e v e l s and t h e r e f o l l o w e d a r a p i d i n c r e a s e i n t h e a m p l i t u d e and number o f PSs i n b o t h t h e FIM and M F -evoked f i e l d p o t e n t i a l s ( F i g . 2 . 9 A i i i , v i ) . 35 F i g . 2.9 C h a nges i n e v o k e d r e s p o n s e s i n CA3 f o l l o w i n g p e r f u s i o n w i t h ACSF c o n t a i n i n g a d e c r e a s e d [ C l ] (7 mM). A. C o n t r o l r e s p o n s e s t o FIM ( i ) and MF ( i v ) s t i m u l a t i o n a r e shown. The i n i t i a l e f f e c t o f l o w [ C l ] was t o a b o l i s h t h e MF o 2 ( v ) and FIM ( i i ) P S s . N o t e t h e p e r s i s t e n c e o f t h e 1 PS i n t h e M F - evoked f i e l d r e s p o n s e . W i t h i n 2-4 m i n u t e s t h e r e s p o n s e s r e c o v e r e d and s h o r t l y t h e r e a f t e r a r a p i d i n c r e a s e i n t h e a m p l i t u d e a n d n u m b e r o f P S s i n t h e e v o k e d p o t e n t i a l s o c c u r r e d ( i i i , v i ) . W i t h t h e o n s e t o f e v o k e d e p i l e p t i f o r m f i r i n g s p o n t a n e o u s a c t i v i t y c o m p r i s e d o f b u r s t s o f PSs ( v i i ) a p p e a r e d . B. The S c h a f f e r c o l l a t e r a l - e v o k e d a c t i v i t y i n l o w [ C l ] s h o w i n g c o n t r o l ( i ) , t r e a t m e n t ( i i ) and r e c o v e r y ( i i i ) r e s p o n s e s . I n ( i i ) t h e a n t i d r o m i c PS was i n c r e a s e d a s were t h e a m p l i t u d e and number o f PSs f o l l o w i n g i t (peak a m p l i t u d e s t r u n c a t e d ) . C. The e f f e c t s o f a p r o l o n g e d t r e a t m e n t w i t h l o w [ C l ] . C o n t r o l r e s p o n s e ( i ) . As i n t h e l o w e r r e c o r d s o f (A) t h e r e was an i n c r e a s e o f t h e a m p l i t u d e and number o f PSs i n MF-evoked r e s p o n s e ( i i ) . S p o n t a n e o u s p o p u l a t i o n b u r s t s ( a s i n A ( v i i ) b u t s h o w n h e r e a t a s l o w e r s w e e p s p e e d ) a l s o d i s -c h a r g e d w i t h c l o c k - l i k e r e g u l a r i t y ( i i i ) b u t t h i s a n d t h e e v o k e d a c t i v i t y ( i v ) c e a s e d a b r u p t l y f o l l o w i n g s e i z u r e - l i k e f i r i n g (• i n i i i ) . J 36 37 C o i n c i d e n t a l l y w i t h t h e o n s e t o f e v o k e d e p i l e p t i f o r m f i r i n g t h e r e a l s o a p p e a r e d , a t r e g u l a r i n t e r v a l s , s p o n t a n e o u s b u r s t s o f PS d i s c h a r g e s ( F i g . 2 . 9 A v i i ) . A t t h e peak o f t h e e f f e c t , t h e MF 1° PS had i n c r e a s e d by 63+11% (n=4) and i n two s l i c e s i n w h i c h i t was m o n i t o r e d , t h e AD PS o f S C - e v o k e d a c t i v i t y a l s o i n c r e a s e d ( F i g . 2 . 9 B ) . P r o l o n g e d p e r f u s i o n (12-20 min) w i t h l o w [ C l ] c a u s e d an e v o k e d o r s p o n t a n e o u s s e i z u r e - l i k e d i s c h a r g e ( F i g . 2 . 9 C i i i ) (Yamamoto and K a w a i , 1968) f o l l o w e d by a p e r i o d o f e l e c t r i c a l s i l e n c e ( F i g . 2 . 9 C i v ) r e s e m b l i n g s p r e a d i n g d e p r e s s i o n (Snow e_t a _ l . , 1 9 8 3 ) a n d f r o m w h i c h r e c o v e r y a f t e r r e t u r n i n g t o n o r m a l medium was s l o w . A l t h o u g h c o n f i r m i n g t h e r e s u l t s o f O g a t a , t h e i n c r e a s e o f t h e AD PS s u g g e s t e d t h a t a c t i o n s o f l o w [ C l ] a d d i t i o n a l t o a r e d u c t i o n o f G A B A e r g i c i n h i b i t i o n s i n c e b i c u c u l l i n e ( B I C ) d o e s n o t c h a n g e t h e AD PS a m p l i t u d e ( c f . C h a p t e r 6 ) . T h e r e f o r e t h e e x p e r i m e n t s were r e p e a t e d i n s l i c e s i n w h i c h 2+ 2 + s y n a p t i c a c t i v i t y was b l o c k e d ( 0 . 2 mM Ca ; 4.7 mM Mg ) ; [ C l ] was r e d u c e d o n l y t o 37 mM i n t h i s c a s e t o r e d u c e t h e o J p o s s i b i l i t y o f s e i z u r e - l i k e a c t i v i t y . As was e x p e c t e d , t h e a d m i n i s t r a t i o n o f 20-25 ml o f t h i s a l t e r e d ACSF b l o c k e d t h e F I M - e v o k e d o r t h o d r o m i c r e s p o n s e ( F i g . 2.10A, 2 . 1 1 A ) ; a n d , a l t h o u g h b o t h t h e p o s i t i v e wave and 2° PS o f t h e MF-evoked p o t e n t i a l were r e d u c e d , t h e 1° PS o f t h i s r e s p o n s e i n c r e a s e d by 244+19% ( n = 8 ) . S i m i l a r i n c r e a s e s were a p p a r e n t i n AD P S s . F o r e x a m p l e , i n t h r e e s l i c e s where t h e r e s p o n s e s were m o n i t o r e d c o n c u r r e n t l y MF 1° a n d AD P S s h a v i n g t h e same p r e - t r e a t m e n t s u b m a x i m a l a m p l i t u d e s ( c a . 2 mV) showed 38 A low CI'/Co2\ high -Mg: S i 2-0 J FIM evoked-OD 10 20 30 55 60 I 8 " CD a 6^  if) o 4-Q-O 0. 2 -low Cr/Co2*,high Mg 2 , MF evoked 4-2 J FIM evoked-AD 10 20 Time (min) 30 F i g . 2.10 In CA3 changes o f PS a m p l i t u d e s f o l l o w i n g e x p o s u r e t o ACSF c o n t a i n i n g low [CI ] (37 mM) , low [ C a 2 + ] (0.2 2 + mM) a n d i n c r e a s e d [Mg ] (4.7 mM) . A. P e r f u s i o n o f t h e a l t e r e d ACSF ( i n d i c a t e d by h o r i z o n t a l b a r ) c a u s e d a r e v e r s i b l e a b o l i t i o n o f t h e o r t h o d r o m i c (OD) PS e v o k e d by FIM s t i m u l i . B. In a n o t h e r s l i c e a l t e r n a t e s t i m u l a t i o n i n t h e h i l u s and t h e FIM r e v e a l e d p a r a l l e l i n c r e a s e s o f t h e FIM AD and MF 1° PSs. R e c o v e r y t o p r e - t r e a t m e n t l e v e l s o c c u r r e d a f t e r r e t u r n i n g t o n o r m a l medium. 39 i n c r e a s e s o f 212+25% and 212+39% r e s p e c t i v e l y ( F i g . 2 . 1 0 B ) . T h e s e r e s u l t s i n d i c a t e d : (1) t h a t t h e i n c r e a s e o f t h e MF 1° and t h e AD PS was a t t r i b u t a b l e t o some f a c t o r o t h e r t h a n a r e d u c t i o n o f a s y n a p t i c a l l y - m e d i a t e d C I - d e p e n d e n t i n h i b i -t i o n and (2) t h a t a t l e a s t a p o r t i o n o f t h e MF 1° PS p r o b a b l y r e f l e c t e d n o n - s y n a p t i c e x c i t a t i o n o f CA3 c e l l s . V o s k y l and t e r K e u r s ( 1 9 7 8 ) have r e p o r t e d t h a t s u b s t i t u t i o n o f [ C I ] w i t h t h e l a r g e r p r o p i o n a t e i o n p r o d u c e s a 16% i n c r e a s e i n t h e s p e c i f i c r e s i s t a n c e o f ACSF. F u r t h e r -m o r e , t h e y s u g g e s t e d a change o f r e s i s t a n c e as t h e b a s i s f o r t h e 33+8% i n c r e a s e i n t h e a m p l i t u d e o f t h e p r e s y n a p t i c f i b r e v o l l e y i n o l f a c t o r y c o r t e x s l i c e s p e r f u s e d w i t h t h i s medium. The p o s s i b l e c o n t r i b u t i o n o f a c h a n g e i n ACSF r e s i s t a n c e i n t h e e f f e c t s r e p o r t e d h e r e was a d d r e s s e d i n t h e f o l l o w i n g manner. I n t h r e e s l i c e s , i n t h e p r e - t r e a t m e n t s i t u a t i o n , a s u p r a m a x i m a l FIM s t i m u l u s e l i c i t e d a m a x i m a l AD PS. I t was r e a s o n e d t h a t any a m p l i t u d e c h a n g e i n l o w [CI ] u n d e r t h i s c o n d i t i o n o f s t i m u l a t i o n may be a t t r i b u t a b l e t o a r e s i s t a n c e c h a n g e r a t h e r t h a n an i n c r e a s e i n t h e number o f n e u r o n s w h i c h w e r e e x c i t e d . F o l l o w i n g t h e p e r f u s i o n o f 2 5 - 3 0 m l o f l o w - 2+ 2 + C I /Ca , h i g h Mg - c o n t a i n i n g ACSF, t h e a m p l i t u d e o f t h i s r e s p o n s e i n c r e a s e d by 30+3%. T h e s e d a t a i n d i c a t e t h a t t h e l a r g e i n c r e a s e s i n t h e a m p l i t u d e o f a s u b m a x i m a l MF 1° o r AD PS a r e a t t r i b u t a b l e o n l y i n p a r t t o a c hange o f t h e e x t r a -c e l l u l a r r e s i s t a n c e . - 2+ P r o l o n g e d p e r f u s i o n (40-120 m l ) o f l o w C I /Ca , h i g h 2 + Mg - c o n t a i n i n g ACSF c a u s e d an i n c r e a s e o f s i n g l e u n i t 40 , .. 40msec (vi) I t 1 I IB I 5 0 sec F i g . 2.11 A. A f t e r c h a n g i n g t o p e r f u s a t e c o n t a i n i n g - 2 + d e c r e a s e d t C l ] (37 mM) and [Ca ] ( 0 . 2 mM) and i n c r e a s e d 2 + [Mg ] (4.7 mM) t h e FIM-evoked o r t h o d r o m i c a c t i v i t y p r e s e n t i n n o r m a l medium ( i ) was e l i m i n a t e d ( i i ) . A h i g h e r i n t e n s i t y FIM s h o c k e l i c i t e d an a n t i d r o m i c and a d d i t i o n a l PSs ( i i i ) . O r t h o d r o m i c t r a n s m i s s i o n r e c o v e r e d i n n o r m a l medium ( i v ) . B. ( i ) T h e c o n t r o l MF r e s p o n s e . T h e i n i t i a l e f f e c t o f t h e a l t e r e d medium was t o i n c r e a s e t h e 1° PS a m p l i t u d e and r e d u c e t h e s i z e o f t h e p o s i t i v e wave w h i c h f o l l o w e d ( n o t s h o w n ) . C o n t i n u e d t r e a t m e n t c a u s e d a f u r t h e r i n c r e a s e i n t h e a m p l i t u d e and number o f PSs i n t h e f i e l d p o t e n t i a l ( i i and i i i ; n o t e c h a n g e o f c a l i b r a t i o n s f o r t h e s e t r a c e s ) . A f t e r 1 h t h e e v o k e d r e s p o n s e was now f o l l o w e d by t r a i n s of a f t e r d i s c h a r g e s ( e . g . i n [ i v ] ) ; one o f t h e s e a f t e r d i s c h a r g e s i s shown i n t h e f i l m e d o s c i l l o s c o p e t r a c e i n ( v ) . C o i n c i d e n t w i t h e v o k e d a f t e r d i s c h a r g e s t h e r e was an o n s e t o f s p o n t a n e o u s f i r i n g o f t r a i n s o f PSs ( v i ) . S i n c e a m p l i t u d e s i n t h e c h a r t r e c o r d i n g s o f ( i v ) and ( v i ) h a v e b e e n a t t e n u a t e d by t h e low r e s p o n s e f r e q u e n c y of the r e c o r d e r , no v o l t a g e c a l i b r a t i o n i s g i v e n . 41 a c t i v i t y a n d a p r o g r e s s i v e i n c r e a s e i n t h e n u m b e r o f P S s e v o k e d by FIM ( F i g . 2 . 1 l A i i i ) a n d MF ( F i g . 2 . 1 l B i i ) s t i m u l i d e s p i t e t h e c o n t i n u e d b l o c k a d e o f s y n a p t i c t r a n s m i s s i o n . I n 3/6 s l i c e s e v o k e d r e s p o n s e s were f o l l o w e d by a f t e r - d i s c h a r g e s ( F i g . 2 . 1 1 B i v ) a n d s p o n t a n e o u s a c t i v i t y c o n s i s t i n g o f PS d i s c h a r g e s l a s t i n g f o r up t o 20 s e c ( F i g . 2 . 1 l B v i ) was a l s o o b s e r v e d . A l l o f t h e s e e f f e c t s were r a p i d l y r e v e r s i b l e a f t e r r e t u r n i n g t o n o r m a l medium. I n c r e a s e d [ K + ] o The e f f e c t s on CA3 a c t i v i t y o f a change o f t h e e x t r a -c e l l u l a r c o n c e n t r a t i o n o f K + ( [ K + ] q ) f r o m 5 t o 10 mM were a l s o e x a m i n e d . I n e a c h o f 3 s l i c e s t h e 1° PS o f t h e M F - e v o k e d r e s p o n s e was u n a f f e c t e d ( s e e a l s o O g a t a , 1975) a l t h o u g h t h e number and a m p l i t u d e o f PSs f o l l o w i n g i t i n c r e a s e d ( F i g . 2.12 i v - v i ) ; p o t e n t i a t i o n o f t h e F I M - e v o k e d o r t h o d r o m i c PS and a r e c r u i t m e n t o f a d d i t i o n a l PSs was o b s e r v e d ( F i g . 2 . 1 2 i - i i i ) . C o i n c i d e n t w i t h t h e o n s e t o f e v o k e d e p i l e p t i f o r m f i r i n g s p o n t a n e o u s a c t i v i t y c o m p r i s e d o f b u r s t s o f PSs a p p e a r e d a t r e g u l a r i n t e r v a l s ( ( F i g . 2 . 1 2 v i i , v i i i ) . T h e s e e f f e c t s o f i n c r e a s e d [ K + ] w e r e r a p i d l y r e v e r s e d a f t e r r e t u r n i n g t o n o r m a l ACSF. I n t r a c e l l u l a r r e c o r d i n g o f CA3 r e s p o n s e s S t a b l e i n t r a c e l l u l a r i m p a l e m e n t s were o b t a i n e d f r o m 15 n e u r o n s , some o f w h i c h were i d e n t i f i e d by AD e x c i t a t i o n f r o m t h e F I M . A l l o f t h e s e c e l l s had s i m i l a r e l e c t r o p h y s i o l o g i c a l 42 F i g . 2.12 R a i s i n g t h e [K ] f r o m 5 t o 10 mM i n c r e a s e d o the number and a m p l i t u d e o f PS i n t h e FIM- and MF-evoked f i e l d p o t e n t i a l , b u t t h e a m p l i t u d e o f t h e MF 1° PS (o) was v i r t u a l l y u n a f f e c t e d . S p o n t a n e o u s a c t i v i t y c o m p r i s e d o f b u r s t s o f PSs ( v i i ) o c c u r r e d a t r e g u l a r i n t e r v a l s ( a p p a r e n t a t t h e s l o w e r sweep s p e e d s u s e d t o o b t a i n t h e r e c o r d s i n [ v i i i ] ). These e f f e c t s were f u l l y r e v e r s i b l e w i t h i n 15 m i n u t e s o f r e t u r n i n g t o n o r m a l p e r f u s a t e . T r a c e s i n e a c h column were o b t a i n e d a t a p p r o x i m a t e l y t h e same t i m e ; c a l i b r a t i o n b a r s t o t h e r i g h t a p p l y t o t h e t r a c e s i n e a c h row. 43 c h a r a c t e r i s t i c s w h i c h were u n l i k e t h o s e o f i n t e r n e u r o n s ( S c h w a r t z k r o i n and M a t h e r s , 1978). Mean v a l u e s ^ (+S.E.M.) f o r the r e s t i n g membrane p o t e n t i a l (RMP), i n p u t r e s i s t a n c e ( R I N ) and s p i k e h e i g h t s o f t h e s e c e l l s were -63+2 mV (n=15), 33+3 megohms ( n = l l ) a n d 84+1 mV (n=12) r e s p e c t i v e l y . I n t h e s e s t u d i e s t h e i n t r a c e l l u l a r p o t e n t i a l s were c o r r e l a t e d w i t h t h e f i e l d r e s p o n s e r e c o r d e d s i m u l t a n e o u s l y w i t h a s e p a r a t e e l e c t r o d e p o s i t i o n e d w i t h i n 50-100^xm o f t h e i m p a l e d c e l l . Shown i n F i g . 2.13A i s a t y p i c a l r e s p o n s e t o low i n t e n s i t y FIM s t i m u l a t i o n i n w h i c h t h e o n s e t o f t h e i n t r a -c e l l u l a r EPSP ( t h e a m p l i t u d e o f w h i c h c o u l d be i n c r e a s e d by a h y p e r p o l a r i z i n g p u l s e ) and the d i s c h a r g e o f an a c t i o n p o t e n t i a l c o r r e s p o n d e d r e s p e c t i v e l y w i t h t h e r e v e r s e d EPSP and t h e PS o f t h e f i e l d r e s p o n s e . The same c o r r e l a t i o n between t h e i n t r a - and e x t r a c e l l u l a r p o t e n t i a l s was o b s e r v e d i n e a c h o f 7 o t h e r c e l l s a c t i v a t e d by t h i s pathway. H i g h e r i n t e n s i t y FIM s hock e v o k e d a s h o r t l a t e n c y AD s p i k e f o l l o w e d by an EPSP ( c f . Dodd e t a l . , 1 9 78). The i n t r a c e l l u l a r c hanges o f p o t e n t i a l p r o d u c e d by a s t i m u l u s a p p l i e d t o t h e MF pathway i n t h e h i l u s were r e c o r d e d i n 13 c e l l s . I n 9 o f t h e s e n e u r o n s h i l a r s t i m u l a t i o n e l i c i t e d an EPSP whose o n s e t and d u r a t i o n p a r a l l e l e d t h e l a t e p o s i t i v e wave i n t h e f i e l d r e s p o n s e . I n 5 o f t h e s e same c e l l s where t h e t h r e s h o l d f o r f i r i n g was r e a c h e d , t h e a c t i o n p o t e n t i a l was a l i g n e d w i t h t h e 2 ° PS s u p e r i m p o s e d on t h e l a t e p o s i t i v e wave ( F i g . 2.13B). In o n l y 4 c e l l s d i d a c t i o n p o t e n t i a l s c o i n c i d e w i t h t h e 44 F i g . 2.13 F I M - and MF-evoked CA3 r e s p o n s e s o b t a i n e d w i t h s i m u l t a n e o u s i n t r a c e l l u l a r ( u p p e r t r a c e s ) a n d e x t r a -c e l l u l a r r e c o r d i n g . A. FIM s t i m u l a t i o n e l i c i t e d i n t h i s c e l l (-66 mV RMP, 36 megohms R I N / 80 mV s p i k e h e i g h t ) an i n t r a -c e l l u l a r EPSP whose o n s e t c o r r e s p o n d e d t o t h a t o f t h e e x t r a -c e l l u l a r p o s i t i v e w a v e ( r e v e r s e d E P S P ) . One o f t h e t w o s t i m u l i p r o d u c e d t h e d i s c h a r g e o f an a c t i o n p o t e n t i a l ( t r u n c a t e d i n r e c o r d ) w h i c h c o i n c i d e d w i t h t h e P S . B. I n a n o t h e r c e l l (-59 mV RMP, 24 megohms R I N / 82 mV s p i k e h e i g h t ) two c o n s e c u t i v e s u p e r i m p o s e d sweeps showed t h e o n s e t o f t h e M F - e v o k e d i n t r a c e l l u l a r E P S P o c c u r r e d a t t h e p e a k o f t h e e x t r a c e l l u l a r 1° PS ( d o t t e d l i n e ) a n d t h e a c t i o n p o t e n t i a l d i s c h a r g e c o i n c i d e d w i t h t h e 2° PS. C. A c t i v a t i o n o f t h e MF p a t h w a y i n t h e h i l u s e l i c i t e d a n i n t r a c e l l u l a r E P S P w h o s e o n s e t i n t h i s c a s e was a l i g n e d w i t h t h a t o f t h e n e g a t i v e - g o i n g wave i n t h e e x t r a c e l l u l a r p o t e n t i a l . D. A 30 V MF s t i m u l u s e l i c i t e d i n t h i s c e l l (-62 mV RMP, 40 megohms R I N / 85 mV s p i k e h e i g h t ) a s u b t h r e s h o l d EPSP whose o n s e t was c o r r e l a t e d a s i n (B) w i t h t h e peak o f t h e e x t r a c e l l u l a r 1° PS ( d o t t e d l i n e ) . A t a h i g h e r i n t e n s i t y o f s t i m u l a t i o n (40 V) t h e a n t i d r o m i c n a t u r e o f t h e a c t i o n p o t e n t i a l was s u g g e s t e d by t h e a b s e n c e o f a p r e c e d i n g s l o w d e p o l a r i z a t i o n . The d a s h e d l i n e i n d i c a t e s t h e c o r r e l a t i o n b e t w e e n t h e a c t i o n p o t e n t i a l and t h e e x t r a -c e l l u l a r 1° PS. j 46 1° PS, and o f t h e s e t h e r e was an u n d e r l y i n g EPSP i n o n l y 2 ( F i g 2 . 1 3 C ) . I n t h e o t h e r s t h e a c t i o n p o t e n t i a l a r o s e a b r u p t l y f r o m t h e membrane p o t e n t i a l ( F i g 2.13D) a n d no e v i d e n c e o f a p r e p o t e n t i a l was o b t a i n a b l e e v e n w i t h h i g h g a i n r e c o r d i n g ( n o t s h o w n ) . D i s c u s s i o n T h e r e c o r d i n g o f f i e l d p o t e n t i a l s c a n p r o v i d e a s u b s t a n t i a l amount o f i n f o r m a t i o n r e g a r d i n g c h a n g e s i n f u n c t i o n o f a p o p u l a t i o n o f n e u r o n s f o l l o w i n g e x p e r i m e n t a l m a n i p u l a t i o n . However, an u n d e r s t a n d i n g i s r e q u i r e d b o t h o f t h e f a c t o r s i n f l u e n c i n g t h e c h a r a c t e r i s t i c s o f t h e r e c o r d e d w a v e f o r m s ( e . g . p o l a r i t y , m a g n i t u d e ) a n d t h e e l e c t r o p h y s -i o l o g i c a l e v e n t s w h i c h u n d e r l i e t h e i r g e n e r a t i o n . The p o t e n t i a l (V) m e a s u r e d w i t h an e x t r a c e l l u l a r e l e c t r o d e i s g e n e r a t e d by t h e f l o w o f c u r r e n t ( I ) t h r o u g h t h e r e s i s t a n c e (R) o f t h e e x t r a c e l l u l a r s p a c e as d e f i n e d by Ohm's Law. The e x t r a c e l l u l a r c u r r e n t r e s u l t s f r o m t h e a c t i v a t i o n o f i o n i c c o n d u c t a n c e s i n n e u r o n a l membrane. The a m p l i t u d e o f a f i e l d p o t e n t i a l i s t h e r e f o r e a f f e c t e d by i ) t h e m a g n i t u d e o f t h e c o n d u c t a n c e c h a n g e and t h e number o f c e l l s a c t i v a t e d ; i i ) t h e s y n c h r o n y o f t h e p o p u l a t i o n r e s p o n s e ; i i i ) t r a n s m e m b r a n e p o t e n t i a l s and c o n c e n t r a t i o n g r a d i e n t s a f f e c t i n g t h e d r i v i n g f o r c e f o r i o n f l u x t h r o u g h t h e i n c r e a s e d membrane p e r m e a b i l -i t y ; i v ) t h e r e s i s t i v i t y o f t h e e x t r a c e l l u l a r f l u i d . The p o l a r i t y o f t h e p o t e n t i a l i s d e p e n d e n t on t h e l o c a t i o n o f t h e e l e c t r o d e r e l a t i v e t o t h e s i t e on t h e n e u r o n s ( e . g . d e n d r i t e s 47 o r s o m a t a ) o f t h e a c t i v e i o n i c c o n d u c t a n c e . A more d e t a i l e d c o n s i d e r a t i o n o f t h i s t o p i c may be f o u n d i n t h e r e p o r t s o f R a i l a n d S h e p h e r d (1968) and N i c h o l s o n and L l i n a s ( 1 9 7 1 ) . S i m p l y on t h e b a s i s o f t h e sh a p e o f t h e e x t r a c e l l u l a r w a v e f o r m i t i s n o t p o s s i b l e t o a s c e r t a i n t h e e v e n t s w h i c h u n d e r l i e t h e g e n e r a t i o n o f i t s v a r i o u s c o m p o n e n t s . I n s t e a d , t h i s i n f o r m a t i o n must be d e d u c e d f r o m t h e r e s u l t s o f v a r i o u s e x p e r i m e n t a l m a n i p u l a t i o n s ( e . g . f r e q u e n c y s t i m u l a t i o n ; r e m o v a l o f e x t r a c e l l u l a r i o n s , e t c . ) and t h e c o r r e l a t i o n w i t h i n t r a c e l l u l a r l y r e c o r d e d p o t e n t i a l s . T h e s e c r i t e r i a h a v e been a p p l i e d by o t h e r s ( S c h w a r t z k r o i n , 1 975; Dudek e_t a _ l . , 1976) i n t h e c h a r a c t e r i z a t i o n o f d e n d r i t i c and s o m a t i c f i e l d p o t e n t i a l s i n t h e C A l and d e n t a t e r e g i o n s and s i n c e t h e r e was v i r t u a l l y no d i f f e r e n c e b e t w e e n t h e r e s p o n s e s d e s c r i b e d i n t h i s r e p o r t a n d t h o s e o f t h e e a r l i e r i n v e s t i g a t i o n s no f u r t h e r d i s c u s s i o n o f t h e s e p o t e n t i a l s i s p r o v i d e d h e r e . T h e r e i s , h o w e v e r , r e l a t i v e l y l i t t l e i n f o r m a t i o n r e g a r d i n g t h e CA3 f i e l d r e s p o n s e s e v o k e d by t h e s t i m u l a t i o n o f v a r i o u s a f f e r e n t and e f f e r e n t p a t h w a y s and s i n c e some o f t h e e x p e r i m e n t s d e s c r i b e d i n t h i s t h e s i s i n v o l v e d an a n a l y s i s by e x t r a c e l l u l a r r e c o r d i n g o f c h a n g e s o f e v o k e d a c t i v i t y i n CA3, a c h a r a c t e r i z a t i o n o f t h e s e f i e l d r e s p o n s e s was deemed n e c e s s a r y . The a m p l i t u d e o f t h e m a x i m a l PS o b s e r v e d i n CA3 f o l l o w i n g a n t i - o r o r t h o d r o m i c a c t i v a t i o n was g e n e r a l l y s m a l l e r t h a n t h a t w h i c h c o u l d be r e c o r d e d i n t h e same s l i c e i n e i t h e r C A l o r t h e d e n t a t e g y r u s . A l t h o u g h t h i s m i g h t h a v e b e e n d u e t o c e l l u l a r d a m a g e , by o t h e r c r i t e r i a t h e c e l l s 48 a p p e a r e d h e a l t h y and d i s p l a y e d e l e c t r o p h y s i o l o g i c a l a c t i v i t i e s c o m p a r a b l e t o t h o s e o b s e r v e d i n C A l and t h e d e n t a t e g y r u s . F o r e x a m p l e , a r o b u s t G A B A - m e d i a t e d i n h i b i t i o n was p r e s e n t ( c f . C h a p t e r 3 ) , p a i r e d p u l s e ( A n d e r s e n , 1960) and l o n g t e r m p o t e n t i a t i o n c o u l d be shown and t h e p a s s i v e and a c t i v e membrane p r o p e r t i e s o f i m p a l e d c e l l s w e r e c o m p a r a b l e t o t h o s e r e p o r t e d as o c c u r r i n g ir\ v i v o and i_n v i t r o ( K a n d e l et. a l . , 1 9 6 1 ; M a c V i c a r a n d D u d e k , 1 9 8 0 ; Wong a n d P r i n c e , 1 9 8 1 ) . D i n g l e d i n e e_t a _ l . , ( 1 9 8 0 ) h a v e s u g g e s t e d t h a t t h e s m a l l e r a m p l i t u d e o f CA3 f i e l d r e s p o n s e s may be r e l a t e d t o t h e l e s s d e n s e p a c k i n g o f c e l l s w i c h i s p a r t i c u l a r l y a p p a r e n t i n CA3a. An e l e c t r i c a l s h o c k a p p l i e d t o t h e FIM c o u l d e v o k e a s h o r t l a t e n c y PS a n d / o r a l o n g e r l a t e n c y PS s u p e r i m p o s e d , on a p o s i t i v e w a v e . I t was u s u a l l y t h e c a s e t h a t t h e s t i m u l u s t h r e s h o l d f o r t h e s h o r t l a t e n c y PS was h i g h e r ( s e e a l s o K a n d e l e t a l . , 1 9 6 1 ; Dodd e_t a l . , 1 9 7 8 ) . T h a t t h e l a t t e r r e s p o n s e r e p r e s e n t e d a n o n - s y n a p t i c e x c i t a t i o n o f CA3 n e u r o n s was s u g g e s t e d by i t s p e r s i s t e n c e ' i n ACSF a l t e r e d t o b l o c k o r t h o d r o m i c t r a n s m i s s i o n and a l s o by a f a i l u r e t o d e m o n s t r a t e e i t h e r p a i r e d p u l s e - o r l o n g t e r m p o t e n t i a t i o n . T h i s PS t h e r e f o r e most l i k e l y r e p r e s e n t s t h e a n t i d r o m i c i n v a s i o n o f CA3 p y r a m i d a l c e l l s f o l l o w i n g t h e e x c i t a t i o n o f e f f e r e n t f i b r e s c o u r s i n g i n t h e F I M . I t i s u n l i k e l y t h a t t h i s p o t e n t i a l r e f l e c t s t h e d i r e c t a c t i v a t i o n o f c e l l s by t h e s p r e a d o f t h e s t i m u l u s c u r r e n t s i n c e t h e r e were s i t u a t i o n s w h e r e h i g h i n t e n s i t y FIM s t i m u l a t i o n e v o k e d t h e l o n g b u t n o t t h e s h o r t l a t e n c y r e s p o n s e ( e . g . F i g . 5 . 1 4 A ) . 49 A s y n a p t i c o r i g i n f o r t h e F I M - e v o k e d p o s i t i v e wave and a s s o c i a t e d PS was s u g g e s t e d by t h e b l o c k i n g e f f e c t s o f l o w 2+ 2 + Ca , h i g h Mg - c o n t a i n i n g medium and t h e p o t e n t i a t i o n o f t h e PS f o l l o w i n g p a i r e d p u l s e and t e t a n i c s t i m u l a t i o n . I n t r a -c e l l u l a r r e c o r d i n g s c o n f i r m e d t h e o r t h o d r o m i c n a t u r e o f t h e r e s p o n s e . The i d e n t i t y o f t h e a f f e r e n t p a t h w a y ( s ) m e d i a t i n g t h e F I M - e v o k e d o r t h o d r o m i c r e s p o n s e i s n o t c e r t a i n . O n l y m i n o r c o n t r i b u t i o n s w o u l d be e x p e c t e d f r o m t h e a c t i v a t i o n o f s e p t a l f i b r e s (Mosko et. aJL. , 1973) s i n c e c h o l i n e r g i c d e p o l a r i z a t i o n s a r e s l o w a n d a r e e v i d e n t i n h i p p o c a m p a l s l i c e s o n l y a f t e r t r a i n s o f h i g h f r e q u e n c y s t i m u l a t i o n ( C o l e and N i c o l l , 1 9 8 3 ) . The r e s p o n s e may a l s o be a t t r i b u t e d t o t h e a c t i v a t i o n o f a r e c u r r e n t e x c i t a t o r y p a t h w a y ( L o r e n t e de No, 1934, L e b o v i t z e t a l . , 1 971; M a c V i c a r and Dudek, 1980; M i l e s and Wong, 1983) b u t t h i s s e e m s an u n l i k e l y e x p l a n a t i o n i n t h o s e c a s e s w h e r e a p r e c e d i n g AD PS was n o t o b s e r v e d . S a v e f o r t h e a b s e n c e o f a p r e s y n a p t i c f i b r e v o l l e y , t h e e l e c t r o p h y s i o l o g i c a l c h a r a c t e r i s t i c s o f o r t h o d r o m i c f i e l d p o t e n t i a l w h i c h c o u l d be e v o k e d by FIM s t i m u l i and w h i c h was u n c o n t a m i n a t e d by p r e c e d i n g AD a c t i v i t y r e s e m b l e d t h a t r e p o r t e d a s o c c u r r i n g in_ v i v o ( C r a g g and H a m l y n , 1957; A n d e r s e n , 1960) a f t e r s t i m u l a t i o n o f t h e c o m m i s s u r a l p a t h w a y ( B l a c k s t a d , 1 9 5 6 ) . A c c o r d i n g l y t h e o r t h o d r o m i c e x c i t a t i o n o b s e r v e d e s p e c i a l l y w i t h low i n t e n s i t i e s o f FIM s t i m u l a t i o n seems l i k e l y t o r e f l e c t t h e a c t i v a t i o n o f t h e s e c o m m i s s u r a l f i b r e s . 5 0 The r e s p o n s e e v o k e d by a s t i m u l a t i n g e l e c t r o d e i n t h e s t r a t u m r a d i a t u m o f C A l o r CA2 c l o s e l y r e s e m b l e d t h a t o b s e r v e d w i t h F I M s t i m u l a t i o n s a v e f o r a l o n g e r l a t e n c y o f o n s e t r e l a t e d p r e s u m a b l y t o t h e l o n g e r c o n d u c t i o n d i s t a n c e . 2+ 2 + E x p e r i m e n t s i n l o w Ca , h i g h Mg - c o n t a i n i n g ACSF i n d i c a t e d t h e s h o r t l a t e n c y PS t o be a n t i d r o m i c and most l i k e l y r e f l e c t i n g t h e a c t i v a t i o n o f S h a f f e r c o l l a t e r a l f i b r e s c o u r s i n g f r o m CA3 t o C A l . The b l o c k a d e o f t h e p o s i t i v e wave and t h e s e c o n d a r y PS w i t h t h e l a t t e r t r e a t m e n t i n d i c a t e d i t s d e p e n d e n c e on t h e e x c i t a t i o n o f a f f e r e n t f i b r e s s u c h as t h o s e s u g g e s t e d t o be i n v o l v e d i n t h e F I M - e v o k e d o r t h o d r o m i c r e s p o n s e . T h e 1° r e s p o n s e e v o k e d by a s t i m u l a t i n g e l e c t r o d e p o s i t i o n e d i n t h e h i l u s has been i n t e r p r e t e d as c o n s i s t i n g o f an EPSP, w h i c h i s n e g a t i v e - g o i n g ( L a n t h o r n and Cotman, 1 9 8 1 ; W h e a l ejt a _ l . , 1 9 8 3 ) b e c a u s e o f t h e p r o x i m i t y o f t h e MF t e r m i n a t i o n t o t h e c e l l body l a y e r ( J o h n s t o n and B r o w n , 1 9 8 3 ) , a n d a s u p e r i m p o s e d PS ( A n d e r s e n e_t a _ l . , 1 9 7 1 b ; Y a m a m o t o , 1972b; O g a t a a n d Ueno, 1 9 7 6 ) . A l t h o u g h i n some s l i c e s t h e 2 + 2 + e f f e c t s o f l o w [ C a ] Q / h i g h [Mg ] q a n d t h e o c c u r r e n c e o f p a i r e d p u l s e and l o n g t e r m p o t e n t i a t i o n were 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 an o r t h o d r o m i c component i n t h e r e s p o n s e , t h e a d d i t i o n a l c o n t r i b u t i o n o f a n o n - s y n a p t i c p r o c e s s was u n m i s t a k e a b l e . Thus t h e 1° PS was p a r t i a l l y b l o c k e d o r w h o l l y u n a f f e c t e d i n medium w h i c h b l o c k e d o t h e r s y n a p t i c e x c i t a t i o n s , was o f t e n r e f r a c t o r y t o p a i r e d p u l s e i n h i b i t i o n o r p o t e n t i a -t i o n a nd r e m a i n e d v i r t u a l l y u n a f f e c t e d i n [ K + l r i w h i c h c a u s e d 51 l a r g e i n c r e a s e s o f FIM o r t h o d r o m i c P S s . The Ca - i n d e p e n d e n t component d i d n o t a p p e a r s i m p l y t o r e p r e s e n t a l a r g e a m p l i t u d e 2+ -p r e s y n a p t i c f i b r e v o l l e y s i n c e i n low [Ca ] Q / [ C 1 ] , h i g h 2 + [Mg ] t h e a m p l i t u d e o f t h e r e s p o n s e i n c r e a s e d up t o 400% and s u b s t a n t i a l r e d u c t i o n s o f t h e 1° PS o f MF^, a c t i v i t y c o u l d be 2+ 2 + o b s e r v e d i n l o w [Ca ] Q , h i g h [Mg ] ( F i g . 2 . 7 B ) . F i n a l l y , s u p p o r t f o r a v a r i a b l e c o n t r i b u t i o n o f o r t h o d r o m i c and n o n - s y n a p t i c ( p r o b a b l y AD) e x c i t a t i o n s i n t h e g e n e r a t i o n o f t h e 1° r e s p o n s e was p r o v i d e d by i n t r a c e l l u l a r a n a l y s e s . A n o n - s y n a p t i c component i n t h e MF 1° r e s p o n s e r e p o r t e d by Yamamoto ( 1 9 7 2 b ) was s u g g e s t e d t o be m e d i a t e d by a n e l e c t r i c a l s y n a p s e b u t no e v i d e n c e t o s u p p o r t o r r e f u t e t h i s h y p o t h e s i s i s c u r r e n t l y a v a i l a b l e . G o t t l e i b and Cowan ( 1 9 7 3 ) and S c h w e r d t f e g e r and S a r v e y ( 1 9 8 3) have shown t h a t a p r o j e c t i o n f r o m CA3 t o t h e h i l u s a n d / o r t h e d e n t a t e g y r u s may e x i s t and t h i s i n f o r m a t i o n t o g e t h e r w i t h t h e e l e c t r o p h y s i o -l o g i c a l d a t a p r e s e n t e d h e r e and by Yamamoto (1972b) s u g g e s t t h a t an AD a c t i v a t i o n o f CA3 c e l l s c a n o c c u r w i t h a s t i m u l u s a p p l i e d i n t h e h i l a r r e g i o n . T h e l a r g e v a r i a t i o n s i n t h e e f f e c t s o f L - 2 - a m i n o -- 4 - p h o s p h o n o b u t y r a t e (L-APB) ( a n a n t a g o n i s t o f g l u t a m a t e e x c i t a t i o n s i n i n v e r t e b r a t e s [ C u l l - C a n d y e t a l . , 1976] ) on t h e 1° PS e v o k e d by h i l a r s t i m u l i ( Y a m a m o t o e t . a l . r 1 9 8 3 ) a r e c o n s i s t e n t w i t h t h e r e b e i n g v a r i a b l e e x t e n t s o f a n t i - a n d o r t h o d r o m i c a c t i v i t y i n i t s g e n e r a t i o n . T h u s L-APB h a d a c t i o n s w h i c h r a n g e d f r o m a c o m p l e t e a b o l i t i o n t o t h e a b s e n c e o f a n y e f f e c t o n t h e 1° P S , w h e r e a s AD r e s p o n s e s w e r e 52 c o n s i s t e n t l y u n a f f e c t e d . A s i m i l a r e x p l a n a t i o n may a l s o u n d e r l i e t h e a p p a r e n t l y c o n t r a d i c t o r y r e p o r t s o f t h e p r e s e n c e ( L a n t h o r n a n d C o t m a n , 1 9 8 1 ) a n d a b s e n c e ( A u l t a n d N a d l e r , 1983) o f a d e p r e s s a n t a c t i o n o f b a c l o f e n on t h e h i l a r - e v o k e d 1° PS i n CA3; b a c l o f e n had no e f f e c t on AD P S s . The r e s u l t s o f e x p e r i m e n t s a s s e s s i n g t h e e f f e c t s o f 2+ 2 + s t i m u l u s f r e q u e n c y and c h a n g e s o f t h e [Ca ] and [Mg ] t o -g e t h e r w i t h t h e i n t r a c e l l u l a r r e c o r d s i n d i c a t e d u n e q u i v o c a l l y t h a t t h e p o s i t i v e wave and t h e a s s o c i a t e d PS w h i c h f o l l o w e d t h e MF 1° r e s p o n s e r e f l e c t t h e s y n a p t i c a c t i v a t i o n o f CA3 n e u r o n s . T h i s o r t h o d r o m i c a c t i v a t i o n may be d u e t o t h e e x c i t a t i o n o f p e r f o r a n t p a t h w a y s , CA3-CA3 a s s o c i a t i o n a l f i b r e s , c o m m i s s u r a l f i b r e s o r a f f e r e n t s f r o m t h e h i l u s ( A m a r a l , 1978; S c h w e r d t f e g e r and S a r v e y , 1 9 8 3 ) . I n n o r m a l ACSF s i n g l e CA3 c e l l s d i s p l a y b u r s t i n g b e h a v i o r e i t h e r s p o n t a n e o u s l y o r w i t h b r i e f i n t r a c e l l u l a r p u l s e s o f d e p o l a r i z i n g c u r r e n t (Wong and P r i n c e , 1 9 8 1 ) . H o w e v e r , i t i s e v i d e n t f r o m t h e i n t r a - a n d e x t r a c e l l u l a r r e c o r d s o f t h i s r e p o r t ( s e e a l s o K a n d e l et_ a _ l . , 1961b; Dodd et_ a l . , 1978; M a c V i c a r and Dudek, 1980; M i s g e l d e t a l . , 1982) t h a t t h e u s u a l r e s p o n s e t o o r t h o d r o m i c s t i m u l a t i o n i s t h e d i s c h a r g e o f a s i n g l e P S . T h a t t h i s was t h e c a s e may be a t t r i b u t a b l e t o t h e r a p i d o n s e t o f a G A B A - m e d i a t e d i n h i b i t i o n ( c f . C h a p t e r 3) w h i c h o v e r r i d e s a n d m a s k s a n y e x c i t a t o r y i n f l u e n c e o f a d e p o l a r i z i n g a f t e r p o t e n t i a l (DAP) ( K a n d e l and S p e n c e r , 1961b; Wong and P r i n c e , 1 9 8 1 ) . The A D - e v o k e d b u r s t 2+ 2 + f i r i n g o b s e r v e d i n low [Ca ] , h i g h [Mg ] may be a t t r i -53 b u t a b l e t o b o t h t h e a b o l i t i o n o f s y n a p t i c i n h i b i t i o n w i t h t h e c o n s e q u e n t e x p r e s s i o n o f t h e DAP and i n c r e a s e d n e u r o n a l 2 + e x c i t a b i l i t y r e l a t e d t o t h e d e c r e a s e o f [ C a ] ( F r a n k e n -h a e u s e r and H o d g k i n , 1957; H o t s o n and P r i n c e , 1 9 8 0 ) . E p i l e p t i f o r m a c t i v i t y e l i c i t e d by A D C s t i m u l i a n d t h e s u b s e q u e n t a c t i v a t i o n o f a l o n g - l a s t i n g a f t e r h y p e r p o l a r i z a t i o n ( H o t s o n a n d P r i n c e , 1980; Wong and P r i n c e , 1981) i s t h e most p r o b a b l e , b a s i s f o r t h e l a r g e r e d u c t i o n s o f A D T a c t i v i t y o b s e r v e d i n t h i s medium ( F i g . 2 . 7 B ) . An u n e x p e c t e d e f f e c t o f low [ C l ] was t h e p o t e n t i a t i o n o f AD P S s w h i c h was a t t r i b u t a b l e n e i t h e r t o t h e l o s s o f G A B A e r g i c i n h i b i t i o n n o r t o a cha n g e i n t h e r e s i s t a n c e o f t h e t i s s u e . O t h e r s (Neumcke, 1983; Ransom and C o n n o r s , 1983) have r e p o r t e d an i n c r e a s e d e x c i t a b i l i t y o f n e u r o n a l membranes f o l l o w i n g t h e r e p l a c e m e n t o f [ C l ] by l e s s p e r m e a n t i o n s ; s i m i l a r e f f e c t s h e r e may h a v e p r o d u c e d a n i n c r e a s e i n t h e number o f e f f e r e n t f i b r e s a c t i v a t e d a n d / o r f a c i l i t a t e d t h e a n t i d r o m i c i n v a s i o n o f s o m a t a . A c h a r a c t e r i s t i c f e a t u r e o f CA3 n e u r o n s f o l l o w i n g t h e i r t r e a t m e n t w i t h c o n v u l s a n t a g e n t s i s t h e a p p e a r a n c e o f s p o n t a n e o u s a c t i v i t y c o m p r i s e d o f t h e s y n c h r o n o u s b u r s t i n g o f a p o p u l a t i o n o f n e u r o n s ( s p o n t a n e o u s p o p u l a t i o n b u r s t , SPB) ( P r i n c e , 1978; S c h w a r t z k r o i n and P r i n c e , 1978; T r a u b a n d Wong, 1982, 1 9 8 3 ) . I n t h e p r e s e n t s t u d y a d e c r e a s e o f [ C l ] c a u s e d e v o k e d and s p o n t a n e o u s b u r s t f i r i n g , a n e f f e c t most l i k e l y a t t r i b u t a b l e t o t h e r e d u c t i o n o f G A B A - m e d i a t e d i n h i b i t i o n ( S c h w a r t z k r o i n and P r i n c e , 1978) a n d , c o n s e q u e n t l y , an 54 i n c r e a s e i n t h e e f f i c a c y o r a r e c r u i t m e n t o f r e c u r r e n t e x c i t a t o r y c i r c u i t s . As in v i v o ( Z u c k e r m a n and G l a s e r , 1968) SPBs w h i c h were i n d i s t i n g u i s h a b l e f r o m t h o s e o b s e r v e d i n l o w [CI ] c o u l d a l s o be i n d u c e d by a p e r f u s i o n o f ACSF i n w h i c h [ K + ] Q was i n c r e a s e d and SPB a c t i v i t y h e r e a g a i n was a s s o c i a t e d w i t h e v o k e d r e s p o n s e s c o m p r i s e d o f m u l t i p l e P S s . A l t h o u g h a s e l e c t i v e i m p a i r m e n t o f s y n a p t i c i n h i b i t i o n c a n n o t be p r e c l u d e d a l t e r n a t i v e m e c h a n i s m s f o r t h e a c t i o n s o f i n c r e a s e d [ K + ] a r e : o ( i ) a d e c r e a s e o f membrane p o t e n t i a l l e a d i n g t o an i n c r e a s e d n e u r o n a l e x c i t a b i l i t y ( S c h w a r t z k r o i n a nd P r i n c e , 1 9 7 8 ) ; ( i i ) a r e d u c t i o n o f t h e K + c o n c e n t r a t i o n g r a d i e n t c a u s i n g an a t t e n u a t i o n o f membrane e v e n t s s u b s e r v e d by a K + c o n d u c t a n c e ; ( i i i ) an e f f e c t on n e u r o t r a n s m i t t e r r e l e a s e (Gage and Q u a s t e l , 1 9 6 5 ; Cooke and Q u a s t e l , 1 9 7 3 ) . I t h a s b e e n s u g g e s t e d by s e v e r a l i n v e s t i g a t o r s t h a t t h e i n i t i a t i o n a n d / o r s y n c h r o n i z a t i o n o f SPB a c t i v i t y i n CA3 i s d e p e n d e n t on r e c u r r e n t s y n a p t i c e x c i t a t i o n ( D i c h t e r and S p e n c e r , 1 9 6 9 b ; A y a l a e t a l . , 1 9 7 3 ; T r a u b a n d Wong, 1 9 8 2 , 1 9 8 3 ) . However i n t h e p r e s e n t s t u d y medium w h i c h was shown t o p r e v e n t s y n a p t i c t r a n s m i s s i o n ( c f . J e f f e r y s and H a a s , 1982) d i d n o t p r e v e n t t h e d e v e l o p m e n t i n some s l i c e s o f SPBs ( s e e a l s o Snow and Dudek, 1 9 8 3 ) . C o n s i s t e n t w i t h t h e i m p l i c a t i o n t h a t n o n - s y n a p t i c p r o c e s s e s .were i n v o l v e d i n t h e i r g e n e r a t i o n t h e c h a r a c t e r i s t i c s ( e . g . b u r s t d u r a t i o n and i n t e r b u r s t i n t e r v a l ) o f t h e s e S P B s w e r e c l e a r l y d i f f e r e n t f r o m t h o s e o b s e r v e d i n s l i c e s e x p o s e d , f o r e x a m p l e , t o i n c r e a s e d 5 5 [K ] b u t r e t a i n i n g i n t a c t s y n a p t i c t r a n s m i s s i o n . T h e s e d a t a i n d i c a t e t h a t as i n t h e C A l r e g i o n ( T a y l o r and Dudek, 1982; 2 + J e f f e r y s and H a a s , 1982; Y a a r i e t a l . , 1983) Ca - i n d e p e n d e n t p r o c e s s e s s u c h as e l e c t r i c a l t r a n s m i s s i o n ( M a c V i c a r and Dudek, 1 9 8 1 ) , e l e c t r i c a l f i e l d ( e p h a p t i c ) i n t e r a c t i o n s ( J e f f e r y s , 1981) and e x t r a c e l l u l a r i o n c h a n g e s ( Y a a r i et_ a _ l . , 1983) c a n p r o d u c e s p o n t a n e o u s a n d h i g h l y s y n c h r o n i z e d n e u r o n a l d i s -c h a r g e s . S i n c e t h e s e e v e n t s w o u l d p r o b a b l y a l s o be o p e r a t i v e i n n o r m a l medium i t i s r e a s o n a b l e t o s u g g e s t t h a t t h e y may a l s o c o n t r i b u t e t o SPB a c t i v i t y i n s l i c e s w e r e s y n a p t i c t r a n s m i s s i o n has n o t been b l o c k e d . 56 CHAPTER 3 POSTACTIVATION I N H I B I T I O N IN THE CA3 REGION I n t r o d u c t i o n F o l l o w i n g t h e i r a c t i v a t i o n by a n t i - o r o r t h o d r o m i c s t i m u l i , p y r a m i d a l and g r a n u l e c e l l s i n t h e h i p p o c a m p a l f o r m a t i o n e x h i b i t a n i n h i b i t i o n w h i c h i s d u e , a t l e a s t i n p a r t , t o t h e e x c i t a t i o n by a r e c u r r e n t p athway o f i n t e r n e u r o n s r e l e a s i n g ^ - a m i n o b u t y r i c a c i d (GABA) ( K a n d e l e t a _ l . , 1 9 6 1 ; A n d e r s e n e t a _ l . , 1964 ; C u r t i s e_t a _ l . , 1970 ; K o h l e r a n d C h a n - P a l a y , 1 9 8 3 ) . T h i s G A B A - m e d i a t e d i n h i b i t i o n i s a t t r i b u t -a b l e t o t h e h y p e r p o l a r i z a t i o n and t h e s h u n t i n g a c t i o n o f a C I c o n d u c t a n c e ( D i n g l e d i n e and Langmoen, 1 9 8 0 ) . I n a d d i t i o n t o t h i s G A B A e r g i c p r o c e s s , t h e r e i s e v i d e n c e f o r an o r t h o d r o m -i c a l l y - a c t i v a t e d i n h i b i t i o n i n C A l ( N i c o l l a n d A l g e r , 1 9 8 1 ; N e w b e r r y and N i c o l l , 1982) and t h e d e n t a t e g y r u s ( T h a l m a n n and A y a l a , 1982) w h i c h i s n o t a t t r i b u t a b l e t o t h e a c t i o n s o f GABA. I n t h i s c h a p t e r t h e r e s u l t s o f an e l e c t r o p h y s i o l o g i c a l a n d p h a r m a c o l o g i c a l e v a l u a t i o n o f t h e i n h i b i t i o n o c c u r r i n g i n t h e CA3 r e g i o n a f t e r a n t i - and o r t h o d r o m i c s t i m u l a t i o n a r e p r e s e n t e d . 57 M e t h o d s Th e 70 h i p p o c a m p a l s l i c e s o b t a i n e d f r o m 29 m a l e a l b i n o r a t s were p r e p a r e d and m a i n t a i n e d as d e s c r i b e d i n C h a p t e r 2 w here t h e t e c h n i q u e s f o r i n t r a - a nd e x t r a c e l l u l a r r e c o r d i n g a r e a l s o o u t l i n e d . An e l e c t r o d e i n C A 3 a (R,-) r e c o r d e d o r e s p o n s e s e v o k e d by s t i m u l i a p p l i e d e i t h e r t o t h e mossy f i b r e (MF) p a t h w a y i n t h e h i l u s ( F i g . 2.5, S_) o r t o t h e f i m b r i a ( S g ) t o p r o d u c e s u b m a x i m a l (2-4mV) o r t h o d r o m i c o r a n t i d r o m i c (AD) p o p u l a t i o n s p i k e s ( P S ) . I n t h e e x p e r i m e n t s , a t e s t (T) s t i m u l a t i o n ( 0 . 0 5 - 0 . 1 Hz) was on a l t e r n a t e o c c a s i o n s p r e c e d e d by a c o n d i t i o n i n g (C) s t i m u l u s t o t h e same ( u s i n g an i d e n t i c a l i n t e n s i t y o f s t i m u l a t i o n ) o r a d i f f e r e n t i n p u t t o t h e ,CA3 r e g i o n o f t h e s l i c e . The i n t e r v a l b e t w e e n t h e c o n d i t i o n i n g and t h e t e s t v o l l e y s (C-T i n t e r v a l ) was v a r i e d b e t w e e n 20-4000 msec. W i t h p a i r e d h o m o s y n a p t i c s t i m u l i i n h i b i t i o n was c a l c u l a t e d as t h e p e r c e n t d i f f e r e n c e b e t w e e n t h e mean a m p l i t u d e s (3-6 d e t e r m i n -a t i o n s ) o f C a n d T r e s p o n s e s ; w i t h p a i r e d h e t e r o s y n a p t i c s t i m u l i t h e p e r c e n t d i f f e r e n c e b e t w e e n t h e c o n d i t i o n e d and u n c o n d i t i o n e d T r e s p o n s e s was c a l c u l a t e d . P a i r e d s t i m u l i were a p p l i e d i n t h e c o m b i n a t i o n s : F I M C + F I M T , MF C + FIM^,, A D C + F I M T , MF C + MF T AND MF^ + AD^. T h e G A B A e r g i c n a t u r e o f t h e i n h i b i t i o n s i n CA3 was e v a l u a t e d by i o n t o p h o r e s i s o f B I C f r o m a s o l u t i o n o f t h e m e t h o c h l o r i d e , 25mM (pH 3.5) i n 150 mM N a C l , o r by p e r f u s i o n ( l - 5 x l 0 _ 6 M , 5 - 1 0 m l ) . 58 R e s u l t s The e f f e c t s o f F ™ c o r MF C s t i m u l i on s u b m a x i m a l F I M T r e s p o n s e s a r e shown i n F i g . 3.1 A,B,C,E. W i t h e i t h e r c o n d i t i o n i n g s h o c k t h e m a g n i t u d e o f t h e i n h i b i t i o n o c c u r r i n g a t 100-1800 msec was s i m i l a r . H o wever, a t C-T i n t e r v a l s <100 m sec, d i s p a r a t e a c t i o n s were a p p a r e n t . Thus w i t h h o m o s y n a p t i c p a i r e d s t i m u l i ( F I M C + FIM^) s e p a r a t e d by 50 msec, i n 3 o f 5 s l i c e s t h e r e was a 3 5 - 7 5 % i n c r e a s e i n t h e a m p l i t u d e o f t h e F I M T r e s p o n s e w h e r e a s i n t h e o t h e r 2 c a s e s i n h i b i t i o n s o f 60% a n d 7 4 % w e r e o b s e r v e d . I n c o n t r a s t , w i t h h e t e r o s y n a p t i c s t i m u l i ( M F C + F I M T ) t h e r e was e i t h e r no e f f e c t (1 s l i c e ) o r i n h i b i t i o n ( 1 1 s l i c e s ) o f FIM^, r e s p o n s e s a t t h e same C-T i n t e r v a l . A c t i v a t i o n o f t h e s e p a t h w a y s a l s o p r o d u c e d a l o n g - l a s t i n g i n h i b i t i o n ( 3 00-1000 msec) o f s p o n t a n e o u s l y a c t i v e CA3 c e l l s ( d a t a n o t s h o w n ) . F i g . 3.4A,B and 3.5C i l l u s t r a t e t h a t t h e i n h i b i t i o n , a t l e a s t t h a t e v o k e d h e t e r o s y n a p t i c a l l y , was n o t a t t r i b u t a b l e t o p o s t - f i r i n g c h a n g e s o f membrane p r o p e r t i e s s i n c e t h e s m a l l a m p l i t u d e o f t h e PS e v o k e d by MF^ a c t i v a t i o n i n d i c a t e s t h a t f e w n e u r o n s w e r e a c t i v a t e d . The f i r s t i n d i c a t i o n t h a t a t l e a s t two m e c h a n i s t i c a l l y d i s t i n c t p r o c e s s e s were i n v o l v e d i n t h e i n h i b i t i o n o f FIM^, r e s p o n s e s a r o s e f r o m two o b s e r v a t i o n s . F i r s t , i n a few s l i c e s i t was p o s s i b l e t o a c t i v a t e d i f f e r -e n t i a l l y e i t h e r t h e e a r l y ( 2 0 m s e c ) o r t h e l a t e r ( 2 0 0 - 3 0 0 msec) p h a s e o f t h e i n h i b i t i o n . S e c o n d l y , i n <5% o f t h e s l i c e s ( w h i c h were e x c l u d e d f r o m f u r t h e r a n a l y s i s ) i t was p o s s i b l e t o e v o k e o n l y t h e e a r l y o r t h e l a t e i n h i b i t i o n b u t n o t b o t h e v e n 59 A. C. D. C -T Intervol (msec) FIM Fig.,. 3.1 Av. Mean (+_S.E.M.) p e r c e n t i n h i b i t i o n e v o k e d by F I M C s t i m u l i on FIM^, a c t i v i t y a t 20 msec and l o n g e r C-T i n t e r v a l s . T he number o f s l i c e s e x a m i n e d f o r e a c h C-T i n t e r v a l i s i n d i c a t e d i n b r a c k e t s . B. O s c i l l o s c o p e t r a c e s o f a F I M C r e s p o n s e ( i ) a n d - i n h i b i t i o n o f FIM^, a c t i v i t y ( i i ) ev o k e d w i t h the same s t i m u l u s i n t e n s i t y 300 msec l a t e r . C, D. C o n v e n t i o n s as f o r A but i n h i b i t i o n o f F I M T r e s p o n s e s e l i c i t e d by MF^ a n d AD^ s t i m u l i , r e s p e c t i v e l y . E . F i l m e d r e c o r d s s h o wing the e f f e c t s o f MF^ , ( i - i i i ) and AD^ ( i v - v ) on F I M T r e s -p o n s e s . T h e u n c o n d i t i o n e d FIM r e s p o n s e i s shown i n ( v i i ) . A l t h o u g h c o m p a r a b l e i n h i b i t i o n o c c u r r e d a t 20 msec w i t h e i t h e r c o n d i t i o n i n g a c t i v a t i o n , a r e d u c t i o n of FIM^ a c t i v i t y a t 400 msec was o b s e r v e d a f t e r MF^ o n l y . In t h i s a n d s u b s e q u e n t f i g u r e s , t r a c e s a r e c o m p r i s e d o f 2 o r 3 c o n s e c u t i v e sweeps; l a b e l l e d a rrow below t h e r e c o r d i n d i c a t e s t h e time and t y p e o f s t i m u l a t i o n ; C-T i n t e r v a l i n msec i s shown i n b r a c k e t s . 60 when FIM o r MF c o n d i t i o n i n g s t i m u l i o f a h i g h i n t e n s i t y w e r e a p p l i e d . F i g . 3.ID i l l u s t r a t e s t h e a c t i o n s on t h e F I M ^ r e s p o n s e o f A D C e l i c i t e d by a f i m b r i a l s h o c k a p p l i e d t h r o u g h a s e p a r a t e e l e c t r o d e . C o m p a r i s o n o f F i g 3. ID t o F i g . 3.1A,C shows t h a t t h e i n h i b i t i o n o f F I M T a t C-T i n t e r v a l s <100 msec was s i m i l a r f o l l o w i n g F I M , MF o r AD c o n d i t i o n i n g s t i m u l i . H o w e v e r , when e l i c i t e d a n t i d r o m i c a l l y t h e i n h i b i t i o n m e a s u r e d a t l o n g e r C-T i n t e r v a l s was l e s s e f f e c t i v e and o f s h o r t e r d u r a t i o n t h a n t h a t o c c u r r i n g w i t h a c o n d i t i o n i n g s h o c k t o t h e FIM o r MF p a t h w a y s . T h e s e d i f f e r e n c e s may h a v e r e f l e c t e d a v a r i a t i o n i n t h e p o t e n c y o f t h e i n h i b i t i o n b e t w e e n s l i c e s . To a d d r e s s t h i s p o s s i b i l i t y a c o m p a r i s o n o f b o t h t h e M F C and A D C i n h i b i t o r y a c t i o n s on F I M T a c t i v i t y was made c o n c u r r e n t l y i n some s l i c e s . A r e p r e s e n t a t i v e e x a m p l e o f one o f t h e s e c o m p a r i s o n s ( F i g . 3.IE) showed t h e i n h i b i t i o n a t 20 msec t o be s i m i l a r w i t h a c o n d i t i o n i n g s t i m u l a t i o n o f e i t h e r t h e MF o r AD i n p u t , b u t a g a i n a r e d u c t i o n o f F I M T a c t i v i t y was e v i d e n t a t 400 msec f o l l o w i n g t h e MF C s t i m u l i o n l y . W i t h p a i r e d MF s t i m u l i t h e r e was a t any C-T i n t e r v a l r e l a t i v e l y l i t t l e i n h i b i t i o n o f t h e 1° PS o f t h e t e s t r e s p o n s e ( F i g . 3.2A) d e s p i t e t h e l a r g e r e d u c t i o n s o f t h e 2° PS w h i c h were o b t a i n a b l e . A l t h o u g h i t was n o t r i g o r o u s l y i n v e s t i g a t e d , t h e p a t t e r n o f t h e i n h i b i t i o n o f t h e 2° MF PS r e s e m b l e d t h a t w h i c h c h a r a c t e r i z e d p a i r e d FIM s t i m u l i . Thus a t 20-50 msec e i t h e r i n h i b i t i o n o r p o t e n t i a t i o n ( e . g . F i g . 3 . 2 B i i ) o f t h e 2° PS c o u l d o c c u r , w h e r e a s a t 50-1800 msec C-T i n t e r v a l s t h i s 61 F i g . 3.2 A. P a i r e d p u l s e s t i m u l a t i o n o f t h e MF i n p u t t o CA3. ; I l l u s t r a t e d i s t h e mean (+S.E.M.) p e r c e n t r e d u c t i o n o f t h e a m p l i t u d e o f t h e 1 ° PS o f t h e t e s t r e s p o n s e e v o k e d 20-900 msec a f t e r the c o n d i t i o n i n g r e s p o n s e . B. T h e s e f i l m e d r e c o r d s i l l u s t r a t e t h e r e f r a c t o r i n e s s o f t h e 1° PS o f t h e MF T r e s p o n s e t o i n h i b i t i o n a t any C-T i n t e r v a l whereas t h e 2 ° PS was p o t e n t i a t e d a n d i n h i b i t e d a t 40 a n d 400 msec i n t e r v a l s r e s p e c t i v e l y . C. ( i ) The u n c o n d i t i o n e d MF r e s p o n s e . When p r e c e d e d by a c o n d i t i o n i n g FIM r e s p o n s e ( i i ) t h e 1° PS was u n a f f e c t e d b u t t h e 2° PS of t h e MF^, f i e l d p o t e n t i a l was s u b s t a n t i a l l y r e d u c e d . D. The i n h i b i t i o n (mean +S.E.M.) o f submaximal AD^ a c t i v i t y p r o d u c e d by a p r e c e d i n g MF^ s t i m u l i . E . T he AD r e s p o n s e o c c u r r i n g w i t h o u t ( i ) a n d w i t h ( i i i ) a p r e c e d i n g MF^ ( i i ) . The c a l i b r a t i o n b ar a p p l i e s t o the r e c o r d s i n B, C and E. 62 2 r e s p o n s e was d e p r e s s e d ( F i g . 3 . 2 B i i i ) . FIM^, s t i m u l i a l s o had s i m i l a r a c t i o n s t o i n h i b i t t h e 2° b u t n o t t h e 1° PS o f t h e MF e v o k e d p o t e n t i a l ( F i g . 3.2 C ) . The l a s t o f t h e d e t e r m i n a t i o n s o f i n p u t i n t e r a c t i o n s i n CA3 e x a m i n e d t h e e f f e c t o f MF C s t i m u l a t i o n on AD^ r e s p o n s e s . F i g . 3.2D shows an a b s e n c e o f any i n h i b i t o r y e f f e c t on t h e A D T r e s p o n s e a t 20 msec w h e r e a s i t was s u b s t a n t i a l l y i n h i b i t e d a t 100-500 msec C-^ T i n t e r v a l s i n e a c h o f 5 s l i c e s t e s t e d . The d u r a t i o n o f t h i s M F ^ - i n d u c e d i n h i b i t i o n o f A D T a c t i v i t y was somewhat s h o r t e r t h a n t h a t o c c u r r i n g w i t h o r t h o d r o m i c (FIM) t e s t r e s p o n s e s (compare F i g . 3.2D and 3 . 1 C ) . S t a b l e i n t r a c e l l u l a r i m p a l e m e n t s o f 11 CA3 c e l l s w i t h m e mbrane p o t e n t i a l s g r e a t e r t h a n -55 mV s h o w e d t h a t t h e s t i m u l a t i o n o f e i t h e r t h e MF o r F I M i n p u t a t a n i n t e n s i t y s u b t h r e s h o l d f o r t h e d i s c h a r g e o f an a c t i o n p o t e n t i a l e l i c i t e d an EPSP f o l l o w e d by a b i p h a s i c h y p e r p o l a r i z a t i o n (HP) o f CA3 n e u r o n s . I n b o t h s i t u a t i o n s t h e e a r l y HP r e a c h e d a peak a t c a . 20 msec b u t r e c o v e r y t o w a r d s t h e p r e - s t i m u l u s membrane p o t e n t i a l was i n t e r r u p t e d by a s e c o n d p h a s e o f HP w h i c h a c h i e v e d a m a x i m a l a m p l i t u d e a t 150-200 msec and p e r s i s t e d f o r up t o 700 msec ( F i g . 3 . 3A,B). T h a t b o t h t h e e a r l y and l a t e p h a s e s c o u l d i n f a c t o c c u r i n t h e a b s e n c e o f a d i s c e r n i b l e EPSP i n t h e M F -evoked r e s p o n s e " i s e v i d e n t i n t h e o s c i l l o s c o p e r e c o r d s o f F i g . 3.3C. T h i s l a t t e r f i g u r e a l s o shows t h a t t h e a m p l i t u d e o f t h e t w o p h a s e s o f t h e HP d i d n o t i n c r e a s e i n p a r a l l e l as t h e s t i m u l u s i n t e n s i t y was i n c r e a s e d . S i n c e a n t i - • and o r t h o d r o m i c a l l y e v o k e d i n h i b i t i o n s o f 63 I I I I I I O 100 200 300 400 500 msec c, 10 v 20 30 40 50 .MF 1.0 mV 40msec F i g . 3.3. M o ving c h a r t r e c o r d i n g s o f t h e i n t r a c e l l u l a r r e s p o n s e i n two CA3 c e l l s t o s u b t h r e s h o l d MF (A) and FIM (B) s t i m u l a t i o n . I n e a c h c a s e t h e r e was f o l l o w i n g t h e EPSP a b i p h a s i c h y p e r p o l a r i z a t i o n (HP). Note the s i m i l a r i t i e s i n the l a t e n c y t o peak f o r e a c h component o f t h e HP. The v a l u e s f o r t h e r e s t i n g membrane p o t e n t i a l (RMP) and the i n p u t r e s i s t a n c e ( R I N ) w h i c h was d e t e r m i n e d by c u r r e n t i n j e c t i o n ( n o t shown) a r e i n d i c a t e d t o t h e r i g h t o f e a c h r e c o r d . C. O s c i l l o s c o p e t r a c e s o f i n t r a c e l l u l a r r e c o r d i n g s o f t h e change i n membrane p o t e n t i a l f o l l o w i n g v a r i o u s i n t e n s i t i e s of MF s t i m u l a t i o n . A f t e r a 20 V s t i m u l u s , b o t h p h a s e s o f t h e HP were p r e s e n t and were not p r e c e d e d by a d e p o l a r i z a t i o n . As the s t i m u l u s was i n c r e a s e d , an EPSP was e l i c i t e d and t h e r e was a n o n - p a r a l l e l i n c r e a s e i n the a m p l i t u d e of each component of the HP. V a l u e s f o r t h e RMP and r e s p e c t i v e l y . R I N i n t h i s c e l l were -58 mV and 33 megohms 64 h i p p o c a m p a l p y r a m i d a l c e l l s h a v e been r e p o r t e d t o be GABA-m e d i a t e d , t h e a c t i o n s o f t h e GABA a n t a g o n i s t b i c u c u l l i n e m e t h o c h l o r i d e ( B I C ) ( C u r t i s e t a _ l . , 1971) a p p l i e d e i t h e r by i o n t o p h o r e s i s w i t h i n 100yam o f t h e c e l l body l a y e r o r by i t s a d d i t i o n t o t h e p e r f u s a t e were e x a m i n e d . B e c a u s e a t e a r l y C-T i n t e r v a l s w i t h h o m o s y n a p t i c p a i r e d s t i m u l i e i t h e r f a c i l i t a t i o n o r i n h i b i t i o n c o u l d o c c u r t h e s e e x p e r i m e n t s w i t h B I C e x a m i n e d o n l y t h e M F ^ - e v o k e d i n h i b i t i o n o f F I M T a c t i v i t y . F o l l o w i n g i t s i o n t o p h o r e t i c (50-200nA f o r 5-30 m i n ) o r b a t h a p p l i c a t i o n B I C i n c r e a s e d t h e a m p l i t u d e o f t h e F I M - e v o k e d o r t h o d r o m i c PS and r e c r u i t e d a d d i t i o n a l P S s ( F i g 3.4A,B; 3.5C) ( S c h w a r t z k r o i n and P r i n c e , 1 9 8 0 ) . S p o n t a n e o u s b u r s t r e s p o n s e s were n o t o b s e r v e d w i t h t h e d o s e s o f B I C e m p l o y e d h e r e ( c f . F i g . 6 . 6 ) . M u l t i p l e P S s c o u l d a l s o a p p e a r f o l l o w i n g MF s t i m u l i i n t h e B I C - t r e a t e d s l i c e , h o w e v e r t h e a m p l i t u d e o f t h e 1° PS r e m a i n e d u n c h a n g e d ( F i g . 3 . 4 A ) . The r e s u l t s o f t h e e x p e r i m e n t s w i t h BIC i n d i c a t e d an a l m o s t c o m p l e t e a b o l i t i o n o f t h e i n h i b i t i o n a t 20 msec ( p r e - t r e a t m e n t i n h i b i t i o n 60.7+7.2%, p o s t - t r e a t m e n t 3.7+8.5%, mean + S.E.M., n = l l ) w h e r e a s t h a t m e a s u r e d a t 200-300 msec was u n a f f e c t e d o r i n c r e a s e d ( p r e - and p o s t - t r e a t m e n t v a l u e s 62.7+9.1% and 78.1+5.6%, r e s p e c t i v e l y , n = l l ) . R e c o v e r y o f i n h i b i t i o n t o n e a r p r e - t r e a t m e n t l e v e l s o c c u r r e d w i t h i n 10-25 m i n o f t h e t e r m i n a t i o n o f d r u g a p p l i c a t i o n . To a p p r o x i m a t e t h e d u r a t i o n o f t h e G A B A e r g i c p r o c e s s t h e c h a n g e s i n t h e p a t t e r n o f t h e i n h i b i t i o n s f o l l o w i n g B I C a d m i n i s t r a t i o n were a s s e s s e d i n 4 s l i c e s . I n e a c h c a s e B I C 6 5 A Pre - treatment FIM MF, Bicuculline treoted (lOOnA.IOn FIMT (20) FIM j (300) Recovery _ F m V 4 msec B. Bicuculline treated (2.5xI0" 6 M. 15ml) 4 .-msec F i g . 3 . 4 -The a c t i o n s o f b Lleucu 1:1 i ne ( B I Q ) on t h e M F - e v o k e d e a r l y and l a t e i n h i b i t i o n o f FIM^ a c t i v i t y . A. The u n o o n d i f c i o n e d FIM; r e s p o n s e i n e a c h o f t h e t h r e e t r e a t m e n t s i t u a t i o n s i s shown i n t h e f i r s t c o l u m n . The MF^ f i e l d p o t e n t i a l shown i n t h e s e c o n d .column i s r e p r e s e n t a t i v e o f t h a t u s e d t o c o n d i t i o n t h e FIM^, r e s p o n s e e l i c i t e d 20 ( t h i r d column) o r 300 msec ( f o u r t h column) l a t e r . T r a c e s f o r e a c h t r e a t m e n t g r o u p w e r e o b t a i n e d o v e r a 2-3 m i n u t e p e r i o d . The FIM s t i m u l u s i n t e n s i t y was d e c r e a s e d from 16 t o 13 V d u r i n g the 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 BIC t o a l l o w a c o m p a r i s o n o f t h e M F ^ - i n d u c e d i n h i b i t i o n o f a p r i m a r y PS s i m i l a r t o t h a t i n t h e p r e - t r e a t m e n t s i t u a t i o n ; the s t r e n g t h o f MF^ s t i m u l i was not c h a n g e d . B. As f o r A e x c e p t p r e - t r e a t m e n t a n d r e c o v e r y r e s p o n s e s a r e n o t shown and BIC was a p p l i e d i n t h e p e r f u s a t e . The p e r s i s t e n c e o f t h e l a t e i n h i b i t i o n i n t h i s c a s e was n o t c o r r e l a t e d w i t h b u r s t f i r i n g i n the MF^ r e s p o n s e ( s e e t e x t f o r e x p l a n a t i o n ) . 66 Pre - treatment 2 msec F i g . 3.5 A, B. P a i r e d pu'l.s:e he t e r ; o s y n a p t i c (MF + i -j ' ' ' F I M T ) i n h i b i t i o n a t d i f f e r e n t C-T i n t e r v a l s b e f o r e ( o - o ) , d u r i n g (*•-•) and a f t e r ( A - A , i n . A o n l y ) t h e i o n t o p h o r e s i s o f BIC (A - 125 nA f o r 6 min; B - 150 nA f o r 25 m i n ) . In b o t h c a s e s , a r e d u c t i o n o f t h e i n h i b i t i o n o c c u r r e d a t C-T i n t e r v a l s o f l e s s t h a n 60 msec w h e r e a s t h e l a t e r i n h i b i t i o n was u n -a f f e c t e d o r p o t e n t i a t e d . C. C o n v e n t i o n s as i n F i g . 3.4A. In t h i s s l i c e i n the c o n t r o l s i t u a t i o n low i n t e n s i t y (15 V) MF s t i m u l i p r o d u c e d an i n h i b i t i o n o f FIM^, a c t i v i t y a t 20 but not 200 msec. BIC r e d u c e d the e a r l y i n h i b i t i o n and FIM^ a c t i v i t y a t t h e 200 msec i n t e r v a l was now i n h i b i t e d . The FIM s t i m u l u s i n t e n s i t y was d e c r e a s e d f r o m 26 t o 23 V d u r i n g t h e d r u g a p p l i c a t i o n . N o t e t h a t t h e s e i n h i b i t i o n s o c c u r r e d i n t h e a b s e n c e o f a f i e l d p o t e n t i a l i n t h e MF^ r e s p o n s e . The a c t i o n s o f BIC were f u l l y r e v e r s i b l e . 67 c a u s e d a v i r t u a l a b o l i t i o n o f t h e i n h i b i t i o n a t 20 msec b u t an a c t i o n o f BIC t o r e d u c e t h e i n h i b i t i o n n e v e r e x t e n d e d b e y o n d t h e 60 msec C-T i n t e r v a l ( F i g . 3.5 A,B) . I n d e e d , B I C o f t e n p o t e n t i a t e d t h e i n h i b i t i o n a t t h e l o n g e r C-T i n t e r v a l s , i n some c a s e s q u i t e d r a m a t i c a l l y . F o r e x a m p l e , i n t h e s l i c e f r o m w h i c h t h e r e c o r d s o f F i g . 3.5C w e re d e r i v e d , i o n t o p h o r e s i s o f B I C r e d u c e d t h e e a r l y i n h i b i t i o n and r e v e a l e d an i n h i b i t i o n a t 200 msec w h i c h was a p p a r e n t n e i t h e r i n t h e p r e - t r e a t m e n t n o r r e c o v e r y r e s p o n s e . T h e d i s c h a r g e o f p y r a m i d a l c e l l s i n b u r s t s o f a c t i o n p o t e n t i a l s c a n e l i c i t a n HP c a u s e d by a c a l c i u m a c t i v a t e d i n c r e a s e o f a p o t a s s i u m c o n d u c t a n c e ( A l g e r and N i c o l l , 1980) w h i c h c a n p r o d u c e a n i n h i b i t i o n o f e v o k e d a c t i v i t y ( c f . C h a p t e r 6 ) . However, a l t h o u g h e v o k e d f i r i n g i n b u r s t s was s o m e t i m e s e v i d e n t i n MF C r e s p o n s e s i n s l i c e s e x p o s e d t o B I C ( e . g . F i g . 3.4A) t h i s d o e s n o t seem t o u n d e r l i e t h e p e r s i s -t e n c e o f t h e l a t e i n h i b i t i o n s i n c e : (1) t h e PSs c o m p r i s i n g t h e b u r s t s were s m a l l i n c o m p a r i s o n w i t h t h e u n c o n d i t i o n e d FIM PS, s u g g e s t i n g t h a t any HP w o u l d o c c u r i n o n l y a few c e l l s and (2) t h e s e b u r s t s w e re n o t o b s e r v e d i n a number o f c a s e s ( e . g . F i g 3.4B, 3. 5C) w h e r e t h e l a t e i n h i b i t i o n was a l s o u n a f f e c t e d f o l l o w i n g t h e a d m i n i s t r a t i o n o f B I C . The c h a n g e s i n t h e i n h i b i t i o n i n CA3 w h i c h f o l l o w e d B I C t r e a t m e n t a r e s u m m a r i z e d and c o m p a r e d w i t h a d i s i n h i b i t o r y a c t i o n o f k a i n i c a c i d i n F i g u r e 5.15. 68 D i s c u s s i o n T h e m a j o r f i n d i n g o f t h e e x p e r i m e n t s d e s c r i b e d i n t h i s c h a p t e r was t h e o c c u r r e n c e i n t h e CA3 r e g i o n o f a l o n g - l a s t i n g i n h i b i t i o n f o l l o w i n g t h e a c t i v a t i o n o f a f f e r e n t i n p u t s . T h i s i n h i b i t i o n was s e p a r a b l e i n t o an e a r l y component w h i c h was b l o c k e d w i t h B I C and a l a t e r p h a s e w h i c h was e i t h e r u n a f f e c t e d o r i n c r e a s e d i n t h e p r e s e n c e o f t h e GABA a n t a g o n i s t . The B I C - s e n s i t i v e e a r l y i n h i b i t i o n r e s e m b l e d t h a t w h i c h o c c u r s a l s o i n C A l and t h e d e n t a t e g y r u s o f t h e h i p p o c a m p a l s l i c e a n d w o u l d a p p e a r t o be d u e t o t h e a c t i v a t i o n o f G A B A e r g i c i n t e r n e u r o n s . The r e s u l t s o f e x p e r i m e n t s a s s e s s i n g t h e c hange i n t h e t i m e c o u r s e o f t h e e x t r a c e l l u l a r l y - d e t e r -m i n e d i n h i b i t i o n f o l l o w i n g BIC t r e a t m e n t s u g g e s t e d a d u r a t i o n f o r t h i s G A B A - m e d i a t e d e v e n t o f l e s s t h a n 60 msec. T h i s i s a l s o c o n s i s t e n t w i t h t h e d u r a t i o n o f t h e i n t r a c e l l u l a r l y -r e c o r d e d e a r l y HP r e p o r t e d h e r e , and w h i c h has b een shown by o t h e r s t o be a C l - d e p e n d e n t p i c r o t o x i n - s e n s i t i v e I P S P ( K n o w l e s et. aT. , 1 9 8 2 ) . On o c c a s i o n i t was p o s s i b l e t o e l i c i t t h e e a r l y i n h i b i t i o n i n t h e a b s e n c e o f any a p p r e c i a b l e PS i n t h e c o n d i t i o n i n g r e s p o n s e . T h i s o b s e r v a t i o n s u g g e s t s t h e e x i s t e n c e o f a f e e d f o r w a r d G A B A e r g i c p a t h w a y s u c h as o c c u r s i n C A l ( A l g e r and N i c o l l , 1 9 8 2 ) . I n t e r e s t i n g l y , e v e n d u r i n g t h e a p p l i c a t i o n o f B I C t h e r e was no e v i d e n c e a t any t i m e f o r a p o t e n t i a t i o n o f F I M ^ a c t i v -i t y by a MF C s t i m u l u s . T h i s i s i n c o n t r a s t t o t h e homosyn-a p t i c p o t e n t i a t i o n s w h i c h w e r e a p p a r e n t o c c a s i o n a l l y w i t h p a i r e d MF b u t e s p e c i a l l y w i t h FIM s t i m u l i ( c f . A n d e r s e n , 1960) 69 i n e i t h e r c o n t r o l o r B I C - t r e a t e d s l i c e s . T h e s e d a t a a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t p a i r e d p u l s e p o t e n t i a t i o n r e f l e c t s a s y n a p t i c change r a t h e r t h a n a g e n e r a l i z e d p o s t -s y n a p t i c e v e n t ( C r e a g e r et_ a _ l . , 1980 ) . The o c c u r r e n c e o f homo- but not h e t e r o s y n a p t i c p a i r e d p u l s e p o t e n t i a t i o n has a l s o been r e p o r t e d w i t h t h e MF and p e r f o r a n t pathways t o CA3 ( M i s g e l d e t a l . , 1 9 79). The c h a n g e o f t h e p a t t e r n o f i n h i b i t i o n a f t e r t h e a d m i n i s t r a t i o n o f BIC i m p l i e d a l o n g l a t e n c y f o r t h e o n s e t o f t h e l a t e i n h i b i t i o n and i n d i c a t e d a l s o t h a t t h e m a j o r p o r t i o n o f t h e h e t e r o s y n a p t i c l o n g - l a s t i n g i n h i b i t i o n was a t t r i b u t a b l e t o t h i s B I C - i n s e n s i t i v e e v e n t . T he f a c t t h a t i n s e v e r a l s l i c e s t h e l a t e i n h i b i t i o n was i n c r e a s e d i n t h e p r e s e n c e o f t h e GABA a n t a g o n i s t may be r e l a t e d t o o t h e r a c t i o n s o f t h e d r u g ( H e y e r e t a l . , 1 9 8 2 ) . A l t h o u g h t h e e f f e c t o f BIC on t h e i n h i b i t i o n o b s e r v e d w i t h p a i r e d FIM s t i m u l i was n o t a s s e s s e d , i t w o u l d a p p e a r t h a t a s i m i l a r mechanism u n d e r l i e s b o t h t h i s and t h e h e t e r o s y n a p t i c i n h i b i t i o n s i n c e t h e i r t i m e c o u r s e s were v e r y s i m i l a r . A d d i t i o n a l l y , a c t i v a t i o n o f t h e MF and FIM i n p u t s e l i c i t e d a b i p h a s i c HP, t h e t i m e c o u r s e o f w h i c h was c o r r e l a t e d w i t h t h e e a r l y and l a t e i n h i b i t i o n ( s e e b e l o w ) . E x t r a - and i n t r a c e l l u l a r a n a l y s e s r e v e a l e d t h a t b o t h the l a t e i n h i b i t i o n and t h e l a t e HP c o u l d o c c u r i n t h e a b s e n c e o f c e l l f i r i n g a n d i n d i c a t e d t h a t t h e s e e v e n t s w e r e n o t a t t r i b u t a b l e t o membrane c u r r e n t s a c t i v a t e d by t h e d e p o l a r -i z a t i o n a c c o m p a n y i n g a c t i o n p o t e n t i a l d i s c h a r g e . F u r t h e r m o r e , t h a t t h e l a t e i n h i b i t i o n was sometimes evok e d i n t h e a b s e n c e 70 o f any PS i n t h e c o n d i t i o n i n g r e s p o n s e s u g g e s t e d t h a t i t c o u l d be a c t i v a t e d by a f e e d f o r w a r d p a t h w a y as a p p e a r e d t o be t h e c a s e a l s o w i t h t h e e a r l y G A B A e r g i c p r o c e s s ( s e e a b o v e ) . To d e t e r m i n e i f t h e l a t e i n h i b i t i o n c o u l d be e v o k e d r e c u r r e n t l y , t h e a c t i o n s o f an AD^ PS, c o n t a m i n a t e d as l i t t l e as p o s s i b l e by o r t h o d r o m i c r e s p o n s e s , on t h e F I M T o r t h o d r o m i c a c t i v i t y w e r e e x a m i n e d . As w o u l d be e x p e c t e d , t h e e a r l y i n h i b i t i o n was s t r o n g l y a c t i v a t e d by A D C s t i m u l i and was c o m p a r a b l e i n i t s p o t e n c y t o t h a t o c c u r r i n g a f t e r MF^ , o r f i m Q a c t i v i t y . H o w ever, t h e t o t a l d u r a t i o n o f t h e A D - e v o k e d i n h i b i t i o n was s h o r t e r t h a n t h a t i n d u c e d w i t h e i t h e r FIM o r MF a c t i v a t i o n s . T h e s e d a t a i m p l y o n l y a r e l a t i v e l y weak a c t i v a t i o n o f t h e l a t e i n h i b i t i o n t h r o u g h a r e c u r r e n t p a t h w a y . S i n c e t h e a c t i v a t i o n o f a f f e r e n t p a t h w a y s s u p p r e s s e d t h e s p o n t a n e o u s f i r i n g o f c e l l s f o r up t o 1 s e c , t h e i n v o l v e -m e n t o f a p o s t s y n a p t i c p r o c e s s i n t h e l a t e i n h i b i t i o n was i m p l i c a t e d . T h i s was a l s o i m p l i e d i n e x p e r i m e n t s a d d r e s s i n g t h e a c t i o n o f MF^ s t i m u l i on AD P S s . A l t h o u g h w i t h t h i s s t i m u l u s p a r a d i g m t h e r e was no e f f e c t on A D T a t s h o r t (20-40 msec) C-T i n t e r v a l s , a t 50-500 msec a m a r k e d r e d u c t i o n o f t h e t e s t r e s p o n s e o c c u r r e d . The r e l a t i v e l y l a r g e r i n t r a c e l l u l a r h y p e r p o l a r i z a t i o n s w h i c h w ere c o r r e l a t e d w i t h t h e l a t e i n h i b i t i o n may e x p l a i n t h e more e f f e c t i v e b l o c k a d e o f a n t i d r o m i c e x c i t a t i o n a t t h e l o n g e r C-T i n t e r v a l s . The b e h a v i o r o f t h e 1° PS o f t h e MF r e s p o n s e was a g a i n , a s i n d i c a t e d i n C h a p t e r 2, u n l i k e t h a t e x p e c t e d o f an o r t h o d r o m i c e x c i t a t i o n ; t h u s i t was r e l a t i v e l y u n a f f e c t e d a t 71 any C-T i n t e r v a l d e s p i t e t h e c o n s i d e r a b l e d e p r e s s i o n s o f t h e 2° PS w h i c h c o u l d o c c u r i n t h e same t e s t r e s p o n s e . The 1° MF PS a p p e a r e d t o be more r e s i s t a n t t o i n h i b i t i o n t h a n was t h e F I M - e v o k e d AD PS. B e c a u s e t h e EPSP was n o t m e a s u r e d , i t i s n o t p o s s i b l e t o comment h e r e on t h e p o s s i b l e i n v o l v e m e n t o f a p r e s y n a p t i c i n h i b i t o r y p r o c e s s i n t h e r e d u c t i o n o f o r t h o d r o m i c t e s t a c t i v i t y . E v i d e n c e f o r a B I C - i n s e n s i t i v e i n h i b i t i o n m e d i a t e d by p r e - s y n a p t i c GABA r e c e p t o r s i n t h e r a t b r a i n h a s b e e n r e p o r t e d ( B owery et_ a _ l . , 1980, H i l l and B o w e r y , 1981) and may a l s o be o p e r a t i v e i n t h e CA3 r e g i o n . However, t h a t a t l e a s t a p o r t i o n o f t h e l a t e i n h i b i t i o n was a t t r i b u t a b l e t o a p o s t -s y n a p t i c e v e n t was i m p l i e d by t h e M F ^ -evoked r e d u c t i o n o f A D T a c t i v i t y and c o n f i r m e d by t h e r e s u l t s o f t h e p r e l i m i n a r y i n t r a c e l l u l a r s t u d i e s . Thus s u b t h r e s h o l d a f f e r e n t s t i m u l a t i o n e l i c i t e d a b i p h a s i c HP — t h e f i r s t p h a s e c o i n c i d i n g w i t h t h e G A B A - m e d i a t e d I P S P a s d i s c u s s e d p r e v i o u s l y . The s e c o n d HP had an o n s e t w h i c h a p p r o x i m a t e d t h a t f o r l a t e i n h i b i t i o n a s d e t e r m i n e d by t h e e x p e r i m e n t s w i t h B I C , and w h i c h l a s t e d f o r up t o 700 msec. F u j i t a ( 1 9 7 9 ) has r e p o r t e d t h e e x i s t e n c e o f s i m i l a r b i p h a s i c HPs i n r a b b i t CA3-CA4 n e u r o n s ijn v i v o a f t e r f i m b r i a l s t i m u l a t i o n . I n t h a t s t u d y , w h e r e a s t h e e a r l y component o f t h e b i p h a s i c HP was a s s o c i a t e d w i t h an i n c r e a s e d c o n d u c t a n c e and was r e v e r s i b l e u p o n i n j e c t i o n o f C l o r h y p e r p o l a r i z a t i o n o f t h e membrane, t h e l a t e HP e x h i b i t e d none o f t h e s e p r o p e r -t i e s . The l a t e HP was i n t e r p r e t e d t o be a d e n d r i t i c I P S P 72 a l t h o u g h t h e e f f e c t s o f GABA a n t a g o n i s t s were n o t i n v e s t i -g a t e d . I n a g r e e m e n t w i t h F u j i t a ' s o b s e r v a t i o n s , K n o w l e s e t a l . ( 1982) f o u n d a s i m i l a r l a t e HP i n g u i n e a p i g CA3 n e u r o n s t o be much l e s s v o l t a g e s e n s i t i v e t h a n t h e e a r l y I P S P and v e r y d i f f i c u l t t o r e v e r s e . T h i s a p p a r e n t v o l t a g e - i n d e p e n d e n c e may r e f l e c t an o r i g i n f o r t h e l a t e HP i n t h e d e n d r i t e s w h i c h was e l e c t r o n i c a l l y r e m o t e f r o m c u r r e n t s i n j e c t e d t h r o u g h a m i c r o e l e c t r o d e i n t h e soma. I n i t i a l l y , K n o w l e s et. a _ l . ( 1 9 8 2) i n f e r r e d a C I - d e p e n d e n t d e n d r i t i c I P S P as t h e b a s i s f o r t h e l a t e HP, b u t more r e c e n t d a t a f r o m t h i s l a b o r a t o r y ( S c h w a r t z -k r o i n , p e r s o n a l c o m m u n i c a t i o n ) s u g g e s t s a p o t a s s i u m c o n d u c t -a n c e t o be i n v o l v e d ( s e e a l s o F i s h e r and A l g e r , 1 9 8 3 ) . An HP w i t h s i m i l a r e l e c t r o p h y s i o l o g i c a l c h a r a c t e r i s t i c s and p h a r m a c o l o g i c a l s e n s i t i v i t y p r o f i l e h a s been r e p o r t e d a s o c c u r r i n g b o t h i n C A l p y r a m i d a l ( N i c o l l a n d A l g e r , 1 9 8 1 ; N e w b e r r y and A l g e r , 1982) and d e n t a t e g r a n u l e c e l l s ( T h a l m a n n and A y a l a , 1 9 8 2 ) . I n t h e d e n t a t e g y r u s , as i n t h e CA3 r e g i o n , t h i s l a t e HP a p p e a r s t o be c o r r e l a t e d b o t h j l n v i v o (McNaughton e t a l . , 1978) and i n v i t r o ( O l i v e r and M i l l e r , 1984) w i t h a p o t e n t a n d l o n g - l a s t i n g i n h i b i t i o n o f r e s p o n s e s e v o k e d by p e r f o r a n t p a t h s t i m u l a t i o n . Of p a r t i c u l a r i n t e r e s t i s t h e o b s e r v a t i o n by T h a l m a n n and A y a l a ( 1 9 8 2 ) o f a p o t e n t i a t i o n o f t h e l a t e HP w i t h p i c r o t o x i n s i n c e an i n c r e a s e o f t h e l a t e i n h i b i t i o n was a l s o e v i d e n t i n t h e p r e s e n t e x p e r i m e n t s a f t e r t h e e x p o s u r e o f CA3 c e l l s t o t h e B I C . E v i d e n c e f o r a l o n g d u r a t i o n n o n - G A B A e r g i c i n h i b i t o r y p o t e n t i a l has a l s o b een r e p o r t e d i n o t h e r n e u r o n s s u c h as t h e 73 t u r t l e m i t r a l c e l l ( M o r i e_t a J . , 1 981), the r a b b i t p y r i f o r m c o r t e x ( S a t o u e t a l . , 1982) and i n t h e o l f a c t o r y c o r t e x o f t h e g u i n e a p i g ( C o n s t a n t i e_t a l . , 1 9 8 0 ) . The e x i s t e n c e o f two d i s t i n c t t y p e s o f IPSPs i n i n v e r t e b r a t e n e u r o n s i s a l s o w e l l documented ( e . g . see r e v i e w by G e r s h e n f e l d , 1 9 7 3 ) . To c o n c l u d e , e v i d e n c e has been p r e s e n t e d h e r e f o r a B l C - i n s e n s i t i v e i n h i b i t i o n w h i c h has a slow l a t e n c y o f o n s e t b u t l o n g - l a s t i n g a c t i o n . T h i s i n h i b i t i o n a p p e a r s t o be a t t r i b u t a b l e , a t l e a s t i n p a r t , t o t h e l a r g e p o s t s y n a p t i c HPs w h i c h c o u l d f o l l o w s u b t h r e s h o l d a f f e r e n t s t i m u l a t i o n . A number o f mechanisms may a c c o u n t f o r t h e l a t e i n h i b i t i o n / H P . F i r s t , t h e s e may be a s c r i b e d t o e v e n t s i n i t i a t e d by n e u r o -t r a n s m i t t e r ( s ) r e l e a s e d from th e a f f e r e n t s ( N i c o l l and A l g e r , 1980; W o j t o w i c z et_ a_l. , 1981; b u t see Newberry and N i c o l l , 1982 ). However, i t s h o u l d be n o t e d t h a t t h e l a t e HP c o u l d a p p e a r i n t h e a b s e n c e o f a p r e c e d i n g EPSP ( e . g . F i g . 3.3C) w h i c h i s t h e c a s e a l s o f o r t h e n o n - G A B A e r g i c l a t e HP i n t h e d e n t a t e g y r u s ( F i g . 1 o f Thalmann and A y a l a , 1 9 8 2 ) . T h a t t h e l a t e HP o c c u r r e d w i t h a s m a l l o r no p r e c e d i n g d e p o l a r i z a t i o n s u g g e s t s t h a t a c t i v a t i o n of e i t h e r a v o l t a g e - d e p e n d e n t 2 + Ca c o n d u c t a n c e o r an e l e c t r o g e n i c pump i n i t i a t e d by a s o d i um i n f l u x ( Ransom e_t a _ l . , 1975; P a d j e n a n d S m i t h , 1983) i s p r o b a b l y n o t i n v o l v e d . S e cond, the i n h i b i t i o n c o u l d a r i s e f r o m i n h i b i t o r y n e u r o t r a n s m i t t e r s r e l e a s e d f r o m e x t r i n s i c p athways c o - a c t i v a t e d w i t h th e m a j o r e x c i t a t o r y i n p u t s t o CA3. However, were t h i s t h e c a s e i t would be d i f f i c u l t t o r e c o n c i l e w i t h t h e d i f f e r e n t i a l i n h i b i t o r y a c t i o n s o f a n t i - and 7 4 o r t h o d r o m i c r e s p o n s e s e v o k e d by s t i m u l i a p p l i e d t o t h e f i m b r i a . T h i r d , t h e l a t e i n h i b i t i o n c o u l d r e f l e c t t h e a c t i v a t i o n o f n o n - G A B A e r g i c i n t e r n e u r o n s f o r w h i c h t h e r e i s a n a t o m i c a l e v i d e n c e ( e . g . Handelmann e_t aT. , 1 9 8 1 ; M o r r i s o n et_ a l . , 1 9 8 2 ) . F u r t h e r m o r e , t h e p o s s i b l e i n v o l v e m e n t o f t h e s e i n t e r n e u r o n s i n a f e e d f o r w a r d p a t h w a y i s s u g g e s t e d s i n c e t h e l a t e i n h i b i t i o n was e l i c i t e d i n t h e a b s e n c e o f a PS i n t h e MF^ r e s p o n s e and b e c a u s e AD s t i m u l a t i o n o n l y w e a k l y a c t i v a t e d t h i s i n h i b i t o r y e v e n t . 75 CHAPTER 4 THE EFFECTS OF ANTAGONISTS ON AMINO ACID- AND SCHAFFER COLLATERAL-COMMISSURAL MEDIATED EXCITATION OF C A l NEURONS I n t r o d u c t i o n T h e f i r s t i n d i c a t i o n o f a r o l e f o r a c i d i c a m i n o a c i d s i n t h e m o d u l a t i o n o f n e u r o n a l e x c i t a b i l i t y was p r o v i d e d by H a y a s h i (1954) who d e s c r i b e d t h e c o n v u l s a n t p r o p e r t y o f L - g l u t a m a t e i n t h e c e r e b r a l c o r t e x . An a c t i o n o f L - g l u t a m a t e and L - a s p a r t a t e t o e x c i t e s p i n a l n e u r o n s was c o n f i r m e d some y e a r s l a t e r i n t h e i o n t o p h o r e t i c s t u d i e s o f C u r t i s and h i s c o l l e a g u e s ( C u r t i s e t a _ l . , 1959; 1960) and t h e i n v e s t i g a t i o n s o f o t h e r s have shown t h a t v i r t u a l l y a l l t y p e s o f n e u r o n s a r e e x c i t e d by t h e s e compounds ( J o h n s o n , 1 9 7 2 ) . B e c a u s e n e u r o g l i a ( K r n j e v i c and S c h w a r t z , 1967) and n e r v e f i b r e s ( K r n j e v i c and P h i l l i s , 1963) a r e n o t d e p o l a r i z e d by L - g l u t a m a t e K r n j e v i c i n f e r r e d t h a t an i n t e r a c t i o n o f t h e a m i n o a c i d w i t h s y n a p t i c r e c e p t o r s was l i k e l y . T h e s e d a t a t o g e t h e r w i t h i n f o r m a t i o n r e g a r d i n g t h e i r d i s t r i b u t i o n s i n t h e c e n t r a l n e r v o u s s y s t e m (Graham et. a l . , 1967) a d v a n c e d t h e p o s s i b i l i t y o f a t r a n s -m i t t e r f u n c t i o n f o r L - g l u t a m a t e and L - a s p a r t a t e a l t h o u g h an o p i n i o n t o t h e c o n t r a r y was o r i g i n a l l y e x p r e s s e d by C u r t i s e_t_ a l . ( 1 9 6 0 ) . I n t h e t w o d e c a d e s w h i c h h a v e e l a p s e d s i n c e t h e s e 76 o r i g i n a l i n v e s t i g a t i o n s a t t e m p t s t o c o n f i r m t h e e x i s t e n c e o f a m i n o a c i d - m e d i a t e d s y n a p t i c e x c i t a t i o n h a v e i n v o l v e d t w o p r i n c i p l e t y p e s o f e x p e r i m e n t a l a p p r o a c h e s . One has e m p l o y e d n e u r o c h e m i c a l a n a l y s e s c o u p l e d w i t h t h e u s e o f s p e c i f i c n e u r o n a l l e s i o n s t o a s s a y p r e s y n a p t i c i n d i c e s s u c h as t i s s u e c o n c e n t r a t i o n s , u p t a k e and r e l e a s e o f a mino a c i d s . The s e c o n d a p p r o a c h has i n v o l v e d t h e a p p l i c a t i o n o f e l e c t r o p h y s i o l o g i c a l r e c o r d i n g t e c h n i q u e s t o d e t e r m i n e t h e p o s t s y n a p t i c c h a n g e s e l i c i t e d by e x o g e n o u s L - g l u t a m a t e o r i t s s t r u c t u r a l a n a l o g u e s v i s a v i s t h o s e c h a n g e s a c c o m p a n y i n g s y n a p t i c a c t i v a t i o n o f n e u r o n s . The f o l l o w i n g b r i e f r e v i e w o f t h e e v i d e n c e f r o m w h i c h a t r a n s m i t t e r r o l e f o r a c i d i c a m i n o a c i d s h a s b e e n a d d u c e d f o c u s e s on d a t a d e r i v e d f r o m t h e h i p p o c a m p u s . F o r a more g e n e r a l c o n s i d e r a t i o n o f a m i n o a c i d - m e d i a t e d s y n a p t i c e x c i t a t i o n t h e r e c e n t r e v i e w s o f W a t k i n s and E v a n s ( 1 9 8 1 ) , P u i l ( 1 9 8 1 ) , M cLennan (1981) and F a g g and F o s t e r ( 1 9 83) a r e recommended. L - g l u t a m a t e and L - a s p a r t a t e a s n e u r o t r a n s m i t t e r s :  N e u r o c h e m i c a l e v i d e n c e A l t h o u g h t h e r e i s no c l e a r d i f f e r e n c e i n t h e c o n c e n -t r a t i o n s o f L - g l u t a m a t e and L - a s p a r t a t e i n t h e d e n d r i t i c and s o m a t i c l a m i n a e o f C A l , CA3 and t h e d e n t a t e g y r u s ( N i t s c h and O k a d a , 1 9 7 9 ) a s i g n i f i c a n t d r o p i n t h e l e v e l s o f t h e s e compounds i s e v i d e n t f o l l o w i n g t h e d e s t r u c t i o n o f p r e s u m e d g l u t a m a t e r g i c o r a s p a r t a t e r g i c a f f e r e n t f i b r e s . F o r e x a m p l e , L - g l u t a m a t e b u t n o t L - a s p a r t a t e l e v e l s d e c l i n e d s i g n i f i c a n t l y f o l l o w i n g r e m o v a l o f t h e c o n t r a l a t e r a l h i p p o c a m p u s and t h e 77 c o n s e q u e n t d e g e n e r a t i o n o f c o m m i s s u r a l f i b r e s ( N i t s c h , 1981; see a l s o N a d l e r et_ a_l. , 1978); and t h e L - g l u t a m a t e c o n t e n t o f f a s c i a d e n t a t a s y n a p t o s o m e s was d e c r e a s e d a f t e r a l e s i o n o f t h e p e r f o r a n t p a t h i n p u t ( N a d l e r and S m i t h , 1981; b u t see a l s o D i L a u r o e t a_l. , 1 9 8 1 ) . The m a j o r d i s a d v a n t a g e o f s t u d i e s o f t i s s u e l e v e l s i s an i n a b i l i t y t o d i s t i n g u i s h between m e t a b o l i c and p r e s u m p t i v e n e u r o t r a n s m i t t e r p o o l s o f t h e s e amino a c i d s . I n t h e i r s t u d i e s , N a d l e r et. a l . (1978) d i s c o u n t e d any v a l u e o f t h e measurement o f h i p p o c a m p a l c o n c e n t r a t i o n s o f L - g l u t a m a t e o r L - a s p a r t a t e o r t h e i r s y n t h e t i c enzymes as r e l i a b l e m a r k e r s o f amino a c i d e r g i c 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 . I n a d d i t i o n , s e c o n d a r y e f f e c t s o f l e s i o n s s u c h as t h e a l t e r a t i o n o f m e t a b o l i c a c t i v i t y and t h e i n d u c t i o n o f g l i o s i s c o m p l i c a t e t h e i n t e r p r e t a t i o n o f d a t a i n s t u d i e s o f t h i s t y p e ( c f . W a t k i n s and E v a n s , 1981). One o f t h e o b j e c t i o n s e x p r e s s e d by C u r t i s e_t a l . (1960) f o r a n e u r o t r a n s m i t t e r r o l e of e x c i t a t o r y amino a c i d s was t h e s i m i l a r t i m e c o u r s e f o r r e c o v e r y f r o m t h e e f f e c t s o f t h e i o n t o p h o r e t i c a l l y a p p l i e d d e x t r o - and l e v o - i s o m e r s . However, i t i s now a p p a r e n t t h a t t h e t e r m i n a t i o n o f t h e a c t i o n s o f a m i n o " a c i d s , r a t h e r t h a n d e p e n d i n g on e n z y m a t i c c a t a b o l i s m , i s a t t r i b u t a b l e t o u p t a k e by p e r i s y n a p t i c c e l l s a n d / o r by d i f f u s i o n f r o m t h e s y n a p t i c c l e f t . A h i g h a f f i n i t y (K^ < 50 ^AM) u p t a k e may be i n v o l v e d w i t h t h e c o n c e n t r a t i o n s p r e s e n t d u r i n g n o r m a l s y n a p t i c e x c i t a t i o n , w h e r e a s a l o w a f f i n i t y s y s t e m a p p e a r s t o t e r m i n a t e t h e e f f e c t s o f h i g h e r l e v e l s o f a m i n o a c i d s s u c h a s o c c u r w i t h i o n t o p h o r e t i c a p p l i c a t i o n s 78 ( W a t k i n s et. aJL., 1977; Lodge et. a l . , 1 9 8 0 ) . T h e s e s o d i u m -d e p e n d e n t u p t a k e , p r o c e s s e s o c c u r p r i m a r i l y i n p r e s y n a p t i c t e r m i n a l s ( S t o r m - M a t h i s e n and I v e r s e n , 1979) and t o a s m a l l e r e x t e n t i n g l i a ( H e n n e_t aJL. , 1974 ) T e r m i n a t i o n s o f t h e e x c i t a t i o n s c a u s e d by compounds s u c h a s D - g l u t a m a t e and t h e g l u t a m a t e a n a l o g u e s k a i n a t e and N - m e t h y l a s p a r t a t e f o r w h i c h t h e r e i s no u p t a k e m e c h a n i s m i s due t o d i f f u s i o n away f r o m n e u r o n a l r e c e p t o r s . By e x a m i n i n g t h e c h a n g e s i n u p t a k e i n a t i s s u e f o l l o w i n g t h e s e l e c t i v e l e s i o n i n g o f an a f f e r e n t p a t h w a y , t h e g l u t a m a t e r g i c o r a s p a r t a t e r g i c n a t u r e o f t h o s e a f f e r e n t t e r m i n a l s may be d e t e r m i n e d . I t i s assumed i n t h e s e s t u d i e s t h a t a n y d e c l i n e o f u p t a k e i s a t t r i b u t a b l e t o t h e l o s s o f p r e s y n a p t i c b o u t o n s r a t h e r t h a n s e c o n d a r y c h a n g e s i n t h e p r o p e r t i e s o f g l i a o r p o s t s y n a p t i c n e u r o n s . S e l e c t i v e u p t a k e 3 o f [ H] l a b e l l e d L - g l u t a m a t e and L - a s p a r t a t e has been r e p o r t e d f o r t h e s y n a p t i c t e r m i n a l s o f t h e p e r f o r a n t p a t h i n p u t t o t h e d e n t a t e g y r u s , t h e mossy f i b r e p a t h w a y f r o m t h e d e n t a t e t o CA3 and t h e c o m m i s s u r a l and S c h a f f e r c o l l a t e r a l a f f e r e n t s t o C A l ( S t o r m - M a t h i s e n , 1977, 1981; S a n d o v a l et_ a l . , 1978; N a d l e r e t a l . , 1 9 7 8 ) . B i l a t e r a l s e c t i o n o f t h e f o r n i x , a m a j o r h i p p o c a m p a l e f f e r e n t p a t h w a y , a l s o p r o d u c e d a r e d u c t i o n o f u p t a k e i n t h e l a t e r a l s e p t u m and t h e m a m m i l l a r y b o d i e s ( S t o r m - M a t h i s e n and W o x e n - O p s a h l , 1978; W a l a a s and Fonnum, 1 9 8 0 ) . B e c a u s e D- and L - a s p a r t a t e and L - g l u t a m a t e a r e a c c u m u l a t e d by t h e same o r s i m i l a r u p t a k e p r o c e s s e s , i t i s n o t p o s s i b l e i n u p t a k e s t u d i e s t o d i s t i n g u i s h b e t w e e n g l u t a m a -79 t e r g i c o r a s p a r t a t e r g i c p a t h w a y s . A d d i t i o n a l s u p p o r t f o r a mino a c i d - m e d i a t e d s y n a p t i c e x c i t a t i o n i n t h e h i p p o c a m p u s has been t h e d e m o n s t r a t i o n o f 2+ Ca - d e p e n d e n t s t i m u l u s - e v o k e d r e l e a s e ( f o r r e v i e w s e e Cox and 2 + B r a d f o r d , 1 9 7 8 ) . A r e q u i r e m e n t f o r C a i n t h i s p r o c e s s d i s t i n g u i s h e s i t f r o m g l i a l r e l e a s e ( b u t see R o b e r t s , 1 9 7 4 ) . 2 + Ca - d e p e n d e n t r e l e a s e o f L - g l u t a m a t e h a s been d e m o n s t r a t e d i n s l i c e s o f d e n t a t e g y r u s f o l l o w i n g d e p o l a r i z a t i o n w i t h i n c r e a s e d t K + l 0 o r v e r a t r i d i n e ( N a d l e r et_ a _ l . , 1976) and by e l e c t r i c a l s t i m u l a t i o n ( W h i t e ejt a _ l . , 1977 ) . R e l e a s e was s i g n i f i c a n t l y d e c r e a s e d f o r s l i c e s i n w h i c h t h e p e r f o r a n t p a t h w a y h a d p r e v i o u s l y b e e n l e s i o n e d , s u g g e s t i n g t h a t t h e L - g l u t a m a t e o r i g i n a t e d m a i n l y f r o m p r e s y n a p t i c t e r m i n a l s . F o r t h e same r e a s o n t h e r e d u c t i o n o f K + - e v o k e d r e l e a s e o f e n d o g e n o u s L - g l u t a m a t e and L - a s p a r t a t e i n t h e i s o l a t e d C A l r e g i o n o f h i p p c a m p a l s l i c e s f r o m c o m m i s s u r o t o m i z e d r a t s was a t t r i b u t e d t o a l o s s o f a m i n o a c i d - u s i n g s y n a p t i c b o u t o n s ( N a d l e r e t a l . , 1976; 1 9 7 8 ) . R e l e a s e o f p r e - l o a d e d D - [ 3 H ] -a s p a r t a t e ( w h i c h i s a c c u m u l a t e d b u t n o t m e t a b o l i z e d ) by e l e c t r i c a l s t i m u l a t i o n o f S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l f i b r e s t o C A l i_n v i t r o h a s a l s o b e e n r e p o r t e d ( M a l t h e -S o r e n s s e n e_t a j _ . , 1979; S k r e d e and M a l t h e - S o r e n s s e n , 1 9 8 1 ) . A 2+ f a i l u r e t o d e m o n s t r a t e Ca - d e p e n d e n t r e l e a s e h a s made c o n t r o v e r s i a l t h e r o l e o f a mino a c i d - m e d i a t e d t r a n s m i s s i o n a t mossy f i b r e t e r m i n a l s ( N a d l e r et. a l . , 1978; b u t s e e u p t a k e s t u d i e s a b o v e . ) T o g e t h e r t h e s e b i o c h e m i c a l d a t a s u p p o r t a r o l e f o r 80 L - g l u t a m a t e i n p e r f o r a n t p a t h - m e d i a t e d t r a n s m i s s i o n i n t h e d e n t a t e g y r u s and L - g l u t a m a t e a n d / o r L - a s p a r t a t e i n t h e S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l i n p u t t o C A l . L - g l u t a m a t e and L - 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 :  E l e c t r o p h y s i o l o g i c a l e v i d e n c e The number o f n a t u r a l l y - o c c u r r i n g and s y n t h e t i c amino a c i d s p r o d u c i n g n e u r o n a l e x c i t a t i o n h a s grown c o n s i d e r a b l y o v e r t h e p a s t two d e c a d e s . Of p a r t i c u l a r i n t e r e s t h a v e been a g o n i s t s w i t h a g r e a t e r p o t e n c y t h a n L - g l u t a m a t e i t s e l f . T h e s e i n c l u d e : k a i n i c a c i d , an a n t h e l m i n t h i c i s o l a t e d f r o m t h e seaweed D i g e n e a s i m p l e x ; q u i s q u a l i c a c i d f r o m t h e s e e d s o f Q u i s q u a l i s i n d i c u s and N - m e t h y l - a s p a r t a t e (NMA), a s y n t h e t i c a m i n o a c i d . E a c h o f t h e s e a n a l o g u e s has a m o l e c u l a r s t r u c t u r e w i t h an a r r a n g e m e n t o f c a t i o n i c and a n i o n i c g r o u p s s i m i l a r t o t h a t o c c u r r i n g i n L - g l u t a m a t e a l t h o u g h t h e s e p a r a t i o n s b e t w e e n t h e s e c h a r g e d g r o u p s may d i f f e r . I n 1960 C u r t i s and W a t k i n s p r o p o s e d t h a t t h e a c i d i c a m i n o a c i d s p r o d u c e t h e i r a c t i o n s by a t h r e e - p o i n t a t t a c h m e n t o f t h e i r c h a r g e d g r o u p s t o a s i n g l e c l a s s o f p o s t s y n a p t i c r e c e p t o r . A l t h o u g h s t u d i e s o f s t r u c t u r e a c t i v i t y r e l a t i o n -s h i p s have s u p p o r t e d t h e c o n c e p t o f a t h r e e - p o i n t a t t a c h m e n t , t h e r e i s e v i d e n c e a v a i l a b l e f o r more t h a n one r e c e p t o r t y p e . T h i s was f i r s t s u g g e s t e d by McLennan ejt a l . ( 1 9 6 8 ) w i t h t h e o b s e r v a t i o n o f a d i f f e r e n t i a l s e n s i t i v i t y o f t h a l a m i c n e u r o n s t o L - g l u t a m a t e , NMA and D L - h o m o c y s t e i c a c i d and l a t e r s u b s t a n t i a t e d by t h e d i f f e r i n g r e s p o n s e p a t t e r n s o f s p i n a l i n t e r n e u r o n s and Renshaw c e l l s t o L - g l u t a m a t e and L - a s p a r t a t e 81 (Duggan, 1974) and k a i n a t e and NMA ( M c C u l l o c h , e t a l . , 1 9 7 4 ) . T h a t more t h a n one c o n d u c t a n c e mechanism may u n d e r l i e t h e d e p o l a r i z i n g a c t i o n s o f amino a c i d e x c i t a n t s a l s o i m p l i e s t h e e x i s t e n c e o f m u l t i p l e r e c e p t o r t y p e s . F o r e x a m p l e , membrane r e s i s t a n c e i s c o n s i s t e n t l y d e c r e a s e d by k a i n a t e and q u i s q u a l a t e w h e r e a s t h e c h a n g e s p r o d u c e d by NMA a r e d o s e d e p e n d e n t . Thus E n g b e r g e_t a l . (1979) r e p o r t e d an i n c r e a s e and d e c r e a s e o f membrane r e s i s t a n c e i n f e l i n e m o t o r n e u r o n s w i t h low and h i g h d o s e s r e s p e c t i v e l y o f NMA. T h i s same e f f e c t h a s now b e e n r e p o r t e d a s o c c u r r i n g i n c e l l s o f t h e c a t n e o c o r t e x ( F l a t m a n e_t a _ l . , 1983), c u l t u r e d s p i n a l c e l l s ( MacDonald and W o j t o w i c z , 1980) and h i p p o c a m p a l C A l p y r a m i d s ( H a b l i t z , 1982; D i n g l e d i n e , 1 9 8 3 a , b ) . R a t h e r t h a n r e d u c i n g a r e s t i n g o u t w a r d p o t a s s i u m c u r r e n t , NMA a p p e a r s t o p r o d u c e an i n c r e a s e o f membrane c o n d u c t a n c e by a c t i v a t i n g a t e t r o d o t o x i n -i n s e n s i t i v e v o l t a g e - d e p e n d e n t i n w a r d c u r r e n t c a r r i e d by + 2 + Na a n d / o r a v o l t a g e - d e p e n d e n t Ca c o n d u c t a n c e ( F l a t m a n e_t a 1., 1983 ; D i n g l e d i n e , 1 9 8 3 a , b ) . P a r a d o x i c a l l y t h i s was measured as an " i n c r e a s e o f membrane r e s i s t a n c e " w i t h c u r r e n t - c l a m p a n a l y s i s ( c f . D i n g l e d i n e , 1 9 8 3 b ) . B o t h L - a s p a r t a t e and L - g l u t a m a t e have s i m i l a r dose r e l a t e d e f f e c t s t o i n c r e a s e o r d e c r e a s e membrane r e s i s t a n c e (Lambert et. a l . , 1981; C o n s t a n t i e t a l . , 1980; MacDonald and W o j t o w i c z , 1982); a n d , a t l e a s t i n c u l t u r e d s p i n a l n e u r o n s t h e i n c r e a s e o f r e s i s t a n c e i s m e c h a n i s t i c a l l y s i m i l a r t o t h a t f o r NMA (MacDonald e t a_l. , 1 9 8 2 ) . D e c r e a s e s o f membrane r e s i s t a n c e h a v e b e e n a t t r i b u t e d t o a v o l t a g e - i n d e p e n d e n t i n c r e a s e o f 82 c o n d u c t a n c e s t o Na , K a n d Ca ( P u i l , 1 9 8 1 ) a n d i o n -d e p e n d e n t amino a c i d u p t a k e ( E n g b e r g e t a _ l . , 1979) e x c e p t i n t h e c a s e s o f NMA and k a i n a t e w h i c h a r e n o t a c t i v e l y t r a n s -p o r t e d . T h e r e f o r e , a l t h o u g h t h e a c i d i c a m i n o a c i d s h a v e a common a c t i o n t o d e p o l a r i z e n e u r o n s , t h i s e f f e c t i s n o t t i e d t o t h e same i o n i c c o n d u c t a n c e e v e n t s . T h i s i n d i c a t e s e i t h e r t h e p r e s e n c e o f s e v e r a l r e c e p t o r t y p e s l i n k e d t o d i f f e r e n t i o n o p h o r e s o r , a l t e r n a t i v e l y , t h e a c t i v a t i o n o f s e v e r a l c o n d u c t a n c e s by a s i n g l e r e c e p t o r . I t i s a l s o e v i d e n t t h a t a d i f f e r e n t i a l b l o c k a d e o f amino a c i d - m e d i a t e d e x c i t a t i o n may be due e i t h e r t o an i n h i b i t i o n o f a g o n i s t - r e c e p t o r i n t e r a c t i o n s a s i s u s u a l l y i n f e r r e d , o r t o a r e d u c t i o n o f r e c e p t o r - l i n k e d e v e n t s a t t h e i o n o p h o r e . An e x a m p l e o f t h e l a t t e r s i t u a t i o n 2 + i s t h e s e l e c t i v e b l o c k a d e by Mg ( A u l t e_t a _ l . , 1 9 8 0 ) o f N M A - i n d u c e d e x c i t a t i o n s ( D i n g l e d i n e , 1 9 8 3 b ) . A n o t h e r c r i t e r i o n f o r t h e i d e n t i f i c a t i o n o f n e u r o -t r a n s m i t t e r s i s t h a t o f " i d e n t i t y o f a c t i o n " w h i c h r e q u i r e s t h a t an e x o g e n o u s a g o n i s t e l i c i t t h e same p o s t s y n a p t i c e f f e c t s as a r e o b s e r v e d f o l l o w i n g t h e a c t i v a t i o n o f t h e p a t h w a y f o r w h i c h t h a t c o m p o u n d i s t h e p u t a t i v e t r a n s m i t t e r . I n t h i s c o n n e c t i o n , H a b l i t z a nd Langmoen (1983) have r e p o r t e d a s i m i l a r i t y i n t h e r e v e r s a l p o t e n t i a l f o r t h e L - g l u t a m a t e r e s p o n s e and t h e S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l e v o k e d EPSP i n C A l h i p p o c a m p a l c e l l s . E v en a s s u m i n g a c o m p l e t e f u l f i l l -ment o f t h e r i g o r o u s t h e o r e t i c a l and p r a c t i c a l p r e r e q u i s i t e s i n s t u d i e s o f t h i s k i n d ( f o r d i s c u s s i o n s e e Brown and 83 J o h n s t o n , 1983) t h e s e e x p e r i m e n t s c a n n o t c o n f i r m a r o l e f o r L - g l u t a m a t e as t h e endogenous t r a n s m i t t e r s i n c e , as f o r example w i t h g l y c i n e and GABA, s t r u c t u r a l l y d i s t i n c t compounds may a c t v i a d i f f e r e n t r e c e p t o r s t o a c t i v a t e t h e same u n d e r -l y i n g i o n i c c o n d u c t a n c e ( s ) . As i n t h e p r e s e n t s t u d y , a p h a r m a c o l o g i c a l a p p r o a c h t o t h e d e m o n s t r a t i o n o f t h e i d e n t i t y o f a c t i o n may a l s o be e m p l o y e d . T h i s r e q u i r e s f i r s t a c h a r a c t e r i z a t i o n o f a n t a g o n -i s t e f f e c t s on amino a c i d - i n d u c e d c e l l f i r i n g , and s u b s e q u e n t -l y a c o m p a r i s o n o f t h e a c t i o n s o f t h e s e a n t a g o n i s t s on s y n a p t i c e x c i t a t i o n so t h a t the n a t u r e o f t h e n e u r o t r a n s m i t t e r and t h e p o s t s y n a p t i c r e c e p t o r s may be d e d u c e d . P h a r m a c o l o g i c a l s t u d i e s i n t h e s p i n a l c o r d ( W a t k i n s , 1981a,b; McLennan, 1981) have d i s t i n g u i s h e d t h r e e t y p e s o f r e c e p t o r s m e d i a t i n g t h e r e s p o n s e s of e x c i t a t o r y amino a c i d s . The f i r s t t y p e i s i d e n t i f i e d by i t s most p o t e n t and s e l e c t i v e a g o n i s t , N - m e t h y 1 - D - a s p a r t a t e (NMDA) (se e A p p e n d i x I f o r c h e m i c a l s t r u c t u r e s ) and i s a n t a g o n i z e d by compounds w h i c h i n c l u d e t h e monoamino d i c a r b o x y l a t e s ( D - ( - ) - o ( - a m i n o s u b e r a t e and D - ( - ) - <?(, - a m i n o a d i p a t e (DAA) (McLennan and Lodge, 1979; D a v i e s and W a t k i n s , 1979); c e r t a i n 6 J - p h o s p h o n a t e compounds s u c h as ( + ) - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e (APV) ( D a v i e s , F r a n c i s e t a l . , 1981), t h e D-(-) i s o m e r o f w h i c h i s one o f t h e most p o t e n t NMA r e c e p t o r a n t a g o n i s t s known ( D a v i e s and W a t k i n s , 1982); t h e p a r t i a l NMA a g o n i s t ( + ) - c i s - 2 , 3 - p i p e r i d i n e d i c a r b o x y l a t e (PDA) ( D a v i e s , Evans e t a l . , 1981; McLennan and L i u , 1 9 8 2 ) ; a n d t h e d i p e p t i d e Y - D - g l u t a m y l g l y c i n e (DGG) 84 ( D a v i e s a n d W a t k i n s , 1 9 8 1 ) . The s e c o n d r e c e p t o r p r e f e r -e n t i a l l y a f f e c t e d by L - g l u t a m a t e i t s e l f and by q u i s q u a l i c a c i d and - a m i n o - 5 - m e t h y l - 3 - h y d r o x y - 4 - i s o x a z o l e - p r o p i o n i c a c i d (AMPA) (Honore e_t a l . , 1981), can be b l o c k e d by L - g l u t a m a t e d i e t h y l e s t e r ( G D E E ) . A t h i r d r e c e p t o r w i t h w h i c h k a i n a t e i n t e r a c t s i s u n i q u e i n r e q u i r i n g t h e p r e s e n c e o f an i s o p r o -p y l e n e s i d e c h a i n ( A p p e n d i x I) as w e l l as t h e u s u a l 3 c h a r g e d g r o u p s . K a i n a t e - i n d u c e d e x c i t a t i o n s a r e a n t a g o n i z e d by DGG ( D a v i e s and W a t k i n s , 1981) and PDA. T h a t L - g l u t a m a t e and L - a s p a r t a t e behave as mixed a g o n i s t s i s i m p l i e d by t h e i r a c t i o n s on membrane c o n d u c t a n c e s ( s e e above) and by t h e v a r y i n g d e g r e e s of b l o c k a d e o f t h e i r a c t i o n s by a n t a g o n i s t s . I n t h e s p i n a l c o r d , f o r e x a m p l e , L - a s p a r t a t e e x c i t a t i o n s a r e more s e n s i t i v e t o NMA b l o c k e r s s u c h a s APV ( W a t k i n s , 1981b) a n d DGG ( D a v i e s a n d W a t k i n s , 1 9 8 1 ) . C o n v e r s e l y , GDEE p r o d u c e s a g r e a t e r r e d u c t i o n o f L - g l u t a m a t e r e s p o n s e s (McLennan and Lodge, 1979). T h e r e f o r e , w h i l e d i f f e r e n t i a l a n t a g o n i s m o f e x c i t a t i o n i n d u c e d e i t h e r s y n a p t i c a l l y o r by a p p l i c a t i o n o f L - g l u t a m a t e o r L - a s p a r t a t e i s o f i n t e r e s t , more i n f o r m a t i v e i s a p r o f i l e o f t h e i n t e r -a c t i o n o f t h e a n t a g o n i s t s w i t h NMA, k a i n a t e and q u i s q u a l a t e r e c e p t o r s . T h i s p h a r m a c o l o g i c a l i n f o r m a t i o n may t h e n be u s e d t o d e t e r m i n e t h e c o n t r i b u t i o n o f e a c h t y p e 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 on s y n a p t i c r e s p o n s e s . D e s p i t e i t s p o t e n t i a l use i n t h e a n a l y s i s o f c o r t i c a l s y n a p t i c f u n c t i o n a n d d y s f u n c t i o n , t h e r e h a s b e e n l i t t l e i n v e s t i g a t i o n o f t h e p h a r m a c o l o g y and p h y s i o l o g y o f t h e 85 r e c e p t o r s m e d i a t i n g e x c i t a t o r y a m ino a c i d r e s p o n s e s i n t h e h i g h e r CNS. I n t h e h i p p o c a m p u s t h e compounds t h a t h a v e been r e p o r t e d t o a f f e c t t h e c o m m i s s u r a l o r S c h a f f e r c o l l a t e r a l p a t h w a y s s u c h a s GDEE, D L - 2 - a m i n o - 4 - p h o s p h o n o b u t y r a t e (APB) and l - h y d r o x y - 3 - a m i n o p y r o l i d - 2 - o n e (HA-966) ( S e g a l , 1976; 1978; W h i t e et. al. , 1979) a r e n o t s u f f i c i e n t l y s e l e c t i v e as a n t a g o n i s t s i n t h e h i g h d o s e s r e q u i r e d t o a c h i e v e e f f e c t s ( B a u d r y a n d L y n c h , 1 9 8 1 ; W a t k i n s a n d E v a n s , 1 9 8 1 ) . The p u r p o s e o f t h e p r e s e n t s t u d y was t o c h a r a c t e r i z e t h e f i r i n g o f C A l n e u r o n s by t h e a c i d i c amino a c i d s and t o d e t e r m i n e t h e e f f e c t s on t h e s e e x c i t a t i o n s o f t h e r e c e n t l y d e v e l o p e d r a n g e o f s u b s t a n c e s w h i c h h a v e been d e s c r i b e d i n t h e s p i n a l c o r d a s p o t e n t and s e l e c t i v e a n t a g o n i s t s . T h i s i n f o r m a t i o n was t h e n a p p l i e d t o t h e a n a l y s i s o f t h e r e c e p t o r t y p e ( s ) m e d i a t i n g t h e S c h a f f e r c o l l a t e r a l - c o m m i s s u r a l (SC) e v o k e d a c t i v a t i o n o f C A l c e l l s . T h e p h e n o m e n o n o f l o n g t e r m p o t e n t i a t i o n ( L T P ) i s e x h i b i t e d by t h e SC p r o j e c t i o n f o l l o w i n g b r i e f h i g h f r e q u e n c y s t i m u l a t i o n ( S c h w a r t z k r o i n and W e s t e r , 1975; A l g e r and T e y l e r , 1 9 7 6 ; D u n w i d d i e a n d L y n c h , 1 9 7 8 ) . A l t h o u g h i t h a s b e e n l o c a l i z e d t o t h e s y n a p s e i t i s n o t c l e a r w h e t h e r t h i s r e f l e c t s c h a n g e s o f p r e - a n d / o r p o s t s y n a p t i c f u n c t i o n . F u r t h e r m o r e , i t i s n o t u n d e r s t o o d what t r i g g e r s LTP a l t h o u g h t h e o b s e r v a t i o n t h a t i t c o u l d be p r e v e n t e d by D L - 2 - a m i n o - 4 - p h o s p h o n o b u t y r a t e ( D u n w i d d i e e_t a _ l . f 1978; Cotman e t a _ l . , 1981) an L - g l u t a m a t e a n t a g o n i s t a t t h e i n v e r t e b r a t e n e u r o m u s c u l a r j u n c t i o n ( C u l l - C a n d y et. a _ l . , 1976) r a i s e s t h e p o s s i b i l i t y t h a t LTP may 86 be p r o d u c e d by a p o s t s y n a p t i c a c t i o n o f an e x c i t a t o r y a m i n o a c i d . T h e r e f o r e some e x p e r i m e n t s a l s o a d d r e s s e d t h e a b i l i t y o f a n t a g o n i s t s t o a f f e c t LTP. M e t h o d s T e c h n i q u e s f o r t h e p r e p a r a t i o n and m a i n t e n a n c e o f t h e i n v i t r o h i p p o c a m p a l s l i c e have been d e s c r i b e d i n C h a p t e r 2. F o r e x t r a c e l l u l a r r e c o r d i n g o f t h e a c t i v i t y o f s i n g l e C A l c e l l s a 4 M N a C l - f i l l e d g l a s s m i c r o e l e c t r o d e was p l a c e d u n d e r v i s u a l c o n t r o l i n s t r a t u m p y r a m i d a l e (R-^, F i g . 2.3) and a d v a n c e d t o m a x i m i z e t h e a c t i o n p o t e n t i a l a m p l i t u d e o f a n e u r o n f i r i n g s p o n t a n e o u s l y o r i n r e s p o n s e t o t h e d e n d r i t i c i o n t o p h o r e s i s o f an e x c i t a t o r y a g o n i s t . A c t i o n p o t e n t i a l s , f i l t e r e d a t 0.1-3 kHz were g a t e d w i t h an a d j u s t a b l e DC l e v e l and i n t e g r a t e d t o o b t a i n a c h a r t r e c o r d o f f i r i n g f r e q u e n c y . A g o n i s t s and a n t a g o n i s t s were a p p l i e d i o n t o p h o r e t i c a l l y f r o m a 7 - b a r r e l e d m i c r o e l e c t r o d e o n t o t h e a p i c a l d e n d r i t e s i n t h e s t r a t u m r a d i a t u m , 100-200j* .m f r o m t h e c e l l body l a y e r ; b a c k i n g c u r r e n t s w e r e g e n e r a l l y + 2 0 - 3 0 n A f o r a l l c o m p o u n d s . The a c t i o n s o f a n t a g o n i s t s were e x a m i n e d i n t h e f o l l o w i n g manner: a t l e a s t 2 c o n t r o l c y c l e s c o m p r i s e d o f a u t o m a t i c a l l y t i m e d a p p l i c a t i o n s o f e x c i t a n t s were r e c o r d e d . S u b m a x i m a l b u t as n e a r l y as p o s s i b l e e q u a l f i r i n g r a t e s were o b t a i n e d by a d j u s t m e n t o f c u r r e n t s e j e c t i n g t h e e x c i t a n t s . T h e n , a s s e r i a l a p p l i c a t i o n o f e x c i t a n t s c o n t i n u e d , t h e a n t a g o n i s t was a p p l i e d t o r e d u c e , by 5 0 - 1 0 0 % , t h e e f f e c t s o f t h e m o s t s e n s i t i v e a g o n i s t . F i n a l l y , a r e c o v e r y r u n was p e r f o r m e d 87 ( e . g . F i g . 4 . 1 ) . The f o l l o w i n g s o l u t i o n s were u s e d : L - g l u t a m a t e (500 mM, pH 8 . 0 ) , L - a s p a r t a t e , (500 mM, pH 8 . 0 ) , k a i n a t e (5-10 mM, pH 8.0 i n 160 mM N a C l ) , q u i s q u a l a t e (5 mM, pH 8.0 i n 160 mM N a C l ) , N - m e t h y l - D L - a s p a r t a t e (NMA) (20-50 mM, pH 8.0 i n 160 mM N a C l ) , a c e t y l c h o l i n e c h l o r i d e (ACh) (500 mM, pH 4 . 0 ) , ( + ) - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e (APV) (20 mM, pH 8.0 i n 160 mM N a C l ) , D - ( - ) - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e (D-APV) ( [ < t f ] D i n 6 N H C 1 - 2 4 . 2 0 ) , L - ( + ) - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e (L-APV) (20 mM, pH 8 i n 160 mM N a C l , [ ] i n 6 N HC1 + 2 4 . 2 ° ) / X-D-glut-a m y l g l y c i n e (DGG) ( 50-100 mM, pH 7.0 i n 100 mM N a C l , [<*] Q i n 6 N HC1 - 2 5 . 2 ° ) , ( + ) - c i s - 2 , 3 - p i p e r i d i n e d i c a r b o x y l a t e (PDA) (100 mM, pH 8.0 i n 100 mM N a C l ) , D - ( - ) - 2 a m i n o - 4 - p h o s p h o n o b u t y r a t e (D-APB) (20 mM, pH 7.5-8.0 i n 160 mM N a C l , i n 6 N HC1 - 2 3 . 5 ° ) , L - ( + ) - 2 - a m i n o - 4 - p h o s p h o n o b u t y r a t e (L-APB) (20 mM, pH 7.5-8.0 i n 160 mM N a C l , [ o C ] D i n 6 N HC1 +20.8°), L - g l u t a m a t e d i e t h y l e s t e r (GDEE) (500 mM, pH 3 . 5 - 4 . 0 ) , D - ( - ) - < - a m i n o a d i p a t e (DAA) (200 mM, pH 8 . 0 ) . The t e c h n i q u e s f o r e v o k i n g , r e c o r d i n g and m e a s u r i n g C A l f i e l d r e s p o n s e s have been d e s c r i b e d i n C h a p t e r 2. B r i e f l y , i n most o f t h e e x p e r i m e n t s a n a l y z i n g t h e 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 on s y n a p t i c e x c i t a t i o n t h e c e n t r a l b a r r e l o f an i o n t o p h o r e t i c e l e c t r o d e p o s i t i o n e d i n s t r a t u m r a d i a t u m 100-300 j>\m f r o m t h e c e l l b o d y l a y e r , (1*2' F ^ 9 * 2.3) r e c o r d e d t h e S C - e v o k e d s u b m a x i m a l EPSP. U s u a l l y t h i s p o t e n t i a l was n o t c o n t a m i n a t e d by a r e v e r s e d PS. S o l u t i o n s f i l l i n g t h e o u t e r b a r r e l s were t h e same as t h o s e u s e d i n s i n g l e u n i t e x p e r i m e n t s 88 w i t h t h e f o l l o w i n g c h a n g e s : q u i s q u a l a t e (50 mM i n 100 mM N a C l , pH 8 ) , k a i n a t e (up t o 50 mM i n 100 mM N a C l , pH 8) and NMA (100 mM i n 100 mM N a C l , pH 8 ) . E x c i t a n t s were a p p l i e d f o r p e r i o d s o f 10-120 s e c w h e r e a s a n t a g o n i s t s were u s u a l l y a d m i n i s t e r e d u n t i l a c o n s t a n t e f f e c t was a c h i e v e d . The a c t i o n o f a n a n t a g o n i s t was c o n s i d e r e d r e a l o n l y i f t h e s i z e o f t h e EPSP m e a s u r e d ( s e e C h a p t e r 2 f o r d e t a i l s ) o v e r a p e r i o d o f 1 m i n u t e (6 c o n s e c u t i v e m e a s u r e m e n t s ) d u r i n g t h e a d m i n i s t r a t i o n o f t h e a n t a g o n i s t was s i g n i f i c a n t l y d i f f e r e n t (p < 0.05) f r o m t h e a m p l i t u d e b e f o r e and a f t e r r e c o v e r y f r o m t h e d r u g a p p l i c a t i o n ( 2 - t a i l e d u n p a i r e d t - t e s t s ) . E f f e c t s w e re h i g h l y r e p r o d u c i b l e when t e s t s were r e p e a t e d u s i n g t h e same s l i c e a n d were n e v e r m i m i c k e d by a p p r o p r i a t e c u r r e n t c o n t r o l s . R e s u l t s p r e s e n t e d a r e means +S.E.M. I n s t u d i e s o f t h e i r e f f e c t s on LTP o f PSs o r EPSPs by h i g h f r e q u e n c y s t i m u l a t i o n (100 Hz f o r 1 s e c ) a n t a g o n i s t s w e re a p p l i e d i n most c a s e s by i o n t o p h o r e s i s t o t h e d e n d r i t e s , b u t i n a f e w e x p e r i m e n t s t h e D - ( - ) - i s o m e r o f APV was p e r f u s e d -5 (10-20 m l o f 5x10 M) . The i n t e n s i t y o f SC a c t i v a t i o n was a d j u s t e d t o p r o d u c e a s u b m a x i m a l PS o f 0.5-4.0 mV and t h e peak n e g a t i v e d e f l e c t i o n f r o m t h e b a s e l i n e o r p r e c e d i n g peak p o s i t i v i t y was m e a s u r e d . R e s u l t s E f f e c t s o f e x c i t a n t s on s i n g l e u n i t a c t i v i t y The u n i d e n t i f i e d C A l n e u r o n s e x a m i n e d by e x t r a c e l l u l a r r e c o r d i n g i n t h i s s t u d y were s e n s i t i v e t o d e n d r i t i c a p p l i c a -89 t i o n o f t h e e x c i t a t o r y a m ino a c i d s NMA, k a i n a t e , q u i s q u a l a t e , L - a s p a r t a t e and L - g l u t a m a t e . E s p e c i a l l y w i t h t h e l a t t e r t h r e e a g o n i s t s i t was p o s s i b l e t o l o c a t e a d e n d r i t i c s i t e w h e r e i o n t o p h o r e t i c e j e c t i o n c u r r e n t s o f 0-50 nA e l i c i t e d t h e f i r i n g o f a c t i o n p o t e n t i a l s , a n d w h e r e v e r t i c a l movement o f t h e e l e c t r o d e t i p 25-50 j^m away f r o m t h e d e n d r i t e a b o l i s h e d o r d r a s t i c a l l y r e d u c e d t h e n e u r o n a l r e s p o n s e . T h i s i m p l i e d t h a t t h e e f f e c t s o b t a i n e d w e r e l o c a l and n o t due t o d i f f u s i o n o f t h e e j e c t e d compounds t o t h e soma 100-200y*.m away. A l t h o u g h a l a r g e number o f c e l l s were e x a m i n e d , o n l y 129 f u l f i l l e d t h e c r i t e r i a f o r s e l e c t i o n w h i c h w e r e : 1) t h a t a c t i o n p o t e n t i a l s c o u l d be s u i t a b l y i s o l a t e d by a d j u s t m e n t o f t h e DC g a t e ; 2) t h a t t h e s p i k e h e i g h t d i d n o t c h a n g e a p p r e c -i a b l y ; a n d 3) t h a t i n p h a r m a c o l o g i c a l t e s t s r e c o v e r i e s t o 80-110% o f c o n t r o l f i r i n g r a t e s were a t t a i n e d . T h e s e c r i t e r i a w e r e e a s i l y a c h i e v e d f o r e x c i t a t i o n e l i c i t e d by L - a s p a r t a t e , L - g l u t a m a t e o r q u i s q u a l a t e , a l l o f w h i c h showed a s h o r t (< 2 s e c ) l a t e n c y t o o n s e t o f f i r i n g and where t h e r a t e r a p i d l y a t t a i n e d a s t a b l e l e v e l w h i c h d e c l i n e d e q u a l l y r a p i d l y w i t h t h e t e r m i n a t i o n o f t h e e j e c t i n g c u r r e n t . On a v e r a g e t h e r e was no o b v i o u s d i f f e r e n c e i n t h e a p p a r e n t p o t e n c y o f L - g l u t a m a t e o r L - a s p a r t a t e as e x c i t a n t s when a p p l i e d t o t h e a p i c a l d e n d r i t e s i n t h e s t r a t u m r a d i a t u m . W i t h few e x c e p t i o n s e q u a l c u r r e n t s e j e c t i n g t h e two a m i n o a c i d s p r o d u c e d n e a r l y e q u i v a l e n t r e s p o n s e s ( s e e a l s o S e g a l , 1 9 8 1 ) . The a c t i o n o f k a i n a t e on t h e o t h e r hand h a d a l o n g e r l a t e n c y o f o n s e t (2-10 s e c ) . O n l y o c c a s i o n a l l y c o u l d a s u b m a x i m a l 90 p l a t e a u o f f i r i n g be a t t a i n e d , a n d more o f t e n t h e r e was a c o n t i n u o u s s l o w r i s e i n t h e n e u r o n a l f i r i n g r a t e d u r i n g k a i n a t e i o n t o p h o r e s i s . The s i t u a t i o n was e v e n more d i f f i c u l t i n t h e c a s e o f NMA. The u s u a l r e s p o n s e t o t h i s s u b s t a n c e , w h i c h a l s o c h a r a c t e r i s t i c a l l y h a d a l o n g l a t e n c y o f o n s e t (2-10 s e c ) ( c f . Yamamoto and Sawada, 1982) was i n i t i a l l y t o c a u s e t h e c e l l t o f i r e i n b u r s t s w h i c h were s u c c e e d e d by c o n t i n u o u s f i r i n g a t a h i g h f r e q u e n c y (>35 H z ) d u r i n g w h i c h t h e a m p l i t u d e o f t h e s p i k e s r a p i d l y d e c l i n e d i n h e i g h t . The t e n d e n c y o f NMA t o r e c r u i t a d j a c e n t n e u r o n s r e q u i r e d t a r g e t c e l l s t o be w e l l -i s o l a t e d a n d c a r e f u l l y g a t e d . I n a d d i t i o n , p a r t i c u l a r l y b e c a u s e o f t h e r a p i d s p i k e i n a c t i v a t i o n , i t was o f t e n d i f f i -c u l t t o o b t a i n a n e q u i l i b r i u m r e s p o n s e w i t h NMA. I n m o s t i n s t a n c e s t h e NMA e j e c t i n g c u r r e n t was s t o p p e d as t h e r e s p o n s e p a t t e r n s h i f t e d f r o m b u r s t s t o t h e r a p i d l y i n a c t i v a t i n g h i g h f r e q u e n c y s p i k e t r a i n . T he s p e c i f i c a g o n i s t s NMA, k a i n a t e and q u i s q u a l a t e a l l w ere c l e a r l y more p o t e n t t h a n e i t h e r L - g l u t a m a t e o r L-a s p a r t a t e , h o w e v e r , s i n c e t r a n s p o r t numbers were n o t d e t e r -m i n e d and w i t h t h e p r o b l e m o f a c h i e v i n g s t a b l e f i r i n g r a t e s a p r e c i s e r a n k o r d e r c o u l d n o t be a s s i g n e d . On t h e b a s i s o f t h e e j e c t i n g c u r r e n t s u s e d and t h e c o n c e n t r a t i o n s i n t h e e l e c t r o d e b a r r e l s t h e s e t h r e e a g o n i s t s a p p e a r e d t o be 20-100 t i m e s more a c t i v e t h a n t h e n a t u r a l l y - o c c u r r i n g compounds. C e l l s c o u l d a l s o be e x c i t e d w i t h ACh. The r e s p o n s e t o ACh a p p l i e d w i t h h i g h c u r r e n t s ( 8 0-100 nA) had a l a t e n c y 5-10 91 t i m e s l o n g e r t h a n t h a t o b s e r v e d w i t h d o s e s o f L - a s p a r t a t e a c h i e v i n g t h e same p l a t e a u f i r i n g r a t e . I n a d d i t i o n , c e l l f i r i n g p e r s i s t e d f o r 2 0 - 8 0 s e c o n d s a f t e r t h e ACh e j e c t i n g c u r r e n t was t e r m i n a t e d ( B i s c o e and S t r a u g h a n , 1966; Dodd et. a l . , 1 9 8 1 ) . A n t a g o n i s m o f a m i n o a c i d - i n d u c e d e x c i t a t i o n s T h e e f f e c t s o f PDA, APV, DAA, DGG, D-APB, L-APB and GDEE on t h e e x c i t a t i o n o f h i p p o c a m p a l C A l n e u r o n s i n d u c e d by NMA, k a i n a t e , q u i s q u a l a t e , L - a s p a r t a t e , L - g l u t a m a t e and ACh a r e s u m m a r i z e d i n T a b l e I . When a p p l i e d w i t h l o w e j e c t i n g c u r r e n t s ( u p t o 20 nA) f o r s h o r t p e r i o d s o f t i m e , PDA e x h i b i t e d p r e f e r e n t i a l a n t a g o n i s m o f t h e NMA r e s p o n s e ( F i g . 4.1A). H i g h e r d o s e s a n d / o r l o n g e r p e r i o d s o f a d m i n i s t r a t i o n o f PDA e l i c i t e d p o t e n t d e p r e s s i o n o f r e s p o n s e s i n d u c e d by a l l t h r e e a g o n i s t s ( D a v i e s , E v a n s e t a l . , 1 9 8 1 ) . T h i s a n t a g o n i s m was, u n f o r t u n a t e l y , c o m p l i c a t e d by an a c c o m p a n y i n g e x c i t a t o r y e f f e c t ( F i g . 4.IB) w h i c h e v e n a t l o w e r d o s e s was m a n i f e s t e d by an e n h a n c e d r e s p o n s e t o q u i s q u a l a t e ( F i g . 4.1A). T h a t t h e e x c i t a t o r y e f f e c t o f PDA c o u l d be b l o c k e d t o a l a r g e e x t e n t w i t h APV ( d a t a n o t shown) i s c o n s i s t e n t w i t h i t s a c t i v i t y as a p a r t i a l NMA a g o n i s t ( D a v i e s , E v a n s et_ a _ l . , 1 9 8 1 ) . The e f f e c t s o f PDA o r o t h e r a n t a g o n i s t s c o u l d n o t be m i m i c k e d by a 100 nA c u r r e n t c a r r i e d by C I ( F i g . 4.1A). Of t h e o t h e r a m i n o a c i d a n t a g o n i s t s t e s t e d , o n l y DAA and APV s e l e c t i v e l y a n t a g o n i z e d N M A - i n d u c e d e x c i t a t i o n s ; and o f t h e two APV was t h e more p o t e n t as has b een r e p o r t e d by 92 A n t a g o n i s t A g o n i s t (mean 1 S . E . o f % change f i r i n g r a t e ) NMA K a i n a t e Q u i s q u a l a t e APV - 9 3 . 512 .2 (n-13 ) + 3 8.318 . 8 (n -10 ) + 2 8 . 0 1 9 . 0 ( n - 1 1 ) DGG - 8 9 . 5 1 2 . 6 ( n - 2 3 ) - 4 9 . 5 1 5 . 5 (n-=28) - - 6 . 2 1 4 . 2 ( n = 3 0 ) * D-APB - 6 8 . 0 1 3 . 6 (n -20 ) - 4 3 . 0 1 8 . 0 (n«r5) - 7 . 5 1 5 . 5 ( n - 1 9 ) * DAA - 5 8 . 9 1 7 . 2 (n=16) + 3 8 . 5 H 7 . 6 (n = 7 ) * + 5 9.418.7 (n=16) PDA* * - 5 2 . 7 1 8 . 1 (n=7) - 9 . 9 H 1 . 7 (n=7) * + 1 4 . 7 1 1 4 . 4 ( n = 7 ) » GDEE - 1 1 . 3 U 4 . 7 ( n = 8 ) * + 9 . 5 1 2 . 9 (n=9) . + 1 4.015.3 ( n - 1 5 ) L - A P B + 5 3 . 9 H 5 . 3 (n-=9) + 1 4.319 . 5 ( n - 4 ) * + 6 6 . 9 * 3 1 . 8 ( n - 9 ) L - • A s p a r t a t e L - G l u t a m a t e ACh APV - 5 3 . 3 i 5 . 3 (n -10 ) - 1 2 . 5 1 2 . 6 (n -10 ) + 1 . 4 H . 8 ( n = 5 ) * DGG - 6 1 . 8 1 1 1 . 2 (n=7) - 1 4 . 2 H 0 . 7 ( n - 9 ) * + 2 . 9 1 4 . 0 ( n - 3 ) * D-APB - 2 9 . 8 1 7 . 0 Kn=8) + 3 . H 7 . 3 (n=8) * + 2 . 8 1 3 . 7 ( n = 4 ) » L - A P B + 1 7 . 4 H 3 . 4 (n=5) * + 3 0 . 4 1 1 0 . 4 ( n = 5 ) * NT • n o t s i g n i f i c a n t l y d i f f e r e n t (p <0.05) f rom z e r o , • • a p p l i e d w i t h i o n t o p h o r e t i c c u r r e n t s < 20 nA ( s e e t e x t ) . NT - n o t t e s t e d . T a b l e I . A c t i o n s o f a m i n o a c i d a n t a g o n i s t s on t h e e x c i t a t o r y r e s p o n s e s t o NMA, k a i n a t e , q u i s q u a l a t e , L - g l u t a -mate, L - a s p a r t a t e and ACh. T h e p e r c e n t (mean +S.E.M.) d e p r e s s i o n (-) o r p o t e n -t i a t i o n (+) c a u s e d by some t e s t d r u g s i n t h e f r e q u e n c y o f s p i k e d i s c h a r g e s o f u n i d e n t i f i e d C A l n e u r o n s e v o k e d by i o n o p h o r e t i c a p p l i c a t i o n s o f e x c i t a n t s . The number o f c e l l s t e s t e d i s i n d i c a t e d i n p a r e n t h e s e s . The a v e r a g e f i r i n g r a t e a t t h e end of a 2 - 4 min a p p l i c a t i o n o f t h e a n t a g o n i s t b e i n g t e s t e d was c o m p a r e d t o t h a t o c c u r r i n g d u r i n g t h e c o n t r o l p e r i o d . A o n e - t a i l e d p a i r e d t - t e s t was use d t o d e t e r m i n e i f ch a n g e s i n f i r i n g r a t e s were s i g n i f i c a n t . 9 3 A. 40-0 J CI JOOnA, 2mm PDA. 2 0 n A , 2 m i n A I A i A 11 f1 1 1 \ 1 SJ S m 52 m m S3 Kam.35nA C M NMA. 12 nA S3 Qu i s . l Z r . A 2 0 sec PDA. 100nA . 2m in 40n 0 J KSJ NMA,15 nA ^ K a i n . 3 0 n A | KSJ I Ouis. l2.5nA 20~sec F i g . 4.1 R a t e m e t e r r e c o r d s showing t h e e f f e c t s o f PDA on h i p p o c a m p a l C A l n e u r o n a l r e s p o n s e s t o NMA, k a i n a t e a n d q u i s q u a l a t e . In (A) r e s p o n s e s t o NMA were r e d u c e d f o l l o w i n g a two m i n u t e a p p l i c a t i o n o f PDA (20 n A ) . In r e c o r d B, o b t a i n e d f r o m a d i f f e r e n t C A l n e u r o n , PDA (100 nA) a n t a g o n i z e d r e s p o n s e s t o e a c h o f t h e t h r e e a g o n i s t s and was i t s e l f e x c i t a t o r y . E x c i t a t i o n s were n o t a f f e c t e d by a 100 nA c u r r e n t c a r r i e d by CI . In t h i s and the f o l l o w i n g r e c o r d s the u p p e r s o l i d b a r i d e n t i f i e s t h e a n t a g o n i s t , i t s e j e c t i n g c u r r e n t and the t i m e e l a p s e d s i n c e t h e s t a r t of a n t a g o n i s t e j e c t i o n . The l o w e r h o r i z o n t a l b a r s i n d i c a t e t h e d u r a t i o n o f a g o n i s t a d m i n i s t r a t i o n and the l e g e n d below i d e n t i f i e s the a g o n i s t s and t h e i r e j e c t i o n c u r r e n t s . 94 o t h e r s ( D a v i e s , F r a n c i s e t aJL. , 1981). The p r e f e r e n t i a l e f f e c t of APV on the NMA response i s i l l u s t r a t e d i n Figure 4.2. P o t e n t i a t i o n of the k a i n a t e - and q u i s q u a l a t e - i n d u c e d responses was an e f f e c t noted with both APV ( F i g . 4.2) and DAA (Table I ) . Experiments i n which the e f f e c t s of the DL mixture and the r e s o l v e d (98%) L-(+)-isomer of APV were compared on the same c e l l s showed t h a t o n l y the DL m i x t u r e was e f f e c t i v e , implying that both the a n t a g o n i s t p r o p e r t i e s of APV and i t s p o t e n t i a t i n g e f f e c t upon q u i s q u a l a t e r e s i d e with the D-(-)-isomer ( c f . Davies, F r a n c i s e_t a_l., 1981; McLennan, 1982). Thus i o n t o p h o r e t i c e j e c t i o n of DL-APV decreased the NMA response by 94.3+3.9% and i n c r e a s e d the q u i s q u a l a t e response by 21.8+4.9% (mean + S.E.M. , n = 4) w h i l e L-APV e j e c t e d w i t h c u r r e n t i n t e n s i t i e s e q u a l to h a l f of those used t o a p p l y DL-APV produced no s i g n i f i c a n t e f f e c t on NMA- or q u i s q u a l a t e -induced responses ( F i g . 4.3A). That antagonism of the NMA r e s p o n s e c o u l d be o b s e r v e d w i t h h i g h (40-100 nA) e j e c t i o n c u r r e n t s of L-APV ( F i g . 4.3A) may be a t t r i b u t e d to the s m a l l contamination of the L-APV sample by the a c t i v e isomer. The d i p e p t i d e DGG produced a marked depression of the NMA-induced response and a l s o antagonized kainate responses, a l b e i t to a l e s s e r extent. Of the three s p e c i f i c a g o n i s t s examined, the response of hippocampal CAl neurons to q u i s -q u alate was l e a s t a f f e c t e d by DGG although a high dose d i d have some a c t i o n ( F i g . 4.2). When a p p l i e d with l a r g e e j e c t i n g c u r r e n t s (75-100 nA) r a t h e r than the 15-50 nA used to o b t a i n 9 5 S D G G . 2 0 n A . 2 m m 2 - A P V . 2 0 n A . 2 m i n ssso N M A 8 5 n A vzzn Komate 3 0 nA Quisqualate !2 5 n A F i g . 4.2 B l o c k a d e o f amino a c i d - i n d u c e d e x c i t a t i o n s by APV and DGG. APV r e d u c e d r e s p o n s e s t o NMA and p o t e n t i a t e d q u i s q u a l a t e - and k a i n a t e - i n d u c e d e x c i t a t i o n s w h i l e DGG a n t a g o n i z e d NMA, k a i n a t e and q u i s q u a l a t e r e s p o n s e s i n t h a t o r d e r o f s e n s i t i v i t y . 96 A 2 0 -B 40 - , m a D.L-APV, 2.5nA.lmin f i a I Ouis. 7.5 nA L-APV. 1.3 nA. 2 mm, L-APV. 50nA,2mm a m a E3 NMA .3nA D.L-APV.I5nA. 2 mm. a m a 20 sec n 0 f t p ,ja 'a I.L-glutamate. 20nA • L-asparitate.-22.5nA a to S3 NMA. l5r,A 20 sec F i g . 4.3 E f f e c t s o f DL-APV and L-APV on r e s p o n s e s o f h i p p o c a m p a l C A l n e u r o n s t o e x c i t a t o r y amino a c i d s . In (A) t h e r a c e m i c m i x t u r e o f APV d e c r e a s e d and i n c r e a s e d the r e s p o n s e s t o NMA a n d q u i s q u a l a t e , r e s p e c t i v e l y . T h e s e a c t i o n s w e r e r e p r o d u c e d o n l y w i t h much h i g h e r d o s e s o f L-APV. In ( B ) DL-APV s e l e c t i v e l y b l o c k e d e x c i t a t i o n s i n d u c e d by NMA and L - a s p a r t a t e . 9 7 t h e d a t a o f T a b l e I , DGG d e c r e a s e d t h e k a i n a t e and q u i s q u a l a t e r e s p o n s e s by 8 0 . 7 + 4 . 9 % ( n= 6 ) and 2 8 . 3 + 8 . 6 % ( n= 6 ) r e s p e c t i v e l y . T h e two i s o m e r s o f APB w h i c h were r e s o l v e d t o a p p r o x -i m a t e l y 9 0 % p u r i t y h a d s t r i k i n g l y d i f f e r e n t a c t i o n s ( F i g . 4 . 4 A ) . D-APB a n t a g o n i z e d t h e r e s p o n s e t o NMA a n d k a i n a t e w h i l e t h e q u i s q u a l a t e r e s p o n s e r e m a i n e d r e l a t i v e l y u n a f f e c t e d . I n c o n t r a s t , t h e L - ( + ) - i s o m e r o f APB p o t e n t i a t e d t h e a c t i o n o f e a c h o f t h e t h r e e a g o n i s t s ( F i g . 4 . 4 A ) and on o c c a s i o n was i t s e l f e x c i t a t o r y . T h i s e x c i t a t o r y e f f e c t o f L-APB was s e n s i t i v e t o b l o c k a d e by D-APB ( d a t a n o t s h o w n ) , a s was r e p o r t e d i n t h e s p i n a l c o r d ( E v a n s , F r a n c i s et. a _ l . , 1 9 8 2 ) . C u r r e n t s up t o 1 0 0 nA e j e c t i n g GDEE had i n c o n s i s t e n t a c t i o n s on e x c i t a t i o n s i n d u c e d by t h e s p e c i f i c a g o n i s t s . GDEE d i d n o t s i g n i f i c a n t l y a n t a g o n i z e any o f t h e e x c i t a t i o n s o f t h e c e l l s r e p o r t e d i n T a b l e I . However, w i t h 7 o t h e r c e l l s w h i c h w e r e e x c l u d e d f r o m T a b l e I b e c a u s e o f i n c o m p l e t e r e c o v e r y ( < 8 0 % o f p r e - t r e a t m e n t f i r i n g r a t e ) , GDEE t e n d e d t o b l o c k r e s p o n s e s t o a l l 3 a g o n i s t s , an e f f e c t w h i c h was sometimes p a r a l l e l e d by a d e c r e a s e i n s p i k e h e i g h t . I n t h e s p i n a l c o r d and h i g h e r c e n t r e s compounds w h i c h a n t a g o n i z e t h e NMA r e c e p t o r t e n d a l s o t o b l o c k L - a s p a r t a t e -i n d u c e d e x c i t a t i o n s , l e a v i n g L - g l u t a m a t e r e s p o n s e s r e l a t i v e l y u n a f f e c t e d ( H i c k s et_ a_l. , 1 9 7 8 ; Lodge e t a l . , 1 9 7 8 ; C o l l i n g -r i d g e and D a v i e s , 1 9 7 9 ; McLennan and Lodge, 1 9 7 9 ) . T a b l e I i n d i c a t e s t h a t t h e same t r e n d o c c u r s a l s o i n the h i p p o c a m p u s . A dose o f e i t h e r APV ( F i g . 4 . 3 B ) o r DGG w h i c h p r o d u c e d a l m o s t c o m p l e t e i n h i b i t i o n o f t h e NMA r e s p o n s e a l s o m a r k e d l y 98 40- , 40 - i 0 J 20 sec 0u i s ' 6nA /M D-APB. 50nA. 2 mm I S O L - A P B . 50nA. 2 mm 0 0 E3 Kainate O nA 40-| 40- , 0 J • a D-APB. 5 0 n A . 2 mm • a .a L - A P B . 5 0 n A . 2 mm. o o • a a 20sec • L-glutamate 2 0 n A • L-osportate 20nA • a S3 NMA 75nA F i g . 4.4 E f f e c t s o f t h e o p t i c a l i s o m e r s o f APB on r e s p o n s e s o f two C A l n e u r o n s t o i o n t o p h o r e s i s o f e x c i t a t o r y a m i n o a c i d s . NMA, L - a s p a r t a t e a n d k a i n a t e r e s p o n s e s w e r e a n t a g o n i z e d by D-APB. W i t h b o t h c e l l s an i n c r e a s e from t h e c o n t r o l f i r i n g r a t e was o b s e r v e d i n t h e p r e s e n c e o f L-APB. 9 9 A DGG. 3nA. 2 min 4 0 i L L L • a a a S 3 N M A - 2nA B a • a a a n • a L-aspartate. 3nA 20 sec DGG. 15 nA DGG. 5 0 nA L-aspartate. 3 nA F i g . 4.5 The d i f f e r e n t i a l a n t a g o n i s m by DGG o f t h e r e s p o n s e s o f C A l n e u r o n s t o NMA, L - a s p a r t a t e and L - g l u t a m a t e . R e c o r d A i l l u s t r a t e s b l o c k a d e o f t h e NMA b u t n o t t h e L -a s p a r t a t e r e s p o n s e by DGG. In (B) h i g h e r d o s e s o f DGG w h i c h r e d u c e d o r a b o l i s h e d t h e L - a s p a r t a t e r e s p o n s e were l e s s e f f e c t i v e a g a i n s t L - g l u t a m a t e - i n d u c e d e x c i t a t i o n s . 100 d e p r e s s e d t h e L - a s p a r t a t e r e s p o n s e b u t c a u s e d l i t t l e o r no chan g e o f t h e L - g l u t a m a t e r e a c t i o n . I n t e r e s t i n g l y , w i t h l o w e r d o s e s o f APV o r DGG ( F i g . 4.5A) i t was o f t e n p o s s i b l e e f f e c t i v e l y t o a n t a g o n i z e t h e N M A - i n d u c e d r e s p o n s e w i t h o u t a p p r e c i a b l y c h a n g i n g t h e L - a s p a r t a t e r e a c t i o n (McLennan and L i u , 1 9 8 2 ) . When DGG was a p p l i e d a t a d o s e w h i c h r e d u c e d b o t h L - g l u t a m a t e - and L - a s p a r t a t e - i n d u c e d e x c i t a t i o n s , t h e l a t t e r w ere more s e n s i t i v e ( F i g . 4 . 5 B ) . T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e s e n s i t i v i t i e s o f NMA a n d q u i s q u a l a t e r e c e p t o r s t o a n t a g o n i s m by DGG. An e f f e c t q u a l i t a t i v e l y s i m i l a r t o t h a t o b s e r v e d w i t h APV a n d DGG was s e e n a l s o w i t h D-APB; t h u s t h e NMA a n d L - a s p a r t a t e , b u t n o t t h e L - g l u t a m a t e , r e s p o n s e s were a n t a g o n -i z e d ( T a b l e I , F i g . 4 . 4 B ) . The L-( + ) - i s o m e r o f APB had no s i g n i f i c a n t e f f e c t on e x c i t a t i o n s i n d u c e d by L - a s p a r t a t e and L - g l u t a m a t e ( T a b l e I , F i g . 4 . 4B ). The f i r i n g o f C A l c e l l s p r o d u c e d by t h e 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 ACh was n o t s i g n i f i c a n t l y a f f e c t e d by 100 nA c u r r e n t s e j e c t i n g DGG, APV and D-APB ( T a b l e I ) . E f f e c t s o f e x c i t a n t s on EPSPs The e x c i t a n t s , a p p l i e d i o n t o p h o r e t i c a l l y a t t h e r e c o r d i n g s i t e i n s t r a t u m r a d i a t u m , d e p r e s s e d e x t r a c e l l u l a r l y r e c o r d e d EPSPs ( T a b l e I I ) . L - G l u t a m a t e and L - a s p a r t a t e were t h e l e a s t p o t e n t a s j u d g e d by t h e i n t e n s i t i e s o f t h e e j e c t i n g c u r r e n t s n e e d e d a nd t h e m a g n i t u d e o f t h e e f f e c t s , and 101 E x c i t a n t E PSPs d e p r e s s e d / EPSPs t e s t e d % r e d u c t i o n (mean +S.E.M. a l l s l i c e s t e s t e d ) NMA K a i n a t e Q u i s q u a l a t e L - a s p a r t a t e L - g l u t a m a t e 24/24 22/22 15/16 5/6 2/4 77.4+3.6* 65.8+4.8** 4 5.4+5.8 31.7+8.7 10.0+5.8 T a b l e I I . C h a n g e s o f t h e S c h a f f e r c o l l a t e r a l - c o m m i s -s u r a l - e v o k e d EPSP f o l l o w i n g i o n t o p h o r e t i c a p p l i c a t i o n s o f e x c i t a t o r y a c i d s . The % d e p r e s s i o n s o f t h e EPSP i n d u c e d by NMA and k a i n a t e were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m one a n o t h e r b u t were g r e a t e r t h a n t h e d e p r e s s i o n i n d u c e d by t h e o t h e r e x c i t a n t s ( p < 0 . 0 1 ) . * * Q u i s q u a l a t e was more p o t e n t t h a n L - g l u t a m a t e (p<0.01) b u t t h e r e was no s i g n i f i c a n t d i f f e r e n c e b e t w e e n L - g l u t a m a t e - a n d L - a s p a r t a t e - i n d u c e d e f f e c t s . 102 r e d u c t i o n s i n EPSPs were f u l l y r e v e r s e d 1-2 m i n a f t e r t e r m i n a t i n g t h e i o n t o p h o r e t i c a p p l i c a t i o n . Q u i s q u a l a t e was more p o t e n t b u t e l i c i t e d q u a l i t a t i v e l y s i m i l a r d e p r e s s i o n s w h i c h u s u a l l y f u l l y r e c o v e r e d w i t h i n 1-3 m i n ( F i g 4 . 6 A ) . More p o t e n t s t i l l was k a i n a t e and i t s e f f e c t s w e r e u s u a l l y l o n g - l a s t i n g ( F i g . 4 . 6 A ) . F u l l r e c o v e r y was o b s e r v e d w i t h 9 s l i c e s w i t h i n 3-25 m i n b u t i n t h e o t h e r 13 r e c o v e r y was i n c o m p l e t e t h r o u g h o u t t h e p e r i o d o f o b s e r v a t i o n (4-20 m i n ) . A l t h o u g h NMA was o f s i m i l a r p o t e n c y t o k a i n a t e i n c a u s i n g d e p r e s s i o n o f t h e EPSP r e c o v e r y was much more r a p i d . I n 18 s l i c e s r e c o v e r y was c o m p l e t e i n 1-3 m i n and was f o l l o w e d by an i n c r e a s e ( 6 - 1 6 5 % ) i n t h e a m p l i t u d e o f t h e EPSP ( F i g . 4.6A,B). T h i s p o t e n t i a t i o n l a s t e d 9 m i n i n 1 s l i c e ( F i g . 4.6A) b u t i n t h e r e m a i n i n g s l i c e s t h e EPSP d i d n o t r e t u r n t o c o n t r o l l e v e l s f o r p e r i o d s up t o 24 m i n ( F i g . 4 . 6 B ) . I n 6 s l i c e s w h e r e p o t e n t i a t i o n o f t h e EPSP was n o t o b s e r v e d , f u l l r e c o v e r y f r o m t h e N M A - i n d u c e d d e p r e s s i o n t o o k 1.5-2 m i n . D e p r e s s i o n o f t h e EPSP was o f t e n a p p a r e n t w i t h o u t any d e t e c t a b l e change i n t h e s i z e o f t h e p r e s y n a p t i c f i b r e v o l l e y , h o w e v e r , w i t h h i g h e r d o s e s o f t h e e x c i t a n t s r e d u c t i o n s i n t h e s i z e o f t h e f i b r e v o l l e y w e r e s o m e t i m e s o b s e r v e d . W i t h k a i n a t e a nd q u i s q u a l a t e any r e d u c t i o n o f t h e f i b r e v o l l e y was a l w a y s s m a l l , w h e r e a s NMA c o m p l e t e l y a b o l i s h e d t h e f i b r e v o l l e y on s e v e r a l o c c a s i o n s . R e c o v e r y o f t h e f i b r e v o l l e y a l w a y s p r e c e d e d t h a t o f t h e EPSP ( F i g s . 4.6B, 4 . 7 A ) . T h e s e e f f e c t s o f t h e e x c i t a n t s on t h e f i e l d p o t e n t i a l s c o u l d be a n t a g o n i z e d a nd t h e s e l e c t i v i t y r e s e m b l e d t h a t 103 F i g . 4.6 E f f e c t o f e x c i t a n t s on t h e S C - e v o k e d f i e l d E P S P . A. I n t h i s r e c o r d t h e e f f e c t s o f NMA, q u i s q u a l a t e ( Q u i s ) and k a i n a t e ( K a i n ) were c o m p a r e d on t h e same s l i c e . The peak a m p l i t u d e o f e a c h EPSP was m e a s u r e d i n r e s p o n s e t o s u c c e s s i v e s t i m u l i d e l i v e r e d a t 0.1 Hz, and t h e v a l u e s p l o t t e d . D r u g s were a p p l i e d i o n t o p h o r e t i c a l l y a t t h e r e c o r d i n g s i t e f o r t h e p e r i o d s i n d i c a t e d by t h e b a r s a b o v e t h e g r a p h . R e p r e s e n t a t i v e r e s p o n s e s c o m p r i s e d o f 5 s u p e r i m p o s e d o s c i l l o s c o p e r e c o r d s were t a k e n a t t h e t i m e s i n d i c a t e d by t h e s m a l l l e t t e r s ( a - e ) . A l l t h r e e e x c i t a n t s d e p r e s s e d t h e EPSP. N o t e t h a t r e c o v e r y f r o m t h e N M A - i n d u c e d d e p r e s s i o n was f o l l o w e d by p o t e n t i a t i o n (b) and t h a t t h e s l o w r e c o v e r y f r o m t h e k a i n a t e - i n d u c e d d e p r e s s i o n was a s s o c i a t e d w i t h t h e g e n e r a t i o n o f a PS ( d ) . B. I n t h i s s l i c e NMA p r o d u c e d a much g r e a t e r p o t e n t i a t i o n o f t h e EPSP f o l l o w i n g r e c o v e r y f r o m t h e d e p r e s s i o n . N o t e a l s o t h e r e d u c e d s i z e b u t b r o a d e n e d p r e s y n a p t i c f i b r e v o l l e y , and t h e v i r t u a l a b o l i t i o n o f t h e EPSP d u r i n g t h e a d m i n i s t r a t i o n o f NMA ( g ) . 104 (a) (b) (c) N M A 50 nA (d) Quis ICOnA (e) _ j 2 m V 10 msec Kain 105 o b s e r v e d when t h e a n t a g o n i s t s w e re e x a m i n e d a g a i n s t t h e e x c i t a t i o n o f s i n g l e u n i t s . Thus APV c o m p l e t e l y p r e v e n t e d t h e N M A - i n d u c e d r e d u c t i o n i n t h e EPSP and f i b r e v o l l e y , as w e l l a s t h e e n s u i n g p o t e n t i a t i o n o f t h e EPSP i n a l l 11 s l i c e s e x a m i n e d ( F i g . 4 . 7 B ) . I n t h e same s l i c e s , q u i s q u a l a t e r e s p o n s e s were e i t h e r u n a f f e c t e d (3/6 s l i c e s ) o r i n c r e a s e d i n d u r a t i o n (3/6 s l i c e s ; F i g . 4.7B) by APV, and k a i n a t e r e s p o n s e s s i m i l a r l y w e r e e i t h e r u n a f f e c t e d (1/3 s l i c e s ) o r s l i g h t l y p o t e n t i a t e d ( 2 / 3 s l i c e s ) . DGG a l s o a b o l i s h e d NMA r e s p o n s e s ( 3 / 3 s l i c e s ) , r e d u c e d o r a b o l i s h e d k a i n a t e r e s p o n s e s ( 2 / 3 s l i c e s ) and s l i g h t l y r e d u c e d q u i s q u a l a t e r e s p o n s e s (3/4 s l i c e s ; F i g . 4 . 8 B ) . E f f e c t s o f a n t a g o n i s t s on EPSPs The 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 , a l s o a d m i n i s -t e r e d a t t h e r e c o r d i n g s i t e i n s t r a t u m r a d i a t u m , had a c t i o n s o n e v o k e d E P S P s w h i c h a r e s u m m a r i z e d i n T a b l e I I I . A PV, a l t h o u g h e x p e l l e d i n d o s e s i n e x c e s s o f t h o s e n e e d e d t o a b o l i s h t h e N M A - i n d u c e d d e p r e s s i o n s , h a d l i t t l e o r no e f f e c t on t h e EPSP a m p l i t u d e ( F i g . 4. 7 B ) . DAA, GDEE and t h e o p t i c a l i s o m e r s o f APB w e r e a l l s i m i l a r l y i n e f f e c t i v e as a n t a g o n i s t s of t h e SC p a t h w a y . By c o n t r a s t DGG was an e f f e c t i v e s y n a p t i c a n t a g o n i s t o f t h i s p a t h w a y when a p p l i e d w i t h d o s e s w h i c h , on t h e b a s i s o f t h e r e s u l t s o f a n a l y s e s w i t h s i n g l e u n i t f i r i n g , w o u l d have b e e n e x p e c t e d t o r e d u c e k a i n a t e and q u i s q u a l a t e r e s p o n s e s as w e l l as t h o s e i n d u c e d by NMA ( F i g . 4 . 8 B ) . I n 14 t e s t s u s i n g 9 106 A n t a g o n i s t DGG L-APB D-APB DAA APV GDEE 21/23 4/9 3/6 2/4 10/26 0/4 EPSPs d e p r e s s e d / EPSPs t e s t e d % r e d u c t i o n 21.3+2 8.2+2 6.4 + 3, 4.2+2, 3.3+1, 0.1+1.9 T a b l e I I I . E f f e c t s o f a n t a g o n i s t s c o l l a t e r a l - c o m m i s s u r a l - e v o k e d E P S P s . on S c h a f f e r C o l u m n 2 g i v e s t h e n u m b e r o f s l i c e s i n w h i c h t h e EPSP was s i g n i f i c a n t l y (p<0.05) d e p r e s s e d c o m p a r e d t o b o t h p r e - and p o s t - d r u g a d m i n i s t r a t i o n , a n d t h e t o t a l n u m b e r o f s l i c e s t e s t e d . The t h i r d c o l u m n p r e s e n t s t h e mean +S.E.M% d e p r e s s i o n c a l c u l a t e d f o r a l l s l i c e s t e s t e d . DGG was s i g n i f i c a n t l y (p<0.05) more p o t e n t t h a n t h e o t h e r compounds. T h e r e were no s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e p o t e n c i e s o f t h e o t h e r 5 s u b s t a n c e s . 107 F i g . 4.7 A. The f i b r e v o l l e y - d e n d r i t i c EPSP ( R 2 ) and t h e s o m a t i c p o t e n t i a l s ( R ^ i n C A l e v o k e d by SC s t i m u l a t i o n a r e i l l u s t r a t e d . R e c o r d s a r e composed o f t h r e e c o n s e c u t i v e s u p e r i m p o s e d s w e e p s ; n e g a t i v i t y i s u p w a r d s . F o l l o w i n g i o n t o -p h o r e s i s o f NMA t o t h e a p i c a l d e n d r i t e s t h e r e was a r e d u c t i o n o f p r e - and p o s t s y n a p t i c a c t i v i t y ( t = l m i n ) . The p r e s y n a p t i c f i b r e v o l l e y r e c o v e r e d f i r s t ( t=2 m i n ) ; f o l l o w e d by t h e EPSP and f i n a l l y t h e PS. B. E f f e c t o f D L - 2 - a m i n o - 5 - p h o s p h o n o v a -l e r a t e on t h e S C - e v o k e d EPSP and on t h e d e p r e s s i o n s o f t h i s r e s p o n s e i n d u c e d by NMA and q u i s q u a l a t e ( Q u i s ) . The g r a p h i s c o n t i n u o u s a n d was c o n s t r u c t e d a s f o r F i g . 4.6. APV com-p l e t e l y a b o l i s h e d t h e d e p r e s s i o n i n d u c e d by NMA and s l i g h t l y p o t e n t i a t e d t h e q u i s q u a l a t e - i n d u c e d d e p r e s s i o n . The a n t a -g o n i s t d i d n o t change t h e a m p l i t u d e o f t h e EPSP. 108 NMA. lOOnA. lmtn . fO t'-2mm t»tmin l'2mm i«5mm I«15 mm 2 - Ammo - 5 - Phosphonovalerole 100 n A 109 s l i c e s , where t h i s d i p e p t i d e c a u s e d d e p r e s s i o n s o f t h e EPSP o f 13 . 0 - 3 0 . 7 % , t h e a m p l i t u d e o f t h e p r e s y n a p t i c f i b r e v o l l e y was s i m u l t a n e o u s l y m e a s u r e d a n d on a l l b u t 1 o c c a s i o n was n o t s i g n i f i c a n t l y a f f e c t e d ( F i g . 4 . 8 A ) . G e n e r a l l y t h e D G G - i n d u c e d d e p r e s s i o n o f t h e EPSP was d e t e c t e d 10-30 s e c a f t e r t h e s t a r t o f t h e a p p l i c a t i o n , r e a c h e d a maximum w i t h i n 3-6 m i n a n d r e c o v e r e d t o c o n t r o l l e v e l s 2-4 m i n a f t e r t e r m i n a t i n g t h e a d m i n i s t r a t i o n o f a n t a g o n i s t . Two o t h e r s u b s t a n c e s w e r e t e s t e d a n d p r o v e d t o be e f f e c t i v e a t r e d u c i n g t h e s y n a p t i c r e s p o n s e . T h e s e were DL-o ( . - a n i i n o a d i p a t e ( 7 / 8 s l i c e s , 21.4 + 5 . 1 % d e p r e s s i o n ) a n d PDA (12/12 s l i c e s , 46.0+6.5%, F i g . 4 . 8 B ) . However, b o t h t h i s PDA ( s e e a b o v e ) a n d L-«(.-aminoadipate ( H a l l e_t a _ l . , 1 9 7 9 ) a r e w e a k l y e x c i t a t o r y , w h i c h c o m p l i c a t e s t h e i n t e r p r e t a t i o n o f t h e s e r e s u l t s . E f f e c t s o f a n t a g o n i s t s on PSs and s y n a p t i c p l a s t i c i t y The e f f e c t s o f APV and DGG a p p l i e d i o n t o p h o r e t i c a l l y t o s t r a t u m r a d i a t u m were e x a m i n e d on t h e PS r e c o r d e d i n s t r a t u m p y r a m i d a l e . C o n s i s t e n t w i t h i t s l a c k o f e f f e c t on t h e EPSP, APV d i d n o t a f f e c t t h e a m p l i t u d e o f t h e PS e v o k e d w i t h l o w f r e q u e n c y ( 0 . 0 5 - 0 . 1 Hz) s t i m u l i ( F i g . 4 . 9 ) . I n c o n t r a s t , DGG r e v e r s i b l y r e d u c e d o r a b o l i s h e d PSs and t h i s a c t i o n p a r a l l e l e d t h e d e c l i n e o f t h e E P S P o n t h e 3 o c c a s i o n s when i t was m o n i t o r e d c o n c u r r e n t l y . B o t h DGG (4/4 s l i c e s ) and APV (8/12 s l i c e s ) c o u l d , h o w e v e r , r e d u c e o r a b o l i s h t h e L T P w h i c h n o r m a l l y o c c u r r e d a f t e r b r i e f t e t a n i c s t i m u l a t i o n . 110 O-a-ferwwxt'pOle C O n A I \ (Ol (01 PTxwfWrwj toOW C O * A i - O G t j t a m y ^ y c . * 0 0 n A < 1 « ( c l n O w a S O n A K o m S O n A s a > - 0 ' C W o n V 9 l y c i n t C O n A C * 2 . ] - P l o c u » n t O c w t n - i t t K 5 0 n A I \ B a F i g . 4.8 E f f e c t s o f a n t a g o n i s t s on t h e S C - e v o k e d f i e l d p o t e n t i a l and on t h e d e p r e s s i o n o f t h i s r e s p o n s e by e x c i t a n t s . A. The u p p e r r e c o r d o f t h e g r a p h i s a p l o t o f EPSP a m p l i t u d e , t h e l o w e r t h a t o f p r e s y n a p t i c f i b r e v o l l e y a m p l i t u d e i n r e s p o n s e t o s u c c e s s i v e s t i m u l i . Of t h e t h r e e a n t a g o n i s t s t e s t e d , o n l y DGG r e d u c e d t h e EPSP b u t was w i t h o u t e f f e c t on t h e f i b r e v o l l e y . O s c i l l o s c o p e r e c o r d s e a c h o f f i v e c o n s e c u t i v e sweeps ( n e g a t i v i t y u p w a r d s ) t a k e n a t t h e t i m e s i n d i c a t e d by ( a - c ) a r e shown a b o v e . The u p p e r m o s t p a r t o f t h e s e r e c o r d s i n d i c a t e s t h e t i m e and s t r e n g t h o f t h e s t i m u l u s . B. I n a n o t h e r s l i c e b o t h DGG a n d PDA r e d u c e d t h e E P S P a n d r e d u c e d o r a b o l i s h e d t h e d e p r e s s i o n s o f t h e EPSP c a u s e d by q u i s q u a l a t e and k a i n a t e . The f i n a l a p p l i c a t i o n s o f q u i s q u a l -a t e and k a i n a i t e w e r e made w i t h t h e i n t e n s i t y o f s t i m u l a t i o n r e d u c e d s o t h a t t h e a m p l i t u d e o f t h e EPSPs m a t c h e d t h o s e w h i c h w e re o b t a i n e d i n t h e p r e s e n c e o f t h e a n t a g o n i s t s . The r e d u c e d e f f e c t i v e n e s s o f t h e t w o e x c i t a n t s i n t h e p r e s e n c e o f t h e a n t a g o n i s t s t h u s i s n o t a t t r i b u t a b l e s i m p l y t o a s m a l l e r EPSP. I l l When i t was b a t h a p p l i e d , D-APV (5X10 M) was i n e f f e c t i v e a s a n a n t a g o n i s t o f t h e S C - e v o k e d E P S P (mean c h a n g e +S.E.M. -0.5+0.3%, n=5) d e s p i t e t h e c o m p l e t e a n t a g o n i s m o f t h e d e p r e s s a n t a c t i o n s o f NMA on s y n a p t i c a c t i v i t y ( F i g . 4 . 1 0 ) . U n d e r t h e s e c o n d i t i o n s a b r i e f h i g h f r e q u e n c y s t i m u l a t i o n h a d no e f f e c t on t h e EPSP a m p l i t u d e . A f t e r w a s h o u t o f t h e d r u g a n d r e c o v e r y o f NMA r e s p o n s e s , a t e t a n i c s h o c k p r o d u c e d a p o t e n t i a t i o n o f t h e EPSP w h i c h p e r s i s t e d t o t h e e n d o f t h e r e c o r d i n g p e r i o d ( F i g . 4 . 1 0 ) . D i s c u s s i o n E f f e c t s o f e x c i t a t o r y a m ino a c i d a g o n i s t s •and a n t a g o n i s t s on s i n g l e u n i t a c t i v i t y The i n v o l v e m e n t o f p o s t s y n a p t i c e x c i t a t o r y a m ino a c i d r e c e p t o r s i n s t r a t u m r a d i a t u m , where t h e SC i n p u t t o t h e C A l p y r a m i d a l c e l l s t e r m i n a t e s ( L a u r b e r g , 1 9 7 9 ) , was i m p l i c a t e d by t h e e x i s t e n c e o f f o c i a t w h i c h i o n t o p h o r e s i s o f L - g l u t a m a t e , L - a s p a r t a t e and q u i s q u a l a t e e l i c i t e d s h o r t l a t e n c y f i r i n g ( c f . B i s c o e and S t r a u g h a n , 1966; D u d a r , 1974; S c h w a r t z k r o i n a n d A n d e r s e n , 1975; S p e n c e r e_t ajL. , 1 9 7 8 ) . Q u a l i t a t i v e l y d i f f e r e n t r e s p o n s e s t o NMA and k a i n a t e were o b s e r v e d a t t h e same s i t e . T h e s l o w c o n t i n u o u s r i s e o f k a i n a t e - m e d i a t e d f i r i n g may be a t t r i b u t a b l e t o t h e g r a d u a l i n c r e a s e o f t h e e x t r a c e l l u l a r c o n c e n t r a t i o n s i n c e g l i a l o r n e u r o n a l u p t a k e s y s t e m s ( J o h n s t o n e t a _ l . , 1979) s u c h as e x i s t f o r L - g l u t a m a t e , L - a s p a r t a t e ( B a l c a r a n d J o h n s t o n , 1 9 7 3 ) a n d p o s s i b l y f o r q u i s q u a l a t e ( L o d g e e_t a _ l . , 1980 ) do n o t e x i s t f o r t h i s a g o n i s t . The a b s e n c e o f an u p t a k e m e c h a n i s m f o r NMA may a l s o 1 1 2 DGG 100 nA 100 Hi 1 sec 100 Hi 1 sec A P V 100 n A 25 m m ! • r i m V •.S . • A ' I n 1 * • 0 ' 100 Hz 1 sec 100 Hi 1 sec • • 1 m m F i g . 4.9 E f f e c t o f a n t a g o n i s t s on t h e PS and on t h e ' g e n e r a t i o n o f LTP. The a m p l i t u d e o f t h e IPS .was r e c o r d e d i n s t r a t u m p y r a m i d a l e i n r e s p o n s e t o 0.1 Hz s t i m u l a t i o n o f t h e SC p r o j e c t i o n and a n t a g o n i s t s were a d m i n i s t e r e d i o n t o p h o r e t i c a l l y i n s t r a t u m r a d i a t u m f o r t h e d u r a t i o n s , i n d i c a t e d by t h e b a r s . The pathway was s t i m u l a t e d a t 100 Hz f o r 1 s e c a t t h e t i m e s shown by t h e a r r o w s b e l o w sthe t r a c e . DGG r e d u c e d t h e a m p l i t u d e o f PSs a n d p r e v e n t e d LTP i n r e s p o n s e t o h i g h f r e q u e n c y s t i m u l a t i o n . F o l l o w i n g r e c o v e r y f r o m th e ..actions o f DGG, L T P was p r o d u c e d u s i n g a p e r i o d o f i d e n t i c a l h i g h f r e q u e n c y s t i m u l a t i o n . A f t e r 17 min, APV was a p p l i e d and had no e f f e c t on the PS. Two m i n u t e s l a t e r t h e s t i m u l u s i n t e n s i t y was r e d u c e d ( l o w e r r e c o r d ) and 3 min l a t e r t h e e f f e c t s o f h i g h f r e q u e n c y s t i m u l a t i o n were a g a i n t e s t e d . A l t h o u g h some s h o r t - l a s t i n g p o s t - t e t a n i c p o t e n t i a t i o n r e s u l t e d , LTP was p r e v e n t e d . A f t e r t h e APV i n j e c t i o n had been t e r m i n a t e d f o r 8 min, 100 Hz s t i m u l a t i o n f o r 1 s e c was a g a i n a b l e t o p r o d u c e LTP w h i c h d i d n o t r e c o v e r c o m p l e t e l y o v e r t h e t i m e t h a t r e s p o n s e s were measured (45 m i n ) . 113 D - 2 • Amino- 5-phosphonovalerote 5% 10" 5M NMA 25nA S 2 -> 0J N S ^ "' *'•'"'.•'• «-• \ ' — • ...... s s s s 10 mm 3-1 t t 100 Hz, I sec 100 Hz, I sec ° J '2 min. F i g . 4.10 The a c t i o n s o f b a t h - a p p l i e d D-APV on t h e a m p l i t u d e s o f S C - e v o k e d d e n d r i t i c EPSPs r e c o r d e d s i m u l -t a n e o u s l y f r o m 2 s l i c e s . A d o s e o f t h e a n t a g o n i s t w h i c h p r e v e n t e d t h e d e p r e s s a n t a c t i o n s o f i o n t o p h o r e t i c a l l y - a p p l i e d NMA ( u p p e r g r a p h ) h a d no e f f e c t on l o w f r e q u e n c y s y n a p t i c e x c i t a t i o n ( b o t h r e c o r d s ) b u t b l o c k e d LTP ( l o w e r g r a p h ) . A f t e r r e t u r n i n g t o n o r m a l p e r f u s a t e and w i t h t h e r e c o v e r y o f NMA r e s p o n s e s a b r i e f t e t a n u s (100 Hz f o r 1 s e c ) p r o d u c e d an e n d u r i n g i n c r e a s e oE t h e EPSP a m p l i t u d e . 114 e x p l a i n i t s t e n d e n c y t o r e c r u i t a d j a c e n t n e u r o n s and e v e n -t u a l l y t o p r o d u c e h i g h f r e q u e n c i e s o f f i r i n g , b u t i s n o t a r e a s o n a b l e e x p l a n a t i o n o f i t s u n i q u e a b i l i t y t o i n d u c e t h e b u r s t i n g p a t t e r n o f f i r i n g o f C A l n e u r o n s . A s i m i l a r r e s p o n s e composed o f b u r s t s o f a c t i o n p o t e n t i a l s h a s been o b s e r v e d n e i t h e r i n d e n t a t e g r a n u l e c e l l s i n o u r l a b o r a t o r y n o r i n s p i n a l m o t o n e u r o n s ( c f . F l a t m a n et_ a l . , 1 9 8 3 ) . However, NMDA has been r e p o r t e d t o e v o k e b u r s t i n g o f s p i n a l i n t e r n e u r o n s i j i v i v o ( M a c D o n a l d and N i s t r i , 1978) and i n f e l i n e n e o c o r t i c a l c e l l s ( F l a t m a n e t a l . , 1 9 8 3 ) . I n t h e l a t t e r s t u d y , i n t r a c e l l u l a r r e c o r d i n g r e v e a l e d t h a t b u r s t 2 + a c t i v i t y was t r i g g e r e d by an N M A - i n d u c e d Ca - d e p e n d e n t p a r o x y s m a l d e p o l a r i z i n g s h i f t ( P D S ) ( s e e a l s o D i n g l e d i n e , 1 9 8 3 a , b ; Wong and P r i n c e , 1978) and t h i s may a l s o be t h e b a s i s f o r t h e r h y t h m i c b u r s t s o f a c t i o n p o t e n t i a l s r e p o r t e d h e r e . I n t h i s c o n n e c t i o n i t i s i n t e r e s t i n g t h a t q u i n o l i n i c a c i d w h i c h i n t e r a c t s w i t h t h e NMA r e c e p t o r ( S t o n e a n d P e r k i n s , 1981) a l s o p r o d u c e s b u r s t f i r i n g o f C A l n e u r o n s (Ganong e_t 2 + a l . , 1 9 8 3 ) . The s i g n i f i c a n c e o f an NMA r e c e p t o r - c o u p l e d Ca i n f l u x f o r t h e m o d u l a t i o n o f c o r t i c a l f u n c t i o n i s d i s c u s s e d b e l o w . As i n o t h e r r e g i o n s , APV was f o u n d t o be a p o t e n t and s e l e c t i v e a n t a g o n i s t o f t h e N M A - i n d u c e d e x c i t a t i o n o f h i p p o c a m p a l n e u r o n s b u t t y p i c a l l y t h e r e was a c o n c o m i t a n t p o t e n t i a t i o n o f t h e e x c i t a t i o n s i n d u c e d by k a i n a t e and q u i s q u a l a t e . B o t h t y p e s o f a c t i o n a p p e a r t o be e x e r t e d by t h e D - ( - ) - i s o m e r . The e f f e c t s o f DAA were q u a l i t a t i v e l y t h e same 115 as t h o s e o b s e r v e d w i t h i t s - p h o s p h o n a t e a n a l o g u e APV. I n o t h e r CNS r e g i o n s D A A has been r e p o r t e d t o enhance t h e e f f e c t o f q u i s q u a l a t e ( E v a n s , F r a n c i s e_t a _ l . , 1979) and L - g l u t a m a t e (Lodge et. a_l. , 1978); t o i n c r e a s e s p o n t a n e o u s a c t i v i t y o f t h e d e n t a t e g y r u s ( H i c k s a n d M c L e n n a n , 1979) a n d s p i n a l c o r d (Lodge et. a_l. , 1978); and t o d e p o l a r i z e f r o g s p i n a l n e u r o n s ( E v a n s , F r a n c i s and W a t k i n s , 1 9 7 8 ) . These o b s e r v a t i o n s i m p l y t h a t D A A has s u b l i m i n a l e x c i t a t o r y e f f e c t s , and t h e e n h a n c e -ment by APV o f q u i s q u a l a t e and k a i n a t e r e p o r t e d h e r e i s a l s o s u g g e s t i v e of a weak e x c i t a t o r y a c t i o n . However, APV d i d n o t p o t e n t i a t e r e s p o n s e s t o ACh, a n d no d i r e c t e v i d e n c e f o r e x c i t a t i o n by APV has been o b t a i n e d . T h a t low i o n t o p h o r e t i c d o s e s o f t h e d i p e p t i d e DGG a n t a g o n i z e d r e s p o n s e s t o k a i n a t e ( a s w e l l a s NMA a n d L -a s p a r t a t e ) b u t n o t q u i s q u a l a t e s u p p o r t s t h e p r o p o s e d i n t e r -a c t i o n o f k a i n a t e and q u i s q u a l a t e w i t h two d i f f e r e n t r e c e p t o r s ( D a v i e s and W a t k i n s , 1 9 8 1 ) . However, t h e r e was a s i g n i f i c a n t r e d u c t i o n o f t h e a c t i o n o f q u i s q u a l a t e when h i g h e r e j e c t i n g c u r r e n t s o f DGG were u s e d , i n d i c a t i n g t h a t t h i s s u b s t a n c e may be u s e f u l as a q u i s q u a l a t e a n t a g o n i s t , e s p e c i a l l y s i n c e o t h e r p o t e n t i a l b l o c k e r s o f t h e s e r e c e p t o r s (GDEE a n d PDA) h a d c o m p l i c a t i n g a c t i o n s w h i c h r e n d e r e d them l e s s u s e f u l as a n t a g o n i s t s i n t h e hi p p o c a m p u s . The d e c l i n e o f t h e L - g l u t a m -a t e r e s p o n s e c o n c o m i t a n t w i t h t h e a n t a g o n i s m by DGG o f q u i s q u a l a t e e x c i t a t i o n s i s c o n s i s t e n t w i t h a p r e f e r e n t i a l r e a c t i o n o f L - g l u t a m a t e w i t h q u i s q u a l a t e r e c e p t o r s (McLennan, H i c k s and L i u , 1 9 82). 116 P r e v i o u s r e p o r t s o f i n h i b i t i o n by APB o f s y n a p t i c r e s p o n s e s i n t h e hippocampus ( D u n w i d d i e e t al_ . , 1978; W h i t e et. a l . , 1979; K o e r n e r and Cotman, 1981) have assumed a n t a g o n i s m o f a m i n o a c i d r e c e p t o r s t o be t h e u n d e r l y i n g c a u s e . O u r r e s u l t s i n d i c a t e t h a t q u i t e d i f f e r e n t a c t i o n s a r e p r o d u c e d by t h e two i s o m e r s o f APB. In agreement w i t h t h e d a t a o f Evans e t a l . (1979) o b t a i n e d w i t h DL-APB i n t h e f r o g s p i n a l c o r d , and t h o s e o f H o r i e_t a_l. (1981) i n t h e r a t o l f a c t o r y c o r t e x , D-APB a n t a g o n i z e d NMA and k a i n a t e t o a s i m i l a r e x t e n t . More r e c e n t s p i n a l c o r d s t u d i e s ( D a v i e s and W a t k i n s , 1982) d e m o n s t r a t e d n o n - s p e c i f i c a n t a g o n i s m by D-APB o f amino a c i d -i n d u c e d r e s p o n s e s w h i c h may s i m p l y r e f l e c t dose d i f f e r e n c e s . As w i t h APV a n d DGG, D-APB a n t a g o n i s m o f t h e NMA r e s p o n s e p a r a l l e l e d a d e c l i n e , a l b e i t s m a l l e r , i n t h e L - a s p a r t a t e r e s p o n s e ( b u t see H o r i e_t al_ . , 1 9 8 1 ) . T y p i c a l l y , t h e e f f e c t o f L-APB was t o e n h a n c e a m i n o a c i d - i n d u c e d r e s p o n s e s a n d o c c a s i o n a l l y t o e x c i t e c e l l s by i t s e l f . The l a t t e r a c t i o n has a l s o b e e n o b s e r v e d i n t h e CA3 r e g i o n ( d a t a n o t s h o w n ) . B l o c k a d e o f t h e a g o n i s t e f f e c t s o f L-APB by D-APB i n t h i s s t u d y a n d by D-APB a n d APV i n t h e i s o l a t e d s p i n a l c o r d p r e p a r a t i o n ( E v a n s e_t a_l. , 1982) i m p l y t h a t L-APB e x c i t a t i o n i s m e d i a t e d t h r o u g h t h e NMA r e c e p t o r . Q u i t e c l e a r l y , i n h i b i t i o n o f s y n a p t i c t r a n s m i s s i o n by L-APB i n t h e h i p p o c a m p a l f o r m a t i o n c a n n o t be a t t r i b u t e d t o a n t a g o n i s m o f any one o f t h e t h r e e ' c o n v e n t i o n a l ' amino a c i d r e c e p t o r s . O u r o b s e r v a t i o n t h a t L-APB d i d n o t a n t a g o n i z e L - g l u t a m a t e - i n d u c e d e x c i t a t i o n s a l s o a r g u e s a g a i n s t i t s 117 i n t e r a c t i o n w i t h a n o v e l g l u t a m a t e r e c e p t o r , b u t d o e s n o t p r e c l u d e a n t a g o n i s m o f t h e r e c e p t o r f o r an u n i d e n t i f i e d n e u r o t r a n s m i t t e r . A l t h o u g h i t was a p o t e n t , n o n - s e l e c t i v e a m i n o a c i d a n t a g o n i s t , PDA a l s o had a d i r e c t A P V - s e n s i t i v e e x c i t a t o r y e f f e c t on C A l n e u r o n s ( c f . S e g a l , 1 9 8 1 ) . P a r t i a l N M A - l i k e a g o n i s t e f f e c t s o f PDA have been o b s e r v e d i n t h e s p i n a l c o r d ( D a v i e s , E v a n s e_t a _ l . , 1 9 8 1 ) . I n t e r p r e t a t i o n o f a c t i o n s on s y n a p t i c m e c h a n i s m s w o u l d be c o m p l i c a t e d by t h i s d e p o l a r i z i n g a c t i o n . A l t h o u g h GDEE h a s a d e m o n s t r a t e d a b i l i t y t o a n t a g o n i z e q u i s q u a l a t e r e s p o n s e s s e l e c t i v e l y i n t h e s p i n a l c o r d in_ v i v o ( D a v i e s and W a t k i n s , 1979; McLennan a nd L o d g e , 1979; b u t s e e E v a n s e_t a _ l . , 1 9 7 9 ) t h e r e s u l t s o f t h i s s t u d y s u p p o r t t h e c o n c l u s i o n o f W h i t e e_t a l . (1979) who d e s c r i b e d i t s a c t i o n s as " c a p r i c i o u s and i n e x p l i c a b l e " . T h i s i s i n c o n t r a s t t o t h e r e p o r t s o f S p e n c e r e t a l . ( 1 9 7 6) f o r C A l n e u r o n s a nd o f H i c k s a n d M c L e n n a n ( 1 9 7 9 ) a n d W h e a l a n d M i l l e r ( 1 9 8 0 ) f o r t h e d e n t a t e g y r u s w here r e a s o n a b l e s p e c i f i c i t i e s were f o u n d . W h a t e v e r t h e e x p l a n a t i o n f o r . t h e s e d i s c r e p a n c i e s may b e , b e c a u s e i t s a c t i o n s a r e u n c l e a r t h e v a l u e o f GDEE a s an a n t a g o n i s t o f e x c i t a t o r y s y n a p t i c i n p u t t o C A l seems l i m i t e d . The e f f e c t s o f t h e a n t a g o n i s t s on amino a c i d - i n d u c e d e x c i t a t i o n s o f C A l n e u r o n s c o n f o r m i n g e n e r a l t o t h o s e r e p o r t e d f o r t h e s p i n a l c o r d . T h e d a t a show t h a t o f t h e a n t a g o n i s t s t e s t e d DGG a n d APV may be u s e f u l i n s y n a p t i c i n v e s t i g a t i o n s , w h i l e o t h e r s s u c h as D- and L-APB, PDA and 118 GDEE may be o f l e s s e r v a l u e b e c a u s e t h e y a r e l e s s p o t e n t a n d / o r h a v e c o m p l i c a t i n g d i r e c t a c t i o n s on C A l c e l l s . E f f e c t s o f e x c i t a t o r y a m i n o a c i d a g o n i s t s  and a n t a g o n i s t s on s y n a p t i c a c t i v i t y The d i s c r e t e e l e c t r i c a l s t i m u l a t i o n u s e d a c t i v a t e d p r e d o m i n a n t l y S c h a f f e r c o l l a t e r a l and p r o b a b l y t o a c o n s i d e r -a b l y s m a l l e r e x t e n t c o m m i s s u r a l f i b r e s ( V o n e i d a e t a _ l . , 1 9 8 1 ) . B o t h p r o j e c t i o n s a p p e a r t o o r i g i n a t e f r o m t h e same CA3 n e u r o n s (Swanson e_t a l _ . , 1980; L a u r b e r g and S0rensen, 1981) and s o p h a r m a c o l o g i c a l l y c a n p r o b a b l y be assumed t o be homogeneous. E f f e c t s p r o d u c e d by o t h e r f i b r e s y s t e m s w h i c h c o u r s e t h r o u g h s t r a t u m r a d i a t u m and r e m a i n i n t a c t i n t r a n s v e r s e l y c u t s l i c e s w o u l d be a t most o f m i n o r c o n s e q u e n c e s i n c e a l a r g e p r o p o r t i o n o f t h e t e r m i n a l s on t h e a p i c a l d e n d r i t e s o f C A l c e l l s o r i g i n a t e f r o m CA3 n e u r o n s ( G o l d o w i t z et_ aT. , 1 9 7 9 ) . 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 e x c i t a n t s r e d u c e d t h e f i e l d EPSP p r e s u m a b l y by d e p o l a r i z i n g a n d / o r i n c r e a s i n g t h e membrane c o n d u c t a n c e w h i c h p r o d u c e d a s h u n t o f p o s t s y n a p t i c c u r r e n t s ; a p r e s y n a p t i c s i t e o f a c t i o n may a l s o h a v e b e e n i n v o l v e d s i n c e i n some c a s e s a r e d u c t i o n o f t h e p r e s y n a p t i c f i b r e v o l l e y was e v i d e n t . The a g o n i s t - i n d u c e d r e d u c t i o n o f EPSPs a l s o e x h i b i t e d a s e n s i t i v i t y t o a n t a g o n i s m w h i c h was e s s e n t i a l l y i d e n t i c a l t o t h a t o f t h e e x c i t a t i o n o f s i n g l e n e u r o n s . T h e b l o c k a d e o f t h e d e p r e s s i o n o f E P S P s i n d u c e d by v a r i o u s e x c i t a n t s may t h e r e f o r e be u s e d t o c h e c k t h e s p e c i -f i c i t y o f an a n t a g o n i s t as i t i s t e s t e d a g a i n s t a s y n a p t i c 1 1 9 r e s p o n s e ( F i g s . 4.7, 4 . 8 B ) . F o r e x a m p l e , when h i g h c u r r e n t s a r e u s e d t o e j e c t p o t e n t a g o n i s t s s u c h a s k a i n a t e o r NMA, EPSPs c a n be a b o l i s h e d and h e n c e t h e e x c i t a n t c a n be assumed t o be a f f e c t i n g t h e e n t i r e r e g i o n t h a t i s g e n e r a t i n g t h e r e c o r d e d s y n a p t i c r e s p o n s e . I n t u r n t h e n , a n t a g o n i s t s s u c h as APV w h i c h c a n p r e v e n t t h e s e l a r g e d e p r e s s i o n s c a n a l s o be a ssumed t o be r e a c h i n g t h e w h o l e a r e a f r o m w h i c h t h e f i e l d p o t e n t i a l i s p r o d u c e d . I f e x o g e n o u s e x c i t a n t s a c t a t t h e same s u b s y n a p t i c s i t e a s d o e s t h e e n d o g e n o u s 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 " * " , t h e n , u n d e r c o n d i t i o n s where s y n a p t i c r e s p o n s e s a r e u n a f f e c t e d , i t c a n be i n f e r r e d t h a t a g i v e n t y p e o f r e c e p t o r i s n o t i n v o l v e d i n m e d i a t i n g t h e r e s p o n s e , r a t h e r t h a n t h a t t h e a n t a g o n i s t i s n o t r e a c h i n g t h e s y n a p t i c r e c e p t o r . The d e p r e s s i o n s i n d u c e d by L - g l u t a m a t e , L - a s p a r t a t e and q u i s q u a l a t e were r e l a t i v e l y weak and s h o r t - l a s t i n g , p r o b a b l y d u e t o u p t a k e o f t h e s e c o m p o u n d s ( s e e I n t r o d u c t i o n ) . I n c o n t r a s t , t h e more p o t e n t a g o n i s t k a i n a t e p r o d u c e d a m a s s i v e and s l o w l y r e v e r s i b l e r e d u c t i o n o f t h e EPSP. C o n c u r r e n t w i t h t h i s a c t i o n t h e r e was t h e i n d u c t i o n o r i n c r e a s e i n s i z e o f t h e PS r e c o r d e d l o c a l l y ( F i g . 4.6A) o r a t t h e c e l l body l a y e r R e c e n t l y L y n c h and h i s c o - w o r k e r s ( F a g n i et. a l . , 1983) r e p o r t e d a r e v e r s i b l e d e n s e n s i t i z a t i o n o f p h y s i o l o g i c a l r e s p o n s e s t o b a t h a p p l i c a t i o n s o f L- ( a n d D-) g l u t a m a t e w h i c h was n o t p a r a l l e l e d by a r e d u c t i o n o f t h e S C - e v o k e d EPSP. T h i s e v i d e n c e s u g g e s t s t h a t e x o g e n o u s g l u t a m a t e may a c t p r i m a r i l y on e x t r a - s y n a p t i c s i t e s a n d / o r t h a t L - g l u t a m a t e i t s e l f d o e s n o t s e r v e a t r a n s m i t t e r f u n c t i o n i n t h e SC p a t h w a y . None-t h e l e s s o t h e r e v i d e n c e p r o v i d e d h e r e i n d i c a t e d a n a c c e s s -a b i l i t y o f i o n t o p h o r e t i c a l l y - a p p l i e d compounds ( e . g . DGG) t o s u b - s y n a p t i c s i t e s . 120 ( c f . . C h a p t e r 5 ) . The mechanisms u n d e r l y i n g t h e p o t e n t i a t i o n o f s y n a p t i c e x c i t a t i o n a r e c o n s i d e r e d i n t h e n e x t c h a p t e r . A l t h o u g h NMA p r o d u c e d a l a r g e d e p r e s s i o n o f t h e EPSP t h e r e w e r e t w o v e r y d i f f e r e n t a s p e c t s t o i t s a c t i o n s i n c o m p a r i s o n w i t h t h o s e o f k a i n a t e . F i r s t , h i g h d o s e s o f NMA p r o d u c e d a p r o n o u n c e d A P V - s e n s i t i v e r e d u c t i o n i n t h e f i b r e v o l l e y w h i c h may h a v e been due t o a d i r e c t i n t e r a c t i o n w i t h p r e s y n a p t i c r e c e p t o r s ( b u t s e e F e r k a n y et a l . , 1982) a n d / o r c h a n g e s o f e x t r a c e l l u l a r i o n c o n c e n t r a t i o n s due t o t h e p o s t s y n a p t i c d e p o l a r i z a t i o n s ( E v a n s , 1 9 8 0 ) . S e c o n d l y , t h i s compound o f t e n p o t e n t i a t e d t h e a m p l i t u d e o f t h e EPSP f o r l o n g p e r i o d s f o l l o w i n g r e c o v e r y f r o m d e p r e s s i o n , an e f f e c t w h i c h was a l s o o b t a i n e d w i t h t h e p e r f o r a n t p a t h - e v o k e d EPSP i n t h e d e n t a t e g y r u s ( d a t a n o t s h o w n ) . T h i s p o t e n t i a t i o n may have been due t o a l o n g - l a s t i n g a l t e r a t i o n i n membrane p o t e n t i a l o r c o n d u c t a n c e . S t u d i e s on m o t o n e u r o n s , n e o c o r t i c a l and h i p p o c a m p a l c e l l s h a v e shown t h e d e p o l a r i z i n g a c t i o n s o f NMA t o be a s s o c i a t e d s o m e t i m e s w i t h an i n c r e a s e d i n p u t r e s i s t a n c e , a n e f f e c t p r o b a b l y d u e t o t h e a c t i v a t i o n o f a v o l t a g e -2+ + d e p e n d e n t i n w a r d Ca a n d / o r Na c u r r e n t ( s e e I n t r o d u c t i o n ) . A r e s i d u a l i n c r e a s e i n membrane r e s i s t a n c e c o u l d e x p l a i n t h e o b s e r v e d i n c r e a s e i n t h e EPSP. A l t e r n a t i v e l y , t h i s a c t i o n may 2+ r e f l e c t e v e n t s t r i g g e r e d by an i n f l u x o f Ca e i t h e r p r e - o r p o s t s y n a p t i c a l l y s i n c e (1) NMA ( a n d L - a s p a r t a t e ) p r o d u c e s an 2+ 2 + i n c r e a s e d i n t r a c e l l u l a r Ca c o n c e n t r a t i o n ( [Ca ]^) ( P a d j e n and S m i t h , 1 9 8 1 ; M a r c i a n i e t a l . , 1982) and (2) an a b i l i t y o f NMA t o p r o d u c e " c a l c i u m s p i k e s " i n h i p p o c a m p a l C A l c e l l s has 121 been r e p o r t e d ( D i n g l e d i n e , 1983a,b; see a l s o F l a t m a n e t a l , , 1 9 8 3 ) . As p o i n t e d o u t by D i n g l e d i n e , t h i s r i s e i n 2+ [Ca ] ^ c o u l d a f f e c t a number o f b i o c h e m i c a l p r o c e s s e s . I n t h i s c o n n e c t i o n , i t i s i n t e r e s t i n g t h a t t h e NMA r e c e p t o r a n t a g o n i s t s DGG and APV p r e v e n t e d LTP s i n c e p o s t s y n a p t i c 2 + Ca e n t r y has b e e n i m p l i c a t e d i n t h i s phenomenon ( B a u d r y and L y n c h , 1981; L y n c h e t a l . , 1 9 8 3 ) . The f i r m e s t c o n c l u s i o n t h a t c a n be d r a w n f r o m t h e a n t a g o n i s t s t u d i e s i s t h a t t h e NMA r e c e p t o r i s n o t i n v o l v e d i n m e d i a t i n g s y n a p t i c e x c i t a t i o n i n t h e SC p a t h w a y . A p p l i c a t i o n s o f APV t h a t w e r e g r e a t e r t h a n t h o s e r e q u i r e d t o a b o l i s h N M A - i n d u c e d d e p r e s s i o n s o f t h e EPSP o r i n c r e a s e s i n C A l f i r i n g p r o d u c e d l i t t l e o r no change o f EPSP i t s e l f . I t i s h i g h l y u n l i k e l y t h a t t h e a b s e n c e o f a n t a g o n i s m w i t h APV was due t o i n a d e q u a t e a c c e s s i b i l i t y t o s y n a p t i c r e c e p t o r s s i n c e , f i r s t , t h e a n t a g o n i s t s w e re a d m i n i s t e r e d a t t h e d e n d r i t i c r e g i o n o f t h e s l i c e w h e r e t h e s t i m u l a t e d f i b r e s t e r m i n a t e d ; s e c o n d , u s i n g t h e same e l e c t r o d e s APV e f f e c t i v e l y a n t a g o n i z e d t h e l a t e r a l p e r f o r a n t p a t h i n p u t i n t o t h e d e n t a t e g y r u s w i t h much l o w e r e j e c t i o n c u r r e n t s ( d a t a n o t s h o w n ) ; and t h i r d , o t h e r s u b s t a n c e s , f o r e x a m p l e DGG, a p p l i e d f r o m t h e same e l e c t r o d e d e p r e s s e d t h e .SC-evoked r e s p o n s e . T h a t DAA d i d n o t a f f e c t t h i s p a t h w a y w h i l e b e i n g a s e l e c t i v e NMA a n t a g o n i s t o f s i n g l e u n i t a c t i v i t y i n t h e h i p p o c a m p u s a l s o i n d i c a t e s t h e a b s e n c e o f any i n t e r a c t i o n o f t h e e n d o g e n o u s t r a n s m i t t e r w i t h t h e NMA r e c e p t o r . F u r t h e r m o r e , t h e s l o w o n s e t o f NMA e x c i t a t i o n s r e p o r t e d h e r e and by o t h e r i n v e s t i g a t o r s i m p l i e s t h a t i t w o u l d 122 n o t be i n v o l v e d i n f a s t s y n a p t i c t r a n s m i s s i o n . U n l i k e t h e s e l e c t i v e NMA a n t a g o n i s t s DAA and APV, DGG c o n s i s t e n t l y d e p r e s s e d t h e s y n a p t i c r e s p o n s e and w i t h t h e h i g h e j e c t i o n c u r r e n t s r e q u i r e d t o do t h i s i t a l s o b l o c k e d k a i n a t e -and q u i s q u a l a t e - i n d u c e d d e p r e s s i o n s o f t h e EPSP and t h e e x c i t a t i o n o f s i n g l e C A l n e u r o n s . 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 e i t h e r t h e k a i n a t e o r q u i s q u a l a t e r e c e p t o r m e d i a t e s s y n a p t i c e x c i t a t i o n i n t h e SC p a t h w a y . The o b s e r v a t i o n t h a t DGG had no a p p r e c i a b l e e f f e c t on t h e f i b r e v o l l e y i n d i c a t e s t h a t t h e s y n a p t i c a n t a g o n i s m i s u n l i k e l y t o be c a u s e d by a s e p a r a t e a c t i o n on t h e e x c i t a b i l i t y o f p r e s y n a p t i c t e r m i n a l s . F u r t h e r m o r e , p o s t s y n a p t i c e f f e c t s u n r e l a t e d t o b l o c k a d e o f n e u r o t r a n s m i t t e r - r e c e p t o r i n t e r a c t i o n s a r e u n l i k e l y t o a c c o u n t f o r t h e a c t i o n s i n c e t h e D G G - i n d u c e d r e d u c t i o n o f t h e EPSP e v o k e d by s t i m u l a t i o n o f t h e m e d i a l p e r f o r a n t p a t h i n t h e d e n t a t e g y r u s was n o t a s s o c i a t e d w i t h any c hange i n membrane p o t e n t i a l , i n p u t r e s i s t a n c e o r n e u r o n a l e x c i t a b i l i t y ( C r u n e l l i e t a l . , 1 9 8 2 ) . F i n a l l y , S e g a l ( 1 9 8 1) has b r i e f l y m e n t i o n e d t h a t t o p i c a l a p p l i c a t i o n o f DGG t o t h e h i p p o c a m p a l s l i c e r e d u c e d t h e S C - e v o k e d EPSP w i t h o u t a f f e c t i n g membrane p o t e n t i a l o r r e s i s t a n c e . C o n f i r m a t i o n t h a t e i t h e r t h e k a i n a t e o r q u i s q u a l a t e r e c e p t o r i s t h e s y n a p t i c r e c e p t o r i n t h i s p a t h w a y must a w a i t t h e d e v e l o p m e n t o f more p o t e n t and s e l e c t i v e a n t a g o n i s t s f o r t h e s e compounds. T h a t GDEE, an e f f e c t i v e q u i s q u a l a t e a n t a g o n i s t i n t h e s p i n a l c o r d ( D a v i e s and W a t k i n s , 1979; McLennan and L o d g e , 1979) d i d n o t a f f e c t t h e s y n a p t i c r e s p o n s e d oes n o t p r e c l u d e a 123 r o l e o f q u i s q u a l a t e r e c e p t o r s i n t h i s p a t h w a y s i n c e s a t i s -f a c t o r y a n t a g o n i s m o f r e s p o n s e s o f s i n g l e C A l n e u r o n s t o t h i s a g o n i s t by GDEE were n e v e r o b t a i n e d . The o p t i c a l i s o m e r s o f APB were e a c h s i m i l a r l y weak d e p r e s s a n t s o f t h e EPS P b u t p r o b a b l y a c t e d by d i f f e r e n t m e c h a n i s m s s i n c e D-APB had a p h a r m a c o l o g i c a l p r o f i l e s i m i l a r t o b u t l e s s p o t e n t t h a n DGG, w h e r e a s L-APB was w e a k l y e x c i t a t o r y a n d d e v o i d o f a n t a g o n i s t a c t i v i t y f o r amino a c i d - i n d u c e d e x c i t a t i o n s o f C A l c e l l s . T h u s , a l t h o u g h t h e d e p r e s s i o n o f t h e SC-e,voked EPSP o b s e r v e d f o l l o w i n g p e r f u s i o n o f h i p p o c a m p a l s l i c e s w i t h h i g h d o s e s o f t h e r a c e m i c m i x t u r e o f APB ( D u n w i d d i e e t a l . , 1978; W h i t e e t a l . , 1979) t h e o r -e t i c a l l y c o u l d be due t o an a c t i o n o f e i t h e r o r b o t h o f t h e s t e r e o i s o m e r s , a n e f f e c t o f D-APB o n t h e NMA r e c e p t o r i s p r e s u m a b l y n o t i n v o l v e d . On t h e o t h e r h a n d , s u c h an a c t i o n m i g h t e x p l a i n why t h e r a c e m a t e was a b l e t o a b o l i s h L T P ( D u n w i d d i e et. a _ l . , 1978; Cotman et_ a _ l . , 1 9 8 1 ) , s i n c e we ha v e shown h e r e t h a t LTP c a n be p r e v e n t e d by APV, an NMA a n t a g o n -i s t . To a t t r i b u t e a r o l e t o e i t h e r L - g l u t a m a t e o r L-a s p a r t a t e as t h e n a t u r a l t r a n s m i t t e r i n t h e SC p r o j e c t i o n i s c o m p l i c a t e d by t h e f a c t t h a t b o t h a m i n o a c i d s may be c a p a b l e o f i n t e r a c t i n g t o v a r y i n g e x t e n t s w i t h t h e t h r e e e x c i t a t o r y a m i no a c i d r e c e p t o r s s o f a r i d e n t i f i e d ; i . e . t h o s e s e l e c t i v e l y a c t i v a t e d by NMA, k a i n a t e and q u i s q u a l a t e ( W a t k i n s and E v a n s , 1 9 8 1 ; M c L e n n a n , 1 9 8 1 ) . I t has h o w e v e r been s u g g e s t e d f r o m t h e r e l a t i v e s e n s i t i v i t i e s o f L - g l u t a m a t e and L - a s p a r t a t e t o 124 b l o c k a d e by a r a n g e o f a n t a g o n i s t s t h a t t h e s e a m i n o a c i d s p r e f e r e n t i a l l y i n t e r a c t i n t h e s p i n a l c o r d w i t h t h e q u i s -q u a l a t e and NMA r e c e p t o r s r e s p e c t i v e l y ( W a t k i n s and E v a n s , 1 9 8 1 ) . S i n c e a s i m i l a r p a t t e r n o f a n t a g o n i s m i s s e e n i n t h e h i p p o c a m p u s , t h e p r e s e n t d a t a , t o g e t h e r w i t h o t h e r e l e c t r o -p h y s i o l o g i c a l and n e u r o c h e m i c a l e v i d e n c e ( s e e I n t r o d u c t i o n ) , f a v o r L - g l u t a m a t e as t h e t r a n s m i t t e r i n t h e SC p a t h w a y . By t h e same r e a s o n i n g , L - a s p a r t a t e c o u l d be m o r e i n v o l v e d i n s y n a p t i c p l a s t i c i t y by a p r e d o m i n a n t a c t i o n on t h e NMA r e c e p t o r . The p o s s i b i l i t y , h o w e v e r , t h a t o t h e r a c i d i c amino a c i d s o r r e l a t e d c o m p o u n d s may be t h e n a t u r a l l i g a n d s f o r t h e s e r e c e p t o r s c a n n o t be e x c l u d e d ( S t o n e and P e r k i n s , 1981; W a t k i n s and E v a n s , 1 9 8 1 ) . I n c o n c l u s i o n , t h e p r e s e n t s t u d y has shown t h a t t h e NMA r e c e p t o r p l a y s no r o l e i n t h e m e d i a t i o n o f s y n a p t i c e x c i t a t i o n b u t may be i n v o l v e d i n t h e g e n e r a t i o n o f LTP i n t h e SC p r o j e c t i o n t o t h e C A l r e g i o n o f t h e r a t h i p p o c a m p u s . However, t h e d e m o n s t r a t i o n t h a t DGG a f f e c t s t h i s p a t h w a y when a p p l i e d i n s u f f i c i e n t a m o u n t s t o a n t a g o n i z e t h e k a i n a t e a n d q u i s -q u a l a t e r e c e p t o r s , w h i l e h a v i n g no d e t e c t a b l e e f f e c t on p r e s y n a p t i c o r p o s t s y n a p t i c e x c i t a b i l i t y , p r o v i d e s s t r o n g e v i d e n c e t h a t a n e x c i t a t o r y a m i n o a c i d i s u t i l i z e d a s t h e n e u r o t r a n s m i t t e r i n t h i s p a t h w a y . 125 CHAPTER 5 EFFECTS OF K AINIC ACID ON SYNAPTIC PROCESSES IN THE HIPPOCAMPAL S L I C E I n t r o d u c t i o n I n i t i a l l y t h e i n t e r e s t i n k a i n i c a c i d , an e x t r a c t o f t h e seaweed D i g e n e a s i m p l e x , was g e n e r a t e d by i t s c l i n i c a l a p p l i c a t i o n i n J a p a n i n t h e k i l l i n g o f A s c a r i s ; t h e l i t e r a l t r a n s l a t i o n o f i t s J a p a n e s e name i s " m o n s t e r o f t h e s e a " . More r e c e n t l y k a i n i c a c i d h a s r e c e i v e d a t t e n t i o n b e c a u s e o f i t s e x c i t a t o r y a n d n e u r o t o x i c p r o p e r t i e s i n t h e m a m m a l i a n ) c e n t r a l n e r v o u s s y s t e m . The e x c i t a t o r y a c t i o n o f k a i n a t e was f i r s t d e m o n s t r a t e d by S h i n o z a k i a nd K o n i s h i (1970) on r a t c o r t i c a l n e u r o n s and i t s p o t e n c y as an e x c i t a n t was s u b s e q u e n t l y shown by o t h e r s t o be 10-200 f o l d g r e a t e r t h a n t h a t o f L - g l u t a m a t e ( B i s c o e et. a l . , 1 9 7 6 ; de M o n t i g n y a n d T a r d i f , 1 9 8 1 ) t o w h i c h i t i s s t r u c t u r a l l y r e l a t e d ( J o h n s t o n e_t a _ l . , 1 9 7 4 ) . An i n t r i g u i n g p r o p e r t y o f k a i n a t e b o t h i n t h e v e r t e b r a t e CNS ( S h i n o z a k i and K o n i s h i , 1970) and t h e i n s e c t n e u r o m u s c u l a r j u n c t i o n (Daoud a n d U s h e r w o o d , 1 9 7 5 ) i s t h e f a c i l i t a t i o n o f r e s p o n s e s t o L - g l u t a m a t e . None o f t h e o t h e r a c i d i c a m i n o a c i d s t e s t e d p r o d u c e d t h i s e f f e c t a n d t h e u n d e r l y i n g m e c h a n i s m r e m a i n s unknown. 126 S t u d i e s o f [ H ] - k a i n i c a c i d b i n d i n g have r e v e a l e d t h e e x i s t e n c e o f a l o w a f f i n i t y ( K D=50yxM) s i t e t h r o u g h o u t t h e r a t b r a i n and a s e c o n d h i g h a f f i n i t y (K D=10y*,M) s i t e o c c u r r i n g p r e d o m i n a n t l y i n f o r e b r a i n r e g i o n s ( L o n d o n and C o y l e , 1 9 7 9 a ; B e r g e r a n d B e n - A r i , 1 9 8 3 ) . C h anges o f b i n d i n g c h a r a c t e r i s t i c s a f t e r l e s i o n s o f a f f e r e n t p a t h w a y s i n d i c a t e a p r e - and p o s t s y n a p t i c l o c a t i o n o f k a i n a t e r e c e p t o r s ( L o n d o n and C o y l e , 1 9 7 9 a ; F o s t e r e_t a _ l . , 1 9 8 1 ) ; t h e e x i s t e n c e o f e x t r a s y n a p t i c r e c e p t o r s f o r k a i n a t e i s a l s o l i k e l y ( D a v i e s e t a l . , 1 9 7 9 ) . The r e s u l t s b o t h o f b i n d i n g ( L o n d o n a n d C o y l e , 1 9 7 9 b ) a n d e l e c t r o p h y s i o l o g i c a l ( J o h n s t o n e_t a l . , 1974) i n v e s t i g a t i o n s h ave shown t h a t t h e i s o p r o p y l e n e s i d e c h a i n as w e l l as t h e u s u a l t h r e e c h a r g e d g r o u p s ( c f . I n t r o d u c t i o n - C h a p t e r 4) a r e r e q u i r e d f o r t h e i n t e r a c t i o n o f k a i n a t e w i t h i t s r e c e p t o r . F u r t h e r m o r e k a i n a t e b i n d i n g s i t e s a r e d i s t i n c t f r o m t h o s e f o r L - g l u t a m a t e o r L - a s p a r t a t e ( B i z i e r e e t a _ l . , 1 9 8 0 ) , an o b s e r v a t i o n w h i c h i s c o n s i s t e n t w i t h t h e c u r r e n t c o n c e p t , d e r i v e d f r o m p h a r m a c o l o g i c a l a n a l y s e s ( M c L e n n a n , 1 9 8 1 ; W a t k i n s , 1 9 8 1 a , b ) , t h a t L - g l u t a m a t e and L - a s p a r t a t e behave a s m i x e d a g o n i s t s a t q u i s q u a l a t e and NMA r e c e p t o r s ( c f . C h a p t e r 4 ) . T o g e t h e r t h e s e d a t a e m p h a s i z e t h e u n i q u e a s p e c t s o f t h e k a i n i c a c i d r e c e p t o r . However i t s r o l e i n n e u r o n a l f u n c t i o n o r d y s f u n c t i o n and i n d e e d t h e n a t u r e o f t h e e n d o g e n o u s l i g a n d a r e q u e s t i o n s w h i c h r e m a i n u n a n s w e r e d . K a i n i c a c i d i s a l s o p o w e r f u l l y n e u r o t o x i c ( O l n e y e t a l . , 1 9 7 4 ) , an e f f e c t w h i c h i s e x e r t e d p r i m a r i l y a g a i n s t n e u r o n a l d e n d r i t e s and p e r i k a r y a ( O l n e y et. a l . , 1 9 7 9 ) ; a x o n s 127 o f p a s s a g e and t e r m i n a l s a r e n o r m a l l y s p a r e d s a v e when l a r g e d o s e s o f t h e c o m p o u n d a r e a d m i n i s t e r e d . O t h e r e x c i t a t o r y a m i n o a c i d s c a u s e s i m i l a r n e u r o n a l l e s i o n s a t t h e s i t e o f t h e i r i n j e c t i o n i n t o t h e b r a i n , t h e n e u r o t o x i c p o t e n c y o f e a c h c o m p o u n d b e i n g i n p a r a l l e l w i t h i t s e f f e c t i v e n e s s a s a n e x c i t a n t ( C o y l e e_t a _ l . , 1978; S c h w a r c z e_t a l . , 1978; N a d l e r e_t a l . , 1 9 8 1 ) . B a s e d on t h e s e o b s e r v a t i o n s , t h e " e x c i t o t o x i c " h y p o t h e s i s , p r o p o s e d o r i g i n a l l y by O l n e y e t ajL. ( 1 9 7 1 ) and s u b s e q u e n t l y s u p p o r t e d by e l e c t r o p h y s i o l o g i c a l and b i n d i n g s t u d i e s , s u g g e s t s t h a t s y n a p t i c r e c e p t o r s l o c a t e d o n t h e d e n d r i t e s and s o m a t a m e d i a t e b o t h t h e t o x i c and e x c i t a t o r y a c t i o n s o f a c i d i c a m ino a c i d s . R a t h e r t h a n t h e d e p o l a r i z a t i o n p e r s e i t i s l i k e l y t h a t t h e i n c r e a s e d membrane c o n d u c t a n c e p r o d u c e s a c h a n g e o f i n t r a c e l l u l a r c o n c e n t r a t i o n s o f i o n s w h i c h l e a d s by unknown p r o c e s s e s t o c e l l d e a t h . B e c a u s e k a i n i c a c i d was amongst t h e most t o x i c o f t h e e x c i t a t o r y a m ino a c i d s and s i n c e i n t r a c e r e b r a l i n j e c t i o n s (1-5 nmole i n ^ a . 1 1) c o u l d p r o d u c e a f o c a l n e u r o n a l l e s i o n w h i l e s p a r i n g a x o n s o f p a s s a g e i t b e c a m e w i d e l y u s e d a n e u r o -b i o l o g i c a l t o o l f o r t h e i n v e s t i g a t i o n o f s t r u c t u r e - f u n c t i o n c o r r e l a t i o n s i n t h e c e n t r a l n e r v o u s s y s t e m and f o r p r o d u c i n g m o d e l s o f n e u r o d e g e n e r a t i v e d i s e a s e s ( c f . M c G e e r e_t a l . , 1 9 7 8 b ) . O n l y some y e a r s l a t e r was i t r e c o g n i z e d t h a t t h e u s e f u l n e s s o f k a i n a t e i n t h i s r e g a r d was c o m p r o m i s e d by an a d d i t i o n a l a c t i o n t o d e s t r o y n e u r o n s r e m o t e f r o m t h e i n j e c t i o n s i t e ( W u e r t h e l e e_t al. , 1 9 7 8 ) . The m e c h a n i s m s u n d e r l y i n g t h e s o - c a l l e d l o c a l and d i s t a n t l e s i o n s a r e p r o d u c e d by mechan-128 i s t i c a l l y d i s t i n c t p r o c e s s e s . The i n v o l v e m e n t o f i n d i r e c t a c t i o n s i n t h e l o c a l d e s t r u c t i o n o f n e u r o n s by k a i n a t e was s u g g e s t e d f i r s t f r o m e x p e r i m e n t s i n t h e s t r i a t u m . T h u s , McGeer e_t a l . ( 1 9 7 8 a ) and B i z i e r e and C o y l e ( 1 9 7 8) d e m o n s t r a t e d t h a t t h e p r i o r d e s t r u c -t i o n o f t h e c o r t i c o s t r i a t a l p r o j e c t i o n , w h i c h h a s been s u g g e s t e d t o u s e L - g l u t a m a t e as i t s n e u r o t r a n s m i t t e r , r e d u c e d t h e n e u r o t o x i c i t y o f k a i n i c a c i d by a t l e a s t two o r d e r s o f m a g n i t u d e . S i n c e t h e p r o p h y l a x i s a f f o r d e d by t h i s d e a f f e r -e n a t i o n was n o t a t t r i b u t a b l e t o a change o f t h e p o s t s y n a p t i c e x c i t a b i l i t y ( M c L e n n a n , 1980) t h e i n v o l v e m e n t o f p r e s y n a p t i c f a c t o r s was i n d i c a t e d . The r e q u i r e m e n t f o r i n t a c t c o r t i c a l a f f e r e n t s i n t h e e x p r e s s i o n o f k a i n a t e n e u r o t o x i c i t y was c o n f i r m e d i n i_n v i t r o s t u d i e s w h e r e c o r t i c a l a n d s t r i a t a l c e l l s w e r e c o - c u l t u r e d ( W h e t s e l l et a l M 1979; P a n n u l a , 1 9 8 0 ) . M c G e e r and McGeer (1978) o r i g i n a l l y p r o p o s e d a c o - o p e r a t i v e i n t e r a c t i o n b e t w e e n k a i n a t e and t r a n s m i t t e r L - g l u t a m a t e t o c a u s e n e u r o n a l d e s t r u c t i o n s , b u t r e c e n t e v i d e n c e a r g u e s a g a i n s t t h i s h y p o t h e s i s ( S c h w a r c z a n d K o h l e r , 1 9 8 0 ) . F o r e x a m p l e , w h i l e i t i s t r u e t h a t k a i n a t e n e u r o t o x i c i t y i n t h e d e a f f e r e n t e d s t r i a t u m i s r e s t o r e d t o some e x t e n t b y c o -i n j e c t i o n o f L - g l u t a m a t e , a s i m i l a r a c t i o n was p r o d u c e d by GABA ( B i z i e r e and C o y l e , 1 9 7 9 ) . I n a d d i t i o n a c h e m i c a l l e s i o n o f t h e s e r o t o n e r g i c i n p u t a l s o i m p a r t s p r o t e c t i o n a g a i n s t k a i n a t e i n t h e s t r i a t u m ( B e r g e r et a _ l . , 1 9 8 2 ) . I n t h e h i p p o c a m p a l f o r m a t i o n p r i o r a b l a t i o n o f t h e r e p u t e d l y g l u t a m a t e r g i c p e r f o r a n t p a t h w a y b l o c k e d t h e d e s t r u c t i o n o f 129 d e n t a t e g r a n u l e c e l l s by l o c a l l y i n j e c t e d k a i n a t e b u t t h e same i m m u n i t y was a l s o c o n f e r r e d by r e m o v a l o f t h e c h o l i n e r g i c s e p t o h i p p o c a m p a l p r o j e c t i o n ( N a d l e r and C u t h b e r t s o n , 1 9 8 0 ) . F u r t h e r m o r e , a l t h o u g h i t a p p e a r s t o be an i n p u t r e l e a s i n g L - g l u t a m a t e a n d / o r L - a s p a r t a t e , a p r i o r l e s i o n o f t h e c o m m i s s u r a l p a t h w a y d i d n o t a t t e n t u a t e k a i n a t e ' s l e t h a l a c t i o n s . I n t h e c o c h l e a r n u c l e u s k a i n a t e t o x i c i t y p e r s i s t s e v e n a f t e r d e s t r u c t i o n o f t h e m a j o r and p o s s i b l y o n l y e x c i t a t o r y a f f e r e n t p a t h w a y w h i c h o r i g i n a t e s i n t h e c o c h l e a and p r o b a b l y u s e s L - g l u t a m a t e o r L - a s p a r t a t e a s i t s t r a n s -m i t t e r ( B i r d and G u l l e y , 1 9 7 9 ) . A b o l i t i o n o f i m p u l s e t r a n s m i s s i o n does n o t a p p e a r i n m o s t c a s e s t o be t h e b a s i s f o r t h e p r o t e c t i o n p r o v i d e d by s p e c i f i c d e a f f e r e n t a t i o n s . T h u s w h i l e a s e p t o h i p p o c a m p a l l e s i o n i m m e d i a t e l y p r o t e c t s d e n t a t e g r a n u l e c e l l s f r o m l o c a l l y i n j e c t e d k a i n a t e w i t h o t h e r i n p u t s i n t h e h i p p o c a m p u s ( N a d l e r and C u t h b e r t s o n , 1 9 8 0 ) , o r i n t h e s t r i a t u m (McGeer and M c G e e r , 1978) and o p t i c t e c t u m ( S t r e i t e_t aJL. , 1980) t h e o n s e t o f an i m m u n i t y t o k a i n a t e - m e d i a t e d n e u r o d e g e n e r a t i o n was s l o w and c o r r e l a t e d w i t h t h e t i m e r e q u i r e d f o r f i b r e a n d t e r m i n a l d e g e n e r a t i o n . From t h e l a t t e r o b s e r v a t i o n i t may be i n f e r r e d t h a t a n i n t e r a c t i o n w i t h a r e c e p t o r l o c a t e d o n t e r m i n a l s ( F e r k a n y et_ a _ l . , 1982; C o l l i n s e_t a l . , 1983) o r by an i n d i r e c t d e p o l a r i z a t i o n ( E v a n s , 1980) k a i n a t e r e c r u i t s an u n i d e n t i f i e d p r e s y n a p t i c f a c t o r ( s ) n e c e s s a r y f o r t h e e x p r e s s i o n o f i t s l e t h a l p o s t s y n a p t i c e f f e c t s . A l t h o u g h o t h e r a c i d i c a m i n o a c i d s a l s o d e s t r o y n e u r o n s 130 when t h e y a r e i n j e c t e d i n t r a c e r e b r a l l y d i f f e r e n c e s i n t h e m e c h a n i s m o f a c t i o n o f t h e s e c o m p o u n d s a n d k a i n a t e seem l i k e l y . I n t h e h i p p o c a m p u s q u i s q u a l a t e , N - m e t h y l a s p a r t a t e (NMA) a n d i b o t e n a t e w h i c h i n t e r a c t w i t h r e c e p t o r s 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 f r o m t h o s e m e d i a t i n g k a i n a t e e x c i t a t i o n s ( s e e I n t r o d u c t i o n o f C h a p t e r 4) e x h i b i t s i m i l a r p o t e n c i e s as n e u r o t o x i n s upon i n j e c t i o n i n t o e a c h h i p p o c a m p a l r e g i o n w h e r e a s a d i f f e r e n t i a l s e n s i t i v i t y t o k a i n a t e e x i s t s w i t h CA3 and d e n t a t e n e u r o n s b e i n g t h e most and l e a s t s e n s i t i v e r e s p e c t i v e l y ( N a d l e r e_t a _ l . , 1978 ) . I n c o n t r a s t t o t h e s i t u a t i o n w i t h k a i n a t e , i b o t e n a t e n e u r o t o x i c i t y i n t h e h i p p o c a m p u s i s n o t a t t e n t u a t e d by a p r i o r l e s i o n o f e i t h e r t h e s e p t o h i p p o c a m p a l o r p e r f o r a n t p a t h p r o j e c t i o n s ( K o h l e r e t a l . , 1979; S c h w a r c z and K o h l e r , 1 9 8 0 ) . The f a c t o r s i n v o l v e d i n t h e l o c a l t o x i c i t y o f k a i n a t e a r e c o m p l e x . Thus on one hand t h e l o c a l n e u r o n a l d e a t h o c c u r s i n d e p e n d e n t l y o f an o b v i o u s p r e s y n a p t i c c o n t r i b u t i o n ( e . g . c o c h l e a r n e u r o n s ) and i n t h i s c o n n e c t i o n t h e r e i s a s i m i l a r i t y w i t h t h e n e u r o t o x i c i t y o f o t h e r e x c i t a t o r y amino a c i d s and t h e e x c i t o t o x i c t h e o r y w o u l d seem t o a p p l y . On t h e o t h e r hand t h e r e q u i r e m e n t i n o t h e r b r a i n r e g i o n s ( e . g . s t r i a t u m ) f o r s p e c i f i c a f f e r e n t s i n d i c a t e s t h a t t h e d e s t r u c t i o n o f n e u r o n s by k a i n a t e i s n o t s i m p l y r e l a t e d t o i t s e x c i t a t o r y a c t i o n s b u t r a t h e r a u n i q u e c o - o p e r a t i v e i n t e r a c t i o n w i t h p r e s y n a p t i c e l e m e n t s i s s u g g e s t e d . T h e d i r e c t i n j e c t i o n o f k a i n a t e i n t o v a r i o u s f o r e b r a i n s t r u c t u r e s s u c h a s t h e a m y g d a l a , s t r i a t u m , t h a l a m u s o r 131 h i p p o c a m p u s p r o d u c e s , i n a d d i t i o n t o t h e l o c a l l e s i o n s , t h e d e s t r u c t i o n o f n e u r o n s r e m o t e f r o m t h e i n j e c t i o n s i t e , p a r t i c u l a r l y w i t h i n t h e l i m b i c s y s t e m , w h i c h h a s a h i g h d e n s i t y o f k a i n i c a c i d r e c e p t o r s ( F o s t e r et. a l . , 1 9 8 1 ; B e r g e r and Ben A r i , 1 9 8 3 ) . F o r e x a m p l e i n t r a h i p p o c a m p a l i n j e c t i o n o f t h e n e u r o t o x i n p r o d u c e s l e s i o n s l o c a l l y and i n t h e a m y g d a l a , p y r i f o r m c o r t e x , c l a u s t r u m - i n s u l a and some t h a l a m i c n u c l e i ( W u e r t h e l e e t a l . , 1978; R u t h , 1 9 8 2 ) . P a r e n t e r a l and i n t r a v e n t r i c u l a r ( 1 - 5 nmol) a d m i n i s t r a t i o n s p r o d u c e a s i m i l a r p a t t e r n o f l i m b i c l e s i o n s , t h e CA3 r e g i o n b e i n g among t h e most v u l n e r a b l e ( N a d l e r et. a _ l . , 1978 ). N o t i n g a s i m i l a r i t y i n t h e d i s t r i b u t i o n o f l e s i o n s a s s o c i a t e d w i t h t e m p o r a l l o b e e p i l e p s y i n man, N a d l e r and h i s c o l l e a g u e s (1978) s u g g e s t e d t h a t t h e d i s t a n t l e s i o n s w e r e a c o n s e q u e n c e o f t h e e l e c t r o e n c e p h a l o -g r a p h i c s e i z u r e s p r o d u c e d by k a i n a t e i n j e c t i o n s . T hey p r o p o s e d t h a t i n t r i n s i c p r o c e s s e s t r i g g e r e d by t h e r e p e a t e d h i g h - f r e q u e n c y d i s c h a r g e o f t h e l i m b i c n e u r o n s d u r i n g s e i z u r e s r a t h e r t h a n t h e d i f f u s i o n o f k a i n a t e f r o m t h e i n j e c t i o n s i t e ( b u t s e e b e l o w ) c a u s e d t h e d i s t a n t damage. T h i s h y p o t h e s i s h a s b e e n s u p p o r t e d by e x p e r i m e n t a l o b s e r v a t i o n s t h a t : ( 1 ) t r e a t m e n t s r e d u c i n g ( e . g . B e n - A r i et. a _ l . , 1979) o r e n h a n c i n g ( F u l l e r and O l n e y , 1979; F u l l e r e t a l . , 1982) s e i z u r e a c t i v i t y a t t e n u a t e and augment r e s p e c t i v e l y t h e d i s t a n t damage; (2) a c u t e t r a n s e c t i o n o f e x c i t a t o r y a f f e r e n t s c a n r e d u c e o r a b o l i s h r e m o t e l e s i o n s i n d i c a t i n g a r e q u i r e m e n t f o r i m p u l s e p r o p a g a t i o n ( N a d l e r and C u t h b e r t s o n , 1 9 8 0 ) ; (3) o t h e r c o n v u l s a n t s s u c h a s a l l y l g l y c i n e ( M e l d r u m e_t a _ l . , 1974 ) , 132 b i c u c u l l i n e ( M e l d r u m and B r i e r l y , 1973) and d i p i p e r i d i n o e t h a n e ( O l n e y e_t a _ l . , 1 9 8 0 ) c a n e l i c i t f o c a l l e s i o n s t h r o u g h o u t l i m b i c s t r u c t u r e s ; and (4) h i g h f r e q u e n c y e l e c t r i c a l s t i m u l a -t i o n s o f t h e p e r f o r a n t p a t h w a y c a n d e s t r o y h i p p o c a m p a l CA3 p y r a m i d s ( S l o v i t e r a n d D a m i a n o , 1 9 8 1 a ) . The d i f f e r e n t i a l p r o t e c t i v e e f f e c t s o f d e a f f e r e n t a t i o n ( N a d l e r and C u t h b e r t s o n , 1980) a r e a l s o c o n s i s t e n t w i t h t h e i n v o l v e m e n t o f s e p a r a t e m e c h a n i s m s i n t h e d e v e l o p m e n t o f d i s t a n t and l o c a l n e u r o n a l d e g e n e r a t i o n s . M o r e r e c e n t e x p e r i m e n t a l e v i d e n c e i n d i c a t e s t h a t s e i z u r e a c t i v i t y a l o n e c a n n o t a d e q u a t e l y e x p l a i n t h e d i s t a n t damage. I n t h i s r e g a r d t h e a s s u m p t i o n t h a t k a i n a t e d o e s n o t r e a c h t h e r e m o t e s i t e s o f damage a f t e r d i r e c t i n j e c t i o n s and p a r t i c u l a r l y w i t h i n t r a v e n t r i c u l a r a d m i n i s t r a t i o n may be i n c o r r e c t s i n c e i t h a s b e e n s h o w n t h a t k a i n a t e d i f f u s e s r a p i d l y f r o m t h e s i t e o f i t s a p p l i c a t i o n and a c h i e v e s a w i d e d i s t r i b u t i o n t h r o u g h o u t t h e b r a i n ( S c h e r e r - S i n g l e r and M c G e e r , 1979; Z a c z e k et. a _ l . , 1980) a l b e i t i n c o n c e n t r a t i o n s t o o s m a l l t o p r o d u c e l o c a l l e s i o n s . I n t r a c e r e b r a l i n j e c t i o n s o f i b o t e n a t e , NMA and q u i s q u a l a t e w h i c h p r o d u c e e l e c t r o e n c e p h a l o -g r a p h i c s e i z u r e s a t l e a s t c o m p a r a b l e t o t h o s e o c c u r r i n g w i t h k a i n a t e i n d u c e o n l y l o c a l d e g e n e r a t i o n ( Z a c z e k a n d C o y l e , 1982; G u l d i n and M a r k o w i t s c h , 1982; A l d i n i o e t al., 1 9 8 3 ) ; and F r e n c h e t a l . ( 1 9 8 2 ) have d e m o n s t r a t e d an a p p a r e n t d i s s o c i a -t i o n b e t w e e n k a i n a t e ' s e p i l e p t o g e n i c and n e u r o t o x i c a c t i o n s . T h e r e f o r e , w h i l e s e i z u r e a c t i v i t y a p p e a r s t o be a p r e r e q u i s i t e t h e p r o d u c t i o n o f d i s t a n t l e s i o n s p r o b a b l y a l s o i n v o l v e s o t h e r 133 c h a n g e s w h i c h amongst t h e e x c i t a t o r y a m ino a c i d c o n v u l s a n t s a r e c a u s e d o n l y by k a i n a t e . T h e h i p p o c a m p u s h a s been s u g g e s t e d t o p l a y a p i v o t a l r o l e i n t h e o n s e t a n d p r o p o g a t i o n o f k a i n i c a c i d - i n d u c e d e p i l e p t i f o r m a c t i v i t y ( B e n - A r i e_t a _ l . , 1981) and h i p p o c a m p a l . p y r a m i d a l c e l l s , e s p e c i a l l y t h o s e i n t h e CA3-CA4 r e g i o n , a r e p a r t i c u l a r l y s e n s i t i v e t o t h e n e u r o t o x i c a c t i o n s o f k a i n a t e . I t has p r e v i o u s l y b e e n shown i n h i p p o c a m p a l s l i c e s t h a t k a i n i c a c i d c a u s e s e p i l e p t i f o r m d i s c h a r g e s o f CA3 c e l l s ( L o t h m a n et. a l . , 1981) and g r e a t l y p o t e n t i a t e s s y n a p t i c e x c i t a t i o n ( C o l l i n g r i d g e a n d M c L e n n a n , 1 9 8 1 ) . T h e r e f o r e i n t h e p r e s e n t s t u d y t h e m e c h a n i s m s o f t h e e p i l e p t o g e n i c and p o t e n t i a t i n g a c t i o n s o f k a i n a t e w e re i n v e s t i g a t e d . To d e t e r m i n e i f t h e enhancement o f s y n a p t i c e x c i t a t i o n m i g h t be r e l a t e d t o i t s d i s t a n t o r l o c a l n e u r o t o x i c i t y t h e e f f e c t s o f o t h e r a c i d i c a m i n o a c i d s on s y n a p t i c p r o c e s s e s were a l s o a s s e s s e d . M e t h ods I n t h i s s t u d y t r a n s v e r s e h i p p o c a m p a l s l i c e s were p r e p a r e d and m a i n t a i n e d as d e s c r i b e d p r e v i o u s l y ( C h a p t e r 2 ) . E a c h e x p e r i m e n t was p e r f o r m e d a t l e a s t 3 t i m e s u s i n g s l i c e s o b t a i n e d f r o m s e p a r a t e a n i m a l s . C o n v e n t i o n a l t e c h n i q u e s o u t l i n e d i n C h a p t e r 2 were u s e d t o s t i m u l a t e and r e c o r d e v o k e d r e s p o n s e s . F o r l o c a l a p p l i c a t i o n k a i n a t e was e j e c t e d f r o m a m u l t i b a r r e l e d e l e c t r o d e u s i n g s t a n d a r d i o n t o p h o r e t i c p r o -c e d u r e s . F o r e x p e r i m e n t s i n C A l and t h e d e n t a t e g y r u s a 50 mM s o l u t i o n i n 100 mM N a C l (pH 8.0) f i l l e d t h e b a r r e l s ; i n CA3 5 134 mM k a i n a t e i n 160 mM N a C l (pH 7.6) was u s e d . N a C l (1-4 M) was u s e d f o r c u r r e n t c o n t r o l s . W i t h b a t h a p p l i c a t i o n s o l u t i o n s o f -8 -4 k a i n a t e (10 -10 M) were f r e s h l y p r e p a r e d i n t h e ACSF. R e s u l t s E x t r a c e l l u l a r l y r e c o r d e d e f f e c t s o f k a i n a t e  on e v o k e d and s p o n t a n e o u s a c t i v i t y C A l When a p p l i e d by i o n t o p h o r e s i s (50-100 nA f o r 10-120 s e c ) e i t h e r t o t h e c e l l b o d i e s i n s t r a t u m p y r a m i d a l e o r t o t h e a p i c a l d e n d r i t e s 100-300 m d i s t a n t i n t h e s t r a t u m r a d i a t u m , k a i n a t e p r o d u c e d a r e d u c t i o n o f t h e S C - e v o k e d EPSP and c o n c u r r e n t l y a l a r g e and l o n g - l a s t i n g i n c r e a s e i n t h e a m p l i t u d e o f t h e o r t h o d r o m i c PS ( F i g . 5 . 1 A ) . P o t e n t i a t i o n o f t h e PS was u s u a l l y p r e c e d e d o r i n t e r r u p t e d by a p e r i o d o f d e p r e s s e d r e s p o n s i v e n e s s b o t h t o a n t i - and o r t h o d r o m i c a c t i v a t i o n . I n most s l i c e s t h e k a i n a t e - i n d u c e d i n c r e a s e o f s y n a p t i c e x c i t a t i o n l a s t e d 20 m i n o r l o n g e r and was r e p r o -d u c i b l e f o l l o w i n g r e c o v e r y . Enhancement o f s y n a p t i c e x c i t a -t i o n , t h e d e g r e e o f w h i c h v a r i e d b e t w e e n s l i c e s ( 5 0 - 3 0 0 % f o r a p r e - t r e a t m e n t PS a m p l i t u d e o f 2-4 mV) , was s o m e t i m e s c o r r e l a -t e d w i t h a d e c r e a s e o f t h e l a t e n c y o f o n s e t f o r t h e PS. A l t h o u g h i t i s a p p e a l i n g t o c o n s i d e r t h a t i t was i n t e r a c t i n g w i t h s y n a p t i c p r o c e s s e s i n t h e d e n d r i t e s t o p r o d u c e t h e s e e l e c t r o p h y s i o l o g i c a l c h a n g e s t h e s i m i l a r e f f e c t s o f k a i n a t e when a p p l i e d e i t h e r t o t h e d e n d r i t e s o r t h e c e l l body l a y e r i n d i c a t e d t h a t e x t r a - s y n a p t i c s i t e s o f a c t i o n m i g h t 135 Koinole 50 nA I Koinote 50 nA • o > E « •to) n -y (b) n (c) n td) ( e ) ' (I) n 10 msec Koinole IO"'M 5 ^ 15mm F i g . 5.1 A. A c t i o n s o f k a i n a t e a d m i n i s t e r e d by i o n t o p h o r e s i s i n t o t h e d e n d r i t i c r e g i o n s of C A l on S C - e v o k e d r e s p o n s e s . The g r a p h s show the a m p l i t u d e o f t h e PS measured i n s t r a t u m p y r a m i d a l e ( u p p e r p l o t ) and o f t h e f i e l d EPSP measured a t t h e s i t e o f i o n t o p h o r e s i s i n s t r a t u m r a d i a t u m . K a i n a t e was a d m i n i s t e r e d f o r t h e t i m e i n d i c a t e d by t h e b a r s . F o l l o w i n g an i n i t i a l d e p r e s s i o n t h e PS was i n c r e a s e d and t h e r e was an a s s o c i a t e d r e d u c t i o n of the EPSP. B. The e f f e c t s o f p e r f u s e d k a i n a t e i n t h e C A l r e g i o n . The PS was p o t e n t i a t e d i n t h i s s l i c e f r o m w h i c h t h e CA3 r e g i o n h a d b e e n s u r g i c a l l y removed. The n o r m a l medium was e x c h a n g e d f o r one c o n t a i n i n g k a i n a t e f o r t h e p e r i o d i n d i c a t e d by t h e b a r ; t h e d e l a y e d o n s e t was due t o t h e l a g t i m e i n t h e p e r f u s i o n s y s t e m . The f i l m e d r e c o r d s (5 c o n s e c u t i v e s u p e r i m p o s e d sweeps, n e g a t i v i t y upwards) were o b t a i n e d a t t h e t i m e s i n d i c a t e d ( a - f ) . D u r i n g t h e two 15 min b r e a k s i n t h e g r a p h measurements were not made. 1 3 6 e x i s t . T h i s p o s s i b i l i t y was a d d r e s s e d i n t h e f o l l o w i n g manner. K a i n a t e was a p p l i e d i o n t o p h o r e t i c a l l y w h i l e f i e l d r e s p o n s e s i n s t r a t u m p y r a m i d a l e o f C A l were r e c o r d e d s i m u -l t a n e o u s l y f r o m t h e c e n t r a l b a r r e l o f t h e i o n t o p h o r e t i c e l e c t r o d e a n d a s e c o n d s i n g l e - b a r r e l e d e l e c t r o d e p o s i t i o n e d 200yx.m a w a y . S i n c e b o t h t h e d e p r e s s i n g a n d p o t e n t i a t i n g a c t i o n s were o b s e r v e d a t e a c h r e c o r d i n g s i t e i t was c o n c l u d e d t h a t t h e e f f e c t s o f k a i n a t e c o u l d e x t e n d a t l e a s t 200ym.m f r o m t h e l o c u s o f i t s a p p l i c a t i o n . F o r t h i s r e a s o n i t c o u l d n o t be d e t e r m i n e d i f t h e p r i m a r y s i t e o f a c t i o n f o r k a i n a t e o c c u r r e d a t t h e d e n d r i t i c o r s o m a t i c l e v e l . A n e n h a n c e m e n t o f S C - e v o k e d e x c i t a t i o n o f C A l n e u r o n s was a l s o p r o d u c e d w i t h b a t h - a p p l i e d k a i n a t e . I n p r e l i m i n a r y e x p e r i m e n t s b r i e f e x p o s u r e s (1-2 m i n , 1-2 m l ) t o p e r f u s a t e c o n t a i n i n g 10 ^-10 ^ M k a i n a t e were u s e d t o m i m i c t h e d u r a t i o n o f t h e i o n t o p h o r e t i c a p p l i c a t i o n s o f t h e d r u g ; i n l a t e r — 7 — 6 s t u d i e s 10-20 ml o f 10 -10 M s o l u t i o n s were p e r f u s e d . W i t h t h e b r i e f a d m i n i s t r a t i o n s 10 ^ M k a i n a t e p r o d u c e d p o t e n -t i a t i o n s (up t o 100% f o r a p r e - t r e a t m e n t r e s p o n s e o f 2-4 mV) -5 w h i c h l a s t e d f o r 10-20 m i n ; i n 10 M s o l u t i o n s i n c r e a s e s o f PS a m p l i t u d e s were l a r g e r (50-1300%) and l o n g e r - l a s t i n g (up t o 1.5 h) ( F i g . 5 . I B ) . On o c c a s i o n w i t h t h e s h o r t d u r a t i o n t r e a t m e n t s w i t h 10 5 M k a i n a t e ( F i g . 5.11A) b u t e s p e c i a l l y — 7 — 6 w i t h t h e l o n g e r e x p o s u r e s t o 10 -10 M k a i n a t e , p o t e n t i a t i o n o f t h e i n i t i a l PS a n d r e c r u i t m e n t o f a d d i t i o n a l P S s was o b s e r v e d . A r e d u c t i o n o f S C - e v o k e d P S s was p r o d u c e d when - •} -4 10-20 m l o f 10 -10 M s o l u t i o n s o f t h e a m m o a c i d w e r e p e r f u s e d and t h i s p e r i o d o f d e p r e s s e d c e l l f i r i n g was f o l l o w e d 137 by a l o n g - l a s t i n g p o t e n t i a t i o n o f s y n a p t i c e x c i t a t i o n ( n o t s h o w n ) . S i m u l t a n e o u s r e c o r d i n g o f t h e d e n d r i t i c and s o m a t i c p o t e n t i a l s r e v e a l e d t h a t p o t e n t i a t i o n o f t h e o r t h o d r o m i c a l l y --5 e v o k e d PS by 1-2 m l p e r f u s i o n s o f 10 M k a i n a t e w e r e a s s o c i a t e d w i t h a r e v e r s i b l e r e d u c t i o n o f t h e EPSP a m p l i t u d e b u t no change o f t h e p r e s y n a p t i c f i b r e v o l l e y was d e t e c t a b l e ; t h e a m p l i t u d e o f A D - e v o k e d PSs r e m a i n e d u n c h a n g e d t h r o u g h o u t t h e e x p e r i m e n t ( F i g . 5 . 2 ) . T h i s same e f f e c t o f k a i n a t e i s shown i n t h e i n p u t / o u t p u t c u r v e s o f F i g . 5.3. A l t h o u g h i n some s l i c e s t h e r e were c l e a r c o n t r i b u t i o n s t o t h e d e n d r i t i c f i e l d p o t e n t i a l o f a r e v e r s e d PS w h i c h c a u s e d an a r t e f a c t u a l r e d u c t i o n o f t h e peak EPSP a m p l i t u d e , i n t h e m a j o r i t y o f t h e s l i c e s t h i s d i d n o t a p p e a r t o e x p l a i n t h e e f f e c t . F u r t h e r m o r e , a d e c r e a s e o f t h e EPSP was a l s o m e a s u r e d a t i n t e n s i t i e s o f s t i m u l a t i o n w h i c h c o n t i n u e d t o be s u b -t h r e s h o l d f o r PS g e n e r a t i o n ( F i g . 5.3C:4 a n d 5 V ) ; a n d a d e c r e a s e o f t h e s l o p e o f t h e EPSP and t h e r e v e r s e d EPSP was e v i d e n t i n d e n d r i t i c and s o m a t i c f i e l d p o t e n t i a l s r e s p e c t i v e l y ( F i g . 5 . 4 ) . An a d d i t i o n a l o b s e r v a t i o n i n C A l i n s l i c e s p e r f u s e d w i t h k a i n a t e was t h e t r a n s i e n t a p p e a r a n c e o f s p o n t a n e o u s p o p u l a t i o n b u r s t s ( S P B ) w h i c h r e s e m b l e d t h o s e r e p o r t e d a s o c c u r r i n g i n t h e p e n i c i l l i n - t r e a t e d C A l r e g i o n ( S c h w a r t z k r o i n and P r i n c e , 1 9 7 8 ) . I t i s l i k e l y t h a t t h e s e SPBs m i r r o r e d t h e s p o n t a n e o u s e p i l e p t i f o r m d i s c h a r g e o f CA3 n e u r o n s ( L o t h m a n e t a l . , 1 9 8 1 ; and s e e b e l o w ) and t h e c o n s e q u e n t t r a n s m i s s i o n o f 138 > 4 e. o J 15 min 25 min I 1 i 1 > E "•'-'.•W.v' Koinote 10 M I m i n F i g . 5.2 Graphs of evoked responses in CAl from above downwards plot the baseline to peak amplitude of the orth- and antidromic PSs recorded in the stratum pyramidale and, from a second electrode positioned in the stratum radiatum, the f i e l d EPSP and the presynaptic f i b r e v o l l e y . The antidromic PS and the f i b r e volley were unchanged by the perfusion of kainate (10~ 5 M, 1 ml) whereas a 4-fold increase of the SC-evoked PS was observed at the peak of the e f f e c t and the EPSP was depressed. 139 Population Spike E P S P Orlhodromc Antidromic A. Control (*) B Kainate 10" 5 M (°) Stimulus Intensity (V ) F i g . 5.3 I n t h i s e x p e r i m e n t o r t h o d r o m i c (S ) a n d a n t i d r o m i c ( S g ) r e s p o n s e s w e r e e v o k e d i n C A l a t v a r i o u s i n t e n s i t i e s o f s t i m u l a t i o n . T h r e e s u p e r i m p o s e d sweeps i n A i n d i c a t e t h e f i e l d EPSP a n d t h e o r t h o d r o m i c PS w h i c h w e r e r e c o r d e d s i m u l t a n e o u s l y f r o m s e p a r a t e e l e c t r o d e s ; s t i m u l i e v o k i n g a n t i d r o m i c r e s p o n s e s were a l t e r n a t e d w i t h o r t h o d r o m i c s t i m u l a t i o n s . T h e a m p l i t u d e s o f . c o n t r o l r e s p o n s e s (A) a t t h e s e and a d d i t i o n a l s t i m u l u s s t r e n g t h s a r e p l o t t e d below ( C ) . R e s p o n s e s e l i c i t e d 10-17 min a f t e r t h e p e r f u s i o n o f 1 ml o f 10 5 M k a i n a t e a r e i l l u s t r a t e d i n B and t h e i r maximum a m p l i t u d e s g r a p h e d i n C ( o ) . The EPSP was d e c r e a s e d a t a l l i n t e n s i t i e s o f s t i m u l a t i o n and t h i s r e d u c t i o n was not c o r r e l a t e d w i t h t h e a p p e a r a n c e of a p o s i t i v e - g o i n g ( r e v e r s e d ) PS i n t h e d e n d r i t i c p o t e n t i a l . A t h i g h e r s t i m u l u s s t r e n g t h s ( 7 , 8 V) t h e a m p l i t u d e o f t h e PS i n c r e a s e d and i t s l a t e n c y d e c r e a s e d as compared t o c o n t r o l r e s p o n s e s . A n t i d r o m i c and f i b r e v o l l e y a m p l i t u d e s were u n a f f e c t e d . 140 E P S P Orthodromic P S 6 v — pre-treatment kainate treated ( I O _ 6 M , I O m l ) 4 v + 2 m V 4 m s e c F i g . 5 . 4 S o l i d l i n e t r a c e s i l l u s t r a t e ( l e f t c o l u m n ) t h e p r e - t r e a t m e n t f i e l d EPSPs r e c o r d e d i n t h e s t r a t u m r a d i a t u m and e l i c i t e d w i t h v a r i o u s i n t e n s i t i e s o f SC s t i m u l a t i o n and ( r i g h t ) t h e f i e l d r e s p o n s e r e c o r d e d s i m u l t a n e o u s l y i n s t r a t u m p y r a m i d a l e . S u p e r i m p o s e d a r e t h e r e s p o n s e s ( d o t t e d l i n e s ) e v o k e d a t t h e e n d o f a 10 m l p e r f u s i o n o f 10 6 M k a i n a t e . N o t e t h e d e c r e a s e o f t h e s l o p e and t h e peak a m p l i t u d e o f t h e EPSP a t e a c h s t i m u l u s s t r e n g t h . The r e s p o n s e s r e c o r d e d a t t h e c e l l body l a y e r show a d e c l i n e o f t h e s l o p e o f t h e r e v e r s e d E P S P a n d an i n c r e a s e o f t h e PS a m p l i t u d e . P r e - a n d p o s t -t r e a t m e n t r e c o r d s a r e c o m p r i s e d o f 3 c o n s e c u t i v e s u p e r i m p o s e d s w e e p s , t h e peak n e g a t i v i t y o f t h e p o s t - t r e a t m e n t PS i s n o t shown. 141 i m p u l s e s i n t h e SC i n p u t t o C A l c e l l s s i n c e : (1) SPBs were n o t o b s e r v e d i n s l i c e s f r o m w h i c h t h e CA3 r e g i o n h ad b e e n removed u s i n g a h a n d - h e l d m i c r o k n i f e and (2) s p o n t a n e o u s b u r s t i n g was n e v e r p r o d u c e d w i t h i o n t o p h o r e t i c a p p l i c a t i o n s o f k a i n a t e . A s i d e f r o m t h i s s p o n t a n e o u s a c t i v i t y t h e a c t i o n s o f k a i n a t e on e v o k e d r e s p o n s e s i n i n t a c t s l i c e s were o t h e r w i s e i n d i s t i n g -u i s h a b l e f r o m t h o s e s l i c e s i n w h i c h t h e CA3 r e g i o n had been r e m o v e d . CA3 W i t h h i g h c o n c e n t r a t i o n s o f k a i n a t e (50 mM i n 100 mM N a C l ) f i l l i n g t h e p e r i p h e r a l b a r r e l s o f i o n t o p h o r e t - i c e l e c t r o d e s i t was p o s s i b l e t o p r o d u c e a l a r g e a n d l o n g - l a s t i n g i n c r e a s e o f s p o n t a n e o u s a c t i v i t y o f CA3a n e u r o n s s i m p l y by l o w e r i n g t h e t i p o f t h e i o n t o p h o r e t i c e l e c t r o d e i n t o t h e s t r a t u m p y r a m i d a l e . T h i s o b s e r v a t i o n was p r o b a b l y r e l a t e d t o t h e g r e a t e r s e n s i t i v i t y o f t h e s e c e l l s t o k a i n a t e ( c f . de M o n t i g n y and T a r d i f , 1981) s i n c e w i t h t h e same e l e c t r o d e s and u s i n g i d e n t i c a l b a c k i n g c u r r e n t s t h e s e e x c i t a t i o n s d i d n o t o c c u r i n e i t h e r t h e C A l r e g i o n o r t h e d e n t a t e g y r u s . T h e r e f o r e i n t h e e x p e r i m e n t s i n CA3 more d i l u t e s o l u t i o n s o f k a i n a t e (5 mM, pH 7.6 i n 160 mM N a C l ) were u s e d t o a l l o w more p r e c i s e c o n t r o l o v e r t h e l e a k a g e o f t h e amino a c i d f r o m t h e i o n t o p h o r e t i c e l e c t r o d e . K a i n a t e a p p l i e d i n o t o p h o r e t i c a l l y (5-20 nA, 10-60 s e c ) t o t h e s t r a t u m p y r a m i d a l e i n C A 3 a p r o d u c e d a l o n g - l a s t i n g ( 1 0 - 2 0 min) i n c r e a s e i n t h e a m p l i t u d e o f MF- ( F i g . 5. 5A and 142 F I M - e v o k e d ( F i g . 5 . 1 3 A i ) o r t h o d r o m i c P S s , an a c t i o n w h i c h was s o m e t i m e s p r e c e d e d by a p e r i o d o f d e p r e s s e d r e s p o n s i v e n e s s . -8 — 7 P e r f u s i o n o f 10-15 ml o f 10 -5x10 M k a i n a t e a l s o p o t e n -t i a t e d s y n a p t i c e x c i t a t i o n p r o d u c e d by MF ( F i g . 5.5B) and FIM s t i m u l i ( F i g . 5 . 6 A ) ; a d e c l i n e o f AD and o r t h o d r o m i c r e s p o n s e s — 6 — 5 was c a u s e d by 10 -10 M s o l u t i o n s . S i n c e t h e MF-evoked 1° PS was c o n s i s t e n t l y i n c r e a s e d w i t h d o s e s o f k a i n a t e p r o d u c i n g no change o f t h e a m p l i t u d e o f AD PSs ( F i g . 5.6B) t h e f o r m e r e f f e c t was a t t r i b u t e d t o t h e enhancement o f o r t h o d r o m i c e x c i t a t i o n m e d i a t e d by t h e MF i n p u t r a t h e r t h a n a change i n t h e AD component o f t h a t r e s p o n s e ( c f . C h a p t e r 2 ) . C o i n c i d e n t a l l y w i t h t h e o n s e t o f e v o k e d e p i l e p t i f o r m f i r i n g i n CA3 f o l l o w i n g b a t h a p p l i c a t i o n s o f k a i n a t e t h e r e was t h e a p p e a r a n c e o f SPBs ( F i g . 5 . 6 B i v , v i ) w h i c h w ere q u a l i t a -t i v e l y i n d i s t i n g u i s h a b l e f r o m t h o s e e l i c i t e d i n medium c o n t a i n i n g l o w [ C I ] q , i n c r e a s e d [ K + ] q ( s e e C h a p t e r 2) o r p e n i c i l l i n ( S c h w a r t z k r o i n and P r i n c e , 1 9 7 8 ) . I n i t i a l l y SPBs w e r e o b s e r v e d i n f r e q u e n t l y and t h e e x t r a c e l l u l a r p o t e n t i a l c o n s i s t e d o f o n l y a few PSs o f s m a l l a m p l i t u d e s . However, w i t h i n 10-15 m i n t h e mumber and s i z e o f PSs c o m p r i s i n g SPBs i n c r e a s e d and t h e s p o n t a n e o u s a c t i v i t y c o n t i n u e d t o a p p e a r a t 5-10 s e c i n t e r v a l s o v e r t h e n e x t 1-4 h. Though n o t r i g o u r -o u s l y a n a l y z e d , a s i m i l a r i t y b e t w e e n s p o n t a n e o u s and e v o k e d e p i l e p t i f o r m d i s c h a r g e s was o f t e n n o t e d ( e . g . F i g . 5 . 6 B i i i -v i ) . L a r g e ( u p t o 100 m s e c ) a n d v a r i a b l e s h i f t s o f t h e l a t e n c i e s o f e v o k e d r e s p o n s e s , p a r t i c u l a r l y w i t h low i n t e n -143 F i g . 5.5 A. ( i ) The p r e - t r e a t m e n t MF-evoked r e s p o n s e r e c o r d e d f r o m t h e c e n t r a l b a r r e l o f an i o n t o p h o r e t i c e l e c t r o d e p o s i t i o n e d i n CA3a. ( i i ) I o n t o p h o r e s i s o f k a i n a t e (5 nA f o r 10 s e c ) c a u s e d an immediate r e d u c t i o n b o t h of t h e 1° PS and t h e p o s i t i v e wave w h i c h f o l l o w e d . A k a i n a t e - r e s i s t a n t c o m p o n e n t o f t h e 1 ° r e s p o n s e i s e v i d e n t . 15 m i n l a t e r a p o t e n t a t i o n o f t h e 1° PS and t h e f i r i n g o f a s e c o n d PS was o b s e r v e d ( i i i ) . B. Changes o f t h e s o m a t i c f i e l d p o t e n t i a l e l i c i t e d by MF s t i m u l i 10, 25 a n d 65 m i n ( i i , i i i a n d i v r e s p e c t i v e l y ) a f t e r the b e g i n n i n g o f a 10 ml p e r f u s i o n w i t h — 8 medium c o n t a i n i n g 10 M k a i n a t e . Note t h e p o t e n t i a t i o n of t h e i n i t i a l PS and t h e d e v e l o p m e n t o f a d d i t i o n a l P S s . A d e p r e s s i o n o f e v o k e d a c t i v i t y was n o t o b s e r v e d . In A and B r e c o r d s a r e c o m p r i s e d o f 2-3 c o n s e c u t i v e s u p e r i m p o s e d t r a c e s . 144 F i g . 5.6 The e f f e c t s o f k a i n a t e on s p o n t a n e o u s and e v o k e d a c t i v i t y i n CA3a. A. F o l l o w i n g b a t h a p p l i c a t i o n o f k a i n a t e o r t h o d r o m i c P Ss e v o k e d by FIM s t i m u l i w e r e i n c r e a s e d and a d d i t i o n a l PSs were r e c r u i t e d ( i i ) . W i t h t h e o n s e t o f SPBs ( e . g . B [ i v ] ) t h e r e w e re l a r g e s h i f t s i n t h e l a t e n c y o f e v o k e d b u r s t s . Shown i n ( i i i ) a r e 3 c o n s e c u t i v e r e s p o n s e s t o FIM s t i m u l i d e l i v e r e d 4 s e c a f t e r SPB d i s c h a r g e s ( c f . F i g . 6 . 5 - 6 . 7 ) . B. ( i ) P r e - t r e a t m e n t r e s p o n s e t o S c h a f f e r c o l l a -t e r a l (SC) s t i m u l i . P e r f u s i o n o f k a i n a t e d i d n o t c h a n g e t h e a n t i d r o m i c PS b u t i n c r e a s e d t h e number and a m p l i t u d e o f PSs w h i c h f o l l o w e d ( i i ) . The a p p e a r a n c e o f m u l t i p l e PSs i n e v o k e d a c t i v i t y ( i i i ) was c o r r e l a t e d w i t h t h e o n s e t o f SPBs ( i v ) . P a r t i a l r e c o v e r y o f e v o k e d r e s p o n s e s h a d o c c u r r e d 2 h l a t e r b u t a s i m i l a r i t y b e t w e e n e v o k e d ( v ) and s p o n t a n e o u s ( v i ) f i e l d p o t e n t i a l s was s t i l l e v i d e n t . C. S p o n t a n e o u s a c t i v i t y c o m p r i s e d o f s i n g l e r a t h e r t h a n m u l t i p l e P S s was o b s e r v e d a f t e r i o n t o p h o r e s i s o f k a i n a t e i n t h e s t r a t u m p y r a m i d a l e o f -7 CA3. D. F o l l o w i n g k a i n a t e t r e a t m e n t (15 m l o f 10 M) i n l o w 2+ 2 + [Ca ] / h i g h [Mg ] a r e t u r n t o n o r m a l ACSF c a u s e d t h e o n s e t o f s p o n t a n e o u s PSs w h i c h were i n d i s t i n g u i s h a b l e f r o m t h o s e e l i c i t e d by i o n t o p h o r e s i s . E. W i t h i n 20 m i n t h e s e s i n g l e PSs were r e p l a c e d by s p o n t a n e o u s l y - o c c u r r i n g f i e l d p o t e n t i a l s c o m p r i s e d o f b u r s t s o f PSs ( s e e t e x t f o r d i s c u s s i o n ) . 145 Koinote 2 5 x l O " ' M . lOml • . . I mV(C.D) , I. 2 - IE) 10 msec (C .E ) 2 0 • (D) 146 s i t i e s o f FIM s t i m u l a t i o n , w e r e c o r r e l a t e d w i t h t h e o n s e t o f S P B s ( F i g . 5 . 6 A i i i ) . T h e f a c t o r s c o n t r i b u t i n g t o t h e s e c h a n g e s o f r e s p o n s e l a t e n c i e s a r e d i s c u s s e d i n t h e n e x t c h a p t e r i n r e l a t i o n t o a s i m i l a r a c t i o n o f f o l a t e and b i c u c u l l i n e . F i e l d p o t e n t i a l s o c c u r r i n g i n t h e a b s e n c e o f e l e c t r i c a l s t i m u l i were s o m e t i m e s e v i d e n t f o l l o w i n g k a i n a t e i o n t o p h o r e s i s p a r t i c u l a r y when l a r g e e j e c t i n g c u r r e n t s w e r e e m p l o y e d . H o w e v e r t h i s s p o n t a n e o u s a c t i v i t y was d i f f e r e n t f r o m t h a t p r o d u c e d w i t h b a t h a p p l i c a t i o n s o f t h e amino a c i d i n t h a t i t was s h o r t - l i v e d ( 2 -5 m i n ) , o c c u r r e d a t 10-50 msec i n t e r v a l s and was c o m p r i s e d o f s i n g l e PSs o f v a r i a b l e a m p l i t u d e s (up t o 5 mV b a s e l i n e t o peak n e g a t i v i t y ) ( F i g . 5 . 6 C ) . T h i s s p o n -t a n e o u s a c t i v i t y was a s s o c i a t e d w i t h b u t o u t l a s t e d by t h e p o t e n t i a t i o n o f s y n a p t i c e x c i t a t i o n . S i n c e a r e c u r r e n t e x c i t a t o r y c i r c u i t r y has been i m p l i c a t e d i n t h e m e c h a n i s m u n d e r l y i n g SPBs i n t h e CA3 r e g i o n —8 ( c f . C h a p t e r 2) t h e e f f e c t s o f k a i n a t e ( c a . 15 m l o f 10 -7 10 M) were e x a m i n e d i n s l i c e s where s y n a p t i c t r a n s m i s s i o n 2+ 2+ had b een b l o c k e d u s i n g l o w [Ca ] Q / i n c r e a s e d [Mg ] q . I n e a c h o f 3 s l i c e s t r e a t e d i n t h i s f a s h i o n s p o n t a n e o u s e p i l e p t i f o r m d i s c h a r g e s d i d n o t o c c u r a n d t h e a n t i d r o m i c a l l y e v o k e d r e s p o n s e was c o m p r i s e d o f a s i n g l e PS ( n o t s h o w n ) . However, w i t h i n 5-10 m i n o f r e t u r n i n g t o n o r m a l m e d i u m s p o n t a n e o u s a c t i v i t y c o m p r i s e d o f s i n g l e PSs and i n d i s t i n g u i s h a b l e f r o m t h o s e r e c o r d e d a f t e r i o n t o p h o r e t i c a d m i n i s t r a t i o n was o b s e r v e d ( F i g . 5.6D). As r e p l e n i s h m e n t o f n o r m a l medium c o n t i n u e d t h i s 147 t y p e o f s p o n t a n e o u s a c t i v i t y was g r a d u a l l y r e p l a c e d by SPBs ( F i g . 5 . 6 E ) , an e f f e c t w h i c h was a s s o c i a t e d w i t h t h e d e v e l -opment o f a n t i - and o r t h o d r o m i c a l l y - e v o k e d b u r s t f i r i n g . D e n t a t e G y r u s I n d o s e s c o m p a r a b l e t o t h o s e r e q u i r e d i n t h e C A l r e g i o n i o n t o p h o r e t i c a p p l i c a t i o n s o f k a i n a t e e i t h e r t o t h e s t r a t u m g r a n u l o s u m o r s t r a t u m m o l e c u l a r e p o t e n t i a t e d t h e PS e v o k e d by s t i m u l a t i o n o f t h e m e d i a l o r l a t e r a l p e r f o r a n t pathways (MPP and LPP r e s p e c t i v e l y ) . T hese p o t e n t i a t i o n s were r e l a t i v e l y g r e a t e r a n d w e r e a s s o c i a t e d w i t h l a r g e r e d u c t i o n s i n t h e l a t e n c y o f PS d i s c h a r g e b u t were s h o r t e r - l a s t i n g t h a n t h o s e r e c o r d e d i n C A l ( F i g . 5 . 1 6 A ) . E x c e p t f o r a b r i e f i n i t i a l d e p r e s s i o n t h e r e was no change o f AD PSs e v o k e d by MF s t i m u l i ( n o t shown). S h i f t s o f t h e i n p u t / o u t p u t c u r v e f o r t h e MPP-evoked PS f o l l o w i n g b a t h a p p l i c a t i o n s of 1-2 ml o f 1 0 ~ 5 M k a i n a t e were a s s o c i a t e d , a g a i n as i n C A l , w i t h r e d u c t i o n s o f EPSPs r e c o r d e d i n t h e m i d d l e m o l e c u l a r l a y e r , t h e r e g i o n o f MPP s y n a p t i c t e r m i n a t i o n s ( F i g . 5.7A). T h i s a c t i o n o f k a i n a t e was p a r t i c u l a r l y s t r i k i n g i n e a c h o f 4 s l i c e s where 10-15 ml o f 10 ^ M s o l u t i o n s o f t h e a m i n o a c i d w e r e p e r f u s e d . F o r example, i n F i g . 5.8 a f t e r k a i n a t e t r e a t m e n t low i n t e n s i t y (5 V) s t i m u l a t i o n s o f t h e MPP e l i c i t e d an 8-10 mV PS e v e n t h o u g h an EPSP was e v i d e n t n e i t h e r i n t h e d e n d r i t i c n o r s o m a t i c f i e l d p o t e n t i a l s . The f i r i n g o f a s e c o n d a r y PS a t h i g h s t i m u l u s i n t e n s i t i e s was an o c c a s i o n a l o b s e r v a t i o n ( F i g . 5.9:30 V ) . 148 > 6 E « 5 "O 2 4 ? 3 o o. 2 H to UJ 0 1 10 I 2 0 3 0 —I 4 0 18 -i | , 6 J cu 14 H "O 1 ' 2 Q . £ 10 o 00 2 0_ Stimulus Intensity (V) - 1 — 10 I 2 0 i 3 0 — i 4 0 > "a Z> Q . E < Q_ a 12 i 10 8 6-4-2-5 10 15 20 25 3 0 Stimulus Intensity (V) 2 0 V t AD 3 0 V »• AD J*4mV 4 msec F i g . 5.7 A. A v e r a g e c u r v e s ( e a c h p o i n t mean o f 3 s u c c e s s i v e r e s p o n s e s , s t a n d a r d d e v i a t i o n s s m a l l e r t h a n s y m b o l s ) r e l a t i n g i n t e n s i t y o f MPP s t i m u l i and a m p l i t u d e o f e v o k e d r e s p o n s e s b e f o r e (o) and a f t e r (•) p e r f u s i o n o f 2 ml o f 10 5 M k a i n a t e i n t h e d e n t a t e g y r u s . A m p l i t u d e s o f EPSP were measured a t a l a t e n c y w h i c h p r e c l u d e d any c o n t a m i n a t i o n o f t h e * d e n d r i t i c f i e l d p o t e n t i a l by a r e v e r s e d PS. Note th e l a r g e s h i f t o f t h e i n p u t / o u t p u t c u r v e f o r t h e PS r e c o r d e d i n t h e s t r a t u m g r a n u l o s u m . B. In a n o t h e r s l i c e , b a s e l i n e t o peak a m p l i t u d e s o f a n t i d r o m i c PSs e v o k e d by v a r i o u s i n t e n s i t i e s o f MF s t i m u l a t i o n b e f o r e (o) and a f t e r t h e p e r f u s i o n o f 10 ml (•) a n d 20 ml (•) o f 10 5 M k a i n a t e . The f i l m e d r e c o r d s f r o m w h i c h p o r t i o n s o f t h e c u r v e s were d e r i v e d i n d i c a t e c o n t r o l r e s p o n s e ( f i r s t column) and t h o s e o b t a i n e d a f t e r 10 and 20 ml p e r f u s i o n s ( s e c o n d and t h i r d columns r e s p e c t i v e l y ) . A f t e r 20 -5 ml o f 10 k a i n a t e had been p e r f u s e d t h e d u r a t i o n of the PS i n c r e a s e d and t h e r e was an i n c r e a s e b o t h o f t h e d u r a t i o n and a m p l i t u d e o f t h e l a t e p o s i t i v e wave, an e f f e c t w h i c h was a l s o a p p a r e n t a f t e r t h e 10 ml p e r f u s i o n . 149 T h a t i n 3/3 o f t h e l a t t e r s l i c e s p r e v i o u s o r s u b s e q u e n t t r e a t m e n t w i t h 10-15 m l o f ACSF c o n t a i n i n g 5, 10 and 15 mM [ K + ] Q c o u l d n o t c a u s e s i m i l a r c h a n g e s o f EPSP and PS a m p l i -t u d e s ( F i g . 5.8) s u g g e s t e d t h a t k a i n a t e ' s , a c t i o n s w e re n o t r e l a t e d s i m p l y t o a d e p o l a r i z a t i o n o f d e n t a t e g r a n u l e c e l l s . I n t h i s c o n n e c t i o n AD PSs were a l s o u n a f f e c t e d by b a t h - a p p l i e d k a i n a t e e x c e p t w i t h p r o l o n g e d e x p o s u r e s where a d e p r e s s a n t a c t i o n was o b s e r v e d ( F i g . 5 . 7 B ) . I n t e r e s t i n g l y , an a d d i t i o n a l e f f e c t o f k a i n a t e was t o e n h a n c e b o t h t h e a m p l i t u d e a n d d u r a t i o n o f t h e p o s i t i v e wave w h i c h f o l l o w e d t h e AD PS ( F i g . 5 . 7 B ) . R e c o v e r y f r o m t h e s e a c t i o n s o f k a i n a t e u s u a l l y o c c u r r e d w i t h i n 1 h o f r e t u r n i n g t o n o r m a l ACSF. E f f e c t s o f o t h e r a m i n o a c i d s i n C A l T h e a c t i o n s o f o t h e r e x c i t a t o r y a m i n o a c i d s a p p l i e d e i t h e r w i t h i o n t o p h o r e t i c c u r r e n t s s i m i l a r t o t h o s e u s e d t o e x p e l k a i n a t e o r by b r i e f p e r i o d s ( 1 - 3 min) o f p e r f u s i o n were i n v e s t i g a t e d on 39 s l i c e s where k a i n a t e a p p l i e d p r e v i o u s l y o r s u b s e q u e n t l y p o t e n t i a t e d t h e S C - e v o k e d PS by a t l e a s t 20% f o r p e r i o d s o f 15 m i n o r l o n g e r . When a p p l i e d by i o n t o p h o r e s i s i n t h e c e l l b o d y o r -5 -4 d e n d r i t i c l a y e r ( F i g . 4.7A) o r i n t h e p e r f u s a t e ( 5 x 1 0 -10 M) NMA p r o d u c e d o n l y a b r i e f o r no i n c r e a s e i n t h e PS f o l l o w e d by a l o n g - l a s t i n g d e p r e s s i o n o f a l l e v o k e d r e s p o n s e s r e c o r d e d b o t h i n t h e s t r a t u m p y r a m i d a l e a n d s t r a t u m r a d i a t u m ( F i g . 5 . 9 C ) . The d e p r e s s a n t a c t i o n o f NMA was m i m i c k e d by p e r f u s e d -5 -4 i b o t e n a t e (5x10 -10 M) . As n o t e d p r e v i o u s l y ( c f . C h a p t e r 150 EPSP Pre-treatment I 0 " 5 M Koinote Recovery 10 mM K * ) J 4 mV 4 msec 4 msec F i g . 5.8 F i l m e d r e c o r d s e a c h c o m p r i s e d o f 3 c o n s e c u -t i v e s u p e r i m p o s e d sweeps showing the changes i n d e n d r i t i c and s o m a t i c f i e l d r e s p o n s e s i n the d e n t a t e g y r u s f o l l o w i n g p e r f u s i o n o f medium c o n t a i n i n g 10 5 M k a i n a t e (10 ml) o r i n c r e a s e d [ K + ] ( 1 5 m l ) . T r e a t m e n t w i t h k a i n a t e c a u s e d a o r e v e r s i b l e d e p r e s s i o n o f t h e d e n d r i t i c EPSP and m a r k e d l y e n h a n c e d s y n a p t i c e x c i t a t i o n . S u b s e q u e n t e x p o s u r e t o medium c o n t a i n i n g 10 mM [ K + ] Q d i d not p r o d u c e c o m p a r a b l e changes o f e i t h e r t h e EPSP o r the PS. In t h e same s l i c e 15 mM [ K + ] de-o p r e s s e d b o t h EPSPs and PSs ( n o t shown). 151 4) t h e a m p l i t u d e o f t h e EPSP was o f t e n p o t e n t i a t e d f o l l o w i n g r e c o v e r y f r o m N M A - i n d u c e d d e p r e s s i o n s . Q u i s q u a l a t e a p p l i e d by i o n t o p h o r e s i s o r p e r f u s i o n - 5 - 4 (10 -10 M) g e n e r a l l y d e p r e s s e d t h e P S , an e f f e c t o c c a s i o n -a l l y p r e c e d e d by a s h o r t - l i v e d e n h a n cement o f t h e S C - e v o k e d PS ( F i g . 5 . 9 C ) . E s p e c i a l l y w i t h t h e h i g h e r d o s e s o f q u i s q u a l a t e r e c o v e r y f r o m d e p r e s s i o n was commonly f o l l o w e d by p o t e n t i a t i o n a l t h o u g h t h i s was b o t h s m a l l e r i n a m p l i t u d e and s h o r t e r i n d u r a t i o n t h a n t h a t e l i c i t e d by k a i n a t e i n t h e same s l i c e . - 3 B o t h L - g l u t a m a t e and L - a s p a r t a t e a t 10 M p r o d u c e d o n l y s h o r t - l a s t i n g c h a n g e s i n t h e PS a m p l i t u d e ( F i g . 5 . 9 A ) ; a t -2 10 M t h e r e s p o n s e was i n i t i a l l y b r i e f l y p o t e n t i a t e d ( 1 0 0 - 4 0 0 % , 20-80 s e c ) and t h e n was d e p r e s s e d f o r l o n g p e r i o d s ( F i g . 5. 9 B ) . P o t e n t i a t i o n was n o t o b s e r v e d f o l l o w i n g r e c o v e r y . K a i n a t e ' s m e c h a n i s m ( s ) o f a c t i o n I t h a s b e e n r e p o r t e d ( S c h w a r t z k r o i n and P r i n c e , 1980; D i n g l e d i n e and G j e r s t a d , 1980) t h a t compounds s u c h a s p e n i c i l l i n and b i c u c u l l i n e ( B I C ) ( W i g s t r o m and G u s t a f f s o n , 1983) w h i c h r e d u c e G A B A e r g i c i n h i b i t i o n c a n p r o d u c e a p o t e n t i a t i o n o f o r t h o d r o m i c a l l y - e v o k e d r e s p o n s e s and c a u s e e p i l e p t i f o r m d i s c h a r g e o f n e u r o n s . S i n c e t h e s e l a t t e r two a c t i o n s a r e a l s o p r o d u c e d by k a i n a t e , t h e c h a n g e s i n p o s t -a c t i v a t i o n i n h i b i t i o n s f o l l o w i n g b a t h o r i o n t o p h o r e t i c a p p l i c a t i o n s o f k a i n a t e were e x a m i n e d e x t r a c e l l u l a r l y i n e a c h h i p p o c a m p a l r e g i o n u s i n g t h e p a i r e d s t i m u l u s p a r a d i g m ( c f . 152 > E LGLU 0 ' 3 M L-GLU O M L A S P 0 ° M '.-'"v'^ -V. lO'^M Ou.s l O ' - M > E C M S3 ...v^r--F i g . 5.9 A. I n t h e C A l r e g i o n p e r f u s i o n f o r 1...min w i t h -3 10 M s o l u t i o n s o f L - g l u t a m a t e ( L - g l u ) o r L - a s p a r t a t e ( L - a s p ) p r o d u c e d b r i e f i n c r e a s e s i n t h e a m p l i t u d e o f t h e S C - e v o k e d PS w h i c h d i d n o t o u t l a s t t h e p e r i o d o f a p p l i c a t i o n . B. L--2 g l u t a m a t e a t 10 M e l i c i t e d a s h o r t - l i v e d p o t e n t i a t i o n f o l l o w e d by a l e n g t h y d e p r e s s i o n o f o r t h o d r o m i c P S s . C. I n a n o t h e r s l i c e a 1 m i n p e r f u s i o n w i t h 10 5 M k a i n a t e p r o d u c e d a l o n g - l a s t i n g p o t e n t i a t i o n o f s y n a p t i c e x c i t a t i o n w h i c h was n o t m i m i c k e d by a s i m i l a r d o s e o f q u i s q u a l a t e ( Q u i s ) a d m i n i s t e r e d c a . 40 m i n l a t e r . S u b s e q u e n t t r e a t m e n t w i t h NMA ( l o w e r g r a p h ) d e p r e s s e d o r t h o d r o m i c a c t i v i t y . 153 C h a p t e r 2) as w e l l as by i n t r a c e l l u l a r a n a l y s i s i n C A l . E x t r a c e l l u l a r a n a l y s i s i n C A l o f k a i n a t e - i n d u c e d c h a n g e s o f r e c u r r e n t i n h i b i t i o n I n t h e p r e l i m i n a r y e x p e r i m e n t s t h e i n t e n s i t i e s o f c o n d i t i o n i n g AD and t e s t o r t h o d r o m i c s t i m u l i r e m a i n e d c o n s t a n t _ 5 a s k a i n a t e was p e r f u s e d ( 1 - 2 m l o f 10 M ) . I n e a c h o f 3 s l i c e s e x a m i n e d i n t h i s manner t h e p o t e n t i a t i o n o f S C - e v o k e d r e s p o n s e s was a s s o c i a t e d i n i t i a l l y w i t h a l o s s o f t h e i n h i b i t i o n a c t i v a t e d by p r e c e d i n g (25 msec) AD s t i m u l i a l t h o u g h t h e a m p l i t u d e o f t h e AD PS r e m a i n e d c o n s t a n t . I t i s u n l i k e l y t h a t t h i s k a i n a t e - i n d u c e d t r a n s i e n t d i s i n h i b i t i o n was due s i m p l y t o t h e l i m i t e d c a p a c i t y o f a c o n s t a n t s t r e n g t h o f i n h i b i t i o n t o a f f e c t a l a r g e r a m p l i t u d e PS ( s e e b e l o w ) s i n c e F i g . 5.10 ( t = 2 0 - 2 5 m i n ) shows a r e c o v e r y o f t h e i n h i b i t i o n w h i l e t h e a m p l i t u d e o f t h e u n c o n d i t i o n e d o r t h o d r o m i c PS was s t i l l p o t e n t i a t e d . I n 6 o t h e r s l i c e s p r e - t r e a t m e n t " i n h i b i t i o n c u r v e s " w e r e g e n e r a t e d by v a r y i n g t h e i n t e n s i t y o f t h e o r t h o d r o m i c s t i m u l i w h i l e t h a t f o r a n t i d r o m i c a c t i v a t i o n s r e m a i n e d c o n s t a n t ( c f . L y n c h et_ a _ l . , 1 9 8 1 ) . U n d e r t h e s e c o n d i t i o n s o f s t i m u l a t i o n r e s p o n s e s i n c o n t r o l s l i c e s r e v e a l e d a s m a l l e r p e r c e n t d e c r e a s e o f t h e c o n d i t i o n e d PS as t h e a m p l i t u d e o f t h e u n c o n d i t i o n e d PS i n c r e a s e d ( F i g . 6 . 9 a ) . A f t e r p e r f u s i o n o f — 6 5-10 m l o f 10 M k a i n a t e p o t e n t i a t i o n o f s y n a p t i c e x c i t a t i o n was a s s o c i a t e d w i t h a t r a n s i e n t a b o l i t i o n o f r e c u r r e n t i n h i b i t i o n ( F i g . 6 . 9 c ) , an a c t i o n w h i c h was p a r a l l e l e d by t h e a p p e a r a n c e o f a d d i t i o n a l PSs i n t h e o r t h o d r o m i c r e s p o n s e ( c f . F i g . 5 . 1 1 A ) . However i n e a c h s l i c e t h e i n h i b i t i o n r e t u r n e d 154 (a) n 10 > 2-(o) (b) v. (c) (d) ( b ) n Kainate IO" 5M, I ml IV. f^—vyv_ (c) + 5 10 20 25 45 Time (min) (d) n (e) 1.2 mV 10 msec 50 60 (e) n — i 65 F i g . 5.10 The a c t i o n s o f k a i n a t e on t h e r e c u r r e n t i nh i b i fc'i on i n C A l . .In t.he ;e :xper i.m.ent an o r t h o d r o m i c t e s t r e s p o n s e was on a l t e r n a t e o c c a s i o n s •;preee!ded (20 msec) by an a n t i d r o m i c s t i m u l u s a c t i v a t i n g t h e r e c u r r e n t i n h i b i t o r y pathway. The g r a p h o f t h e b a s e l i n e t o peak a m p l i t u d e s o f t h e u n i n h i b i t e d ( f i l l e d c i r c l e s ) and i n h i b i t e d (open c i r c l e s ) PSs i n d i c a t e s a- b r i e f p e r i o d f o l l o w i n g -the b a t h a p p l i c a t i o n o f 1 ml o f 10 ^ M k a i n a t e i n w h i c h t h e r e was an a p p a r e n t l o s s o f t h e i n h i b i t i o n . T h i s e f f e c t was c o r r e l a t e d w i t h t h e t r a n s i e n t a p p e a r a n c e o f a s e c o n d a r y PS (up t o 3 mV) i n the o r t h o d r o m i c f i e l d p o t e n t i a l ( n o t shown). A t t=20-25 min t h e u n c o n d i t i o n e d PS r e m a i n e d p o t e n t i a t e d whereas a s u b s t a n t i a l r e d u c t i o n o f t h e c o n d i t i o n e d PS was now e v i d e n t . T h e f i l m e d r e c o r d s (3 c o n s e c u t i v e s u p e r i m p o s e d sweeps) o b t a i n e d a t the t i m e s i n d i c a t e d (a-e) show t h e u n c o n d i t i o n e d r e s p o n s e s ( t o p row) and the c o n d i t i o n i n g a n t i d r o m i c and t e s t o r t h o d r o m i c PSs ( l o w e r r o w ) ; t h e peak p o s i t i v i t i e s i n t h e f i e l d r e s p o n s e s were not p h o t o g r a p h e d . Note th e a b s e n c e of any change i n the a m p l i t u d e o f t h e a n t i d r o m i c PS. 155 f a r i n a d v a n c e o f t h e r e c o v e r y f r o m t h e p o t e n t i a t i n g e f f e c t s o f k a i n a t e ( F i g . 6 . 9 d ) . I n 2 s l i c e s ( n o t shown) p o t e n t i a t i o n was a s s o c i a t e d w i t h no change o r an a p p a r e n t i n c r e a s e o f t h e r e c u r r e n t i n h i b i t i o n . I n t r a c e l l u l a r a n a l y s i s o f t h e a c t i o n s o f k a i n a t e on C A l p y r a m i d a l c e l l s S t a b l e i n t r a c e l l u l a r i m p a l e m e n t s h e l d f o r 30 m i n - 3 h w ere o b t a i n e d f r o m 16 C A l p y r a m i d a l c e l l s i d e n t i f i e d by AD a c t i v a t i o n f r o m t h e a l v e u s . I n t h e e x p e r i m e n t s c h a n g e s o f p a s s i v e and a c t i v e membrane p r o p e r t i e s were d e t e r m i n e d as t h e medium was s w i t c h e d t o one c o n t a i n i n g k a i n a t e ; u s u a l l y 1-2 ml -5 o f a 10 M s o l u t i o n were p e r f u s e d . E x t r a c e l l u l a r r e c o r d i n g s w e r e o b t a i n e d s i m u l t a n e o u s l y w i t h an e l e c t r o d e p o s i t i o n e d i n t h e s t r a t u m p y r a m i d a l e w i t h i n c a . lOO^utm o f t h e i m p a l e d c e l l . Mean (+S.E.M.) v a l u e s f o r t h e r e s t i n g membrane p o t e n t i a l (RMP) (-64+lmV), i n p u t r e s i s t a n c e ( R I N ) (24jf2 megohms) and a c t i o n p o t e n t i a l ( A P ) a m p l i t u d e s ( 8 8 + 1 mV) i n n o r m a l m e d i u m w e r e s i m i l a r t o t h o s e r e p o r t e d by o t h e r s ( S c h w a r t z k r o i n , 1 9 7 5 ; D i n g l e d i n e e t a _ l . , 1980; H o t s o n and P r i n c e , 1 9 8 0 ) . P o t e n -t i a t i o n o f t h e S C - e v o k e d PS ( 1 0 0 - 1 0 0 0 % l a s t i n g 30 m i n o r l o n g e r ) was c o r r e l a t e d i n t r a c e l l u l a r l y w i t h a s m a l l d e p o l a r -i z a t i o n o f t h e RMP ( 4 . 3 + 0 . 3 mV, n = 1 3 ) . I n t h o s e c e l l s i n w h i c h i t was m e a s u r e d R I N was e i t h e r u n c h a n g e d ( n = 4) o r d e c r e a s e d by 4.8+1.0 megohms ( n = 8 ) . No change i n t h e d u r a t i o n o r a m p l i t u d e o f APs was d e t e c t a b l e . E x c e p t i n t h o s e s l i c e s w h ere t h e CA3 r e g i o n had been r e m o v e d , k a i n a t e i n i t i a l l y c a u s e d t h e t r a n s i e n t a p p e a r a n c e 156 ( 1 - 3 m i n ) o f s p o n t a n e o u s membrane d e p o l a r i z a t i o n s l a s t i n g 20-100 msec w h i c h w e r e e n h a n c e d by h y p e r p o l a r i z a t i o n a n d s y n c h r o n i z e d w i t h e x t r a c e l l u l a r f i e l d p o t e n t i a l s . T hese random d e p o l a r i z a t i o n s most l i k e l y r e f l e c t e d s y n a p t i c a c t i v a t i o n as a r e s u l t of t h e k a i n a t e - i n d u c e d e p i l e p t i f o r m d i s c h a r g e o f CA3 c e l l s . A f t e r t r e a t m e n t w i t h k a i n a t e 5/13 c e l l s d i s c h a r g e d 1 o r more APs ( a s s o c i a t e d w i t h m u l t i p l e PSs) a t an i n t e n s i t y o f o r t h o d r o m i c s t i m u l a t i o n w h i c h was p r e v i o u s l y s u b t h r e s h o l d ( F i g . 5.11A). The peak a m p l i t u d e s o f t h e SC-evoked i n t r a -c e l l u l a r EPSPs m e a s u r e d e i t h e r a t RMP o r s u p e r i m p o s e d on a 5-10 mV h y p e r p o l a r i z i n g p u l s e i n 6 c e l l s were unchanged o v e r t h e c o u r s e o f t h e e x p e r i m e n t : v a l u e s f o r p r e - a n d p o s t -t r e a t m e n t EPSPs were 10.9+2.4 mV and 11.1+2.5 mV r e s p e c t i v e l y ( F i g . 5.11A). In 7 o t h e r c e l l s t h e EPSP measured i n t h e same manner d e c l i n e d f r o m c o n t r o l v a l u e s o f 13.0+1.5 mV t o 8.9+1.2 mV a f t e r p e r f u s i o n w i t h k a i n a t e . A l t h o u g h t h e r e d u c t i o n o f t h e R I N may have e x p l a i n e d p a r t o f t h e e f f e c t , t h e c o n t r i -b u t i o n o f o t h e r f a c t o r s was s u g g e s t e d i n r e c o r d i n g s where t h e EPSP was r e d u c e d d i s p r o p o r t i o n a t e l y more t h a n th e R J N ( F i g . 5 . 1 1 B ) . I n c e l l s h e l d f o r more t h a n 1 h t h e EPSP c o u l d r e c o v e r f u l l y . A more c o n s i s t e n t a c t i o n o f k a i n a t e was t h e r e d u c t i o n o f t h e IPSP e v o k e d e i t h e r by o r t h o - o r a n t i d r o m i c s t i m u l a t i o n a n d m e a s u r e d a t t h e RMP a t a l a t e n c y o f 30 msec f r o m t h e s t i m u l u s a r t e f a c t ( F i g s . 5.11A, 5.12A,B). Thus i n 13/13 c e l l s h y p e r p o l a r i z i n g I P S P s were r e d u c e d by 79+6.3% f r o m a p r e -.15 7 F i g . 5.11 A. The e x t r a c e l l u l a r ( t o p row) and i n t r a -c e l l u l a r r e s p o n s e s e v o k e d a t r e s t i n g p o t e n t i a l ( m i d d l e ) and s u p e r i m p o s e d on a 120 m s e c h y p e r p o l a r i z i n g p u l s e ( 0 . 8 nA) ( l o w e r ) . P e r f u s i o n o f C£. 2 ml o f k a i n a t e c a u s e d a p o t e n -t i a t i o n o f t h e S C - e v o k e d PS, an a c t i o n w h i c h was c o r r e l a t e d w i t h a s m a l l r e d u c t i o n o f t h e i n t r a c e l l u l a r EPSP (t=5 m i n ) . The a p p e a r a n c e o f a s e c o n d PS (t=7 min) was c o r r e l a t e d i n t r a -c e l l u l a r ^ w i t h t h e d i s c h a r g e o f two a c t i o n p o t e n t i a l s and t h e a b o l i t i o n o f t h e I P S P . The n o t c h e s i n t h e EPSP i n t h e l o w e r r e c o r d s a r e due t o t h e l a r g e e x t r a c e l l u l a r f i e l d s b e i n g " s e e n " a c r o s s t h e m e m b r a n e . A t t=37 m i n s y n a p t i c e x c i t a t i o n was s t i l l e n h a n c e d a l t h o u g h t h e I P S P had r e c o v e r e d . The g r a p h r e l a t i n g s t e a d y - s t a t e membrane p o t e n t i a l t o t h e m a g n i t u d e o f i n t r a c e l l u l a r c u r r e n t p u l s e s i n d i c a t e s t h a t k a i n a t e d e c r e a s e d t h e i n p u t r e s i s t a n c e ; t h e r e s t i n g p o t e n t i a l d e c l i n e d by o n l y 2 mV. B. L o w e r t r a c e s show t h e S C - e v o k e d ( a r r o w ) E P S P s u p e r i m p o s e d on a h y p e r p o l a r i z i n g p u l s e p r o d u c e d by i n j e c t i o n o f c u r r e n t ( u p p e r t r a c e s ) . 10 m i n a f t e r p e r f u s i o n o f k a i n a t e t h e EPSP was s u b s t a n t i a l l y r e d u c e d b u t R I N was o n l y s l i g h t l y d e c r e a s e d . P a r t i a l r e c o v e r y o f t h e EPSP was e v i d e n t 20 m i n l a t e r . 158 A. Control Koinale (IO~ 5M.2 mm. 1 = 0 mm) 40 msec 40 mvf 40 msec 1 = 5 3 7 i L Kainote o 2 min nA -i.o - 5 0 - 60 - 7 0 mV - 8 0 L - 9 0 B. Control Kamate ( I 0 " 5 M , 2 mm, t=0) 10 2 0 nA * J 159 A 4 m V + [ Control Kainate ( I 0 " 5 M . 150 sec. t = 0 mm t = 7 13 18 4 0 m V [ B 4 0 m V + [ Kainate ( I 0 ~ 5 M , 120 sec. .t=0 mm ) t= l7 24 100 msec F i g . 5.12 A. E x t r a c e l l u l a r ( u p p e r r e c o r d s ) and i n t r a c e l l u l a r r e s p o n s e s r e c o r d e d s i m u l t a n e o u s l y i n the C A l r e g i o n . In the c o n t r o l s i t u a t i o n SC s t i m u l i d e l i v e r e d 60 msec a f t e r t h e b e g i n n i n g o f a 200 msec d e p o l a r i z i n g p u l s e e l i c i t e d a h y p e r p o l a r i z i n g IPSP. 7 min a f t e r t h e b e g i n n i n g o f b a t h a p p l i c a t i o n o f k a i n a t e t h e PS was g r e a t l y p o t e n t i a t e d . A t t h e same t i m e t h e I P S P p e r s i s t e d and t h e r e was an e n h a n c e d i n h i b i t i o n o f c u r r e n t - i n d u c e d c e l l f i r i n g . . A t t = 13 min t h e I P S P a n d i t s a b i l i t y t o i n h i b i t c e l l f i r i n g was r e d u c e d . R e c o v e r y o f t h e I P S P a t t=18 m i n was a s s o c i a t e d w i t h t h e c o n t i n u e d p o t e n t i a t i o n o f s y n a p t i c e x c i t a t i o n . B. As f o r A bu t w i t h a n t i d r o m i c s t i m u l i . A g a i n k a i n a t e c a u s e d a r e v e r s i b l e r e d u c t i o n o f IPSPs and t h e i r i n h i b i t o r y a c t i o n s on c u r r e n t - i n d u c e d c e l l f i r i n g . In A and B f i l m e d r e c o r d s a r e c o m p r i s e d o f 10 c o n s e c u t i v e s u p e r i m p o s e d sweeps. 160 t r e a t m e n t v a l u e o f 5.9+0.8 mV. The d e p r e s s i o n o f t h e I P S P was g r e a t e r t h a n c o u l d be a t t r i b u t e d t o t h e change o f R I N « As was s u g g e s t e d by t h e r e s u l t s o f e x t r a c e l l u l a r a n a l y s e s t h e r e was n o t a p a r a l l e l i s m b e t w e e n k a i n a t e ' s d i s i n h i b i t o r y and p o t e n -t i a t i n g a c t i o n s . Thus a p r o m i n e n t I P S P p r o d u c i n g an i n h i b i -t i o n o f c u r r e n t i n d u c e d c e l l f i r i n g c o u l d p e r s i s t a t a t i m e when s y n a p t i c e x c i t a t i o n was s t i l l e n h a n c e d ( F i g . 5 . 1 2 A ) . The o n s e t o f o r t h o d r o m i c a l l y - e v o k e d f i r i n g o f 2 o r more APs was c o r r e l a t e d h o w e v e r w i t h a d e p r e s s i o n o f t h e I P S P ( F i g . 5 . 1 1 A ) , a c o n c l u s i o n r e a c h e d a l s o i n t h e e x p e r i m e n t s w i t h p a i r e d s t i m u l i ( s e e a b o v e ) . The r e s p o n s e o f k a i n a t e - t r e a t e d C A l c e l l s t o 200-300 msec p u l s e s o f d e p o l a r i z i n g c u r r e n t c o n t i n u e d t o be t h e d i s c h a r g e o f t r a i n s o f s i n g l e APs ( F i g . 5 . 1 2 A , B ) . CA3 I n a d d i t i o n t o an e a r l y G A B A - m e d i a t e d B I C - s e n s i t i v e i n h i b i t i o n t h e r e i s i n t h e CA3 r e g i o n an i n h i b i t i o n w h i c h h a s a l a t e o n s e t b u t l o n g - l a s t i n g e f f e c t and i s i n s e n s i t i v e t o BIC ( c f . C h a p t e r 3 ) . U s i n g t h e same s t i m u l a t i n g p r o c e d u r e s a s d e s c r i b e d i n C h a p t e r 3 t h e c h a n g e s o f t h e F I M Q and p a r t i c -u l a r l y t h e MF^-evoked i n h i b i t i o n o f F I M ^ o r t h o d r o m i c PS were a s s e s s e d a t 20 and 200-300 msec C-T i n t e r v a l s a f t e r i o n t o -p h o r e t i c o r b a t h a d m i n i s t r a t i o n s o f k a i n a t e . When i t was a p p l i e d i o n t o p h o r e t i c a l l y ( 2 . 5 - 1 0 nA f o r up t o 3 m i n ) t o t h e s t r a t u m p y r a m i d a l e i n d o s e s p r o d u c i n g p o t e n t i a t i o n o f F I M - e v o k e d o r t h o d r o m i c P S s , k a i n a t e h a d no e f f e c t on t h e i n h i b i t i o n o c c u r r i n g 20 msec a f t e r MF s t i m u l i ; 161 p r e - , p o s t - t r e a t m e n t and r e c o v e r y v a l u e s f o r t h e e a r l y i n h i b i t i o n (mean +S.E.M.) were 72.7+6% ( n = 9 ) , 80.7+9% (n=9) and 72.5+11% ( n = 8 ) . F i g . 5 . 1 3 A i i i n d i c a t e s an i n c r e a s e o f t h e e a r l y i n h i b i t i o n f o l l o w i n g k a i n a t e t r e a t m e n t , h o w e v e r t h i s e f f e c t may h a v e b e e n r e l a t e d t o t h e s m a l l i n c r e a s e i n t h e a m p l i t u d e o f t h e MF^ 1° P S . I n c o n t r a s t , when m e a s u r e d c o n c u r r e n t l y i n t h e same s l i c e s , t h e p r e - t r e a t m e n t i n h i b i t i o n o f 71.6+4.2% ( n = l l ) m e a s u r e d a t a 300 msec C-T i n t e r v a l was r e d u c e d t o o n l y 3.5+3% ( n = l l ) f o l l o w i n g k a i n a t e a d m i n i s t r a t i o n ( F i g . 5 . 1 3 A i i i ) . R e c o v e r y o f t h e l a t e i n h i b i t i o n o c c u r r e d w i t h i n 1 5 - 2 5 m i n o f t h e t e r m i n a t i o n o f t r e a t m e n t . T h e s e a c t i o n s o f k a i n a t e w e re n o t m i m i c k e d by c u r r e n t c o n t r o l s ( F i g . — 8 5 . 1 3 B ) . I n 2 o t h e r s l i c e s b a t h - a p p l i e d k a i n a t e ( 2 - 5 x 1 0 M) v i r t u a l l y a b o l i s h e d t h e l a t e i n h i b i t i o n p r o d u c e d w i t h p a i r e d F I M s t i m u l i w h i l e o n l y s l i g h t l y r e d u c i n g t h e e a r l y GABA-m e d i a t e d p r o c e s s ( F i g . 5 . 1 4 A ) . F i g . 5.14B i n d i c a t e s t h a t t h e r e d u c t i o n o f t h e l a t e i n h i b i t i o n c o u l d be p r o d u c e d i n d e p e n d e n t l y o f any e n h a ncement o f s y n a p t i c e x c i t a t i o n s u g g e s t i n g t h e a b s e n c e o f a c a u s a l r e l a t i o n s h i p b e t w e e n t h e two a c t i o n s o f k a i n a t e . I n c o n t r a s t t o t h e s i t u a t i o n s w h e r e B I C r e d u c e d t h e e a r l y G A B A e r g i c i n h i b i t i o n ( F i g . 3.4A,B) t h e e l i m i n a t i o n o f t h e l a t e n o n -G A B A e r g i c e v e n t was n o t c o r r e l a t e d w i t h b u r s t f i r i n g i n t h e u n c o n d i t i o n e d r e s p o n s e ( F i g . 5.13B; 5 . 1 4 A ) . The e f f e c t s o f BIC and k a i n a t e on t h e e a r l y and l a t e i n h i b i t i o n s i n t h e CA3 r e g i o n a r e s u m m a r i z e d i n F i g . 5.15. 162 F i g . 5.13 A. ( i ) The s t i m u l u s i n t e n s i t y / P S a m p l i t u d e i n p u t / o u t p u t c u r v e s f o r t h e F I M - e v o k e d o r t h o d r o m i c r e s p o n s e i n C A 3 a a n d t h e i n h i b i t i o n c u r v e s ( s e e t e x t f o r e x p l a n a t i o n ) g e n e r a t e d 20 msec ( i i ) and 300 msec ( i i i ) a f t e r " c o n d i t i o n i n g MF s t i m u l i . C o n t r o l r e s p o n s e s (o) r e v e a l c o m p a r a b l e e a r l y and l a t e i n h i b i t i o n s . F o l l o w i n g t h e i o n t o p h o r e t i c e j e c t i o n o f k a i n a t e w i t h a 10 nA c u r r e n t f o r 3 m i n t h e r e was a p o t e n -t i a t i o n o f t h e e v o k e d r e s p o n s e s r e v e a l e d as a s h i f t t o t h e l e f t o f t h e i n p u t / o u t p u t c u r v e i n ( i ) . A t t h e same t i m e t h e e a r l y i n h i b i t i o n was i n c r e a s e d ( [•] i n i i ) w h e r e a s t h e l a t e i n h i b i t i o n was a b o l i s h e d ( i i i ) . A f t e r 25 m i n t h e r e was a r e c o v e r y ( a ) t o p r e - t r e a t m e n t v a l u e s . B. F i l m e d r e c o r d s o f t h e c h a n g e s i n t h e e a r l y ( 2 5 m s e c ) a n d l a t e ( 2 2 5 m s e c ) i n h i b i t i o n f o l l o w i n g t h e 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 k a i n a t e i n t h e c e l l body l a y e r o f CA3a. C o n v e n t i o n s a s f o r F i g . 3.4. The i n t e n s i t y o f FIM s t i m u l a t i o n was d e c r e a s e d f r o m 6 t o 5 V a f t e r k a i n a t e t r e a t m e n t t o e v o k e an u n c o n d i t i o n e d PS o f an a m p l i t u d e c o m p a r a b l e t o t h a t i n t h e p r e - t r e a t m e n t s i t u a t i o n . K a i n a t e r e v e r s i b l y a b o l i s h e d t h e l a t e i n h i b i t i o n w h i l e p r o d u c i n g o n l y a s m a l l r e d u c t i o n o f t h e e a r l y i n h i b i t i o n . A 2.5 nA c u r r e n t c a r r i e d by C I d i d n o t m i m i c k a i n a t e ' s a c t i o n s . 164 F i g . 5.14 A. The c h a n g e s o f t h e i n h i b i t i o n p r o d u c e d a t 20 m s e c ( c e n t r e c o l u m n ) a n d 300 m s e c ( r i g h t c o l u m n ) w i t h p a i r e d F I M s t i m u l i f o l l o w i n g p e r f u s i o n o f k a i n a t e ( 2 x 1 0 M, 17 m l ) . To a v o i d c o m p l i c a t i o n s i n t r o d u c e d by t h e p o t e n t i a t i n g a c t i o n s o f k a i n a t e a s u p r a m a x i m a l s t i m u l u s (15 V) was u s e d t h r o u g h o u t t h e e x p e r i m e n t . As w i t h i o n t o p h o r e t i c a p p l i c a t i o n s o f k a i n a t e t h e r e was a s e l e c t i v e r e d u c t i o n o f t h e l a t e i n h i b i t i o n w h i c h was r e v e r s i b l e a f t e r w a s h - o u t w i t h n o r m a l medium. B. I n a n o t h e r s l i c e i o n t o p h o r e s i s o f k a i n a t e c a u s e d an i m m e d i a t e p o t e n t i a t i o n o f t h e F I M - e v o k e d o r t h o d r o m i c PS ( f i l l e d c i r c l e s ) w h i l e h a v i n g no e f f e c t on t h e i n h i b i t i o n o f t e s t PSs p r e c e d e d (20 msec) by MF C s t i m u l i . A f t e r c h a n g i n g t h e s t i m u l a t i o n p a r a m e t e r s ( b r e a k i n r e c o r d ) t o a s s e s s t h e i n h i b i t i o n a t 300 m s e c a s i m i l a r d o s e o f k a i n a t e b r i e f l y d e p r e s s e d t h e u n c o n d i t i o n e d r e s p o n s e and d e c r e a s e d t h e l a t e i n h i b i t i o n . A l a r g e r d o s e o f k a i n a t e a p p l i e d 15 m i n l a t e r ( t =30 m in) p r o d u c e d a s u b s t a n t i a l . d e p r e s s i o n o f e v o k e d a c t i v i t y and c a u s e d a l o n g e r - l a s t i n g d e p r e s s i o n o f t h e l a t e i n h i b i t i o n . 165 166 100 5 0 LU c o OJ E o la _c 0 L 100 5 0 0 L Pre-treatment (10) JL 2 0 2 0 0 - 3 0 0 Pre-treatment 19> ( I I ) BIC treated (10) (10) A . 2 0 2 0 0 - 3 0 0 Kainate treated ( 9 ) 1 2 0 3 0 0 2 0 (ill) 3 0 0 Recovery (3) 2 0 2 0 0 - 3 0 0 Recovery ( 8 ) JL (©) 2 0 3 0 0 F i g . 5.15 Summary of the e f f e c t s o f b i c u c u l l i n e ( t o p ) and k a i n a t e ( b o t t o m ) on t h e i n h i b i t i o n of FIM-evoked o r t h o -d r o m i c PSs e l i c i t e d 20 and 200-300 msec f o l l o w i n g a MF C s t i m -u l i ( s e e t e x t f o r e x p l a n a t i o n ) . The number of s l i c e s i n e a c h g r o u p i s i n d i c a t e d i n b r a c k e t s above t h e e r r o r b a r s . 167 D e n t a t e G y r u s T h e r e was e v i d e n c e i n 3 s l i c e s f o r a b r i e f d e p r e s s i o n by k a i n a t e o f t h e i n h i b i t i o n o c c u r r i n g a t 20 msec C-T i n t e r v a l s w i t h p a i r e d MPP s t i m u l i . F o r e x a m p l e , i n F i g . 5.16C 1-2 m i n a f t e r t h e t e r m i n a t i o n o f t h e i o n t o p h o r e s i s o f k a i n a t e i n t h e s t r a t u m g r a n u l o s u m s t i m u l i o f a r e d u c e d i n t e n s i t y b u t e v o k i n g t h e same a m p l i t u d e c o n d i t i o n i n g P Ss p r o d u c e d a p o t e n t i a t i o n o f t h e t e s t o r t h o d r o m i c PS ( F i g . 5 . 1 6 C i i ) w h e r e a s i n h i b i t i o n o f t e s t a c t i v i t y was o b s e r v e d i n t h e p r e - t r e a t m e n t s i t u a t i o n . A s i m i l a r s e q u e n c e o f e v e n t s i s s e e n iri v i v o f o l l o w i n g t h e s y s t e m i c a d m i n i s t r a t i o n o f k a i n a t e ( S l o v i t e r a n d Damiano, 1 9 8 1 b ) . A p p r o x i m a t e l y 3 m i n l a t e r t h e p o t e n t i a t i o n o f s y n a p t i c e x c i t a t i o n c o n t i n u e d w h i l e t h e p o s t a c t i v a t i o n i n h i b i t i o n h a d r e c o v e r e d a n d was i n f a c t i n c r e a s e d ( F i g . 5 . 1 6 C i i i ) . I n 4 s l i c e s t h e p o t e n t i a t i n g a c t i o n s o f i o n t o p h o r e t -i c a l l y - a p p l i e d k a i n a t e were n o t c o r r e l a t e d a t any t i m e w i t h a c l e a r d i s i n h i b i t o r y a c t i o n . S i m i l a r l y t h e s u b s t a n t i a l i n c r e a s e o f M P P - e v o k e d c e l l f i r i n g p r o d u c e d b y 1 0 - 1 5 m l -5 p e r f u s i o n s o f 10 M k a i n a t e w e re c o r r e l a t e d w i t h t h e p e r s i s t e n c e o f t h e i n h i b i t i o n o f t e s t r e s p o n s e s e l i c i t e d a t 20 msec C-T i n t e r v a l s ( F i g . 5 . 1 6 B i ) . S u b s e q u e n t t r e a t m e n t w i t h B I C (10 ^ M) a b o l i s h e d t h e i n h i b i t i o n and a l s o c a u s e d b u r s t f i r i n g i n b o t h t h e c o n d i t i o n i n g a n d t e s t r e s p o n s e s ( F i g . 5 . 1 6 B i i ) . 168 F i g . 5.16 A. P o t e n t i a t i o n o f t h e PS r e c o r d e d i n t h e s t r a t u m g r a n u l o s u m i n r e s p o n s e t o m e d i a l p e r f o r a n t p a t h (MPP) s t i m u l a t i o n f o l l o w i n g i o n t o p h o r e s i s o f k a i n a t e i n t h e c e l l body l a y e r . B. ( i ) MPP-evoked c o n d i t i o n i n g (C) and t e s t (T) a c t i v i t y a t a 20 msec C-T i n t e r v a l a f t e r p e r f u s i o n o f 10 m l o f -5 10 M k a i n a t e s h o w i n g a p e r s i s t e n c e o f i n h i b i t i o n o f t e s t r e s p o n s e s . N o t e i n t h e c o n d i t i o n i n g r e s p o n s e t h e d i s c h a r g e o f a 14 mV PS i n t h e a b s e n c e o f a d e t e c t a b l e r e v e r s e d EPSP. ( i i ) A d d i t i o n o f b i c u c u l l i n e ( B I C , 5 x 1 0 ^ M) t o t h e k a i n a t e -c o n t a i n i n g medium a b o l i s h e d p a i r e d p u l s e i n h i b i t i o n and c a u s e d e p i l e p t i f o r m f i r i n g . C. I n a n o t h e r s l i c e p a i r e d MPP s t i m u l i r e v e a l a p o t e n t i n h i b i t i o n o f t e s t a c t i v i t y i n t h e c o n t r o l s i t u a t i o n ( i ) . 1-2 m i n a f t e r k a i n a t e i o n t o p h o r e s i s ( i i ) a l o w e r i n t e n s i t y c o n d i t i o n i n g s t i m u l a t i o n (7 V) e v o k e d a PS a t a s h o r t e r l a t e n c y o f o n s e t b u t c o m p a r a b l e i n a m p l i t u d e t o t h e p r e - t r e a t m e n t PS; t h e t e s t r e s p o n s e was p o t e n t i a t e d . ( i i i ) 6-7 m i n l a t e r p o t e n t i a t i o n o f c o n d i t i o n i n g PSs c o n t i n u e d (11 V s t i m u l u s ) b u t i n h i b i t i o n o f t e s t a c t i v i t y h ad r e c o v e r e d . A l a r g e d e p r e s s i o n o f t h e p eak a m p l i t u d e o f r e v e r s e d EPSP i n t h e t e s t f i e l d p o t e n t i a l i s a l s o e v i d e n t . ( i v ) 25 m i n l a t e r f u l l r e c o v e r y f r o m k a i n a t e ' s a c t i o n s had o c c u r r e d . A l l r e c o r d s a r e c o m p r i s e d o f 3 c o n s e c u t i v e s u p e r i m p o s e d s w eeps. 169 170 D i s c u s s i o n A l t h o u g h t h e t h r e s h o l d d o s e s v a r i e d b e t w e e n t h e m a j o r h i p p o c a m p a l r e g i o n s k a i n i c a c i d p r o d u c e d c o m p a r a b l e e l e c t r o -p h y s i o l o g i c a l c h a n g e s i n C A l , CA3 and t h e d e n t a t e g y r u s . Thus i n l o w c o n c e n t r a t i o n s k a i n a t e r e d u c e d i n h i b i t i o n s and e n h a n c e d s y n a p t i c e x c i t a t i o n , an e f f e c t w h i c h was r e f l e c t e d as l a r g e and l o n g - l a s t i n g i n c r e a s e s i n t h e a m p l i t u d e s o f o r t h o d r o m i c P S s . A t 50-1000 f o l d g r e a t e r l e v e l s a d e p r e s s i o n o f e v o k e d a c t i v i t y was o b s e r v e d , t h e t h r e s h o l d d o s e w i t h 1 0 - 1 5 m l -6 -5 -5 -4 p e r f u s i o n s were 10 -10 M i n CA3 and 10 -10 M i n C A l and t h e d e n t a t e g y r u s . R o b i n s o n and D e a d w y l e r ( 1 9 8 1 ) h a v e r e p o r t e d a l a r g e and s u s t a i n e d d e p o l a r i z a t i o n o f CA3 and C A l -6 -5 p y r a m i d a l c e l l s a t 10 M a n d 10 M c o n c e n t r a t i o n s o f k a i n a t e , r e s p e c t i v e l y . W h i l e an i n t r a c e l l u l a r a n a l y s i s o f k a i n a t e ' s e f f e c t s on d e n t a t e g r a n u l e c e l l s i s n o t a v a i l a b l e , i t seems l i k e l y t h a t t h e d e p r e s s i o n o f e v o k e d a c t i v i t y i n t h i s r e g i o n was due a l s o t o a l a r g e d e p o l a r i z a t i o n o f n e u r o n s . I n t h i s r e g a r d t h e l o w e r t h r e s h o l d d o s e f o r t h e d e p r e s s i o n o f e v o k e d r e s p o n e s i n CA3 n e u r o n s v i s a v i s C A l i s c o n s i s t e n t w i t h t h e g r e a t e r s e n s i t i v i t y o f t h e CA3 n e u r o n s t o e x c i t a t i o n by i o n t o p h o r e t i c a l l y - a p p l i e d k a i n a t e (de M o n t i g n y and T a r d i f , 1 9 8 1 ) . The t r a n s i e n t d e p r e s s i o n s b o t h o f AD a n d s y n a p t i c r e s p o n s e s f o l l o w i n g k a i n a t e i o n t o p h o r e s i s were p r o b a b l y r e l a t e d t o an e x c e s s i v e d e p o l a r i z a t i o n . T h e r e were s e v e r a l o b s e r v a t i o n s w h i c h i m p l i e d t h a t t h e d i s i n h i b i t i n g a nd p o t e n t i a t i n g a c t i o n s o f much l o w e r c o n c e n -t r a t i o n s o f k a i n a t e were n o t r e l a t e d t o a d e p o l a r i z a t i o n o f 171 n e u r o n s . F i r s t , t h e r e was no c h a n g e o f t h e a m p l i t u d e o f AD P S s . S e c o n d , NMA and q u i s q u a l a t e , w h i c h e x h i b i t an e x c i t a t o r y p o t e n c y c o m p a r a b l e t o t h a t o f k a i n a t e , d i d n o t c a u s e a l o n g - l a s t i n g e n h a n c e m e n t o f s y n a p t i c e x c i t a t i o n , b u t d i d c a u s e d e p r e s s i o n s o f e v o k e d a c t i v i t y . T h i r d , i n t h e d e n t a t e g y r u s , k a i n a t e p o t e n t i a t i o n s were n o t m i m i c k e d by i n c r e a s e d [ K + ] ( 8 -15 mM). F i n a l l y , t h e d o s e s a t w h i c h k a i n a t e a f f e c t s s y n a p t i c p r o c e s s e s i n C A l ( 1 0 - 1 5 ml o f 1 0 ~ 7 - 1 0 ~ 6 M; 1-2 m l o f 1 0 ~ 5 M) — 8 — 7 a n d i n CA3 ( 1 0 - 1 5 m l o f 10 -10 M) h a v e b e e n s h o w n by i n t r a c e l l u l a r r e c o r d i n g t o c a u s e l i t t l e o r no membrane d e p o l a r i z a t i o n ( t h i s s t u d y i n C A l o n l y and s e e R o b i n s o n and D e a d w y l e r 1 9 8 1; W e s t b r o o k and L o t h m a n , 1983; F i s h e r a n d A l g e r , 1983 ) . I n t h e C A l r e g i o n k a i n a t e b l o c k e d t h e I P S P w h i c h has b e e n s h o w n t o be m e d i a t e d by a r e c u r r e n t a c t i v a t i o n o f G A B A e r g i c i n t e r n e u r o n s ( A n d e r s o n e t a l - . , 1964; D i n g l e d i n e and Langmoen, 1980 ). The d e c l i n e o f t h e IP S P was g r e a t e r t h a n t h a t w h i c h c o u l d be a s c r i b e d t o t h e d e c r e a s e o f R-r . . . An I N a n t a g o n i s m by k a i n a t e o f p o s t s y n a p t i c e v e n t s a t t h e GABA r e c e p t o r - C l i o n o p h o r e i s an u n l i k e l y b a s i s f o r t h e e f f e c t s i n c e : ( 1 ) n e u r o c h e m i c a l i n v e s t i g a t i o n s h a v e i n d i c a t e d a n 3 a b s e n c e o f a n y i n h i b i t i o n by k a i n a t e o f [ H]-GABA b i n d i n g ( R u c k e_t a l . r 1980 ) ; a n d ( 2 ) F i s h e r a n d A l g e r ( 1 983 ) h a v e r e p o r t e d t h a t t h e k a i n a t e - i n d u c e d d e p r e s s i o n o f t h e I P S P was n o t p a r a l l e l e d by a d e c l i n e o f h y p e r p o l a r i z i n g r e s p o n s e s 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 GABA. The l a t t e r o b s e r v a t i o n a l s o i m p l i e s t h a t a cha n g e o f t h e e q u i l i b r i u m p o t e n t i a l f o r C l was 172 n o t i n v o l v e d . T h e r e f o r e , by e l i m i n a t i o n i t w o u l d a p p e a r t h a t a p r e s y n a p t i c b l o c k a d e , e i t h e r a t t h e s y n a p s e s i n t h e d i s y n a p t i c p a t h w a y o r by an o v e r d e p o l a r i z a t i o n o f G A B A e r g i c b a s k e t c e l l s , may be i n v o l v e d i n k a i n a t e 1 s d i s i n h i b i t o r y a c t i o n s . I n t h i s r e g a r d , s i n c e i n k a i n i c a c i d - l e s i o n e d h i p p o c a m p i t h e r e i s a v i r t u a l a b s e n c e o f I P S P s ( L a n c a s t e r e t a l . , 1 983; F r a n c k and S c h w a r t z k r o i n , 1983) i t i s a p p e a l i n g t o i n f e r t h a t a g r e a t e r s e n s i t i v i t y t o k a i n a t e o f G A B A e r g i c i n t e r n e u r o n s v i s \ v i s p y r a m i d a l c e l l s may p r e d i s p o s e t h e f o r m e r t o t h e d e p o l a r i z i n g and c o n s e q u e n t l y t h e n e u r o t o x i c a c t i o n s o f t h e a m ino a c i d . I n t h e CA3 r e g i o n t h e B I C - i n s e n s i t i v e l a t e i n h i b i t i o n was v i r t u a l l y a b o l i s h e d by i o n t o p h o r e t i c a l l y - a p p l i e d k a i n a t e . I n C h a p t e r 3 i t was s u g g e s t e d , b e c a u s e o f t h e i r s i m i l a r e l e c t r o p h y s i o l o g i c a l and p h a r m a c o l o g i c a l c h a r a c t e r i s t i c s , t h a t t h e s l o w o n s e t , i n h i b i t o r y h y p e r p o l a r i z a t i o n b o t h i n C A l and CA3 s h a r e d a common m e c h a n i s t i c o r i g i n . C o n s i s t e n t w i t h t h i s h y p o t h e s i s i t h a s b e e n r e p o r t e d r e c e n t l y t h a t t h e " s l o w , p o t a s s i u m d e p e n d e n t I P S P " i n C A l was a l s o b l o c k e d by k a i n a t e ( F i s h e r and A l g e r , 1 9 8 3 ) . The s e l e c t i v e b l o c k a d e i n CA3 o f t h e l a t e i n h i b i t i o n i m p l i e s t h a t a n a l t e r a t i o n o f p a s s i v e membrane p r o p e r t i e s was n o t i n v o l v e d . By a n a l o g y w i t h t h e d i s i n h i b i t o r y a c t i o n s o f k a i n a t e i n C A l i t i s p o s s i b l e t h a t an i m p a i r m e n t o f t h e f u n c t i o n o f i n t e r n e u r o n s may a l s o u n d e r l i e t h e d e p r e s s i o n o f t h e l a t e i n h i b i t i o n i n CA3. T h a t no d e c l i n e o f t h e e a r l y G A B A - m e d i a t e d i n h i b i t i o n i n t h e CA3 r e g i o n was d e t e c t a b l e may h a v e b e e n due t o 173 d i f f e r e n c e s i n t h e d u r a t i o n and m a g n i t u d e o f k a i n a t e c o n c e n -t r a t i o n s a c h i e v e d by i o n t o p h o r e t i c v s b a t h a d m i n i s t r a t i o n s . B e c a u s e t h e i n d u c t i o n o f e p i l e p t i f o r m f i r i n g w o u l d have p r e v e n t e d a m e a n i n g f u l i n t e r p r e t a t i o n o f t h e d a t a , no a t t e m p t was made t o d e t e r m i n e t h e e f f e c t s o f v a r i o u s d o s e s o f b a t h - a p p l i e d k a i n a t e o n p a i r e d p u l s e i n h i b i t i o n . I n t r a -c e l l u l a r a n a l y s e s by F i s h e r and A l g e r (1983) have r e v e a l e d a r e v e r s i b l e d e p r e s s i o n by p e r f u s e d k a i n a t e o f t h e G A B A - m e d i a t e d I P S P i n CA3. No e x p l a n a t i o n may be o f f e r e d h e r e f o r t h e r e f r a c t o r -i n e s s o f t h e d e n t a t e g y r u s t o t h e d i s i n h i b i t o r y a c t i o n s o f k a i n a t e w h i c h w ere n o t e x p r e s s e d e v e n a f t e r p e r f u s i o n o f 10-15 -5 ml o f 10 M s o l u t i o n s . S l o v i t e r and Damiano ( 1 9 8 1 b ) h a v e r e p o r t e d k a i n a t e - i n d u c e d e p i l e p t i f o r m d i s c h a r g e s o f d e n t a t e g r a n u l e c e l l s i n . v i v o . K a i n i c a c i d a l s o e n h a n c e d n e u r o n a l r e s p o n s i v e n e s s t o s y n a p t i c e x c i t a t i o n s ( C o l l i n g r i d g e and M c L e n n a n , 1981; W e s t b r o o k a n d L o t h m a n , 1 9 8 3 ) . T h i s a c t i o n was m a n i f e s t e x t r a c e l l u l a r l y i n C A l , CA3 and t h e d e n t a t e g y r u s as a s h i f t t o t h e l e f t o f t h e i n p u t / o u t p u t c u r v e p l o t t i n g s t i m u l u s i n t e n s i t y a g a i n s t PS a m p l i t u d e ; i n t r a c e l l u l a r l y , c e l l s c o u l d f i r e APs t o o r t h o d r o m i c s t i m u l i w h i c h p r e v i o u s l y were s u b t h r e s h o l d . A r e d u c t i o n o f t h e EPSP r e c o r d e d e i t h e r i n t r a -o r e x t r a c e l l u l a r l y was u s u a l l y b u t n o t i n v a r i a b l y a s s o c i a t e d w i t h t h e s e p o t e n t i a t i o n s . The s i m p l e s t e x p l a n a t i o n f o r t h e i n c r e a s e o f t h e PS c o n c o m m i t a n t w i t h a d e c l i n e o f t h e EPSP w o u l d be a s u b -174 t h r e s h o l d d e p o l a r i z a t i o n o f c e l l s b r i n g i n g them c l o s e r t o t h e t h r e s h o l d f o r f i r i n g ( a s s u m i n g no a c c o m m o d a t i o n ) b u t a t t h e same t i m e r e d u c i n g t h e d r i v i n g f o r c e f o r s y n a p t i c c o n d u c -t a n c e s . However, as d i s c u s s e d p r e v i o u s l y , t h e s e c h a n g e s o f o r t h o d r o m i c r e s p o n s e s w e re p r o d u c e d w i t h c o n c e n t r a t i o n s o f k a i n a t e h a v i n g l i t t l e o r no e f f e c t on t h e RMP m e a s u r e d w i t h i n t r a c e l l u l a r i m p a l e m e n t s o f n e u r o n a l s o m a t a . I n a d d i t i o n , o t h e r d e p o l a r i z i n g t r e a t m e n t s a t b e s t o n l y w e a k l y m i m i c k e d k a i n a t e ' s p o t e n t i a t i n g a c t i o n s . I n s l i c e s o f t h e o l f a c t o r y c o r t e x A u k e r e t a l . ( 1 9 8 2) n o t e d f o l l o w i n g k a i n i c a c i d t r e a t m e n t t h a t " a s t h e E P S P a p p r o a c h e d t h e p o i n t o f a b o l i s h m e n t t h e ( o r t h o d r o m i c a l l y -e v o k e d ) p o p u l a t i o n s p i k e i n c r e a s e d t o a maximum s i z e " and i n t h e p r e s e n t i n v e s t i g a t i o n t h i s s e q u e n c e o f e v e n t s was p a r t i c u l a r l y e v i d e n t i n t h e d e n t a t e g y r u s . The s u b s t a n t i a l r e d u c t i o n o f t h e E P S P by k a i n a t e may h a v e b e e n d u e t o a p r e f e r e n t i a l d e p o l a r i z a t i o n and i n c r e a s e o f c o n d u c t a n c e i n t h e d e n d r i t e s s i n c e e v i d e n c e f o r a g r e a t e r d e n s i t y o f d e n d r i t i c vs 3 s o m a t i c k a i n a t e r e c e p t o r s h a s been i n d i c a t e d by [ H ] - k a i n a t e b i n d i n g s t u d i e s i n t h e h i p p o c a m p u s ( F o s t e r e t a l _ . , 1 9 8 1 ) . A l t h o u g h no c h a n g e o f t h e p r e s y n a p t i c f i b r e v o l l e y was d e t e c t e d t h i s d o e s n o t p r e c l u d e t h e p o s s i b i l i t y o f a r o l e f o r a d e c r e a s e o f t r a n s m i t t e r r e l e a s e i n t h e r e d u c t i o n o f t h e EPSP. The m e c h a n i s m ( s ) u n d e r l y i n g t h e i n c r e a s e d e x c i t a b i l i t y o f n e u r o n s e x p o s e d t o d o s e s o f k a i n a t e c a u s i n g l i t t l e o r no membrane d e p o l a r i z a t i o n and d e c r e a s i n g t h e EPSP i s n o t c l e a r . 175 The r e d u c t i o n o f G A B A - m e d i a t e d i n h i b i t i o n w o u l d i n i t s e l f be e x p e c t e d t o c a u s e an i n c r e a s e o f o r t h o d r o m i c e x c i t a t i o n ( e . g . W i g s t r o m and G u s t a f s s o n , 1 9 8 3 ) . However, t h e r e was a p o o r c o r r e l a t i o n b e t w e e n t h e d i s i n h i b i t o r y and p o t e n t i a t i n g a c t i o n s o f k a i n a t e . F o r e x a m p l e , i n t h e C A l r e g i o n t h e e n h a n c e m e n t o f o r t h o d r o m i c e x c i t a t i o n c o u l d p e r s i s t d e s p i t e t h e a p p a r e n t r e c o v e r y o f t h e r e c u r r e n t i n h i b i t i o n and t h e I P S P . I n d e e d , i n t h e d e n t a t e g y r u s and CA3 t h e p o t e n t i a t i o n was a s s o c i a t e d w i t h no c h a n g e o r i n some c a s e s an i n c r e a s e o f t h e p o s t a c t i v a t i o n G A B A - m e d i a t e d i n h i b i t i o n . Q u i t e c o n c e i v a b l y t h e i n c r e a s e o f i n h i b i t i o n i n t h e s e c a s e s c o u l d be due t o an e n h a n c e d d i s c h a r g e o f b a s k e t c e l l s t o s y n a p t i c i n p u t f r o m c o l l a t e r a l p a t h w a y s . One m e c h a n i s m by w h i c h k a i n a t e c o u l d i n c r e a s e t h e p r o b a b i l i t y o f c e l l f i r i n g t o o r t h o d r o m i c s t i m u l i i s by p r o d u c i n g a l o n g - l a s t i n g d e p o l a r i z a t i o n i n t h e d e n d r i t e s w h i c h t h e n f a c i l i t a t e s c o u p l i n g b e t w e e n d e n d r i t e s and s o m a t a p o s s i b l y v i a a n e f f e c t o r r e g e n e r a t i v e d e n d r i t i c s p i k i n g m e c h a n i s m s ( T r a u b and L l i n a s , 1979; Wong e t a _ l . , 1979; B e n a r d o e t a l . , 1 9 8 2 ) . C o r r e l a t i o n b e t w e e n e l e c t r o p h y s i o l o g i c a l and  n e u r o t o x i c a c t i o n s o f k a i n a t e I b o t e n a t e , NMA a n d i n h i g h e r d o s e s L - g l u t a m a t e a n d L - a s p a r t a t e d e p r e s s e d t h e S C - e v o k e d PS i n C A l f o r l o n g p e r i o d s , an e f f e c t w h i c h was a s s o c i a t e d w i t h l a r g e n e u r o n a l d e p o l a r i z a t i o n s ( i n t r a c e l l u l a r d a t a n o t s h o w n ) . The p o t e n c y o f t h e s e compounds as d e p r e s s a n t s o f e v o k e d r e s p o n s e s 176 p a r a l l e l s t h e i r a b i l i t y t o c a u s e l o c a l l e s i o n s when i n j e c t e d d i r e c t l y i n t o t h e h i p p o c a m p u s ( N a d l e r e_t a _ l . , 1 9 8 1 ) a n d a c c o r d i n g l y i s c o n s i s t e n t w i t h t h e e x c i t o t o x i c m e c h a n i s m o r i g i n a l l y p r o p o s e d by O l n e y e_t a _ l . , 1 9 7 1 . I n d o s e s com-p a r a b l e t o t h o s e r e q u i r e d w i t h NMA ( p r e s u m a b l y r e f l e c t i n g t h e s i m i l a r e x c i t a t o r y p o t e n c i e s o f t h e two compounds [ c f . C h a p t e r 4] ) k a i n a t e a l s o d e p o l a r i z e d C A l c e l l s and d e p r e s s e d e v o k e d r e s p o n s e s . T h i s o b s e r v a t i o n i m p l i e s t h a t 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 t h e l o c a l t o x i c i t y o f k a i n a t e may i n some c a s e s be a t t r i b u t a b l e t o a d i r e c t d e p o l a r i z i n g a c t i o n as a p p e a r s t o be t h e c a s e w i t h i b o t e n a t e a n d NMA ( K o h l e r e_t a _ l . , 1 9 7 9 ) . T h a t i n b o t h t h e h i p p o c a m p u s ( N a d l e r e t a _ l . , 1 9 8 1 ) a n d s t r i a t u m ( Z a c z e k a n d C o y l e , 1 9 8 2 ) t h e t h r e s h o l d d o s e f o r k a i n a t e - i n d u c e d l o c a l damage i s ca. 2 o r d e r s o f m a g n i t u d e b e l o w t h a t o f NMA has been a s c r i b e d t o a u n i q u e b u t u n d e f i n e d i n t e r a c t i o n o f k a i n a t e w i t h s y n a p t i c t e r m i n a l s s i n c e t r a n s e c -t i o n o f c o r t i c a l p a t h w a y s t o t h e s t r i a t u m i n c r e a s e s t h e d o s e o f k a i n a t e r e q u i r e d f o r l o c a l damage t o a l e v e l s i m i l a r t o t h a t f o r NMA (McGeer e t a l . , 1978b and T a b l e 1 i n Z a c z e k and C o y l e , 1 9 8 2 ) . I n t h e h i p p o c a m p u s t h e i n t e r p r e t a t i o n o f m e c h a n i s m s o f t o x i c i t y may be c o m p l i c a t e d by t h e s u p e r -i m p o s i t i o n o f e p i l e p t o g e n i c and l o c a l damage. Some o f t h e n e u r o t o x i c a c t i o n s o f k a i n a t e a r e m o d u l a t e d by o n g o i n g e x c i t a t o r y i n p u t a s i n d i c a t e d by t h e a b i l i t y o f i n c r e a s e d a u d i t o r y s t i m u l a t i o n t o a u g m e n t t h e t o x i c i t y o f k a i n a t e i n t h e c o c h l e a r n u c l e u s ( M a t t o x e t a _ l . , 1980) and by t h e s p a r i n g e f f e c t s o f a c u t e l e s i o n s o f a f f e r e n t p a t h w a y s ( s e e 177 I n t r o d u c t i o n ) . The r e s u l t s o f t h e p r e s e n t s t u d y show t h a t o f t h e e x c i t a t o r y a m i n o a c i d s t e s t e d o n l y k a i n a t e c a u s e s a p r o l o n g e d e nhancement o f s y n a p t i c e x c i t a t i o n i n e a c h o f t h e m a j o r h i p p o c a m p a l r e g i o n s and t h i s a c t i o n i s p a r a l l e l e d by a u n i q u e a b i l i t y o f i n t r a c e r e b r a l i n j e c t i o n s o f k a i n a t e t o i n d u c e f o c a l l e s i o n s t h r o u g h o u t t h e l i m b i c s y s t e m . T h a t t h e r e may be a c o r r e l a t i o n b e t w e e n t h e s e e l e c t r o p h y s i o l o g i c a l and d i s t a n t n e u r o t o x i c a c t i o n s o f k a i n a t e i s an i n t r i g u i n g p o s s i b i l i t y . G i v e n t h a t n e u r o n a l d i s c h a r g e e n h a n c e s k a i n a t e ' s n e u r o t o x i c i t y i t w o u l d seem r e a s o n a b l e t o s u g g e s t t h a t low c o n c e n t r a t i o n s o f k a i n a t e (10-100 n m o l e s / 1 ) w o u l d augment t h e n e u r o t o x i c c a p a b i l i t i e s o f t h e amino a c i d by p r o m o t i n g t h e s y n a p t i c a 1 l y - e v o k e d d i s c h a r g e o f c e l l s . T h i s s e q u e n c e o f e v e n t s s e e m s p a r t i c u l a r l y a p p l i c a b l e t o t h e l i m b i c s y s t e m where c e l l s a r e p r o n e t o e p i l e p t i f o r m f i r i n g and h a v e a h i g h d e n s i t y o f k a i n a t e r e c e p t o r s ( F o s t e r e_t a l . , 1981) and where t h e n e u r o n a l c i r c u i t r y w o u l d f a v o r a p r o g r e s s i v e i n c r e a s e o f r e c u r r e n t s y n a p t i c e x c i t a t i o n . I n s u p p o r t o f t h i s t h e o r y i t h a s been shown t h a t f o l l o w i n g i t s i n t r a c e r e b r a l i n j e c t i o n (2-5 n m o l e s i n c_a. 1 jxl) k a i n a t e d i f f u s e s r a p i d l y a n d w i d e l y t h r o u g h o u t t h e b r a i n ( S c h e r e r - S i n g l e r and McGeer, 1979; Zac'zek e t a l . , 1980 ) . M e c h a n i s m ( s ) o f k a i n a t e ' s e p i l e p t o g e n i c i t y P r e v i o u s s t u d i e s w i t h c o n v u l s a n t s s u c h as p e n i c i l l i n ( D i n g l e d i n e and G j e r s t a d , 1980) and B I C (Wong and T r a u b , 1983) h a v e shown t h a t a r e d u c t i o n o f G A B A - m e d i a t e d i n h i b i t i o n may be 178 a c r u c i a l f a c t o r i n t h e o n s e t o f e p i l e p t i f o r m a c t i v i t y . C o n s i s t e n t w i t h t h i s h y p o t h e s i s t h e r e s u l t s o f e x t r a - a n d i n t r a c e l l u l a r r e c o r d i n g s ( s e e a l s o F i s h e r and A l g e r , 1983) showed t h e o n s e t o f b u r s t f i r i n g i n C A l t o be a s s o c i a t e d w i t h a n a b o l i t i o n o f G A B A - m e d i a t e d i n h i b i t i o n . I t a l s o s e e m s l i k e l y t h a t d i s i n h i b i t i o n may be i n v o l v e d i n t h e k a i n a t e -i n d u c e d s p o n t a n e o u s a n d e v o k e d e p i l e p t i f o r m d i s c h a r g e s o f p o p u l a t i o n s o f CA3 n e u r o n s ( F i s h e r and A l g e r , 1 9 8 3 ) . C o n s i s t e n t w i t h t h i s h y p o t h e s i s i n t h e d e n t a t e g y r u s b a t h -a p p l i e d k a i n a t e d i d n o t r e d u c e r e c u r r e n t i n h i b i t i o n and o r t h o d r o m i c r e s p o n s e s , a l t h o u g h g r e a t l y p o t e n t i a t e d , c o n t i n u e d t o be c o m p r i s e d o f s i n g l e P S s ; s u b s e q u e n t b l o c k a d e o f G A B A - m e d i a t e d i n h i b i t i o n w i t h B I C d i d c a u s e e p i l e p t i f o r m f i r i n g ( c f . M i s g e l d e t a l . , 1 9 8 2 ) . W h i l e d i s i n h i b i t i o n may be a m a j o r f a c t o r u n d e r l y i n g k a i n a t e ' s e p i l e p t o g e n i c ! t y t h e c o n t r i b u t i o n o f o t h e r a c t i o n s c a n n o t be d i s c o u n t e d . Thus t h e s i m u l t a n e o u s e n h a n c e m e n t o f r e c u r r e n t e x c i t a t i o n ( T r a u b and Wong, 1983) and t h e a t t e n -u a t i o n o f r e c u r r e n t i n h i b i t i o n may e x p l a i n t h e r e q u i r e m e n t f o r l o w d o s e s o f k a i n a t e t o e v o k e e p i l e p t i f o r m a c t i v i t y i n CA3 n e u r o n s . F i n a l l y c h a n g e s i n membrane b i o p h y s i c a l p r o p e r t i e s l e a d i n g t o b u r s t f i r i n g ( P r i n c e , 1 9 7 8 ; H o t s o n a n d P r i n c e , 1981) a r e p o s s i b l e s i n c e W e s t b r o o k and Lothman ( 1 9 8 3 ) have n o t e d t h e i n d u c t i o n o f c u r r e n t - e v o k e d b u r s t f i r i n g i n k a i n a t e - t r e a t e d C A l b p y r a m i d s . However t h a t a n t i d r o m i c s t i m u l i i n CA3 d i d n o t c a u s e b u r s t f i r i n g i n k a i n a t e - t r e a t e d s l i c e s i n w h i c h s y n a p t i c t r a n s m i s s i o n had b een b l o c k e d 179 s u g g e s t s t h a t c h a n g e s o f p a s s i v e membrane c h a r a c t e r i s t i c s a r e r e l a t i v e l y u n i m p o r t a n t . 180 CHAPTER 6 FOLIC ACID-INDUCED CHANGES OF NEURONAL E X C I T A B I L I T Y I n t r o d u c t i o n K a i n i c a c i d i s a p o w e r f u l n e u r o n a l e x c i t a n t w h i c h e l i c i t s a c o m p l e x s p e c t r u m o f r e a c t i o n s when i n j e c t e d i n t r a c e r e b r a l l y . T h e s e i n c l u d e t h e p r o d u c t i o n o f e l e c t r o -e n c e p h a l o g r a p h i c and b e h a v i o r a l s e i z u r e s i n a d d i t i o n t o t h e d e s t r u c t i o n o f n e u r o n s b o t h a t s i t e o f t h e i n j e c t i o n and i n d i s t a n t b r a i n s t r u c t u r e s p a r t i c u l a r l y t h o s e i n t h e l i m b i c s y s t e m . A number o f n e u r o d e g e n e r a t i v e d i s e a s e s i n humans a r e a l s o c h a r a c t e r i z e d by l o c a l i z e d o r d i f f u s e l e s i o n s o f s p e c i f i c c l a s s e s o f n e u r o n s . F o r e x a m p l e t h e n e u r o p a t h o l o g i e s e v i d e n t a t a u t o p s y i n p a t i e n t s w i t h t e m p o r a l l o b e e p i l e s p y , H u n t i n g -t o n ' s d i s e a s e o r c e r e b e l l a r d e g e n e r a t i o n b e a r a s t r i k i n g s i m i l a r i t y t o t h o s e w h i c h may be i n d u c e d by k a i n i c a c i d . I m p l i c i t i n t h e s e o b s e r v a t i o n s i s t h e p o s s i b i l i t y t h a t k a i n i c a c i d r e c e p t o r s may be i n v o l v e d i n t h e e t i o l o g y o f some n e u r o l o g i c a l d i s o r d e r s and t h e r e f o r e t h e i d e n t i f i c a t i o n o f t h e en d o g e n o u s l i g a n d f o r t h e r e c e p t o r i s o f more t h a n h e u r i s t i c i n t e r e s t . A l t h o u g h s h a r i n g s t r u c t u r a l s i m i l a r i t i e s w i t h k a i n a t e i t seems u n l i k e l y t h a t L - g l u t a m a t e may f u l f i l l t h e r o l e o f t h e e n d o g e n o u s a g o n i s t f o r k a i n a t e r e c e p t o r s . Thus a n t a g o n i s t s o f 181 L - g l u t a m a t e e x c i t a t i o n s do n o t a f f e c t k a i n a t e - i n d u c e d r e s p o n s e s (McLennan, 1981; W a t k i n s and E v a n s , 1981); L-3 g l u t a m a t e w e a k l y i n h i b i t s s p e c i f i c [ H ] - k a i n a t e b i n d i n g t o n e u r o n a l membranes (London and C o y l e , 1979); t h e i o n i c c o n d u c t a n c e s u n d e r l y i n g t h e d e p o l a r i z i n g a c t i o n s o f the two e x c i t a n t s d i f f e r (Evans et. a_l. , 1977; MacDonald and W o j t o w i c z , 1980); t h e e x c i t a t o r y p o t e n c y o f k a i n a t e r e q u i r e s i n a d d i t i o n t o t h e t h r e e c h a r g e d g r o u p s an i n t a c t i s o p r o p y l e n e s i d e c h a i n ( J o h n s t o n e t a _ l . , 1974 ); a n d t h e n e u r o t o x i c p r o p e r t i e s o f k a i n a t e and L - g l u t a m a t e a r e d i s s i m i l a r ( N a d l e r et. a_l. , 1 9 81). N o n e t h e l e s s s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s s u g g e s t t h a t t h e l i g a n d f o r t h e k a i n a t e r e c e p t o r i s l i k e l y t o i n c o r p o r a t e a g l u t a m a t e - l i k e c o n f o r m a t i o n . F o l i c a c i d ( p t e r o y 1 - L - g l u t a m i c a c i d ) c o n s i s t s o f a m o l e c u l e o f L - g l u t a m a t e c o u p l e d by an N - a c y l bond t o p t e r o i c a c i d ; t h e r e d u c e d f o r m o f t h e m o l e c u l e , 5 , 6 , 7 , 8 - t e t r a h y d r o -f o l i c a c i d (THF) a c t s as a m e t h y l g r o u p a c c e p t o r t o y i e l d t h e 5 5 co-enzymes N - f o r m y l - T H F ( f o l i n a t e ) and N -m e t h y l - T H F (MTHF). A number o f s t u d i e s have s u g g e s t e d a r o l e f o r f o l a t e i n t h e m o d u l a t i o n o f n e u r o n a l e x c i t a b i l i t y . H a y a s h i (1959) f i r s t r e p o r t e d the p o t e n t , c o n v u l s a n t a c t i o n s o f 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 s o f f o l i c a c i d i n d o g s a n d t h i s e f f e c t h a s b e e n c o n f i r m e d by o t h e r s u s i n g s y s t e m i c o r i n t r a c e r e b r a l a d m i n i -s t r a t i o n s o f f o l a t e , f o l i n a t e and MTHF i n r a t s ( N o e l l et. a l . , 1960; S p e c t o r , 1971; H o m e s and Obbens, 1972). An i n c r e a s e o f b r a i n f o l a t e c o n c e n t r a t i o n s has a l s o been c o r r e l a t e d w i t h an enhancement o f k i n d l i n g ( G o f f e t a_l. , 1978) o r s o u n d - i n d u c e d 182 s e i z u r e s ( C o o k e , 1978) and an e l e v a t i o n o f f o l a t e l e v e l s i s m e a s u r a b l e i n c o b a l t - i n d u c e d e p i l e p t i c f o c i ( M a y e r s d o r f e_t a l . , 1 9 7 1 ) . O r i g i n a l l y a m e c h a n i s m o f a c t i o n i n v o l v i n g a ch a n g e o f n e u r o t r a n s m i t t e r m e t h y l a t i o n r e a c t i o n s was p r o p o s e d , h o w e v e r t h i s h y p o t h e s i s h a s s i n c e b een p r o v e n i n c o r r e c t ( M e l l e r e t a l . , 1974 ) . R e c e n t l y Ruck e t a l . (1 9 8 0 ) r e p o r t e d t h a t MTHF i n h i b i t e d (K.= 0.88jXM) t h e b i n d i n g o f [ 3 H ] - k a i n a t e t o r a t 3 c e r e b e l l a r membranes; MTHF o n l y w e a k l y a n t a g o n i z e d [ H ] -g l u t a m a t e b i n d i n g (K^ = 83yA.M). Aware o f t h i s f i n d i n g , O l n e y e t a l . ( 1 9 8 1 , a , b ) c o m p a r e d t h e n e u r o t o x i c i t y o f i n t r a c e r e b r a l i n j e c t i o n s o f f o l a t e and i t s d e r i v a t i v e s w i t h t h e n e u r o n a l d e a t h p r o d u c e d b y k a i n a t e . T h e y f o u n d t h a t i n t r a s t r i a t a l i n j e c t i o n o f f o l i c a c i d ( 5 0 - 1 0 0 n m o l e s ) i n d u c e d p r o l o n g e d s e i z u r e a c t i v i t y a n d p r o d u c e d t h e k a i n i c a c i d - l i k e r e m o t e l e s i o n s . F o l i c a c i d d i d n o t d e s t r o y n e u r o n s l o c a l l y and i t s p o t e n c y a s a n e u r o t o x i n was g r e a t e r t h a n t h a t o f MTHF b u t 1-2 o r d e r s o f m a g n i t u d e l e s s t h a n t h a t f o r k a i n a t e . T o g e t h e r t h e s e d a t a s u g g e s t t h a t t h e a c t i o n s o f f o l a t e may be m e d i a t e d a t l e a s t i n p a r t by a n i n t e r a c t i o n w i t h k a i n a t e r e c e p t o r s . A c c o r d i n g l y i n t h e p r e s e n t s t u d y f o l i c a c i d was a p p l i e d t o e a c h o f t h e t h r e e m a j o r r e g i o n s o f t h e r a t h i p p o c a m p a l s l i c e t o d e t e r m i n e c h a n g e s i n n e u r o n a l a c t i v i t y w i t h t h e a i m s o f (1) u n d e r s t a n d i n g t h e e l e c t r o p h y s i o l o g i c a l b a s i s f o r i t s c o n v u l s a n t p r o p e r t i e s (2) a l l o w i n g a c o m p a r i s o n 183 w i t h k a i n a t e - i n d u c e d a l t e r a t i o n s o f s y n a p t i c p r o c e s s e s and (3) r e l a t i n g t h e e l e c t r o p h y s i o l o g i c a l and n e u r o t o x i c a c t i o n s o f f o l a t e . M e t h o d s F o r t h e e x p e r i m e n t s 73 h i p p o c a m p a l s l i c e s o b t a i n e d f r o m 52 m a l e a l b i n o r a t s were m a i n t a i n e d in_ v i t r o u s i n g s t a n d a r d i n c u b a t i o n c o n d i t i o n s d e s c r i b e d i n C h a p t e r 2 where t h e t e c h n i q u e s f o r e v o k i n g and r e c o r d i n g e x t r a - and i n t r a c e l l u l a r r e s p o n s e s a r e a l s o o u t l i n e d . A f t e r a 5-10 m i n p e r i o d o f r e c o r d i n g s t a b l e s u b m a x i m a l r e s p o n s e s t h e p e r f u s i n g medium was c h a n g e d t o one c o n t a i n i n g - 4 - 3 10 -10 M f o l a t e a n d i n m o s t c a s e s 1 0 - 1 5 m l w e r e a d m i n -i s t e r e d b e f o r e r e t u r n i n g t o n o r m a l p e r f u s a t e . The e f f e c t s o f — 6 B I C ( 1 - 5 x 1 0 M) w e r e a l s o e x a m i n e d i n some e x p e r i m e n t s . A l l d r u g s o l u t i o n s were f r e s h l y p r e p a r e d and a d j u s t e d t o a f i n a l pH o f 7.3-7.4. R e s u l t s E f f e c t s o f f o l a t e i n C A l and t h e d e n t a t e g y r u s B e c a u s e f o l a t e e l i c i t e d i n CA3 n e u r o n s s p o n t a n e o u s a c t i v i t y w h i c h p r o d u c e d v i a S c h a f f e r c o l l a t e r a l f i b r e s a s y n a p t i c a c t i v a t i o n o f C A l c e l l s ( s e e b e l o w ) , t h e f o l a t e -i n d u c e d c h a n g e s i n C A l r e s p o n s e s w e re a s s e s s e d i n s l i c e s f r o m w h i c h t h e CA3 r e g i o n had been r e m o v e d . P e r f u s i o n o f f o l a t e -3 (10 M, 10-15 ml) p r o d u c e d a l a r g e and l o n g - l a s t i n g (up t o 184 Pre - treatment Folate I Recovery A , J 2 m V 4 msec B. — 3 V , , , , i * i —, -5 0 -10 2 0 . 3 0 4 5 5 0 T . m e ( m m ) F i g . 6.1 The a c t i o n s o f f o l a t e on r e s p o n s e s i n C A l evoke d by . s t i m u l a t i o n o f the S c h l a t t e r c o l l a t e . r a l - c o m m i s s u r a l (SC) pathway. A. R e c o r d s (3 c o n s e c u t i v e s u p e r i m p o s e d sweeps) f r o m s t r a t u m p y r a m i d a l e b e f o r e , a t t h e p e a k o f e f f e c t a n d -3 f o l l o w i n g p e r f u s i o n w i t h f o l a t e (10 M), s t i m u l u s 6 V. B. R e c o r d s (3 c o n s e c u t i v e s u p e r i m p o s e d sweeps) o f EPSPs r e c o r d e d f r o m s t r a t u m r a d i a t u m , e v o k e d w i t h t h r e e d i f f e r e n t i n t e n s i t i e s o f s t i m u l a t i o n . In t h i s and s u b s e q u e n t f i g u r e s t h e t i m e o f s t i m u l a t i o n i s i n d i c a t e d by an arrow below t h e r e c o r d s and n e g a t i v i t y i s downwards. C. In a n o t h e r s l i c e t i m e c o u r s e s o f p o t e n t i a t i o n o f t h e PS and d e p r e s s i o n o f t h e EPSP r e c o r d e d s i m u l t a n e o u s l y i n s t r a t a p y r a m i d a l e and r a d i a t u m r e s p e c t i v e l y . F o l a t e was p e r f u s e d d u r i n g t h e time i n d i c a t e d a t a r a t e o f c a . 1 ml/min. 185 F i g . 6.2 A c t i o n s o f f o l a t e i n a r e a d e n t a t a . Conven-t i o n s as f o r F i g . 6.1. A. R e c o r d s from s t r a t u m g r a n u l o s u m ; s t i m u l a t i o n o f t h e m e d i a l p e r f o r a n t p a t h (6 V ) . B. R e c o r d s f r o m t h e m i d d l e p o r t i o n o f s t r a t u m m o l e c u l a r e ; n o t e t h e a b s e n c e o f any c h a n g e i n t h e EPSP i n t h i s s l i c e . C. T i m e c o u r s e s o f t h e changes e l i c i t e d by f o l a t e . 186 Stimulus Intensity (V) B. -—- 8 -i > E -4 -Q_ 00 Q_ L U o -4 " T ~ 5 7 S t imu lus Intens i ty ( V ) P r e - t reatment F o l a t e (10 M , 10 m l ) F i g . 6.3 A v e r a g e c u r v e s ( e a c h p o i n t mean of 3 s u c c e s s i v e r e s p o n s e s , s t a n d a r d d e v i a t i o n s s m a l l e r t h a n s y m b o l s ) r e l a t i n g i n t e n s i t y o f s t i m u l a t i o n and a m p l i t u d e s of e v o k e d r e s p o n s e s b e f o r e a n d a t t h e p e a k o f t h e e f f e c t o f f o l a t e , A i n C A l , B i n a r e a d e n t a t a . Note t h e r e d u c t i o n o f the EPSP i n C A l a t a l l s t i m u l u s i n t e n s i t i e s , i n c l u d i n g t h o s e t o o s m a l l t o e l i c i t a PS; and t h e l e s s e r a c t i o n o f f o l a t e on d e n t a t e EPSPs. 187 1.5 h) p o t e n t i a t i o n o f t h e PS (maximum 467+59%, mean +S.E.M., n=8) e v o k e d by s t i m u l a t i o n o f t h e SC p a t h w a y . The t i m e c o u r s e o f t h e c h a n g e s i n t h e a m p l i t u d e s o f t h e PS and EPSP t o g e t h e r w i t h t y p i c a l r e s p o n s e s a r e i l l u s t r a t e d i n F i g . 6.1. The r e d u c t i o n o f t h e EPSP i n F i g . 6.IC may have been a t t r i b u t a b l e i n p a r t t o t h e a p p e a r a n c e o f a s m a l l r e v e r s e d PS i n t h e f i e l d p o t e n t i a l r e c o r d e d i n t h e s t r a t u m r a d i a t u m ( F i g . 6.1B:7 V) ; h o w e v e r s i m i l a r r e d u c t i o n s were a l s o o b s e r v e d i n EPSPs e v o k e d by s t i m u l i w h i c h w e re s u b t h r e s h o l d f o r PS g e n e r a t i o n ( F i g . 6.1B:3 V ) . I n t h e d e n t a t e g y r u s s i m i l a r d o s e s o f f o l a t e m a r k e d l y i n c r e a s e d t h e a m p l i t u d e o f t h e PS e v o k e d by MPP s t i m u l a t i o n , i n c r e a s e s o f 760+177% (n=3) h a v i n g been o b s e r v e d ( F i g . 6 . 2 ) . D e p r e s s i o n o f t h e EPSP r e c o r d e d s i m u l t a n e o u s l y i n t h e m i d d l e m o l e c u l a r l a y e r , t h e r e g i o n o f MPP s y n a p t i c t e r m i n a t i o n , was o b s e r v e d i n o n l y one o f t h r e e s l i c e s ( F i g . 6.2C) and was l e s s t h a n t h a t r e c o r d e d i n C A l . F i g . 6.3 shows t h e c h a n g e o f t h e i n p u t / o u t p u t r e l a t i o n s b e t w e e n t h e s t i m u l u s i n t e n s i t y a n d t h e E P S P a n d PS i n C A l ( F i g . 6. 3A) a n d t h e d e n t a t e g y r u s ( F i g . 6. 3B) . I n b o t h h i p p o c a m p a l a r e a s t h e t r a n s i e n t a p p e a r a n c e o f a s e c o n d PS was s o m e t i m e s n o t e d d u r i n g t h e peak o f t h e a c t i o n o f f o l a t e and t h e l a t e n c y o f t h e f i r s t s p i k e was r e d u c e d . The a m p l i t u d e and w a v e f o r m o f r e s p o n s e s e v o k e d by AD s t i m u l i were u n a f f e c t e d ( d a t a n o t s h o w n ) . E f f e c t s i n CA3 -4 W i t h l o w e r d o s e s (10 M, 10-15 ml) f o l a t e i n c r e a s e d t h e a m p l i t u d e and d e c r e a s e d t h e l a t e n c y o f o r t h o d r o m i c P Ss 188 e v o k e d by FIM s t i m u l i ( F i g . 6.4b,d; 6 . 5 A b , e ) . T h e r e a f t e r a p r o g r e s s i v e i n c r e a s e i n t h e number and a m p l i t u d e o f PSs w h i c h i m m e d i a t e l y f o l l o w e d i t was o b s e r v e d . I n c o n t r a s t n e i t h e r t h e M F - e v o k e d 1° PS n o r t h e AD PS e l i c i t e d by S c h a f f e r c o l l a t e r a l s t i m u l a t i o n was a f f e c t e d b u t t h e n u m b e r a n d a m p l i t u d e o f f o l l o w i n g P S s i n c r e a s e d ( F i g s . 6 . 4 a , c , e , g ; 6 . 5 c , f ) . The a p p e a r a n c e o f m u l t i p l e PSs i n e v o k e d r e s p o n s e s a l s o c o i n c i d e d w i t h t h e o n s e t o f s p o n t a n e o u s p o p u l a t i o n b u r s t s ( S P B s ) ( F i g . 6.5g; 6 . 8 ) . I n i t i a l l y S P B s w e r e i n f r e q u e n t a n d o f s m a l l a m p l i t u d e and d u r a t i o n ; h o w e v e r 5-10 m i n l a t e r t h e SPBs were o f 6-12 mV a m p l i t u d e (peak p o s i t i v i t y t o peak n e g a t i v i t y ) and d i s c h a r g e d w i t h a c l o c k - l i k e r e g u l a r i t y w i t h i n t e r b u r s t i n t e r v a l s v a r y i n g f r o m 5-10 s e c i n d i f f e r e n t s l i c e s ( F i g . 6 . 8 a - l o w e r t r a c e ) . SPBs o c c u r r e d i n e v e r y s u b r e g i o n o f CA3 and p e r s i s t e d u n t i l t h e e x p e r i m e n t was t e r m i n a t e d (up t o 4 h _ 3 l a t e r ) . When 10 M s o l u t i o n s o f f o l a t e were p e r f u s e d t h e o n l y d e t e c t a b l e d i f f e r e n c e was t h e f a s t e r o n s e t o f e v o k e d and s p o n t a n e o u s e p i l e p t i f o r m a c t i v i t y . A d i r e c t d e p r e s s i o n o f CA3 e v o k e d r e s p o n s e s by f o l a t e was n e v e r o b s e r v e d . W i t h t h e a p p e a r a n c e o f l a r g e a m p l i t u d e S P B s l a r g e v a r i a t i o n s ( u p t o 100 m s e c ) o f t h e l a t e n c y o f o r t h o d r o m i c r e s p o n s e s e v o k e d by low i n t e n s i t i e s ( 5 - 1 5 V) o f FIM s t i m u l i w e r e o b s e r v e d ( F i g . 6.5h; 6.6A; 6 . 7 ) . The l a t e n c y o f t h e d e l a y e d b u r s t s a p p e a r e d t o be r e l a t e d t o t h e t i m i n g o f t h e s t i m u l u s f o l l o w i n g a n SPB a n d t o t e s t t h i s h y p o t h e s i s t h e f i r s t s p i k e o f an SPB was g a t e d w i t h an a d j u s t a b l e DC l e v e l and t h e o u t p u t s i g n a l o f t h e d i s c r i m i n a t o r was u s e d t o t r i g g e r 189 F i g . 6.4 E f f e c t s o f f o l a t e ( 1 0 M) i n CA3 o n r e s p o n s e s r e c o r d e d i n s t r a t u m p y r a m i d a l e and e v o k e d by s t i m u l a t i o n o f MF o r FIM o r t h o d r o m i c i n p u t s . The g r a p h s , f r o m b e l o w u p w a r d s , p l o t t h e t i m e c o u r s e o f c h a n g e s o f a m p l i t u d e o f t h e p r i m a r y , s e c o n d a r y , t e r t i a r y a n d q u a t e r n a r y P S s ; a n d s a m p l e r e c o r d s ( s i n g l e sweeps) t a k e n a t t h e t i m e s i n d i c a t e d i n t h e l o w e r m o s t g r a p h a r e shown a b o v e . MF-evoked r e s p o n s e s : a , c , e , g , ; F I M - e v o k e d : b , d , f , h . The i n i t i a l 1.5 msec o f t h e s a m p l e r e c o r d s a r e c o n t a m i n a t e d by " r i n g i n g " o f t h e s t i m u l u s a r t e f a c t i n t h e t a p e r e c o r d i n g s y s t e m . F o l a t e a p p l i c a t i o n was t e r m i n a t e d b e f o r e s p o n t a n e o u s b u r s t i n g a c t i v i t y d e v e l o p e d . N o t e t h e l a c k o f p o t e n t i a t i o n o f t h e p r i m a r y PS e v o k e d by MF s t i m u l a t i o n . P e a k - t o - p e a k a m p l i t u d e s o f t h e p o p u l a t i o n s p i k e s w e r e m e a s u r e d i n t h i s e x p e r i m e n t . 190 191 ' F i g . 6.5 Changes i n s p o n t a n e o u s , FIM-evoked and AD-evoked a c t i v i t y ( f i r s t , s e c o n d and t h i r d column o f t r a c e s r e s p e c t i v e l y ) i n t h e CA3 r e g i o n o f a f q . l a . t e - t r e a t e d h i p p o -campal s l i c e . F i l m e d r e c o r d s ( s i n g l e sweeps e x c e p t i n [b] and [ c ] ) i n e a c h h o r i z o n t a l row were o b t a i n e d a t a p p r o x i m a t e l y the same t i m e . The i n i t i a l e f f e c t of f o l a t e was t o i n c r e a s e the F I M-evoked PS and r e c r u i t a d d i t i o n a l PSs ( e ) ; AD r e s p o n s e s were s i m i l a r l y a f f e c t e d s a v e f o r the a b s e n c e o f any change i n t h e AD PS (• i n f ) . A f t e r t h e a d m i n i s t r a t i o n o f ca_. 13 ml o f f o l a t e SPBs a p p e a r e d (g) and c o i n c i d e n t a l l y l a r g e s h i f t s o f t h e l a t e n c y o f t h e FIM-(h) b u t not the AD-evoked r e s p o n s e ( i ) o c c u r r e d . I f the i n t e n s i t y o f AD s t i m u l a t i o n was d e c r e a s e d so a s t o be s u b t h r e s h o l d f o r an AD PS d e l a y e d b u r s t s were o b s e r v e d ( j ) . S p o n t a n e o u s and e v o k e d e p i l e p t i f o r m d i s c h a r g e s were v i r t u a l l y i d e n t i c a l ( g , h , i , j ) . A (o) 192 8. (a) (b) (d) (e) 2.6 sec 3.6 sec ( C ) - ^ v V ^ -4.6 sec 5 6 sec 3 sec (b) 5 sec (c) 6 sec (d) 8 sec (e) (f> 10 m V 10 m s e c (b-e) •20 •  (o) 10 s e c ( f ) (f) 10 mV 10'msec (o-d) 20 (e) 10 sec ( f ) F i g . 6.6 Changes i n t h e l a t e n c y o f FIM-evoked r e s p o n s e s i n CA3 a t v a r i o u s b u r s t - s h o c k i n t e r v a l s i n f o l a t e (A.) and b i c u c u l l i n e ' (B) : t r e a t e d s l i c e s . A. (a) Two s u c c e s s i v e and r e p r e s e n t a t i v e t r a c e s showing no r e s p o n s e t o a FIM shock (9.0 V) g i v e n 2.6 s e c a f t e r a s p o n t a n e o u s b u r s t . A t a 3.6 s e c b u r s t - s h o c k i n t e r v a l and w i t h the same i n t e n s i t y o f s t i m u l a -t i o n a d e l a y e d e p i l e p t i f o r m d i s c h a r g e o c c u r r e d ( b ) ; t h e b u r s t l a t e n c y f u r t h e r d e c r e a s e d as t h e i n t e r v a l was i n c r e a s e d ( c , d ) . The l a t e n c y i n t h e c o n t r o l ( p r e - d r u g ) a c t i v a t i o n was 2.8 msec f o r a 2.5 mV PS ( n o t i l l u s t r a t e d ) . B. S t i m u l a t i o n c o n v e n t i o n s as f o r A; s t i m u l u s i n t e n s i t y c o n s t a n t a t 5.3 V. In b o t h A and B t h e e v o k e d b u r s t s were c o m p a r a b l e one t o a n o t h e r , r e g a r d l e s s o f t h e d i f f e r e n c e s i n o n s e t l a t e n c y , and r e s e m b l e d t h o s e w h i c h o c c u r r e d s p o n t a n e o u s l y ( [ e ] , and a t much s l o w e r sweep s p e e d s , [f ] ) . 193 a s i n g l e s t i m u l u s c y c l e . The i n t e r v a l b e t w e e n t h e b e g i n n i n g o f t h i s c y c l e a n d t h e d e l i v e r y o f t h e s t i m u l u s o f a l o w (3-15 V) b u t c o n s t a n t i n t e n s i t y was t h e n v a r i e d ( b u r s t - s h o c k i n t e r v a l ) . F i g . 6.6Aa i l l u s t r a t e s t h a t up t o 3 s e c f o l l o w i n g t h e SPB i t was n o t p o s s i b l e t o e v o k e any r e s p o n s e . As t h e b u r s t s h o c k i n t e r v a l was i n c r e a s e d t h e l a t e n c y o f t h e d e l a y e d b u r s t d e c r e a s e d ( F i g . 6 .6Ab-d). R e g a r d l e s s o f t h e r e s p o n s e l a t e n c y e v o k e d b u r s t s were c o m p a r a b l e one t o a n o t h e r and were i n d i s t i n g u i s h a b l e f r o m SPBs i n . t h e i r a m p l i t u d e o r d u r a t i o n ( F i g . 6 . 6 A e ) . When t h e c o n d i t i o n s o f s t i m u l a t i o n were c h a n g e d so t h a t t h e b u r s t - s h o c k i n t e r v a l r e m a i n e d c o n s t a n t and t h e i n t e n s i t y o f s t i m u l a t i o n was v a r i e d a d i r e c t r e l a t i o n s h i p b e t w e e n t h e s t i m u l u s s t r e n g t h a n d t h e l a t e n c y o f d e l a y e d b u r s t s c o u l d be d e m o n s t r a t e d . A t t h e h i g h e r i n t e n s i t i e s o f s t i m u l a t i o n t h e l a t e n c y o f t h e e v o k e d r e s p o n s e was t h e same as t h a t o b s e r v e d i n t h e p r e - t r e a t m e n t s i t u a t i o n ( F i g . 6.7:10.5 V ) . When r e s p o n s e s w e r e e l i c i t e d b y a s h o c k a p p l i e d i n CA1/CA2 o r t h e h i l u s c o m p a r a b l e v a r i a t i o n s i n t h e r e s p o n s e l a t e n c y w e r e a p p a r e n t o n l y a t i n t e n s i t i e s o f s t i m u l a t i o n w h i c h w e r e s u b t h r e s h o l d f o r t h e g e n e r a t i o n o f a n AD PS ( F i g . 6 . 5 i , j ) . I n any c a s e t h e s h i f t s o f l a t e n c y d i d n o t a p p e a r t o r e p r e s e n t any c o i n c i d e n t a l a p p e a r a n c e o f an SPB s i n c e f o r a g i v e n b u r s t - s h o c k i n t e r v a l t h e y were r e l a t i v e l y c o n s t a n t a n d o c c u r r e d i n some c a s e s f a r i n a d v a n c e o f t h e e x p e c t e d SPB. I n 3 e x p e r i m e n t s t h e s p o n t a n e o u s a c t i v i t y i n f o l a t e -t r e a t e d s l i c e s was s t u d i e d b e f o r e a n d a f t e r t h e c o n n e c t i o n s b e t w e e n t h e d e n t a t e g y r u s , f i e l d s CA3 and C A l were c u t u s i n g a 194 F i g . 6.7 The changes i n t h e l a t e n c y o f t h e FIM-evoked o r t h o d r o m i c r e s p o n s e a t v a r i o u s b u r s t - s h o c k i n t e r v a l s a r e p l o t t e d f o r s e v e r a l i n t e n s i t i e s a t s t i m u l a t i o n . E p i l e p t i f o r m - 3 a c t i v i t y was i n d u c e d by f o l a t e (10 M, 15 m l ) . Each mean (+S.E.M.) was b a s e d on t h e r e s u l t s o f f i v e s u c c e s s i v e t e s t s ( e x c e p t where i n d i c a t e d by b r a c k e t e d numbers). A t a c o n s t a n t s t i m u l u s i n t e n s i t y t h e l a t e n c y was i n v e r s e l y p r o p o r t i o n a l t o th e b u r s t - s h o c k i n t e r v a l ; when t h e b u r s t - s h o c k i n t e r v a l was c o n s t a n t t h e l a t e n c y o f t h e e v o k e d b u r s t d e c r e a s e d as t h e s t i m u l u s s t r e n g t h i n c r e a s e d . 195 h a n d - h e l d m i c r o k n i f e . E v o k e d r e s p o n s e s w e re u n c h a n g e d f o l l o w i n g t h i s s u r g i c a l i n t e r v e n t i o n . S t i m u l a t i o n on t h e CA3 s i d e o f t h e c u t b e t w e e n CA3 and C A l p r o d u c e d an AD PS f o l l o w e d by m u l t i p l e P S s i n CA3 b u t no r e s p o n s e was e v i d e n t i n C A l i n d i c a t i n g t h e c o m p l e t e n e s s o f t h e a x o n a l t r a n s e c t i o n . S t i m u l a t i o n o n t h e C A l s i d e o f t h e c u t e v o k e d a s i n g l e o r t h o d r o m i c PS and i n 1 o f 3 s l i c e s a s m a l l e r s e c o n d a r y PS. SPBs p e r s i s t e d o n l y i n t h e i s o l a t e d CA3 r e g i o n w h e r e a s i n t h e i n t a c t s l i c e t h e y w e r e r e c o r d e d b o t h i n CA3 a n d C A l ( F i g . 6 . 8 d - f ) . To d e t e r m i n e i f SPBs were d e p e n d e n t on s y n a p t i c t r a n s m i s s i o n t h e e f f e c t s o f a p e r f u s i n g m e d i u m a l t e r e d t o 2+ 2 + c o n t a i n 0.5 mM Ca /8 mm Mg w e r e e x a m i n e d . F i g . 6 . 8 a - c i n d i c a t e s t h e e l i m i n a t i o n SPBs c o i n c i d e n t a l l y w i t h t h e b l o c k a d e o f M F -evoked e p i l e p t i f o r m f i r i n g ; s p o n t a n e o u s and o r t h o d r o m i c a l l y - e v o k e d a c t i v i t y i n C A l was a l s o a b o l i s h e d by t h e l a t t e r t r e a t m e n t ( d a t a n o t s h o w n ) . T h e s e e f f e c t s o f low 2+ 2 + Ca / i n c r e a s e d Mg w e re o b s e r v e d i n e a c h o f 4 s l i c e s t e s t e d and were r e v e r s i b l e w i t h i n 15-20 m i n o f r e t u r n i n g t o n o r m a l medium. ;' I n t r a c e l l u l a r r e c o r d i n g s were o b t a i n e d d u r i n g f o l a t e p e r f u s i o n f r o m 10 CA3 n e u r o n s w i t h r e s t i n g p o t e n t i a l s g r e a t e r t h a n -60 mV and s p i k e a m p l i t u d e s o f a t l e a s t 80 mV. I n e a c h c e l l s p o n t a n e o u s f i r i n g o f b u r s t s o f APs was s y n c h r o n i z e d w i t h SPBs r e c o r d e d e x t r a c e l l u l a r l y w i t h an a d j a c e n t e l e c t r o d e ( F i g . 6.8g) . I n j e c t i o n o f h y p e r p o l a r i z i n g c u r r e n t r e v e a l e d s u d d e n s p o n t a n e o u s membrane d e p o l a r i z a t i o n s ( F i g . 6.8h) ( a s s o c i a t e d 196 7* (a) (b) (c) 10 msec 10 sec (d) (e) ( f ) -^WiYrr , my CA I | * 4 mV CA3 40msec (e) 10 sec (d.f) 1 mV 40msec(q.h) 100 - ( i ) F i g . 6.8 ( a , b , c ) : The e f f e c t s o f p e r f u s i o n w i t h a 2+ 2 + medium c o n t a i n i n g 0.5 mM Ca and 8 mM Mg on e v o k e d ( u p p e r t r a c e s ) and s p o n t a n e o u s b u r s t s ( l o w e r t r a c e s ) o c c u r r i n g i n CA3 2+ 2 + a f t e r f o l a t e . , (a) b e f o r e ; (b) d u r i n g low [Ca ] / h i g h [Mg ] p e r f u s i o n ; ( c ) p a r t i a l r e c o v e r y f o l l o w i n g r e s t o r a t i o n o f t h e n o r m a l medium. Note the p e r s i s t e n c e o f t h e MF 1° PS i n t h e t o p t r a c e i n ( b ) . (d) and ( e ) : i n a d i f f e r e n t s l i c e s p o n -t a n e o u s b u r s t s r e c o r d e d i n s t r a t u m p y r a m i d a l e i n C A l ( u p p e r t r a c e s ) , and CA3 ( l o w e r t r a c e s ) a f t e r f o l a t e t r e a t m e n t a-re a b o l i s h e d i n C A l f o l l o w i n g t r a n s e c t i o n o f the CA3-CA1 a x o n a l pathway ( f ) . The l a t e n c y between a CA3 b u r s t and a f o l l o w e r b u r s t i n C A l i s shown i n the f a s t e r r e c o r d s ( e ) . (See t e x t f o r e x p l a n a t i o n . ) ( g ) : I n t r a c e l l u l a r l y - r e c o r d e d b u r s t f i r i n g o f a c t i o n p o t e n t i a l s i n f o l a t e - t r e a t e d CA3 n e u r o n ( l o w e r t r a c e ) was s y n c h r o n i z e d w i t h SPB. ( h ) : H y p e r p o l a r i z a t i o n o f t h i s c e l l t o -80 mV RMP r e v e a l e d a PDS u n d e r l y i n g t h e s p o n t a n e o u s d i s c h a r g e . ( i ) : a s p o n t a n e o u s b u r s t s u p e r i m p o s e d on a b a s e l i n e t o show the p r o l o n g e d h y p e r p o l a r i z a t i o n w h i c h f o l l o w s e p i l e p t i f o r m d i s c h a r g e s . 197 e x t r a c e l l u l a r l y w i t h S P B s ) w h i c h r e s e m b l e d t h e p a r o x y s m a l d e p o l a r i z a t i o n s h i f t s ( P D S s ) f i r s t d e s c r i b e d i n t h e p e n -i c i l l i n - t r e a t e d h i p p o c a m p u s i n v i v o by M a t s u m o t o and Ajmone M a r s a n ( 1 9 6 4 ) . S p o n t a n e o u s b u r s t s were f o l l o w e d by p r o l o n g e d ( 0 . 5 - 1 s e c ) h y p e r p o l a r i z a t i o n s w i t h a m p l i t u d e s o f up t o 10 mV ( F i g . 6 . 8 i ) . A c t i o n s o f f o l a t e on r e c u r r e n t i n h i b i t i o n F o l a t e has b een f o u n d t o have no o r a t b e s t v e r y weak e x c i t a t o r y p r o p e r t i e s on c o r t i c a l n e u r o n s and t h e a b s e n c e o f any d e p r e s s a n t a c t i o n s o f f o l a t e on e v o k e d r e s p o n s e s s u g g e s t e d t h a t i t was, i f a t a l l , a weak e x c i t a n t i n t h e h i p p o c a m p u s . T h e r e f o r e i t seemed p o s s i b l e t h a t t h e p o t e n t i a t i n g and e p i l e p t o g e n i c a c t i o n s o f f o l a t e m i g h t be due t o a r e d u c t i o n o f i n h i b i t o r y p r o c e s s e s . T h i s p o s s i b i l i t y was a d d r e s s e d by (1) c o m p a r i n g t h e a c t i o n s o f f o l a t e w i t h t h o s e o f B I C a n d ( 2 ) d e t e r m i n i n g t h e c h a n g e s i n r e c u r r e n t i n h i b i t i o n i n C A l w h i c h a c c o m p a n i e d f o l a t e - i n d u c e d p o t e n t i a t i o n s . A l t h o u g h E P S P s w e r e n o t m o n i t o r e d p e r f u s i o n o f B I C — 6 ( 1 - 5 x 1 0 M) p o t e n t i a t e d o r t h o d r o m i c PSs and c a u s e d e v o k e d e p i l e p t i f o r m d i s c h a r g e s i n t h e d e n t a t e g y r u s and C A l as has b e e n r e p o r t e d p r e v i o u s l y ( S c h w a r t z k r o i n a n d P r i n c e , 1 9 7 8 ; M i s g e l d e_t a _ l . , 1 9 8 2 ) . I n t h e CA3 r e g i o n B I C r e p r o d u c e d a l l o f t h e e f f e c t s o f f o l a t e . T h u s t h e a m p l i t u d e o f t h e F I M -e v o k e d o r t h o d r o m i c PS i n c r e a s e d and a d d i t i o n a l PSs were r e c r u i t e d ; r e s p o n s e s c o m p r i s e d o f b u r s t s o f P S s w e r e a l s o r e c o r d e d a f t e r MF a n d S c h a f f e r c o l l a t e r a l s t i m u l i b u t i n 198 e i t h e r s i t u a t i o n t h e a m p l i t u d e o f t h e f i r s t PS was u n c h a n g e d ( d a t a n o t s h o w n ) . I n a d d i t i o n , l a r g e v a r i a t i o n s i n t h e l a t e n c i e s o f o r t h o d r o m i c r e s p o n s e s e v o k e d by low i n t e n s i t i e s o f FIM s t i m u l a t i o n w e r e o b s e r v e d and t h e s e were c o r r e l a t e d as d i s c u s s e d p r e v i o u s l y w i t h t h e b u r s t - s h o c k i n t e r v a l and t h e s t i m u l u s s t r e n g t h ( F i g . 6.6B). C h a n g e s o f r e c u r r e n t i n h i b i t i o n i n C A l f o l l o w i n g f o l a t e t r e a t m e n t were d e t e r m i n e d e x t r a c e l l u l a r l y u s i n g p a i r e d s t i m u l i ( c f . C h a p t e r 2 ) . B r i e f l y on a l t e r n a t e o c c a s i o n s S C - e v o k e d r e s p o n s e s w e r e p r e c e d e d ( 2 0 - 3 0 m s e c ) by a c o n d i t i o n i n g AD s t i m u l u s a n d t h e i n h i b i t i o n 'was c a l c u l a t e d a s t h e p e r c e n t d i f f e r e n c e i n a m p l i t u d e b e t w e e n c o n d i t i o n e d and u n c o n d i t i o n e d o r t h o d r o m i c PSs and t h e p e r c e n t i n h i b i t i o n was p l o t t e d a g a i n s t t h e a m p l i t u d e o f t h e u n c o n d i t i o n e d PS ( e . g . F i g . 6 . 9 a ) . I n 5/5 s l i c e s f r o m w h i c h t h e CA3 r e g i o n had been r e m o v e d , a f t e r -3 t h e p e r f u s i o n o f 10-15 ml o f 10 M f o l a t e t h e r e was e i t h e r no c h a n g e o r an a p p a r e n t e n h a ncement o f t h e i n h i b i t i o n a t a t i m e when t h e u n c o n d i t i o n e d PS was p o t e n t i a t e d ( F i g . 6.9b) (maximum 5 0 9 + 7 2 % f o r a mean p r e - t r e a t m e n t a m p l i t u d e o f 1.7+0.2 mV, n = 5) . I n t h e s l i c e f r o m w h i c h t h e d a t a o f F i g . 6.9 w e r e d e r i v e d s u b s e q u e n t e x p o s u r e t o k a i n a t e c a u s e d a t r a n s i e n t d i s i n h i b i t i o n ( F i g . 6 . 9 c ) b u t a s n o t e d i n C h a p t e r 5 t h e p o t e n t i a t i n g a c t i o n o f k a i n a t e was a l s o a p p a r e n t w hen t h e i n h i b i t i o n h a d r e c o v e r e d ( F i g . 6 . 9 d ) . S i n c e n e i t h e r a c i d a f f e c t e d t h e a m p l i t u d e o f t h e AD PS any change o f i n h i b i t i o n c a n n o t be a t t r i b u t e d t o an a l t e r a t i o n o f c o n d i t i o n i n g r e s p o n s e s . 199 8CH S 4 0 -0 -(o) 80-, 40 (c) 4 6 8 2 4 6 8 Population Spike Amplitude (mV) > 6 (o) » Folo 1 6 , 1 0 % miiiiuiiiHii (b) Koinate. I0'DM (c) (d) » 6 < 5 a o CL 0 J + 10 r V / n 20 50 60 75 Time (min) 85 100 - r V A , — 110 120 130 F i g . 6.9 The e f f e c t s o f f o l a t e and k a i n a t e on r e c u r r e n t i n h i b i t i o n o f C A l n e u r o n s . .The p e r c e n t i n h i b i t i o n ( s e e t e x t f o r d e t a i l s ) o f an S C - e v o k e d t e s t r e s p o n s e when p r e c e d e d (20 msec) by a c o n d i t i o n i n g AD s t i m u l u s e v o k i n g a 3 mV a n t i d r o m i c PS i n p l o t t e d on . t h e o r d i n a t e . The s t i m u l u s i n t e n s i t y a n d t h u s t h e a m p l i t u d e o f t h e a n t i d r o m i c PS was c o n s t a n t t h r o u g h o u t t h e e x p e r i m e n t , whereas t h e s t i m u l u s v o l t a g e f o r t h e u n c o n d i t i o n e d t e s t r e s p o n s e s was v a r i e d t o p r o d u c e t h e PS a m p l i t u d e s i n d i c a t e d on t h e a b s c i s s a . T he g r a p h s (above) show t h e d e g r e e of i n h i b i t i o n , (a) c o n t r o l ; (b) -3 a f t e r p e r f u s i o n w i t h f o l a t e (10 M); (c) a f t e r p e r f u s i o n w i t h k a i n a t e (10 ^ M); (d) r e c o v e r y . The i n h i b i t i o n was r e d u c e d o n l y by k a i n a t e . The g r a p h below shows t h e time c o u r s e of t h e c h a n g e s , w i t h l e t t e r e d a r r o w s i n d i c a t i n g the t i m e s a t w h i c h t h e u p p e r g r a p h s w e r e c o n s t r u c t e d . S o l i d c i r c l e s u n c o n -d i t i o n e d o r t h o d r o m i c PS; open c i r c l e s i n h i b i t e d r e s p o n s e s . 200 D i s c u s s i o n I n C A l and t h e d e n t a t e g y r u s p e r f u s i o n o f s i m i l a r d o s e s o f f o l a t e i n c r e a s e d t h e p r o b a b i l i t y o f s y n a p t i c a l l y - e v o k e d n e u r o n a l d i s c h a r g e , an e f f e c t w h i c h was m a n i f e s t a s an i n c r e a s e i n t h e a m p l i t u d e o f t h e PS and a s s o c i a t e d w i t h no c h a n g e o r a r e d u c t i o n i n t h e peak a m p l i t u d e o f t h e d e n d r i t i c f i e l d E P S P . S i m i l a r c h a n g e s o f s y n a p t i c e x c i t a b i l i t y a r e p r o d u c e d by k a i n a t e , t h e t h r e s h o l d d o s e w i t h b a t h a p p l i c a t i o n s o f s i m i l a r d u r a t i o n s (10-15 m i n ) b e i n g c_a. 1 0 0 0 - 1 0 0 - f o l d l e s s t h a n f o r f o l a t e . I n t h e CA3 r e g i o n a 1 0 - f o l d l o w e r d o s e o f f o l a t e p o t e n t i a t e d t h e F I M - e v o k e d o r t h o d r o m i c r e s p o n s e b u t t h e M F - e v o k e d 1° PS was u n a l t e r e d . E v e n t u a l l y s t i m u l a t i o n o f e i t h e r i n p u t e l i c i t e d a f i e l d p o t e n t i a l c o m p r i s e d o f m u l t i p l e PSs and t h i s was a s s o c i a t e d w i t h t h e a p p e a r a n c e o f S P B s . The a b o l i t i o n o f s p o n t a n e o u s and a n t i d r o m i c a l l y - e v o k e d s y n c h r o n o u s b u r s t d i s c h a r g e o f CA3 n e u r o n s i n medium w h i c h b l o c k e d o r t h o d r o m i c t r a n s m i s s i o n i n d i c a t e d t h a t f o l a t e - i n d u c e d e p i l e p t o g e n e s i s was r e l a t e d t o a m o d u l a t i o n o f s y n a p t i c p r o c e s s e s . C o n s i s t e n t w i t h t h i s h y p o t h e s i s l a r g e i n t r a -c e l l u l a r P D S s , w h i c h r e p r e s e n t summed E P S P s ( J o h n s t o n a n d B r o w n , 1981; L e b e d a e_t a _ l . , 1982) w e r e o b s e r v e d t o u n d e r l i e t h e s p o n t a n e o u s b u r s t s o f CA3 n e u r o n s . S i n c e r e c u r r e n t i n h i b i t i o n i n C A l was n o t a f f e c t e d by f o l a t e i t seems u n l i k e l y t h a t t h e o n s e t o f e p i l e p t i f o r m a c t i v i t y was due t o d i s i n h i b i -t i o n , as a p p e a r s t o be t h e c a s e w i t h k a i n a t e ( C h a p t e r 5 ) , and p e n i c i l l i n ( S c h w a r t z k r o i n and P r i n c e , 1 9 7 8 ) . However, t h i s 201 p o s s i b i l i t y c a n n o t be e x c l u d e d w i t h o u t an i n t r a c e l l u l a r a s s e s s m e n t o f c h a n g e s o f I P S P s i n CA3 p y r a m i d s . I t i s e n t i r e l y p l a u s i b l e t h a t t h e e p i l e p t o g e n i c i t y o f f o l a t e may i n s t e a d l i e i n i t s c a p a b i l i t y t o e n h a n c e r e c u r r e n t e x c i t a t i o n . Compounds s u c h as 4 - a m i n o p y r i d i n e , t e t r a e t h y l a m m o n i u m , d i i s o p r o p y l p h o s p h o f l u o r i d a t e ( L e b e d a e t a _ l . , 1983) and p o s s i b l y i n c r e a s e d t K + l 0 ( C h a p t e r 2) a p p e a r a l s o t o p r o d u c e t h e i r b u r s t f i r i n g o f CA3 n e u r o n s by p r o c e s s e s o t h e r t h a n d i s i n h i b i t i o n . The t r i g g e r i n g m e c h a n i s m f o r SPBs was i n t r i n s i c t o t h e CA3 r e g i o n s i n c e t h i s a c t i v i t y p e r s i s t e d i n t h e s u r g i c a l l y i s o l a t e d CA3 i s l a n d . T h a t t h e s p o n t a n e o u s b u r s t s i n C A l were due t o s y n a p t i c e x c i t a t i o n v i a S c h a f f e r c o l l a t e r a l f i b r e s was i n d i c a t e d by t h e i r a p p e a r a n c e a t a c o n s t a n t l a t e n c y f o l l o w i n g e a c h CA3 d i s c h a r g e ( F i g . 6.8e) and t h e i r i m m e d i a t e d i s a p p e a r -a n c e f o l l o w i n g t r a n s e c t i o n o f t h e s y n a p t i c c o n n e c t i o n f r o m CA3 t o C A l . The same r e l a t i o n b e t w e e n b u r s t s o c c u r r i n g i n C A l and CA3 h a s been r e p o r t e d i n b i c u c u l l i n e - (Wong and T r a u b , 1983) and p e n i c i l l i n - t r e a t e d ( S c h w a r t z k r o i n and P r i n c e , 1978) h i p p o c a m p a l s l i c e s . An i n t r i g u i n g f e a t u r e o b s e r v e d o n l y i n t h e CA3 r e g i o n was t h e l a r g e , v a r i a b l e s h i f t s o f t h e l a t e n c y o f o r t h o -d r o m i c a l l y - e v o k e d b u r s t f i r i n g f o l l o w i n g f o l a t e , b i c u c u l l i n e o r k a i n a t e ( F i g . 5 . 6 A i i i ) t r e a t m e n t . D e l a y e d s y n c h r o n i z e d b u r s t s h a v e been r e p o r t e d as o c c u r r i n g i n t h e h i p p o c a m p u s b o t h i n v i v o ( M a t s u m o t o et_ a l . , 1969; D i c h t e r and S p e n c e r , 1969a) a n d m o r e r e c e n t l y ir\ v i t r o (Wong a n d T r a u b , 1 9 8 3 ) . I n 2 0 2 a g r e e m e n t w i t h t h e r e s u l t s o f Wong and T r a u b (1983) d e l a y e d b u r s t s c o u l d be e l i c i t e d w i t h l o w i n t e n s i t y a c t i v a t i o n o f v a r i o u s a f f e r e n t i n p u t s , a p p e a r e d i n an a l l - o r - n o n e f a s h i o n and w e r e i n d i s t i n g u i s h a b l e f r o m S P B s . I n a d d i t i o n t o c o n f i r m i n g t h e r e l a t i o n s h i p o f t h e o n s e t l a t e n c y o f d e l a y e d b u r s t s t o t h e s t i m u l u s s t r e n g t h , a c o r r e l a t i o n w i t h t h e t i m i n g o f t h e s t i m u l u s f o l l o w i n g an SPB was d e m o n s t r a t e d . I t w o u l d a p p e a r t h a t t h e d e l a y e d o n s e t o f b u r s t f i r i n g i s a t t r i b u t a b l e t o t h e t i m e r e q u i r e d f o r s i g n a l p r o c e s s i n g w i t h i n c o m p l e x l o c a l n e t w o r k s ( T r a u b and Wong, 1 9 8 3 ) . M e c h a n i s m o f A c t i o n I t i s u n l i k e l y t h a t t h e p o t e n t i a t i o n o f o r t h o d r o m -i c a l l y - e v o k e d P S s by f o l a t e i s due t o i t s r o l e i n c e l l u l a r m e t a b o l i s m . F i r s t , f o l i c a c i d i s v e r y s l o w l y t a k e n u p by s l i c e s o f b r a i n t i s s u e ( M a k u l u e_t a _ l . , 1973; S p e c t o r e t a l . , 1977) a n d t h e r e d u c t i o n o f f o l a t e t o i t s m e t a b o l i c a l l y a c t i v e f o r m s (THF) p r o b a b l y o c c u r s i n t r a c e l l u l a r l y . S e c o n d , a m e t h o p t e r i n w h i c h b l o c k s t h e e n z y m a t i c r e d u c t i o n o f f o l a t e h a s no e f f e c t on e i t h e r t h e e l e c t r o p h y s i o l o g i c a l ( C l i f f o r d and F e r r e n d e l l i , 1983) o r n e u r o t o x i c a c t i o n s ( O l n e y e t a l _ . , 1981a) o f t h e v i t a m i n . I n d e e d , a m e t h o p t e r i n i t s e l f c a u s e s s e i z u r e a c t i v i t y ( N o e l l e_t aJL. , 1960 ; a n d s e e a l s o C l i f f o r d a n d F e r r e n d e l l i , 1983) a l b e i t i n h i g h e r d o s e s t h a n a r e r e q u i r e d w i t h f o l a t e . A d i r e c t d e p o l a r i z a t i o n i s a l s o an i m p r o b a b l e e x p l a n -a t i o n o f t h e l a r g e i n c r e a s e s o f s y n a p t i c e x c i t a t i o n . Thus 203 e v e n w i t h p r o l o n g e d p e r f u s i o n s o f m i l l i m o l a r c o n c e n t r a t i o n s o f f o l a t e AD P S s w e r e u n a f f e c t e d a n d a d i r e c t d e p r e s s i o n o f o r t h o d r o m i c PS was n o t o b s e r v e d i n a n y o f t h e h i p p o c a m p a l r e g i o n s . D a v i e s and W a t k i n s ( 1 973) and McLennan ( 1 9 8 2 ) have a l s o r e p o r t e d a weak e x c i t a t i o n o f r a t c e n t r a l n e u r o n s by f o l a t e , f o l i n a t e o r MTHF, an o b s e r v a t i o n w h i c h i s i n a g r e e m e n t w i t h s t r u c t u r e - a c t i v i t y s t u d i e s o f N - a c y l a t e d a m i n o a c i d s ( C u r t i s and W a t k i n s , 1 9 6 5 ) . B e c a u s e f o l i c a c i d c o n t a i n s a g l u t a m a t e m o i e t y w i t h i n i t s s t r u c t u r e an i n t e r a c t i o n w i t h NMA o r q u i s q u a l a t e r e c e p t o r s t o p r o d u c e i t s e f f e c t s may be i m p l i e d . However, L - g l u t a m a t e i t s e l f r e p r o d u c e s n e i t h e r t h e e x c i t a b i l i t y c h a n g e s i n C A l ( s e e C h a p t e r 5) o r CA3 ( d a t a n o t shown) n o r t h e p a t t e r n o f c e l l d e a t h c a u s e d by f o l a t e ( N a d l e r e t a l . , 1 9 8 1 ) . E v i d e n c e h a s been p r e s e n t e d f o r a b l o c k a d e o f amino a c i d u p t a k e by f o l a t e i n c e r e b e l l u m ( R o b e r t s , 1974; R o b e r t s e t a l . , 1981) and s u c h an a c t i o n c o u l d c o n c e i v a b l y e x p l a i n t h e enh a n c e m e n t o f PS r e s p o n s e s s i n c e n e u r o c h e m i c a l e v i d e n c e ( s e e I n t r o d u c t i o n , C h a p t e r 4) i n d i c a t e s t h a t L - g l u t a m a t e may be a h i p p o c a m p a l n e u r o t r a n s m i t t e r . However t h e a m p l i t u d e o f t h e EPSP m e a s u r e d e i t h e r i n C A l o r t h e d e n t a t e g y r u s was n e v e r o b s e r v e d t o i n c r e a s e as m i g h t be e x p e c t e d . F u r t h e r m o r e , no a c t i o n o f f o l a t e , f o l i n a t e o r MTHF on L - g l u t a m a t e u p t a k e i n h i p p o c a m p a l t i s s u e h a s been m e a s u r a b l e (H. Mc L e n n a n , unpub-l i s h e d o b s e r v a t i o n s ) . Thus a b l o c k a d e o f n e u r o t r a n s m i t t e r u p t a k e as a m e c h a n i s m o f a c t i o n f o r f o l a t e w o u l d seem t o be p r e c l u d e d . 204 S i n c e t h e a c t i o n s o f f o l a t e t o p o t e n t i a t e o r t h o d r o m i c PSs and i n d u c e s p o n t a n e o u s and e v o k e d e p i l e p t i f o r m f i r i n g w e r e d u p l i c a t e d w i t h b i c u c u l l i n e a r e d u c t i o n o f G A B A - m e d i a t e d e v e n t s by f o l a t e may be p r o p o s e d . On t h e m a t t e r o f d i s i n h i b i -t i o n by f o l a t e t h e r e i s some d i s c r e p a n c y i n t h e l i t e r a t u r e . A n t a g o n i s m o f G A B A e r g i c p r e s y n a p t i c i n h i b i t i o n i n t h e r a t c u n e a t e n u c l e u s by i o n t o p h o r e t i c a l l y - a p p l i e d f o l a t e h a s been r e p o r t e d ( H i l l and M i l l e r , 1974) and D a v i e s and W a t k i n s ( 1 9 7 3 ) s u g g e s t e d t h a t a b l o c k a d e o f i n h i b i t i o n p r o d u c e d by e x o g e n o u s GABA may have o c c u r r e d on c a t c o r t i c a l n e u r o n s . On t h e o t h e r h a n d e l e c t r o p h y s i o l o g i c a l e v i d e n c e a r g u i n g a g a i n s t a d i s i n h i b -i t o r y m e c h a n i s m h a s been p r e s e n t e d ( H i l l et. a _ l . , 1974) and 3 f o l a t e s a f f e c t n e i t h e r H-GABA b i n d i n g (Ruck e_t a l . , 1980) n o r u p t a k e ( R o b e r t s e_t a _ l . , 1 9 8 1 a n d H. M c L e n n a n , u n p u b l i s h e d o b s e r v a t i o n s ) . The r e s u l t s o f e x p e r i m e n t s i n t h e p r e s e n t s t u d y a s s e s s i n g c h a n g e s i n r e c u r r e n t i n h i b i t i o n a r e a l s o c o n s i s t e n t w i t h t h e a b s e n c e o f any d i s i n h i b i t o r y e f f e c t o f f o l a t e . I n v i e w o f t h e s h i f t s o f t h e E P S P / P S i n p u t / o u t p u t c u r v e s w h i c h were e s p e c i a l l y l a r g e i n t h e d e n t a t e g y r u s ( F i g . 6 . 3 B ) , a m e c h a n i s m o f f o l a t e i n v o l v i n g an i n c r e a s e d c o u p l i n g b e t w e e n d e n d r i t i c d e p o l a r i z a t i o n and s p i k e i n i t i a t i o n i n t h e soma seems a r e a s o n a b l e p o s s i b i l i t y . L y n c h e t a l . ( 1 9 8 1 ) have s u g g e s t e d t h a t a s i m i l a r p r o c e s s may u n d e r l i e t h e p o t e n -t i a t i o n s p r o d u c e d by e n k e p h a l i n i n C A l , and t h e r e i s g r o w i n g e v i d e n c e f o r a c t i v e as w e l l as p a s s i v e c o n d u c t i o n o f h i p p o -c a m p a l d e n d r i t i c d e p o l a r i z i n g e v e n t s ( T r a u b and L l i n a s , 1979; 205 B e n a r d o e t a _ l . , 1 9 8 2 ) . F o l a t e may a l t e r t h e s e a c t i v e d e n d r i t i c p r o c e s s e s , a l t h o u g h no d i r e c t e v i d e n c e f o r t h i s i s c u r r e n t l y a v a i l a b l e . T h e e l e c t r o p h y s i o l o g i c a l d a t a o f t h e p r e s e n t s t u d y may p r o v i d e an i n s i g h t i n t o t h e d i f f e r e n c e s i n t h e p a t t e r n s o f n e u r o p a t h o l o g i e s p r o d u c e d by i n t r a c e r e b r a l i n j e c t i o n s o f f o l a t e and k a i n a t e . The f a c t t h a t f o l a t e , i n c o n t r a s t t o t h e s i t u a t i o n w i t h k a i n a t e , c a u s e s no l o c a l c e l l d e a t h i n t h e s t r i a t u m ( O l n e y et_ aT. , 1981b) w o u l d a p p e a r t o be due t o i t s weak e x c i t a t o r y a c t i o n s , an o b s e r v a t i o n w h i c h i s c o n s i s t e n t w i t h O l n e y ' s e x c i t o t o x i c h y p o t h e s i s ( O l n e y et_ a l . , 1 9 7 1 ) . I n t h e c e r e b e l l u m MTHF does d e s t r o y P u r k i n j e c e l l s and r e d u c e s b i o c h e m i c a l m a r k e r s f o r GABA ( R o b e r t s e_t a _ l . , 1 9 8 1 ) . However t h e much l o n g e r t i m e c o u r s e f o r t h i s e f f e c t s u g g e s t s t h a t m e t a b o l i c a l t e r a t i o n s s u c h as o c c u r i n t h e n e u r o p a t h o l o g y o f v i t a m i n B 1 2 d e f i c i e n c y ( B r e n n a n et. a l . , 1 9 8 1 ) , r a t h e r t h a n a d i r e c t d e p o l a r i z a t i o n o f n e u r o n s , i s i n v o l v e d . The d i s t a n t l e s i o n s p r o d u c e d by f o l i c a c i d a p p e a r t o be c o r r e l a t e d w i t h t h e i n d u c t i o n o f s t a t u s e p i l e p t i c u s s i n c e t r e a t m e n t w i t h d i a z e p a m r e d u c e s b o t h i t s c o n v u l s i v e and n e u r o t o x i c a c t i o n s ( F u l l e r e_t aJL., 1 9 8 1 ) . As f o r k a i n a t e i t i s n o t p o s s i b l e t o d e t e r m i n e w h e t h e r a d i f f u s i o n o f f o l a t e , f o r w h i c h t h e r e i s v i r t u a l l y no u p t a k e p r o c e s s i n t h e b r a i n ( s e e a b o v e ) , c o n t r i b u t e s t o t h e o n s e t a n d m a i n t e n a n c e o f e p i l e p t i f o r m a c t i v i t y w i t h i n v a r i o u s r e g i o n s o f t h e l i m b i c s y s t e m . U n e x p e c t e d l y t h e r e was a c o n s i d e r a b l e v a r i a t i o n i n t h e d o s e s o f k a i n a t e and f o l a t e c a u s i n g e p i l e p t i f o r m a c t i v i t y 206 i n v i v o (3 n m o l e s and 50-100 n m o l e s r e s p e c t i v e l y ) and j l n v i t r o ( 1 0 ~ 8 - 1 0 ~ 7 M and 1 0 ~ 4 - 1 0 ~ 3 M). T h e r e s u l t s o f o t h e r s l e a v e l i t t l e d o u b t t h a t t h e c o n v u l s i v e and d i s t a n t c y t o t o x i c e f f e c t s o f k a i n a t e and f o l a t e a r e b r o a d l y s i m i l a r and t h e p r e s e n t i n v e s t i g a t i o n has shown t h a t t h e i r a c t i o n s on e x c i t a t o r y s y n a p t i c p r o c e s s e s a r e c o m p a r a b l e . However i n c o n t r a s t t o t h e s i t u a t i o n w i t h k a i n a t e , f o l a t e d i d n o t p o t e n t i a t e t h e MF-evoked 1° PS, c a u s e d no d e t e c t a b l e c h a n g e o f r e c u r r e n t i n h i b i t i o n i n C A l and was n e v e r o b s e r v e d t o d e p r e s s e v o k e d r e s p o n s e s . T h i s a p p a r e n t d i s c r e p a n c y b e t w e e n 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 u l t s and t h e b i n d i n g d a t a of Ruck e_t a _ l . ( 1 9 8 0) may r e f l e c t t h e e x i s t e n c e o f d i f f e r e n t t y p e s o f k a i n i c a c i d r e c e p t o r s i n d i f f e r e n t b r a i n r e g i o n s . I n d e e d L o n g o n i e_t a _ l . ( 1982) r e p o r t e d t h a t c o m p a r e d 3 t o c e r e b e l l a r t i s s u e t h e b i n d i n g o f [ H ] - k a i n a t e i n t h e h i p p o c a m p u s was 1 0 0 0 - f o l d l e s s s e n s i t i v e t o i n h i b i t i o n by f o l a t e . C o n c e i v a b l y t h e r e may be two k a i n i c a c i d r e c e p t o r t y p e s , one w h i c h b i n d s k a i n a t e b u t n o t f o l a t e and m e d i a t e s t h e p o w e r f u l n e u r o n a l e x c i t a t i o n s and a n o t h e r w h i c h b i n d s b o t h a g o n i s t s and t r i g g e r s membrane c h a n g e s o t h e r t h a n d e p o l a r -i z a t i o n , p o s s i b l y t h o s e l e a d i n g t o an e nhancement o f s y n a p t i c e x c i t a t i o n . A n o t h e r p r o p o s a l w h i c h must be c o n s i d e r e d i s t h a t t h e e l e c t r o p h y s i o l o g i c a l a c t i o n s o f f o l a t e a r e e n t i r e l y u n r e l a t e d t o i t s a b i l i t y t o i n h i b i t k a i n a t e b i n d i n g . I n t h i s c o n n e c t i o n A u k e r e_t a _ l . ( 1982 ) f o u n d t h a t f o l a t e n e i t h e r c h a n g e d e v o k e d r e s p o n s e s n o r b l o c k e d t h e p o t e n t i a t i n g a c t i o n s o f k a i n a t e i n t h e o l f a c t o r y c o r t e x . 207 Q u e s t i o n s r e g a r d i n g t h e r e c e p t o r h o m o l o g i e s o f k a i n a t e and f o l a t e n o t w i t h s t a n d i n g , t h e p o s s i b i l i t y o f t h e i n v o l v e m e n t o f f o l a t e o r a r e l a t e d compound i n t h e m o d u l a t i o n o f n e u r o n a l e x c i t a b i l i t y e s p e c i a l l y w i t h r e g a r d t o t h e e t i o l o g y o f e p i l e p s y w o u l d seem t o w a r r a n t f u r t h e r i n v e s t i g a t i o n . I n t h i s c o n n e c t i o n B r e n n a n e_t a _ l . (1981) have d e m o n s t r a t e d a " p a r t i a l -l y c a l c i u m - d e p e n d e n t " r e l e a s e o f f o l a t e a n d i t s r e d u c e d d e r i v a t i v e s f r o m s l i c e s o f r a t c e r e b e l l u m by d e p o l a r i z a t i o n w i t h p o t a s s i u m . F u r t h e r m o r e a n i m a l s w i t h e l e v a t e d b r a i n f o l a t e l e v e l s a r e p r e d i s p o s e d t o s e i z u r e a c t i v i t y ( M i l l e r a n d W e b s t e r , 1975) and a r e more a m e n a b l e t o k i n d l i n g ( G o f f et_ a l . , 1 9 7 8 ) . F o l a t e i s a l s o 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 s y n a p t o s o m a l f r a c t i o n s ( B r i d g e r s and M c C l a i n , 1972; C a r l e_t a l . , 1980 ). F i n a l l y , i t has l o n g b een known t h a t s e r u m and CSF l e v e l s o f f o l a t e a r e r e d u c e d by a n t i c o n v u l s a n t s s u c h as d i p h e n y l h y d a n t o i n and p h e n o b a r b i t o n e , h o w e v e r t h e h y p o t h e s i s t h a t t h e t h e r a p e u t i c e f f e c t s o f t h e s e d r u g s i s r e l a t e d t o t h i s d e c l i n e o f f o l a t e r e m a i n s c o n t r o v e r s i a l ( R e y n o l d s , 1 9 7 2 ) . 208 R e f e r e n c e s A L D I N I O , C , FRENCH, E.D. & SCHWARCZ, R. 1983. The e f f e c t s o f i n t r a h i p p o c a m p a l i b o t e n i c a c i d and t h e i r b l o c k a d e by ( - ) 2 - a m i n o - 7 - p h o s p h o h e p t a n o i c a c i d : m o r p h o l o g i c a l and e l e c t r o e n c e p h a l o g r a p h i c a l a n a l y s i s . E x p . B r a i n R e s . 51:36-44. ALGER, B.E. & NICOLL, R.A. 1980. 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