UBC Theses and Dissertations

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UBC Theses and Dissertations

Electrical and chemical responsiveness of trigeminal root ganglion neurons of the guinea pig in… Spigelman, Igor 1986

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O / ELECTRICAL AND CHEMICAL RESPONSIVENESS OF TRIGEMINAL ROOT GANGLION NEURONS OF THE GUINEA PIG IN VITRO by IGOR SPIGELMAN B . S c , U n i v e r s i t y Of Toronto, 1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department Of Neurosci'ence We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA February 1986 © Igor Spigelman, 1986 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 of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 that p e r m i s s i o n f o r e x t e n s i v e copying of 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 of my Department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Pharmacology & Therapeut ics The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: 11 A p r i l 1986 i i A b s t r a c t I n t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s were u s e d t o i n v e s t i g a t e t h e e l e c t r i c a l and c h e m i c a l membrane p r o p e r t i e s o f n e u r o n a l somata i n g u i n e a p i g t r i g e m i n a l r o o t g a n g l i o n (TRG) s l i c e s . A l l i m p a l e d n e u r o n s (150) r e m a i n e d q u i e s c e n t p r i o r t o s q u a r e wave c u r r e n t p u l s e i n j e c t i o n t h r o u g h t h e i n t r a c e l l u l a r r e c o r d i n g e l e c t r o d e . The m a j o r i t y of n e u r o n s (=* 100) e x h i b i t e d v o l t a g e -and t i m e - d e p e n d e n t r e c t i f i c a t i o n i n r e s p o n s e t o h y p e r p o l a r i z i n g c u r r e n t p u l s e s . Two g r o u p s o f n e u r o n s were d i s t i n g u i s h e d on t h e b a s i s o f a c t i o n p o t e n t i a l c h a r a c t e r i s t i c s . In one g r o u p , a c t i o n p o t e n t i a l s had a p l a t e a u (hump) i n t h e r e p o l a r i z a t i o n p h a s e . A c t i o n p o t e n t i a l s e v o k e d f r o m t h e s e c o n d g r o u p o f n e u r o n s d i d not have t h i s c h a r a c t e r i s t i c . A v e r a g e a m p l i t u d e and d u r a t i o n o f s p i k e s and a f t e r h y p e r p o l a r i z a t i o n s was l a r g e r i n t h e f i r s t g r o u p o f n e u r o n s . B a t h a p p l i c a t i o n o f S - g l u t a m a t e ( 1 0 " 2 M) c a u s e d s m a l l (1-2 mV) d e p o l a r i z a t i o n s i n 2 o f 6 n e u r o n s t e s t e d . T h e s e d e p o l a r i z a t i o n s were n o t a s s o c i a t e d w i t h c h a n g e s i n membrane c o n d u c t a n c e . 5 - h y d r o x y t r y p t a m i n e ( 1 0 " 3 M) p r o d u c e d a s m a l l c o n d u c t a n c e i n c r e a s e and d e p o l a r i z a t i o n i n 1 o f 6 n e u r o n s t e s t e d . S i m i l a r a p p l i c a t i o n of 7 - a m i n o b u t y r i c a c i d (GABA; 10""-10" 2 M) p r o d u c e d d e c r e a s e s i n membrane r e s i s t a n c e (up t o 63%) a s s o c i a t e d w i t h membrane d e p o l a r i z a t i o n (2-14 mV) i n 30 of 36 n e u r o n s t e s t e d . R e d u c t i o n o f s p i k e a m p l i t u d e was o b s e r v e d d u r i n g GABA-mediated d e p o l a r i z a t i o n . T h e s e i n v e s t i g a t i o n s s u g g e s t t h a t membrane p r o p e r t i e s o f g u i n e a p i g TRG n e u r o n s a r e s i m i l a r t o t h o s e r e p o r t e d f o r o t h e r mammalian s e n s o r y g a n g l i o n n e u r o n s . I l l T a b l e o f C o n t e n t s A b s t r a c t i i C h a p t e r I INTRODUCTION 1 C h a p t e r I I METHODS 7 A. S L I C E PREPARATION 7 B. ELECTROPHYSIOLOGICAL RECORDING SET-UP 8 C h a p t e r I I I RESULTS 11 A. ELECTRICAL MEMBRANE PROPERTIES OF TRG NEURONS 11 I . INPUT RESISTANCE MEASUREMENTS 11 I I . MEMBRANE VOLTAGE- AND TIME-DEPENDENT RECTIFICATION 19 I I I . ACTION POTENTIALS . .• 23 IV. MEMBRANE OSCILLATORY TENDENCY 31 B. PHARMACOLOGICAL PROPERTIES OF TRG NEURONS 36 I . EFFECTS OF S-GLUTAMATE 36 I I . EFFECTS OF 5-HYDROXYTRYPTAMINE (5-HT) 36 I I I . EFFECTS OF GAMMA-AMINOBUTYRIC ACID (GABA) 37 C h a p t e r IV DISCUSSION 45 A. PHYSIOLOGICAL ROLE OF THE SOMATA OF PRIMARY AFFERENT NEURONS 45 B. ACTION POTENTIAL CHARACTERISTICS OF PRIMARY AFFERENT NEURONS 49 C. VOLTAGE- AND TIME-DEPENDENT RECTIFICATION IN SENSORY NEURONS 51 D. ACTIONS OF GLUTAMATE IN SENSORY GANGLION NEURONS 54 E . ROLE OF 5-HYDROXYTRYPTAMINE IN SENSORY TRANSMISSION ...55 F. ACTION OF GABA ON PRIMARY SENSORY NEURONS 58 G. MEMBRANE OSCILLATORY BEHAVIOUR AND ACCOMODATION 62 H. DIRECTIONS FOR FUTURE RESEARCH 66 C h a p t e r V SUMMARY AND CONCLUSIONS 68 BIBLIOGRAPHY 73 V L i s t o f F i g u r e s 1. C u r r e n t - v o l t a g e r e l a t i o n s h i p o f a TRG n e u r o n 14 2. E l e c t r o d e r e c t i f i c a t i o n 15 3. I n p u t r e s i s t a n c e o f TRG n e u r o n s 18 4. V a r i a t i o n i n membrane r e s p o n s e s 21 5. V o l t a g e - and t i m e - d e p e n d e n t r e c t i f i c a t i o n 22 6. Examples o f two t y p e s of a c t i o n p o t e n t i a l s 26 7. Sub and s u p r a t h r e s h o l d r e s p o n s e s o f a TRG n e u r o n 27 8. E f f e c t s ' o f membrane p o t e n t i a l on AHP s i z e 30 9. Membrane o s c i l l a t o r y b e h a v i o u r 34 10. R e p e t i t i v e f i r i n g 35 11. E f f e c t s o f n e u r o t r a n s m i t t e r c a n d i d a t e s ..39 12. E f f e c t of GABA on a c t i o n p o t e n t i a l a m p l i t u d e 44 v i L i s t of T a b l e s E f f e c t s of b a t h a p p l i e d GABA on some membrane p r o p e r t i e s of TRG n e u r o n s 1 '.I. INTRODUCTION V e r t e b r a t e p r i m a r y s e n s o r y n e u r o n s have been t h e s u b j e c t of i n t e n s i v e i n v e s t i g a t i o n d u r i n g t h e l a s t c e n t u r y ( c f . r e v i e w by L i e b e r m a n , 1976). The axon f i b e r s o f t h e s e n e u r o n s c o n v e y i n f o r m a t i o n f r o m s p e c i a l i z e d r e c e p t o r s t h a t s e n s e c h a n g e s i n t h e p h y s i c a l o r e l e c t r i c a l e n v i r o n m e n t o f t h e o r g a n i s m ( G r a y , 1959). T h e s e c h a n g e s may be p e r c e i v e d a s d i f f e r e n t m o d a l i t i e s of s e n s a t i o n i . e . , t o u c h , p r e s s u r e , t e m p e r a t u r e , p a i n , s i g h t , h e a r i n g , t a s t e and s m e l l . I m p u l s e s w h i c h a r e g e n e r a t e d by r e c e p t o r s t h a t may be r e p r e s e n t a t i v e of s e p a r a t e c e l l t y p e s or d i f f e r e n t s p e c i a l i z e d e n d i n g s of p r i m a r y s e n s o r y n e u r o n s a r e t r a n s m i t t e d v i a t h e i r axons t o t h e c e n t r a l n e r v o u s s y s t e m (CNS). W i t h few e x c e p t i o n s , somata o f t h e p r i m a r y s e n s o r y n e u r o n s of v e r t e b r a t e s l i e o u t s i d e t h e CNS i n g a n g l i a . T h e s e a r e l o c a t e d on t h e d o r s a l r o o t s between t h e i r p o i n t s o f j u n c t i o n w i t h t h e v e n t r a l r o o t and e n t r a n c e of t h e d o r s a l r o o t i n t o t h e s p i n a l c o r d ( K r i e g , 1966). G a n g l i a o f c r a n i a l n e r v e s w h i c h have a s e n s o r y component a l s o a r e l o c a t e d o u t s i d e t h e CNS. D o r s a l r o o t and c r a n i a l g a n g l i a a r e c o l l e c t i v e l y r e f e r r e d t o as c r a n i o s p i n a l , c e r e b r o s p i n a l o r s e n s o r y g a n g l i a . T h r e e e x c e p t i o n s t o t h i s a r r a n g e m e n t a r e t h e p r i m a r y s e n s o r y n e u r o n s of v i s u a l and o l f a c t o r y s y s t e m s and t h e m e s e n c e p h a l i c n u c l e u s o f t h e t r i g e m i n a l n e r v e w h i c h l i e s w i t h i n t h e b r a i n s t e m ( L i e b e r m a n , 1976). 2 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 i n v e s t i g a t i o n s of s e n s o r y n e u r o n membrane p r o p e r t i e s a r e i m p o r t a n t f o r s e v e r a l r e a s o n s . S i g n a l t r a n s m i s s i o n o f s e n s o r y i n f o r m a t i o n from p e r i p h e r a l r e c e p t o r s i s m e d i a t e d by i o n i c mechanisms of t h e n e u r o n a l membrane; t h e a l t e r a t i o n o f i t s e l e c t r i c a l p r o p e r t i e s c a n i n f l u e n c e , p r o f o u n d l y , s i g n a l t r a n s m i s s i o n o f s e n s o r y i n f o r m a t i o n t o t h e CNS. B o t h endogenous s u b s t a n c e s and t h e r a p e u t i c a l l y u s e d d r u g s may e x e r t t h e i r e f f e c t s t h r o u g h t h e s e i o n i c mechanisms. In a d d i t i o n , many o f human p a t h o l o g i c a l c o n d i t i o n s a p p e a r t o i n v o l v e a d i s r u p t i o n of n o r m a l membrane f u n c t i o n and s t u d y o f a l t e r a t i o n s i n membrane p r o p e r t i e s t h u s may be c o n s i d e r e d t o be c l i n i c a l l y i m p o r t a n t . The membrane e l e c t r i c a l p r o p e r t i e s of d o r s a l r o o t g a n g l i o n n e u r o n s were among t h e f i r s t t o be s t u d i e d u s 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 t e c h n i q u e s ( S v a e t i c h i n , 1951). S e v e r a l f e a t u r e s o f t h e s e n s o r y n e u r o n s o f t h e d o r s a l r o o t g a n g l i o n (DRG) have made them r e a d i l y a c c e s s i b l e t o e l e c t r o p h y s i o l o g i c a l a n a l y s e s . The c e l l b o d i e s o f t h e s e n e u r o n s a r e c o n v e n i e n t l y i s o l a t e d f r o m o t h e r t y p e s o f n e u r o n s t h u s a l l o w i n g f o r an unambiguous i d e n t i f i c a t i o n . D u r i n g e m b r y o l o g i c a l d e v e l o p m e n t , somata o f p r i m a r y s e n s o r y n e u r o n s may p o s s e s s s e v e r a l p r o c e s s e s . However, o n l y a s i n g l e p r o c e s s i n t h e a d u l t a n i m a l e x t e n d s from t h e soma t o an axon w h i c h r u n s from t h e p e r i p h e r y t o t h e CNS, e f f e c t i v e l y making a T - s h a p e d c o n n e c t i o n ( K r i e g , 1966). The p r e s e n c e o f o n l y a s i n g l e stem p r o c e s s w h i c h i s a t t a c h e d t o a s p h e r i c a l o r o v o i d soma f a c i l i t a t e s t h e a n a l y s i s o f e l e c t r o p h y s i o l o g i c a l membrane p r o p e r t i e s when i n t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s a r e 3 em p l o y e d . T h i s r e l a t i v e l y s i m p l e g eometry o b v i a t e s some o f t h e d i f f i c u l t i e s i n i n t e r p r e t a t i o n of t h e v o l t a g e r e s p o n s e s t o i n t r a c e l l u l a r c u r r e n t i n j e c t i o n w h i c h i s u s e d t o a s s e s s c e r t a i n membrane p r o p e r t i e s . S y n a p t i c c o n t a c t s o r i n p u t s a r e v i r t u a l l y a b s e n t and t h i s f u r t h e r s i m p l i f i e s a n a l y s i s and i n t e r p r e t a t i o n o f r e s u l t s . R a y a h a r a e t a l (1984) have shown t h a t s y n a p t i c c o n t a c t s o f s p i n a l c o r d f i b r e s w i t h c e l l s o f t h e c a t DRG do o c c u r , b u t t h e numbers a r e v e r y s m a l l . In c o n t r a s t t o DRG n e u r o n s w h i c h have been s u b j e c t e d t o e x t e n s i v e e l e c t r o p h y s i o l o g i c a l e x a m i n a t i o n s , n e u r o n s of t h e t r i g e m i n a l r o o t ('Gasserian o r s e m i l u n a r ) g a n g l i o n (TRG) have r e c e i v e d v e r y l i t t l e a t t e n t i o n . T h i s may have been due i n p a r t t o t h e a n a t o m i c a l i n a c c e s s i b i l i t y o f t h e t r i g e m i n a l g a n g l i o n r e l a t i v e t o t h e DRG and i n p a r t , t o t h e g e n e r a l a s s u m p t i o n t h a t t h e membrane p r o p e r t i e s o f c r a n i a l s e n s o r y g a n g l i o n n e u r o n s a r e t h e same a s t h e p r o p e r t i e s of DRG n e u r o n s , an a s s u m p t i o n t h a t h i t h e r t o has n o t been v e r i f i e d e x p e r i m e n t a l l y . In humans (and i n mammals g e n e r a l l y ) , t h e t r i g e m i n a l r o o t g a n g l i o n i s t h e l a r g e s t s e n s o r y g a n g l i o n i n t h e body and i s composed o f t h r e e d i v i s i o n s : o p h t h a l m i c , m a x i l l a r y and m a n d i b u l a r b r a n c h e s . E a c h TRG i s formed a t t h e p o i n t of c o n v e r g e n c e o f i t s t h r e e d i v i s i o n s on t h e f l o o r of t h e c r a n i a l v a u l t , w i t h t h e l a r g e s t o r m a n d i b u l a r b r a n c h i n t h e most l a t e r a l p o s i t i o n . T h e s e b i l a t e r a l b r a n c h e s p r o v i d e s e n s o r y i n n e r v a t i o n t o t h e m u c o s a l l i n i n g o f t h e o r a l c a v i t i e s , t h e s k i n , and m u s c l e s o f m a s t i c a t i o n ( t h e l a t t e r a r e i n n e r v a t e d by n e u r o n s whose somata l i e w i t h i n t h e t r i g e m i n a l m e s e n c e p h a l i c n u c l e u s of 4 t h e CNS). In a d d i t i o n , t h e m y s t a c i a l v i b r i s s a e ( w h i s k e r s ) p o s s e s s e d by most mammals a l s o a r e s e r v e d by f i b e r s o f t h e c e l l b o d i e s i n t h e TRG. A l s o , s e n s o r y i n n e r v a t i o n of t o o t h p u l p , s u r r o u n d i n g g i n g i v a and p e r i o d o n t a l membrane i s m e d i a t e d by t h e t r i g e m i n a l n e r v e ( K e l l y , 1981). The need f o r i n t r a c e l l u l a r s t u d i e s o f TRG n e u r o n s a r i s e s n o t o n l y f r o m t h e f a c t t h a t t h e i r s t u d y has been n e g l e c t e d i n c o m p a r i s o n t o DRG n e u r o n s b ut a l s o b e c a u s e o f t h e t r i g e m i n a l g a n g l i o n ' s r e l e v a n c e t o c e r t a i n human p a t h o l o g i c a l c o n d i t i o n s . F o r example, t r i g e m i n a l n e u r a l g i a , a l s o known a l s o a s t i c d o l o u r e u x i s a d i s o r d e r i n w h i c h t h e a f f l i c t e d p a t i e n t e x p e r i e n c e s what may be d e s c r i b e d a s l i g h t n i n g - l i k e a t t a c k s o f p a i n o f e x t r e m e i n t e n s i t y t h a t c a n be p r e c i p i t a t e d by v e r y l i g h t s t i m u l i f r o m t h e s k i n , f a c e o r gums, i n an a p p a r e n t a b s e n c e o f n e u r o l o g i c a l d e f i c i t . A l t h o u g h p r e v i o u s l y c o n s i d e r e d t o be a d i s o r d e r of t h e CNS, r e c e n t and more s u b s t a n t i a l e v i d e n c e p o i n t s t o p e r i p h e r a l o r i g i n s o f t r i g e m i n a l n e u r a l g i a , w i t h a more l i k e l y s i t e o f p a t h o l o g y b e i n g w i t h i n t h e t r i g e m i n a l , g a n g l i o n i t s e l f or i n i t s s e n s o r y r o o t ( K e r r , 1979). P r e v i o u s i n v e s t i g a t i o n s 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 t o m o n i t o r n e u r o n a l d i s c h a r g e a c t i v i t y of t h e TRG n e u r o n s have p r o v i d e d a w e a l t h o f i n f o r m a t i o n on t h e f u n c t i o n a l s o m a t o t o p i c o r g a n i z a t i o n of p r i m a r y a f f e r e n t n e u r o n s i n a number of mammalian s p e c i e s ( K e r r and L y s a k , 1964; D a r i a n - S m i t h e t . a l . , 1965; Z u c k e r and W e l k e r , 1969). O t h e r i n v e s t i g a t o r s have u s e d e x t r a c e l l u l a r r e c o r d i n g s f r o m t r i g e m i n a l g a n g l i o n n e u r o n s f o r o b s e r v a t i o n s o f p o s s i b l e e f f e c t s o f e p i l e p t o g e n i c a g e n t s on t h e 5 e x c i t a b i l i t y o f t h e i r n e u r o n a l membrane ( B u r c h i e l e t a l . , 1978). However, e x t r a c e l l u l a r r e c o r d i n g s p r o v i d e i n s u f f i c i e n t i n f o r m a t i o n f o r t h e u n d e r s t a n d i n g of mechanisms of membrane e x c i t a b i l i t y . Hence, an i n t r a c e l l u l a r a n a l y s i s i s r e q u i r e d . P r e v i o u s i n t r a c e l l u l a r s t u d i e s o f TRG n e u r o n s have been r a t h e r p r e l i m i n a r y i n s c o p e and have d e a l t w i t h e i t h e r c u l t u r e d (Fukuda and Kameyama, 1980) or d i s s o c i a t e d and i n t e r n a l l y p e r f u s e d c e l l s ( K r i s h t a l and P i d o p l i c h k o , 1980, 1981; K r i s h t a l and M archenko, 1984). T h e s e e x p e r i m e n t a l p r e p a r a t i o n s a r e not i d e a l b e c a u s e e x t e n s i v e m o d i f i c a t i o n s o f p h y s i o l o g i c a l membrane p a r a m e t e r s l i k e l y have o c c u r r e d and hence t h e ' n a t u r a l ' p h y s i o l o g i c a l e n v i r o n m e n t of t h e n e u r o n has been o n l y a p p r o x i m a t e d . The e x t r a p o l a t i o n o f r e s u l t s t o t h e no r m a l c e l l f u n c t i o n i s d u b i o u s , b e c a u s e o f t h e u n c e r t a i n t y • i n i n t e r p r e t a t i o n of r e s u l t s r e l a t i v e t o t h o s e o b t a i n e d from i n t a c t p h y s i o l o g i c a l s y s t e m s . To d a t e , t h e o n l y i n t r a c e l l u l a r i n v e s t i g a t i o n i n w h i c h TRG n e u r o n s were n o t s u b j e c t e d t o e x t e n s i v e m a n i p u l a t i o n was t h a t by P u i l e t a l ( i n p r e s s ) i n w h i c h membrane e l e c t r i c a l p r o p e r t i e s of TRG n e u r o n s were examined i n i n v i v o c o n d i t i o n s . A l t h o u g h t h e i r g u i n e a p i g p r e p a r a t i o n c l o s e l y a p p r o x i m a t e d p h y s i o l o g i c a l c o n d i t i o n s , i t d i d not p e r m i t a p p l i c a t i o n o f p h a r m a c o l o g i c a l a g e n t s i n known c o n c e n t r a t i o n s d i r e c t l y t o t h e n e u r o n a l membrane. On t h e o t h e r hand, t h e r e a r e many a d v a n t a g e s and d i s a d v a n t a g e s i n s t u d y i n g t h e b e h a v i o u r o f n e u r o n s i n v i t r o . Some o f t h e e x p e r i m e n t a l a d v a n t a g e s a l s o may be c o n s i d e r e d 6 d i s a d v a n t a g e s . F o r example, i s o l a t i o n f r o m t h e u s u a l p h y s i o l o g i c a l e n v i r o n m e n t , p o s s i b l e m e c h a n i c a l d e f o r m a t i o n o f t h e c e l l body and l o s s o f t h e l o n g p e r i p h e r a l and c e n t r a l p o r t i o n s of t h e n e u r o n a l p r o c e s s e s r e n d e r s u c h p r e p a r a t i o n s somewhat u n p h y s i o l o g i c a l . However, t h e a d v a n t a g e s o u t w e i g h t h e n e g a t i v e c o n s i d e r a t i o n s , e s p e c i a l l y when t h e r e s u l t s o f s t u d i e s i n t h e i n v i t r o p r e p a r a t i o n complement t h o s e o b t a i n e d f r o m i n  v i v o p r e p a r a t i o n s . S e c o n d l y , p r o b l e m s t h a t c a n n o t be a d d r e s s e d a d e q u a t e l y by t h e l a t t e r methods o f t e n may be a n s w e r e d by s i m i l a r i n v e s t i g a t i o n s i n v i t r o . An i n v i t r o s l i c e p r e p a r a t i o n of t h e TRG of t h e g u i n e a p i g w ould f a c i l i t a t e s t a b l e i n t r a c e l l u l a r r e c o r d i n g s from n e u r o n s whose c e l l b o d i e s have n o t been m o d i f i e d e x t e n s i v e l y by e x p e r i m e n t a l m a n i p u l a t i o n . T h e r e f o r e , t h i s s i t u a t i o n may be c l o s e r p h y s i o l o g i c a l l y t o i n v i v o c o n d i t i o n s w h i l e r e t a i n i n g o t h e r a d v a n t a g e s i n h e r e n t t o i n v i t r o e x p e r i m e n t a t i o n . . The p r e s e n t s t u d y d e s c r i b e s , f o r t h e f i r s t t i m e , b o t h p a s s i v e and a c t i v e membrane p r o p e r t i e s o f g u i n e a p i g TRG n e u r o n s i n v i t r o a l l o w i n g a c o m p a r i s o n t o p u b l i s h e d r e p o r t s on t h e membrane p r o p e r t i e s of o t h e r p r i m a r y s e n s o r y n e u r o n s . The m o d u l a t i o n o f t h e membrane p r o p e r t i e s of TRG n e u r o n s by n e u r o a c t i v e s u b s t a n c e s a l s o i s p r e s e n t e d . 7 I I . METHODS A. S L I C E PREPARATION A l b i n o g u i n e a p i g s o f e i t h e r sex w e i g h i n g 200-400g were u s e d f o r a l l e x p e r i m e n t s . A n a e s t h e s i a was i n d u c e d w i t h 4% h a l o t h a n e or i s o f l u r a n e a d m i n i s t e r e d i n a -2 l i t e r chamber. The t r a c h e a was c a n n u l a t e d and t h e a n i m a l ' s s k u l l h a i r removed w i t h s c i s s o r s . The a n i m a l t h e n was p l a c e d i n a s t e r e o t a x i c head h o l d e r . A c r a n i o t o m y was p e r f o r m e d w h i l e a n a e s t h e s i a was m a i n t a i n e d e n d o t r a c h e a l l y w i t h 2% h a l o t h a n e . A f t e r m id-c o l l i c u l a r d e c e r e b r a t i o n w i t h a s c a l p e l b l a d e , t h e e n c e p h a l o n was a s p i r a t e d t o r e v e a l t h e u n d e r l y i n g p o s t e r i o r f o s s a e . At t h i s s t a g e , t h e a d m i n i s t e r e d h a l o t h a n e c o n c e n t r a t i o n was r e d u c e d t o 0.5%. H e a r t r a t e was m o n i t o r e d v i a e l e c t r o d e s i n s e r t e d s u b d e r m a l l y i n t h e t h o r a x r e g i o n t h r o u g h o u t t h e s u r g i c a l d i s s e c t i o n p r o c e d u r e i n m a j o r i t y o f e x p e r i m e n t s . The l e f t t r i g e m i n a l g a n g l i o n was c a r e f u l l y i s o l a t e d from t h e s u r r o u n d i n g c o n n e c t i v e t i s s u e and c a r t i l a g e n o u s bone 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 and f i n e i n s t r u m e n t s ( e . g . No. 5 j e w e l e r ' s f o r c e p s ) . C a r e was t a k e n n o t t o d i s t u r b b l o o d v e s s e l s i n t h e v i c i n i t y o f t h e g a n g l i o n f o r two r e a s o n s : (1) a m i n i m i z a t i o n o f t h e d u r a t i o n o f h y p o x i a was c o n s i d e r e d d e s i r a b l e , a n d (2) p r o f u s e b l e e d i n g made v i s u a l i z a t i o n o f t h e g a n g l i o n and t h e r e f o r e , d i s s e c t i o n , d i f f i c u l t . The c e n t r a l and p e r i p h e r a l ends o f t h e g a n g l i o n were s e v e r e d s u c h t h a t t h e t o t a l l e n g t h o f t h e e x c i s e d t i s s u e was 1 t o 1.3 c e n t i m e t e r s . U s u a l l y , 8 t h e c e n t r a l end was c u t as c l o s e as p o s s i b l e t o i t s e n t r y i n t o t h e b r a i n s t e m . . F o l l o w i n g t o t a l e x c i s i o n from t h e p o s t e r i o r f o s s a , t h e g a n g l i o n was immersed q u i c k l y i n t o c o l d (4°C) 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 (Yamamoto's) s o l u t i o n w h i c h was o x y g e n a t e d w i t h 95/5% g a s e o u s m i x t u r e o f 0 2 / C 0 2 . The r e m a i n i n g c o n n e c t i v e t i s s u e , a r a c h n o i d a l and d u r a l ( c a p s u l a r ) s h e a t h s s u r r o u n d i n g t h e g a n g l i o n , and i t s c e n t r a l and p e r i p h e r a l r o o t stumps, were removed from t h e g a n g l i o n w i t h t h e a i d o f a d i s s e c t i n g m i c r o s c o p e . The g a n g l i o n was p l a c e d on a T e f l o n s t a g e of a m e c h a n i c a l t i s s u e c h o p p e r w h i c h was c o v e r e d w i t h a r t i f i c i a l C S F - s a t u r a t e d f i l t e r p a p e r and t h e n c u t i n t o t h i n (300-500 urn) l o n g i t u d i n a l l y o r i e n t e d s l i c e s ; t h i s p r o c e d u r e p r e s e r v e d some o f t h e axon b u n d l e s i n t h e stumps of b o t h c e n t r a l and p e r i p h e r a l b r a n c h e s . S l i c e s were t r a n s f e r r e d i m m e d i a t e l y i n t o an i n c u b a t i n g chamber where t h e y were m a i n t a i n e d i n o x y g e n a t e d Yamamoto's s o l u t i o n a t 37°C u n t i l needed f o r r e c o r d i n g . The p r o c e d u r e s f o r d i s s e c t i o n , s l i c i n g , e t c . o f t h e s e c o n d g a n g l i o n were i d e n t i c a l t o t h o s e d e s c r i b e d above and t h e s e were c a r r i e d o u t w i t h i n 15 min a f t e r p r e p a r a t i o n o f s l i c e s o f t h e f i r s t g a n g l i o n . B. ELECTROPHYSIOLOGICAL RECORDING SET-UP F o r r e c o r d i n g , s l i c e s were t r a n s f e r r e d t o a s u p e r p e r f u s i o n chamber w i t h a g l a s s b o t t o m . A N y l o n mesh was u s e d f o r r e t e n t i o n o f t h e s l i c e s i n o r d e r t o p r e v e n t t h e i r movement o r f l o a t i n g i n t h e p e r f u s i o n f l u i d . The r e c o r d i n g chamber was f i x e d t o t h e s t a g e o f a L e i t z m i c r o s c o p e e q u i p p e d w i t h Hoffman m o d u l a t i o n c o n t r a s t o p t i c s w h i c h a l l o w e d v i s u a l i z a t i o n o f 9 i n d i v i d u a l c e l l s a t t h e edges o f t h e s l i c e . A g e n t l e s t r e a m o f a i r was blown o v e r t h e l e n s i n o r d e r t o p r e v e n t c o n d e n s a t i o n o f v a p o u r on t h e o b j e c t i v e l e n s . Sodium S - g l u t a m a t e , 7-a m i n o b u t r y i c a c i d and 5 - h y d r o x y t r y p t a m i n e c r e a t i n i n e s u l f a t e ( a l l f r o m Sigma) were a p p l i e d i n a p p r o p r i a t e d i l u t i o n s w i t h t h e s t a n d a r d p e r f u s i o n s o l u t i o n w h i c h had t h e f o l l o w i n g c h e m i c a l c o m p o s i t i o n ( i n mM): N a C l , 124; KC1, 3.75; KH 2PO a, 1.25; M g S O „ , 2.0; C a C l 2 , 2.0; NaHC0 3, 26; and d e x t r o s e , 10. C o n t i n u o u s b u b b l i n g w i t h a 95% 0 2 and 5% C 0 2 m i x t u r e e n s u r e d a d e q u a t e o x y g e n a t i o n and a pH o f 7.4. An aluminum h e a t i n g b l o c k , i . e . a P e l l e t i e r d e v i c e , was u s e d t o keep t h e b a t h f l u i d t e m p e r a t u r e a t 36 °C t h r o u g h o u t t h e e x p e r i m e n t . The t o t a l chamber volume was 3 ml i n t h e i n i t i a l e x p e r i m e n t s , b u t a f t e r s e v e r a l e x p e r i m e n t s , was r e d u c e d t o 1 ml, t o f a c i l i t a t e a f a s t e r e x c h a n g e o f s o l u t i o n , p a r t i c u l a r l y i n t h e c a s e of d r u g a p p l i c a t i o n s . A s t e a d y e f f l u x of f l u i d f r o m t h e chamber was a c h i e v e d by means o f a f i l t e r p a p e r s t r i p w h i c h c o n n e c t e d t h e r e c o r d i n g chamber t o a s m a l l a d j a c e n t w e l l f r o m w h i c h t h e f l u i d was removed by a s p i r a t i o n . The m i c r o s c o p e and m i c r o m a n i p u l a t o r a s s e m b l y were h o u s e d on a v i b r a t i o n - d a m p i n g t a b l e t h a t o p e r a t e d by an a i r -c u s h i o n a r r a n g e m e n t . I n t r a c e l l u l a r r e c o r d i n g s were made u s i n g p o t a s s i u m c h l o r i d e (3M) o r p o t a s s i u m s u l p h a t e (4M) f i l l e d g l a s s m i c r o p i p e t t e s . R e s i s t a n c e s o f KC1 e l e c t r o d e s r a n g e d from 10 t o 40 MJ2 w h i c h were m e a s u r e d i n t i s s u e p r i o r t o impalement of c e l l s . E l e c t r o d e s f i l l e d w i t h K 2SO f t had h i g h e r v a l u e s o f t i p r e s i s t a n c e s 28-55 Mfi). The m i c r o p r o b e s y s t e m (M701, WP I n s t r u m e n t s ) w h i c h was 10 u s e d t o measure p o t e n t i a l s a l l o w e d i n j e c t i o n o f c u r r e n t v i a a b r i d g e - b a l a n c e c i r c u i t f o r c o n v e n t i o n a l measurement of membrane r e s i s t a n c e w i t h c o n s t a n t c u r r e n t - p u l s e s . R e s t i n g membrane p o t e n t i a l of t h e c e l l was c o n t i n u o u s l y m o n i t o r e d w i t h a c h a r t r e c o r d e r ( G o u l d ) ; t h e z e r o v o l t a g e l e v e l was d e t e r m i n e d upon w i t h d r a w a l o f an e l e c t r o d e from t h e c e l l . A m p l i f i e d p o t e n t i a l s were s t o r e d on t a p e u s i n g a H e w l e t t - P a c k a r d FM t a p e r e c o r d e r (Model 3968A; f r e q u e n c y range f l a t t o 5 KHz a t 7.5 i p s ) and p r i n t e d o n - l i n e on c h a r t r e c o r d e r p a p e r . F o r d e t a i l e d d a t a a n a l y s i s , i n t r a c e l l u l a r l y r e c o r d e d p o t e n t i a l s were p l a y e d back i n t o t h e o s c i l l o s c o p e ( T e k t r o n i x RM565) v i a a waveform r e c o r d e r ( B i o m a t i o n 8 0 5 ) . T h i s m i c r o c o m p u t e r d i g i t i z e d t h e i n p u t waveform "sweep" ( r a t e 1/sec) i n t o 2048 p o i n t s and p e r m i t t e d manual c o n t r o l of d i g i t a l - t o - a n a l o g u e c o n v e r s i o n f o r d i s p l a y on t h e o s c i l l o s c o p e and p h o t o g r a p h y w i t h a P o l a r o i d camera e q u i p p e d w i t h a 75mm l e n s ( T e k t r o n i x ) , o r r e p r o d u c t i o n o f t h e sweep on p a p e r of an X-Y pen r e c o r d e r ( H e w l e t t - P a c k a r d 7015B). 11 I I I . RESULTS A. ELECTRICAL MEMBRANE PROPERTIES OF TRG NEURONS I . INPUT RESISTANCE MEASUREMENTS I n t r a c e l l u l a r r e c o r d i n g s were o b t a i n e d f r o m a t o t a l o f 150 TRG n e u r o n s . O n l y c e l l s t h a t had s t a b l e r e s t i n g membrane p o t e n t i a l s more n e g a t i v e t h a n -50 mV were s e l e c t e d f o r o f f - l i n e a n a l y s i s ' . S t a b l e r e c o r d i n g s c o u l d be m a i n t a i n e d f o r a s l o n g a s 2.5 h o u r s i n some n e u r o n s . However, most c e l l i m p a l e m e n t s were s u c c e s s f u l f o r s h o r t e r p e r i o d s a t r e a s o n a b l y n e g a t i v e membrane p o t e n t i a l l e v e l s , d e p e n d i n g on t h e e x p e r i m e n t a l c i r c u m s t a n c e s . In many i n s t a n c e s , t h e i n p u t impedance o f t h e e l e c t r o d e i n c r e a s e d d u r i n g s t a b l e i n t r a c e l l u l a r r e c o r d i n g ( p r e s u m a b l y due t o c l o g g i n g o f t h e e l e c t r o d e t i p ) t o an e x t e n t t h a t c u r r e n t s w h i c h were r e q u i r e d f o r a d e s i r e d v o l t a g e d i s p l a c e m e n t ( e . g . , 5-20 mV) of t h e membrane p o t e n t i a l c o u l d n o t be i n j e c t e d w i t h o u t e x c e s s i v e i n t e r f e r e n c e f r o m e l e c t r o d e r e c t i f i c a t i o n . I n i t i a l l y , v a l u e s o f membrane i n p u t r e s i s t a n c e ( R i ) were o b t a i n e d d i r e c t l y f r o m c h a r t r e c o r d e r t r a c e s . T h i s was a c h i e v e d by m e a s u r i n g t h e s l o p e o f t h e l i n e a r p o r t i o n i n t h e I/V c u r v e o f t h e membrane v o l t a g e r e s p o n s e s t o h y p e r p o l a r i z i n g c u r r e n t p u l s e s . However, t h e m a j o r i t y o f TRG n e u r o n s e x h i b i t e d a v o l t a g e - and t i m e - d e p e n d e n t r e c t i f i c a t i o n when h y p e r p o l a r i z i n g c u r r e n t p u l s e s (0.5-10 nA) were i n j e c t e d i n t r a c e l l u l a r l y . F i g u r e 1 i l l u s t r a t e s a c u r r e n t - v o l t a g e r e l a t i o n s h i p o f one s u c h c e l l . I t i s r e a d i l y a p p a r e n t from t h e v o l t a g e r e c o r d i n g t r a c e s 12 ( i n s e t ) t h a t o n l y v e r y s m a l l membrane p o t e n t i a l d i s p l a c e m e n t s may p r o v i d e a c c u r a t e e s t i m a t e s of i n p u t r e s i s t a n c e , i . e . , v o l t a g e - and t i m e - d e p e n d e n t r e c t i f i c a t i o n becomes a p p a r e n t w i t h t h e l a r g e r membrane p o t e n t i a l d i s p l a c e m e n t s . Thus t h e % v a l u e s e s t i m a t e d f r o m t h e s l o p e of t h e peak v o l t a g e r e s p o n s e v a l u e s a r e a s s o c i a t e d w i t h a l a r g e e r r o r , and t h e r e f o r e a d i f f e r e n t method f o r o b t a i n i n g R e s t i m a t e s was u s e d . A r e l i a b l e e s t i m a t e of R^ c a n be o b t a i n e d f r o m measurements of t h e p o t e n t i a l n e a r t h e end o f r e s p o n s e s t o h y p e r p o l a r i z i n g c u r r e n t p u l s e s (50-100 ms d u r a t i o n : d o t t e d l i n e i n F i g . 1 ) . A l t h o u g h t h i s method was s a t i s f a c t o r y i n some c e l l s , c o m p l i c a t i o n s a r o s e i n t h o s e c e l l s where s t e a d y - s t a t e v a l u e s o f membrane v o l t a g e d i s p l a c e m e n t c o u l d not be r e a c h e d a t t h e t e r m i n a t i o n o f - t h e 50 ms c u r r e n t p u l s e . In a d d i t i o n , many e l e c t r o d e s p o s s e s s e d s i g n i f i c a n t c a p a c i t a n c e and r e c t i f i c a t i o n w h i c h c o u l d mask a sag i n t h e membrane r e s p o n s e of a n e u r o n w h i c h e x h i b i t e d t i m e - d e p e n d e n t r e c t i f i c a t i o n t o a h y p e r p o l a r i z i n g c u r r e n t p u l s e i n j e c t i o n ; t h i s a l s o r e s u l t e d i n o v e r e s t i m a t e s o f t h e s t e a d y - s t a t e v o l t a g e d i s p l a c e m e n t v a l u e s . F i g u r e 2 i l l u s t r a t e s an example o f p r o m i n e n t e l e c t r o d e r e c t i f i c a t i o n t h a t c o u l d r e s u l t i n s u c h o v e r e s t i m a t e s . B e c a u s e o f t h e above c o n s i d e r a t i o n s , R^ i n a m a j o r i t y o f n e u r o n s was o b t a i n e d u s i n g v o l t a g e d i s p l a c e m e n t s o f l e s s t h a n 10 mV. An e x c e p t i o n was made f o r o b s e r v a t i o n s o f R^ c h a n g e s due t o d r u g a p p l i c a t i o n s . In s u c h c a s e s , c h a n g e s i n Ri due t o d r u g a d m i n i s t r a t i o n were c o n s i d e r e d t o be more i m p o r t a n t t h a n a b s o l u t e R^ v a l u e s b e c a u s e t h e y r e p r e s e n t e d d r u g a c t i o n . 1 3 F i g u r e 1. C u r r e n t - v o l t a g e r e l a t i o n s h i p o f a TRG n e u r o n . Open c i r c l e s r e p r e s e n t s t e a d y s t a t e v o l t a g e r e s p o n s e s t o 50 ms h y p e r p o l a r i z i n g c u r r e n t p u l s e s . C l o s e d c i r c l e s i n d i c a t e peak v o l t a g e r e s p o n s e s t o c u r r e n t i n j e c t i o n . E l e c t r o d e r e c t i f i c a t i o n becomes e v i d e n t w i t h l a r g e membrane p o t e n t i a l d i s p l a c e m e n t s . T h i s c a n be s e e n i n t h e i n s e t w h i c h d i s p l a y s t h e n e u r o n a l h y p e r p o l a r i z i n g r e s p o n s e s w h i c h were measured f o r t h e g r a p h . F i g u r e 2. C o n t r o l v o l t a g e t r a c e s o b t a i n e d w i t h a 50 Mfl, 3 M KC1 f i l l e d e l e c t r o d e t h a t showed p r o n o u n c e d r e c t i f i c a t i o n d u r i n g p a s s a g e o f h y p e r p o l a r i z i n g c u r r e n t p u l s e s . A l t h o u g h t h e a c t i v e b r i d g e o f t h e a m p l i f i e r was w e l l b a l a n c e d a t t h e o n s e t o f t h e p u l s e s , e l e c t r o d e t i p r e s i s t a n c e i n c r e a s e d d u r i n g t h e c o u r s e o f c u r r e n t a p p l i c a t i o n as seen by t h e c o n t i n u i n g i n c r e a s e s i n n e g a t i v i t y o f p o t e n t i a l . T h i s r e c t i f i c a t i o n d i d n o t i n c l u d e any i n c r e a s e i n e l e c t r o d e c a p a c i t a n c e b e c a u s e no l a r g e c a p a c i t i v e e f f e c t ( i . e . a l a r g e v o l t a g e t r a n s i e n t ) was o b s e r v e d a t p u l s e t e r m i n a t i o n . 14 F i g u r e 1 current (nA) o 1 0> a C/i 0) 24 o > h32 1 5 F i g u r e 2 1 nA 1 20 mV 1 6 F i g u r e 3 i s a h i s t o g r a m of R i v a l u e s o b t a i n e d from 80 TRG n e u r o n s . About 65% o f t h i s p o p u l a t i o n of n e u r o n s f a l l w i t h i n t h e r a n g e o f 3 t o 15 Mfi. The s m a l l number of n e u r o n s w i t h h i g h R i v a l u e s i s a t l e a s t p a r t l y a r e s u l t o f s i g n i f i c a n t e l e c t r o d e s a m p l i n g b i a s . A l a r g e number o f n e u r o n s w i t h h i g h R i v a l u e s were i m p a l e d , but s u c h c e l l s c o u l d n o t be r e c o r d e d from f o r l o n g p e r i o d s ( e . g . g r e a t e r t h a n 10 m i n ) , p r e s u m a b l y b e c a u s e o f poor s o m a t i c i m p a l e m e n t s of s m a l l c e l l s w i t h c o a r s e e l e c t r o d e t i p s . The d a t a f r o m s u c h n e u r o n s were n o t u s e d i n a n a l y s e s o f i n p u t r e s i s t a n c e . Use of e l e c t r o d e s w i t h f i n e r t i p s and, t h e r e f o r e , h i g h e r impedances l i k e l y w ould have f a c i l i t a t e d s t a b l e o r p r o l o n g e d r e c o r d i n g s from t h e s e n e u r o n s . However, h i g h impedance e l e c t r o d e s (> 40 Mfi) d i d n o t have a d e q u a t e c u r r e n t p a s s i n g c a p a b i l i t y t o a l l o w g e n e r a t i o n o f a c t i o n p o t e n t i a l s i n most n e u r o n s t h a t were p e n e t r a t e d w i t h s u c h e l e c t r o d e s . T h e r e f o r e , e l e c t r o d e s w i t h t i p r e s i s t a n c e s l e s s t h a n 40 MS2 were u s e d t o a c h i e v e a compromise between t h e a b i l i t y t o s u c c e s s f u l l y i m p a l e c e l l s f o r p r o l o n g e d r e c o r d i n g s and t h e a b i l i t y t o i n j e c t s u f f i c i e n t amounts o f c u r r e n t f o r a n a l y s i s of membrane p r o p e r t i e s . 1 7 F i g u r e 3 . D i s t r i b u t i o n o f measured i n p u t r e s i s t a n c e s i n TRG n e u r o n s . V a l u e s of R i were o b t a i n e d by m e a s u r i n g t h e membrane v o l t a g e r e s p o n s e s t o s m a l l h y p e r p o l a r i z i n g c u r r e n t p u l s e s (< 10 mV) o r by o b t a i n i n g t h e s l o p e o f t h e l i n e a r p o r t i o n o f t h e s t e a d y - s t a t e r e s p o n s e s i n t h e I/V c u r v e s of n e u r o n s t h a t showed a l a r g e amount of membrane r e c t i f i c a t i o n . 1 8 F i g u r e 3 20-16-8 12 E z 8-4-3 9 15 21 27 33 R j ( M 1 2 ) 19 I I . MEMBRANE VOLTAGE- AND TIME-DEPENDENT RECTIFICATION The m a j o r i t y of TRG n e u r o n s e x h i b i t e d v a r y i n g d e g r e e s o f membrane r e c t i f i c a t i o n upon h y p e r p o l a r i z i n g c u r r e n t p u l s e i n j e c t i o n . An example of t h i s p r o n o u n c e d " s a g g i n g " b e h a v i o u r o f v o l t a g e r e s p o n s e s i s shown i n F i g u r e 4a. An o p p o s i t e e x t r e m e i n membrane b e h a v i o u r o f a n o t h e r n e u r o n i s shown i n F i g u r e 4b. O n l y 3 o u t o f 80 n e u r o n s showed no d e t e c t a b l e s a g a s s o c i a t e d w i t h h y p e r p o l a r i z i n g membrane d i s p l a c e m e n t s o f up t o 35 mV. The " s a g g i n g " b e h a v i o u r of most o t h e r n e u r o n s was l e s s p r o n o u n c e d t h a n t h a t shown i n F i g u r e 4a. A p o s s i b i l i t y o f e x t e n s i v e c e l l damage was e x c l u d e d s i n c e t h e s e c e l l s p r o d u c e d a c t i o n p o t e n t i a l s i n r e s p o n s e t o r e p e t i t i v e i n j e c t i o n s o f 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 o t h e r w i s e had s t a b l e membrane p o t e n t i a l s . E l e c t r o d e r e c t i f i c a t i o n and an i n a b i l i t y t o p a s s ' a d e q u a t e ' amounts o f c u r r e n t may have r e s u l t e d i n a m a s k i n g of a f a s t c o n d u c t a n c e i n c r e a s e i n some n e u r o n s . T h i s s u g g e s t i o n was e x c l u d e d i n many c a s e s where t h e e l e c t r o d e was c a p a b l e o f p a s s i n g c u r r e n t s o f up t o 7 nA w i t h o u t n o t i c e a b l e r e c t i f i c a t i o n . Some h y p e r p o l a r i z i n g r e s p o n s e s o f one n e u r o n a r e shown on an e xpanded t i m e base i n F i g u r e 5. T h e s e r e c o r d s e x h i b i t a c h a r a c t e r i s t i c v o l t a g e -d e p endence o f t h e t i m e - d e p e n d e n t r e c t i f i c a t i o n , o r " s a g " i n t h e v o l t a g e r e s p o n s e . I t i s r e a d i l y a p p a r e n t t h a t an i n c r e a s e i n t h e amount o f h y p e r p o l a r i z i n g c u r r e n t c a u s e s a f a s t e r a p p r o a c h t o t h e s t e a d y - s t a t e v a l u e o f membrane v o l t a g e . d i s p l a c e m e n t . 2 0 F i g u r e 4 . Two examples o f e x t r e m e s i n membrane r e s p o n s e s o f TRG n e u r o n s . C a l i b r a t i o n p u l s e ( 2 0 mV, 10 ms) i n A a p p l i e s t o t r a c e s i n A and B. B o t h n e u r o n s f i r e d a c t i o n p o t e n t i a l s w i t h o v e r s h o o t s . F i g u r e 5. Response o f a n e u r o n e t o h y p e r p o l a r i z i n g c u r r e n t p u l s e i n j e c t i o n w h i c h was r e c o r d e d w i t h a K 2 S O „ f i l l e d e l e c t r o d e . Note t h e v a r i a t i o n of r a t e o f v o l t a g e s a g w i t h i n c r e a s i n g amounts o f i n j e c t e d c u r r e n t . The b r i d g e o f t h e WPI a m p l i f i e r was w e l l b a l a n c e d a t b o t h o n s e t and t e r m i n a t i o n o f p u l s e . E l e c t r o d e c o n t r o l upon w i t h d r a w a l f r o m t h e c e l l showed n e g l i g i b l e r e c t i f i c a t i o n . 21 F i g u r e 4 22 F i g u r e 5 1 nA 23 I I I . ACTION POTENTIALS I n j e c t i o n of s u p r a t h r e s h o l d d e p o l a r i z i n g c u r r e n t t h r o u g h t h e r e c o r d i n g e l e c t r o d e r e s u l t e d i n . g e n e r a t i o n o f one o r more a c t i o n p o t e n t i a l s . In most c a s e s and as m e n t i o n e d p r e v i o u s l y , a b i l i t y t o e l i c i t a c t i o n p o t e n t i a l s was l i m i t e d by t h e c u r r e n t p a s s i n g p r o p e r t y o f t h e e l e c t r o d e . In many i n s t a n c e s , t h e s p i k e a m p l i t u d e s were s m a l l a f t e r i n i t i a l p e n e t r a t i o n of t h e c e l l , b u t l a t e r t h e s e were i n c r e a s e d i n a m p l i t u d e . T h i s i n c r e a s e was p r e s u m a b l y a r e s u l t o f s e a l i n g of t h e c e l l membrane a r o u n d t h e e l e c t r o d e , s i n c e membrane h y p e r p o l a r i z a t i o n o c c u r r e d c o n c o m i t a n t l y w i t h i n c r e a s e o f i n p u t r e s i s t a n c e . In some n e u r o n s , however, t h e a c t i o n p o t e n t i a l a m p l i t u d e r e m a i n e d s m a l l e ven t h o u g h r e s t i n g membrane p o t e n t i a l was a t s t a b l e p o t e n t i a l s o f -60 mV o r b e t t e r . T y p i c a l a c t i o n p o t e n t i a l s e l i c i t e d f r o m two d i f f e r e n t TRG n e u r o n s a r e shown i n F i g u r e 6. A f t e r an i n i t i a l membrane r e s p o n s e , a f a s t - r i s i n g and p r e s u m a b l y s o m a t i c a c t i o n p o t e n t i a l o c c u r r e d ; t h i s was f o l l o w e d by r e p o l a r i z a t i o n and an a f t e r h y p e r p o l a r i z a t i o n (AHP). Two t y p e s o f a c t i o n p o t e n t i a l s were r e a d i l y d i s t i n g u i s h a b l e on t h e b a s i s o f s p i k e d u r a t i o n s and a p r e s e n c e o r a b s e n c e of an i n f l e c t i o n i n t h e r e p o l a r i z a t i o n p h a s e . The s p i k e i n F i g u r e 6b had a l o n g e r d u r a t i o n and i n a d d i t i o n a "hump" a p p e a r e d on t h e r e p o l a r i z i n g p h a s e . C e l l s w h i c h e x h i b i t e d a c t i o n p o t e n t i a l s o f t h i s t y p e a l s o t e n d e d t o d i s c h a r g e a c t i o n p o t e n t i a l s i n a r e p e t i t i v e manner i n r e s p o n s e t o l a r g e 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 . I n c o n t r a s t , c e l l s 24 e x h i b i t i n g a c t i o n p o t e n t i a l s o f t h e t y p e i n F i g . 6a t e n d e d t o d i s c h a r g e o n l y s i n g l e a c t i o n p o t e n t i a l s , i n r e s p o n s e t o i n j e c t e d c u r r e n t p u l s e s , a l t h o u g h some r e p e t i t i v e f i r i n g sometimes was o b s e r v e d ( s e e F i g u r e 7 a ) . N e u r o n s w h i c h e x h i b i t e d a c t i o n p o t e n t i a l a m p l i t u d e s o f l e s s t h a n 50 mV were n o t u s e d f o r a n a l y s i s of a c t i v e p r o p e r t i e s . The mean a m p l i t u d e o f s p i k e s w i t h o u t a p l a t e a u i n t h e r e p o l a r i z a t i o n p h ase was 65 mV ± 1.9 mV S. E. ( n = 3 l ) . The mean a m p l i t u d e of s p i k e s t h a t p o s s e s s e d a hump d u r i n g r e p o l a r i z a t i o n was 78 mV" ± 2.3 mV S. E . (n=7), s i g n i f i c a n t l y l a r g e r t h a n i n t h e p r e v i o u s g r o u p (P< 0.01). The a m p l i t u d e and d u r a t i o n o f AHPs i n n e u r o n s where AHPs c o u l d be m e a s u r e d w i t h o u t e x c e s s i v e i n t e r f e r e n c e f r o m e l e c t r o d e r e c t i f i c a t i o n were a l s o d i f f e r e n t f o r t h e two k i n d s o f a c t i o n p o t e n t i a l s . T hus, AHPs f o l l o w i n g s p i k e s w i t h a hump had a mean a m p l i t u d e and d u r a t i o n o f 16.8 mV and 6 ms r e s p e c t i v e l y (n=6). AHPs f o l l o w i n g a c t i o n p o t e n t i a l s i n t h e s e c o n d g r o u p had a mean a m p l i t u d e o f 14.1 mV and a mean d u r a t i o n o f 2.9 ms (n=6). 25 F i g u r e 6. Examples of a c t i o n p o t e n t i a l s evoked from two d i f f e r e n t TRG neurons. Arrows i n d i c a t e the onset of a 50 ms d e p o l a r i z i n g c u r r e n t p u l s e which was used to evoke the sp i k e s i n A and B. Note the presence of a hump du r i n g the r e p o l a r i z i n g phase of the s p i k e i n B. F i g u r e 7. A c t i o n p o t e n t i a l s and su b t h r e s h o l d behaviour of a t r i g e m i n a l g a n g l i o n neuron. A: responses to 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 . Note the o s c i l l a t i o n s of the membrane v o l t a g e with i n j e c t i o n of a 0.4 nA pulse of c u r r e n t . B: anodal break e x c i t a t i o n of the same neuron r e s u l t e d i n genera t i o n of a s p i k e . 26 F i g u r e 6 A 27 F i g u r e 7 10 ms J 1 O O.SnA ^ , . 0.4 nA ^ 0.2 nA "~ ^ 28 However, t h e s e v a l u e s may not be v e r y good a p p r o x i m a t i o n s of t h e AHP p a r a m e t e r s . Measurements of AHPs were made d i f f i c u l t by t h e s u p e r p o s i t i o n of a c t i o n p o t e n t i a l s w i t h t h e i n t r a c e l l u l a r i n j e c t i o n o f a c u r r e n t p u l s e (50-100 ms). The mechanism u n d e r l y i n g t h e AHP would be compromised by t h e d e p o l a r i z i n g c u r r e n t p u l s e . However, i f t h e s p i k e was e v o k e d a t t h e a n o d a l b r e a k ( e . g . F i g u r e 7b,) t h e r e s u l t i n g AHP would be e x p e c t e d t o be s i m i l a r t o t h a t p r o d u c e d by an a c t i o n p o t e n t i a l t h a t i n v a d e s t h e soma f r o m t h e p e r i p h e r a l axon ( i . e . i n t h e a b s e n c e o f i n t r a s o m a t i c c u r r e n t i n j e c t i o n ) . The s i z e of t h e AHP was o b s e r v e d t o be d e p e n d e n t on t h e membrane p o t e n t i a l l e v e l . I n n e u r o n s w i t h a c t i o n p o t e n t i a l s t h a t c o u l d be e v o k e d by i n j e c t i o n of 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 t h e AHP s i z e was s e e n c o n s i s t e n t l y t o i n c r e a s e w i t h d e p o l a r i z a t i o n . T h i s phenomenon i s i l l u s t r a t e d i n F i g u r e 8 w h i c h shows o s c i l l o s c o p e t r a c e s o b t a i n e d f r o m a TRG n e u r o n a t two d i f f e r e n t v a l u e s of r e s t i n g membrane p o t e n t i a l . T h i s n e u r o n e x h i b i t e d ' f a s t ' a c t i o n p o t e n t i a l s w i t h o u t a p l a t e a u i n t h e r e p o l a r i z i n g p h a s e . At a more h y p e r p o l a r i z e d membrane p o t e n t i a l , t h e a m p l i t u d e o f t h e e v o k e d a c t i o n p o t e n t i a l was l a r g e r t h a n a t d e p o l a r i z e d l e v e l s and t h e peak a m p l i t u d e o f t h e AHP was r e d u c e d . An i n c r e a s e d t e n d e n c y t o g e n e r a t e s p i k e s a l s o i s e v i d e n t ; t h e r e s p o n s e s i n A and B were e l i c i t e d w i t h i d e n t i c a l c u r r e n t p u l s e s . T h i s was s i m i l a r t o t h e i n c r e a s e i n membrane o s c i l l a t o r y b e h a v i o u r a t s u b t h r e s h o l d v o l t a g e s , but h e r e , a c t i o n p o t e n t i a l s ( w i t h l a r g e AHPs) were e v o k e d and p o s s i b l y a r e s u p e r i m p o s e d on t h e o s c i l l a t i o n s . 29 F i g u r e 8. E f f e c t o f membrane p o t e n t i a l on s i z e o f s p i k e a f t e r h y p e r - p o l a r i z a t i o n (AHP). In b o t h A and B, r e s p o n s e s were e l i c i t e d w i t h a 2.7 nA d e p o l a r i z i n g c u r r e n t p u l s e . In A, a c t i o n p o t e n t i a l h e i g h t was s m a l l e r t h a n i n B, w h i l e t h e AHP i s l a r g e r . A l s o n o t e t h a t two s p i k e s were e v o k e d a t more d e p o l a r i z e d membrane p o t e n t i a l i n A, whereas an i d e n t i c a l c u r r e n t p u l s e e v o k e d o n l y a s i n g l e a c t i o n p o t e n t i a l a t h y p e r p o l a r i z e d membrane p o t e n t i a l i n B. F i g u r e 8 31 IV. MEMBRANE OSCILLATORY TENDENCY Many TRG n e u r o n s d i s p l a y e d o s c i l l a t o r y f l u c t u a t i o n s o f membrane p o t e n t i a l i n r e s p o n s e 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 . F i g u r e 7 ' i l l u s t r a t e s t h e p o t e n t i a l r e s p o n s e s t o c u r r e n t p u l s e i n j e c t i o n s i n one su c h n e u r o n . O s c i l l a t o r y b e h a v i o u r i s e v o k e d a t c u r r e n t s j u s t s u b t h r e s h o l d t o f u l l s p i k e g e n e r a t i o n . I n s u c h c e l l s , l a r g e h y p e r p o l a r i z i n g c u r r e n t p u l s e s a l s o e v o k e d o s c i l l a t i o n s o f membrane p o t e n t i a l f o l l o w i n g t e r m i n a t i o n of t h e c u r r e n t p u l s e . I f membrane p o t e n t i a l d i s p l a c e m e n t ( o r i n j e c t e d c u r r e n t ) was l a r g e enough, an " a n o d a l b r e a k r e s p o n s e " o r s p i k e f o l l o w e d t e r m i n a t i o n of t h e p u l s e , a s i l l u s t r a t e d i n F i g u r e 7b. O s c i l l a t o r y membrane b e h a v i o u r was o b s e r v e d most r e a d i l y i n c e l l s w h i c h had a r e l a t i v e l y h i g h A t e n d e n c y o f n e u r o n s t o e x h i b i t g r e a t e r p o t e n t i a l o s c i l l a t i o n s a t t h e more d e p o l a r i z e d membrane p o t e n t i a l s a l s o was o b s e r v e d . N eurons t h a t showed s t e a d y membrane d e p o l a r i z a t i o n w i t h t i m e , p r e s u m a b l y due t o a p o o r impalement o r a s h u n t a r o u n d t h e c e l l - e l e c t r o d e s e a l , were more l i k e l y t o d i s p l a y o s c i l l a t o r y r e s p o n s e s a t d e p o l a r i z e d p o t e n t i a l s . An example of o s c i l l a t o r y b e h a v i o u r a t a d e p o l a r i z e d membrane p o t e n t i a l c an be s e e n i n F i g u r e 9 where t h e b e h a v i o u r i s much more p r o n o u n c e d a t -52 mV t h a n a t -60 mV. R e p e t i t i v e f i r i n g c o u l d be e v o k e d i n some n e u r o n s w i t h a s u f f i c i e n t l y l a r g e d e p o l a r i z i n g c u r r e n t p u l s e . The l a t e n c y o f o n s e t a s w e l l a s t h e number o f a c t i o n p o t e n t i a l s c o u l d be c h a n g e d by v a r y i n g t h e a m p l i t u d e o f a p p l i e d c u r r e n t p u l s e . An example o f t h i s b e h a v i o u r i s shown i n F i g u r e 10. Note t h e 32 c h a n g e s i n l a t e n c y o f o n s e t o f s p i k e s w i t h s y s t e m a t i c i n c r e a s e s i n i n t e n s i t y o f i n j e c t e d c u r r e n t . I t i s r e a d i l y a p p a r e n t t h a t t h e f a s t r i s i n g and r e p o l a r i z i n g p h a s e s o f t h e s o m a t i c a c t i o n p o t e n t i a l a r e unchanged, but t h e i n i t i a l p h ase of t h e a c t i o n p o t e n t i a l s becomes l o n g e r w i t h e a c h s u c e s s i v e a c t i o n p o t e n t i a l . 33 F i g u r e 9. Example of i n c r e a s e d o s c i l l a t o r y behaviour of a t r i g e m i n a l g a n g l i o n neuron at d e p o l a r i z e d membrane p o t e n t i a l s . A: Sub and s u p r a t h r e s h o l d responses at -60 mV r e s t i n g membrane p o t e n t i a l . B: Subthreshold response of the same neuron at a d e p o l a r i z e d membrane p o t e n t i a l of -52 mV evoked with i n j e c t i o n of the same amount of c u r r e n t as i n the case of the su b t h r e s h o l d response in A. F i g u r e 10. R e p e t i t i v e f i r i n g i n a t r i g e m i n a l g a n g l i o n neuron. Increase i n amount of d e p o l a r i z i n g c u r r e n t l e d to an i n c r e a s e i n spike d i s c h a r g e frequency. The magnitudes of d e p o l a r i z i n g c u r r e n t s were 0.16, 0.21, 0.32, 0.41 nA i n A, B, C and D r e s p e c t i v e l y . Increase i n l a t e n c y of somatic spike i n i t i a t i o n can be r e a d i l y seen i n C and D. A l s o , note the su b t h r e s h o l d membrane o s c i l l a t i o n s p r i o r to spike g e n e r a t i o n . 34 F i g u r e 9 15 ms J 1 o -60 mV B -52 mV 35 F i g u r e 10 I 0.16 nA 0.21 nA 0.32 nA 0.41 nA 36 B. PHARMACOLOGICAL PROPERTIES OF TRG NEURONS I . EFFECTS OF S-GLUTAMATE E f f e c t s of S - g l u t a m a t e were s t u d i e d i n 6 TRG n e u r o n s . B a t h a p p l i c a t i o n s o f g l u t a m a t e a t a c o n c e n t r a t i o n o f 10" 2 M d i d not e l i c i t c h a n g e s i n membrane p o t e n t i a l o r i n p u t r e s i s t a n c e i n f o u r n e u r o n s . In t h e o t h e r two n e u r o n s , s u c h g l u t a m a t e a p p l i c a t i o n s e v o k e d s m a l l d e p o l a r i z a t i o n s (1-2 mV) w h i c h were no t a s s o c i a t e d w i t h n o t i c e a b l e c h a n g e s i n R^. An example of t h i s r e s p o n s e i s shown f o r one n e u r o n i n F i g u r e 11a. In b o t h c a s e s , g l u t a m a t e - e v o k e d d e p o l a r i z a t i o n s were f u l l y r e v e r s i b l e upon r e t u r n t o t h e c o n t r o l p e r f u s a t e . I I . EFFECTS OF 5-HYDROXYTRYPTAMINE (5~HT) R e s p o n s e s t o 5-HT ( 1 0 " 3 M) were s t u d i e d i n 6 n e u r o n s . In f i v e o f t h e s e n e u r o n s no c h a n g e s i n membrane p o t e n t i a l o r i n p u t r e s i s t a n c e c o u l d be d e t e c t e d w i t h up t o 5 m i n u t e s o f d r u g p e r f u s i o n . In one n e u r o n ( F i g u r e 11b), a p p l i c a t i o n o f 5-HT r e s u l t e d i n membrane c o n d u c t a n c e d e c r e a s e , m e a s u r e d w i t h h y p e r p o l a r i z i n g c u r r e n t p u l s e s , c o n c o m i t a n t l y w i t h a s m a l l d e p o l a r i z a t i o n o f t h e membrane. R e t u r n t o t h e c o n t r o l p e r f u s a t e a b o l i s h e d t h i s e f f e c t . 37 I I I . EFFECTS OF GAMMA-AMINOBUTYRIC ACID (GABA) GABA i n c o n c e n t r a t i o n s r a n g i n g f r o m 10"" t o 10" 2 M was a p p l i e d t o 36 n e u r o n s . In s i x n e u r o n s , GABA a p p l i c a t i o n d i d not e l i c i t d e t e c t a b l e c h a n g e s i n e i t h e r r e s t i n g membrane p o t e n t i a l o r R j . GABA a p p l i c a t i o n t o t h e t h i r t y o t h e r n e u r o n s (some of w h i c h f a i l e d t o r e s p o n d t o g l u t a m a t e a p p l i c a t i o n s ) p r o d u c e d a d e c r e a s e i n Rj w h i c h o c c u r r e d c o n c o m i t a n t l y w i t h membrane d e p o l a r i z a t i o n ( r a n g e : 2-14 mV). The r e s p o n s e o f one n e u r o n t o an a p p l i c a t i o n of GABA i s r e p r o d u c e d i n F i g u r e 11c. F o l l o w i n g t h e i n i t i a l membrane d e p o l a r i z a t i o n and c o n d u c t a n c e i n c r e a s e w h i c h r e a c h e d t h e i r maxima, s l i g h t h y p e r p o l a r i z a t i o n f r o m peak membrane d e p o l a r i z a t i o n was o b s e r v e d , p o s s i b l y due t o r e c e p t o r d e s e n s i t i z a t i o n . T h i s d e c r e a s e i n maximum r e s p o n s e o c c u r r e d i n a l l c a s e s where GABA was a p p l i e d f o r p r o l o n g e d t i m e p e r i o d s . Upon r e t u r n i n g t h e p e r f u s i o n back t o one o f c o n t r o l s o l u t i o n , t h e r e i s a g r a d u a l r e t u r n o f b o t h R and membrane p o t e n t i a l t o l e v e l s o b s e r v e d p r i o r t o a p p l i c a t i o n of GABA. Time f o r r e c o v e r y d e pended i n p a r t on t h e c o n c e n t r a t i o n of a p p l i e d d r u g and on t h e r a t e of p e r f u s a t e f l o w . The l a t e n c y o f maximal r e s p o n s e t o GABA a p p l i c a t i o n was d e c r e a s e d when h i g h e r c o n c e n t r a t i o n s o f GABA were a p p l i e d . 38 F i g u r e 11. R e s p o n s e s of t h r e e n e u r o n s t o b a t h a p p l i c a t i o n s o f g l u t a m a t e O 0 " 2 M) , 5-HT ( 1 0 " 3 M) and GABA (10" 3' M) . L i n e s were drawn t h r o u g h t h e r e s t i n g membrane p o t e n t i a l s i n t r a c e s of c h a r t r e c o r d s o f b o t h A and B. No c h a n g e s i n membrane i n p u t r e s i s t a n c e were o b s e r v e d i n A and t h e h y p e r p o l a r i z i n g p u l s e s t h e r e f o r e were o m i t t e d f r o m t h e t r a c e f o r b e t t e r v i s u a l i z a t i o n o f t h e change i n membrane p o t e n t i a l . The r e s t i n g membrane p o t e n t i a l p r i o r t o d r u g a p p l i c a t i o n was -60 mV i n a l l t h r e e n e u r o n s . Note t h e r e d u c t i o n i n membrane d e p o l a r i z a t i o n t h a t o c c u r s p r i o r t o r e t u r n t o c o n t r o l p e r f u s a t e i n C. i 39 F i g u r e 11 glu 1 0 " M J 1 0 mV t r-2 3min B 5-HT 10~ M t TTTT 10 mV TTTTT 1 mm 2 GABA 1C\W\ t 10 mV r 0 3 min 4 40 T a b l e I - E f f e c t s o f b a t h a p p l i e d GABA on some membrane p r o p e r t i e s o f TRG n e u r o n s C e l l # [GABA] AV m(mV) AR t(MO) % A R i V m(mV) 1 1 0" 2M 5 0.9 19 -70.5 2 1 0" 2M 3.5 4.0 25 -60 3 1 0" 2M 1 2 5.4 56 -60 3 1 0" 2M 9 5.4 53 -57.5 4 1 0" 3M 7.5 2.6 51 -57 5 1 0- 3M 3 2.6 57 -60 6 1 0" 3M 2.5 1 .3 23 -66 7 1 0" 3M 5 5.9 54 -55 7 10" 3M 6 7.7 63 -59 8 1 0" 3M 3.5 T 8 . 6 51 -60 9 1 0" 3M 1 4 2.8 27 -54 10 1 0" 3M 3 4.9 51 -65 1 1 1 0" 3M 5 2.6 35 -62 12 1 0 - 4M 2 2.0 35 -59 12 1 0" 3M 2.6 3.7 51 -61 1 3 1 0" 3M 2 1 . 1 19 -55 1 4 1 0- 4M 2.5 1 .2 34 -63.5 14 1 0' 3M 5 2.0 51 -65 15 1 0" 3M 4 1 .9 40 -61 16 10" "M 3.5 1.4 25 -65 16 10" 3M 4 4.0 42 -74 16 1 0" 2M 5.5 3.5 31 -79 4 1 T a b l e 1 summarizes t h e e f f e c t s o f GABA a p p l i e d i n t h r e e d i f f e r e n t c o n c e n t r a t i o n s t o 16 TRG n e u r o n s . B e c a u s e e s t i m a t e s o f R^ o b t a i n e d d u r i n g e x p e r i m e n t s w i t h GABA a p p l i c a t i o n s a r e n o t v e r y a c c u r a t e a p p r o x i m a t i o n s o f t r u e ( i . e . i d e a l ) R^  , t h e %AR^ g i v e s a b e t t e r i n d i c a t i o n o f t h e e f f e c t o f GABA t h a n A R^ . I t can be seen from t h e t a b u l a t e d r e s u l t s t h a t t h e r e i s no c l e a r c o r r e l a t i o n of t h e r e s t i n g membrane p o t e n t i a l w i t h t h e a m p l i t u d e of maximal r e s p o n s e t o GABA. In a d d i t i o n , a l t h o u g h t h e r e i s a d e f i n i t e r e l a t i o n between %ARi a n d maximal d e p o l a r i z a t i o n p r o d u c e d a t d i f f e r e n t GABA c o n c e n t r a t i o n s i n t h e same c e l l , t h i s does n o t seem t o be t h e c a s e when a c o m p a r i s o n i s made between d i f f e r e n t n e u r o n s . F o r example, i n an extreme c a s e , a 27 % change o f i n c e l l number 9 p r o d u c e d a 14 mV d e p o l a r i z a t i o n w h i l e a 54 %AR t i n c e l l number 7 p r o d u c e d o n l y a 5 mV d e p o l a r i z a t i o n . In b o t h c a s e s , a 1 0 " 3 M c o n c e n t r a t i o n o f GABA was a p p l i e d when t h e c e l l s h ad s i m i l a r r e s t i n g membrane p o t e n t i a l s and i n p u t r e s i s t a n c e s . T h e s e r e s u l t s a r e s u g g e s t i v e o f a d i f f e r e n t i a l s e n s i t i v i t y t o GABA i n TRG n e u r o n s . The ' i n c r e a s e i n membrane c o n d u c t a n c e e v o k e d by a p p l i c a t i o n of GABA r e s u l t e d i n d i m i n u t i o n o f t h e 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 p r o d u c e d by i n j e c t i o n o f 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 . The d e c r e a s e i n membrane p o t e n t i a l c a n n o t a c c o u n t f o r t h e much l a r g e r d e c r e a s e i n a c t i o n p o t e n t i a l h e i g h t b e c a u s e a m p l i t u d e s of s p i k e s g e n e r a t e d by 50 ms 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 , c o n c o m i t a n t l y w i t h DC c u r r e n t i n j e c t i o n t o d e p o l a r i z e t h e r e s t i n g membrane p o t e n t i a l , a r e n o t r e d u c e d t o t h e e x t e n t seen w i t h GABA-mediated d e p o l a r i z a t i o n s . E f f e c t s o f GABA on t h e 42 s p i k e h e i g h t a r e i l l u s t r a t e d i n F i g u r e 12. A t a maximal membrane d e p o l a r i z a t i o n o f 10.5 mV e v o k e d w i t h a 10' 3 M c o n c e n t r a t i o n o f GABA, t h e c o r r e s p o n d i n g d e c r e a s e i n s p i k e a m p l i t u d e i s a p p r o x i m a t e l y 50 mV. T h e r e f o r e d e p o l a r i z a t i o n o f t h e membrane a l o n e a p p a r e n t l y was not e n t i r e l y r e s p o n s i b l e f o r t h e r e d u c t i o n i n s i z e of t h e a c t i o n p o t e n t i a l . I t i s i m p o r t a n t t o n o t e t h a t i n a l l c a s e s where d r u g a p p l i c a t i o n s were made, none o f t h e i m p a l e d n e u r o n s were o b s e r v e d t o d i s c h a r g e a c t i o n p o t e n t i a l s t h a t had a hump i n t h e r e p o l a r i z a t i o n p hase of t h e s p i k e . N e u r o n s t h a t were s a m p l e d d u r i n g t h e p h a r m a c o l o g i c a l t e s t s e i t h e r c o u l d n o t be i n d u c e d t o f i r e s p i k e s due t o l i m i t e d c u r r e n t p a s s i n g a b i l i t y o f t h e e l e c t r o d e o r d i s c h a r g e d s p i k e s w i t h o u t a hump i n t h e r e p o l a r i z i n g p h a s e of t h e s p i k e . 43 F i g u r e 12. P l o t of membrane p o t e n t i a l and a c t i o n p o t e n t i a l a m p l i t u d e c h a n g e s r e s u l t i n g f r o m b a t h a p p l i c a t i o n of 10" 3 M GABA. A c l e a r c o r r e l a t i o n c a n be o b s e r v e d between t h e GABA-m e d i a t e d membrane d e p o l a r i z a t i o n and t h e d e c r e a s e i n t h e a m p l i t u d e of t h e s p i k e s e v o k e d by s u p r a t h r e s h o l d (1.5 X) 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 . N o t e t h a t t h e d e c r e a s e i n s p i k e h e i g h t i s much g r e a t e r t h a n t h e c o r r e s p o n d i n g d e c r e a s e i n membrane p o t e n t i a l s u g g e s t i n g a s h u n t i n g e f f e c t o f t h e GABA-e v o k e d i n c r e a s e i n membrane i n p u t c o n d u c t a n c e . V m ( m V ) s p i k e ampl i tude (mV) 45 IV. DISCUSSION A. PHYSIOLOGICAL ROLE OF THE SOMATA OF PRIMARY AFFERENT NEURONS The o f f s t r e a m a n a t o m i c a l p o s i t i o n o f t h e p e r i k a r y a of t h e c e r e b r o s p i n a l g a n g l i a w i t h r e s p e c t t o t h e i r a x o n a l p r o c e s s e s t h a t t r a n s m i t s e n s o r y . i n f o r m a t i o n f r o m p e r i p h e r y t o t h e CNS has l e d t o t h e v i e w t h a t g a n g l i o n c e l l b o d i e s a r e of l i m i t e d s i g n i f i c a n c e i n t e r m s o f an e l e c t r o p h y s i o l o g i c a l r o l e i n t h e i n t a c t a n i m a l ( L i e b e r m a n , 1976). T h i s v i e w i s s u p p o r t e d by t h e e v i d e n c e t h a t t h e m a j o r i t y of somata i n c e r e b r o s p i n a l g a n g l i a a p p e a r t o be d e v o i d of s y n a p t i c c o n t a c t s . However, a s m a l l number of s y n a p t i c b o u t o n t e r m i n a t i o n s have been d e m o n s t r a t e d c o n c l u s i v e l y t o i m p i n g e on p e r i k a r y a i n t h e c a t DRG. The o r i g i n o f a x o n s t h a t s y n a p s e on t h e s e s e n s o r y c e l l b o d i e s have been l o c a t e d i n t h e s p i n a l c o r d ( K a y a h a r a e t a l , 1984). S p o n t a n e o u s e l e c t r i c a l a c t i v i t y was n o t o b s e r v e d i n o u r i n t r a c e l l u l a r r e c o r d i n g s f r o m f r e s h l y i s o l a t e d TRG c e l l s w h i c h i n v a r i a b l y were q u i e s c e n t p r i o r t o p a s s a g e of c u r r e n t t h r o u g h t h e i n t r a c e l l u l a r e l e c t r o d e o r s t i m u l a t i o n of a x o n a l p r o c e s s e s . A l s o , D a r i a n -S m i t h (1973) has shown t h a t t h e c e n t r a l l y l o c a t e d t e r m i n a l s o f l a r g e m y e l i n a t e d axons a r e d e p o l a r i z e d f o l l o w i n g p e r i p h e r a l s t i m u l a t i o n , b e f o r e e x c i t a t i o n i s o b s e r v e d w i t h i n t h e g a n g l i o n . T h u s , most o f t h e a v a i l a b l e e v i d e n c e would n o t i n d i c a t e t h a t s y n a p t i c a c t i v i t y i s s i g n i f i c a n t i n s e n s o r y g a n g l i o n f u n c t i o n . However, t h e p r e s e n c e o f r e c e p t o r s f o r GABA as i n d i c a t e d i n o u r 46 i n v e s t i g a t i o n o f TRG c e l l s and s y n a p t i c c o n t a c t s i n DRG c e l l s w ould s u g g e s t t h a t c a u t i o n s h o u l d be e x e r c i s e d b e f o r e r u l i n g out a s y n a p t i c m o d u l a t i n g f u n c t i o n i n e i t h e r g a n g l i a . I n a d d i t i o n t o t h e above m e n t i o n e d o b s e r v a t i o n s , t h e p e r i k a r y a o f t h e p r i m a r y s e n s o r y n e u r o n s s y n t h e s i z e compounds n e c e s s a r y f o r t h e o v e r a l l f u n c t i o n i n g of t h e c e l l , i n c l u d i n g i t s c e n t r a l and p e r i p h e r a l t e r m i n a l s . S u b s t a n c e s s y n t h e s i z e d i n t h e p e r i k a r y o n t r a v e l v i a f a s t and slow a x o p l a s m i c t r a n s p o r t t o b o t h t e r m i n a l ends where t h e y may be u t i l i z e d f o r t h e v a r i o u s f u n c t i o n s i n c l u d i n g n e u r o t r a n s m i t t e r r e l e a s e . The s y n t h e s i z i n g o r " f a c t o r y " r o l e of t h e soma i s s u p p o r t e d by t h e r e p o r t e d p r e s e n c e o f p u t a t i v e t r a n s m i t t e r s s u c h a s S - g l u t a m a t e and s u b s t a n c e P i n t h e somata o f p r i m a r y s e n s o r y n e u r o n s . The p r e s e n c e o f t h e s e s u b s t a n c e s h a s a l s o been d e m o n s t r a t e d i n n e u r o n s o f t h e TRG ( H o k f e l t e t a l , 1975). In s p i t e o f t h e e v i d e n c e s u g g e s t i n g an e l e c t r o p h y s i o l o g i c a l ^ p a s s i v e r o l e of t h e s e n s o r y n e u r o n c e l l b o d i e s , some e v i d e n c e i n d i c a t e s a p o s s i b l e m o d u l a t o r y r o l e i n s e n s o r y s i g n a l t r a n s m i s s i o n . An i n v a r i a b l e f i n d i n g o f i n t r a c e l l u l a r e l e c t r o p h y s i o l o g i c a l s t u d i e s o f DRG i s t h a t an a c t i o n p o t e n t i a l a t t h e p e r i p h e r y a l w a y s i n v a d e s t h e soma r e g a r d l e s s o f t h e s p e c i e s and t h e d e g r e e of c o m p l e x i t y o f t h e i n i t i a l g l o m e r u l a r segment o f t h e stem p r o c e s s ( S v a e t i c h i n , 1951; I t o , 1957; S a t o and A u s t i n , 1961; S c o t t e t a l , 1969). A l s o , t h e r e i s a d e l a y o f i m p u l s e t r a n s m i s s i o n t h a t c o n s i s t e n t l y o c c u r s f o r a r r i v a l o f a s p i k e i n t h e g a n g l i o n , p r e s u m a b l y a t t h e j u n c t i o n of p e r i p h e r a l , c e n t r a l , and stem p r o c e s s e s (Dun, 1955). 47 More i m p o r t a n t l y , some e x p e r i m e n t a l e v i d e n c e i m p l i e s t h a t t h e c e l l body and stem p r o c e s s may f u n c t i o n as a f i l t e r and m o d u l a t o r o f i n c o m i n g s i g n a l s by damping s i g n a l s t h a t l i e o u t s i d e t h e n a t u r a l r e s o n a n t f r e q u e n c i e s ( c f . P u i l e t a l , i n p r e s s ) . T a g i n i and Camino (1973) have shown t h a t u nder c o n d i t i o n s o f f a t i g u e i n d u c e d by h i g h f r e q u e n c y s t i m u l a t i o n a s e c o n d a c t i o n p o t e n t i a l i s g e n e r a t e d i n t h e v i c i n i t y o f t h e g a n g l i o n f o l l o w i n g t h e e l e c t r i c a l l y i n d u c e d s p i k e , measured i n t r a - a x o n a l l y i n t h e f r o g s p i n a l n e r v e . S u r p r i s i n g l y , t h e s e c o n d a c t i o n p o t e n t i a l t r a v e l l e d a n t i d r o m i c a l l y t o w a r d s t h e p e r i p h e r y , a p p a r e n t l y w i t h o u t i n v a d i n g t h e c e n t r a l p r o c e s s . T h i s phenomenon s u b s e q u e n t l y was shown t o o c c u r n o t o n l y a s a r e s u l t o f a r t i f i c i a l l y i n d u c e d f a t i g u e b u t a l s o a s a c o n s e q u e n c e of a c t i o n p o t e n t i a l p r o p a g a t i o n t h r o u g h nonhomogeneous r e g i o n s (Ramon e t a l , 1975). Rebound a c t i o n p o t e n t i a l s o f l o n g d u r a t i o n were d e m o n s t r a t e d when a s p i k e t r a v e l l e d from a r e g i o n o f f a s t c o n d u c t i o n , t o a r e g i o n o f slow c o n d u c t i o n v e l o c i t y . P e r i k a r y a of a m p h i b i a n DRG n e u r o n s do not p o s s e s s e x t e n s i v e stem p r o c e s s c o n v o l u t i o n s ( g l o m e r u l i ) , w h i c h a r e much more p r o m i n e n t i n mammals, e s p e c i a l l y c a r n i v o r e s ( C hase, 1909). T h i s would s u g g e s t t h a t s e c o n d a r y 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 would be much more p r o m i n e n t i n mammalian s e n s o r y n e u r o n s . U n f o r t u n a t e l y , no s t u d i e s have been y e t made t o t e s t t h i s p o s s i b i l i t y . In t h e t r i g e m i n a l s y s t e m , a n t i d r o m i c s t i m u l a t i o n o f t h e s e n s o r y f i b e r s i s known t o p r o d u c e v a s o d i l a t i o n and p l a s m a e x t r a v a s a t i o n as i t d o e s i n o t h e r n e u r o n a l s y s t e m s ( C o u t u r e and C u e l l o , 1984). I t seems q u i t e p o s s i b l e t h a t t h i s 48 phenomenon may o c c u r as a r e s u l t o f r e b o u n d a c t i o n p o t e n t i a l s t h a t t r a v e l a n t i d r o m i c a l l y , f o l l o w i n g i n t e n s i v e s e n s o r y r e c e p t o r a c t i v i t y , i n n o r m a l p h y s i o l o g i c a l c i r c u m s t a n c e s . N o r m a l l y , c e l l b o d i e s of n e u r o n s i n c e r e b r o s p i n a l g a n g l i a do n o t g e n e r a t e a c t i o n p o t e n t i a l s i n a b s e n c e o f i n p u t , b u t s p o n t a n e o u s a c t i v i t y has been r e c o r d e d i n d o r s a l r o o t s o f c a t and r a b b i t s p i n a l g a n g l i a one or more d a y s a f t e r t r a n s e c t i o n o f t h e s p i n a l n e r v e r o o t d i s t a l t o t h e g a n g l i o n ( K i r k , 1974). U n l i k e t h e f i n d i n g s o f T a g i n i and Camino ( 1 9 7 4 ) , t h e a c t i o n p o t e n t i a l s s u b s e q u e n t t o axotomy, p r o p a g a t e d o n l y a l o n g t h e c e n t r a l l y d i r e c t e d p r o c e s s e s and n o t t o t h e p e r i p h e r y . I t i s c o n c e i v a b l e t h a t i n c e r t a i n c o n d i t i o n s s u c h a s t r i g e m i n a l n e u r a l g i a , d e g e n e r a t i o n o f t h e p e r i p h e r a l axons may be i n v o l v e d i n t h e i n d u c t i o n o f a b n o r m a l a c t i v i t y i n s e n s o r y g a n g l i o n c e l l s . A l t e r n a t i v e l y , e x c e s s i v e r e d u c t i o n i n c o n d u c t i o n v e l o c i t y o f stem p r o c e s s e s c o u l d r e s u l t i n a p r o d u c t i o n o f a b n o r m a l r e b o u n d a c t i v i t y . T h i s i n t u r n may i n v o k e a r e l e a s e of a l g o g e n i c s u b s t a n c e s as a r e f l e x c o n s e q u e n c e o f c u t a n e o u s v a s o d i l a t i o n and e x t r a v a s a t i o n , t h e r e b y e l i c i t i n g p a i n f u l s e n s a t i o n s . The l a t t e r s u g g e s t i o n seems l e s s l i k e l y s i n c e t h e p a i n e x p e r i e n c e d by p a t i e n t s a f f l i c t e d w i t h t r i g e m i n a l n e u r a l g i a i s f a s t i n o n s e t ; i n d e e d , i t s t e r m i n a t i o n i s u n l i k e t h e s l o w l y d e v e l o p i n g p r o t r a c t e d p a i n e x p e r i e n c e d a f t e r p e r i p h e r a l a d m i n i s t r a t i o n o f compounds s u c h as h i s t a m i n e o r 5-HT. 49 B. ACTION POTENTIAL CHARACTERISTICS OF PRIMARY AFFERENT NEURONS Many p r e v i o u s s t u d i e s have c o n t r i b u t e d t o a b e t t e r u n d e r s t a n d i n g of membrane p r o p e r t i e s and f u n c t i o n o f t h e p r i m a r y s e n s o r y n e u r o n s o f d i f f e r e n t mammalian s p e c i e s . Use o f i n t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s combined w i t h e x t r a c e l l u l a r s t i m u l a t i o n of t h e p e r i p h e r a l p r o c e s s e s o f t h e n e u r o n s has a l l o w e d c l a s s i f i c a t i o n of d i f f e r e n t t y p e s o f s e n s o r y n e u r o n s a c c o r d i n g t o t h e i r c o n d u c t i o n v e l o c i t i e s and a c t i o n p o t e n t i a l c h a r a c t e r i s t i c s ; t h e s e s t u d i e s would i n c l u d e DRG ( B e s s o u e t a l , 1971), nodose g a n g l i o n ( G a l l e g o and E y z a g u i r r e , 1978), and p e t r o s s a l g a n g l i o n o f t h e c a t (B e l m o n t e and G a l l e g o , 1983), as w e l l a s mouse DRG ( Y o s h i d a and Matsuda, 1979), o r r a t DRG ( H a r p e r and Lawson, 1985). In our i n v e s t i g a t i o n s , two t y p e s o f a c t i o n p o t e n t i a l s c o u l d be d i s t i n g u i s h e d r e a d i l y on t h e b a s i s of t h e i r d u r a t i o n s and t h e a b s e n c e o r p r e s e n c e o f a hump i n t h e r e p o l a r i z i n g p h a s e . O t h e r i n v e s t i g a t o r s have shown t h a t an i n f l u x of c a l c i u m , i n a d d i t i o n t o t h a t o f sodium, p l a y s a r o l e i n t h e g e n e s i s o f a c t i o n p o t e n t i a l s w h i c h e x h i b i t l o n g d u r a t i o n s w i t h a hump i n t h e r e p o l a r i z i n g p hase ( Y o s h i d a e t a l , 1978). Such a c t i o n p o t e n t i a l s i n r a t DRG a r e p r o d u c e d i n u n m y e l i n a t e d G - f i b e r s w i t h c o n d u c t i o n v e l o c i t i e s l e s s t h a n 1.4 m/s ( H a r p e r and Lawson, 1985). The sodium component i n t h e s e t y p e s o f a c t i o n p o t e n t i a l s i s o n l y p a r t l y s e n s i t i v e t o b l o c k a d e w i t h t e t r o d o t o x i n ( T T X ) , whereas t h e s p i k e s w i t h o u t a hump a r e b l o c k e d c o m p l e t e l y by TTX a d m i n i s t r a t i o n ( Y o s h i d a e t a l , 1978; G a l l e g o , 1983). The 50 p r o p o r t i o n of n e u r o n s w i t h T T X - r e s i s t a n t t o T T X - s e n s i t i v e a c t i o n p o t e n t i a l s v a r i e s g r e a t l y between d i f f e r e n t s p e c i e s and p r e p a r a t i o n s . The main c a u s e of t h i s v a r i a b i l i t y i s t h e d i f f e r e n c e s i n p r i m a r y a f f e r e n t n e u r o n p o p u l a t i o n s . F o r example, i n mouse DRG, T T X - r e s i s t a n t s p i k e s a r e p r e s e n t i n 33% of s a m p l e d n e u r o n s ( Y o s h i d a e t a l , 1978), whereas i n c a t p e t r o s s a l g a n g l i o n , 90% o f c e l l s w i t h m y e l i n a t e d a x o n s have s p i k e s r e s i s t a n t t o TTX ( G a l l e g o , 1983). B e c a u s e s t i m u l a t i o n o f c e n t r a l o r p e r i p h e r a l a x o n s was n o t e m ployed i n t h e p r e s e n t s t u d y , i t was p o s s i b l e t o c l a s s i f y t h e TRG n e u r o n s o n l y i n 2 g r o u p s on t h e b a s i s o f a c t i o n p o t e n t i a l d i f f e r e n c e s . S u r g i c a l and a n a t o m i c a l c o n s t r a i n t s l i m i t t h e l e n g t h o f t h e TRG s l i c e s t h a t c a n be o b t a i n e d . F u r t h e r c l a s s i f i c a t i o n w o u l d r e q u i r e a x o n a l s t i m u l a t i o n and measurements of c o n d u c t i o n v e l o c i t i e s . T h i s w o u l d be a c c o m p l i s h e d most e a s i l y under i n v i v o e x e r i m e n t a l c o n d i t i o n s where i n t a c t a x o n s ca n be s t i m u l a t e d a t t h e i r p e r i p h e r a l r e c e p t o r s o r a t t h e c e n t r a l p r o c e s s e s . One a l s o may e x p e c t t h e s a m p l e d p o p u l a t i o n s o f s e n s o r y n e u r o n s i n TRG t o be more homogeneous t h a n t h o s e f o u n d i n t h e d o r s a l r o o t g a n g l i a o f mammalian s p e c i e s , p a r t l y b e c a u s e t h e c e l l b o d i e s o f m y e l i n a t e d , f a s t c o n d u c t i n g f i b e r s of s p i n d l e s and t e n d o n o r g a n s of t h e t h e jaw m u s c u l a t u r e a r e l o c a t e d i n t h e m e s e n c e p h a l i c n u c l e u s i n t h e b r a i n s t e m ( L i e b e r m a n , 1976). Thus, t h e r e s h o u l d be p r o p o r t i o n a l l y fewer c e l l b o d i e s of t h e f a s t e s t c o n d u c t i n g f i b e r s i n t h e g a n g l i o n p r o p e r . The mean s i z e and d u r a t i o n o f AHPs was d i f f e r e n t i n TRG 51 n e u r o n s w h i c h d i s c h a r g e d s p i k e s w i t h a hump compared t o n e u r o n s whose a c t i o n p o t e n t i a l s d i d n o t have t h i s p r o p e r t y . In t h e f r o g DRG, a c t i o n p o t e n t i a l s o f C - n e u r o n s a r e f o l l o w e d by AHPs w i t h a m p l i t u d e s and d u r a t i o n s w h i c h e x c e e d t h o s e o f A - n e u r o n s ( H o l z e t a l , 1985). In t h e r a b b i t n o d o s e g a n g l i o n , AHP a m p l i t u d e i s s i m i l a r i n C - n e u r o n s and A - n e u r o n s , b ut t h e d u r a t i o n o f AHPs i s much g r e a t e r i n C - n e u r o n s ( S t a n s f i e l d and W a l l i s , 1985). A c a l c i u m component i s p r e s e n t i n c a t p e t r o s a l g a n g l i o n n e u r o n s t h a t e x h i b i t a c t i o n p o t e n t i a l s w i t h a hump, and b e c a u s e C a + 2 c h a n n e l b l o c k e r s d e c r e a s e t h e a m p l i t u d e and d u r a t i o n o f t h e l o n g AHPs i n t h e s e n e u r o n s , i t was p r o p o s e d t h a t t h e l o n g AHPs a r e p r o d u c e d by a c t i v a t i o n o f t h e C a + 2 - d e p e n d e n t K + c o n d u c t a n c e ( G a l l e g o , 1983). However, i t r e m a i n s t o be shown whether a C a + 2 - d e p e n d e n t K + c o n d u c t a n c e i s r e s p o n s i b l e f o r t h e AHPs i n t h o s e TRG n e u r o n s t h a t have l o n g d u r a t i o n s p i k e s w i t h a hump. In TRG, t h e o b s e r v a t i o n t h a t AHP s i z e i n s p i k e s w i t h o u t a hump i n c r e a s e s w i t h membrane d e p o l a r i z a t i o n i m p l i e s a dependence of t h e s e AHPs on K + c o n d u c t a n c e . C. VOLTAGE- AND TIME-DEPENDENT. RECTIFICATION IN SENSORY NEURONS Most of t h e TRG s e n s o r y n e u r o n s were shown t o p o s s e s s membrane r e c t i f i c a t i o n i n r e s p o n s e t o h y p e r p o l a r i z i n g c u r r e n t p u l s e s . T h i s phenomenon, f i r s t d e s c r i b e d i n f r o g DRG n e u r o n s by I t o (1957) h a s been c a l l e d " t i m e - d e p e n d e n t r e c t i f i c a t i o n " . C z e h , Kudo and Kuno (1977) p r o p o s e d an i n c r e a s e i n sodium c o n d u c t a n c e ( g N a ) p r o d u c e d by t h e r e m o v a l o f p a r t i a l 52 i n a c t i v a t i o n o f Na an d / o r Ca c h a n n e l s , t o a c c o u n t f o r t h i s phenomenon. An i n v o l v e m e n t o f g N a a l s o has been s u p p o r t e d by t h e d a t a o f G a l l e g o (1983) who showed t h e r e c t i f i c a t i o n t o be e i t h e r p a r t i a l l y o r c o m p l e t e l y a b o l i s h e d i n sodium f r e e s o l u t i o n s i n t h e p r e s e n c e o f TTX. Mayer and Westb r o o k (1984) who u s e d a v o l t a g e - c l a m p a n a l y s i s o f c u l t u r e d mouse DRG n e u r o n s f o u n d t i m e - d e p e n d e n t r e c t i f i c a t i o n t o be due t o a "mixed" N a + and K + c u r r e n t . 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 t h a t t h e r e c t i f i c a t i o n i n TRG n e u r o n s may be b o t h t i m e - and v o l t a g e -d e p e n d e n t . T h i s p r o p o s i t i o n stems f r o m two o b s e r v a t i o n s made c o n s i s t e n t l y i n t h i s s t u d y . F i r s t , t h e t i m e c o u r s e of membrane d e p o l a r i z a t i o n f r o m t h e i n t i t a l peak h y p e r p o l a r i z i n g r e s p o n s e t o a s t e a d y s t a t e v a l u e depended l a r g e l y on t h e amount o f membrane d i s p l a c e m e n t by c u r r e n t p u l s e i n j e c t i o n i n a g i v e n n e u r o n . S e c o n d l y , t h e e x t e n t o f membrane r e c t i f i c a t i o n a l s o seemed t o be d e p e n d e n t on t h e r e s t i n g membrane p o t e n t i a l of t h e n e u r o n . Thus membrane r e c t i f i c a t i o n was e a s i e r t o e l i c i t w i t h h y p e r p o l a r i z i n g c u r r e n t p u l s e s i n c e l l s w i t h v e r y n e g a t i v e r e s t i n g membrane p o t e n t i a l s . To v e r i f y t h e s e o b s e r v a t i o n s , c u r r e n t p u l s e s of 50 ms d u r a t i o n were s u p e r i m p o s e d on t o n i c DC c u r r e n t s and t h e d e g r e e o f membrane r e c t i f i c a t i o n was compared f o r d e p o l a r i z e d and h y p e r p o l a r i z e d membrane p o t e n t i a l v a l u e s on t h e same n e u r o n . L a r g e r s a g g i n g h y p e r p o l a r i z i n g r e s p o n s e s c o u l d be o b t a i n e d a t r e l a t i v e l y h y p e r p o l a r i z e d r e s t i n g p o t e n t i a l s . T h i s may be e x p l a i n e d p a r t i a l l y on a b a s i s t h a t r e s t i n g c o n d u c t a n c e i s h i g h e r i n TRG n e u r o n s a t d e p o l a r i z e d membrane p o t e n t i a l s ( P u i l 53 e t a l , u n p u b l i s h e d o b s e r v a t i o n s ) t h u s r e q u i r i n g l a r g e r h y p e r p o l a r i z i n g c u r r e n t i n p u t s f o r membrane p o t e n t i a l d i s p l a c e m e n t . A n o t h e r p o s s i b l e c o n t r i b u t i n g f a c t o r may be t h a t s o d i u m c h a n n e l a c t i v a t i o n o c c u r s more q u i c k l y a t more n e g a t i v e membrane p o t e n t i a l s o r t h a t t h e r e i s s i m p l y a l a r g e r d r i v i n g f o r c e f o r s o d i um i o n s [ d e f i n e d by ( V m - E ^ a ) ] t h a t a c c o u n t s f o r a f a s t e r t i m e c o u r s e of t h e sag a t more n e g a t i v e r e s t i n g p o t e n t i a l s . Sag b e h a v i o u r a p p e a r e d t o be a b s e n t i n a s m a l l p r o p o r t i o n o f TRG n e u r o n s but t h i s d o e s not mean n e c e s s a r i l y t h a t t h e s e c e l l s r e p r e s e n t a d i s t i n c t o r s e p a r a t e g r o u p o f s e n s o r y n e u r o n s . C z e h , Kudo and Kuno (1977) have shown t h a t somata of DRG n e u r o n s u n d e r g o s i g n i f i c a n t c h a n g e s i n membrane e l e c t r i c a l p r o p e r t i e s f o l l o w i n g axotomy of t h e p e r i p h e r a l f i b e r s . In p a r t i c u l a r , a t two weeks f o l l o w i n g axotomy, t h e number of c e l l s e x h i b i t i n g s a g b e h a v i o u r was r e d u c e d d r a s t i c a l l y . The amount of c u r r e n t r e q u i r e d t o i n d u c e r e c t i f i c a t i o n a l s o was much g r e a t e r on t h e a v e r a g e i n c e l l s w h i c h showed r e c t i f i c a t i o n . I t i s not c l e a r , however, i f t h e c h ange i n membrane p r o p e r t i e s o c c u r s o v e r a s h o r t p e r i o d o f t i m e , i . e . w i t h i n h o u r s o f axotomy w h i c h was u s u a l l y w i t h i n t h e t i m e p e r i o d t h a t i n t r a c e l l u l a r r e c o r d i n g s were made f o l l o w i n g e x c i s i o n from t h e a n i m a l i n t h e p r e s e n t i n v e s t i g a t i o n s . I n t e r e s t i n g l y , n e u r o n s t h a t showed no sag b e h a v i o u r a l s o a r e e n c o u n t e r e d on o c c a s i o n d u r i n g r e c o r d i n g s f r o m c e l l s i n i n v i v o e x p e r i m e n t s where t h e p e r i p h e r a l a x o n s a r e p r e s u m a b l y i n t a c t ( P u i l , e t a l . , i n p r e s s ) . 54 D. ACTIONS OF GLUTAMATE IN SENSORY GANGLION NEURONS S - G l u t a m a t e i s r e c o g n i z e d a s t h e major impermeant i n t r a c e l l u l a r a n i o n i n t h e CNS o f v e r t e b r a t e s ( P u i l , 1981). In t h e s p i n a l c o r d i t was p r o p o s e d as a n e u r o t r a n s m i t t e r o f t h e p r i m a r y a f f e r e n t f i b e r s i m p l i c a t e d i n m o n o s y n a p t i c e x c i t a t i o n (Graham e t a l , 1967). The h i g h e s t c o n c e n t r a t i o n s of g l u t a m a t e i n t h e s p i n a l c o r d o c c u r s i n t h e d o r s a l h o r n s , b u t t h e d o r s a l r o o t s and t h e DRG a l s o c o n t a i n h i g h e r c o n c e n t r a t i o n s t h a n t h o s e f o u n d i n t h e v e n t r a l h o r n s ( J o h n s t o n and A p r i s o n , 1970). I t was of i n t e r e s t t o o b s e r v e p o s s i b l e e f f e c t s o f g l u t a m a t e on TRG n e u r o n s e s p e c i a l l y i n view o f p a s t r e p o r t s where g l u t a m a t e was w i t h o u t e f f e c t on DRG ( D e G r o a t e t a l , 1972) .and s u p e r i o r c e r v i c a l g a n g l i o n (Bowery and Brown, 1974) n e u r o n s . A l s o , o t h e r i n v e s t i g a t o r s have r e p o r t e d weak e f f e c t s i n t h e DRG ( G a l l a g h e r e t a l , 1978) as w e l l a s i n t h e p r i m a r y a f f e r e n t m e s e n c e p h a l i c n e u r o n s o f t h e t r i g e m i n a l n e r v e ( H e n d e r s o n e t a l , 1982). In o u r i n v e s t i g a t i o n s , g l u t a m a t e a p p l i c a t i o n s r e s u l t e d i n e i t h e r s m a l l membrane d e p o l a r i z a t i o n s or no e f f e c t on membrane e l e c t r i c a l p r o p e r t i e s o f s i x TRG n e u r o n s . The r e s u l t s i n d i c a t e a s i m i l a r i t y between s o m a t i c r e s p o n s e s t o g l u t a m a t e o f TRG n e u r o n s and n e u r o n s i n t h e t r i g e m i n a l m e s e n c e p h a l i c n u c l e u s o r i n t h e DRG. An a b s e n c e o f s i g n i f i c a n t e f f e c t s o f g l u t a m a t e on membrane e l e c t r i c a l p r o p e r t i e s o f t h e s e n e u r o n s s u g g e s t s an a b s e n c e o f g l u t a m a t e r e c e p t o r s s u c h a s t h o s e d e m o n s t r a t e d i n t h e CNS. A l s o , p e r i k a r y a o f r a t DRG n e u r o n s have been shown t o l a c k 55 an u p t a k e s y s t e m f o r g l u t a m a t e . I n s t e a d , g l u t a m a t e i s t a k e n up by t h e s u r r o u n d i n g g l i a l c e l l s (Schon and K e l l y , 1974), w h i l e t h e p e r i k a r y a o f DRG n e u r o n s e x h i b i t h i g h a f f i n i t y u p t a k e f o r g l u t a m i n e (Duce and Keen, 1983). S i m i l a r o b s e r v a t i o n s were made w i t h i n n e r h a i r c e l l s and g l i a l c e l l s i n t h e g u i n e a p i g c o c h l e a ( E y b a l i n and P u j o l , 1983). T h e s e i n v e s t i g a t i o n s p r o v i d e s t r o n g e v i d e n c e f o r t h e p r e s e n c e o f a n e u r o n a l - g l i a l g l u t a m i n e c y c l e i n s e n s o r y n e u r o n g a n g l i a ( c f . Shank and A p r i s o n , 1981). T h i s c y c l e p r o v i d e s n e u r o n s w i t h a s u b s t r a t e f o r s y n t h e s i s o f g l u t a m a t e and i t s s u b s e q u e n t u t i l i z a t i o n i n v a r i o u s c e l l u l a r f u n c t i o n s , i n c l u d i n g t h e f o r m a t i o n o f n e u r o t r a n s m i t t e r p o o l s . I n t e r e s t i n g l y , t h e s m a l l n e u r o n s t h a t have been shown i m m u n o h i s t o c h e m i c a l l y t o c o n t a i n o t h e r t r a n s m i t t e r c a n d i d a t e s s u c h a s s u b s t a n c e P or s o m a t o s t a t i n ( H o k f e l t e t a l , 1976) t a k e up g l u t a m i n e a t s i x t i m e s t h e r a t e seen i n l a r g e d i a m e t e r DRG n e u r o n s (Duce and Keen, 1983). T h i s o b s e r v a t i o n seems t o c o n t r a d i c t t h e p r o p o s e d n e u r o t r a n s m i t t e r c a n d i d a c y of g l u t a m a t e i n l a r g e d i a m e t e r p r i m a r y a f f e r e n t n e u r o n s t h a t a r e i n v o l v e d i n m o n o s y n a p t i c r e f l e x e s (Graham e t a l , 1967) b e c a u s e one w o u l d e x p e c t g r e a t e r u p t a k e o f a s u b s t r a t e f o r g l u t a m a t e s y n t h e s i s i n n e u r o n s t h a t u t i l i z e i t as t r a n s m i t t e r . E. ROLE OF 5-HYDROXYTRYPTAMINE IN SENSORY TRANSMISSION An a l g o g e n i c compound, 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) , has been i m p l i c a t e d i n a v a r i e t y o f b i o l o g i c a l r e a c t i o n s , f o r example, i t c a u s e s v a s o c o n s t r i c t i o n when r e l e a s e d f r o m p l a t e l e t s f o l l o w i n g 56 i n j u r y ( M i l l s e t a l , 1968). 5-HT a l s o i s t h o u g h t t o be t h e n e u r o t r a n s m i t t e r of n e u r o n s whose c e l l b o d i e s a r e g r o u p e d m a i n l y i n t h e m i d b r a i n r a p h e n u c l e i ( C o n s o l a z i o n e and C u e l l o , 1982). When i n j e c t e d b e n e a t h t h e s k i n , 5-HT c a u s e s a p r o n o u n c e d and l o n g - l a s t i n g d i s c h a r g e i n s e n s o r y n e r v e s of t h e r a t . Mast c e l l s o f t h e r a t c o n t a i n 5-HT i n a d d i t i o n t o h i s t a m i n e w h i c h d i d n o t c a u s e n e u r o n a l d i s c h a r g e (van G e l d e r , 1962). A p p l i c a t i o n of 5-HT t o i s o l a t e d b u l l f r o g DRG has been shown t o p r o d u c e s l o w m a i n t a i n e d and f a s t t r a n s i e n t d e p o l a r i z a t i o n s i n b o t h f a s t c o n d u c t i n g A - n e u r o n s and slow C - n e u r o n s ( H o l z e t a l , 1985). A l s o , slow c o n d u c t i n g C - n e u r o n s i n t h e r a b b i t nodose g a n g l i o n r e c e n t l y have been shown t o p r o d u c e p r e d o m i n a n t l y d e p o l a r i z i n g r e s p o n s e s i n r e s p o n s e t o 5-HT a p p l i c a t i o n , whereas A - c e l l s were not a f f e c t e d ( S t a n s f i e l d and W a l l i s , 1985). I n t h e g u i n e a p i g TRG, 5-HT has been b i o c h e m i c a l l y and i m m u n o h i s t o c h e m i c a l l y l o c a t e d i n t h e mast c e l l s , but n o t i n t h e n e u r o n a l c e l l b o d i e s ( L e h t o s a l o e t a l , 1983). I f t h e r e l e a s e of c h e m i c a l s s u c h as 5-HT from t h e mast c e l l s o c c u r s i n t h e s k i n i t i s p o s s i b l e t h a t s i m i l a r r e l e a s e o f 5-HT from mast c e l l s i n t h e TRG may p r o d u c e r e s p o n s e s i n t h e c e l l b o d i e s , p r o v i d e d r e c e p t o r s f o r 5-HT a r e p r e s e n t on TRG n e u r o n s . A n o t h e r i m p o r t a n t r e a s o n f o r s t u d y i n g 5-HT e f f e c t s on t h e s e n e u r o n s i s t h e p r o p o s e d m o d u l a t i o n of s e n s o r y i n f o r m a t i o n by p r i m a r y a f f e r e n t t e r m i n a l s o f n e u r o n s c o n t a i n i n g s u b s t a n c e P and 5-HT. D e m o n s t r a t i o n o f 5-HT and s u b s t a n c e P i n t h e c e l l b o d i e s o f r a p h e n u c l e i n e u r o n s p r o v i d e d t h e f i r s t example o f c o e x i s t e n c e 57 o f n e u r o p e p t i d e and n e u r o t r a n s m i t t e r i n t h e CNS ( C h a n - P a l a y e t a l , 1978; H o k f e l t e t a l , 1978). S t i m u l a t i o n i n t h e r e g i o n o f m e d u l l a r y n u c l e u s r a p h e magnus and d o r s a l r a p h e n u c l e u s r e s u l t s i n p r o f o u n d s u r f a c e a n a l g e s i a , whereas i n t e r r u p t i o n o f d e s c e n d i n g a x o ns of t h e s e n e u r o n s b l o c k s t h e a n a l g e s i c e f f e c t o f s t i m u l a t i o n ( K e l l y , 1981). The a n a l g e s i c e f f e c t i s v e r y s p e c i f i c i n t h a t o t h e r s e n s o r y m o d a l i t i e s r e m a i n u n a f f e c t e d d u r i n g s t i m u l a t i o n , s u g g e s t i n g a c l o s e i n t e r a c t i o n of r a p h e n e u r o n s w i t h p a i n p athways, w h i c h l e d t o t h e model p r o p o s e d by I v e r s e n ( 1 9 7 8 ) . A c c o r d i n g t o t h i s m o d e l, 5 - H T - c o n t a i n i n g f i b e r s e x c i t e t h e e n k e p h a l i n - c o n t a i n i n g i n t e r n e u r o n s w h i c h i n t u r n i n h i b i t t h e r e l e a s e o f t r a n s m i t t e r ( s u b s t a n c e P) f r o m t h e t e r m i n a l s o f t h e p a i n f i b e r s . A p o s s i b i l i t y s t i l l e x i s t s t h a t i n h i b i t i o n o f n o c i c e p t i v e pathways i s m e d i a t e d by d i r e c t i n f l u e n c e o f t h e r a p h e n e u r o n s on p r i m a r y a f f e r e n t t e r m i n a l s . U n l i k e n e u r o n s i n f r o g DRG o r C - n e u r o n s i n r a b b i t nodose g a n g l i o n , t h e s i x p r i m a r y a f f e r e n t n e u r o n s i n t h e TRG t h a t were t e s t e d seemed t o be l i t t l e a f f e c t e d by b a t h a p p l i c a t i o n o f 1mM 5-HT. In v i e w o f t h e f a c t t h a t 5-HT d e p o l a r i z e s o n l y C - n e u r o n s i n t h e nodose g a n g l i o n , i t i s q u i t e l i k e l y t h a t t h i s a l s o may be t h e c a s e i n TRG. The number o f s t a b l e r e c o r d i n g s from c e l l s w i t h l o n g d u r a t i o n s p i k e s was l i m i t e d , p r e s u m a b l y b e c a u s e o f t h e s m a l l c e l l s i z e w h i c h makes i t more d i f f i c u l t t o s u c c e s s f u l l y r e c o r d f r o m C n e u r o n s u s i n g r e l a t i v e l y c o a r s e t i p p e d m i c r o e l e c t r o d e s . The o n l y r e s p o n s e o b s e r v e d f o l l o w i n g 5-HT a p p l i c a t i o n was a membrane d e p o l a r i z a t i o n w i t h a r e l a t i v e l y f a s t r e c o v e r y 58 f o l l o w i n g d i s c o n t i n u a t i o n of 5-HT p e r f u s i o n . T h i s t y p e o f r e s p o n s e i s s i m i l a r t o t h a t o b s e r v e d f o r g l u t a m a t e a d m i n i s t r a t i o n on TRG n e u r o n s . I t i s p o s s i b l e t h a t t h e s l i g h t d e p o l a r i z a t i o n s o b s e r v e d w i t h g l u t a m a t e and 5-HT a r e a c o n s e q u e n c e o f e l e c t r o g e n i c u p t a k e o f t h e s e a g e n t s by t h e c e l l r a t h e r t h a n due t o any a c t i o n on s p e c i f i c r e c e p t o r s f o r g l u t a m a t e o r 5-HT. However, i f TRG n e u r o n s p o s s e s s an u p t a k e mechanism f o r 5-HT, t h e ' compound must be r a p i d l y m e t a b o l i z e d w i t h i n t h e c e l l s i n c e no 5-HT c a n be l o c a l i z e d w i t h i n t h e n e u r o n s ( L e h t o s a l o , 1983). An a l t e r n a t i v e e x p l a n a t i o n would s u g g e s t a l a c k of an u p t a k e s y s t e m f o r 5-HT i n TRG n e u r o n s and a b s e n c e o f s p e c i f i c r e c e p t o r s . F. ACTION OF GABA ON PRIMARY SENSORY NEURONS Gamma-aminobutyric a c i d h as been i m p l i c a t e d a s a p o s s i b l e n e u r o t r a n s m i t t e r i n a number o f n e u r o n a l pathways i n t h e CNS o f v e r t e b r a t e s . In t h e mammalian CNS, p r e s y n a p t i c i n h i b i t i o n i s a s s o c i a t e d w i t h a d e p o l a r i z a t i o n o f p r i m a r y a f f e r e n t t e r m i n a l s ( E c c l e s , 1964). R e d u c t i o n o f p r e s y n a p t i c i n h i b i t i o n by GABA a n t a g o n i s t s p i c r o t o x i n and b i c u c u l l i n e l e d t o t h e s u g g e s t i o n t h a t GABA may be a m e d i a t o r o f p r e s y n a p t i c i n h i b i t i o n ( E c c l e s , e t a l , 1963; S c h m i d t , 1964). The r o l e o f GABA i n p r i m a r y a f f e r e n t d e p o l a r i z a t i o n h as been shown c o n c l u s i v e l y i n numerous s t u d i e s ( c f . r e v i e w by L e v y , 1977). S e v e r a l i n d e p e n d e n t i n v e s t i g a t i o n s have r e v e a l e d t h a t exogenous GABA a l s o d e p o l a r i z e s t h e somata o f a m p h i b i a n and mammalian DRG p r i m a r y 59 a f f e r e n t n e u r o n s ( F e l t z and R a s m i n s k y , 1974; G a l l a g h e r e t a l , 1974; N i s h i e t a l , 1974). In DRG n e u r o n s , GABA-mediated d e p o l a r i z a t i o n s a r e c a u s e d by i n c r e a s e s i n c h l o r i d e c o n d u c t a n c e ( N i s h i e t a l , 1974; G a l l a g h e r e t a l , 1978). In t h e p r e s e n t s t u d y , GABA d e p o l a r i z e d a m a j o r i t y o f TRG n e u r o n s t o w h i c h i t was a p p l i e d ( 3 0 / 3 6 ) . A l l d e p o l a r i z a t i o n s were a s s o c i a t e d w i t h d e c r e a s e s i n i n p u t r e s i s t a n c e . T hese r e s u l t s i n d i c a t e t h a t somata o f TRG p r i m a r y a f f e r e n t n e u r o n s a r e s i m i l a r t o t h o s e o f DRG i n t h e i r r e s p o n s e s t o GABA a p p l i c a t i o n s . D e c r e a s e s i 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 d u r i n g GABA-m e d i a t e d d e p o l a r i z a t i o n s f u r t h e r c o r r o b o r a t e t h i s c o n t e n t i o n . D e p o l a r i z a t i o n of t h e membrane a p p a r e n t l y was n o t e n t i r e l y r e s p o n s i b l e f o r s h u n t i n g o f t h e a c t i o n p o t e n t i a l . The r e d u c t i o n i n s p i k e a m p l i t u d e c o u l d i n v o l v e e i t h e r i n a c t i v a t i o n o f v o l t a g e -d e p e n d e n t c h a n n e l s r e s p o n s i b l e f o r s p i k e g e n e r a t i o n o r , more l i k e l y , be a r e s u l t o f an e f f e c t i v e membrane " v o l t a g e c l amp" t h a t o c c u r s when 10" 3 M GABA i n c r e a s e s membrane c o n d u c t a n c e w h i c h " f i x e s " t h e membrane p o t e n t i a l n e a r t h e c h l o r i d e e q u i l i b r i u m p o t e n t i a l . B e c a u s e t h e r e l a t i o n s h i p between d e p o l a r i z a t i o n s and c o n d u c t a n c e i n c r e a s e s was n o t t h e same f o r a l l c e l l s t e s t e d , t h a t i s s m a l l c o n d u c t a n c e c h a n g e s sometimes r e s u l t e d i n l a r g e d e p o l a r i z a t i o n s , and l a r g e c o n d u c t a n c e c h a n g e s sometimes p r o d u c e d s m a l l d e p o l a r i z a t i o n s , a p o s s i b i l i t y e x i s t s t h a t r e c e p t o r s e n s i t i v i t y f o r GABA i s n o t t h e same f o r d i f f e r e n t p o p u l a t i o n s o f TRG n e u r o n s . A l t h o u g h some r e c o r d i n g s were made w i t h p o t a s s i u m s u l p h a t e e l e c t r o d e s , a m a j o r i t y o f GABA 60 a p p l i c a t i o n s were p e r f o r m e d d u r i n g r e c o r d i n g w i t h p o t a s s i u m c h l o r i d e f i l l e d e l e c t r o d e s . T h i s may have i n c r e a s e d a p p r e c i a b l y i n t r a c e l l u l a r c h l o r i d e c o n c e n t r a t i o n s , d e p e n d i n g on t h e amount of l e a k a g e due t o i o n i c c o n c e n t r a t i o n d i f f e r e n c e s o f e l e c t r o l y t e and i n t r a c e l l u l a r m i l i e u and a l s o on t h e i n t e n s i t y o f a p p l i e d h y p e r p o l a r i z i n g c u r r e n t p u l s e s . More i m p o r t a n t l y , s i g n i f i c a n t l e a k a g e would o c c u r f r o m t h e e l e c t r o d e t i p due t o h y d r o s t a t i c p r e s s u r e , a l t h o u g h t h i s may be somewhat r e d u c e d i n t h i s s t u d y b e c a u s e o f t h e a c u t e a n g l e w h i c h t h e e l e c t r o d e makes w i t h t h e chamber f l o o r 5 0 ° ) . A n o t h e r p o s s i b l e , b u t l e s s l i k e l y r e a s o n f o r t h e d i f f e r e n c e s i n s e n s i t i v i t y t o GABA c o u l d be due t o a compromised c h l o r i d e pump. In view o f t h e f a c t t h a t GABA-m e d i a t e d d e p o l a r i z a t i o n i s c h l o r i d e - d e p e n d e n t , the. i n t e r n a l c h l o r i d e c o n c e n t r a t i o n o f DRG p r i m a r y a f f e r e n t n e u r o n s must be h i g h e r t h a n t h a t o f t h e o u t s i d e and an i n w a r d l y d i r e c t e d c h l o r i d e pump mechanism s h o u l d be p r e s e n t i n t h e s e n e u r o n s ( N i s h i e t a l , 1974). T h e r e f o r e , i f t h e i n t e r n a l c h l o r i d e c o n c e n t r a t i o n i s low i t w i l l r e s u l t i n a r e d u c e d d e p o l a r i z i n g r e s p o n s e t o GABA. A l t e r n a t i v e l y , s e n s i t i v i t y d i f f e r e n c e s may be, a s F e l t z and R a s m i n s k y p r o p o s e d ( 1 9 7 4 ) , due t o t h e s u r r o u n d i n g s a t e l i t e c e l l s a c t i n g p e r h a p s a s a s i t e o f GABA u p t a k e . The p r e s e n c e i n n e u r o n s o f two t y p e s o f r e c e p t o r s f o r GABA was shown f i r s t i n c u l t u r e d e m b r y o n i c s e n s o r y n e u r o n s o f t h e c h i c k ( D u n l a p and F i s c h b a c h , 1978) and s u b s e q u e n t l y i n mammalian p e r i p h e r a l ( D e s a r m e n i e n e t a l , 1982) and c e n t r a l (Bowery e t a l , 1980) n e u r o n s . In s e n s o r y n e u r o n s , t h e s e c o n d t y p e o f GABA 61 e f f e c t was a d e c r e a s e i n t h e s o m a t i c C a + 2 a c t i o n p o t e n t i a l d u r a t i o n t h a t r e s u l t e d f r o m a s e l e c t i v e d e c r e a s e i n v o l t a g e -s e n s i t i v e Ca c h a n n e l c o n d u c t a n c e w h i c h d i d n o t i n v o l v e a change i n r e s t i n g membrane p e r m e a b i l i t y ( D u n l a p and F i s c h b a c h , 1981). B a c l o f e n , a GABA a n a l o g u e u s e d c l i n i c a l l y a s a m u s c l e r e l a x a n t , r e c e n t l y has been f o u n d t o be e f f i c a c e o u s i n t h e t r e a t m e n t of t r i g e m i n a l n e u r a l g i a (Fromm e t a l , 1980; S t e a r d o e t a l , 1984); t h i s a n a l o g u e i s s e l e c t i v e f o r t h e s e c o n d e f f e c t w h i c h i s not a n t a g o n i z e d by p i c r o t o x i n o r b i c u c u l l i n e . In TRG n e u r o n s , a C a + 2 d e p e n d e n t component o f t h e a c t i o n p o t e n t i a l may be p r e s e n t o n l y i n n e u r o n s w h i c h d i s c h a r g e s p i k e s of l o n g d u r a t i o n . In o r d e r t o a d e q u a t e l y e s t i m a t e e f f e c t s o f GABA ( o r b a c l o f e n ) on t h e shape and d u r a t i o n o f a c t i o n p o t e n t i a l s i n t h e s e n e u r o n s one would need t o a r t i f i c i a l l y p r o l o n g t h e C a + 2 -component o f t h e s p i k e by b l o c k a d e o f K c h a n n e l s a n d / o r t o i n c r e a s e t h e e x t r a c e l l u l a r C a + 2 c o n c e n t r a t i o n . A l t e r n a t i v e l y , a v o l t a g e c l a m p a n a l y s i s c o u l d be u s e d f o r more e x a c t e s t i m a t i o n o f c h a n g e s i n C a + 2 c u r r e n t . A p h y s i o l o g i c a l r o l e f o r G A B A - i n d u c e d r e d u c t i o n of C a + 2 component of s o m a t i c a c t i o n p o t e n t i a l i s u n c l e a r . In s y m p a t h e t i c g a n g l i a , GABA d e p o l a r i z e s b o t h p r e - and p o s t -g a n g l i o n i c a x o n s . However, when d e p o l a r i z a t i o n i s b l o c k e d e n t i r e l y w i t h b i c u c u l l i n e , no change i n t r a n s m i t t e r r e l e a s e c a n be d e t e c t e d e i t h e r w i t h GABA o r b a c l o f e n ( K o k e t s u e t a l , 1974; K a t o and Kuba, 1980). A l s o , GABA and i t s s y n t h e s i z i n g enzyme 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 a r e b o t h a b s e n t i n s y m p a t h e t i c g a n g l i a ( N a g a t a e t a l , 1966; M c B r i d e an d K l i n g m a n , 1972). 62 Whereas a f u n c t i o n a l r o l e o f GABA-mediated d e p o l a r i z a t i o n s i n p r e s y n a p t i c i n h i b i t i o n s i s i m p l i e d by s e n s i t i v i t y of p r i m a r y a f f e r e n t s t o b i c u c u l l i n e ( C u r t i s e t a l , 1971), no s e l e c t i v e a n t a g o n i s t of b a c l o f e n i n d u c e d e f f e c t s i s a v a i l a b l e t o a s s e r t a p h y s i o l o g i c a l r o l e of t h e s e c o n d GABA r e c e p t o r t y p e i n p r e s y n a p t i c i n h i b i t i o n . G. MEMBRANE OSCILLATORY BEHAVIOUR AND ACCOMODATION S u b t h r e s h o l d o s c i l l a t o r y b e h a v i o u r i s o b s e r v e d i n a v a r i e t y of e x c i t a b l e membranes i n c l u d i n g s q u i d g i a n t axon ( C h a n d l e r e t a l , 1962) and e m b r y o n i c c u l t u r e d h e a r t c e l l a g g r e g a t e s (Clapham and De F e l i c e , 1982). U s i n g e q u i v a l e n t e l e c t r i c a l c i r c u i t m o d e l s t h i s o s c i l l a t o r y a c t i v i t y c a n be d e s c r i b e d by t h e r e s i s t i v e , c a p a c i t i v e a n d i n d u c t i v e c h a r a c t e r i s t i c s of t h e membrane (Mauro e t a l , 1970). When o s c i l l a t o r y c u r r e n t s a r e a p p l i e d t o e x c i t a b l e membranes t h e y may e x h i b i t r e s o n a n t c h a r a c t e r i s t i c s , i . e. o s c i l l a t o r y i n p u t s i g n a l s t h a t most c l o s e l y r e s e m b l e t h e n a t u r a l f r e q u e n c y o f o s c i l l a t i o n a r e more e f f i c a c e o u s i n p r e t u r b i n g t h e membrane p o t e n t i a l t h a n t h e s i g n a l f r e q u e n c i e s t h a t l i e o u t s i d e t h e n a t u r a l r e s o n a n t f r e q u e n c y ( i e s ) o f t h e membrane. The o b s e r v a t i o n made i n t h e p r e s e n t s t u d y u s i n g t i m e - d o m a i n a n a l y s i s t e c h n i q u e s was t h a t o s c i l l a t o r y b e h a v i o u r i s more p r o n o u n c e d a t d e p o l a r i z e d l e v e l s of membrane p o t e n t i a l . T h e s e f i n d i n g s a r e f u l l y c o r r o b o r a t e d u s i n g f r e q u e n c y - d o m a i n a n a l y s i s t e c h n i q u e s , where r e s o n a n t b e h a v i o u r was shown t o depend 63 d r a m a t i c a l l y on t h e r e s t i n g membrane p o t e n t i a l ( P u i l e t a l , i n p r e s s ) . The a b i l i t y of somata of TRG n e u r o n s t o a c t as a f i l t e r o f s u b t h r e s h o l d i n p u t s i g n a l s by damping t h o s e s i g n a l s t h a t l i e o u t s i d e t h e n a t u r a l r e s o n a n t f r e q u e n c y p o s e s an i n t r i g u i n g p r o b l e m . The somata o f TRG n e u r o n s a r e n o t known t o p o s s e s s s y n a p t i c c o n t a c t s and b e i n g l o c a t e d away from t h e main axon do n o t seem t o impede a c t i o n p o t e n t i a l t r a n s m i s s i o n , e x c e p t f o r a s m a l l d e l a y a t t h e j u n c t i o n of t h e stem p r o c e s s and t h e main axon (Dun, 1955). O s c i l l a t o r y membrane b e h a v i o u r i s d e p e n d e n t on t h e a c t i v i t y o f v o l t a g e - d e p e n d e n t i o n i c c h a n n e l s t h a t g o v e r n membrane e x c i t a b i l i t y . A n o t h e r phenomenon t h a t i s d e p e n d e n t on t h e i o n i c c h a n n e l a c t i v i t y i s t h a t o f s p i k e a c c o m o d a t i o n . A c c o m o d a t i o n i s an i m p o r t a n t p r o p e r t y of n e u r o n s w h i c h i s u s u a l l y d e f i n e d a s an i n c r e a s e i n a c t i o n p o t e n t i a l t h r e s h o l d due t o a p p l i c a t i o n of s l o w l y r i s i n g c u r r e n t ( K o e s t l e r , 1981). A c c o m o d a t i o n has been shown t o be much l e s s p r o m i n e n t i n s p i n a l m o t o n e u r o n s t h a n i n p e r i p h e r a l n e r v e ( A r a k i and O t a n i , 1959; F r a n k and F u o r t e s , 1960). I t would seem t h a t i n m o t o n e u r o n s , d e c r e a s e d a c c o m o d a t i o n i s due t o t h e a c t i v i t y o f s y n a p s e s i m p i n g i n g on t h e n e u r o n ( c f . H u b b a r d e t a l , 1969). L a t e r i n v e s t i g a t i o n s have shown t h e p r e s e n c e o f a p e r s i s t e n t i n w a r d sodium c u r r e n t i n m o t o n e u r o n s ( S c h w i n d t and C r i l l , 1980; 1982) and n e o c o r t i c a l n e u r o n s ( S t a f s t r o m e t a l , 1982) w h i c h c o u n t e r e d t h e o u t w a r d p o t a s s i u m c u r r e n t and t h u s may e f f e c t i v e l y r e d u c e a c c o m o d a t i o n . 64 P e r i k a r y a o f p r i m a r y s e n s o r y n e u r o n s a r e m o s t l y d e v o i d o f s y n a p s e s and s h o u l d e x h i b i t more a c c o m o d a t i o n t h a n m o t o n e u r o n s . A c c o m o d a t i o n can be o b s e r v e d r e a d i l y i n TRG n e u r o n s t h a t d i s c h a r g e r e p e t i t i v e l y i n r e s p o n s e t o i n j e c t i o n o f 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 . I t i s e v i d e n t f r o m F i g u r e 10 o f R e s u l t s t h a t t h e f a s t r i s i n g component o f t h e s p i k e s seems t o r e m a i n unchanged but t h e i n i t i a l p h a s e becomes more p r o l o n g e d w i t h e a c h s u c c e s s i v e a c t i o n p o t e n t i a l . A number o f m o dels have been p u t f o r w a r d t o e x p l a i n t h i s phenomenon. E a r l y work on s q u i d axon ( H o d g k i n and H u x l e y , 1952) and f r o g m y e l i n a t e d f i b e r s ( F r a n k e n h a u s e r and V a l l b o , 1965) i m p l i c a t e d d i f f e r e n c e s i n s odium c u r r e n t i n a c t i v a t i o n r a t e s t o be r e s p o n s i b l e f o r v a r i a t i o n s i n a c c o m o d a t i o n seen i n t h e e x c i t a b l e n e u r o n s . I n m o t o n e u r o n s , i t i s b a s e d m a i n l y on t h e r a t e o f r e c o v e r y from p o t a s s i u m c o n d u c t a n c e s i n c e r e c o v e r y from sodium i n a c t i v a t i o n i n t h e s e n e u r o n s i s r a p i d and t h e r e f o r e c a n be n e g l e c t e d ( c f . J a c k e t a l , 1983). The r e l a t i v e c o n t r i b u t i o n s of s o d i u m and p o t a s s i u m c u r r e n t s t o t h e a c c o m o d a t i o n o b s e r v e d i n TRG n e u r o n s r e m a i n t o be shown. The p o s s i b l e p r e s e n c e o f non-homogeneous membrane i n t h e p e r i k a r y a l r e g i o n of TRG n e u r o n s may f u r t h e r c o m p l i c a t e e x p e r i m e n t a l i n t e r p r e t a t i o n s . S h i f t s i n t h e l o c a t i o n of i m p u l s e g e n e r a t i o n s i t e s have been shown t o o c c u r upon e l e c t r i c a l s t i m u l a t i o n i n s e v e r a l t y p e s o f e x c i t a b l e membranes. F o r example, Ringham (1971) showed t h a t a t h i g h e r c u r r e n t i n j e c t i o n s t r e n g t h s , t h e s i t e of i m p u l s e g e n e r a t i o n moved c l o s e r t o t h e soma o f t h e c r a y f i s h s t r e t c h r e c e p t o r . In TRG n e u r o n s , 65 a c c o m o d a t i o n c h a r a c t e r i s t i c s may be d i f f e r e n t f o r t h e stem segment compared t o t h e r e s t o f t h e s o m a t i c membrane ( i . e . t h e stem p r o c e s s may be a n a l a g o u s t o t h e s p i k e i n i t i a t i n g zone of t h e m o t o n e u r o n ) . T h e r e i s no good r e a s o n t o assume t h a t t h e s u b t h r e s h o l d o s c i l l a t o r y phenomenon t h a t o c c u r r e d as a r e s u l t o f v o l t a g e -d e p e n d e n t i o n i c c h a n n e l a c t i v i t y i s not p r e s e n t d u r i n g t h e g e n e r a t i o n o f s u p r a t h r e s h o l d e v e n t s w h i c h a r e p r o b a b l y m e d i a t e d by t h e s e same c h a n n e l s . T h e r e f o r e , i t i s n o t s u f f i c i e n t t o e x c l u d e a m o d u l a t o r y r o l e o f t h e c e l l body on t h e b a s i s o f an e l e c t r o p h y s i o l o g i c a l o b s e r v a t i o n t h a t l a r g e h y p e r p o l a r i z a t i o n s o f t h e c e l l body membrane do n o t impede t h e p a s s a g e o f an a c t i o n p o t e n t i a l f r o m t h e p e r i p h e r y t o t h e c e n t r a l t e r m i n a l s . I t i s c o n c e i v a b l e t h a t t h e m o d u l a t o r y r o l e o f TRG n e u r o n c e l l body becomes i m p o r t a n t d u r i n g t h e t r a n s m i s s i o n o f r e p e t i t i v e a c t i o n p o t e n t i a l s , where a c c o m o d a t i o n i s an i m p o r t a n t f a c t o r i n d e t e r m i n i n g f r e q u e n c y and d u r a t i o n o f t h e d i s c h a r g e . Thus h y p e r p o l a r i z a t i o n o f t h e c e l l body w h i c h c a n r e s u l t i n l a r g e d e c r e a s e s o f o s c i l l a t o r y t e n d e n c y ( r e s o n a n c e ) and an i n c r e a s e i n a c c o m o d a t i o n may s i g n i f i c a n t l y r e d u c e t h e d u r a t i o n o r f r e q u e n c y o f t h e a c t i o n p o t e n t i a l d i s c h a r g e . C o n v e r s e l y , d e p o l a r i z a t i o n o f c e l l body membrane may e n h a n c e s p i k e d i s c h a r g e by i n c r e a s i n g o s c i l l a t i o n and r e d u c i n g a c c o m o d a t i o n i n a manner a n a l o g o u s t o t h e d e c r e a s e o f a c c o m o d a t i o n i n t h e m o t o n e u r o n s and n e o c o r t i c a l c e l l s by s y n a p t i c i n p u t s . 66 H. DIRECTIONS FOR FUTURE RESEARCH One compound t h a t a l s o has been p r o p o s e d as a p u t a t i v e n e u r o t r a n s m i t t e r i n p r i m a r y t e r m i n a l s of a f f e r e n t n e u r o n s i s s u b s t a n c e P. T h i s u n d e c a p e p t i d e w h i c h was d i s c o v e r e d by E u l e r and Gaddum (1931) has been s i n c e shown t o be l o c a l i z e d i n s p e c i f i c n e u r o n a l t r a c t s of t h e mammalian n e r v o u s s y s t e m s , p a r t i c u l a r l y i n DRG n e u r o n s and i n a r e a s o f t h e s p i n a l c o r d where t e r m i n a l s o f t h e s e n e u r o n s a r e l o c a l i z e d ( T a k a h a s h i e t a l , 1974). S p e c i f i c a l l y , S u b s t a n c e P was f o u n d a l m o s t e x c l u s i v e l y i n t h e s m a l l c e l l b o d i e s of t h e DRG and TRG n e u r o n s as w e l l as i n t h e i r c e n t r a l and p e r i p h e r a l p r o c e s s e s ( H o k f e l t e t a l , 1977). N e u r o p h y s i o l o g i c a l s t u d i e s of e x c i t a t o r y a c t i o n s o f s u b s t a n c e P i n s p i n a l n e u r o n s p r o v i d e d e v i d e n c e f o r f a c i l i t a t i o n of d i s c h a r g e s i n d u c e d by n o c i c e p t i v e s t i m u l a t i o n i n t h e s p i n a l c o r d w i t h i o n t o p h o r e t i c s u b s t a n c e P a p p l i c a t i o n ( H e nry, 1976). In a d d i t i o n , n e u r o n s i n t h e o p h t h a l m i c d i v i s i o n o f c a t t r i g e m i n a l n e r v e were shown t o c o n t a i n s u b s t a n c e P and t o i n n e r v a t e t h e a r t e r i e s of t h e c i r c l e o f W i l l i s , one of t h e few p a i n s e n s i t i v e a r e a s w i t h i n t h e c r a n i u m (Mayberg e t a l , 1981). T h e s e f i n d i n g s p o i n t t o t h e p o s s i b i l i t y t h a t s u b s t a n c e P may be a n e u r o t r a n s m i t t e r a t CNS t e r m i n a l s o f n o c i c e p t i v e p r i m a r y a f f e r e n t n e u r o n s i n t h e t r i g e m i n a l and s p i n a l g a n g l i a , i n a d d i t i o n t o i t s p o s s i b l e r o l e a s a v a s o d i l a t o r s u b s t a n c e a t t h e p e r i p h e r a l s e n s o r y t e r m i n a l s (Lembeck e t a l , 1977). I t w o u l d be of i n t e r e s t t o t e s t s u b s t a n c e P on t h e TRG 67 s l i c e s t o see whether t h e compound can m o d u l a t e t h e s o m a t i c membrane p r o p e r t i e s o f s e n s o r y n e u r o n s . F e t a l t r i g e m i n a l g a n g l i o n n e u r o n s of t h e r a t , s t u d i e d under c o n d i t i o n s of i n t e r n a l p e r f u s i o n and v o l t a g e c l a m p were o b s e r v e d t o r e p o n d (40/110) w i t h i n w a r d N a + / K + c u r r e n t s t o r a p i d pH c h a n g e s i n t h e e x t r a c e l l u l a r medium. P r e l i m i n a r y t e s t s showed s u c h n e u r o n s t o be i n s e n s i t i v e t o p u t a t i v e t r a n s m i t t e r s s u c h a s GABA, h i s t a m i n e , g l u t a m a t e , 5-HT and s u b s t a n c e P a p p l i e d a t pH 7.4 ( K r i s h t a l and P i d o p l i c h k o , 1981). O b s e r v a t i o n s made w i t h f e t a l n e u r o n s and t h e p r e s e n t s t u d y of S - g l u t a m a t e and 5-HT e f f e c t s a r e r a t h e r p r e l i m i n a r y . A more e x t e n s i v e s t u d y i s needed t o e v a l u a t e 5-HT a c t i o n on TRG n e u r o n s . . I n view o f r e c e n t r e p o r t s o f 5-HT a c t i o n on t h e C-n e u r o n s o f t h e c a t nodose g a n g l i o n , use o f f i n e - t i p p e d e l e c t r o d e s , f o r p r o l o n g e d r e c o r d i n g s f r o m n e u r o n s t h a t e x h i b i t a c t i o n p o t e n t i a l s w i t h a r e p o l a r i z a t i o n p h a s e p l a t e a u , i s i n d i c a t e d . 68 V. SUMMARY AND CONCLUSIONS 1. The m e t h o d o l o g y f o r o b t a i n i n g v i a b l e t h i n s l i c e s of t h e t r i g e m i n a l r o o t g a n g l i o n o f t h e g u i n e a p i g was d e v e l o p e d i n o r d e r t o a l l o w p r o l o n g e d ( s t a b l e ) i n t r a c e l l u l a r r e c o r d i n g s w i t h e l e c t r o p h y s i o l o g i c a l t e c h n i q u e s , f r o m somata of p r i m a r y a f f e r e n t n e u r o n s . T h i s i n v i t r o s l i c e p r e p a r a t i o n p e r m i t t e d t h e b a t h a p p l i c a t i o n o f p h a r m a c o l o g i c a l a g e n t s a t known c o n c e n t r a t i o n s w h i c h f a c i l i t a t e d i n v e s t i g a t i o n s o f t h e i n f l u e n c e of e n d o g e n o u s l y f o u n d compounds and d r u g s on membrane e l e c t r i c a l p r o p e r t i e s o f t r i g e m i n a l r o o t g a n g l i o n n e u r o n s . 2. T h e s e i n v e s t i g a t i o n s have p e r m i t t e d , f o r t h e f i r s t t i m e , a d e s c r i p t i o n o f b o t h p a s s i v e and a c t i v e ( v o l t a g e - and t i m e -d e p e n d e n t ) membrane p r o p e r t i e s o f t r i g e m i n a l r o o t g a n g l i o n n e u r o n s i n v i t r o . The m o d u l a t o r y a c t i o n s o f s e v e r a l n e u r o a c t i v e s u b s t a n c e s on t h e s e membrane p r o p e r t i e s a l s o were s t u d i e d i n t h e s e i n v e s t i g a t i o n s . 3. A l l 150 n e u r o n s w h i c h were i m p a l e d w i t h m i c r o e l e c t r o d e s r e m a i n e d q u i e s c e n t p r i o r t o i n t r a c e l l u l a r i n j e c t i o n of d e p o l a r i z i n g c u r r e n t s quare-wave p u l s e s . O n l y c e l l s t h a t m a i n t a i n e d s t e a d y membrane p o t e n t i a l s more n e g a t i v e t h a n 50 mV and d i s p l a y e d a c t i o n p o t e n t i a l s g r e a t e r t h a n 50 mV were u s e d i n t h e d e t a i l e d a n a l y s i s o f membrane p r o p e r t i e s . The mean r e s t i n g membrane p o t e n t i a l o f TRG n e u r o n s was -61.7 ± 0.4 mV S. E . (n=50). I n j e c t i o n o f h y p e r p o l a r i z i n g c u r r e n t p u l s e s (0.5-10 nA) 69 e v o k e d " t i m e - d e p e n d e n t r e c t i f i c a t i o n " i n a m a j o r i t y o f sample d n e u r o n s ( a b o u t 1 0 0 ) . T h i s membrane r e s p o n s e of TRG n e u r o n s , d e s c r i b e d p r e v i o u s l y i n a number o f i n v e s t i g a t i o n s o f o t h e r t y p e s of p r i m a r y s e n s o r y n e u r o n s , was f o u n d t o have a marked v o l t a g e d e p e n d e n c y . O s c i l l a t o r y b e h a v i o u r of t h e membrane p o t e n t i a l o f TRG n e u r o n s was o b s e r v e d d u r i n g i n t r a c e l l u l a r i n j e c t i o n s o f 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 o r f o l l o w i n g t e r m i n a t i o n o f s i m i l a r i n j e c t i o n s of h y p e r p o l a r i z i n g p u l s e s . T h i s phenomenon a l s o was shown t o be v o l t a g e - d e p e n d e n t , w i t h more p r o n o u n c e d o s c i l l a t i o n s e v i d e n t a t d e p o l a r i z e d l e v e l s o f membrane p o t e n t i a l r e l a t i v e t o t h e i n i t i a l r e s t i n g s t a t e . 4. Two g r o u p s o f n e u r o n s c o u l d be d i s t i n g u i s h e d on t h e b a s i s o f t h e i r a c t i o n p o t e n t i a l c h a r a c t e r i s t i c s . A g r o u p w h i c h t y p i f i e d a m a j o r i t y o f sampled n e u r o n s e x h i b i t e d f a s t - r i s i n g s p i k e s w i t h mean a m p l i t u d e o f 65 ± 1.9 mV S. E. (n = 31) and a f t e r h y p e r p o l a r i z a t i o n s t h a t r a n g e d f r o m 4.5 mV t o 25 mV i n s i z e (mean 14.1 ± 1.1 mV S. E.; n = 2 2 ) . Peak a m p l i t u d e o f t h e a f t e r h y p e r p o l a r i z a t i o n was p a r t l y d e p e n d e n t on t h e v a l u e of t h e r e s t i n g membrane p o t e n t i a l , and was l a r g e s t a t d e p o l a r i z e d l e v e l s o f membrane p o t e n t i a l . A c t i o n p o t e n t i a l s e v o k e d by i n t r a c e l l u l a r i n j e c t i o n o f 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 i n t h e s e c o n d g r o u p o f n e u r o n s had a p l a t e a u i n t h e i r r e p o l a r i z a t i o n p h a s e . T h e s e a c t i o n p o t e n t i a l s had a mean a m p l i t u d e of 78 ± 2.3 mV S. E. (n = 7) and a f t e r h y p e r p o l a r i z a t i o n s r a n g i n g f r o m 13 t o 25 mV (mean 16.8 ± 2.7 mV S . E . n = 6) i n a m p l i t u d e . R e p e t i t i v e f i r i n g c o u l d be e v o k e d i n b o t h g r o u p s o f n e u r o n s i f 70 a d e q u a t e s u p r a t h r e s h o l d amounts of d e p o l a r i z i n g c u r r e n t were a p p l i e d t h r o u g h t h e r e c o r d i n g e l e c t r o d e . A c c o m o d a t i o n of a c t i o n p o t e n t i a l s o f t e n was o b s e r v e d i n s u c h c a s e s . 5. B a t h a p p l i c a t i o n s of S - g l u t a m a t e ( 1 0 " 2 M) r e s u l t e d i n s m a l l (1-2 mV) membrane d e p o l a r i z a t i o n s i n two of t h e s i x n e u r o n s w h i c h were t e s t e d f o r r e s p o n s e s t o S - g l u t a m a t e . T h e s e d e p o l a r i z a t i o n s were n o t a s s o c i a t e d w i t h n o t i c e a b l e c h a n g e s i n membrane c o n d u c t a n c e and d i s a p p e a r e d c o m p l e t e l y upon r e t u r n t o c o n t r o l p e r f u s a t e . S i m i l a r l y , 5 - h y d r o x y t r y p t a m i n e (5-HT) had l i t t l e e f f e c t on s i x TRG n e u r o n s . A s m a l l (1-2 mV) membrane d e p o l a r i z a t i o n n o t u n l i k e t h a t o b s e r v e d f o r S - g l u t a m a t e was seen .in one n e u r o n , a l t h o u g h a s l i g h t i n c r e a s e i n membrane c o n d u c t a n c e was o b s e r v e d . 6. B a t h p e r f u s i o n o f gamma-aminobutyric a c i d (GABA 10"* - 10~ 2 M) r e s u l t e d i n membrane d e p o l a r i z a t i o n (2-14 mV) i n 83% of t e s t e d n e u r o n s (n = 3 6 ) . R e d u c t i o n o f maximal r e s p o n s e , p r e s u m a b l y due t o r e c e p t o r d e s e n s i t i z a t i o n , was o b s e r v e d r e a d i l y when 10" 3 M c o n c e n t r a t i o n s o f GABA were a p p l i e d . GABA-mediated c o n d u c t a n c e i n c r e a s e s r e s u l t e d i n a d e c r e a s e i n t h e h e i g h t o f a c t i o n p o t e n t i a l s e v o k e d by i n t r a c e l l u l a r i n j e c t i o n s of 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 . T h i s e f f e c t c o u l d n o t be a c c o u n t e d f o r by membrane d e p o l a r i z a t i o n a l o n e and a l s o was a t t r i b u t e d t o a G A B A - i n d u c e d s h u n t o f t h e s p i k e - g e n e r a t i n g mechanism. 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