Substance P (SP) Inputs to the Nucleus Tractus Solitarius (NTS) of the Rat: Colocalization with Serotonin (5HT) in Raphe-NTS Projection Neurons

183 S u b s t a n c e P (SP) I n p u t s to t h e N u c l e u s T r a c t u s S o l i t a r i u s (NTS) of t h e Rat: C o l o c a l i z a t i o n w i t h S e r o t o n i n (5HT) in R a p h e - N T S P r o j e c t i o n N e u r o n s . K.B. Thor, K.M. Hill, C. H a r r o d , a n d C.J. Helke. Dept. of P h a r m a c o l . , USUHS, 4301 J o n e s Bridge Rd., B e t h e s d a , MD, 2 0 8 1 4 As m u c h a s 2 5 % of t h e SP in t h e NTS is c o n t a i n e d in t e r m i n a l s o r i g i n a t i n g f r o m n o d o s e a n d p e t r o s a l g a n g l i a n e u r o n s I. SP-containing n e u r o n s a r e a l s o i n t r i n s i c to t h e NTS 2. However, it is u n k n o w n if t h e s e a r e t h e o n l y n e u r o n s t h a t c o n t r i b u t e to t h e SP c o n t e n t of t h e NTS. Therefore, i n j e c t i o n s of r h o d a m i n e - f i l l e d m i c r o s p h e r e s (RMs), a f l u o r e s c e n t t r a c e r , w e r e i n j e c t e d i n t o t h e NTS of r a t s t h a t w e r e s u b s e q u e n t l v t r e a t e d w i t h e o l c h i c i n e a n d p r o c e s s e d for S P - i m m u n o f l u o r e s c e n c e (SP-IF) ~. N e u r o n s t h a t c o n t a i n e d b o t h RMs a n d S P - I F were p r i m a r i l y l o c a l i z e d to t h e n u c l e u s r a p h e p a l l i d u s (NRP), n u c l e u s r a p h e o b s c u r u s (NRO), n u c l e u s r a p h e m a g n u s (NRM), a n d t h e p a r a o l i v a r y n u c l e u s (PON). Over a t h i r d of t h e d o u b l y - l a b e l l e d n e u r o n s w e r e l o c a t e d in t h e NRP, w i t h t h e r e m a i n d e r e q u a l l y d i v i d e d a m o n g t h e o t h e r t h r e e regions. In t h e NRP, over 50% of t h e R M - l a b e l l e d n e u r o n s were f o u n d to be IF for SP, while in the NRP a n d PON 2 0 - 2 5 % w e r e IF, a n d only 7% of t h e R M - l a b e l l e d n e u r o n s in NRM w e r e IF. In a d j a c e n t s e c t i o n s from t h i s s e r i e s of e x p e r i m e n t s , it w a s a l s o often f o u n d t h a t o v e r h a l f of t h e R M - l a b e l l e d n e u r o n s in t h e NRP w e r e 5HT-IF. Therefore, t h e p o s s i b i l i t y of S P - 5 H T c o l o c a l i z a t i o n w a s e x a m i n e d 4. S P - 5 H T c o l o c a l i z a t i o n in t e r m i n a l s w a s e x a m i n e d u s i n g d u a l - c o l o r IF with rat anti-SP detected by r h o d a m i n e - c o n j u g a t e d s e c o n d a r y a n t i s e r a and rabbit anti-5HT detected by fluorescein-conjugated secondary antisera. D o u b l y - I F t e r m i n a l s were a b u n d a n t in t h e m e d i a l a n d c o m m i s s u r a l s u b n u c l e i of t h e NTS, a n d in t h e d o r s a l m o t o r n u c l e u s of t h e v a g u s n e r v e (DMN). The l a t e r a l NTS a n d a r e a p o s t r e m a c o n t a i n e d fewer d o u b l y - I F t e r m i n a l s . In t h e m e d i a l a n d c o m m i s s u r a l s u b n u c l e i of t h e NTS, t h e d e n s i t y of d o u b l y - I F t e r m i n a l s w a s c o m p a r a b l e to t h a t in t h e h y p o g l o s s a l n u c l e u s a n d t h e v e n t r a l h o r n . However, b e c a u s e of t h e g r e a t e r n u m b e r of SP t e r m i n a l s in t h e NTS, t h e p r o p o r t i o n of t h e SP t e r m i n a l s t h a t were 5HT-IF w a s small. To q u a n t i f y t h e p r o p o r t i o n of SP t h a t w a s c o n t a i n e d in 5HT t e r m i n a l s , r a d i o i m m u n o a s s a y of SP levels were d e t e r m i n e d in c o n t r o l r a t s a n d 5,7D H T - t r e a t e d r a t s . In t h e s e e x p e r i m e n t s , only a n i n s i g n i f i c a n t d e c r e a s e in SP w a s d e t e c t e d a t c a u d a l a n d i n t e r m e d i a t e levels of t h e NTS in 5,7-DHTt r e a t e d r a t s , w h i c h e m p h a s i z e s t h e s m a l l p r o p o r t i o n of d o u b l y - I F v a r i c o s i t i e s that were seen. However, a 4 5 % d e c r e a s e in SP l e v e l s (p<0.05) w a s d e t e c t e d a t r o s t r a l levels of t h e NTS of 5 , 7 - D H T - t r e a t e d r a t s . T h i s d e c r e a s e w a s s u b j e c t i v e l y verified in b r a i n s e c t i o n s t a k e n from 5 , 7 - D H T - t r e a t e d r a t s t h a t w e r e p r o c e s s e d for i m m u n o h i s t o c h e m i s t r y . The a n a t o m i c a l s o u r c e of t h e d o u b l y - I F v a r i c o s i t i e s t h a t were d e t e c t e d in t h e NTS w a s d e t e r m i n e d b y p r o c e s s i n g b r a i n s e c t i o n s , from r a t s t h a t h a d received RM i n j e c t i o n s into t h e NTS, for d u a l - c o l o r IF, as above, b u t u s i n g 7amino-4-methylcoumarin-3-acetate-conjugated secondary antibodies, which p r o d u c e a b l u e f l u o r e s c e n c e , to d e t e c t 5HT-IF. N e u r o n s t h a t were t r i p l y l a b e l l e d w e r e f o u n d in all f o u r a r e a s of t h e CNS t h a t c o n t a i n e d SP-IF NTSafferent n e u r o n s , i.e. NRP, NRO, NRM, a n d PON. No n e u r o n s in t h e n o d o s e or p e t r o s a l g a n g l i a w e r e f o u n d to colocalize 5HT a n d SP, e l i m i n a t i n g t h e s e cells a s a p o s s i b l e s o u r c e of d o u b l y - I F v a r i c o s i t i e s in t h e NTS. 1. Helke et al., (1980) P e p t i d e s 1 : i - 9 2. L j u n g d a h l et al., (1978) N e u r o s c i e n c e 3 : 8 6 1 - 9 4 3 3. T h o r a n d Helke (1987) J. Comp. Neurol. 2 6 5 : 2 7 5 - 2 9 3 4. T h o r et al., (1988) S y n a p s e (In Press)