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Role of norepinephrine in the hippocampal CA 1 region of the rat : inhibition and disinhibition Leung, Pak-Huen Patrick 1984

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ROLE OF NOREPINEPHRINE IN THE HIPPOCAMPAL CA1 REGION OF THE RAT: INHIBITION AND DISINHIBITION by PAK-HUEN PATRICK LEUNG B . S c , U n i v e r s i t y of B r i t i s h C o l u m b i a , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Depa 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 as. c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1984 © Pak-Huen P a t r i c k Leung, 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f % frfy^/QCO'tfY The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date .'/foXU S0y ,?<Pf i i A b s t r a c t The e f f e c t of n o r e p i n e p h r i n e (NE) p e r f u s i o n on 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 of t h e CA1 r e g i o n e v o k e d by s t r a t u m r a d i a t u m s t i m u l a t i o n were examined i n t h e r a t h i p p o c a m p a l s l i c e p r e p a r a t i o n . B o 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 h e 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) and t h e p o p u l a t i o n s p i k e , were shown t o e x h i b i t a r e v e r s i b l e d e c r e a s e i n t h e p r e s e n c e of 50M M NE. R e c u r r e n t i n h i b i t i o n , p r e s u m a b l y m e d i a t e d by b a s k e t c e l l i n t e r n e u r o n s , was a s s e s s e d by e x a m i n i n g the e f f e c t of a p r e c e d i n g c o n d i t i o n i n g p u l s e a p p l i e d t o t h e a l v e a r pathway ( a n t i d r o m i c ) on s t r a t u m r a d i a t u m e v o k e d r e s p o n s e s . NE p e r f u s i o n p r o d u c e d a r e v e r s i b l e d e c r e a s e i n r e c u r r e n t i n h i b i t o r y p r o c e s s e s r e s u l t i n g i n a d i s i n h i b i t i o n o f p y r a m i d a l c e l l e v o k e d r e s p o n s e s . E x p e r i m e n t s c a r r i e d out w i t h i n t r a c e l l u l a r r e c o r d i n g s showed c l o s e l y s i m i l a r r e s u l t s as t h e e x t r a c e l l u l a r c o r r e l a t e s . The e f f e c t s o f NE on t h e e x t r a c e l l u l a r f i e l d r e s p o n s e s r e c o r d e d i n s l i c e s o b t a i n e d f r o m a n i m a l s i n w h i c h endogenous NE had been d e p l e t e d , by i n t r a c e r e b r a l o r i n t r a v e n t r i c u l a r i n j e c t i o n s of 6 -hydroxydopamine (6-OHDA) were a l s o e x amined. P e r f u s i o n of NE on t h e s e s l i c e s r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n t h e i n h i b i t i o n of t h e EPSP r e s p o n s e and an enhancement of t h e p o p u l a t i o n s p i k e as w e l l as m u l t i p l e s p i k e d i s c h a r g e s c h a r a c t e r i s t i c o f o n - g o i n g e p i l e p t i f o r m a c t i v i t y . i i i I t i s p r o p o s e d t h a t NE e x e r t s i n h i b i t o r y i n f l u e n c e s d i r e c t l y on b o t h p y r a m i d a l and b a s k e t c e l l i n t e r n e u r o n s , t h e l a t t e r o f w h i c h would r e s u l t i n d i s i n h i b i t i o n of p y r a m i d a l c e l l d i s c h a r g e . An i m b a l a n c e between t h e s e two i n f l u e n c e s , as p e r h a p s o c c u r s f o l l o w i n g 6-OHDA l e s i o n s , may c o n t r i b u t e t o t h e g e n e r a t i o n of e p i l e p t i f o r m a c t i v i t y . i v T a b l e of C o n t e n t s A b s t r a c t i i T a b l e of c o n t e n t s i v L i s t o f f i g u r e s v i L i s t of a b b r e v i a t i o n s v i i i A c k n owledgements i x CHAPTER I . INTRODUCTION , .1 A. C e l l u l a r O r g a n i z a t i o n and I n t r i n s i c Pathways 1 B. E x t r i n s i c A f f e r e n t Pathways 5 1. E n t o r h i n a l c o r t e x / P e r f o r a n t p a t h 6 2. S e p t a l i n p u t ..7 3. C o m m i s s u r a l a f f e r e n t s 8 4. B r a i n stem a f f e r e n t s 9 a. s e r o t o n i n (5-HT) 9 b. n o r e p i n e p h r i n e (NE) 10 C. The P r e s e n t S t u d y 13 CHAPTER I I . MATERIALS AND METHODS 16 A. In V i t r o S l i c e P r e p a r a t i o n 16 B. 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 P r o c e d u r e s 17 C. P a i r e d P u l s e P a r a d i g m 19 D. 6-Hydroxydopamine L e s i o n s 22 CHAPTER I I I . RESULTS 23 A. The E x t r a c e l l u l a r F i e l d s P o t e n t i a l s 23 B. E f f e c t o f NE on F i e l d R e s p o n s e s 25 C. E f f e c t o f NE on R e c u r r e n t I n h i b i t i o n 28 1. E x t r a c e l l u l a r r e s p o n s e s 28 2. I n t r a c e l l u l a r r e c o r d i n g s 32 V D. 6-Hydroxydopamine L e s i o n s . . 35 CHAPTER IV. DISCUSSION 39 A. E f f e c t of NE on P y r a m i d a l C e l l s 39 B. E f f e c t of NE on R e c u r r e n t I n h i b i t i o n 42 C. 6-Hydroxydopamine L e s i o n and R o l e of NE i n E p i l e p t o g e n i s i s 44 D. G e n e r a l D i s c u s s i o n 50 R e f e r e n c e s 53 v i L i s t o f F i g u r e s F I G . 1. S c h e m a t i c D i a g r a m o f t h e H i p p o c a m p a l S l i c e P r e p a r a t i o n 2 F I G . 2. S c h e m a t i c D i a g r a m of t h e P a i r e d P u l s e P a r a d i g m 20 F I G . 3. C h a r a c t e r i s t i c E x t r a c e l l u l a r F i e l d R e s p o n s e s of t h e H i p p o c a m p a l CA1 R e g i o n 24 F I G . 4. Time C o u r s e o f NE A c t i o n on t h e CA1 E v o k e d R e s p o n s e s 26 F I G . 5. EPSP A m p l i t u d e v s S t i m u l u s I n t e n s i t y 27 F I G . 6. E f f e c t of NE on P a i r e d P u l s e I n h i b i t i o n 29 F I G . 7. R e l a t i o n s h i p between P a i r e d P u l s e I n h i b i t i o n and P o p u l a t i o n S p i k e A m p l i t u d e i n t h e P r e s e n c e and A b s e n c e of NE 30 F I G . 8. % I n h i b i t i o n vs A m p l i t u d e of C o n t r o l , T e s t R e s p o n s e s 31 F I G . 9. C h a r a c t e r i s t i c I n t r a c e l l u l a r R e s p o n s e s of t h e H i p p o c a m p a l CA1 P y r a m i d a l N eurons 33 F I G . 10. I n t r a c e l l u l a r C o r r e l a t e s of P a i r e d P u l s e I n h i b i t i o n 34 F I G . 11. E f f e c t of NE on t h e E x t r a c e l l u l a r EPSP R e s p o n s e s i n S l i c e s O b t a i n e d f r o m 6-OHDA-. t r e a t e d A n i m a l s -. . .36 v i i F I G . 12. E f f e c t of NE on t h e ' E x t r a c e l l u l a r P o p u l a t i o n S p i k e R e s p o n s e s i n S l i c e s O b t a i n e d from 6-O H D A - t r e a t e d A n i m a l s 37 F I G . 13. S c h e m a t i c D i a g r a m of t h e D i s t r i b u t i o n of a-and j 3 - r e c e p t o r s i n Normal and 6-0HDA-t r e a t e d C o n d i t i o n s 47 v i i i L i s t of A b b r e v i a t i o n s ACh a c e t y l c h o l i n e AChE a c e t y l c h o l i n e s t e r a s e A l v a l v e u s , a l v e a r C c o n d i t i o n i n g p u l s e of a p a i r e d p u l s e p a r a d i g m CA 1 c o r n u ammonis ( s u b f i e l d ) 1 CA2 c o r n u ammonis ( s u b f i e l d ) 2 CA3 c o r n u ammonis ( s u b f i e l d ) 3 CA4 c o r n u ammonis ( s u b f i e l d ) 4 CNS c e n t r a l n e r v o u s s y s t e m CSF c e r e b r o - s p i n a l f l u i d C-T c o n d i t i o n i n g - t e s t C-AMP c y c l i c a d e n o s i n e mono-phosphate DG d e n t a t e g y r u s EPSP 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 GABA gamma-amino-butyric a c i d GAD g l u t a m i c a c i d d e c a r b o x y l a s e G l u g l u t a m i c a c i d 5-HT 5 - h y d r o x y t r y p t a m i n e ( s e r o t o n i n ) Hz h e r t z IPSP i n h i b i 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 i .p. i n t r a - p e r i t o n e a l l y kg k i l o g r a m kHz k i l o h e r t z M m o l a r Mfi megohm ug m i c r o g r a m M l m i c r o l i t r e MM m i c r o m o l a r Mm m i c r o m e t e r , m i c r o n mg m i l l i g r a m min m i n u t e mM mi 1 1 i m o l a r msec m i l l i s e c o n d mV m i l l i v o l t NE n o r e p i n e p h r i n e NS not s i g n i f i c a n t 6-OHDA 6-hydroxydopamine RMP r e s t i n g membrane p o t e n t i a l r c o r r e l a t i o n c o e f f i c i e n t Sch S c h a f f e r c o l l a t e r a l S.D. s t a n d a r d d e v i a t i o n S.E. s t a n d a r d e r r o r S.E.M. s t a n d a r d e r r o r of t h e mean SL s t r a t u m l a c u n o s u m SM s t r a t u m m o l e c u l a r e SO s t r a t u m o r i e n s SP s t r a t u m p y r a m i d a l e SR s t r a t u m r a d i a t u m Tc c o n t r o l , t e s t p u l s e Tp t e s t p u l s e o f a p a i r e d p u l s e p a r a d i g m V v o l t i x A c knowledgements The a u t h o r w o u l d l i k e t o t hank most s i n c e r e l y t h e p a t i e n t g u i d a n c e and i n v a l u a b l e s u g g e s t i o n s of Dr. J . J . M i l l e r i n t h e c o m p l e t i o n of t h i s t h e s i s . The a u t h o r i s a l s o i n d e b t e d t o Is'tvan Mody f o r d i s c u s s i o n and c o o p e r a t i o n , and t o J o e Tay and K u r t Henze f o r t e c h n i c a l a s s i s t a n c e . I am a l s o f o r e v e r i n d e b t e d t o my p a r e n t s f o r t h e i r p a t i e n t and e n d l e s s s u p p o r t s and t h e i r i n t e r e s t i n t h i s t h e s i s . 1 CHAPTER I INTRODUCTION A. C e l l u l a r O r g a n i z a t i o n and I n t r i n s i c Pathways P h y l o g e n e t i c a l l y , t h e hippocampus i s c l a s s i f i e d as a p o r t i o n of t h e a r c h i c o r t e x i n t h a t i t s n e u r o n a l a r r a n g e m e n t i s i n t e r m e d i a t e between t h e n o n - l a m i n a r p a l e o c o r t e x and t h e m u l t i - l a y e r e d n e o c o r t e x . The b a s i c c e l l u l a r a r r a n g e m e n t of t h e s i n g l e l a y e r e d h i p p o c a m p a l f o r m a t i o n and i t s c o n n e c t i o n s have been d e s c r i b e d i n d e t a i l by C a j a l (1911) and l a t e r by L o r e n t e de No ( 1 9 3 4 ) . B r i e f l y , t h e p y r a m i d - s h a p e d p r i n c i p a l n e u r o n s a r e o r g a n i z e d i n a t i g h t l y p a c k e d l a y e r ( s t r a t u m p y r a m i d a l e ) and d i v i d e d i n t o s u b f i e l d s CA1 ( r e g i o s u p e r i o r ) , CA2 ( i n t e r m e d i a t e z o n e ) , CA3/CA4 ( r e g i o i n f e r i o r ) and t h e h i l u s ( F i g u r e 1 ) . T h e s e c e l l s have a d e n d r i t i c a r b o r i z a t i o n w h i c h e x t e n d s t o w a r d t h e h i p p o c a m p a l f i s s u r e and forms t h e a p i c a l d e n d r i t i c l a y e r w h i c h i s s u b d i v i d e d into, t h e s t r a t u m r a d i a t u m ( S R ) , s t r a t u m l a c u n o s u m (SL) and s t r a t u m m o l e c u l a r e (SM). S h o r t e r b a s i l a r d e n d r i t e s p r o j e c t i n t o t h e s t r a t u m o r i e n s (SO) a l o n g t h e s u p e r f i c i a l s u r f a c e o f t h e hip p o c a m p u s . In a d d i t i o n t o t h e p r i n c i p a l n e u r o n s d e s c r i b e d a bove, t h e r e a r e a v a r i e t y of i n t e r n e u r o n s i n t h e s e r e g i o n s w h i c h do n o t p r o j e c t o u t of t h i s s t r u c t u r e . The b e s t known of t h e s e a r e t h e c l a s s i c a l b a s k e t c e l l s f i r s t d e s c r i b e d by 2 FIGURE 1. S c h e m a t i c D i a g r a m of t h e H i p p o c a m p a l S l i c e P r e p a r a t i o n The p r i n c i p a l n e u r o n s ( p y r a m i d a l c e l l s ) of t h e hippocampus a r e a r r a n g e d i n a l a m i n a r f a s h i o n w i t h t h e c e l l b o d i e s a g g r e g a t e d i n t h e s t r a t u m p y r a m i d a l e (SP) and a p i c a l d e n d r i t e s e x t e n d i n g i n t o t h e s t r a t u m r a d i a t u m (SR) and 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 (SL-M) and b a s a l d e n d r i t e s e x t e n d i n g i n t o t h e s t r a t u m o r i e n s ( S O ) . 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 SR (SR S t i m ) a c t i v 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 s (Sch) from t h e CA3 p y r a m i d a l n e u r o n s , w h i c h form 'en p a s s a g e ' s y n a p t i c c o n t a c t s w i t h t h e d e n d r i t e s of CA1 p y r a m i d a l c e l l s , e l i c i t s t h e c h a r a c t e r i s t i c EPSP and p o p u l a t i o n s p i k e r e s p o n s e s from SR and SP, r e s p e c t i v e l y . CA1 p y r a m i d a l n e u r o n s , i n t u r n , p r o j e c t o ut of t h e h i p p o c a m p a l f o r m a t i o n i n t o t h e s u b i c u l u m (Sub) v i a t h e f i m b r i a (Fm) and v i a r e c u r r e n t c o l l a t e r a l s , a c t i v a t e t h e i n h i b i t o r y b a s k e t c e l l s . A l v e a r s t i m u l a t i o n ( A l v S t i m ) a c t i v a t e s b o t h t h e p y r a m i d a l c e l l s a n t i d r o m i c a l l y and t h e b a s k e t c e l l s o r t h o d r o m i c a l l y v i a t h e r e c u r r e n t c o l l a t e r a l s . DG, d e n t a t e g y r u s . 2.1 3 L o r e n t e de No i n 1934. The n e u r o n s have a v a r i e t y of s h a p e s i n c l u d i n g h o r i z o n t a l , p o l y g o n a l o r even p y r a m i d a l . E a c h of t h e s e i n t e r n e u r o n s r e c e i v e s e x t e n s i v e i n p u t s from axon c o l l a t e r a l s o f t h e p y r a m i d a l c e l l s and t h e y i n t u r n form d e n s e a x o n a l r a m i f i c a t i o n s t e r m i n a t i n g i n s t r a t u m p y r a m i d a l e (SP) and s t r a t u m r a d i a t u m ( B l a c k s t a d & F l o o d , 1963). The de n s e t e r m i n a l p l e x i t e n d t o e n v e l o p t h e p y r a m i d a l c e l l s much l i k e t h e b a s k e t c e l l s i n t h e c e r e b e l l u m and a r e b e l i e v e d t o m e d i t a t e i n h i b i t o r y i n f l u e n c e s on t h e p r i n c i p a l n e u r o n s . I t has been w e l l e s t a b l i s h e d t h a t g amma-amino-butyric a c i d (GABA) i s t h e n e u r o t r a n s m i t t e r m e d i a t i n g t h e i n h i b i t i o n . The t o p o g r a p h i c a l d i s t r i b u t i o n of G A B A - e r g i c n e u r o n a l e l e m e n t s has been v e r i f i e d by f o u r d i f f e r e n t t e c h n i q u e s . B i o c h e m i c a l a s s a y s ( S t o r m - M a t h i s e n & Fonnum, 1972), i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n of g l u t a m i c a c i d d e c a r b o x y l a s e (GAD) ( S a i t o e t a l , 1974), GABA u p t a k e s t u d i e s ( I v e r s e n & Schon, 1973) and measurements o f endogenous GABA (Okada & Shimada, 1975) a l l d e m o n s t r a t e a d i s t r i b u t i o n p a t t e r n t h a t c o r r e s p o n d s t o t h e p l e x i of t h e i n h i b i t o r y i n t e r n e u r o n s a r o u n d t h e p y r a m i d a l l a y e r . The f a c t t h a t d e a f f e r e n t a t i o n does n o t a l t e r GAD a c t i v i t y o r GABA u p t a k e f u r t h e r s u p p o r t s t h e i d e a t h a t GABA i s m e d i a t i n g s y n a p t i c t r a n s m i s s i o n o f i n t e r n e u r o n s i n d i g e n o u s t o t h e hippocampus ( S t o r m - M a t h i s e n , 1975). P h a r m a c o l o g i c a l l y , i t has been shown t h a t GABA a b o l i s h e s s p o n t a n e o u s o r g l u t a m a t e - i n d u c e d d i s c h a r g e s o f h i p p o c a m p a l n e u r o n s ( C u r t i s e t a l , 1971). I n t h e same s t u d y , i t was a l s o shown t h a t b o t h t h e s y n a p t i c a l l y and G A B A - i n d u c e d i n h i b i t i o n s were 4 a n t a g o n i z e d by b i c u c u l l i n e , a GABA b l o c k e r . I t i s a l s o w e l l known t h a t p r o l o n g e d e x p o s u r e t o b i c u c u l l i n e r e s u l t s i n c h a r a c t e r i s t i c s e i z u r e - l i k e m u l t i p l e s p i k e a c t i v i t y ( e . g . Wong & T r a u b , 1983). The p r i n c i p a l n e u r o n s o f t h e d e n t a t e g y r u s ( g r a n u l e c e l l s ) a r e a l s o o r g a n i z e d i n a l a m i n a r f a s h i o n l o c a t e d i n t h e s t r a t u m g r a n u l o s u m , w i t h an e x t e n s i v e a p i c a l d e n d r i t i c a r b o r i z a t i o n e x t e n d i n g t h r o u g h o u t t h e s t r a t u m m o l e c u l a r e . B a s k e t c e l l i n t e r n e u r o n s a r e a l s o f o u n d i n t h i s r e g i o n , most l i k e l y m e d i a t i n g i n h i b i t o r y i n f l u e n c e s on t h e g r a n u l e c e l l s . On t h e b a s i s o f a n a t o m i c a l s t u d i e s , t h e r e i s a w e l l d e s c r i b e d t r i - s y n a p t i c l o o p i n t e r c o n n e c t i n g t h e v a r i o u s components of t h e d e n t a t e g y r u s and hippocampus p r o p e r . Axons o f t h e g r a n u l e c e l l s form t h e mossy f i b r e b u n d l e w h i c h p r o j e c t s t o t h e CA3 r e g i o n where t h e y make s y n a p t i c c o n t a c t s w i t h t h e d e n d r i t e s of CA3 p y r a m i d a l n e u r o n s p r o x i m a l t o t h e c e l l l a y e r . E f f e r e n t p r o c e s s e s of t h e CA3 p y r a m i d a l c e l l s p r o j e c t i n t o t h e f i m b r i a , t h e major o u t p u t f i b r e b u n d l e of t h e hippocampus, and v i a u n m y e l i n a t e d c o l l a t e r a l s ( S c h a f f e r c o l l a t e r a l s ) i n t o t h e s t r a t u m r a d i a t u m of CA1 t o form 'en p a s s a g e ' s y n a p t i c c o n t a c t s w i t h t h e d i s t a l d e n d r i t e s of p y r a m i d a l c e l l s ( L o r e n t e de No, 1934; Swanson e t a l , 1978). The a x o n a l p r o j e c t i o n s of CA1 p y r a m i d a l c e l l s r u n t o w a r d t h e s t r a t u m o r i e n s and b r a n c h o f f i n t o t h e s u b i c u l u m or j o i n t h e f i m b r i a v i a t h e a l v e u s . A number o f e l e c t r o p h a r m a c o l o g i c a l s t u d i e s have d e m o n s t r a t e d g l u t a m a t e ( G l u ) t o have p o t e n t e x c i t a t o r y a c t i o n s on a wide range of n e u r o n s i n c l u d i n g t h e 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 ( Dudar, 1974). The d e n d r i t i c 5 r e g i o n of t h e p y r a m i d a l c e l l s seemed t o be more r e s p o n s i v e t o G I u t h a n t h e c e l l b o d i e s t h e m s e l v e s ( S c h w a r t z k r o i n e t a l , 1974) and t h e s e n s i t i v i t y i s d i s t r i b u t e d i n a c c o r d a n c e t o t h e shape of t h e d e n d r i t i c t r e e ( S p e n c e r et_ a_l, 1978). A u t o r a d i o g r a p h i c s t u d i e s u s i n g t r i t i a t e d - G l u showed a c o n c e n t r a t i o n i n SR and SO of t h e CA1-CA3 r e g i o n s ( I v e r s e n & S t o r m - M a t h i s e n , 1976). S u r g i c a l l e s i o n of t h e CA3 a f f e r e n t s t o t h e i p s i l a t e r a l CA1 r e g i o n r e s u l t s i n a h i g h l y s i g n i f i c a n t d r o p i n GIu u p t a k e i n t h i s r e g i o n , s u g g e s t i n g t h a t GIu m i g h t be t h e t r a n s m i t t e r s u b s t a n c e 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 pathway ( S t o r m - M a t h i s e n , 1977). B. E x t r i n s i c A f f e r e n t Pathways In a d d i t i o n t o t h e t r i - s y n a p t i c c i r c u i t d e s c r i b e d a b ove, t h e h i p p o c a m p a l f o r m a t i o n a l s o r e c e i v e s o t h e r a f f e r e n t i n n e r v a t i o n f r o m b o t h c o r t i c a l and s u b c o r t i c a l s o u r c e s , i n c l u d i n g e n t o r h i n a l c o r t e x , p y r i f o r m c o r t e x , c i n g u l u m , septum, amygdala, l o c u s c o e r u l e u s and a l s o 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 a l f o r m a t i o n v i a t h e h i p p o c a m p a l commi s s u r e s . 6 1. E n t o r h i n a l c o r t e x / P e r f o r a n t p a t h The f i r s t s y n a p s e of t h e t r i - s y n a p t i c l o o p p r e v i o u s l y d e s c r i b e d r e c e i v e s i t s p r i n c i p a l a f f e r e n t s from t h e e n t o r h i n a l c o r t e x v i a t h e p e r f o r a n t p a t h (Ramon y C a j a l , 1911; L o r e n t e de No, 1934). The axons o f t h e e n t o r h i n a l c o r t i c a l n e u r o n s p e r f o r a t e t h e h i p p o c a m p a l f i s s u r e and form 'en p a s s a g e ' s y n a p s e s w i t h t h e d e n d r i t e s of t h e g r a n u l e c e l l s i n t h e SM ( L a a t s c h & Cowan, 1966). The p r o j e c t i o n s from 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 i s t o p o g r a p h i c a l l y s p e c i f i c . The m e d i a l and l a t e r a l e n t o r h i n a l c o r t i c e s p r o j e c t v i a t h e m e d i a l and l a t e r a l p e r f o r a n t p a t h s t o 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 SM of t h e d e n t a t e g y r u s , 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 & J e u n e , 1972; S t e w a r d & S c o v i l l e , 1976). The m e d i a l and l a t e r a l e n t o r h i n a l c o r t i c e s a l s o p r o j e c t t o p o g r a p h i c a l l y o n t o 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 of t h e CA1 and CA3 r e g i o n s of t h e hippocampus ( S t e w a r d & S c o v i l l e , 1976). 7 2. S e p t a l i n p u t D e n e r v a t i o n s t u d i e s s h owing s u b s e q u e n t . d e c r e a s e i n s t a i n i n g of b o t h a c e t y c h o l i n e s t e r a s e (AChE) ( e . g . L e w i s & S h u t e , 1967) and c h o l i n e a c e t y l t r a n s f e r a s e (ChAT) ( O d e r f e l d - N o w a k e t a l , 1974) s u g g e s t e d t h a t c h o l i n e r g i c n e u r o n s from t h e m e d i a l s e p t a l n u c l e u s and d i a g o n a l band or from p r o j e c t i o n s p a s s i n g t h r o u g h t h e m e d i a l septum v i a t h e m e d i a l f o r e b r a i n b u n d l e i n n e r v a t e t h e h i ppocampus v i a t h e f i m b r i a . H i s t o c h e m i c a l s t a i n i n g by ChAT, c o n s i d e r e d a s u p e r i o r marker f o r c h o l i n e r g i c n e u r o n s ( S t o r m - M a t h i s e n & Fonnum, 1973), c o n f i r m e d t h e e a r l i e r d i s t r i b u t i o n p a t t e r n of AChE s t a i n i n g b e i n g l o c a l i z e d m a i n l y t o r e g i o n s j u s t above and below th e p y r a m i d a l and g r a n u l e c e l l l a y e r s ( S h u t e & L e w i s , 1961). Most i n v e s t i g a t o r s r e p o r t e d m a i n l y an e x c i t a t o r y a c t i o n of a c e t y l c h o l i n e (ACh) on h i p p o c a m p a l n e u r o n s ( e . g . B l a n d , 1974; Wheal & M i l l e r , 1980). S p e n c e r e t a l (1978) u s i n g a m i c r o i o n t o p h o r e t i c t e c h n i q u e have shown a d i f f e r e n t i a l p a t t e r n of ACh s e n s i t i v i t y i n a r e a s a r o u n d t h e p y r a m i d a l c e l l l a y e r s i m i l a r t o t h o s e of t h e AChE and ChAT s t u d i e s . 8 3. C o m m i s s u r a l a f f e r e n t s Two c o m m i s s u r e s , t h e d o r s a l and v e n t r a l p s a l t e r i a , c a r r y t h e f i b r e s of r e c i p r o c a l i n n e r v a t i o n s from t h e h i p p o c a m p a l f o r m a t i o n s of e a c h h e m i s p h e r e . The d o r s a l p s a l t e r i u m c o n t a i n s axons t h a t p r o j e c t t o t h e e n t o r h i n a l c o r t e x , p a r a - s u b i c u l u m , s u b i c u l u m , 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 o f CA1-CA3 and t h e d e n t a t e g y r u s ( B l a c k s t a d , 1956; G o l d o w i t z e t a l , 1975). The v e n t r a l p s a l t e r i u m c o n t a i n s i n t e r h e m i s p h e r i c p r o j e c t i o n s from t h e h i p p o c a m p a l r e g i o n t o t h e hippocampus, d e n t a t e g y r u s , p r e - s u b i c u l u m and p o s s i b l y a l s o t h e p a r a - s u b i c u l u m , and t h e e n t o r h i n a l c o r t e x ( L o r e n t e de No, 1934; B l a c k s t a d , 1956; G o t t l i e b & Cowan, 1973; Swanson e t a l , 1978). The CA1 r e g i o n of t h e hippocampus r e c e i v e s c o n t r a l a t e r a l a f f e r e n t s t o SO m a i n l y from CA3, t o SR m a i n l y from t h e h i l u s ( G o t t l i e b & Cowan, 1973; Swanson e t a l , 1978) and t o 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 a minor p r o j e c t i o n from t h e e n t o r h i n a l c o r t e x ( B l a c k s t a d , 1956). C o m m i s s u r a l p r o j e c t i o n s t o t h e CA3 r e g i o n a r i s e m a i n l y from t h e c o n t r a l a t e r a l CA3 and h i l a r r e g i o n ( G o t t l i e b & Cowan, 1973; Swanson e_t a l , 1978) w i t h t h e i n n e r v a t i o n b e i n g more p r o n o u n c e d i n SO t h a n SR (Swanson e_t a l , 1978). The r e c i p r o c a l i n n e r v a t i o n o f t h e c o n t r a l a t e r a l CA3 r e g i o n s a r e m o r e - o r - l e s s h o m o t o p i c i n d e n s i t y ( G o t t l i e b & Cowan, 1973; Swanson e t a l , 1978) . The d e n t a t e g y r u s 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 from 9 t h e c o n t r a l a t e r a l h i l u s and e n t o r h i n a l c o r t e x (Swanson e t a l , 1978; B e r g e r e_t aJL, 1981). The e n t o r h i n a l c o r t i c a l p r o j e c t i o n s t o t h e c o n t r a l a t e r a l d e n t a t e g y r u s have s i m i l a r t e r m i n a t i o n p a t t e r n s as t h e i p s i l a t e r a l p e r f o r a n t p a t h p r o j e c t i o n s : m e d i a l t o t h e m i d d l e o n e - t h i r d and l a t e r a l t o t h e o u t e r o n e - t h i r d of SM ( S t e w a r d , 1976), and a more i n t e n s e i n n e r v a t i o n t o t h e lo w e r b l a d e t h a n t h e upper b l a d e ( G o l d o w i t z e_t a l , 1975; S t e w a r d , 1976). The c o m m i s s u r a l p r o j e c t i o n f r o m t h e h i l u s t o t h e c o n t r a l a t e r a l d e n t a t e g y r u s t e r m i n a t e s i n t h e i n n e r o n e - t h i r d of t h e SM ( B l a c k s t a d , 1956; L a a t s c h & Cowan, 1967; G o t t l i e b & Cowan, 1973). 4. B r a i n stem a f f e r e n t s a. s e r o t o n i n (5-HT) The median raphe n u c l e u s has been shown t o be t h e s o u r c e of 5-HT i n n e r v a t i o n t o t h e h i p p o c a m p a l f o r m a t i o n by a s e l e c t i v e l e s i o n i n g t e c h n i q u e ( L o r e n s & G u l d b e r g , 1974). An a u t o r a d i o g r a p h i c s t u d y by Moore & H a l a r i s (1975) s u g g e s t e d t h a t axons from t h e median raphe c o u r s e t h r o u g h t h e m e d i a l f o r e b r a i n b u n d l e and a r r i v e 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 v i a t h e f o r n i x and c i n g u l u m . The same s t u d y a l s o s u g g e s t e d t h e i n n e r v a t i o n p a t t e r n of 5-HT a f f e r e n t s t o t h e hippocampus i s s i m i l a r t o t h e n o r a d r e n e r g i c i n n e r v a t i o n w h i c h w i l l be 10 d i s c u s s e d i n d e t a i l i n t h e f o l l o w i n g s e c t i o n . S e r o t o n e r g i c t e r m i n a 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 a r e m o s t l y c o n c e n t r a t e d i n a band a b o u t 65*xm wide a l o n g t h e v e n t r a l b o r d e r of t h e d e n t a t e g y r u s (Moore, 1975). S e g a l & Bloom (1974) r e p o r t e d a d e p r e s s a n t a c t i o n of 5-HT t o n e u r o n a l d i s c h a r g e of p y r a m i d a l c e l l s but t h e mechanism and f u n c t i o n a l r o l e of 5-HT i n t h i s r e g i o n o f t h e hippocampus r e m a i n u n c l e a r . b. n o r e p i n e p h r i n e (NE) E a r l y i m m u n o h i s t o c h e m i c a l s t u d i e s d e m o n s t r a t e d t h e p r e s e n c e of n o r a d r e n e r g i c t e r m i n a l s i n a l l r e g 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 w i t h t h e d e n t a t e g y r u s b e i n g more d e n s e l y i n n e r v a t e d t h a n t h e hippocampus p r o p e r (Fuxe, 1965; B l a c k s t a d e_t a l , 1967). B i o c h e m i c a l i n v e s t i g a t i o n s of NE c o n t e n t (Anden e t a_l, 1966), NE u p t a k e (Gage & Thompson, 1980) and t y r o s i n e h y d r o x y l a s e ( U r e t s k y & I v e r s e n , 1970) g e n e r a l l y a g r e e d w i t h t h e above f i n d i n g s . More r e c e n t s t u d i e s ( U n g e r s t e d t , 1971; Koda e t a l , 1978) r e v e a l e d t h a t n o r a d r e n e r g i c i n n e r v a t i o n of t h e hippocampus a r i s e s a l m o s t e x c l u s i v e l y from t h e l o c u s c o e r u l e u s . The p r o j e c t i o n , w h i c h i s m a i n l y i p s i l a t e r a l , a r r i v e s 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 r i n c i p a l l y v i a t h e v e n t r a l a m y g d a l o i d b u n d l e - a n s a p e d u n c u l a r i s (Loy e t a l , 1980). Moore & Bloom (1979) r e p o r t e d a d i f f u s e p l e x u s of n o r a d r e n e r g i c t e r m i n a l s c o n c e n t r a t i n g i n t h e SR o f CA1, but f i b r e s a r e a l s o o b s e r v e d 11 t o c o u r s e a l o n g t h e d e n d r i t i c s h a f t s of p y r a m i d a l c e l l s and b r a n c h o f f t o ,run l a t e r a l l y a l o n g SP. Loy e t a l (1980) d e s c r i b e d f l u o r e s c e n t t e r m i n a l v a r i c o s i t i e s s i t u a t e d m a i n l y a l o n g t h e d e n d r i t e s of p y r a m i d a l n e u r o n s but a few were a l s o f o u n d t o a p p o s e somata o f i n t e r n e u r o n s . The q u e s t i o n of w hether t h e s e two t y p e s of p u t a t i v e t e r m i n a l i n n e r v a t i o n s a r e t r u l y f u n c t i o n a l o r n o t w i l l be i n v e s t i g a t e d and d i s c u s s e d i n t h e p r e s e n t t h e s i s . R e c e p t o r b i n d i n g s t u d i e s d e m o n s t r a t e d s i g n i f i c a n t c o n c e n t r a t i o n s o f b o t h a-( U ' P r i c h a r d e_t a l , 1977) and / ^ - a d r e n o c e p t o r s ( C r u t c h e r & D a v i s , 1980) i n t h e h i p p o c a m p a l r e g i o n . The l a t t e r s t u d y a l s o showed, u s i n g a s p e c i f i c a - r e c e p t o r l i g a n d , t r i t i a t e d - W B 4 1 0 1 , t h a t t h e d e n s i t y o f a - r e c e p t o r b i n d i n g s i t e s i n t h e d e n t a t e g y r u s i s t w i c e t h a t f o u n d i n t h e h ippocampus p r o p e r . U s i n g t h e i o n t o p h o r e t i c t e c h n i q u e , S e g a l & Bloom (1974) f i r s t showed t h a t NE e x e r t s an i n h i b i t o r y e f f e c t on t h e s p o n t a n e o u s and A C h - i n d u c e d f i r i n g o f h i p p o c a m p a l p y r a m i d a l n e u r o n s . I n t r a c e l l u l a r r e c o r d i n g s d e m o n s t r a t e d t h a t NE m i g h t e x e r t i t s i n h i b i t o r y a c t i o n by a s l i g h t h y p e r p o l a r i z a t i o n a c c o m p a n i e d by a c o n d u c t a n c e i n c r e a s e w h i c h seemed t o d e c r e a s e t h e r e s p o n s e t o b o t h c o n s t a n t h y p e r p o l a r i z i n g and d e p o l a r i z i n g c u r r e n t p u l s e s (Langmoen e t a l , 1981). U s i n g e x t r a c e l l u l a r r e c o r d i n g p o t e n t i a l s , M u e l l e r e t al_ (1981) r e p o r t e d t h a t a l t h o u g h NE has no s i g n i f i c a n t e f f e c t on 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 EPSP, i t does a f f e c t t h e 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 a d o s e - d e p e n d e n t manner. P e r f u s i o n w i t h NE a t 5M M r e s u l t e d i n 1 2 a 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 of t h e p o p u l a t i o n s p i k e , 25/iM NE r e s u l t e d i n a b i p h a s i c p o t e n t i a t i o n - i n h i b i t i o n t y p e o f r e s p o n s e whereas p e r f u s i o n w i t h 50M M r e s u l t e d i n an i n h i b i t i o n . The same s t u d y a l s o showed t h a t c l o n i d i n e , an ( ^ " a d r e n e r g i c a g o n i s t , had an i n h i b i t o r y e f f e c t ; on t h e o t h e r hand, i s o p r o t e r e n o l , a ^ - a d r e n e r g i c a g o n i s t , e x e r t e d a p o t e n t i a t i n g e f f e c t on t h e p o p u l a t i o n s p i k e r e s p o n s e . The a u t h o r s s u g g e s t e d t h a t a - r e c e p t o r s m i g h t m e d i a t e t h e i n h i b i t o r y e f f e c t of NE whereas 0 - r e c e p t o r s m i g h t m e d i a t e t h e p o t e n t i a t i n g e f f e c t on p y r a m i d a l c e l l e x c i t a b i l i t y . T h i s o p p o s i n g r e l a t i o n s h i p c o u l d e x p l a i n t h e r e v e r s a l o f NE a c t i o n s a t low and h i g h c o n c e n t r a t i o n s . 1 3 C. The P r e s e n t S t u d y The f u n c t i o n a l r o l e of n o r e p i n e p h r i n e i n t h e CA1 r e g i o n o f t h e hippocampus was i n v e s t i g a t e d i n t h e p r e s e n t t h e s i s by a d d r e s s i n g t h r e e major q u e s t i o n s : 1. What i s t h e e f f e c t of NE on t h e 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 ? T h i s i n i t i a l s t u d y was an a t t e m p t t o c o n f i r m t h e i n h i b i t o r y a c t i o n o f NE on CA1 p y r a m i d a l c e l l a c t i v i t y o b s e r v e d by p r e v i o u s i n v e s t i g a t o r s . S p e c i a l emphasis was p l a c e d on t h e e f f e c t o f NE on t h e p o p u l a t i o n d e n d r i t i c r e s p o n s e (EPSP) i n view of t h e s u r p r i s i n g r e p o r t by M u e l l e r e t a l (1981) t h a t t h e p o p u l a t i o n EPSP i s n o t a f f e c t e d by NE i n s p i t e o f t h e a n a t o m i c a l / i m m u n o h i s t o c h e m i c a l e v i d e n c e t h a t n o r a d r e n e r g i c i n n e r v a t i o n i s c o n c e n t r a t e d i n t h e d e n d r i t i c r e g i o n of CA 1 ( B l a c k s t a d , 1967; Moore & Bloom, 1979; Loy et_ a l , 1980). 2. What i s t h e e f f e c t of NE on r e c u r r e n t b a s k e t c e l l  i n h i b i t i o n ? The e f f e c t of NE on i n h i b i t o r y p r o c e s s e s i n t h e hi p p o c a m p u s , l i k e many p a r t s of t h e CNS, has s c a r c e l y been e x a m i n e d . E n g b e r g & T h a l l e r (1970) f i r s t r e p o r t e d t h a t NE r e d u c e d t h e i n h i b i 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 s ( I P S P s ) i n s p i n a l m o t o n e u r o n s . J o r d a n & McCrea (1976) r e p o r t e d a d e p r e s s a n t a c t i o n on most of t h e s p i n a l i n t e r n e u r o n s 1 4 r e s p o n s i v e t o NE t r e a t m e n t . F l u o r e s c e n c e h i s t o c h e m i c a l s t u d i e s have shown t h a t many s m a l l i n t e r n e u r o n s f o u n d n e a r m o t o n e u r o n s a r e i n v e s t e d w i t h N E - c o n t a i n i n g f i b r e s ( J o r d a n e t a l , 1977). In t h e o l f a c t o r y b u l b , NE was r e c e n t l y d e m o n s t r a t e d t o a t t e n u a t e t h e d e n d r o d e n d r i t i c i n h i b i t o r y f e e d b a c k o f t h e g r a n u l e c e l l s o n t o t h e m i t r a l c e l l s . The IPSPs of t h e m i t r a l c e l l s were d e c r e a s e d i n b o t h a m p l i t u d e and d u r a t i o n , t h u s f a c i l i t a t i n g t h e i r f i r i n g ( J a h r & N i c o l l , 1982). In t h e h ippocampus, Loy e t a l ( 1 9 8 0 ) , u s i n g t h e f l u o r e s c e n c e h i s t o c h e m i c a l t e c h n i q u e , r e v e a l e d N E - c o n t a i n i n g v a r i c o s i t i e s a p p o s e d t o t h e somata of i n t e r n e u r o n s i n a d d i t i o n t o t h e d e n d r i t e s of t h e p y r a m i d a l c e l l s . Langmoen e t a l (1981) and M a d i s o n & N i c o l l (1982a) m e n t i o n e d t h a t NE i n v a r i a b l y d e c r e a s e d t h e s y n a p t i c a l l y e v o k e d IPSPs of t h e CA1 p y r a m i d a l c e l l s . I t seems l i k e l y t h a t NE not o n l y e x e r t s i n h i b i t o r y i n f l u e n c e s on t h e p r i n c i p a l n e u r o n s but a l s o on t h e r e c u r r e n t b a s k e t c e l l s y s t e m . In t h e p r e s e n t s t u d y , t h i s p o s s i b i l i t y was i n v e s t i g a t e d u s i n g t h e p a i r e d p u l s e p a r a d i g m , by a c t i v a t i n g t h e b a s k e t c e l l s y s t e m v i a t h e r e c u r r e n t c o l l a t e r a l s and m o n i t o r i n g i t s i n h i b i t o r y i n f l u e n c e on t h e CA1 p y r a m i d a l c e l l s i n t h e a b s e n c e or p r e s e n c e of NE. 3. What i s t h e e f f e c t o f NE on f i e l d r e s p o n s e s  f o l l o w i n g 6-hydroxydopamine l e s i o n o f t h e  h i p p o c a m p a l n o r a d r e n e r g i c i n p u t s ? On t h e b a s i s e l e c t r o p h a r m a c o l o g i c a l of p r e v i o u s h i s t o c h e m i c a l s t u d i e s , i t seems l i k e l y t h a t and NE i s 1 5 i n v o l v e d i n r e g u l a t i n g t h e a c t i v i t i e s of b o t h t h e p y r a m i d a l n e u r o n s and t h e i n h i b i t o r y i n t e r n e u r o n s of t h e h ippocampus, and t h e p r o p e r f u n c t i o n i n g of t h e hippocampus i s l i k e l y t o depend on t h e b a l a n c e between t h e s e two p r o c e s s e s . In o r d e r t o examine t h i s p o s s i b i l i t y , endogenous l e v e l s o f h i p p o c a m p a l NE were a l t e r e d by s e l e c t i v e l e s i o n s of t h e a s c e n d i n g n o r a d r e n e r g i c s y s t e m i n n e r v a t i n g t h e h i p p o c a m p a l f o r m a t i o n . A number of n e u r o t o x i n s has been r e p o r t e d t o l e s i o n c a t e c h o l a m i n e r g i c n e u r o n s s e l e c t i v e l y . I n t r a v e n t r i c u l a r i n j e c t i o n of 6 -hydroxydopa p r o d u c e d a d o s e - d e p e n d e n t r e d u c t i o n i n NE i n a wide a r e a of t h e CNS w h i l e l e a v i n g d o p a m i n e r g i c n e u r o n s i n t a c t ( R i c h a r d s o n & J a c o b o w i t z , 1973). B j o r k l u n d e t a l (1975, 1979) u s i n g 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 of 5 , 7 - d i h y d r o x y t r y p t a m i n e showed a s i m i l a r r e s u l t , i n c l u d i n g a 70% d e c r e a s e i n NE c o n t e n t i n t h e f o r e b r a i n r e g i o n . A l l of t h e above . s t u d i e s showed, u s i n g f l u o r e s c e n c e h i s t o c h e m i s t r y , a x o n a l and t e r m i n a l d e g e n e r a t i o n of t h e s e p r o c e s s e s by s p r o u t i n g as e a r l y as f i v e d a y s a f t e r t r e a t m e n t . A more p o p u l a r n e u r o t o x i n , 6-hydroxydopamine (6-OHDA), showed s i m i l a r b ut more c o m p l e t e l e s i o n s of n o r a d r e n e r g i c n e u r o n s ( U r e t s k y & I v e r s e n , 1969). In t h e p r e s e n t s t u d y , t h e e f f e c t of NE on t h e p h y s i o l o g y o f t h e h i p p o c a m p a l s l i c e p r e p a r a t i o n d e p r i v e d of i t s endogenous NE by 6-OHDA t r e a t m e n t s was i n v e s t i g a t e d . The p o s s i b i l i t y of d i s r u p t e d f u n c t i o n i n g of t h e s y s t e m due t o d i f f e r e n t i a l s u p e r s e n s i t i v i t y t o NE between t h e p y r a m i d a l c e l l s and t h e i n h i b i t o r y p r o c e s s e s w i l l be d i s c u s s e d . 16 CHAPTER I I MATERIALS AND METHODS A. In V i t r o S l i c e P r e p a r a t i o n E x p e r i m e n t s were p e r f o r m e d on 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 male W i s t a r r a t s . A n i m a l s were s a c r i f i c e d by d e c a p i t a t i o n and t h e i r b r a i n s removed and p l a c e d i n i c e - c o l d , o x y g e n a t e d 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 (CSF) c o n t a i n i n g ( i n mM): N a C l 124; KC1 5; C a C l 2 and MgS0 4 2; K 2 H P 0 4 1.25; NaHC0 3 24; D - g l u c o s e 10. F o l l o w i n g d i s s e c t i o n of t h e hippocampus from s u r r o u n d i n g t i s s u e s , t r a n s v e r s e s e c t i o n s (450£tm i n t h i c k n e s s ) were c u t p e r p e n d i c u l a r t o t h e s e p t o - t e m p o r a l a x i s u s i n g a S o r v a l l t i s s u e c h o p p e r . The s l i c e s were t h e n t r a n s f e r r e d t o a n y l o n mesh i n a r e c o r d i n g chamber and p e r f u s e d a t a r a t e of 2ml/min of o x y g e n a t e d , a r t i f i c i a l CSF, warmed t o a t e m p e r a t u r e of 34.0 ± 1.0°C. Warmed and h u m i d i f i e d 95% 0 2 , 5% C 0 2 gas m i x t u r e a l s o f l o w e d o v e r t h e s l i c e s i n t h e chamber t o e n s u r e a d e q u a t e o x y g e n a t i o n . The s l i c e s were a l l o w e d t o e q u i l i b r a t e and r e c u p e r a t e from s u r g i c a l trauma f o r a t l e a s t 45min t o an hour b e f o r e r e c o r d i n g commenced. 1 7 B. 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 P r o c e d u r e s B i p o l a r s t i m u l a t i n g e l e c t r o d e s (62 jum d i a m e t e r ) were v i s u a l l y p o s i t i o n e d , w i t h t h e a i d of a d i s s e c t i n g m i c r o s c o p e , i n t h e SR of t h e h i p p o c a m p a l CA1 r e g i o n f o r o r t h o d r o m i c a c t i v a t i o n of p y r a m i d a l c e l l s , and i n t h e a l v e u s t o evoke a n t i d r o m i c r e s p o n s e s ( F i g . 1 ) . S t i m u l i c o n s i s t e d of s i n g l e s q u a r e wave p u l s e s (1-15V, 0.1msec) d e l i v e r e d a t a f r e q u e n c y of 0.1Hz t h r o u g h an i s o l a t i o n u n i t . G l a s s m i c r o p i p e t t e s f i l l e d w i t h 2M N a C l and h a v i n g t i p r e s i s t a n c e s o f 2-6 megohms (Mfl) were u s e d t o r e c o r d t h e e x t r a c e l l u l a r EPSP r e s p o n s e i n t h e SR and t h e p o p u l a t i o n s p i k e r e s p o n s e i n t h e SP o f CA1 ( F i g . 1 ) . The a m p l i t u d e of t h e EPSP was measured f r o m b a s e l i n e t o peak, t h e a m p l i t u d e of 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 r e s p o n s e f rom t h e f i r s t 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 t h e a n t i d r o m i c s p i k e from b a s e l i n e t o peak. E l e c t r i c a l a c t i v i t y was a m p l i f i e d , f i l t e r e d ( 1 - 0 . 1 k H z ) , and d i s p l a y e d on an o s c i l l o s c o p e o r l e d t o a PDP 11/23 computer f o r o n - l i n e s i g n a l a v e r a g i n g . I n t r a c e l l u l a r r e c o r d i n g s were p e r f o r m e d w i t h g l a s s m i c r o p i p e t t e s (50-80MO) f i l l e d w i t h 1M p o t a s s i u m a c e t a t e s o l u t i o n . C e l l p e n e t r a t i o n was a i d e d by a sudden b r e a k t o h i g h c a p a c i t a n c e c o m p e n s a t i o n w h i c h c r e a t e s h i g h f r e q u e n c y o s c i l l a t i o n and v i b r a t e s t h e e l e c t r o d e t i p a t h i g h f r e q u e n c y . Once p e n e t r a t i o n was s u c c e s s f u l and s t a b i l i z e d , t h e e l e c t r o d e t i p r e s i s t a n c e was b a l a n c e d out by e l e c t r o n i c 18 s u b t r a c t i o n w h i c h i s f u n c t i o n a l l y e q u i v a l e n t t o a Whe a t s t o n e b r i d g e . Membrane p r o p e r t i e s were a s s e s s e d by m e a s u r i n g t h e r e s t i n g membrane p o t e n t i a l (RMP) and t h e i n p u t r e s i s t a n c e by c u r r e n t p u l s e i n j e c t i o n s . O n l y c e l l s t h a t met t h e f o l l o w i n g c r i t e r i a were c h o s e n f o r f u r t h e r e x p e r i m e n t a t i o n : a m p l i t u d e o f a c t i o n p o t e n t i a l s > 75mV; r e s t i n g membrane p o t e n t i a l (RMP) more n e g a t i v e t h a n -60mV and i n p u t r e s i s t a n c e > 20MO. Once s t a b i l i z e d c o n t r o l r e s p o n s e s were o b t a i n e d , NE was added t o t h e a r t i f i c i a l CSF p e r f u s a t e v i a a th r e e - w a y v a l v e . P e r f u s i o n t i m e l a s t e d f o r 15min and c o n c e n t r a t i o n s o f NE r a n g e d from 10 t o 50uM, a l t h o u g h 50;iM was u s e d i n most e x p e r i m e n t s t o e n s u r e maximal e f f e c t . Changes i n 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 s d u r i n g or f o l l o w i n g d r u g exposure-were m o n i t o r e d and e x p r e s s e d a s a p e r c e n t a g e of t h e p r e - e x p o s u r e c o n t r o l s . F o r i n t r a c e l l u l a r r e c o r d i n g s , c h a n g e s were m o n i t o r e d by a c t i o n p o t e n t i a l g e n e r a t i o n , RMP and i n p u t r e s i s t a n c e measurements. 1 9 C. P a i r e d P u l s e P a r a d i g m The p a i r e d p u l s e p a r a d i g m c o n s i s t e d of a s t i m u l a t i o n c y c l e o f a c o n t r o l , t e s t ( T c ) p u l s e f o l l o w e d by a p a i r i n g o f a c o n d i t i o n i n g (C) and t e s t p u l s e (Tp) a t p a r t i c u l a r C-T i n t e r v a l s . D a t a was c o l l e c t e d a t 3 min p e r i o d s and c o n s i s t e d of e i g h t Tc p u l s e s (0.2Hz) d e l i v e r e d t o t h e SR e l e c t r o d e t o s y n a p t i c a l l y a c t i v a t e t h e CA1 p o p u l a t i o n s p i k e . T h e s e p u l s e s were i m m e d i a t e l y f o l l o w e d by e i g h t a d d i t i o n a l t e s t p u l s e s (Tp) e a c h of w h i c h was p r e c e d e d by an a n t i d r o m i c c o n d i t i o n i n g p u l s e (C) a p p l i e d t o t h e a l v e a r ( A l v ) e l e c t r o d e a t C-T i n t e r v a l s o f 20-30msec ( F i g . 2 A ) . A l v e a r s t i m u l a t i o n n o t o n l y e v o k e s t h e c h a r a c t e r i s t i c a n t i d r o m i c p o p u l a t i o n s p i k e but a l s o o r t h o d r o m i c a l l y a c t i v a t e s t h e r e c u r r e n t c o l l a t e r a l i n h i b i t i o n v i a b a s k e t c e l l i n t e r n e u r o n s . O n - l i n e s i g n a l a v e r a g i n g was o b t a i n e d f r o m t h e l a s t 5 p u l s e s o f e i t h e r t h e t e s t r e s p o n s e s a l o n e ( T c ) o r when p a i r e d w i t h t h e c o n d i t i o n i n g A l v p u l s e ( C - T p ) . The d e g r e e of i n h i b i t i o n p r o d u c e d by t h e c o n d i t i o n i n g (C) p u l s e was d e t e r m i n e d by t a k i n g t h e d i f f e r e n c e between t h e a m p l i t u d e s o f t h e SR-evoked c o n t r o l t e s t ( T c ) r e s p o n s e and t h a t o b t a i n e d when p a i r e d w i t h t h e a n t i d r o m i c c o n d i t i o n i n g p u l s e ( T p ) . The d e g r e e of i n h i b i t i o n was e x p r e s s e d a s a p e r c e n t a g e o f t h e c o n t r o l t e s t p u l s e a m p l i t u d e : % i n h i b i t i o n = (Tc - Tp / T c ) x 100% The p e r c e n t i n h i b i t i o n ( % i n h i b i t i o n ) of t h e p o p u l a t i o n s p i k e was compared b e f o r e and d u r i n g 50MM NE p e r f u s i o n s . S i n c e , t h e a m p l i t u d e of t h e p o p u l a t i o n s p i k e i t s e l f was i n f l u e n c e d 20 FIGURE 2. S c h e m a t i c D i a g r a m of t h e P a i r e d P u l s e P a r a d i g m A. In t h e c o n t r o l c o n d i t i o n (no d r u g ) , a s t a b l e c o n t r o l , t e s t (Tc) r e s p o n s e i s f i r s t e s t a b l i s h e d by s t i m u l a t i n g SR a l o n e . Then t h e d e g r e e o f r e c u r r e n t i n h i b i t i o n i s a s s e s s e d by p a i r i n g t h i s t e s t p u l s e (Tp) w i t h a p r e c e d i n g , c o n d i t i o n i n g p u l s e (C) d e l i v e r e d t o t h e a l v e u s ( A l v ) . B. In t h e p r e s e n c e of NE (50/LIM), t h e r e s p o n s e t o t h e c o n t r o l , t e s t p u l s e would be s m a l l e r ( d o t t e d t r a c e ; a c c o r d i n g t o M u e l l e r e_t a_l, 1981). In o r d e r t o a v o i d t h e argument t h a t t h e d e g r e e of i n h i b i t i o n i s d e p e n d e n t on t h e a m p l i t u d e of t h e c o n t r o l r e s p o n s e , i t would be r e a d j u s t e d t o i t s pre-NE a m p l i t u d e ( s o l i d t r a c e ) b e f o r e p a i r e d p u l s e i n h i b i t i o n i s r e a s s e s s e d . 21 by NE (as r e p o r t e d by M u e l l e r e_t a l , 1981) and i n o r d e r t o a v o i d t h e argument t h a t t h e d e g r e e o f r e c u r r e n t i n h i b i t i o n m i g h t somehow depend on t h e a m p l i t u d e of t h e p o p u l a t i o n s p i k e i t s e l f , t h e a m p l i t u d e of t h e c o n t r o l t e s t p u l s e (Tc) r e s p o n s e d u r i n g NE was r e a d j u s t e d t o t h e same a m p l i t u d e as t h e pre-NE c o n t r o l r e s p o n s e ( F i g . 2 B ) . F o r t h e same r e a s o n , a % i n h i b i t i o n y_s a m p l i t u d e c u r v e was e s t a b l i s h e d i n w h i c h t h e d e g r e e of 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 a r a n g e o f a m p l i t u d e s (1-5mV) o f t h e Tc r e s p o n s e s b o t h b e f o r e and d u r i n g NE (50/xM) p e r f u s i o n s . However, i n a l l t h e e x p e r i m e n t s , t h e a l v e a r c o n d i t i o n i n g (C) p u l s e s were u n a l t e r e d t o keep t h e r e c u r r e n t i n h i b i t o r y i n t e r n e u r o n a c t i v a t i n g i n t e n s i t i e s c o n s t a n t . I n t r a c e l l u l a r c o r r e l a t e s of t h e p a i r e d p u l s e p a r a d i g m were a l s o e x amined. P y r a m i d a l c e l l s were s y n a p t i c a l l y a c t i v a t e d a t 1.2x t h r e s h o l d by SR s t i m u l a t i o n and t h e i n t e n s i t y of t h e c o n d i t i o n i n g A l v p u l s e was a d j u s t e d t o i n h i b i t t h e p a i r e d , t e s t (Tp) p u l s e 50% of t h e t i m e . The d e g r e e of i n h i b i t i o n p r o d u c e d by t h e c o n d i t i o n i n g (C) .pulse was t h e n a s s e s s e d i n t h e p r e s e n c e o f 50MM NE. 22 D. 6-Hydroxydopamine Lesions T h i s set of experiments was performed on f i f t e e n in v i t r o s l i c e p r e p a r a t i o n s obtained from male Wistar r a t s s u b j e c t e d to 6-hydroxydopamine (6-OHDA) l e s i o n s . P r i o r to the 6-OHDA i n j e c t i o n s , the animals were i n j e c t e d with a monoamine oxidase i n h i b i t o r , p a r g y l i n e (50mg/kg i.p.) to prevent premature o x i d a t i o n of the 6-OHDA. Both i n t r a v e n t r i c u l a r (n=5) (25M1 of lOuq/ul) and u n i - (n=3) or b i l a t e r a l (n=5) i n t r a c e r e b r a l i n j e c t i o n of 6-OHDA i n t o the d o r s a l noradrenergic bundle (2M1 of 2vg/uD were used to dep l e t e c e n t r a l NE l e v e l s . Although NE l e v e l s were not measured, both types of l e s i o n s have p r e v i o u s l y been shown to r e s u l t in a c o n s i s t e n t 85-95% r e d u c t i o n of hippocampal NE (Assaf, 1978; Assaf et a_l, 1979). A l l in v i t r o r e c o r d i n g on 6-OHDA-treated animals was performed 6-8 weeks f o l l o w i n g the l e s i o n . The experimental paradigm d e s c r i b e d i n s e c t i o n B was used in t h i s set of experiments as w e l l . Both the e x t r a c e l l u l a r f i e l d responses of EPSP and p o p u l a t i o n spike were monitored. 23 CHAPTER I I I RESULTS A. The E x t r a c e l l u l a r F i e l d R e s p o n s e s S t i m u l a t i o n of t h e SR e v o k e d t h e c h a r a c t e r i s t i c n e g a t i v e - g o i n g EPSP i n t h e a p i c a l d e n d r i t i c r e g i o n of CA1 w h i c h was maximal i n t h e m i d d l e t h i r d o f t h i s r e g i o n ( F i g . 1 & 3 ) . In o r d e r t o s t a n d a r d i z e between e x p e r i m e n t s , i n t e n s i t i e s were a d j u s t e d t o evoke an EPSP a m p l i t u d e of 1mV as p r e - N E c o n t r o l s . The s t i m u l u s - t o - p e a k l a t e n c y f o r t h i s r e s p o n s e a v e r a g e d 5.5 ± 1 . 1 m s e c (± S.D.; n = 3 l ) . The i n f l e c t i o n on t h e downward s l o p e of t h e EPSP r e s p o n s e i s a r e f l e c t i o n of t h e f i b r e v o l l e y ( F i g . 3 A ) . Due t o t h e s o u r c e - s i n k r e l a t i o n s h i p of d i p o l e c h a r a c t e r i s t i c s of t h e p y r a m i d a l c e l l s , a r e f l e c t i o n of t h e EPSP r e c o r d e d i n t h e SR i s m a n i f e s t e d as a p o s i t i v e - g o i n g wave i n t h e SP. At h i g h e r i n t e n s i t i e s , a s h a r p 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 i s s u p e r i m p o s e d on t h e p o s i t i v i t y and r e f l e c 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 a p o p u l a t i o n o f p y r a m i d a l n e u r o n s ( A n d e r s e n e t a l , 1971). A g a i n , i n o r d e r t o s t a n d a r d i z e between e x p e r i m e n t s , an a m p l i t u d e of 2mV, measured f r o m t h e f i r s t p o s i t i v e peak t o t h e n e g a t i v e peak was u s e d f o r most e x p e r i m e n t s . The l a t e n c y of s t i m u l u s - t o - p e a k ( n e g a t i v e ) of t h e p o p u l a t i o n s p i k e r e s p o n s e a v e r a g e d 6.0 ± 0 . 8 m s e c ( n = l 7 ) . A l v e a r s t i m u l a t i o n e v o k e d t h e c h a r a c t e r i s t i c s h o r t l a t e n c y 24 FIGURE 3. C h a r a c t e r i s t i c E x t r a c e l l u l a r F i e l d R e s p o n s e s of t h e H i p p o c a m p a l CA1 R e g i o n S t r a t u m r a d i a t u m (SR) s t i m u l a t i o n e l i c i t s a t t h e l e v e l of SR t h e n e g a t i v e - g o i n g EPSP r e s p o n s e (A) w h i c h i s o c c a s i o n a l l y i n t e r r u p t e d by t h e s l i g h t i n f l e c t i o n o f t h e f i b r e v o l l e y ( a r r o w h e a d ) . At s u p r a t h r e s h o l d i n t e n s i t i e s , t h e p o s i t i v i t y o f t h e s o m a t i c EPSP r e c o r d e d a t t h e l e v e l of t h e s t r a t u m p y r a m i d a l e (SP) i s s u p e r i m p o s e d by t h e s h a r p 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 (B) 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 d i s c h a r g e of a p o p u l a t i o n o f p y r a m i d a l n e u r o n s . A l v e a r ( A l v ) s t i m u l a t i o n e l i c i t s t h e s h o r t l a t e n c y n e g a t i v e -g o i n g a n t i d r o m i c p o p u l a t i o n s p i k e a r i s i n g s t r a i g h t o f f t h e b a s e l i n e ( C ) . 25 n e g a t i v e - g o i n g a n t i d r o m i c p o p u l a t i o n s p i k e r e c o r d e d i n t h e SP. The s t i m u l u s - t o - p e a k l a t e n c y a v e r a g e d 1.6 ± 0.2msec (n=l0) ( F i g . 3 ) . B. E f f e c t of NE on F i e l d R e s p o n s e s A l t h o u g h most of t h e e x p e r i m e n t s were c a r r i e d o u t w i t h 50MM NE, l o w e r c o n c e n t r a t i o n s were a l s o t e s t e d . P e r f u s i o n w i t h 10MM NE p r o d u c e d a c o n s i s t e n t enhancement of t h e p o p u l a t i o n s p i k e r e s p o n s e ( a b o u t 50%) but no s i g n i f i c a n t change i n t h e EPSP r e s p o n s e . However, p e r f u s i o n w i t h 50MM NE r e s u l t e d i n a c o n s i s t e n t r e d u c t i o n i n t h e a m p l i t u d e of b o t h t h e EPSP (25-28%) and t h e p o p u l a t i o n s p i k e (20-23%) r e s p o n s e s . T h i s e f f e c t was u s u a l l y maximal f o l l o w i n g 4~7min of d r u g p e r f u s i o n . B o t h t h e EPSP and p o p u l a t i o n s p i k e r e t u r n e d t o c o n t r o l l e v e l s f o l l o w i n g t e r m i n a t i o n of d r u g a p p l i c a t i o n . M o r e o v e r , t h e p o p u l a t i o n s p i k e r e s p o n s e c o n s i s t e n t l y e x h i b i t e d a r e b o u n d e x c i t a t i o n , s u g g e s t i n g t h e p o s s i b i l t y o f a d u a l a c t i o n o f NE on p y r a m i d a l c e l l s ( F i g . 4 ) . S t i m u l u s i n t e n s i t y v s EPSP a m p l i t u d e p l o t s were o b t a i n e d . As shown i n F i g . 5, NE had a more p r o n o u n c e d i n h i b i t o r y e f f e c t a t low s t i m u l u s i n t e n s i t i e s whereas a t h i g h e r l e v e l s t h e i n h i b i t i o n became l e s s marked. N e i t h e r t h e a n t i d r o m i c p o p u l a t i o n s p i k e e v o k e d by s t i m u l a t i o n of t h e a l v e u s nor t h e o r t h o d r o m i c a l l y e v o k e d f i b r e v o l l e y r e s p o n s e 26 FIGURE 4. Time C o u r s e of NE A c t i o n on t h e CA1 E v o k e d R e s p o n s e s NE (50/uM) p r o d u c e d a c o n s i s t e n t r e d u c t i o n i n b o t h t h e p o p u l a t i o n s p i k e ( A ) and EPSP ( • ) r e s p o n s e s (n=11). (At l e a s t s i x c o n t r o l r e s p o n s e s were r e c o r d e d o v e r more t h a n 30min and an a v e r a g e of t h e s e i s t a k e n as "100% c o n t r o l " ) . Note t h e c o n s i s t e n t r e b o u n d e x c i t a t i o n o f t h e p o p u l a t i o n s p i k e but n o t t h e EPSP d u r i n g r e c o v e r y . ( I n t h i s and a l l s u b s e q u e n t g r a p h s , e r r o r bar = ± S.E.M.). T r a c e s show t h e c o m p u t e r - a v e r a g e d r e s p o n s e s from p re-NE c o n t r o l , 15min d u r i n g NE and 30min a f t e r t e r m i n a t i o n of d r u g p e r f u s i o n . Note t h e l a c k of e f f e c t of NE on t h e f i b r e v o l l e y i n t h e EPSP r e s p o n s e s ( a r r o w h e a d s ) . i 26.1 + 10 20 30 Time (min) 4 0 5 0 6 0 CONTROL Pop. Sp. EPSPwfl 15 min 45 min i m V 0-2mV 5 m s 27 FIGURE 5. EPSP A m p l i t u d e v s S t i m u l u s I n t e n s i t y In t h e p r e s e n c e of 50MM NE ( A ), t h e c u r v e was s h i f t e d t o t h e r i g h t of t h a t of t h e pre-NE c o n t r o l ( • ) (n=4). S t u d e n t ' s t - t e s t d e m o n s t r a t e d t h a t t h e c h a n g e s were h i g h l y s i g n i f i c a n t a t low s t i m u l u s i n t e n s i t i e s (***, p < 0 . 0 l ; *, p<0.05) but n o t s i g n i f i c a n t (N.S.) a t h i g h i n t e n s i t i e s . The i n c r e a s i n g s i z e of t h e SEM w i t h i n c r e a s i n g s t i m u l u s i n t e n s i t i e s i s however n o t r e a d i l y e x p l i c a b l e . ( r = c o r r e l a t i o n c o e f f i c i e n t f o r t h e l i n e a r i t y o f t h e c u r v e ) . Bottom t r a c e s show t h e c o r r e s p o n d i n g s t i m u l u s - r e s p o n s e p r o f i l e s of a v e r a g e d f i e l d EPSPs b e f o r e and d u r i n g 50/nM NE p e r f u s i o n . 27.1 0.8 - i 0.6 H > E ^ 0.4 H Q. E < CO Q_' UJ 0.2 0.0 T 0 1 2 3 4 S t i m u l u s Intensity ( Inc rements in V o l t s ) Control NE 5 0 p M .4mV 10 msec 28 were a l t e r e d by s i m i l a r c o n c e n t r a t i o n s of NE. C. E f f e c t of NE on R e c u r r e n t I n h i b i t i o n 1. E x t r a c e l l u l a r r e s p o n s e s F o l l o w i n g a c o n d i t i o n i n g p u l s e a p p l i e d t o t h e a l v e a r s t i m u l a t i n g e l e c t r o d e , t h e SR-evoked p o p u l a t i o n s p i k e was c o n s i s t e n t l y i n h i b i t e d f o r p e r i o d s of 20-60msec, p r e s u m a b l y by a c t i v a t i o n of r e c u r r e n t i n h i b i t o r y i n t e r n e u r o n s ( F i g . 6 A ) . The p a i r e d p u l s e i n h i b i t i o n was most p o t e n t a t t h e c o n d i t i o n i n g - t e s t i n t e r v a l of 20-30msec and t h i s p e r i o d was t h e r e f o r e c h o s e n f o r t h e p r e s e n t e x p e r i m e n t a l p a r a d i g m . In t h e c o n t r o l s i t u a t i o n (no d r u g ) , t h e d e g r e e o f i n h i b i t i o n was i n v e r s e l y r e l a t e d t o t h e a m p l i t u d e of t h e p o p u l a t i o n s p i k e , s u c h t h a t t h e p e r c e n t i n h i b i t i o n was g r e a t e r when t h e p o p u l a t i o n s p i k e was s m a l l ( F i g . 7 & 8 ) . In t h e p r e s e n c e of 50MM NE, a c o n s i s t e n t 63% r e d u c t i o n i n p a i r e d p u l s e i n h i b i t i o n ( d i s i n h i b i t i o n ) was o b s e r v e d when th e c o n t r o l p u l s e ( T c ) was p r e - s e t a t 2mV ( F i g . 7 & 8 ) . As shown i n F i g . 8, t h e i n h i b i t i o n was r e d u c e d o v e r t h e whole a m p l i t u d e r a n g e i n t h e p r e s e n c e of 50MM NE and t h i s d e c r e a s e a p p e a r e d t o be more e f f e c t i v e a s t h e a m p l i t u d e of t h e p o p u l a t i o n 29 FIGURE 6. E f f e c t of NE on P a i r e d P u l s e I n h i b i t i o n A. In t h e c o n t r o l c o n d i t i o n (no d r u g ) , a s t a b l e SR-e v o k e d p o p u l a t i o n s p i k e , c o n t r o l ( T c ) r e s p o n s e was e s t a b l i s h e d (upper t r a c e ) . When p r e c e d e d by t h e a l v e a r -e v o k e d , c o n d i t i o n i n g (C) a n t i d r o m i c s p i k e , w h i c h p r e s u m a b l y a l s o a c t i v a t e d t h e r e c u r r e n t i n h i b i t o r y s y s t e m , r e d u c e d t h e p a i r e d , t e s t (Tp) r e s p o n s e . B . In t h e p r e s e n c e of 50MM NE, t h e c o n t r o l , t e s t r e s p o n s e was r e d u c e d ( d o t t e d t r a c e ) and was r e a d j u s t e d t o t h e p r e-NE a m p l i t u d e ( s o l i d t r a c e ) . As e v i d e n t f r o m t h e SR-e v o k e d (Tp) r e s p o n s e of t h e b o t t o m t r a c e , t h e p a i r e d p u l s e i n h i b i t i o n was s i g n i f i c a n t l y r e d u c e d as compared w i t h c o n t r o l . Note t h e l a c k of e f f e c t of NE on t h e a l v e a r - e v o k ' e d a n t i d r o m i c s p i k e r e s p o n s e . 29.1 30 FIGURE 7. R e l a t i o n s h i p between P a i r e d P u l s e I n h i b i t i o n and P o p u l a t i o n S p i k e A m p l i t u d e i n t h e P r e s e n c e and A b s e n c e of NE C o m p u t e r - a v e r a g e d t r a c e s show s u p e r i m p o s e d r e s p o n s e s of t h e c o n t r o l , t e s t p u l s e (Tc) and t h e a l v e a r - c o n d i t i o n e d , t e s t p u l s e (Tp) of t h e p a i r e d p u l s e p a r a d i g m ( c f F i g . 6 ) . As shown i n t h e upper t r a c e s and t h e c o r r e s p o n d i n g % i n h i b i t i o n , p a i r e d p u l s e i n h i b i t i o n was more p o t e n t a t l o w e r 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 . In t h e p r e s e n c e of 50MM NE ( b o t t o m t r a c e s ) , % i n h i b i t i o n was r e d u c e d o v e r t h e e n t i r e a m p l i t u d e , r a n g e . Note t h a t p a i r e d p u l s e i n h i b i t i o n was a l m o s t c o m p l e t e l y a b o l i s h e d a t t h e Tc a m p l i t u d e of 5mV. The N E - i n d u c e d r e d u c t i o n i n % i n h i b i t i o n seemed t o be more e f f e c t i v e a t h i g h e r r e s p o n s e a m p l i t u d e s . C O N T R O L ^U/y2^ M E A N v. INHIBITION ( n - 5 ) 6 7 - 5 D U R I N G N E J M E A N v. INHIBITION 2 6 8 R E D U C T I O N IN INHIBITION (%•) 6 0 - 3 C O N T R O L P U L S E A M P L I T U D E (mV) 1 31 FIGURE 8. % I n h i b i t i o n vs A m p l i t u d e of C o n t r o l , Test Response The mean % i n h i b i t i o n (± S.E.) from F i g . 7 i s p l o t t e d a g a i n s t the Tc a m p l i t u d e s from both c o n t r o l ( • ) and NE-t r e a t e d ( O ) c o n d i t i o n s . Curves r e p r e s e n t b e s t - f i t t i n g e x p o n e n t i a l c u r v e s . NE s h i f t e d the c u r v e t o the l e f t o v e r , the e n t i r e a m p l i t u d e range. 80 32 s p i k e was i n c r e a s e d . A t t h e h i g h e r a m p l i t u d e s ( 5 m V ) , t h e N E - i n d u c e d d i s i n h i b i t i o n was a l m o s t c o m p l e t e ( F i g . 7 ) . T h i s e f f e c t was r e v e r s i b l e i n a p p r o x i m a t e l y 3 0 m i n f o l l o w i n g t e r m i n a t i o n o f t h e NE p e r f u s i o n . 2 . I n t r a c e l l u l a r r e c o r d i n g s I n 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 f r o m t w e l v e CA1 p y r a m i d a l c e l l s a l l o f w h i c h d i s p l a y e d s t a b l e membrane c h a r a c t e r i s t i c s : R M P , 66 ± 2 . 2 m V (± S . E . ) , s p i k e a m p l i t u d e s o f 9 7 . 7 ± 4 . 7 m V a n d i n p u t r e s i s t a n c e s , 2 5 . 7 ± 1 . 9 M f l . The c h a r a c t e r i s t i c E P S P r e s p o n s e a n d t h e s y n a p t i c s p i k e g e n e r a t i o n w e r e e l i c i t e d f o l l o w i n g SR s t i m u l a t i o n ( F i g . 9A & B ) . A l v e a r s t i m u l a t i o n p r o d u c e d a n a n t i d r o m i c a c t i o n p o t e n t i a l w h i c h a r o s e s t r a i g h t o f f t h e b a s e l i n e r e f l e c t i n g r e t r o g r a d e s o m a t i c i n v a s i o n o f a n a c t i o n p o t e n t i a l ( F i g . 9 C ) . B o t h o r t h o d r o m i c a l l y a n d a n t i d r o m i c a l l y a c t i v a t e d s p i k e s w e r e f o l l o w e d by h y p e r p o l a r i z i n g , i n h i b i 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 s ( I P S P s ) ( F i g . 9B & C ) . T h e s e I P S P s r e p r e s e n t a c t i v a t i o n o f t h e r e c u r r e n t b a s k e t c e l l i n h i b i t o r y s y s t e m . The e f f e c t i v e n e s s o f t h i s i n h i b i t o r y i n f l u e n c e on i n t r a c e l l u l a r s p i k e a c t i v a t i o n was e x a m i n e d u s i n g t h e p a i r e d p u l s e p a r a d i g m ( F i g . 1 0 ) . A p y r a m i d a l n e u r o n was s y n a p t i c a l l y a c t i v a t e d 100% o f t h e t i m e ( 8 / 8 ) a t a n i n t e n s i t y o f 1 . 2 x t h r e s h o l d . A l v e a r s t i m u l a t i o n , w h i c h a l s o a c t i v a t e d t h e c e l l 33 FIGURE 9. C h a r a c t e r i s t i c I n t r a c e l l u l a r R e s p o n s e s of t h e H i p p o c a m p a l CA1 P y r a m i d a l Neurons S u b - t h r e s h o l d SR s t i m u l a t i o n e v o k e d t h e c h a r a c t e r i s t i c EPSP (A) and s u p r a - t h r e s h o l d s t i m u l a t i o n r e s u l t e d i n s p i k e g e n e r a t i o n s u p e r i m p o s e d on t h e u n d e r l y i n g EPSP ( B ) . A l v e a r s t i m u l a t i o n r e s u l t e d i n an a c t i o n p o t e n t i a l w i t h no u n d e r l y i n g EPSP ( C ) . Note t h e s u b s e q u e n t h y p e r p o l a r i z a t i o n s i n B & C a f t e r s p i k e g e n e r a t i o n , t h e s o - c a l l e d i n h i b i 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 s ( I P S P s ) r e s u l t e d p r e s u m a b l y from t h e a c t i v a t i o n o f t h e r e c u r r e n t b a s k e t c e l l s y s t e m . 33.1 |20mV 20ms 34 FIGURE 10. I n t r a c e l l u l a r C o r r e l a t e s of P a i r e d P u l s e I n h i b i t i o n a. A l v e a r ( A l v ) s t i m u l a t i o n e v o k e d t h e a n t i d r o m i c s p i k e and t h e c h a r a c t e r i s t i c IPSP. At t h e peak of t h e IPSP ( a r r o w s ) , a C-T i n t e r v a l of 30msec, t h e p a i r e d , t e s t p u l s e (Tp) was d e l i v e r e d t o a s s e s s t h e p o t e n c y o f t h e i n h i b i t o r y i n f l u e n c e . b. In t h e c o n t r o l c o n d i t i o n (no d r u g ) , SR s t i m u l a t i o n a l o n e ( l e f t t r a c e s ) r e s u l t e d i n s p i k e g e n e r a t i o n e i g h t t i m e s out of e i g h t s t i m u l a t i o n s . When p r e c e d e d by t h e A l v c o n d i t i o n i n g p u l s e , t h e c e l l d i s c h a r g e d o n l y f o u r t i m e s out of t h e e i g h t s t i m u l a t i o n s . Note t h e s l i g h t h y p e r p o a r i z a t i o n i n d i c a t i n g t h e IPSP. c . In t h e p r e s e n c e of 50MM NE, d e s p i t e t h e s l i g h t h y p e r p o l a r i z a t i o n due p r o b a b l y t o a c o m b i n a t i o n o f t h e e f f e c t of NE on t h e p y r a m i d a l c e l l and t h e IPSP, t h e c e l l d i s c h a r g e d e i g h t t i m e s out of e i g h t s t i m u l a t i o n s r e s u l t i n g i n a c o m p l e t e d i s i n h i b i t i o n . a. Alv b. Control -60--100 J SR Freq. ot Discharge ft, to SR Stim. 8 20ms 35 a n t i d r o m i c a l l y ( F i g . 10A), was a d j u s t e d t o i n h i b i t t h e o r t h o d r o m i c a c t i v a t i o n h a l f t h e t i m e ( F i g . 10B). In t h e p r e s e n c e o f 50MM NE, t h e i n h i b i t o r y i n f l u e n c e o f t h e a l v e a r c o n d i t i o n i n g p u l s e was g r e a t l y a t t e n u a t e d . The N E - m e d i a t e d d i s i n h i b i t i o n a l l o w e d t h e c e l l t o d i s c h a r g e s e v e n or e i g h t t i m e s out o f e i g h t s t i m u l a t i o n s ( F i g . 10C). In a d d i t i o n t o t h i s d i s i n h i b i t o r y i n f l u e n c e , NE a l s o p r o d u c e d a s l i g h t h y p e r p o l a r i z a t i o n (3-4mV) of t h e R M P ( F i g . 10C) and an e q u i v o c a l c o n d u c t a n c e i n c r e a s e ( d a t a not shown). D. 6-Hydroxydopamine L e s i o n s E x t r a c e l l u l a r r e c o r d i n g s o b t a i n e d from h i p p o c a m p a l s l i c e s removed from a n i m a l s w h i c h had r e c e i v e d 6-OHDA l e s i o n s 6-8 weeks p r e v i o u s l y , d i d not d i f f e r f r o m n o r m a l s a l t h o u g h t h r e s h o l d i n t e n s i t i e s r e q u i r e d f o r a c t i v a t i o n were somewhat l o w e r . Q u a n t i t a t i v e measurements of t h i s p a r a m e t e r change were not a n a l y z e d . P e r f u s i o n w i t h 50MM NE r e s u l t e d i n s i g n i f i c a n t a l t e r a t i o n s i n b o t h t h e EPSP and p o p u l a t i o n s p i k e r e s p o n s e s i n t h e s e p r e p a r a t i o n s . W h i l e t h e d e g r e e of i n h i b i t i o n of t h e EPSP was r e d u c e d by a p p r o x i m a t e l y 50% as compared t o n o r m a l c o n t r o l s ( F i g . 11), NE p r o d u c e d an enhancement of t h e p o p u l a t i o n s p i k e r e s p o n s e ( F i g . 1 2 ) . In a d d i t i o n , s e c o n d a r y s p i k e s and l a t e r t e r t i a r y d i s c h a r g e s were r e c o r d e d , n e i t h e r of w h i c h was e v i d e n t i n n o r m a l 36 FIGURE 11. E f f e c t of NE on t h e E x t r a c e l l u l a r EPSP R e s p o n s e s i n S l i c e s O b t a i n e d from 6-OHDA-treated A n i m a l s A r e d u c t i o n of t h e i n h i b i t o r y e f f e c t of NE on t h e EPSPs i n 6 - OHDA-treated p r e p a r a t i o n s ( V ) (n=5) was o b s e r v e d as compared w i t h n o r m a l s l i c e s ( • ) (n=11). V a l u e s a t I0~25min were 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 ( S t u d e n t ' s t - t e s t ) . 37 FIGURE 12. E f f e c t o f NE on t h e E x t r a c e l l u l a r P o p u l a t i o n S p i k e R e s p o n s e s i n S l i c e s O b t a i n e d f r o m 6-OHDA-t r e a t e d A n i m a l s E nhancement o f t h e p o p u l a t i o n s p i k e s f o l l o w i n g NE p e r f u s i o n s i n 6 - O H D A - t r e a t e d p r e p a r a t i o n s ( A ) (n=9) was o b s e r v e d a s c o m p a r e d w i t h t h e n o r m a l i n h i b i t i o n ( A ) . V a l u e s a t l 0 - 2 5 m i n were s i g n i f i c a n t l y d i f f e r e n t , p<0.fJ1. O c c u r r e n c e o f s e c o n d a r y ( O ) and t e r t i a r y ( • ) d i s c h a r g e s i s r e p r e s e n t e d as a p e r c e n t a g e o f t h e p r e - p e r f u s i o n c o n t r o l s . T r a c e s show t y p i c a l r e c o r d i n g s o f t h e p o p u l a t i o n s p i k e a n d m u l t i p l e d i s c h a r g e s a t i n d i c a t e d t i m e p e r i o d s d u r i n g a n d f o l l o w i n g NE p e r f u s i o n . 37.1 150-i + 10 20 30 Time (min.) 40 50 60 Control 15 min. 4 0 min. 2 mV 10 msec 38 c o n t r o l s l i c e s r e g a r d l e s s of t h e p e r i o d of NE p e r f u s i o n ( F i g . 1 2). F o l l o w i n g r e t u r n t o t h e c o n t r o l medium, t h e m u l t i p l e s p i k e d i s c h a r g e s p e r s i s t e d f o r p e r i o d s i n e x c e s s of 1 h o u r . In one p r e p a r a t i o n , n e u r o n a l f i r i n g p a t t e r n s r e s e m b l i n g i n t e r i c t a l b u r s t d i s c h a r g e s were o b s e r v e d d u r i n g , and f o l l o w i n g NE p e r f u s i o n . NE had no e f f e c t on e i t h e r f i b r e v o l l e y r e s p o n s e or t h e a n t i d r o m i c p o p u l a t i o n s p i k e i n 6-OHDA-treated p r e p a r a t i o n s . The e f f e c t s of 50MM NE on e v o k e d r e s p o n s e s i n s l i c e s o b t a i n e d from i n t r a v e n t r i c u l a r 6-OHDA l e s i o n e d a n i m a l s were e s s e n t i a l l y t h e same as t h o s e o b t a i n e d f r o m a n i m a l s r e c e i v i n g b i l a t e r a l 6-OHDA l e s i o n s of t h e d o r s a l n o r a d r e n e r g i c b u n d l e . In a l l p r e p a r a t i o n s s u b j e c t e d t o u n i l a t e r a l i n t r a c e r e b r a l i n j e c t i o n s of 6-OHDA, NE p e r f u s i o n o f t h e i p s i l a t e r a l h ippocampus p r o d u c e d t h e c h a r a c t e r i s t i c m u l t i p l e s p i k e d i s c h a r g e , w h i l e t h e c o n t r a l a t e r a l h i ppocampus ( w i t h t h e e x c e p t i o n of one, w h i c h was p r o b a b l y c o n t a m i n a t e d by t h e c o n t r a l a t e r a l 6-OHDA i n j e c t i o n ) e x h i b i t e d n o r m a l c o n t r o l - l i k e r e s p o n s e s . 39 CHAPTER IV DISCUSSION A. E f f e c t of NE on P y r a m i d a l C e l l s As o r i g i n a l l y d e m o n s t r a t e d by M u e l l e r e t a l ( 1 9 8 1 ) , t h e r e s u l t s o f t h e p r e s e n t s t u d y i n d i c a t e d a d i f f e r e n t i a l e f f e c t of NE a t low (10MM) and h i g h (50MM) c o n c e n t r a t i o n s . The p o s s i b l e mechanism u n d e r l y i n g t h e s e c h a n g e s i s d i s c u s s e d i n t h e c o n t e x t of d i s i n h i b i t i o n . However, s i n c e 50juM NE was u s e d m a i n l y i n t h e p r e s e n t s t u d y , t h e e f f e c t of 50yM NE on h i p p o c a m p a l n e u r o n s i s d i s c u s s e d f i r s t . In c o n t r a s t t o t h e l a c k o f e f f e c t o f NE on t h e EPSP r e s p o n s e r e p o r t e d by M u e l l e r e_t a l ( 1 9 8 1 ) , t h e p r e s e n t s t u d y showed t h a t t h e a m p l i t u d e s o f b o t h t h e p o p u l a t i o n s p i k e and EPSP were r e d u c e d by 50MM NE p e r f u s i o n s ( F i g . 4 ) . T h i s d i s c r e p a n c y may be a c c o u n t e d f o r i f one t a k e s i n t o c o n s i d e r a t i o n t h e f a c t t h a t h i g h s t i m u l a t i o n i n t e n s i t i e s were employed t o evoke b o t h t h e p o p u l a t i o n s p i k e and t h e EPSP r e s p o n s e s i n t h e i r s t u d y . As shown i n F i g . 5, t h e h i g h e r t h e i n t e n s i t y , t h e l e s s t h e r e l a t i v e d e g r e e of i n h i b i t i o n p r o d u c e d by NE. T h e r e f o r e , a t n e a r l y maximal a m p l i t u d e s f o r t h e EPSP r e s p o n s e , t h e i n h i b i t i o n would be i n s i g n i f i c a n t and p r o b a b l y n o t e a s i l y d e t e c t e d . 40 T h e r e a r e a number of ways i n w h i c h N E - i n d u c e d i n h i b i t i o n of p y r a m i d a l n e u r o n s c o u l d be i n i t i a t e d : a. r e d u c t i o n i n t h e e x c i t a b i l i t y o f t h e a c t i v a t i n g f i b r e s ; b. r e d u c t i o n i n s y n a p t i c e f f i c a c y , and; c . r e d u c t i o n i n t h e s y n c h r o n y o f p y r a m i d a l c e l l d i s c h a r g e s . The f i r s t p o s s i b i l t y i s u n l i k e l y i n v i e w o f t h e l a c k of e f f e c t o f NE on t h e p r e s y n p a t i c f i b r e v o l l e y and t h e a n t i d r o m i c s p i k e a m p l i t u d e s . In t h e c a s e o f r e d u c t i o n of s y n a p t i c e f f i c a c y , t h e N E - i n d u c e d d e c r e a s e i n EPSP a m p l i t u d e w o u l d s u g g e s t a change i n t h e e f f i c i e n c y o f ' i n f o r m a t i o n t r a n s f e r ' o f t h e d e n d r i t e s . T h i s change c o u l d p r o b a b l y be e f f e c t e d by t h e s l i g h t h y p e r p o l a r i z a t i o n (mean: 2.7 mV) and t h e c o n d u c t a n c e i n c r e a s e r e s u l t i n g from NE a p p l i c a t i o n r e p o r t e d by Langmoen et_ a l ( 1 9 8 1 ) , S e g a l (1981) and e q u i v o c a l l y d e m o n s t r a t e d by t h e p r e s e n t s t u d y . The s l i g h t h y p e r p o l a r i z i n g r e s p o n s e m i g h t a c c o u n t f o r t h e i n h i b i t o r y e f f e c t of NE on s p o n t a n e o u s l y f i r i n g p y r a m i d a l n e u r o n s as r e p o r t e d by S e g a l & Bloom ( 1 9 7 4 ) . However, more i m p o r t a n t l y , t h e N E - i n d u c e d c o n d u c t a n c e i n c r e a s e a t t e n u a t e d t h e r e s p o n s e s t o d e p o l a r i z i n g c u r r e n t p u l s e s and t h e number of a c c o m p a n y i n g s p i k e s p o s s i b l y by w o r k i n g as an i o n i c s h u n t (Langmoen et. a_l, 1981). T h i s s h u n t m i g h t not a f f e c t t h e f a s t i o n i c f l u x e s as i n t h e c a s e of an a n t i d r o m i c a l l y e v o k e d a c t i o n p o t e n t i a l ; but i t m ight be h i g h l y e f f e c t i v e i n s h u n t i n g slow i o n i c f l u x e s o f l o c a l s y n a p t i c p o t e n t i a l s 41 o r t h o d r o m i c a l l y a c t i v a t e d as i n t h e c a s e of t h e p r e s e n t s t u d y . In t h e c a s e o f t h e l a c k of s y n c h r o n y of d i s c h a r g e of t h e p y r a m i d a l c e l l s ( w h i c h m i g h t a f f e c t t h e a m p l i t u d e of t h e p o p u l a t i o n s p i k e r e s p o n s e ) , i t would d e p e n d on t h e ' r e a d i n e s s ' of e a c h p y r a m i d a l c e l l t o d i s c h a r g e when a s y n c h r o n o u s i n p u t a r r i v e s , a s i n t h e c a s e o f an e l e c t i c a l s t i m u l a t i o n t o t h e SR. I t i s b e l i e v e d t h a t t h i s ' r e a d i n e s s ' t o d i s c h a r g e m ight depend on t h e ' p r e - s e t t i n g ' of t h e p y r a m i d a l c e l l s by c h r o n i c i n h i b i t o r y i n f l u e n c e s from e i t h e r t h e r e c u r r e n t i n h i b i t o r y b a s k e t c e l l s y s t e m or t h e a l l e g e d ' f e e d - f o r w a r d ' i n h i b i t o r y i n t e r n e u r o n s . Due t o t h e i n h e r e n t d i s a d v a n t a g e o f t h e b a t h p e r f u s i o n t e c h n i q u e , t h e s e i n t e r n e u r o n s would a l s o come i n t o c o n t a c t w i t h and q u i t e l i k e l y be i n f l u e n c e d by NE a s w e l l . Hence when one examines t h e p o p u l a t i o n s p i k e r e s p o n s e , one i s n o t r e a l l y m o n i t o r i n g t h e e x c i t a b i l i t y of t h e p y r a m i d a l c e l l s a l o n e but t h e o v e r a l l o u t p u t o f t h e h i p p o c a m p a l CA1 r e g i o n a s i n f l u e n c e d by NE. 42 B. E f f e c t of NE on R e c u r r e n t I n h i b i t i o n B e f o r e d i s c u s s i n g t h e e f f e c t of NE on p a i r e d p u l s e i n h i b i t i o n i n t h e h i p p o c a m p a l CA1 r e g i o n , t h e a p p a r e n t dependency of % i n h i b i t i o n on t h e a m p l i t u d e of t h e p o p u l a t i o n s p i k e r e s p o n s e s h o u l d be c o n s i d e r e d . The d e c r e a s e i n % i n h i b i t i o n w i t h i n c r e a s i n g 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 e e 'CONTROL' i n F i g . 8) c o u l d be e x p e c t e d i f one c o n s i d e r s t h a t t h e c o n d i t i o n i n g p u l s e i n t e n s i t y was kept c o n s t a n t . Hence, o n l y a c e r t a i n number o f b a s k e t c e l l s were a c t i v a t e d d e s p i t e r e c r u i t m e n t of more p y r a m i d a l c e l l s a t i n c r e a s i n g t e s t p u l s e i n t e n s i t i e s . T h e r e f o r e , t h e d e g r e e of p a i r e d p u l s e i n h i b i t i o n a p p e a r s t o d e c r e a s e a t h i g h e r r e s p o n s e a m p l i t u d e s . F u r t h e r m o r e , a s s u m i n g t h a t t h e same number o f p y r a m i d a l c e l l s c o n t r i b u t e s t o t h e e x t r a c e l l u l a r p o p u l a t i o n s p i k e a t i d e n t i c a l c o n t r o l ( T c ) r e s p o n s e a m p l i t u d e s b e f o r e and d u r i n g NE p e r f u s i o n , one c o u l d c o n c l u d e t h a t t h e lo w e r p e r c e n t i n h i b i t i o n i n t h e p r e s e n c e of NE r e f l e c t s a l o w e r number o f p y r a m i d a l c e l l s b e i n g i n h i b i t e d by t h e i r c o r r e s p o n d i n g b a s k e t c e l l s . Hence a lo w e r p e r c e n t a g e of t h e b a s k e t c e l l s i n v o l v e d were a c t i v a t e d i n t h e p r e s e n c e of NE. As d e m o n s t r a t e d by two d i f f e r e n t e x p e r i m e n t a l a p p r o a c h e s ( F i g . 7 & 10), t h e r e s u l t s s u g g e s t t h a t NE not o n l y e x e r t s an i n h i b i t o r y e f f e c t on t h e p r i n c i p a l ( p y r a m i d a l ) n e u r o n s but a l s o s i g n i f i c a n t l y i n h i b i t s t h e r e c u r r e n t i n h i b i t o r y i n t e r n e u r o n s ( b a s k e t c e l l s ) r e s u l t i n g i n a d i s i n h i b i t o r y i n f l u e n c e on t h e f o r m e r . In view o f t h e 43 d i f f u s e i n n e r v a t i o n of n o r a d r e n e r g i c f i b r e s from t h e l o c u s c o e r u l e u s , t h i s g e n e r a l i n h i b i t o r y a c t i o n of NE seems l i k e l y t o e x t e n d t o o t h e r i n t e r n e u r o n 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 , p r o b a b l y i n c l u d i n g t h e a l l e g e d ' f e e d - f o r w a r d ' i n t e r n e u r o n s ( A l g e r & N i c o l l , 1982). A c c o r d i n g t o t h e s t u d y of M u e l l e r e t a l ( 1 9 8 1 ) , t h e a - a g o n i s t m e d i a t e d i n h i b i t i o n and 0 - a g o n i s t m e d i a t e d enhancement of t h e p o p u l a t i o n s p i k e s u g g e s t t h a t t h e f o r m e r m i g h t be r e s p o n s i b l e f o r t h e i n h i b i t i o n o f t h e p r i n c i p a l n e u r o n s and t h e l a t t e r f o r t h e i n h i b i t o r y i n t e r n e u r o n s . S u p p o s i n g t h a t a s y n c h r o n o u s p u l s e of a c t i o n p o t e n t i a l s a r r i v e s 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 f r o m t h e l o c u s c o e r u l e u s , i t would p r o b a b l y r e s u l t i n a s y n c h r o n o u s i n h i b i t i o n of t h e i n h i b i t o r y i n t e r n e u r o n s and hence t h e p y r a m i d a l c e l l s would be more e x c i t a b l e t o o t h e r a f f e r e n t i n p u t s . M o r e o v e r , s i n c e t h e i n h i b i t o r y i n t e r n e u r o n s were i n h i b i t e d m o r e - o r - l e s s s i m u l t a n e o u s l y , t h e y p r o b a b l y would r e c o v e r from t h e i n h i b i t i o n a t a s i m i l a r i n s t a n t i n t i m e r e s u l t i n g i n a s y n c h r o n o u s i n h i b i t i o n of t h e p y r a m i d a l c e l l s w h i c h would t h e n be i n a q u i e t but r e a d y s t a t e t o r e s p o n d t o t h e n e x t s y n c h r o n o u s d i s c h a r g e and so on. T h i s c y c l e would p r o b a b l y c o n t i n u e w i t h i m p r o v i n g s y n c h r o n y a f t e r e a c h c y c l e i n a p o s i t i v e f e e d b a c k f a s h i o n . The f i n a l r e s u l t c o u l d be a l t e r n a t e r h y t h m i c d i s c h a r g e s of t h e r e c u r r e n t b a s k e t c e l l s y s t e m and t h e p y r a m i d a l c e l l s p r o d u c i n g an o u t p u t s i m i l a r t o a rhythm g e n e r a t o r . The h i p p o c a m p a l f o r m a t i o n i s i n d e e d w e l l known as a t h e t a - r h y t h m g e n e r a t o r e s p e c i a l l y a f t e r s t i m u l a t i o n o f t h e l o c u s c o e r u l e u s ( e . g . Macadar e t a l , 44 1974). N a t u r a l l y , t h e more p o t e n t t h e i n h i b i t o r y i n f l u e n c e of NE on t h e i n h i b i t o r y p r o c e s s e s , t h e more e f f e c t i v e t h e r hythm g e n e r a t o r would be. The p r o p e r f u n c t i o n i n g o f t h i s r h y t hm g e r e n a t o r would t h e n depend on t h e b a l a n c e of NE a c t i o n on t h e i n t e r n e u r o n s and t h e p r i n c i p a l n e u r o n s , p o s s i b l y w i t h t h e dampening e f f e c t of NE on t h e l a t t e r w o r k i n g as a s a f e t y mechanism t o p r e v e n t t h e s y s t e m from g o i n g i n t o h y p e r s y n c h r o n y o r p o s s i b l y e p i l e p t o g e n i c s t a t e s . C. 6-Hydroxydopamine L e s i o n and R o l e of NE i n  E p i l e p t o g e n e s i s I t m i g h t a t f i r s t seem s u r p r i s i n g and even p a r a d o x i c a l t h a t a s y s t e m d e p l e t e d o f i t s endogenous NE, w h i c h seems t o e x e r t an i n h i b i t o r y i n f l u e n c e on t h e p y r a m i d a l c e l l s , would upon r e p l e n i s h m e n t of t h e same d r u g by e xogenous a p p l i c a t i o n a c t u a l l y e x h i b i t an enhancement of a c t i v i t y and e v e n t u a l l y e p i l e p t i f o r m m u l t i p l e s p i k e d i s c h a r g e s . The n o t i o n of i n v o l v e m e n t of NE i n i n h i b i t o r y p r o c e s s e s , p a r t i c u l a r y i n e p i l e p t o g e n e s i s and t h e k i n d l i n g model has been w e l l documented ( e . g . K i l i a n & F r e y , 1973). D e p l e t i o n of endogenous NE has been shown t o f a c i l i t a t e e p i l e p t o g e n e s i s i n d u c e d p h a r m a c o l o g i c a l l y (Mason e_t a l , 1978), a u d i o g e n i c a l l y (Bourn e t a_l, 1977; S h a y w i t z e_t a l , 1978; and H o r t o n e t a l , 1980) o r by k i n d l i n g ( E h l e r s e t a l , 1980; 45 C o r c o r a n e_t a l , 1 9 8 0 ) . H o w e v e r , i n a l l o f t h e s e s t u d i e s , NE s e e m s o n l y t o p l a y a m o d u l a t o r y r o l e . T h e r e seems t o be o n l y one b e h a v i o r a l c o r r e l a t e o f t h e p r e s e n t f i n d i n g s w h e r e NE p l a y s a m o r e d i r e c t r o l e i n e p i l e p t o g e n e s i s . B l u m e_t al_ ( 1 9 7 7 ) s h o w e d t h a t e x o g e n o u s NE a c t u a l l y p r o d u c e d c o n v u l s i v e s e i z u r e s i n 6 - O H D A - t r e a t e d b u t p r e v i o u s l y i n s e n s i t i v e a n i m a l s . The f a c t t h a t t h e e f f e c t o f NE on t h e E P S P r e s p o n s e o f 6 - O H D A - t r e a t e d p r e p a r a t i o n s i s s t i l l i n h i b i t o r y t h o u g h r e d u c e d ( F i g . 11) s u g g e s t s t h a t t h e p o t e n t i a t i o n o f t h e p o p u l a t i o n s p i k e r e s p o n s e i s d u e t o f a c t o r s o t h e r t h a n a c h a n g e i n t h e b a s i c n a t u r e o f n o r a d r e n e r g i c i n f l u e n c e on t h e p y r a m i d a l c e l l s p o s t - s y n a p t i c a l l y . I n f a c t , t h i s d i s s o c i a t i o n o f t h e t w o f i e l d r e s p o n s e s s t r o n g l y s u p p o r t s t h e h y p o t h e s i s t h a t t h e p o t e n t i a t i o n o f t h e p o p u l a t i o n s p i k e r e s p o n s e i s a r e s u l t o f ' d i s i n h i b i t i o n ' o f t h e p y r a m i d a l c e l l s f r o m t h e i n h i b i t o r y p r o c e s s e s , w h i c h a r e l i k e l y t o be s o m a t i c i n s t e a d o f d e n d r i t i c . A s m e n t i o n e d i n t h e p r e v i o u s s e c t i o n , a n i m b a l a n c e b e t w e e n t h e N E - i n d u c e d i n h i b i t o r y i n f l u e n c e s on t h e p y r a m i d a l c e l l s a n d t h e i n h i b i t o r y p r o c e s s e s c o u l d d r i v e t h e n o r m a l r h y t h m g e n e r a t o r i n t o h y p e r s y n c h r o n y o r e p i l e p t o g e n i c i t y . T h i s i m b a l a n c e c o u l d r e s u l t f r o m : a . p r e f e r e n t i a l r e g e n e r a t i v e s p r o u t i n g o f t e r m i n a l s o n t o i n h i b i t o r y i n t e r n e u r o n s ; a n d / o r , b . d i f f e r e n t i a l d e n e r v a t i o n s u p e r s e n s i t i v i t y b e t w e e n t h e p y r a m i d a l c e l l s a n d t h e i n h i b i t o r y i n t e r n e u r o n s a f t e r 6 -OHDA l e s i o n s . 46 R e g e n e r a t i v e s p r o u t i n g of n o r a d r e n e r g i c f i b r e s and b o u t o n s a f t e r c h e m i c a l l e s i o n s by 6-OHDA and o t h e r a d r e n e r g i c t o x i n s has been r e p o r t e d , as r e v e a l e d by b i o c h e m i c a l a s s a y s ( S c h m i d t e t a l , 1979, and 1980) and f l u o r e s c e n c e h i s t o c h e m i s t r y ( B j o r k l u n d e t a l , 1975 and 1979). The l a t t e r s t u d y a l s o showed t h a t a l t h o u g h r e g e n e r a t i o n t e n d s t o r e s t o r e t h e o r i g i n a l f i b r e a r c h i t e c t u r e , h y p e r i n n e r v a t i o n p a t t e r n s and a b n o r m a l t e r m i n a l a r r a n g e m e n t s a r e o f t e n f o r m e d . S i n c e a c c o r d i n g t o Loy e_t a l ( 1 9 8 0 ) , t h e r e a r e more f l u o r e s c e n t v a r i c o s i t i e s i n n e r v a t i n g t h e d e n d r i t e s o f t h e p y r a m i d a l c e l l s t h a n t h e i n t e r n e u r o n s i n n o r m a l a n i m a l s , any random s p r o u t i n g d u r i n g t h e r e g e n e r a t i v e p r o c e s s would p r o b a b l y r e s u l t i n a c o m p a r a t i v e l y more e x t e n s i v e i n n e r v a t i o n t o t h e l a t t e r t h a n n o r m a l and s u b s e q u e n t l y a h i g h e r d e g r e e o f i n h i b i t o r y i n f l u e n c e on t h e i n h i b i t o r y i n t e r n e u r o n s as compared t o n o r m a l . The above c o n j e c t u r e i s , however, h i g h l y s p e c u l a t i v e . P o s t - s y n a p t i c s u p e r s e n s i t i v i t y a f t e r d e n e r v a t i o n i n t h e CNS has been w e l l documented. S e v e r a l s t u d i e s have shown t h a t 6-OHDA-induced d e p l e t i o n o f NE, a p p l i e d by d i f f e r e n t methods, r e s u l t s i n a 50-64% i n c r e a s e i n ^ - a d r e n e r g i c r e c e p t o r b i n d i n g i n t h e r a t c e r e b r a l c o r t e x ( S p o r n e_t a l , 1977; Minneman e_t a l , 1979). In g e n e r a l , t h e e x t e n t of d e n e r v a t i o n s u p e r s e n s i t i v i t y f o r a - a d r e n o c e p t o r s i s not as g r e a t as t h e ^ - a d r e n o c e p t o r s t h r o u g h o u t t h e CNS ( U ' P r i c h a r d e t a l , 1980). In p a r t i c u l a r , t h e h i p p o c a m p a l f o r m a t i o n has u n d e r g o n e a 34-50% i n c r e a s e i n 0 - a d r e n o c e p t o r d e n s i t y but 47 FIGURE 13. S c h e m a t i c D i a g r a m o f t h e D i s t r i b u t i o n of a- and j 3 - r e c e p t o r s i n Normal and 6-OHDA-treated C o n d i t i o n s As p r o p o s e d i n t h e d i s c u s s i o n , t h e a- ( c=> ) and 0-( ) a d r e n o c e p t o r s a r e d i f f e r e n t i a l l y d i s t r i b u t e d on t h e p y r a m i d a l c e l l ( c l e a r ) and t h e r e c u r r e n t i n h i b i t o r y i n t e r n e u r o n ( s h a d e d ) i n t h e n o r m a l s i t u a t i o n ( A ) . Note t h a t o n l y t h e n o r a d r e n e r g i c i n n e r v a t i o n i s d e p i c t e d h e r e f o r s i m p l i c i t y . As a r e s u l t o f 6-OHDA t r e a t m e n t ( B ) , t h e i n h i b i t o r y n e u r o n might be p r e f e r e n t i a l l y i n h i b i t e d due t o p r o b a b l e random s p r o u t i n g and more i m p o r t a n t l y , t o d i f f e r e n t i a l s u p e r s e n s i t i v i t y o f t h e /3 o v e r t h e a - r e c e p t o r s , e s p e c i a l l y i n t h e p r e s e n c e o f exogenous NE. 47 -1 A. NORMAL B. POST - 6 - O H D A 48 i n s i g n i f i c a n t and e q u i v o c a l c h a n g e s i n a - a d r e n o c e p t o r d e n s i t y a f t e r 6-OHDA l e s i o n i n g of l o c u s c o e r u l e u s - d o r s a l b u n d l e ( U ' P r i c h a r d e t a l , 1980; Sharma e l a l , 1981). S c a t c h a r d a n a l y s e s have a l s o shown t h a t t h e i n c r e a s e i n b i n d i n g o f j 3 - a g o n i s t s was due t o an i n c r e a s e i n t h e d e n s i t y of ^ - a d r e n o c e p t o r b i n d i n g s i t e s (Sharma e t a_l, 1981). I t i s p r o b a b l e t h a t t h i s d i f f e r e n t i a l d e n e r v a t i o n s u p e r s e n s i t i v i t y of t h e two a d r e n o c e p t o r s c o u l d be t h e b a s i s o f t h e a f o r e m e n t i o n e d i m b a l a n c e o f i n h i b i t i o n between t h e p y r a m i d a l c e l l s a nd t h e i n h i b i t o r y p r o c e s s e s . S e v e r a l o t h e r f i n d i n g s a l s o l e n d c r e d e n c e t o t h i s p o s s i b i l i t y . D e s p i t e t h e f a c t t h a t a few s t u d i e s showed t h a t t h e i n h i b i t o r y a c t i o n o f NE on p y r a m i d a l c e l l s i s s l i g h t l y a n t a g o n i z e d b u t not b l o c k e d by t h e ^ - a n t a g o n i s t , s o t o l o l (Langmoen e_t a_l, l 9 8 l ; S e g a l e t a l , 1980; S e g a l , 1981), t h e s t u d y of M u e l l e r e t a l (1981) s u g g e s t s t h a t t h e i n h i b i t o r y a c t i o n of NE on t h e CA1 p y r a m i d a l c e l l s i s m e d i a t e d p r e d o m i n a n t l y by a - a d r e n o c e p t o r s . The h y p o t h e s i s i s f u r t h e r s u p p o r t e d by t h e f a c t t h a t b o t h S e g a l (1981) and de M o n t i g n y e_t a l (1980) f a i l e d t o d e t e c t any s i g n s of d e n e r v a t i o n s u p e r s e n s i t v i t y a f t e r 6-OHDA t r e a t m e n t i n t h e CA1 and CA3 p y r a m i d a l n e u r o n s , r e s p e c t i v e l y , w h i c h i s i n agreement w i t h t h e l a c k o f 6-OHDA-induced a - a d r e n o c e p t o r s u p e r s e n s i t v i t y i n t h e h i p p o c a m p u s . C o n v e r s e l y , t h e p o t e n t i a t i o n o f t h e p o p u l a t i o n s p i k e r e s p o n s e by ^ - a g o n i s t s ( M u e l l e r e t a l , 1981), i n c o n j u n c t i o n w i t h t h e d i s i n h i b i t o r y e f f e c t o f NE v i a t h e r e c u r r e n t 49 i n h i b i t o r y i n t e r n e u r o n s , s u g g e s t t h a t t h e /3-adrenoceptor m e d i a t e s t h e i n h i b i t o r y i n f l u e n c e on t h e i n h i b i t o r y i n t e r n e u r o n s i n t h e h i p p o c a m p a l CA1 r e g i o n . Hence, t h e 34-50% i n c r e a s e i n /3-adrenoceptor d e n s i t y a f t e r 6-OHDA-induced d e n e r v a t i o n would r e s u l t i n an i m b a l a n c e of a p o t e n t i n h i b i t o r y i n f l u e n c e on t h e i n h i b i t o r y p r o c e s s e s w h i c h i s not o f f s e t by a s i m i l a r l y s u p e r s e n s i t i z e d a - a d r e n o c e p t o r p o p u l a t i o n on t h e p y r a m i d a l c e l l s . (See F i g . 1 3 ) . As d i s c u s s e d p r e v i o u s l y , t h i s t y p e o f i m b a l a n c e c o u l d d r i v e t h e s y s t e m t o h y p e r s y n c h r o n y i n t h e p r e s e n c e of NE. T h i s t h e o r y i s f u r t h e r s u p p o r t e d by t h e r e c e n t r e p o r t of M u e l l e r & D u n w i d d i e (1983) t h a t / 3 - a g o n i s t s f a c i l i t a t e w hereas a - a g o n i s t s r e d u c e e p i l e p t i f o r m a c t i v i t y i n d u c e d by p e n i c i l l i n and h i g h K + i n t h e jjn v i t r o h i p p o c a m p a l s l i c e p r e p a r a t i o n s . The p r e s e n t s t u d y i s t h e f i r s t p h y s i o l o g i c a l r e p o r t t h a t i n h i p p o c a m p i d e p l e t e d of t h e i r endogenous NE, r e p l e n i s h m e n t of NE i t s e l f a c t u a l l y r e s u l t s i n g e n e r a t i o n of e p i l e p t i f o r m a c t i v i t y p r o b a b l y as a c o n s e q u e n c e of d i f f e r e n t i a l s u p e r s e n s i t i v i t i e s o f t h e a d r e n e r g i c r e c e p t o r s . 50 D. G e n e r a l D i s c u s s i o n I t seems t o be g e n e r a l l y t r u e t h a t NE e x e r t s an i n h i b i t o r y i n f l u e n c e i n most a r e a s of t h e CNS and as shown i n t h e p r e s e n t s t u d y , i n h i b i t o r y i n t e r n e u r o n s a r e no e x c e p t i o n . F u r t h e r m o r e , even N E - i n d u c e d enhancement of r e s p o n s e s c o u l d l i k e l y be a t t r i b u t e d t o a ' d i s i n h i b i t o r y ' t y p e o f a c t i o n w i t h NE a c t i n g on t h e c o r r e s p o n d i n g i n h i b i t o r y p r o c e s s e s ( J a h r & N i c o l l , 1982). In t h e h i p p o c a m p u s , t h e p o t e n t i a t i n g e f f e c t of ^0^M. NE ( M u e l l e r e t a l , 1981; and t h e p r e s e n t s t u d y ) and t h e c o n s i s t e n t r e b o u n d e x c i t a t i o n of t h e p o p u l a t i o n s p i k e a f t e r 50MM c o u l d a l s o be e x p l a i n e d w i t h t h e same ' d i s i n h i b i t i o n ' h y p o t h e s i s . The key t o t h i s h y p o t h e s i s i s b a s e d on a d i f f e r e n t i a l d i s t r i b u t i o n o f a d r e n o c e p t o r s on t h e p y r a m i d a l c e l l s ( a l p h a ) and i n h i b i t o r y i n t e r n e u r o n s ( b e t a ) ( F i g . 1 3 ) . I t i s p o s s i b l e t h a t ^ - a d r e n o c e p t o r s c o u l d be more s e n s i t i v e t o NE a t a l o w e r c o n c e n t r a t i o n t h a n t h e a - r e c e p t o r s r e s u l t i n g i n a p r e d o m i n a n t d i s i n h i b i t o r y i n f l u e n c e on t h e p y r a m i d a l c e l l s . However, c o m p a r a t i v e s t u d i e s o f t h e s e n s i t i v i t y of t h e a-and / 3 - a d r e n o c e p t o r s t o NE have n o t been a v a i l a b l e i n t h e l i t e r a t u r e . S e c o n d l y , s i n c e any one i n h i b i t o r y i n t e r n e u r o n i s l i k e l y t o c o n t r o l more t h a n one p y r a m i d a l c e l l , any i n h i b i t i o n on t h e i n t e r n e u r o n s would be a m p l i f i e d w i t h t h e d i s i n h i b i t o r y i n f l u e n c e b e i n g much more s e n s i t i v e t h a n t h a t of t h e d i r e c t i n h i b i t i o n on t h e p y r a m i d a l c e l l s . C e r t a i n models o f e p i l e p t i f o r m d i s c h a r g e g e n e r a t i o n have been b a s e d on a r e d u c e d l e v e l o f GABA-mediated 51 i n h i b i t i o n (Meyer & P r i n c e , 1973; Wong & P r i n c e , 1979; D i n g l e d i n e & G j e r s t a d , 1980; S c h w a r t z k r o i n & W y l e r , 1980). O t h e r - s t u d i e s have a l s o shown t h a t when GABA t r a n s m i s s i o n was d i s r u p t e d by p e n i c i l l i n p e r f u s i o n s ( O l i v e r e_t a l , 1977) or i n low CI medium ( S e g a l , 1981), t h e r e s u l t i n g e x t r a c e l l u l a r m u l t i p l e s p i k e d i s c h a r g e s r e f l e c t o n - g o i n g e p i l e p t i f o r m a c t i v i t y . L i k e w i s e , t h e p r e s e n t f i n d i n g o f N E - i n d u c e d g e n e r a t i o n o f s i m i l a r e p i l e p t i f o r m a c t i v i t y i n 6-OHDA-treated p r e p a r a t i o n s s u g g e s t s t h a t NE r e d u c e s t h e l e v e l of GABA-mediated i n h i b i t i o n a s w e l l . M a d i s o n & N i c o l l (1982a) have shown t h a t NE r e d u c e s t h e a m p l i t u d e o f t h e IPSPs i n t h e h i p p o c a m p a l CA1 r e g i o n w i t h o u t a f f e c t i n g t h e r e s p o n s e 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. T h i s f i n d i n g i n d i c a t e s t h a t NE doe s n o t i n t e r f e r e d i r e c t l y on GABA t r a n s m i s s i o n a t t h e s y n a p t i c l e v e l but i s l i k e l y t o a c t d i r e c t l y on G A B A - e r g i c n e u r o n s . The p r o p o s e d s u p e r s e n s i t i z e d / 3 - r e c e p t o r s on t h e i n h i b i t o r y i n t e r n e u r o n s c o u l d , i n t h e p r e s e n c e of NE, r e s u l t i n a p o t e n t i n h i b i t i o n o f t h e s e G A B A - e r g i c n e u r o n s , meaning a s u b s t a n t i a l r e d u c t i o n i n GABA r e l e a s e upon t h e p y r a m i d a l c e l l s . However, one c a n n o t t o t a l l y i g n o r e t h e f a c t t h a t t h e r e a r e f u n c t i o n a l / ^ - a d r e n o c e p t o r s on t h e p y r a m i d a l c e l l s . They p r o b a b l y c o n t r i b u t e s l i g h t l y t o t h e i n h i b i t o r y i n f l u e n c e on t h e s e c e l l s , i n a d d i t i o n t o t h a t m e d i a t e d by a - a d r e n o c e p t o r s , by a s m a l l c-AMP-mediated h y p e r p o l a r i z a t i o n . On t h e o t h e r hand, r e c e n t f i n d i n g s by 52 M a d i s o n & N i c o l l (1982b) showed t h a t , i n t h e p r e s e n c e of NE, t h e r e p e t i t i v e d i s c h a r g e s of t h e CA1 p y r a m i d a l c e l l s , e l i c i t e d by e i t h e r c u r r e n t i n j e c t i o n or G l u a p p l i c a t i o n , i s s i g n i f i c a n t l y p r o l o n g e d . T h i s e f f e c t i s p r e s u m a b l y i n d u c e d by a ^ - r e c e p t o r - m e d i a t e d i n h i b i t i o n of t h e C a + + - a c t i v a t e d , K + c o n d u c t a n c e . T h i s d u a l ^ - r e c e p t o r - m e d i a t e d e f f e c t on t h e p y r a m i d a l c e l l s f u r t h e r s u p p o r t s t h e ' q u i e t r e a d i n e s s ' o f p y r a m i d a l c e l l s upon p r i m i n g by NE t o r e s p o n d t o s u b s e q u e n t s y n a p t i c a c t i v a t i o n w i t h a b u r s t i n g s y n c h r o n y . The r e s u l t i n g c o m b i n e d a c t i o n of s u p p r e s s i n g t h e weak i n p u t s and e n h a n c i n g s t r o n g a c t i v a t i o n s i s i n a c c o r d w i t h t h e c o n c e p t p r o p o s e d by Woodward e t a l (1979) t h a t NE i m p r o v e s t h e ' s i g n a l - t o - n o i s e ' r a t i o i n CNS n e u r o n s . M o r e o v e r , i t i s p o s s i b l e t h a t a f u r t h e r r e d u c t i o n of t h e C a + + - a c t i v a t e d , K + c o n d u c t a n c e by NE, due t o s u p e r s e n s i t i v i t y , i s r e s p o n s i b l e f o r a p o t e n t b l o c k o f t h e accommodation r e s p o n s e r e s u l t i n g i n rampant r e p e t i t i v e d i s c h a r g e s and e p i l e p t i f o r m a c t i v i t y i n t h e 6-OHDA-treated p r e p a r a t i o n s . Even t h o u g h f u r t h e r s t u d i e s a r e r e q u i r e d t o examine, f o r i n s t a n c e , t h e e f f e c t o f low NE c o n c e n t r a t i o n s and ^ - a n t a g o n i s t s on t h e r e c u r r e n t i n h i b i t i o n and d i r e c t r e c o r d i n g from i n t e r n e u r o n s i n t h e p r e s e n c e of NE and o t h e r s y m p athomimetic d r u g s , one c o u l d c o n c l u d e q u i t e c o n f i d e n t l y t h a t NE e x e r t s a g e n e r a l i n h i b i t o r y i n f l u e n c e on most p r o c e s s e s i n t h e CNS, i n c l u d i n g t h e i n h i b i t o r y ones t h a t m i g h t r e s u l t i n an i n d i r e c t ' d i s i n h i b i t i o n ' . 53 R e f e r e n c e s A l g e r , B.E., and N i c o l l , R.A. 1982. F e e d - f o r w a r d d e n d r i t i c i n h i b i t i o n i n r a t 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 s t u d i e d i_n v i t r o . J . P h y s i o l . 328 : 105-123. 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