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The role of type-2 serotonin receptors in morphine-produced analgesia Paul, Dennis John 1987

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THE ROLE OF TYPE-2 SEROTONIN RECEPTORS IN MORPHINE-PRODUCED ANALGESIA By DENNIS JOHN PAUL B.A., University of C i n c i n n a t i , 1980 M.A., University of B r i t i s h Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PSYCHOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1987 Q Dennis John Paul, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) A B S T R A C T I t i s g e n e r a l l y a c c e p t e d t h a t t h e n e u r o t r a n s m i t t e r , s e r o t o n i n m e d i a t e s m o r p h i n e - p r o d u c e d a n a l g e s i a , h o w e v e r , i t i s n o t c l e a r w h e t h e r t h i s m e d i a t i o n o c c u r s a t b r a i n o r s p i n a l c o r d s e r o t o n i n r e c e p t o r s . A n i s s u e t h a t h a s n o t o f t e n b e e n c o n s i d e r e d i s t h e d i f f e r e n t i a l r o l e t h a t s e r o t o n i n r e c e p t o r t y p e s may p l a y i n m o r p h i n e - p r o d u c e d a n a l g e s i a . P a u l a n d P h i l l i p s ( 1 9 8 6 ) o b s e r v e d t h a t p i r e n p e r o n e , a s e r o t o n i n a n t a g o n i s t w i t h a p r e f e r e n t i a l a f f i n i t y f o r t h e S2 r e c e p t o r , a t t e n u a t e s m o r p h i n e -p r o d u c e d a n a l g e s i a . T h i s r e s u l t i s p a r t i c u l a r l y i n t e r e s t i n g b e c a u s e t h e r e a r e r e p o r t e d l y no S2 r e c e p t o r s i n t h e s p i n a l c o r d . T h e p u r p o s e s o f t h i s d i s s e r t a t i o n w e r e : t o c o n f i r m t h e f i n d i n g o f P a u l a n d P h i l l i p s , t o l o c a l i z e t h e S2 r e c e p t o r s t h a t m e d i a t e t h e a n t i - a n a l g e s i c e f f e c t o f p i r e n p e r o n e , a n d t o t e s t t h e h y p o t h e s i s t h a t p i r e n p e r o n e may e x e r t i t s a n t i - a n a l g e s i c e f f e c t t h r o u g h a l p h a - a d r e n e r g i c r e c e p t o r s . I n e a c h o f f i v e e x p e r i m e n t s , t a i l - f l i c k l a t e n c i e s ( t h e t i m e t h a t i t t a k e s f o r e a c h r a t t o w i t h d r a w i t s t a i l f r o m a 52 C w a t e r b a t h ) w e r e m e a s u r e d 0 , 3 0 , 6 0 , 9 0 , a n d 120 m i n a f t e r d r u g i n j e c t i o n . I n E x p e r i m e n t 1 , t h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e was c h a l l e n g e d w i t h 0 . 0 8 , 0 . 1 6 , a n d 0 . 2 4 m g / k g o f p i r e n p e r o n e . E a c h d o s e o f p i r e n p e r o n e a t t e n u a t e d m o r p h i n e -p r o d u c e d a n a l g e s i a . M o r e o v e r , e a c h d o s e o f p i r e n p e r o n e p r o d u c e d h y p e r a l g e s i a i n r a t s r e c e i v i n g n o m o r p h i n e . I n E x p e r i m e n t 2 , m o r p h i n e - p r o d u c e d a n a l g e s i a was c h a l l e n g e d w i t h 1 , 3 , a n d 10 m g / k g o f k e t a n s e r i n H C I . O n l y t h e v e r y h i g h 10 m g / k g d o s e o f ketanserin s i g n i f i c a n t l y attenuated morphine-produced analgesia. Because ketanserin i s pharmacologically s i m i l a r to pirenperone but does not re a d i l y enter the central nervous system, t h i s r e s u l t indicates that central S2 receptors mediate the a n t i -analgesic e f f e c t of pirenperone and ketanserin. A t h i r d experiment demonstrated that 10 mg/kg of ketanserin did not block the analgesia produced by ketamine. Ketamine i s thought to produce analgesia by a d i f f e r e n t mechanism than morphine. Thus, the attenuation of analgesia by S2 receptor blockers i s not a general phenomenon, and i t may be s p e c i f i c to morphine-produced analgesia and other analgesics that act on t h i s system. Experiment 4 was designed to assess whether i t i s S2 receptors i n the brain or i n the spinal cord that mediate the anti-analgesic e f f e c t of S2 receptor blockade. The analgesic e f f e c t of morphine on t a i l - f l i c k latencies was challenged with pirenperone i n rats with spinal cords transected at the lower thoracic l e v e l and i n sham-surgery comparison r a t s . Pirenperone attenuated morphine-produced analgesia i n the sham-surgery group but not i n the rats with transected spinal cords. These r e s u l t s indicate that brain S2 receptors mediate the attenuation of morphine-produced analgesia by pirenperone. In the f i f t h and f i n a l experiment, morphine-produced analgesia was challenged with 10 mg/kg of LY53857. LY53857 i s an S2 antagonist which unlike pirenperone and ketanserin has no action at alpha-adrenergic receptors. Like pirenperone and ketanserin, LY53857 attenuated morphine-produced analgesia. This r e s u l t supports the view that S2 receptors mediate the anti-analgesic e f f e c t s of pirenperone and ketanserin. Together, the r e s u l t s of these f i v e experiments indicate that S2 receptors i n the brain are important for opioid-mediated analgesia. This conclusion challenges the widely held view that only spinal cord serotonin receptors mediate morphine-produced analgesia. V T A B L E OF CONTENTS A b s t r a c t i i T a b l e o f C o n t e n t s v L i s t o f T a b l e s v i i i L i s t o f F i g u r e s i x A c k n o w l e d g e m e n t x I . I n t r o d u c t i o n 1 1 . M e t h o d s o f M e a s u r i n g P a i n P e r c e p t i o n i n L a b o r a t o r y A n i m a l s 2 A . T h e T a i l - F l i c k T e s t 2 B . T h e H o t - P l a t e T e s t 3 C . T h e F l i n c h - J u m p T e s t 3 D . F o r m a l i n W r i t h i n g T e s t 4 E . N o x i o u s S t i m u l i U s e d i n E l e c t r o p h y s i o l o g i c a l R e c o r d i n g 4 2 . M e c h a n i s m s o f M o r p h i n e A n a l g e s i a . 7 A . B r a i n s t e m - t o - s p i n a l c o r d a n a l g e s i a s y s t e m 7 B . S p i n a l M e c h a n i s m s o f O p i a t e A n a l g e s i a 12 3 . T h e I n v o l v e m e n t o f S e r o t o n i n i n M o r p h i n e A n a l g e s i a . . . . 1 4 A . A n t a g o n i s m o f S e r o t o n e r g i c T r a n s m i s s i o n a n d M o r p h i n e A n a l g e s i a 14 B . S e r o t o n i n A g o n i s t s a n d A n a l g e s i a 16 C . A n a t o m i c a l L o c a l i z a t i o n o f S e r o t o n i n R e c e p t o r s t h a t a r e T h o u g h t t o M e d i a t e M o r p h i n e - P r o d u c e d A n a l g e s i a 17 v i 4. S e r o t o n i n R e c e p t o r T y p e s 22 A . The S 1 - S 2 D i s t i n c t i o n 22 B . D i s t r i b u t i o n o f S e r o t o n i n B i n d i n g S i t e s . . - ; 23 C . B e h a v i o r a l C o r r e l a t e s o f S1 a n d S2 B i n d i n g S i t e s . 2 5 5 . P a u l a n d P h i l l i p s (1 9 8 6 ) 26 6 . G e n e r a l R a t i o n a l e a n d P u r p o s e s 28 I I . G e n e r a l M e t h o d 30 1 . S u b j e c t s 30 2 . A p p a r a t u s 30 3 . P r o c e d u r e 30 4. S t a t i s t i c a l A n a l y s i s 31 I I I . E x p e r i m e n t 1: E f f e c t o f P i r e n p e r o n e o n M o r p h i n e A n a l g e s i a 35 M e t h o d 35 R e s u l t s 36 D i s c u s s i o n 39 I V . E x p e r i m e n t 2 : E f f e c t o f K e t a n s e r i n o n M o r p h i n e A n a l g e s i a 41 M e t h o d 41 R e s u l t s 44 D i s c u s s i o n 47 V . E x p e r i m e n t 3 : E f f e c t o f K e t a n s e r i n o n K e t a m i n e - P r o d u c e d A n a l g e s i a 49 M e t h o d 49 R e s u l t s 50 D i s c u s s i o n 53 v i i V I . E x p e r i m e n t 4: E f f e c t o f P i r e n p e r o n e o n M o r p h i n e - P r o d u c e d A n a l g e s i a i n S p i n a l R a t s . . 54 M e t h o d ~ 55 R e s u l t s 56 D i s c u s s i o n 59 V I I . E x p e r i m e n t 5: E f f e c t o f LY53857 o n M o r p h i n e - P r o d u c e d A n a l g e s i a 61 M e t h o d 61 R e s u l t s 62 D i s c u s s i o n 65 V I I I . D i s c u s s i o n 67 1 . Summary a n d G e n e r a l D i s c u s s i o n o f t h e E x p e r i m e n t s 67 A . G e n e r a l D i s c u s s i o n o f E x p e r i m e n t s 1, 2, 3, a n d 4 68 B . G e n e r a l D i s c u s s i o n o f E x p e r i m e n t 5 71 2. R e s u l t s o f a P i l o t E x p e r i m e n t t o D i r e c t l y T e s t W h e t h e r C e n t r a l o r S p i n a l S2 R e c e p t o r s M e d i a t e t h e B l o c k a d e o f M o r p h i n e - P r o d u c e d A n a l g e s i a b y S2 R e c e p t o r B l o c k e r s 72 3. I m p l i c a t i o n s o f t h e P r e s e n t F i n d i n g s f o r T h e o r i e s o f P a i n P e r c e p t i o n a n d A n a l g e s i a 73 4. G e n e r a l I m p l i c a t i o n s a n d F u t u r e D i r e c t i o n s 77 I X . R e f e r e n c e s 83 v i i i L I S T OF T A B L E S T a b l e 1 . T h e v e h i c l e , c o n c e n t r a t i o n , i n j e c t i o n v o l u m e , d o s e , r o u t e o f a d m i n i s t r a t i o n , a n d s o u r c e o f e a c h — d r u g u s e d i n t h i s d i s s e r t a t i o n 3 2 - 3 3 T a b l e 2 . T h e i n h i b i t i o n c o n s t a n t s o f k e t a n s e r i n a n d p i r e n p e r o n e f o r S 1 , S 2 , h i s t a m i n e t y p e - 1 ( H 1 ) , a l p h a , d o p a m i n e ( D A ) , a n d m u s c a r i n i c a c e t y l c h o l i n e ( A c h - m ) r e c e p t o r s a s d e t e r m i n e d b y i n v i t r o r e c e p t o r b i n d i n g i x L I S T OF F I G U R E S F i g u r e 1 . The a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h t h r e e d o s e s o f p i r e n p e r o n e . . . . 3 7 - 3 8 F i g u r e 2 . T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h t h r e e d o s e s o f k e t a n s e r i n H C I . 4 5 - 4 6 F i g u r e 3 . The a n a l g e s i c e f f e c t o f 100 m g / k g o f k e t a m i n e H C I c h a l l e n g e d w i t h 10 m g / k g o f k e t a n s e r i n H C I 5 1 - 5 2 F i g u r e 4 . The a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h 0 . 2 4 m g / k g o f p i r e n p e r o n e i n r a t s w i t h t r a n s e c t e d s p i n a l c o r d s a n d i n s h a m -s u r g e r y c o n t r o l s 5 7 - 5 8 F i g u r e 5 . T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h 10 m g / k g o f L Y 5 3 8 5 7 6 3 - 6 4 X ACKNOWLEDGEMENT I t h a n k D r . J o h n P . J . P i n e l f o r h i s s u p p o r t , f o r h i s c o n f i d e n c e , a n d f o r h i s i n s i g h t f u l c r i t i c i s m s t h r o u g h o u t t h i s p r o j e c t . I a l s o t h a n k J . P f a u s , M . M a n a , L . S y m o n s , J . ~ D r u h a n , K . K i m , B . C h r i s t i a n s o n , D . P e t r o v i c , S . H a n c o c k , C . J o n e s , S . M e n d e l s o n , a n d C Y a c u r a f o r t h e i r a s s i s t a n c e i n t h e c o n d u c t o f t h e s e e x p e r i m e n t s . T h i s r e s e a r c h was s u p p o r t e d b y a g r a n t t o D r . J o h n P i n e l f r o m t h e M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a a n d u n k n o w i n g l y b y g r a n t s t o s e v e r a l o t h e r f a c u l t y m e m b e r s . T h i s d i s s e r t a t i o n i s d e d i c a t e d t o s p l e n d i d a n d s t e r i l e D o l o r e s . 1 I. INTRODUCTION One of the most important discoveries of modern neuroscience i s that there are neural c i r c u i t s whose primary function i s to suppress the e f f e c t s of normally painful s t i m u l i . These "analgesia c i r c u i t s " have been shown to be activated by opiates such as morphine, but the fact that several serotonin antagonists attenuate the analgesic e f f e c t s of opiates suggests that serotonin i s also involved. Further support for t h i s notion comes from the recent finding that serotonin agonists can by themselves produce analgesia s i m i l a r to that produced by morphine. Recently, i t has been shown that there are two types of serotonin receptors. The d i s t i n c t i o n between these two types of serotonin receptors i s made on the basis of t h e i r r e l a t i v e a f f i n i t y for serotonin and the serotonin antagonist, spiperone. Some serotonin receptors, c a l l e d S1 receptors, have a high a f f i n i t y for serotonin and a low a f f i n i t y for spiperone; whereas, other serotonin receptors, now c a l l e d S2 receptors, have a much lower a f f i n i t y for serotonin, and a much higher a f f i n i t y for spiperone than do S1 receptors. This d i s s e r t a t i o n focuses on the contribution of S2 receptors to morphine-produced analgesia. The general introduction of t h i s topic i s divided into f i v e major sections: The f i r s t describes several methods of measuring nociception i n laboratory animals; the second comprises a d e s c r i p t i o n of the neural c i r c u i t s that are presumed to mediate morphine analgesia; 2 t h e t h i r d f o c u s e s o n t h e e v i d e n c e t h a t s e r o t o n i n i s i n v o l v e d i n t h e s e c i r c u i t s ; t h e f o u r t h r e v i e w s t h e e v i d e n c e t h a t t h e r e a r e t w o d i f f e r e n t c l a s s e s o f s e r o t o n i n r e c e p t o r s ; t h e f i f t h " d e s c r i b e s t h e s t u d y ( P a u l & P h i l l i p s , 1986) f r o m w h i c h t h i s s e r i e s o f e x p e r i m e n t s e v o l v e d ; a n d t h e s i x t h , a n d f i n a l , s e c t i o n o f t h e I n t r o d u c t i o n i s a s t a t e m e n t o f t h e g e n e r a l r a t i o n a l e . 1 . M e t h o d s o f M e a s u r i n g P a i n P e r c e p t i o n i n L a b o r a t o r y A n i m a l s F o r e t h i c a l a n d p r a c t i c a l r e a s o n s , t h e n e u r a l s u b s t r a t e s o f p a i n p e r c e p t i o n a r e m o s t c o m m o n l y i n v e s t i g a t e d i n l a b o r a t o r y a n i m a l s . The m e t h o d s u s e d t o m e a s u r e p a i n p e r c e p t i o n v a r y g r e a t l y i n t e r m s o f b o t h t h e t y p e o f n o x i o u s s t i m u l a t i o n e m p l o y e d a n d w h e r e o n t h e b o d y i t i s a p p l i e d . T h e m o s t w i d e l y u s e d n o x i o u s s t i m u l i a r e h e a t a p p l i e d t o t h e t a i l o r p a w s ; e l e c t r i c s h o c k a p p l i e d t o t h e t a i l s , f e e t , o r s k i n ; a n d n o x i o u s c h e m i c a l s a p p l i e d s u b c u t a n e o u s l y . B e c a u s e l a b o r a t o r y r a t s h a v e b e e n t h e m o s t common s u b j e c t s o f p a i n r e s e a r c h a n d b e c a u s e t h e y w e r e t h e s u b j e c t s i n e a c h o f t h e e x p e r i m e n t s o f t h i s t h e s i s , t h e m o s t common m e t h o d s o f m e a s u r i n g p a i n p e r c e p t i o n i n r a t s a r e b r i e f l y d e s c r i b e d i n t h i s s e c t i o n . A . The T a i l - F l i c k T e s t . T h e m o s t common b e h a v i o r a l t e s t o f n o c i c e p t i o n u s e d i n a n i m a l r e s e a r c h , a n d t h e t e s t t o b e u s e d i n t h e e x p e r i m e n t s o f t h i s t h e s i s , i s t h e t a i l - f l i c k t e s t o f D ' A m o u r a n d S m i t h (1941; s e e a l s o J a n s s e n , N i e m e g e e r s , & D o r y , 1963) . I n t h i s t e s t , a s o u r c e o f h e a t , u s u a l l y a f o c u s e d l i g h t beam o r h o t 3 w a t e r , i s a p p l i e d t o t h e t a i l o f e a c h r a t , a n d t h e t i m e t h a t i t t a k e s e a c h r a t t o w i t h d r a w i t s t a i l ( i . e . t h e t a i l - f l i c k l a t e n c y ) i s m e a s u r e d . T h e r e l a t i v e p o t e n c y o f a n a l g e s i c d r u g s o~h t h i s m e a s u r e c o r r e l a t e s w e l l w i t h t h e i r r e l a t i v e a n a l g e s i c p o t e n c y i n humans ( D * A m o u r & S m i t h , 1 9 4 1 ; S m i t h , D ' A m o u r , & D 1 A m o u r , 1 9 4 3 ) . F o l l o w i n g t r a n s e c t i o n o f t h e s p i n a l c o r d , t h e t a i l - f l i c k r e s p o n s e r e m a i n s i n t a c t ( I r w i n e t a l . , 1 9 5 1 ) , i n d i c a t i n g t h a t i t i s a s p i n a l l y - o r g a n i z e d r e f l e x . B e c a u s e t h e t a i l c a n b e w i t h d r a w n a f t e r a p r e s e t t i m e l i m i t , t h e r e i s u s u a l l y n o t t i s s u e damage a n d r e p e a t e d t e s t i n g d o e s n o t s i g n i f i c a n t l y a f f e c t t a i l - f l i c k l a t e n c i e s ( F e n n e s s y & L e e , 1 9 7 5 ) . B . The H o t - P l a t e T e s t . A s e c o n d common t e s t o f n o c i c e p t i o n i s t h e h o t - p l a t e t e s t ( W o o l f e & M a c D o n a l d , 1 9 4 4 ) , i n w h i c h t h e s u b j e c t i s p l a c e d o n a h e a t e d s u r f a c e , a n d t h e t i m e t h a t i t t a k e s f o r t h e a n i m a l t o r a i s e a n d l i c k t h e b o t t o m o f a h i n d p a w i s m e a s u r e d . R e p e a t e d t e s t i n g w i t h t h e h o t - p l a t e t e s t i s f r e q u e n t l y c o n f o u n d e d b y t h e s u b j e c t s l e a r n i n g t o j u m p a s s o o n a s t h e y a r e p l a c e d o n t h e s u r f a c e , a n d b y damage t o t h e paws ( F e n n e s s y & L e e , 1 9 7 5 ) . C . T h e F l i n c h - J u m p T e s t . I n t h e f l i n c h - j u m p t e s t o f n o c i c e p t i o n ( E v a n s , 1 9 6 1 ) , a n a n i m a l i s p l a c e d o n a g r i d t h r o u g h w h i c h d i s c r e t e s h o c k s o f v a r i o u s i n t e n s i t i e s a r e a d m i n i s t e r e d . F i r s t , t h e m i n i m a l l e v e l o f c u r r e n t i n t e n s i t y n e c e s s a r y t o e l i c i t a f l i n c h i s d e t e r m i n e d , f o l l o w e d b y d e t e r m i n a t i o n o f t h e m i n i m a l l e v e l o f c u r r e n t n e c e s s a r y t o e l i c i t a j u m p . T h e s e f l i n c h a n d j u m p t h r e s h o l d s a r e u s u a l l y d e t e r m i n e d u s i n g r e p e a t e d s t e p w i s e 4 i n c r e a s e s , a n d t h e n d e c r e a s e s , i n s h o c k i n t e n s i t y . T h e e n t i r e p r o c e d u r e t y p i c a l l y r e q u i r e s a b o u t 90 m i n t o c o m p l e t e . T h u s , t h i s m e t h o d i s i m p r a c t i c a l f o r u s e i n t i m e - c o u r s e s t u d i e s o r w i t h s h o r t - a c t i n g a n a l g e s i c s . T h e f l i n c h a n d j u m p r e s p o n s e s a r e a s s u m e d t o r e f l e c t s p i n a l l y - a n d c o r t i c a l l y - o r g a n i z e d r e s p o n s e s , r e s p e c t i v e l y . H o w e v e r , t h e r e h a s b e e n no s y s t e m a t i c t e s t o f t h e s e a s s u m p t i o n s . D . F o r m a l i n W r i t h i n g T e s t . T h e i n j e c t i o n o f f o r m a l i n i n t o a r a t paw p r o d u c e s a s e r i e s o f c h a r a c t e r i s t i c r e s p o n s e s t h a t a r e t y p i c a l l y e l i c i t e d b y p a i n f u l s t i m u l i . T h e t i m e t h a t a r a t e n g a g e s i n e a c h o f t h e s e b e h a v i o r s c a n be r e c o r d e d a n d u s e d a s a m e a s u r e o f t h e s e v e r i t y o f t h e p a i n . M e l z a c k a n d h i s c o l l e a g u e s ( e . g . D e n n i s & M e l z a c k , 1 9 7 9 ) h a v e a r g u e d t h a t t h i s t e s t i s a m o d e l o f c h r o n i c p a i n , a s o p p o s e d t o t h e s h a r p , p h a s i c p a i n i n v o l v e d i n o t h e r a n i m a l t e s t s . O p i a t e d r u g s i n d u c e a n a l g e s i a a t a much l o w e r d o s e i n t h e f o r m a l i n t e s t t h a n i n t h e t a i l - f l i c k o r h o t - p l a t e t e s t s ( A b b o t t , M e l z a c k & L e b e r , 1 9 8 2 ) . H o w e v e r , f o r m a l i n d o e s p r o d u c e t i s s u e damage a t t h e i n j e c t i o n s i t e , a n d r e p e a t e d t e s t i n g i s t h u s i m p r a c t i c a l . E . N o x i o u s S t i m u l i U s e d i n E l e c t r o p h y s i o l o g i c a l R e c o r d i n g . I t i s o f t e n u s e f u l t o r e c o r d t h e r e s p o n s e s o f s i n g l e n e u r o n s t o n o x i o u s s t i m u l i . V a r i o u s s t i m u l i t h a t a r e p r e s u m e d t o b e p a i n f u l a r e u s e d t o i d e n t i f y n e u r o n s t h a t m e d i a t e p a i n p e r c e p t i o n . T h e s e s t i m u l i i n c l u d e a p i n c h o f t h e s k i n , a n e l e c t r i c s h o c k , a weak a c i d a p p l i e d t o t h e s k i n , o r a f o c u s e d l i g h t beam a p p l i e d t o t h e s k i n . N e u r o n s t h a t r e s p o n d t o t h e s e s t i m u l i a r e c o n s i d e r e d t o be 5 "nociceptive" neurons. The various procedures for measuring pain i n laboratory animals are d i f f e r e n t i a l l y s e n s i t i v e to experimental ~ manipulations. Of p a r t i c u l a r relevance to the present experiments i s the finding of Dennis and Melzack (1979, 1980) that the e f f e c t s of serotonergic drugs on morphine-produced analgesia are dependent upon the measure of analgesia. Thus, the adoption of the t a i l - f l i c k test as the basic procedure i n each of the present experiments requires j u s t i f i c a t i o n . The following are the reasons why i t rather than the numerous available a l t e r n a t i v e s , was employed. Several considerations led to the s e l e c t i o n of the t a i l -f l i c k test as the most appropriate model of nociception. F i r s t , the t a i l - f l i c k test has been the most commonly used method of measuring pain i n laboratory animals. This was an important consideration because a primary motivation behind t h i s series of experiments was to determine whether d i f f e r e n t i a l involvement of SI and S2 receptors i n morphine-produced analgesia could account for many of the inconsistent r e s u l t s reported using t h i s procedure. A second reason for the selection of the t a i l - f l i c k test was that the designs of the experiments incorporated repeated t e s t i n g . Consequently, tests that are greatly influenced by repeated testing were unsuitable. Rats subjected to repeated hot-plate t e s t i n g learn to avoid the noxious heat stimulus by jumping as soon as they are placed i n the apparatus, and formalin i n j e c t i o n for the writhing test causes severe tissue 6 d a m a g e , m a k i n g t h e s e t w o common t e s t s i m p r a c t i c a l . I n c o n t r a s t , t h e r e i s no s y s t e m a t i c c h a n g e i n t a i l - f l i c k l a t e n c y w i t h r e p e a t e d d a i l y t e s t i n g , o r e v e n w i t h r e p e a t e d t e s t i n g w i t h i n a s e s s i o n ( D 1 A m o u r & S m i t h , 1 9 4 1 ) . A l l o f t h e m e t h o d s o f m e a s u r i n g p a i n p e r c e p t i o n i n a n i m a l s d e s c r i b e d i n t h i s i n t r o d u c t i o n a r e p r e d i c t i v e m o d e l s o f a n a l g e s i a . T h a t i s , t h e e f f e c t i v e n e s s o f v a r i o u s d r u g s i n a l t e r i n g t h e m e a s u r e i s c o r r e l a t e d w i t h t h e d r u g s ' p o t e n c i e s i n r e l i e v i n g human p a i n . B u t p a i n i s n o t a u n i t a r y p h e n o m e n o n . I n h u m a n s , t h e s u b j e c t i v e f e e l i n g o f p a i n seems t o v a r y w i t h d i f f e r e n t n o x i o u s s t i m u l i . I n t h e a n i m a l l i t e r a t u r e , t y p e s o f p a i n h a v e b e e n d i s t i n g u i s h e d a l o n g a t e m p o r a l d i m e n s i o n ( e . g . D e n n i s & M e l z a c k , 1 9 7 9 ) . D i f f e r e n t t y p e s o f p a i n may be m e d i a t e d b y d i f f e r e n t n e u r a l s u b s t r a t e s ( A b b o t t , G r i m e s , & M e l z a c k , 1 9 8 4 ; C o d e r r e , A b b o t t , & M e l z a c k ; D e n n i s & M e l z a c k , 1 9 7 9 ) . When we e m p l o y t h e t a i l - f l i c k t e s t , w h a t t y p e o f p a i n a r e we m e a s u r i n g ? W h i c h n e u r a l s u b s t r a t e s a r e we a c t i v a t i n g ? How d o e s t h i s m o d e l r e l a t e t o t y p e s o f human p a i n ? B e c a u s e o u r k n o w l e d g e o f t h e n e u r a l s u b s t r a t e o f p a i n a n d a n a l g e s i a i s i n c o m p l e t e , i t i s n o t p o s s i b l e t o t e l l w h e t h e r t h e s e m o d e l s a r e s u b s e r v e d b y n e u r a l s y s t e m s t h a t a r e a n a l a g o u s t o s y s t e m s t h a t m e d i a t e human p a i n a n d a n a l g e s i a . T h e v a l u e o f t h e t a i l - f l i c k t e s t , o r a n y t e s t o f a n a l g e s i a , a s a m o d e l o f p a i n p e r c e p t i o n may b e v e r i f i e d o n l y when t h e c i r c u i t r y i n v o l v e d i s m o r e c o m p l e t e l y u n d e r s t o o d . 7 2. Mechanisms of Morphine A n a l g e s i a The o b s e r v a t i o n t h a t s p i n a l t r a n s e c t i o n o n l y p a r t i a l l y b l o c k s morphine-produced a n a l g e s i a ( I r w i n et a l . , 1951;~Takagi, Matsumura, Yanai, & Ogiu, 1955) has l e d many to p o s t u l a t e t h a t morphine produces a n a l g e s i a through two d i f f e r e n t systems. One system mediating morphine-produced a n a l g e s i a i s assumed to o r i g i n a t e i n the b r a i n and to i n h i b i t the incoming p a i n messages v i a connections between the b r a i n and the s p i n a l cord segment at which the messages en t e r the c e n t r a l nervous system. A second morphine-analgesia system i s assumed to e x i s t e n t i r e l y w i t h i n the s p i n a l c o r d . A c c o r d i n g to t h i s t h e o r y , s p i n a l t r a n s e c t i o n only p a r t i a l l y b l o c ks morphine-produced a n a l g e s i a because i t d i s c o n n e c t s the f i r s t mechanism, but not the second. The f o l l o w i n g two s e c t i o n s summarize the evidence f o r the e x i s t e n c e of these p u t a t i v e a n a l g e s i a mechanisms. A. B r a i n s t e m - t o - s p i n a l c o r d a n a l g e s i a system. An important step i n determining where morphine a c t s to produce a n a l g e s i a was the i s o l a t i o n and l o c a l i z a t i o n of the nervous system's o p i o i d r e c e p t o r s . E a r l y i n v i t r o b i n d i n g s t u d i e s performed on b r a i n t i s s u e homogenates found marked d i f f e r e n c e s i n the number of o p i o i d b i n d i n g s i t e s i n v a r i o u s r e g i o n s of r a t and monkey nervous systems (Kuhar, P e r t , & Snyder, 1973; H i l l a r , Pearson, & Simon, 1973; P e r t & Snyder, 1973). Some g e n e r a l a r e a s , such as the medulla and s p i n a l c o r d were found t o have few b i n d i n g s i t e s , whereas high c o n c e n t r a t i o n s of b i n d i n g s i t e s were found i n v a r i o u s l i m b i c s t r u c t u r e s and i n the s t r i a t u m . The main 8 shortcoming of these i n v i t r o binding studies was that because they were based on the analysis of homogenized gross structures dissected from the brain, t h e i r powers of s p a t i a l resolution were low. These studies thus, provided only a very general i n d i c a t i o n of the location of opioid receptors i n the brain. The technique of autoradiography (Atweh & Kuhar, 1977a,b,c; Pert, Kuhar, & Snyder, 1975; 1976), proved to be a much more e f f e c t i v e method of determining the d i s t r i b u t i o n of opioid binding s i t e s i n the central nervous system. Rat brain and spinal cord s l i c e s were incubated in solutions of r a d i o a c t i v e l y l a b e l l e d drugs that bind to opioid receptors. Subsequent autoradiographs revealed high l e v e l s of r a d i o a c t i v i t y i n the periaqueductal gray (PAG), interpeduncular nucleus, i n f e r i o r c o l l i c u l u s , median raphe, amygdala, nucleus accumbens, area postrema, several diencephalic n u c l e i , and the dorsal horn of the spinal gray matter (Goodman, Snyder, Kuhar, & Young, 1980; see Atweh & Kuhar, 1983 for a review). These then are the l i k e l y s i t e s of opioid receptors i n the central nervous system. Next, to e s t a b l i s h unequivocally that these binding s i t e s i d e n t i f i e d by autoradiography r e f l e c t the presence of opioid receptors, several investigators showed that functional changes are associated with the binding of the drug to the s i t e . Generally speaking, i n j e c t i o n s of opiate drugs d i r e c t l y into these receptor-rich areas have been found to produce subsets of the e f f e c t s of peripheral opiate i n j e c t i o n s . For example, morphine injected d i r e c t l y into the striatum was found to produce 9 t h e m o t o r r i g i d i t y c h a r a c t e r i s t i c o f p e r i p h e r a l m o r p h i n e a d m i n i s t r a t i o n ; w h e r e a s , i n j e c t i o n o f m o r p h i n e i n t o t h e a r e a p o s t r e m a was f o u n d t o p r o d u c e n a u s e a a n d v o m i t i n g ( A t w e h & K u h a r , 1 9 8 3 ) . O f d i r e c t r e l e v a n c e t o t h i s d i s s e r t a t i o n a r e t h o s e s t u d i e s o f o p i o i d r e c e p t o r f u n c t i o n t h a t f o c u s e d o n a n a l g e s i a . S m a l l a m o u n t s o f m o r p h i n e o r e n d o g e n o u s o p i o i d s i n j e c t e d d i r e c t l y i n t o v a r i o u s r e g i o n s o f t h e PAG o f r a t s , c a t s , a n d m o n k e y s h a v e b e e n s h o w n t o p r o d u c e s t r o n g a n a l g e s i c e f f e c t s ( F o s t e r , J e n d e n , & L o m a x , 1 9 6 7 ; M a l i c k & G o l d s t e i n , 1 9 7 7 ; P e r t & Y a k s h , 1 9 7 5 ; T s u o & J a n g , 1 9 6 4 ) . The f a c t t h a t t h e a n a l g e s i a p r o d u c e d b y o p i a t e i n j e c t i o n s i n t o t h e PAG was r e v e r s e d b y t h e o p i a t e r e c e p t o r b l o c k e r , n a l o x o n e p r o v i d e d f u r t h e r e v i d e n c e t h a t t h e a n a l g e s i a was m e d i a t e d b y t h e a c t i o n o f t h e o p i a t e s o n o p i o i d r e c e p t o r s . M o r e o v e r , n a l o x o n e i n j e c t e d i n t o t h e PAG was f o u n d t o r e v e r s e t h e a n a l g e s i c e f f e c t o f m o r p h i n e when i t was i n j e c t e d s y s t e m i c a l l y ( Y e u n g & R u d y , 1 9 8 0 a ) . M o r e r e c e n t l y , i t h a s b e e n s h o w n t h a t a n a l g e s i a c a n a l s o be p r o d u c e d b y i n j e c t i n g m o r p h i n e i n t o e i t h e r t h e n u c l e u s r a p h e m a g n u s , t h e n u c l e u s r e t i c u l a r i s p a r a g i g a n t o -c e l l u l a r i s , o r t h e n u c l e u s r e t i c u l a r i s p a r a g i g a n t o c e l l u l a r i s l a t e r a l i s o f t h e r o s t r a l v e n t r a l m e d u l l a ( A k a i k e , S h i b a t a , S a t o h , & T a k a g i , 1 9 7 8 ; A z a m i , L l e w e l y n , & R o b e r t s , 1 9 8 2 ; D i c k e n s o n , O l i v e r a s , & B e s s o n , 1 9 7 9 ) . I t t h u s a p p e a r s t h a t t h e PAG i s n o t t h e o n l y b r a i n s t e m n u c l e u s w i t h m o r p h i n e r e c e p t o r s i n v o l v e d i n t h e m e d i a t i o n o f a n a l g e s i a ( A t w e h & K u h a r , 1 9 8 3 ; B a s b a u m & F i e l d s , 1 9 8 4 ) ; h o w e v e r , i t i s c o n s i d e r e d t o b e t h e p r i m a r y o n e . 10 It has been hypothesized (Abols & Basbaura, 1981; Behbehani & Pomeroy, 1978; Behbehani, Pomeroy & Mack, 1981; Gallager & Pert, 1978; Mantyh, 1 983) that the contribution of the PAG to" morphine-produced analgesia i s mediated vi a connections between the PAG and the nucleus raphe magnus, commonly referred to as the NRM. Support for t h i s theory comes from several l i n e s of evidence: Proudfit and Anderson (1975) found that lesions of the NRM attenuate analgesia produced by i n j e c t i o n of morphine into the PAG. Pomeroy and Behbehani (1979) found that e l e c t r i c a l stimulation of the PAG produced e x c i t a t i o n of neurons i n the NRM. Behbehani and Pomeroy (1978) found that i n j e c t i o n s of morphine into the PAG a l t e r the f i r i n g rate of NRM neurons. F i n a l l y , Beitz (1982a,b) showed that some of the neurons that project from the PAG to the NRM contain either serotonin or neurotensin. On the basis of t h i s combined evidence, Basbaum and F i e l d s (1978, 1984) suggested that the analgesic e f f e c t of morphine i s mediated by i t s binding to opioid receptors i n the PAG and the subsequent a c t i v a t i o n of the NRM v i a serotonergic and/or neurotensinergic neurons. Studies demonstrating that the analgesia produced by i n j e c t i o n s of morphine into the PAG can be s u b s t a n t i a l l y attenuated by transection of the spinal cord or lesions of the dorsolateral funiculus, a descending t r a c t of the spinal cord, i s strong evidence that a major component of morphine analgesia i s mediated by brain-to-spinal cord connections (Jurna & Grossman, 1976; Kitahata, Yosaka, Taub, Bonikos, & Hoffert, 1974; LeBars, 11 M e n e t r e y , C o n s e i l l e r , & B e s s o n , 1 9 7 5 ; M u r p h i n , B e n n e t t , & M a y e r , 1 9 7 6 ) . A l s o c o n s i s t e n t w i t h t h i s v i e w i s t h e o b s e r v a t i o n t h a t t h e N R M , a s w e l l a s t w o o t h e r n u c l e i o f t h e r a t r o s t r a l " v e n t r a l m e d u l l a , t h e n u c l e u s r e t i c u l a r i s p a r a g i g a n t o c e l l u l a r i s a n d t h e n u c l e u s r e t i c u l a r i s p a r a g i g a n t o c e l l u l a r i s l a t e r a l i s , t h a t r e c e i v e i n p u t f r o m t h e PAG ( B e i t z , 1 9 8 2 a , b ; M a n t y h , 1 9 8 3 ) a l l p r o j e c t t o t h e s p i n a l c o r d v i a t h e d o r s o l a t e r a l f u n i c u l u s i n s e v e r a l d i f f e r e n t s p e c i e s ( B a s b a u m & F i e l d s , 1 9 7 9 ; L e i c h n e t z , W a t k i n s , G r i f f i n , M a r t i n , & M a y e r , 1 9 7 8 ; M a r t i n , J o r d a n , & W i l l i s , 1 9 7 8 ) . E v i d e n c e f o r a s e c o n d , more d i r e c t , b r a i n - t o - s p i n a l c o r d a n a l g e s i a p a t h w a y i s p r o v i d e d b y a r e c e n t d e m o n s t r a t i o n t h a t t h e r e a r e a l s o e x t e n s i v e p r o j e c t i o n s f r o m t h e PAG d i r e c t l y t o t h e s p i n a l c o r d o f r a t s , c a t s , a n d m o n k e y s ( M a n t y h & P e s c h a n s k i , 1 9 8 2 ) . A x o n s d e s c e n d i n g f r o m c e l l b o d i e s i n t h e PAG a n d f r o m t h o s e i n t h e NRM a n d t h e o t h e r r o s t r a l v e n t r a l m e d u l l a n u c l e i t e r m i n a t e i n t h e m a r g i n a l l a y e r a n d s u b s t a n t i a g e l a t i n o s a o f t h e d o r s a l h o r n o f t h e s p i n a l c o r d ( B a s b a u m , C l a n t o n , & F i e l d s , 1 9 7 8 ; M a n t y h & P e s c h a n s k i , 1 9 8 2 ) . T h e s e t w o a r e a s o f t h e d o r s a l h o r n a r e w h e r e t h e a x o n s o f p r i m a r y n o c i c e p t i v e n e u r o n s e n t e r t h e c e n t r a l n e r v o u s s y s t e m a n d s y n a p s e o n a s c e n d i n g s p i n a l n e u r o n s ( s e e W i l l i s , 1985 f o r a r e v i e w ) . T h e r a p i d - c o n d u c t i n g A o n o c i c e p t i v e p r i m a r i e s , w h i c h t e n d t o c a r r y i n f o r m a t i o n a b o u t m e c h a n i c a l p a i n , p r o j e c t t o t h e m a r g i n a l l a y e r o f t h e d o r s a l h o r n ; w h e r e a s , t h e s l o w - c o n d u c t i n g C - p o l y m o d a l f i b e r s s y n a p s e i n t h e s u b s t a n t i a g e l a t i n o s a o f t h e d o r s a l h o r n ( e . g . B e a l & B i c k n e l l , 1 9 8 1 ; 12 R e t h e l y i & Capowski, 1977). T h i s convergence of the descending axons of the a n a l g e s i a c i r c u i t s w i t h the incoming n o c i c e p t i v e f i b e r s i n the marginal l a y e r and s u b s t a n t i a g e l a t i n o s a "of the d o r s a l horn i m p l i e s t h a t i t i s a t these s i t e s t h a t the a n a l g e s i a c i r c u i t s e x e r t t h e i r i n h i b i t o r y e f f e c t s (Basbaum & F i e l d s , 1984; F i e l d s & Basbaum, 1978; Melzack, 1973; Melzack & W a l l , 1965). Supporting t h i s view i s the o b s e r v a t i o n t h a t a c t i v a t i o n of the NRM o r other r o s t r a l v e n t r a l medulla n u c l e i by i n t r a c e r e b r a l i n j e c t i o n o f morphine o r by e l e c t r i c a l s t i m u l a t i o n i n h i b i t s the responses o f d o r s a l horn neurons t h a t have been i d e n t i f i e d to t r a n s m i t n o c i c e p t i v e i n f o r m a t i o n from the s p i n a l c o r d to the b r a i n ( L i e b e s k i n d , Guilbaud, Besson, & O l i v e r a s , 1973; W i l l i s , Haber, & M a r t i n , 1977), but has no e f f e c t on the f i r i n g r a t e s of neurons t h a t are o n l y r e s p o n s i v e to non-noxious t a c t i l e s t i m u l i (see Besson & Le Bars, 1978 f o r a r e v i e w ) . B. S p i n a l Mechanisms of O p i a t e A n a l g e s i a . As p r e v i o u s l y mentioned, t r a n s e c t i o n of the s p i n a l cord s i g n i f i c a n t l y a t t e n u a t e s , but does not e l i m i n a t e morphine a n a l g e s i a i n those areas of the body t h a t are served by s p i n a l c o r d segments t h a t are below the t r a n s e c t i o n ( I r w i n et a l . , 1951; Takagi et a l . , 1955). T h i s o b s e r v a t i o n has l e d to the c o n c l u s i o n (e.g. Soja & S i n c l a i r , 1983) t h a t o p i a t e s may a l s o produce a n a l g e s i a by a c t i n g d i r e c t l y on the o p i o i d r e c e p t o r s of the d o r s a l horn. T h i s theory i s supported by the f a c t t h a t i n t r a t h e c a l a d m i n i s t r a t i o n of morphine, t h a t i s , i n j e c t i o n of morphine i n t o the s p i n a l subarachnoid space, a l s o has s t r o n g a n a l g e s i c e f f e c t s (Yaksh, 13 1 9 8 1 ; Y a k s h & R e d d y , 1 9 8 1 ; Y a k s h & R u d y , 1 9 7 7 ) . O p i o i d r e c e p t o r s h a v e b e e n i d e n t i f i e d o n t h e p r e s y n a p t i c t e r m i n a l s o f p r i m a r y a f f e r e n t a x o n s e n t e r i n g t h e d o r s a l h o r n ( A t w e h & K u h a r , _ 1 9 7 7 a ; F i e l d s , E m s o n , L e i g h , G i l b e r t , & I v e r s e n , 1 9 8 0 ; H i l l e r , S i m o n , C r a i n , & P e t e r s o n , 1 9 7 8 ; L a M o t t e , P e r t , & S n y d e r , 1 9 7 6 ) . M o r e o v e r , i o n t o p h o r e t i c a d m i n i s t r a t i o n o f m o r p h i n e o n t o t h e s e p r e s y n a p t i c o p i o i d r e c e p t o r s h a s b e e n s h o w n t o p r o d u c e c h a n g e s i n e x c i t a b i l i t y o f b o t h A o a n d C p r i m a r y a f f e r e n t s ( B e l c h e r & R y a l l , 1 9 7 8 ; C a l v i l l o , H e n r y , & N e u m a n , 1 9 7 4 ; D o s t r o v s h y & P o m e r a n z , 1 9 7 6 ; D u g g a n , H a l l , & H e d l e y , 1 9 7 7 , D u g g a n , J o h n s o n , & M o r t o n , 1 9 8 1 ; Z i e g l g a n s b e r g e r & B a y e r l , 1 9 7 6 ) . H e n c e , t h e r e i s s u p p o r t f o r a p r e s y n a p t i c i n h i b i t o r y r o l e f o r o p i o i d s i n t h e s p i n a l c o r d . A n a l t e r n a t i v e t o t h i s " p r e s y n a p t i c " i n t e r p r e t a t i o n i s t h a t d i r e c t p o s t s y n a p t i c i n h i b i t i o n o f n o c i c e p t i v e t r a n s m i s s i o n b y m o r p h i n e c a n p r o d u c e a n a l g e s i a b y b l o c k i n g t h e t r a n s m i s s i o n o f n o c i c e p t i v e i n p u t t o t h e s p i n o t h a l a m i c t r a c t ( B a s b a u m & F i e l d s , 1 9 8 4 ) . C o n s i s t e n t w i t h t h i s v i e w i s t h e d e m o n s t r a t i o n o f R u d a ( 1 9 8 0 ) t h a t n e u r o n s t h a t c o n t a i n e n d o g e n o u s o p i o i d p e n t a p e p t i d e s c o n t a c t s p i n o t h a l a m i c t r a c t n e u r o n s . A c c o r d i n g l y , m o r p h i n e may a c t w i t h i n t h e d o r s a l h o r n o f t h e s p i n a l c o r d i n o n e o f t w o w a y s , o r p e r h a p s i n b o t h , b y p r e s y n a p t i c i n h i b i t i o n o f t h e n o c i c e p t i v e p r i m a r i e s o r b y d i r e c t p o s t s y n a p t i c i n h i b i t i o n o f a s c e n d i n g s e n s o r y n e u r o n s . 1 4 3. T h e I n v o l v e m e n t o f S e r o t o n i n i n M o r p h i n e A n a l g e s i a O v e r t h e p a s t 20 y e a r s t h e r e h a s b e e n g r o w i n g e v i d e n c e t h a t t h e n e u r o t r a n s m i t t e r s e r o t o n i n p l a y s a n i m p o r t a n t r o l e " i n t h e m e d i a t i o n o f m o r p h i n e a n a l g e s i a ( s e e M e s s i n g & L y t l e , 1977 f o r a r e v i e w ) . T h e f i r s t t w o p a r t s o f t h i s s e c t i o n w i l l r e v i e w t h e e f f e c t s o n m o r p h i n e - p r o d u c e d a n a l g e s i a o f s e r o t o n i n a n t a g o n i s t s a n d a g o n i s t s , r e s p e c t i v e l y . T h e t h i r d p a r t r e v i e w s d a t a r e l e v a n t t o t h e q u e s t i o n o f w h e t h e r t h e c r i t i c a l s e r o t o n i n r e c e p t o r s a r e l o c a t e d i n t h e s p i n a l c o r d o r i n t h e b r a i n . A . A n t a g o n i s m o f S e r o t o n e r g i c A c t i v i t y a n d M o r p h i n e  A n a l g e s i a . T h e i d e a t h a t s e r o t o n i n i s a c r i t i c a l n e u r o t r a n s m i t t e r i n t h e m e d i a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a i s p r i m a r i l y b a s e d o n e v i d e n c e t h a t m o r p h i n e - p r o d u c e d a n a l g e s i a i s a t t e n u a t e d b y m a n i p u l a t i o n s t h a t d e c r e a s e t h e a c t i o n o f s e r o t o n i n . T e n n e n (1968) was t h e f i r s t t o p r o p o s e a r o l e f o r s e r o t o n i n i n m o r p h i n e - p r o d u c e d a n a l g e s i a . He b a s e d h i s h y p o t h e s i s o n h i s f i n d i n g s t h a t b l o c k a d e o f s e r o t o n i n b i o s y n t h e s i s w i t h p a r a - c h l o r o p h e n y l a l a n i n e a t t e n u a t e d t h e a n a l g e s i c e f f e c t o f m o r p h i n e a n d t h a t t h e b l o c k a d e was r e v e r s e d b y r e s t o r a t i o n o f s e r o t o n i n l e v e l s w i t h i n j e c t i o n s o f 5-h y d r o x y t r y p t o p h a n , a s e r o t o n i n p r e c u r s o r . A l t h o u g h t h e s e f i n d i n g s h a v e b e e n f r e q u e n t l y r e p l i c a t e d ( B e r g e , H o l e , & O g r e n , 1983; F e n n e s s y & L e e , 1970; G o r l i t z & F r e y , 1972; R e i g l e & B a r k e r , 1983; T i l s o n & R e c h , 1974; T u l u n a y , Y a n o , & T a k e m o r i , 1976; V o g t , 1974) , some i n v e s t i g a t o r s h a v e n o t o b s e r v e d t h e u s u a l i n h i b i t o r y e f f e c t o f p a r a - c h l o r o p h e n y l a l a n i n e o n m o r p h i n e 1 5 a n a l g e s i a ( B u x b a u m , Y a r b o r o u g h , & C a r t e r , 1 9 7 3 ; F e n n e s s y & L e e , 1 9 8 0 ; H a r v e y , S c h l o s b e r g , & Y u n g e r , 1 9 7 4 ; S u g r u e , 1 9 7 9 ) . T h i s i n c o n s i s t e n c y h a s r e c e i v e d c o n s i d e r a b l e f o r m a l d i s c u s s i o n ( B e r g e e t a l . , 1 9 8 3 ; F e n n e s y & L e e , 1 9 7 0 ; T i l s o n & R e c h , 1 9 7 4 ) , b u t i t h a s y e t t o be c o n v i n c i n g l y r e s o l v e d . T h e s e l e c t i v e d e s t r u c t i o n o f s e r o t o n i n - c o n t a i n i n g n e u r o n s h a s a l s o b e e n f o u n d t o a t t e n u a t e t h e a n a l g e s i c e f f e c t o f m o r p h i n e . F o r e x a m p l e , t h e a m p h e t a m i n e d e r i v a t i v e s p a r a -c h l o r o a m p h e t a m i n e a n d f e n f l u r a m i n e h a v e b o t h b e e n s h o w n t o p r o d u c e a s e v e r e a n d p e r m a n e n t r e d u c t i o n i n b r a i n s e r o t o n i n b y d e s t r o y i n g s e r o t o n e r g i c n e u r o n s . R a t s e x p o s e d t o e i t h e r o f t h e s e d r u g s f o r a s u f f i c i e n t l y l o n g t i m e t o d e s t r o y s e r o t o n e r g i c n e u r o n s no l o n g e r e x h i b i t m o r p h i n e - p r o d u c e d a n a l g e s i a ( B e r g e e t a l . , 1 9 8 3 ; D u n c a n & S p e n c e r , 1 9 7 3 ; S u g r u e , 1 9 7 9 ; T a k e m o r i , T u l u n a y , & Y a n o , 1 9 7 5 ; T u l u n a y e t a l . , 1 9 7 6 ) A n o t h e r s e l e c t i v e s e r o t o n e r g i c n e u r o t o x i n , 5 , 6 - d i h y d r o x y t r y p t a m i n e h a s a l s o b e e n s h o w n t o a t t e n u a t e m o r p h i n e - p r o d u c e d a n a l g e s i a ( V o g t , 1 9 7 4 ) . B e c a u s e 5 , 6 - d i h y d r o x y t r y p t a m i n e c a u s e s a l o n g - l a s t i n g l o s s o f s e r o t o n i n f r o m t h e s p i n a l c o r d b u t h a s o n l y a t e m p o r a r y e f f e c t o n b r a i n s e r o t o n i n ( B a u m g a r t e n , E v e t t s , H o l m a n , I v e r s e n , V o g t , & W i l s o n , 1 9 7 2 ) , t h i s f i n d i n g s u g g e s t s t h a t s e r o t o n e r g i c n e u r o n s i n t h e s p i n a l c o r d m e d i a t e m o r p h i n e - p r o d u c e d a n a l g e s i a . S u g r u e ( 1 9 7 9 ) r e p o r t e d t h a t t h e s i m i l a r n e u r o t o x i n 5 , 7 -d i h y d r o x y t r y p t a m i n e p r o d u c e d a 60% r e d u c t i o n i n b r a i n s e r o t o n i n w i t h o u t s i g n i f i c a n t l y d i s r u p t i n g m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e s u p p o r t f o r t h e h y p o t h e s i s t h a t s e r o t o n i n m e d i a t e s 1 6 m o r p h i n e - p r o d u c e d a n a l g e s i a t h a t h a s come f r o m t h e u s e o f s e r o t o n i n r e c e p t o r b l o c k e r s h a s b e e n i n c o n s i s t e n t . C y p r o h e p t i d e n e , a s e r o t o n i n a n t a g o n i s t , w h i c h i s a l s o sr p o t e n t b l o c k e r o f h i s t a m i n e r e c e p t o r s ( S t o n e , W e n g e r , L u d d e n , S t a v o r s k i , & R o s s , 1 9 6 1 ) , h a s b e e n s h o w n t o b l o c k t h e a n a l g e s i a p r o d u c e d b y 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 m o r p h i n e i n m i c e ( G o r l i t z & F r e y , 1 9 7 2 ) ; w h e r e a s , t h e more s e l e c t i v e s e r o t o n i n r e c e p t o r b l o c k e r s m e t h y s e r g i d e , m i a n s e r i n a n d m e t e r g o l i n e h a v e n o t ( B e r g e , F a s m e r , & K j e l l , 1 9 8 3 ; F e n n e s s y & L e e , 1 9 7 0 ) . H o w e v e r , t h e s e s e l e c t i v e s e r o t o n i n r e c e p t o r b l o c k e r s h a v e b e e n f o u n d t o a t t e n u a t e t h e a n a l g e s i a p r o d u c e d b y i n j e c t i o n s o f m o r p h i n e i n t o t h e PAG o f t h e r a t ( Y a k s h , D u C h a t e a u , & R u d y , 1 9 7 6 ) , o r NRM ( D i c k e n s o n e t a l . , 1 9 7 2 ) . I n s u m m a r y , p r o c e d u r e s t h a t d e p l e t e s e r o t o n i n s t o r e s , d e s t r o y s e r o t o n i n n e u r o n s , o r b l o c k s e r o t o n i n r e c e p t o r s h a v e b e e n f o u n d t o a t t e n u a t e m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e r e h a v e , h o w e v e r , b e e n n o t a b l e e x c e p t i o n s t o t h i s g e n e r a l r u l e . B . S e r o t o n i n A g o n i s t s a n d A n a l g e s i a . T h e o b s e r v a t i o n t h a t a n t a g o n i s m o f s e r o t o n e r g i c a c t i v i t y t e n d s t o r e d u c e t h e a n a l g e s i c e f f e c t o f m o r p h i n e h a s l e d many t o a s s e s s t h e a n a l g e s i c e f f e c t s o f s e r o t o n i n a g o n i s t s . F o r e x a m p l e , q u i p a z i n e a n d 5 - m e t h o x y - N , N - d i a m e t h y l t r y p t a m i n e , w h i c h a c t a s s e r o t o n i n a g o n i s t s b y s t i m u l a t i n g c e n t r a l n e r v o u s s y s t e m s e r o t o n i n r e c e p t o r s , w e r e f o u n d t o h a v e a n a l g e s i c p r o p e r t i e s i n r a t s ( B e r g e , H o l e , & D a h l e , 1 9 8 0 ; S a m a n i n , B e r n a s c o n i , & Q u a t t r o n e , 1 9 7 6 ) . Q u i p a z i n e -p r o d u c e d a n a l g e s i a i s r e v e r s e d b y m e t e r g o l i n e , a s e r o t o n i n 1 7 r e c e p t o r b l o c k e r ( S a m a n i n e t a l . , 1 9 7 6 ) . H o w e v e r , t h e a n a l g e s i c e f f e c t o f 5 - m e t h o x y - N , N - d i a m e t h y l t r y p t a m i n e i s r e v e r s e d b y n o r a d r e n a l i n d e p l e t i o n , b u t n o t b y s e r o t o n i n d e p l e t i o n , -s u g g e s t i n g t h a t t h i s d r u g p r o d u c e s a n a l g e s i a v i a a l p h a - a d r e n e r g i c r e c e p t o r s ( A r c h e r , M i n o r , & P o s t , 1 9 8 5 ) . A n o t h e r s e r o t o n i n a g o n i s t , f l u o x e t i n e , w h i c h f a c i l i t a t e s t r a n s m i s s i o n a t s e r o t o n e r g i c s y n a p s e s b y i n h i b i t i n g u p t a k e o f s e r o t o n i n f r o m t h e s y n a p s e , a l s o h a s b e e n s h o w n t o p r o d u c e a n a l g e s i a b y i t s e l f ( M e s s i n g , F i s h e r , P h e b u s , & L y t l e , 1 9 7 6 ; M e s s i n g , P h e b u s , F i s h e r , & L y t l e , 1 9 7 5 ) a n d t o f a c i l i t a t e t h e a n a l g e s i a p r o d u c e d b y m o r p h i n e ( L a r s o n & T a k e m o r i , 1 9 7 7 ) . T h e d e m o n s t r a t i o n t h a t s e r o t o n i n a g o n i s t s p r o d u c e a n a l g e s i a i s n o t b y i t s e l f s t r o n g e v i d e n c e f o r t h e s e r o t o n e r g i c m e d i a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a . S e r o t o n i n c o u l d b e i n v o l v e d i n a n i n d e p e n d e n t , n o n - o p i o i d a n a l g e s i a s y s t e m w i t h o u t b e i n g a c t i v e i n t h e c i r c u i t r y t h a t i s a c t i v a t e d b y m o r p h i n e . S t r o n g e r e v i d e n c e f o r t h e i n v o l v e m e n t o f s e r o t o n i n i n m o r p h i n e - p r o d u c e d a n a l g e s i a i s p r o v i d e d b y t h e f i n d i n g t h a t t h e s e r o t o n i n p r e c u r s o r 5 - h y d r o x y t r y p t o p h a n p o t e n t i a t e s m o r p h i n e - p r o d u c e d a n a l g e s i a w i t h o u t h a v i n g a n y a n a l g e s i c a c t i o n o f i t s own ( G a r d i n e r & E b e r h a r t , 1 9 7 0 ; T a k a g i , T a k a s h i m a , & K i m u r a , 1 9 6 4 ; T u l u n a y e t a l . , 1 9 7 6 ) . C . A n a t o m i c a l L o c a l i z a t i o n o f S e r o t o n i n R e c e p t o r s t h a t a r e  T h o u g h t t o M e d i a t e M o r p h i n e - P r o d u c e d A n a l g e s i a . B e c a u s e t h e NRM i s p a r t o f t h e n e u r a l c i r c u i t m e d i a t i n g t h e a n a l g e s i c e f f e c t o f m o r p h i n e a n d i s a l s o t h e s o u r c e o f s e r o t o n e r g i c n e u r o n s t h a t 1 8 p r o j e c t to the s p i n a l cord (Basbaum et a l . , 1976; Bowker, Westlund, & C o u l t e r , 1981; Dahlstrom & Fuxe, 1965), F i e l d s & Basbaum (1978) proposed t h a t s e r o t o n i n ' s r o l e i n morphine-produced a n a l g e s i a i s mediated by the s e r o t o n i n r e c e p t o r s on the d o r s a l horn neurons t h a t r e c e i v e i n p u t from axons descending from the NRM. Others have claimed (Berge et a l . , 1983; Genovese, Zonta, & Mantegazza, 1973; Roberts, 1984) t h a t i t i s s e r o t o n e r g i c synapses i n the b r a i n t h a t are important f o r morphine-produced a n a l g e s i a . Two main s t r a t e g i e s have been.used to l o c a l i z e the s e r o t o n i n r e c e p t o r s mediating the a n a l g e s i c e f f e c t s of morphine: (1) the comparison of i n t r a t h e c a l and i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n s of s e r o t o n i n a g o n i s t s and a n t a g o n i s t s , and (2) the s e l e c t i v e d e p l e t i o n of s p i n a l c o r d or brainstem s e r o t o n i n . I n t r a t h e c a l i n j e c t i o n s of n e i t h e r 5,6-dihydroxytryptamine or 5,7-dihydroxytryptamine, which have been found to s e l e c t i v e l y d e s t r o y s p i n a l cord s e r o t o n i n - c o n t a i n i n g neurons without a f f e c t i n g f o r e b r a i n neurons, have been shown to block the a n a l g e s i c e f f e c t of morphine (Deakin & Dostrovsky, 1978; K u r a i s h i , Harada, A r a t a n i , Satoh, & T a k a g i , 1983). In c o n t r a s t , i n j e c t i o n s of 5,7-dihydroxytryptamine i n t o the d o r s a l raphe n u c l e i of r a t s a p p a r e n t l y has no e f f e c t on a n a l g e s i a (Deakin & Dostrovsky, 1978). Taken t o g e t h e r , these r e s u l t s are commonly used as evidence f o r the r o l e of s p i n a l c o r d s e r o t o n i n i n the m e d i a t i o n of morphine-produced a n a l g e s i a . However, Romandi, E s p o s i t o , and Samanin (1985) r e p o r t t h a t 5,7-dihydroxytryptamine i n j e c t i o n s i n t o n e i t h e r the v e n t r o m e d i a l tegmentum, which d e p l e t e 1 9 f o r e b r a i n s e r o t o n i n , n o r t h e v e n t r a l r a p h e , w h i c h d e p l e t e s p i n a l s e r o t o n i n , a f f e c t m o r p h i n e - p r o d u c e d a n a l g e s i a . M o r e o v e r , some i n v e s t i g a t o r s h a v e f a i l e d t o c o n f i r m t h a t l e s i o n s o f s p i n a l c o r d s e r o t o n e r g i c n e u r o n s b y i n t r a t h e c a l i n j e c t i o n o f 5 , 6 -d i h y d r o x y t r y p t a m i n e r e s u l t i n a b l o c k a d e o f m o r p h i n e - p r o d u c e d a n a l g e s i a ( K u r a i s h i e t a l . , 1 9 8 3 ; P r o u d f i t & Y a k s h , 1 9 8 0 ) , a n d i n j e c t i o n s o f 5 , 6 - d i h y d r o x y t r y p t a m i n e i n t o t h e c e r e b r a l v e n t r i c l e s a l s o h a v e b e e n s h o w n t o b l o c k m o r p h i n e - p r o d u c e d a n a l g e s i a ( G e n o v e s e e t a l . , 1 9 7 3 ) . B e r g e e t a l . ( 1 9 8 3 ) h a v e p r o p o s e d t h a t many c o n f l i c t i n g r e s u l t s o b t a i n e d w i t h v a r i o u s m e t h o d s o f s e r o t o n i n d e p l e t i o n may be a c c o u n t e d f o r b y c o n s i d e r i n g t h a t p a r a - c h l o r o p h e n y l a l a n i n e t r e a t m e n t p r o d u c e s a d e p l e t i o n o f b o t h a s c e n d i n g a n d d e s c e n d i n g s e r o t o n i n p a t h w a y s , w h e r e a s p a r a - c h l o r o a m p h e t a m i n e t r e a t m e n t p r e f e r e n t i a l l y d e s t r o y s c e r e b r a l s e r o t o n i n n e r v e t e r m i n a l s ( K o h l e r , R o s s , S r e b r o , & O g r e n , 1 9 7 8 ; O g r e n e t a l . , 1 9 8 1 ) , l e a v i n g s p i n a l n e r v e t e r m i n a l s i n t a c t . T h e y r e p o r t e d t h a t p r e t r e a t m e n t w i t h e i t h e r p a r a - c h l o r o p h e n y l a l a n i n e o r p a r a -c h l o r o a m p h e t a m i n e b l o c k s m o r p h i n e - p r o d u c e d a n a l g e s i a , w h i c h w o u l d i m p l i c a t e f o r e b r a i n s e r o t o n i n . O g r e n e t a l . ( 1 9 8 1 ) a l s o r e p o r t e d t h a t t h e s e r o t o n i n u p t a k e i n h i b i t o r , z i m e l e d i n e i n j e c t e d b e f o r e p a r a - c h l o r o a m p h e t a m i n e p r e f e r e n t i a l l y p r o t e c t s a g a i n s t t h e n e u r o t o x i c e f f e c t s i n f o r e b r a i n t e r m i n a l s , b u t h a s o n l y a weak p r o p h y l a c t i c e f f e c t o n b r a i n s t e m t e r m i n a l s . T h u s , t r e a t m e n t w i t h z i m e l i d i n e a n d p a r a - c h l o r o a m p h e t a m i n e p r o d u c e s a r a t w i t h a s e l e c t i v e b r a i n s t e m s e r o t o n i n l e s i o n . B e r g e e t a l . ( 1 9 8 3 ) 20 r e p o r t e d t h a t s u c h l e s i o n s a l s o b l o c k m o r p h i n e - p r o d u c e d a n a l g e s i a , a n d t h e y t h u s c o n c l u d e d t h a t b r a i n s t e m s e r o t o n e r g i c s y n a p s e s c o n t r i b u t e t o m o r p h i n e - p r o d u c e d a n a l g e s i a . ~ L l e w e l y n , A z a m i , a n d R o b e r t s ( 1 9 8 3 ) p r o v i d e d f u r t h e r e v i d e n c e t h a t b r a i n s t e m s e r o t o n e r g i c r e c e p t o r s c o n t r i b u t e t o n o c i c e p t i o n a n d a n a l g e s i a b y d e m o n s t r a t i n g t h a t s e r o t o n i n i n j e c t e d i n t o t h e NRM p r o d u c e s a n a l g e s i a . F u r t h e r m o r e , t h e s e r o t o n i n u p t a k e b l o c k e r z i m e l i d i n e o r t h e s e r o t o n i n r e l e a s i n g a g e n t , f e n f l u r a m i n e , b o t h p r o d u c e p o t e n t a n a l g e s i a w h e n i n j e c t e d i n t o t h e NRM ( L l e w e l y n , A z a m i , & R o b e r t s , 1 9 8 4 ) . Y a k s h ( 1 9 7 9 ) a n d Y a k s h e t a l . ( 1 9 7 6 ) f o u n d t h a t i n t r a t h e c a l a d m i n i s t r a t i o n o f t h e s e r o t o n i n a n t a g o n i s t s , c i n a n s e r i n o r m i a n s e r i n b l o c k t h e a n a l g e s i a p r o d u c e d b y i n j e c t i o n s o f m o r p h i n e i n t o t h e PAG ( Y a k s h , 1 9 7 9 ; Y a k s h e t a l . , 1 9 7 6 ) . H o w e v e r , P r o u d f i t a n d Hammond ( 1 9 8 1 ) f o u n d t h a t i n t r a t h e c a l i n j e c t i o n s o f m e t h y s e r g i d e , a s e r o t o n i n a n t a g o n i s t , d i d n o t s i g n i f i c a n t l y a t t e n u a t e a n a l g e s i a p r o d u c e d b y s u b c u t a n e o u s i n j e c t i o n s o f m o r p h i n e . T h i s l a t t e r r e s u l t s u g g e s t s t h a t s p i n a l s e r o t o n i n r e c e p t o r s d o n o t p l a y a n m a j o r r o l e i n a n a l g e s i a p r o d u c e d b y p e r i p h e r a l l y i n j e c t e d m o r p h i n e , b u t l e a v e s o p e n t h e p o s s i b i l i t y o f a r o l e f o r s u p r a s p i n a l s e r o t o n i n r e c e p t o r s . C o n s i d e r e d t o g e t h e r , t h e Y a k s h e t a l . a n d t h e P r o u d f i t a n d Hammond f i n d i n g s e m p h a s i z e t h e d a n g e r i n t h e common p r a c t i c e o f g e n e r a l i z i n g f r o m d a t a c o l l e c t e d f o l l o w i n g c e n t r a l i n j e c t i o n s o f m o r p h i n e t o t h e a n a l g e s i a p r o d u c e d b y p e r i p h e r a l m o r p h i n e i n j e c t i o n s . I n t r a t h e c a l a d m i n i s t r a t i o n o f s e r o t o n i n o r t h e s e r o t o n i n 21 agonists, quipazine and MK 212 produce strong analgesia that i s reversed by serotonin antagonists (Wang, 1977; Yaksh & Wilson, 1979), but th i s does not necessarily implicate serotonin receptors i n morphine-produced analgesia. These int r a t h e c a l i n j e c t i o n s might have activated an independent analgesia system unrelated to morphine. Peripheral i n j e c t i o n s of serotonin antagonists have been shown to increase the responsiveness of dorsal horn neurons to noxious stimulation (Rivot, Calvino, & Besson, 1 987) . These re s u l t s implicate a t o n i c a l l y active ( i . e . , normally f i r i n g at a high rate) serotonergic system that i n h i b i t s nociceptive afferent neurons i n ra t s . Similar i n j e c t i o n s have been shown to block the i n h i b i t i o n of nociceptive neurons by e l e c t r i c a l stimulation of the PAG or NRM (Yezierski, Wilcox & W i l l i s , 1982; Carstens, Fraunhoffer, & Zimmerman, 1981). In summary, the evidence from the study of serotonin antagonists and agonists supports the notion that serotonergic neurons are involved i n some forms of analgesia. Although the evidence i s far from unequivocal, there i s support from studies involving s e l e c t i v e lesions and l o c a l i n j e c t i o n s that both spinal and cerebral serotonergic mechanisms are involved. Although serotonin i s widely believed to be involved i n the analgesic e f f e c t s of systemically administered morphine—see the i n f l u e n t i a l recent texts of Kandel and Schwartz (1981) and Carlson (1981)—there i s i n f a c t l i t t l e d i r e c t evidence for t h i s view. 22 4 . S e r o t o n i n R e c e p t o r T y p e s ~ T h e r e c e n t d i s c o v e r y t h a t t h e r e a r e a t l e a s t t w o t y p e s o f s e r o t o n i n r e c e p t o r s , S1 a n d S2 ( P e r o u t k a , L e b o v i t z , & S n y d e r , 1 9 8 1 ; P e r o u t k a & S n y d e r , 1 9 7 9 ) r a i s e d t h e q u e s t i o n o f w h i c h r e c e p t o r t y p e m e d i a t e s m o r p h i n e a n a l g e s i a . T h e f o l l o w i n g t h r e e p a r t s o f t h i s s e c t i o n : A ) s u m m a r i z e t h e e v i d e n c e f o r t h e d i s t i n c t i o n b e t w e e n S1 a n d S2 r e c e p t o r s , B ) r e v i e w t h e c u r r e n t k n o w l e d g e o f t h e l o c a l i z a t i o n o f e a c h o f t h e r e c e p t o r t y p e s w i t h i n t h e c e n t r a l n e r v o u s s y s t e m , a n d C) d i s c u s s t h e f u n c t i o n s t h a t a r e t h o u g h t t o be m e d i a t e d b y e a c h o f t h e t y p e s . A . The S 1 - S 2 D i s t i n c t i o n . R a d i o a c t i v e l y l a b e l l e d s e r o t o n i n h a s b e e n s h o w n t o b i n d t o h o m o g e n i z e d m e m b r a n e s f r o m t h e c e n t r a l n e r v o u s s y s t e m . B e c a u s e t h i s b o u n d r a d i o a c t i v e s e r o t o n i n i s d i s p l a c e d b y u n l a b e l l e d s e r o t o n i n o r b y d r u g s t h a t a r e t h o u g h t t o b i n d t o s e r o t o n i n r e c e p t o r s , b u t i s n o t e a s i l y d i s p l a c e d b y o t h e r t r a n s m i t t e r s , i t h a s b e e n p r o p o s e d t h a t t h e s e b i n d i n g s i t e s r e p r e s e n t r e c e p t o r s t h a t a r e s p e c i f i c t o s e r o t o n i n . T h e f i n d i n g t h a t r a d i o a c t i v e l y - l a b e l e d s p i p e r o n e a l s o b i n d s t o t h e s e same s i t e s was a n i m p o r t a n t s t e p t o w a r d t h e d i s c o v e r y o f S1 a n d S2 s e r o t o n i n r e c e p t o r t y p e s . P e r o u t k a a n d S n y d e r ( 1 9 7 9 ) o b s e r v e d t h a t a f t e r w a s h i n g s p i p e r o n e - l a b e l e d m e m b r a n e s w i t h a c o n c e n t r a t i o n o f s e r o t o n i n t h a t c o m p l e t e l y e l i m i n a t e d l a b e l l e d s e r o t o n i n b i n d i n g , a p p r o x i m a t e l y h a l f o f t h e l a b e l l e d s p i p e r o n e r e m a i n e d . A l o w c o n c e n t r a t i o n o f s p i p e r o n e e a s i l y d i s p l a c e d t h i s 23 remaining l a b e l l e d spiperone, but i t required a 100-fold increase over the o r i g i n a l serotonin concentration i n the wash to accomplish the same task. The opposite was true of l a b e l l e d serotonin that was incubated with spiperone. Thus, to put i t concisely, Peroutka and Snyder found that serotonin binds with high a f f i n i t y to some serotonin receptors, which they named S1 receptors; whereas spiperone binds with high a f f i n i t y to others, which they referred to as S2 receptors. Further evidence for the idea that there are two d i s t i n c t serotonin receptor types comes from the observation that some drugs that are active at serotonin receptors e a s i l y displace l a b e l l e d serotonin, but not l a b e l l e d spiperone; whereas other drugs e a s i l y displace l a b e l l e d spiperone but not la b e l l e d serotonin (Peroutka & Snyder, 1979). For example, the concentration of the serotonin agonist, 5-methoxytryptamine that i s required to displace spiperone i s 250 times greater than that required to displace serotonin. On the other hand, cinanserin i s 100 times more e f f e c t i v e i n di s p l a c i n g spiperone than serotonin. Because both serotonin and spiperone bind to the same s i t e s , but are d i f f e r e n t i a l l y displaced from subgroups of the s i t e s , Peroutka and Snyder (1979) concluded that there must be at least two d i s t i n c t populations of serotonin receptors. B. D i s t r i b u t i o n of Serotonin Binding S i t e s . Early information from studies comparing membrane binding i n homogenized tissue indicated that S1 and S2 binding s i t e s are d i f f e r e n t i a l l y d i s t r i b u t e d within various central nervous system 24 a r e a s . The hippocampus, s t r i a t u m , raphe n u c l e i and s p i n a l gray matter showed dense S1 b i n d i n g ( B l a c k s h e a r , Steranka, & Sanders-Bush, 1981; Peroutka & Snyder, 1981), and S2 b i n d i n g was found to be p a r t i c u l a r l y dense i n f r o n t a l and o c c i p i t a l c o r t e x , accumbens, s t r i a t u m , and o l f a c t o r y t u b e r c l e , with no d e t e c t a b l e b i n d i n g i n the hypothalamus or s p i n a l gray matter (Blackshear e t a l . , 1981; Leysen, Niemegeers, Van Nueten, & Laduron, 1982; Monroe & Smith, 1983; Peroutka & Snyder, 1981). Autoradiography of s e c t i o n s of r a t b r a i n t i s s u e incubated i n r a d i o a c t i v e l y - l a b e l l e d s e r o t o n i n (Biegon, Rainbow, & McEwan, 1982; Young & Kuhar, 1980) has shown t h a t s e r o t o n i n b i n d i n g s i t e s are most conc e n t r a t e d i n the hippocampus, septum, medial and d o r s a l raphe, and i n t e r p e d u n c u l a r nucleus. There i s a l s o dense b i n d i n g i n the f r o n t a l c o r t e x , some amygdaloid and hypothalamic n u c l e i , d o r s a l tegmentum, caudate, o l f a c t o r y t u b e r c l e , median c e n t r a l gray, and i n the d o r s a l horn of the s p i n a l c o r d . But, because r a d i o l a b e l e d s e r o t o n i n binds to a l l s e r o t o n i n r e c e p t o r s , these s t u d i e s d i d not d i s t i n g u i s h between s e r o t o n i n r e c e p t o r types. R e f i n e d a u t o r a d i o g r a p h i c techniques t h a t used drugs t h a t are s e l e c t i v e f o r s e r o t o n i n r e c e p t o r types allowed the v i s u a l i z a t i o n of S1 and S2 b i n d i n g (Desamukh, Yamamura, Woods, & Nelson, 1983; Laduron, Janssen, & Leysen, 1982; M a r c i n k i e w i c z , Verge, Gozlan, P i c h a t , & Hamon, 1984; Pazos, C o r t e s , & P a l a c i o s , 1985; Pazos & P a l a c i o s , 1985; S l a t e r & P a t e l , 1983). R e s u l t s from these s t u d i e s have g e n e r a l l y confirmed and extended r e s u l t s o b tained w i t h t h e l e s s p r e c i s e membrane b i n d i n g t e c h n i q u e s . F o r e x a m p l e , t h e b i n d i n g s i t e s i n t h e m e d i a n r a p h e h a v e b e e n s h o w n t o b e p r i m a r i l y S1 s i t e s , a n d t h e s i t e s i n t h e d o r s a l r a p h e a r e p r i m a r i l y S2 s i t e s . T h e S1 b i n d i n g s e e n i n t h e s p i n a l g r a y m a t t e r i s a l m o s t e n t i r e l y r e s t r i c t e d t o t h e d o r s a l h o r n , w i t h t h e b i n d i n g s i t e s m o s t c o n c e n t r a t e d i n t h e s u b s t a n t i a g e l a t i n o s a . T h u s , b i n d i n g t o b o t h S1 a n d S2 r e c e p t o r s h a s b e e n l o c a l i z e d t o a r e a s t h o u g h t t o be a s s o c i a t e d w i t h a n a l g e s i a . C . B e h a v i o r a l C o r r e l a t e s o f S1 a n d S2 B i n d i n g S i t e s . A l t h o u g h t h e m e d i a t i o n o f many b e h a v i o r a l e f f e c t s o f s e r o t o n i n r e c e p t o r s t i m u l a t i o n h a v e b e e n a t t r i b u t e d t o e i t h e r S1 o r S2 r e c e p t o r s , t h e r e a r e f e w r e l e v a n t d a t a . A d m i n i s t r a t i o n o f s e r o t o n i n o r s e r o t o n i n a g o n i s t s p r o d u c e s a v a r i e t y o f b e h a v i o r a l e f f e c t s , w h i c h i n c l u d e h e a d - t w i t c h i n g , f o r e p a w t r e a d i n g , t r e m o r , h i n d l i m b a b d u c t i o n , a n d S t r a u b t a i l ( J a c o b s , 1 9 7 6 ) . T h i s e n t i r e s y n d r o m e i s b l o c k e d b y m e t e r g o l i n e a n d m e t h y s e r g i d e , t w o s e r o t o n i n a n t a g o n i s t s w i t h p a r t i a l a c t i o n a t b o t h S1 a n d S2 r e c e p t o r s ( C o l p a e r t & J a n s s e n , 1 9 8 3 ; O r t m a n n , B i s c h o f f , R a d e k e , B u e c h , & D e l i n i - S t u l a , 1 9 8 2 ) . I n c o n t r a s t , t h e s e l e c t i v e S2 a n t a g o n i s t s k e t a n s e r i n , p i r e n p e r o n e , a n d p i p a m p e r o n e b l o c k e d t h e h e a d t w i t c h e s ( C o l p a e r t & J a n s s e n , 1 9 8 3 ; G r e e n , 1 9 8 4 ; L u c k i , N o b l e r , & F r a s e r , 1 9 8 4 ; O r t m a n n e t a l . , 1 9 8 2 ) b u t h a d no e f f e c t o n t h e o t h e r b e h a v i o r a l s y m p t o m s ( G r e e n , 1 9 8 4 ; L u c k i e t a l . , 1 9 8 4 ) . M e n d e l s o n a n d G o r z a l k a ( 1 9 8 5 a , b ; 1 9 8 6 b ) f o u n d t h a t b l o c k a d e o f S2 r e c e p t o r s w i t h s e l e c t i v e a n t a g o n i s t s i n h i b i t s t h e s e x u a l 26 behavior of female ra t s ; whereas, quipazine, an agonist with r e l a t i v e l y high a f f i n i t y for S2 receptors, was found to f a c i l i t a t e i t . In contrast, S1 agonists have been shown to i n h i b i t female sexual behavior (Mendelson & Gorzalka, 1986a). Moreover, Mendelson & Gorzalka (1986a) found that S1 receptors may serve an opposite function i n the sexual behavior of male rats than i n female rats; the S1 agonist 8-hydroxy-2,dipropylaminotetralin i n h i b i t e d the sexual behavior of female rats, but f a c i l i t a t e d that of males. On the basis of t h e i r research, Mendelson and Gorzalka have speculated that serotonin receptor types appear to play antagonistic roles i n females, but work in concert i n males. 5. Paul and P h i l l i p s (1986) L i t t l e attention has been given to the r e l a t i v e roles of S1 and S2 receptors i n morphine-produced analgesia. Because i t has been hypothesized that spinal serotonin receptors mediate morphine-produced analgesia (Basbaum & F i e l d s , 1984; F i e l d s & Basbaum, 1978; Messing & L y t l e , 1977; Samanin et a l . , 1978) and because spinal cord serotonin receptors are almost exclusively S1 receptors (Monroe & Smith, 1983; Pazos et a l . , 1985), many have assumed that spinal S1 receptors mediate morphine-produced analgesia (e.g. Pazos et a l . , 1985). However, the data of Zemlan, Kow, and Pfaff (1983) suggest that stimulation of spinal S1 receptors produces hyperalgesia. They found that systemic administration of the serotonin agonists quipazine and 5-methoxy-N,N-diamethyltryptamine to rats with transected spinal cords f a c i l i t a t e d responding to noxious s t i m u l i . Furthermore, cinanserin, a serotonin antagonist with a p r e f e r e n t i a l " a f f i n i t y for S2 receptors, has been shown to block analgesia produced by inje c t i o n s of morphine into the PAG (Yaksh, 1979; Yaksh et a l . , 1976). However, recent data indicate that cinanserin may have agonistic properties at S1 receptors as well as i t s S2 receptor blocking e f f e c t (Janssen, 1983). Because v i r t u a l l y no S2 receptors have been found i n the spinal cord, the blockade seen by Yaksh et a l . may have been produced by stimulation of S1 receptors within a system that f a c i l i t a t e s nociceptive transmission, and would thus support the hypothesis of Zemlan et a l . that S1 receptors i n the spinal dorsal horn f a c i l i t a t e nociceptive transmission. Thus, although a d i r e c t implication of the widely accepted PAG-NRM-spinal cord model i s that serotonin d i r e c t l y or i n d i r e c t l y i n h i b i t s ascending nociceptive information, there may be two spinal serotonergic systems: the f i r s t i n h i b i t i n g ascending nociceptive f i b e r s , and the second f a c i l i t a t i n g l o c a l spinal withdrawal r e f l e x e s . I have recently published (Paul & P h i l l i p s , 1986) a f i r s t attempt to assess the e f f e c t of S2 receptor blockade on the analgesia produced by systemic i n j e c t i o n of morphine. Rats were injected with 0.04, 0.08, or 0.16 mg/kg (SC) of the se l e c t i v e S2 receptor blocker, pirenperone, followed 60 min l a t e r by 10 mg/kg (IP) of morphine s u l f a t e . Each rat was tested for analgesia 15 min a f t e r the morphine administration by measuring the amount of 28 time that i t took to remove i t s t a i l from a hot water bath, that i s by measuring i t s t a i l - f l i c k latency. As expected, morphine by i t s e l f s u b s t a n t i a l l y increased t a i l - f l i c k l a t e n c i e s , thus ind i c a t i n g strong morphine-produced analgesia. When morphine administration was preceded by i n j e c t i o n of 0.16 mg/kg of pirenperone, but not 0.04 or 0.08 mg/kg, there was a s i g n i f i c a n t attenuation of morphine-produced analgesia. These results suggest that S2 receptors mediate morphine-produced analgesia. 6. General Rationale and Purposes Clearly, our theories of how the neurotransmitter serotonin i s involved i n the mediation of pain and morphine-produced analgesia are far from unequivocal. The large number of contradictory findings suggest that some c r u c i a l variable has not been considered. The recent findings that S1 receptors may serve to enhance nociception (Zemlan et a l . , 1983), whereas S2 receptors may mediate i n h i b i t i o n (Paul & P h i l l i p s , 1986), led me to conclude that the study of nociception and analgesia may also benefit from a closer examination of the d i f f e r e n t i a l roles of S1 and S2 receptors. This approach has recently proven successful i n resolving some of the inconsistencies i n the study of serotonin's role i n sexual behavior (Mendelson & Gorzalka, 1985a,b; 1986a,b). The experiments i n the present thesis had two general purposes. The f i r s t was to more c l o s e l y determine where i n the nervous system the s e l e c t i v e S2 receptor blocker pirenperone acts to block morphine-produced analgesia. The second general purpose of t h i s series of experiments was to test the a l t e r n a t i v e hypothesis that the anti-analgesic e f f e c t of pirenperorie and other S2 antagonists i s due to the reported action of these drugs at alpha-adrenergic receptors (Janssen, 1983). I I . GENERAL METHOD T h i s s e c t i o n d e s c r i b e s t h e m e t h o d s common t o a l l f i v e e x p e r i m e n t s o f t h i s t h e s i s . A n y s p e c i f i c m o d i f i c a t i o n s o r a d d i t i o n s t o t h i s g e n e r a l m e t h o d o l o g y a r e d e s c r i b e d i n t h e m e t h o d s e c t i o n o f e a c h e x p e r i m e n t . 1 . S u b j e c t s S e r v i n g a s s u b j e c t s i n e a c h e x p e r i m e n t w e r e 3 0 0 - t o - 4 5 0 g m a l e r a t s h o u s e d i n d i v i d u a l l y w i t h f r e e a c c e s s t o P u r i n a l a b chow a n d w a t e r . 2 . A p p a r a t u s A l l t a i l - f l i c k t e s t s w e r e c o n d u c t e d i n a 6 . 5 x 6 . 5 x 20 cm c h a m b e r . E a c h r a t ' s t a i l was d r a w n t h r o u g h a 2 cm w i d e o p e n i n g a t t h e r e a r o f t h e c h a m b e r , a n d a p p r o x i m a t e l y 5 cm o f t h e t a i l was s u b m e r g e d i n a 52 C w a t e r b a t h . T h e t i m e t h a t i t t o o k e a c h r a t t o r e m o v e i t s t a i l f r o m t h e b a t h , t h a t i s t h e t a i l - f l i c k l a t e n c y , was r e c o r d e d e l e c t r o n i c a l l y . On t h e f e w t r i a l s t h a t a s u b j e c t d i d n o t r e s p o n d w i t h i n 10 s e c , i t s t a i l was r e m o v e d f r o m t h e b a t h b y t h e e x p e r i m e n t e r t o p r e v e n t t i s s u e d a m a g e , a n d a t a i l - f l i c k l a t e n c y o f 10 s e c was a s s i g n e d . A l l t e s t i n g o c c u r r e d i n t h e c o l o n y r o o m d u r i n g t h e l a s t 5 h r o f t h e l i g h t p h a s e o f t h e 1 2 / 1 2 h r l i g h t / d a r k c y c l e . 3 . P r o c e d u r e B a s e l i n e t a i l - f l i c k l a t e n c i e s w e r e r e c o r d e d o n 5 o r 6 c o n s e c u t i v e d a y s . E a c h d a i l y t e s t s e s s i o n c o n s i s t e d o f f i v e t a i l - f l i c k t e s t s a d m i n i s t e r e d a t 3 0 - m i n i n t e r v a l s . E a c h s u b j e c t s p e n t t h e 3 0 - m i n i n t e r t e s t i n t e r v a l s i n i t s home c a g e . T h e f i r s t 31 o f t h e f o u r d r u g - t e s t s e s s i o n s o c c u r r e d o n t h e d a y a f t e r t h e l a s t b a s e l i n e s e s s i o n , a n d t h e r e m a i n i n g t h r e e o c c u r r e d a t 4 - d a y i n t e r v a l s t h e r e a f t e r . B a s e l i n e s e s s i o n s w e r e c o n d u c t e d " o n e a c h o f t h e 3 d a y s b e t w e e n c o n s e c u t i v e d r u g - t e s t d a y s . T h e d r u g - t e s t s e s s i o n s w e r e i d e n t i c a l t o t h e b a s e l i n e s e s s i o n s e x c e p t t h a t i m m e d i a t e l y a f t e r t h e f i r s t t a i l - f l i c k t e s t o n e a c h d r u g - t e s t d a y , e a c h r a t was i n j e c t e d w i t h e i t h e r t h e a p p r o p r i a t e d o s e o f a s e r o t o n i n a n t a g o n i s t o r i t s v e h i c l e , f o l l o w e d e i t h e r b y a n a n a l g e s i c o r i t s s a l i n e v e h i c l e . T h u s , t h e r e w e r e f o u r b a s i c c o n d i t i o n s i n e a c h s t u d y : v e h i c l e - v e h i c l e , a n a l g e s i c - v e h i c l e , v e h i c l e - a n t a g o n i s t , a n d a n a l g e s i c - a n t a g o n i s t . I n e a c h s t u d y , e a c h s u b j e c t was t e s t e d u n d e r a l l f o u r o f t h e s e t r e a t m e n t c o m b i n a t i o n s i n a c o u n t e r b a l a n c e d s e q u e n c e . T a b l e 1 l i s t s e a c h d r u g u s e d i n t h e s e e x p e r i m e n t s w i t h i t s s o u r c e , v e h i c l e , c o n c e n t r a t i o n , i n j e c t i o n v o l u m e , d o s e , a n d r o u t e o f a d m i n i s t r a t i o n . 4 . S t a t i s t i c a l A n a l y s i s F o r e a c h d o s e o f t h e a n t a g o n i s t a n ANOVA was u s e d t o a s s e s s t h e s i g n i f i c a n c e o f t h e w i t h i n - g r o u p d i f f e r e n c e s i n t a i l - f l i c k l a t e n c i e s f o r t h e f o u r p o s t - i n j e c t i o n i n t e r v a l s . I n a l l c a s e s , t h e m a i n e f f e c t s f o r t e s t i n t e r v a l a n d t r e a t m e n t , a n d t h e i n t e r v a l x t r e a t m e n t i n t e r a c t i o n w e r e s i g n i f i c a n t a t t h e . 0 5 a l p h a l e v e l . N e w m a n - K e u l ' s p o s t - h o c c o m p a r i s o n s w e r e t h e n u s e d t o a s s e s s t h e s i g n i f i c a n c e o f t r e a t m e n t d i f f e r e n c e s a n d d i f f e r e n c e s a t s p e c i f i c t e s t i n t e r v a l s . T h e a l p h a l e v e l was . 0 5 f o r a l l p o s t - h o c c o m p a r i s o n s . A 2 x 2 ANOVA was a l s o u s e d t o 32 T a b l e 1 . T h e v e h i c l e , c o n c e n t r a t i o n , i n j e c t i o n v o l u m e , d o s e , r o u t e o f a d m i n i s t r a t i o n , a n d s o u r c e o f e a c h d r u g u s e d i n t h i s s e r i e s o f e x p e r i m e n t s . T h e pH was a d j u s t e d t o b e t w e e n ~6 a n d 7 w i t h NaOH when n e c e s s a r y . DRUG VEHICLE CONCENTRATION (mg/ml) VOLUME (ml/kg) DOSE (mg/kg) ROUTE SOURCE I ANALGESICS ! 1 Morphine Sulphate I Saline 1 10 1 10 IP BDH | 1 Ketamine HCI 1 Saline I 50 2 100 IP Parke-Davis | |S2 ANTAGONISTS 1 1 Pirenperone 1 Citrate 1 0.4 1 0.8 1 1 1.2 1 0.2 0.2 1 0.2 1 0.08 0.1G 0.24 SC Janssen I 1 Ketanserin HCI 1 Citrate 1 5 | 1 15 | 1 50 | 0.2 0.2 | 0.2 1 1 3 10 SC Janssen I I LY53857 1 Saline 1 10 | 1 | 10 IP L i l l y | I LO LO 34 assess the p o s s i b i l i t y that a s h i f t i n baseline caused by the antagonist may account for any observed attenuation of analgesia. This analysis was used on data at the peak of the analgesic (60 min for morphine and 30 min for ketamine). 35 I I I . EXPERIMENT 1: E F F E C T OF P IRENPERONE ON MORPHINE A N A L G E S I A T h e f i r s t e x p e r i m e n t was d e s i g n e d t o c o n f i r m t h e p r e l i m i n a r y f i n d i n g ( P a u l & P h i l l i p s , 1986) t h a t t h e s e l e c t i v e S2 a n t a g o n i s t , p i r e n p e r o n e ( C o l p a e r t & J a n s s e n , 1982; C o l p a e r t e t a l . , 1983; L e y s e n e t a l . , 1981) b l o c k s m o r p h i n e - p r o d u c e d a n a l g e s i a . C o n f i r m a t i o n o f t h i s f i n d i n g was a p a r t i c u l a r l y i m p o r t a n t s t e p i n t h i s t h e s i s b e c a u s e i t was i n c o n s i s t e n t w i t h c u r r e n t t h e o r i e s o f m o r p h i n e - p r o d u c e d a n a l g e s i a a n d b e c a u s e e a c h o f t h e e x p e r i m e n t s o f t h e t h e s i s was b a s e d o n i t . A n o t h e r r e a s o n f o r " r e p l i c a t i n g " t h e P a u l a n d P h i l l i p s (1986) s t u d y was t h a t i t was n o t c o n d u c t e d a c c o r d i n g t o t h e p r o c e d u r e s a d o p t e d f o r t h i s s e r i e s o f e x p e r i m e n t s . B r i e f l y , i n t h e p r e s e n t e x p e r i m e n t s , p i r e n p e r o n e a n d m o r p h i n e w e r e i n j e c t e d c o n c u r r e n t l y , a n d t h e r a t s w e r e t e s t e d r e p e a t e d l y t h r o u g h o u t t h e 2 h r s e s s i o n ; w h e r e a s , i n t h e P a u l a n d P h i l l i p s e x p e r i m e n t , p i r e n p e r o n e was i n j e c t e d 60 m i n b e f o r e m o r p h i n e , a n d t h e r a t s were t e s t e d o n l y o n c e , 15 m i n a f t e r t h e m o r p h i n e i n j e c t i o n . M e t h o d F o l l o w i n g 5 d a y s o f b a s e l i n e t e s t i n g t h e 39 r a t s s e r v i n g a s s u b j e c t s w e r e r a n d o m l y d i v i d e d i n t o t h r e e g r o u p s . T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e o r s a l i n e i n j e c t e d i n t r a p e r i t o n e a l l y was c h a l l e n g e d w i t h 0.08 (n=12), 0.16 (n=14), o r 0.24 m g / k g (n=13) o f p i r e n p e r o n e o r i t s c i t r a t e v e h i c l e a d m i n i s t e r e d s u b c u t a n e o u s l y . R e s u l t s F i g u r e 1 i l l u s t r a t e s t h e r e s u l t s o f E x p e r i m e n t 1 . I n j e c t i o n o f 10 m g / k g o f m o r p h i n e s u l p h a t e b y i t s e l f p r o d u c e d p o t e n t a n a l g e s i a , a s i n d i c a t e d b y s u b s t a n t i a l l y i n c r e a s e d t a i l - f l i c k l a t e n c i e s a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s i n t h e m o r p h i n e - v e h i c l e c o n d i t i o n c o m p a r e d t o t h e v e h i c l e - v e h i c l e c o n d i t i o n a t a l l t h r e e d o s e s o f p i r e n p e r o n e . T h e m a j o r f i n d i n g o f t h i s s t u d y was t h a t when 0 . 0 8 , 0 . 1 6 o r 0 . 2 4 m g / k g o f p i r e n p e r o n e was i n j e c t e d w i t h t h e m o r p h i n e , t h e m o r p h i n e - p r o d u c e d a n a l g e s i a was a t t e n u a t e d . A s e c o n d i m p o r t a n t f i n d i n g was t h a t e a c h o f t h e t h r e e d o s e s o f p i r e n p e r o n e p r o d u c e d h y p e r a l g e s i a when a d m i n i s t e r e d b y t h e m s e l v e s ; t h a t i s , t h e t a i l - f l i c k l a t e n c i e s i n t h e v e h i c l e - p i r e n p e r o n e c o n d i t i o n w e r e s i g n i f i c a n t l y s h o r t e r t h a n t h o s e i n t h e v e h i c l e - v e h i c l e c o n t r o l c o n d i t i o n . F o r a l l t h r e e g r o u p s , t h e o v e r a l l ANOVAs r e v e a l e d s i g n i f i c a n t m a i n e f f e c t s f o r t r e a t m e n t ( 0 . 0 8 m g / k g g r o u p , F ( 3 , 3 3 ) = 1 5 . 1 , p < . 0 0 0 1 ; 0 . 1 6 m g / k g g r o u p , F ( 3 , 3 9 ) = 1 7 . 0 , p < 0 . 0 0 0 1 ; 0 . 2 4 m g / k g g r o u p , F ( 3 , 3 6 ) = 3 1 . 7 p < . 0 0 0 1 ) . T h e a n a l g e s i c e f f e c t o f m o r p h i n e was c o n f i r m e d b y t h e s i g n i f i c a n c e o f t h e o v e r a l l d i f f e r e n c e b e t w e e n t h e v e h i c l e - v e h i c l e t r e a t m e n t a n d t h e m o r p h i n e - v e h i c l e c o n d i t i o n f o r e a c h o f t h e t h r e e g r o u p s ( a l l t h r e e N e w m a n - K e u l 1 s p < . 0 5 ) . I n e a c h g r o u p , m o r p h i n e p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n mean t a i l - f l i c k l a t e n c y a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s ( a l l n i n e N e w m a n - K e u l ' s p < . 0 5 ) . T h e t a i l - f l i c k l a t e n c i e s o f r a t s a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s w e r e s i g n i f i c a n t l y s h o r t e r when t h e y w e r e i n j e c t e d F i g u r e 1 . T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h t h r e e d o s e s o f p i r e n p e r o n e . M e a n t a i l - f l i c k l a t e n c i e s w e r e a s s e s s e d a t 0 , 3 0 , 6 0 , 9 0 , a n d 120 m i n a l r t e r t h e i n j e c t i o n s i n e a c h o f t h e f o u r t r e a t m e n t s . T h e t h r e e g r a p h s i l l u s t r a t e t h e e f f e c t s o f 0 . 0 8 , 0 . 1 6 , a n d 0 . 2 4 m g / k g d o s e s o f p i r e n p e r o n e ( n = 1 2 , 1 4 , a n d 1 3 , r e s p e c t i v e l y ) . T h e a n a l g e s i c e f f e c t o f m o r p h i n e i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d M O R - V E H c o n d i t i o n s . T h e e f f e c t o f p i r e n p e r o n e b y i t s e l f o n mean t a i l - f l i c k l a t e n c y i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d V E H - P I R c o n d i t i o n s . The a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y p i r e n p e r o n e i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e M O R - V E H a n d M O R - P I R c o n d i t i o n s . 0.08 0.16 J 1 1 L_ 1 I 1 | | • • 0 30 60 90 120 0 30 60 90 120 TIME POST-INJECTION (min) 0.24 ' ' l I l 0 30 60 90 120 OJ oo 39 w i t h m o r p h i n e a n d a n y o f t h e t h r e e d o s e s o f p i r e n p e r o n e t h a n w h e n t h e y w e r e t r e a t e d w i t h m o r p h i n e b y i t s e l f ( a l l N e w m a n - K e u l ' s p < . 0 5 ) . A l t h o u g h t h e mean t a i l - f l i c k l a t e n c i e s o f r a t s t r e a t e d w i t h e a c h o f t h e t h r e e d o s e s o f p i r e n p e r o n e b y i t s e l f w e r e s h o r t e r t h a n when t h e y r e c e i v e d t h e v e h i c l e - v e h i c l e i n j e c t i o n s a t e v e r y i n t e r v a l ( e x c l u d i n g t h e 0 - m i n i n t e r v a l ) , t h i s e f f e c t r e a c h e d s t a t i s t i c a l s i g n i f i c a n c e ( N e w m a n - K e u l 1 s p < . 0 5 ) o n l y a t t h e 3 0 -a n d 6 0 - m i n t e s t i n t e r v a l s a f t e r t h e 0 . 0 8 m g / k g d o s e o f p i r e n p e r o n e a n d a t a l l f o u r i n t e r v a l s a f t e r t h e 0 . 2 4 m g / k g d o s e . T h e 2 X 2 ANOVAs r e v e a l e d t h a t t h e s h i f t s i n b a s e l i n e f o l l o w i n g t h e v e h i c l e - p i r e n p e r o n e i n j e c t i o n s may a c c o u n t f o r t h e o b s e r v e d a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a . A l t h o u g h t h e m a i n e f f e c t f o r p i r e n p e r o n e was s i g n i f i c a n t a t t h e 0 . 0 8 a n d 0 . 2 4 m g / k g d o s e s ( 0 . 0 8 m g / k g g r o u p , F ( 1 , 1 1 ) = 1 7 . 2 1 , p < . 0 0 5 ; 0 . 1 6 m g / k g g r o u p , F ( 1 , 1 3 ) = 3 . 8 5 , p > . 0 5 ; 0 . 2 6 m g / k g g r o u p , F ( 1 , 1 2 ) = 2 7 . 9 9 , p < . 0 0 1 ) , t h e m o r p h i n e x p i r e n p e r o n e i n t e r a c t i o n s w e r e n o t ( F ( 1 , 1 1 ) = 0 . 5 6 ; F ( 1 , 1 3 ) = 0 ; F ( 1 , 1 2 ) = 0 . 7 1 , r e s p e c t i v e l y ; a l l t h r e e p > . 0 5 ) . D i s c u s s i o n T h e r e s u l t s o f E x p e r i m e n t 1 c o n f i r m t h e f i n d i n g o f P a u l a n d P h i l l i p s ( 1 9 8 6 ) t h a t t h e s e l e c t i v e S2 r e c e p t o r b l o c k e r , p i r e n p e r o n e a t t e n u a t e s t h e a n a l g e s i c e f f e c t o f m o r p h i n e . M o r e o v e r , p i r e n p e r o n e , w h e n a d m i n i s t e r e d b y i t s e l f , p r o d u c e d h y p e r a l g e s i a . B e c a u s e p i r e n p e r o n e h a s a p r e f e r e n t i a l a f f i n i t y f o r S2 r e c e p t o r s , t h i s p a t t e r n o f r e s u l t s p r o v i d e s f u r t h e r evidence that S2 receptors play a s i g n i f i c a n t role i n the perception of pain and i n the analgesic e f f e c t of morphine. Considering the observation that S2 binding s i t e s "nave not been demonstrated i n the spinal cord (Monroe & Smith, 1983; Pazos et a l . , 1985), these r e s u l t s are s u r p r i s i n g . Serotonin i s generally thought to exert control over nociception and analgesia through spinal cord receptors (e.g. Carlson, 1981; Kandel & Schwartz, 1981). Accordingly, the fact that a s e l e c t i v e S2 blocker such as pirenperone can attenuate morphine-produced analgesia suggests that serotonin may be producing t h i s e f f e c t through blockade of S2 receptors i n the brain or peripheral nervous system. It i s , however, possible that the reduction i n baseline t a i l - f l i c k latencies following vehicle-pirenperone i n j e c t i o n s may account for the observed anti-analgesic e f f e c t of pirenperone. 41 IV. EXPERIMENT 2: EFFECT OF KETANSERIN ON MORPHINE ANALGESIA Experiment 2 was designed to assess the e f f e c t of the sel e c t i v e S2 antagonist ketanserin on morphine-produced analgesia. Pirenperone i s a de r i v a t i v e of ketanserin and these two drugs have comparable a f f i n i t i e s f or the same receptors. Although ketanserin and pirenperone have a f f i n i t i e s for the same receptors (Table 2), ketanserin does not e a s i l y enter the central nervous system (Laduron et a l . , 1982). Because i t i s only at very high doses that ketanserin a f f e c t s receptors i n the central nervous system, any eff e c t s of low doses can be reasonably attributed to i t s action at peripheral receptors. The d i f f e r e n t i a l a b i l i t i e s of pirenperone and ketanserin to enter the central nervous system provided a convenient way to test whether morphine-produced analgesia i s mediated by S2 receptors i n the central or peripheral nervous system. Blockade of morphine-produced analgesia by a low doses of ketanserin would implicate peripheral S2 receptors, whereas, blockade r e s t r i c t e d to high doses would support a ro l e for central receptors. Method Following 6 days of baseline t e s t i n g , the 32 rats serving as subjects were randomly divided into three groups. The analgesic e f f e c t of 10 mg/kg of morphine or sa l i n e injected i n t r a p e r i t o n e a l l y was challenged with 1 (n=10), 3 (n=11), or 10 mg/kg (n=11) of ketanserin HCI or i t s c i t r a t e vehicle administered subcutaneously. Table 2. The i n h i b i t i o n constants of ketanserin and pirenperone for S1, S2, histamine type-1 (H1), alpha, dopamine (DA), and muscarinic acetylcholine (Ach-m) receptors as determined by i n v i t r o receptor binding techniques (adapted from Janssen, 1983). Values are expressed i n nanomoles. 43 Drug S2 S I H a l p h a DA Ach -m K e t a n s e r i n 2 . 1 - 10 10 220 P i r e n p e r o n e 2 . 0 - 14 6 .8 16 R e s u l t s F i g u r e 2 i l l u s t r a t e s t h e r e s u l t s o f E x p e r i m e n t 2 . I n j e c t i o n o f 10 m g / k g o f m o r p h i n e s u l p h a t e b y i t s e l f p r o d u c e d p o t e n t a n a l g e s i a , a s s e e n b y s u b s t a n t i a l l y i n c r e a s e d t a i l - f l i c k l a t e n c i e s a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s i n t h e m o r p h i n e - v e h i c l e c o n d i t i o n c o m p a r e d t o t h e v e h i c l e - v e h i c l e c o n d i t i o n a t a l l t h r e e d o s e s o f k e t a n s e r i n . T h e m a j o r f i n d i n g o f t h i s s t u d y was t h a t 10 m g / k g o f k e t a n s e r i n , b u t n o t 1 o r 3 m g / k g , a t t e n u a t e d m o r p h i n e - p r o d u c e d a n a l g e s i a . F o r a l l t h r e e g r o u p s , t h e o v e r a l l ANOVAs r e v e a l e d s i g n i f i c a n t m a i n e f f e c t s f o r t r e a t m e n t (1 m g / k g g r o u p , F ( 3 , 2 7 ) = 1 0 . 6 , p < . 0 0 0 1 ; 3 m g / k g g r o u p , F ( 3 , 3 0 ) = 1 7 . 5 , p < . 0 0 0 1 ; 10 m g / k g g r o u p , F ( 3 , 3 0 ) = 1 9 . 3 , p < . 0 0 0 1 ) . T h e a n a l g e s i c e f f e c t o f m o r p h i n e was c o n f i r m e d b y t h e s i g n i f i c a n c e o f t h e o v e r a l l d i f f e r e n c e b e t w e e n t h e v e h i c l e - v e h i c l e c o n d i t i o n a n d t h e m o r p h i n e - v e h i c l e t r e a t m e n t f o r a l l t h r e e g r o u p s ( a l l t h r e e N e w m a n - K e u l 1 s p < . 0 5 ) . I n t h e 3 a n d 10 m g / k g g r o u p s , m o r p h i n e p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n mean t a i l - f l i c k l a t e n c y a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s ; a n d i n t h e 1 m g / k g g r o u p , i t p r o d u c e d s i g n i f i c a n t i n c r e a s e s a t t h e 3 0 - a n d 6 0 - m i n i n t e r v a l s ( a l l e i g h t N e w m a n - K e u l 1 s p < . 0 5 ) . T h e t a i l - f l i c k l a t e n c i e s o f r a t s a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s w e r e s i g n i f i c a n t l y s h o r t e r when t h e y w e r e i n j e c t e d w i t h m o r p h i n e a n d 10 m g / k g o f k e t a n s e r i n t h a n when t h e y w e r e t r e a t e d w i t h m o r p h i n e b y i t s e l f ( a l l t h r e e N e w m a n - K e u l ' s p < . 0 5 ) . I n c o n t r a s t , 1 o r 3 m g / k g o f k e t a n s e r i n d i d n o t s i g n i f i c a n t l y 45 F i g u r e 2 . T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h t h r e e d o s e s o f k e t a n s e r i n H C I . Mean t a i l - f l i c k l a t e n c i e s w e r e a s s e s s e d a t 0 , 3 0 , 6 0 , 9 0 , a n d 120 m i n a f t e r i n j e c t i o n s o f r a t s i n t h e f o u r t r e a t m e n t c o n d i t i o n s . T h e t h r e e g r a p h s i l l u s t r a t e t h e 1 , 3 , a n d 10 m g / k g d o s e s o f k e t a n s e r i n ( n = 1 0 , 1 1 , a n d 1 1 , r e s p e c t i v e l y ) . T h e a n a l g e s i c e f f e c t o f m o r p h i n e i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d M O R - V E H c o n d i t i o n s . The e f f e c t o f k e t a n s e r i n b y i t s e l f o n mean t a i l - f l i c k l a t e n c y i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d V E H - K E T c o n d i t i o n s . T h e a t t e n u a t i o n o f m o r p h i n e -p r o d u c e d a n a l g e s i a b y k e t a n s e r i n i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e M O R - V E H a n d M O R - K E T c o n d i t i o n s . 47 r e d u c e t h e t a i l - f l i c k , l a t e n c i e s o f m o r p h i n e - t r e a t e d s u b j e c t s a t a n y o f t h e t e s t i n t e r v a l s . A l t h o u g h t h e mean t a i l - f l i c k l a t e n c i e s o f r a t s t r e a t e d w i t h e a c h o f t h e t h r e e d o s e s o f k e t a n s e r i n a n d t h e v e h i c l e w e r e c o n s i s t e n t l y s h o r t e r t h a n w h e n t h e y r e c e i v e d t h e v e h i c l e - v e h i c l e i n j e c t i o n s , t h i s e f f e c t n e v e r r e a c h e d s t a t i s t i c a l s i g n i f i c a n c e . The 2 X 2 ANOVAs r e v e a l e d t h a t t h e s h i f t s i n b a s e l i n e f o l l o w i n g t h e v e h i c l e - k e t a n s e r i n i n j e c t i o n s d o n o t a c c o u n t f o r t h e o b s e r v e d a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e m a i n e f f e c t f o r k e t a n s e r i n was s i g n i f i c a n t a t t h e h i g h e s t d o s e (1 m g / k g g r o u p , F ( 1 , 9 ) = 0 . 5 7 , p > . 0 5 ; 3 m g / k g g r o u p , F ( 1 , 1 0 ) = 1 . 8 6 , p > . 0 5 ; 10 m g / k g g r o u p , F ( 1 , 1 0 ) = 2 4 . 0 5 , p < . 0 0 1 ) , a s was t h e m o r p h i n e x p i r e n p e r o n e i n t e r a c t i o n ( F ( 1 , 9 ) = 1 . 1 1 , p > . 0 5 ; F ( 1 , 1 0 ) = 0 . 0 5 , p > . 0 5 ; F ( 1 , 1 0 ) = 5 . 0 2 , p < . 0 5 , r e s p e c t i v e l y ) . D i s c u s s i o n The f a c t t h a t k e t a n s e r i n s i g n i f i c a n t l y a t t e n u a t e d m o r p h i n e -p r o d u c e d a n a l g e s i a a t t h e h i g h 10 m g / k g d o s e p r o v i d e s f u r t h e r e v i d e n c e t h a t S2 r e c e p t o r s a r e i m p o r t a n t f o r t h e m e d i a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a . I n E x p e r i m e n t 1 , p i r e n p e r o n e , a d r u g t h a t i s p h a r m a c o l o g i c a l l y s i m i l a r t o k e t a n s e r i n ( s e e T a b l e 2 ) , b l o c k e d m o r p h i n e - p r o d u c e d a n a l g e s i a a t a d o s e much l o w e r t h a n t h e d o s e s o f k e t a n s e r i n t h a t f a i l e d t o b l o c k i t i n t h i s e x p e r i m e n t . B e c a u s e p i r e n p e r o n e e a s i l y e n t e r s t h e c e n t r a l n e r v o u s s y s t e m , w h e r e a s k e t a n s e r i n d o e s n o t , t h i s p a t t e r n o f r e s u l t s p r o v i d e s e v i d e n c e t h a t t h e S2 r e c e p t o r s t h a t a r e i m p o r t a n t f o r m o r p h i n e - p r o d u c e d a n a l g e s i a a r e i n t h e c e n t r a l 48 nervous system. In Experiment 1, pirenperone by i t s e l f caused a decrease i n baseline t a i l - f l i c k l a t e n c i e s . This r e s u l t raised the _ p o s s i b i l i t y that the observed anti-analgesic e f f e c t of the S2 antagonist i s nothing more than a r e f l e c t i o n of the baseline s h i f t . The res u l t s of Experiment 2 , however, challenge t h i s hypothesis. Because 10 mg/kg of ketanserin s i g n i f i c a n t l y attenuated morphine-produced analgesia but produced only a s l i g h t , nonsignificant decrease i n baseline t a i l - f l i c k latency, i t seems l i k e l y that the attenuation of morphine-produced analgesia and the hyperalgesia produced by these S2 antagonists are d i s s o c i a b l e . 49 V . E X P E R I M E N T 3 : E F F E C T OF K E T A N S E R I N ON K E T A M I N E A N A L G E S I A E x p e r i m e n t 3 was d e s i g n e d t o a s s e s s t h e e f f e c t o f ^S2 r e c e p t o r b l o c k a d e o n a n a l g e s i a t h a t i s n o t m e d i a t e d b y a d e s c e n d i n g i n h i b i t o r y s y s t e m a c t i v a t e d b y s u p r a s p i n a l o p i o i d r e c e p t o r s . The a n a l g e s i c , k e t a m i n e a p p e a r s t o e x e r t i t s a n a l g e s i c e f f e c t b y a c t i n g d i r e c t l y o n s p i n a l r e c e p t o r s ( O k u d a , 1 9 8 6 ; S m i t h , P e r r o t t i , M a n s e l l , & M o n r o e , 1 9 8 5 ) . T h r e e f i n d i n g s s u g g e s t t h a t i t d o e s n o t p r o d u c e i t s a n a l g e s i c e f f e c t b y a c t i v a t i n g a d e s c e n d i n g i n h i b i t o r y p a t h w a y . F i r s t , a l t h o u g h t r a n s e c t i o n o f t h e s p i n a l c o r d a t t e n u a t e s m o r p h i n e - p r o d u c e d a n a l g e s i a ( I r w i n e t a l . , 1 9 5 1 ; T a k a g i e t a l . , 1 9 5 5 ) , s p i n a l c o r d t r a n s e c t i o n p r o d u c e s a 9 - f o l d i n c r e a s e i n t h e p o t e n c y o f k e t a m i n e a s a n a n a l g e s i c ( P e k o e & S m i t h , 1 9 8 2 ) . S e c o n d , s p i n a l c o r d t r a n s e c t i o n e n h a n c e s t h e i n h i b i t o r y e f f e c t o f k e t a m i n e o n d o r s a l h o r n n o c i c e p t i v e n e u r o n s ( O k u d a , 1 9 8 6 ) . A n d t h i r d , n a l o x o n e i n j e c t e d i n t o t h e PAG a t t e n u a t e s m o r p h i n e - p r o d u c e d a n a l g e s i a b u t n o t k e t a m i n e - p r o d u c e d a n a l g e s i a ( S m i t h e t a l . , 1 9 8 5 ) . M e t h o d T h e 15 r a t s s e r v i n g a s s u b j e c t s r e c e i v e d 5 d a y s o f b a s e l i n e t e s t i n g . T h e n t h e a n a l g e s i c e f f e c t o f 100 m g / k g k e t a m i n e h y d r o c h l o r i d e was c h a l l e n g e d w i t h 10 m g / k g k e t a n s e r i n , t h e d o s e o f k e t a n s e r i n t h a t was f o u n d i n E x p e r i m e n t 2 t o b l o c k m o r p h i n e -p r o d u c e d a n a l g e s i a . 50 R e s u l t s F i g u r e 3 i l l u s t r a t e s t h e r e s u l t s o f E x p e r i m e n t 3 . I n j e c t i o n o f 100 m g / k g o f k e t a m i n e p r o d u c e d a p o t e n t a n a l g e s i a a t r t h e 3 0 -a n d 6 0 - m i n t e s t i n t e r v a l s . T h e m a j o r f i n d i n g o f t h i s e x p e r i m e n t was t h a t a d o s e o f k e t a n s e r i n t h a t a t t e n u a t e d m o r p h i n e - p r o d u c e d a n a l g e s i a i n E x p e r i m e n t 2 , d i d n o t s i g n i f i c a n t l y a t t e n u a t e k e t a m i n e - p r o d u c e d a n a l g e s i a . T h e o v e r a l l ANOVA r e v e a l e d a s i g n i f i c a n t m a i n e f f e c t o f t r e a t m e n t ( F ( 3 , 4 5 ) = 1 3 . 8 , p < . 0 0 0 1 ) . T h e a n a l g e s i c e f f e c t o f k e t a m i n e was c o n f i r m e d b y t h e s i g n i f i c a n c e o f t h e o v e r a l l d i f f e r e n c e b e t w e e n t h e v e h i c l e - v e h i c l e a n d t h e k e t a m i n e - v e h i c l e c o n d i t i o n s ( N e w m a n - K e u l ' s p < . 0 5 ) . K e t a m i n e p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n t a i l - f l i c k l a t e n c y a t t h e 3 0 - a n d 6 0 - m i n t e s t i n t e r v a l s ( b o t h N e w m a n - K e u l 1 s p < . 0 5 ) . T h e l a c k o f s i g n i f i c a n t e f f e c t o f k e t a n s e r i n o n k e t a m i n e -p r o d u c e d a n a l g e s i a was i n d i c a t e d b y t h e l a c k o f s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e mean t a i l - f l i c k l a t e n c i e s o f t h e r a t s i n t h e k e t a m i n e - k e t a n s e r i n c o n d i t i o n a n d t h e k e t a m i n e - v e h i c l e c o n d i t i o n ( a l l f o u r N e w m a n - K e u l ' s p > . 0 5 ) . A s i n E x p e r i m e n t 2 t h e mean t a i l - f l i c k l a t e n c i e s o f r a t s i n t h e v e h i c l e - k e t a n s e r i n c o n d i t i o n w e r e a l l s h o r t e r t h a n t h o s e i n t h e v e h i c l e - v e h i c l e c o n d i t i o n a l t h o u g h n o n e o f t h e s e d i f f e r e n c e s was s t a t i s t i c a l l y s i g n i f i c a n t ( a l l f i v e N e w m a n - K e u l ' s p > . 0 5 ) . 51 Figure 3 . The analgesic e f f e c t of 1 0 0 mg/kg of ketamine HCI challenged by 1 0 mg/kg of ketanserin HCI. Mean t a i l - f l i c k latencies ( N = 1 5 ) were assessed 0 , 3 0 , 6 0 , 9 0 , and 1 2 0 mrn a f t e r injections i n the four treatment conditions. The analgesic e f f e c t of ketamine i s i l l u s t r a t e d by the difference between the VEH-VEH and KTA-VEH conditions. The e f f e c t of ketanserin by i t s e l f on mean t a i l - f l i c k latency i s i l l u s t r a t e d by the difference between the VEH-VEH and VEH-KET conditions. The attenuation of ketamine-produced analgesia by ketanserin i s i l l u s t r a t e d by the difference between the KTA-VEH and KTA-KET conditions. 'O' VEH-VEH KTA -VEH VEH-KET KTA -KET 1 I J 0 30 60 90 120 TIME POST - INJECTION (min) T h e 2 X 2 ANOVA r e v e a l e d t h a t t h e s h i f t i n b a s e l i n e f o l l o w i n g t h e v e h i c l e - k e t a n s e r i n i n j e c t i o n s d o e s n o t a c c o u n t f o r t h e o b s e r v e d a t t e n u a t i o n o f k e t a m i n e - p r o d u c e d a n a l g e s i a s T h e m a i n e f f e c t f o r k e t a n s e r i n was n o t s i g n i f i c a n t ( F ( 1 , 1 5 ) = 0 . 5 6 , p > . 0 5 ; ) . L i k e w i s e , t h e k e t a m i n e x p i r e n p e r o n e i n t e r a c t i o n was n o t s i g n i f i c a n t ( F ( 1 , 1 5 ) = 1 . 6 5 , p > . 0 5 ) . D i s c u s s i o n T h e r e s u l t s o f E x p e r i m e n t 3 show t h a t b l o c k a d e o f S2 r e c e p t o r s w i t h k e t a n s e r i n d o e s n o t a t t e n u a t e k e t a m i n e - p r o d u c e d a n a l g e s i a . T h i s f i n d i n g e s t a b l i s h e d t h a t t h e a n t i - a n a l g e s i c e f f e c t o f k e t a n s e r i n i s n o t c o m p l e t e l y g e n e r a l a n d s u g g e s t s t h a t i t m i g h t be s p e c i f i c t o m o r p h i n e - p r o d u c e d a n a l g e s i a . 54 VI. EXPERIMENT 4: EFFECT OF PIRENPERONE ON MORPHINE ANALGESIA IN SPINAL RATS Because S2 receptors have not been found i n the spinal cord, the finding of Experiment 1 that pirenperone blocks morphine-produced analgesia suggests that S2 receptors that mediate the ef f e c t are i n the brain or the peripheral nervous system, or both. In Experiment 2, ketanserin, a pirenperone-like drug that does not readily enter the central nervous system, attenuated morphine-produced analgesia only at high doses, thus suggesting that the S2 receptors that mediate morphine-produced analgesia are i n the central nervous system. Together, the res u l t s of Experiments 1 and 2 provide evidence that S2 receptors that mediate morphine-produced analgesia are i n the brain. The finding of Experiment 3 that even a high dose of ketanserin did not attenuate the analgesic e f f e c t of ketamine, a drug that produces i t s analgesic e f f e c t by acting d i r e c t l y on spinal cord receptors, provided further support for a t h i s hypothesis. Experiment 4 was designed to test the cerebral S2 hypothesis of morphine-produced analgesia by assessing the e f f e c t of S2 receptor blockade with pirenperone on morphine-produced analgesia i n rats with transected spinal cords. Transection of the spinal cord i s o l a t e s the opioid analgesia c i r c u i t r y of the spinal cord from the influence of descending i n h i b i t i o n or e x c i t a t i o n o r i g i n a t i n g i n the brain. If the hypothesis that pirenperone produces i t s anti-analgesic e f f e c t v i a supraspinal receptors i s correct, then pirenperone should have no e f f e c t on morphine-55 p r o d u c e d a n a l g e s i a i n r a t s w i t h s p i n a l c o r d t r a n s e c t i o n s . On t h e o t h e r h a n d , i f p i r e n p e r o n e i s f o u n d t o b l o c k m o r p h i n e - p r o d u c e d a n a l g e s i a , t h e n a r o l e f o r s p i n a l r e c e p t o r s w o u l d be i m p l i c a t e d . M e t h o d T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e o r s a l i n e i n j e c t e d i n t r a p e r i t o n e a l l y was a s s e s s e d i n r a t s w i t h o r w i t h o u t s p i n a l c o r d t r a n s e c t i o n s t h a t w e r e i n j e c t e d s u b c u t a n e o u s l y w i t h 0 . 2 4 m g / k g o f p i r e n p e r o n e o r i t s c i t r a t e v e h i c l e . F o l l o w i n g 6 d a y s o f b a s e l i n e t e s t i n g , t h e 141 r a t s s e r v i n g a s s u b j e c t s w e r e a s s i g n e d t o e i t h e r t h e s p i n a l c o r d t r a n s e c t i o n c o n d i t i o n o r t h e s h a m - s u r g e r y c o n d i t i o n a s n e e d e d . S u r g e r y was c a r r i e d o u t u n d e r p e n t o b a r b i t o l a n e s t h e s i a . S p i n a l c o r d s w e r e t r a n s e c t e d a t t h e T 1 2 - L 1 l e v e l b y e x p o s i n g t h e v e r t e b r a e , r e m o v i n g t h e s p i n o u s p r o c e s s , a n d t r a n s e c t i n g t h e c o r d w i t h a s p a t u l a . C o m p l e t e t r a n s e c t i o n w a s c o n f i r m e d v i s u a l l y b e f o r e s u t u r i n g t h e m u s c l e a n d c l o s i n g t h e i n c i s i o n w i t h w o u n d c l i p s . The v e r t e b r a e o f t h e s h a m - s u r g e r y c o n t r o l s w e r e e x p o s e d , b u t t h e s p i n o u s p r o c e s s was l e f t i n t a c t . A l l t e s t i n g was c o n d u c t e d n o t l e s s t h a n 6 h r b u t n o t m o r e t h a n 12 h r a f t e r s u r g e r y . E l e v e n r a t s d i e d f r o m t h e a n e s t h e t i c a n d f o r t y - f o u r r a t s d i e d o r d i d n o t r e s p o n d t o t h e h o t w a t e r a f t e r s p i n a l c o r d t r a n s e c t i o n . T h e e i g h t y - s i x r a t s s u r v i v i n g t h e o p e r a t i o n w e r e a s s i g n e d t o o n e o f f o u r t r e a t m e n t c o n d i t i o n s i n e a c h o f t h e t w o s u r g i c a l c o n d i t i o n s . T h e r e w e r e 10 r a t s w i t h t r a n s e c t i o n s i n t h e v e h i c l e - v e h i c l e a n d t h e v e h i c l e - p i r e n p e r o n e g r o u p s a n d 11 r a t s i n t h e m o r p h i n e - v e h i c l e a n d t h e m o r p h i n e - p i r e n p e r o n e g r o u p s . T h e r e 56 w e r e 11 s h a m - o p e r a t e d r a t s i n e a c h o f t h e f o u r t r e a t m e n t g r o u p s . R e s u l t s ~~ T h e r e s u l t s o f E x p e r i m e n t 4 a r e i l l u s t r a t e d i n F i g u r e 4 . I n j e c t i o n o f 10 m g / k g o f m o r p h i n e s u l p h a t e b y i t s e l f p r o d u c e d p o t e n t a n a l g e s i a , a s i n d i c a t e d b y t h e s u b s t a n t i a l l y g r e a t e r t a i l -f l i c k l a t e n c i e s o f t h e r a t s i n t h e m o r p h i n e - v e h i c l e g r o u p r e l a t i v e t o r a t s i n t h e v e h i c l e - v e h i c l e g r o u p a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s . P i r e n p e r o n e h a d t w o m a j o r e f f e c t s . F i r s t , i t s i g n i f i c a n t l y a t t e n u a t e d t h e m o r p h i n e - p r o d u c e d a n a l g e s i a i n t h e s h a m - s u r g e r y a n i m a l s b u t n o t i n t h e r a t s w i t h s p i n a l c o r d t r a n s e c t i o n s . S e c o n d , p i r e n p e r o n e b y i t s e l f p r o d u c e d a s i g n i f i c a n t h y p e r a l g e s i a i n t h e s h a m - s u r g e r y a n i m a l s , b u t a g a i n i t d i d n o t d o s o i n t h e s u b j e c t s w i t h s p i n a l c o r d t r a n s e c t i o n s . F o r b o t h s u r g e r y c o n d i t i o n s , t h e o v e r a l l ANOVAs r e v e a l e d s i g n i f i c a n t m a i n e f f e c t s f o r t r e a t m e n t ( s h a m - s u r g e r y c o n d i t i o n , F ( 3 , 4 0 ) = 1 4 . 0 , p < . 0 0 0 1 ; t r a n s e c t i o n c o n d i t i o n , F ( 3 , 3 8 ) = 5 . 8 , p < 0 . 0 1 ) . T h e a n a l g e s i c e f f e c t o f m o r p h i n e was c o n f i r m e d b y t h e s i g n i f i c a n c e o f t h e o v e r a l l d i f f e r e n c e b e t w e e n t h e v e h i c l e -v e h i c l e g r o u p a n d t h e m o r p h i n e - v e h i c l e g r o u p f o r e a c h o f t h e t w o c o n d i t i o n s ( b o t h N e w m a n - K e u l ' s p < . 0 5 ) . I n b o t h c o n d i t i o n s , m o r p h i n e p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n mean t a i l - f l i c k l a t e n c y a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s ( a l l s i x N e w m a n - K e u l 1 s p < . 0 5 ) . F o r r a t s i n t h e s h a m - s u r g e r y c o n d i t i o n , t h e t a i l - f l i c k l a t e n c i e s a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s w e r e F i g u r e 4 . T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h 0 . 2 4 m g / k g o f p i r e n p e r o n e i n r a t s w i t h t r a n s e c t e d s p i n a l c o r d s a n d i n s h a m - s u r g e r y c o n t r o l s . M e a n t a i l - f l i c k l a t e n c i e s w e r e a s s e s s e d a t 0 , 3 0 , 6 0 , 9 0 , a n d 120 m i n a f t e r i n j e c t i o n s i n e a c h o f t h e f o u r t r e a t m e n t c o n d i t i o n s . T h e g r a p h l a b e l e d SHAM r e p r e s e n t s t h e r a t s i n t h e s h a m - s u r g e r y g r o u p a n d t h e g r a p h l a b e l e d S P I N A L r e p r e s e n t s t h e r a t s w i t h t r a n s e c t e d s p i n a l c o r d s (n=10 f o r t h e V E H - V E H a n d V E H - P I R t r e a t m e n t s i n t h e S P I N A L c o n d i t i o n , n=11 f o r a l l o t h e r t r e a t m e n t s ) . T h e a n a l g e s i c e f f e c t o f m o r p h i n e i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d M O R - V E H g r o u p s . T h e e f f e c t o f p i r e n p e r o n e b y i t s e l f o n mean t a i l - f l i c k l a t e n c y i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d V E H - P I R g r o u p s . T h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y p i r e n p e r o n e i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e M O R - V E H a n d M O R - P I R g r o u p s . SHAM © O z 111 6 g LL z < UJ 8 « £ " O " V E H - V E H M O R - V E H - A - V E H - P I R - A - M O R - P I R I I SPINAL X J . ± ± J 30 60 90 120 0 30 TIME POST-INJECTION (min) 60 90 120 Oo 59 s i g n i f i c a n t l y shorter i n animals injected with morphine and pirenperone than i n animals treated with morphine by i t s e l f ( a l l three Newman-Keul's p<.05). In contrast, at no test i n t e r v a l were the t a i l - f l i c k latencies of rats i n the transection condition that were injected with morphine and pirenperone s i g n i f i c a n t l y d i f f e r e n t than the latencies of those injected with morphine alone ( a l l four Newman-Keul's p>.05). Although i n the sham-surgery condition, the mean t a i l - f l i c k latencies of rats that were treated with pirenperone alone were s i g n i f i c a n t l y shorter than those of rats receiving the vehicle-vehicle combination of in j e c t i o n s ( a l l four Newman-Keul's p<.05), rats i n the spinal-cord-transection condition injected with t h i s dose of pirenperone did not d i f f e r s i g n i f i c a n t l y from those i n the vehicle-vehicle treatment group at any of the po s t - i n j e c t i o n i n t e r v a l s ( a l l four Newman-Keul1s p>.05). The 2 X 2 ANOVA revealed that the s h i f t i n baseline following the vehicle-pirenperone inje c t i o n s i n the sham-surgery group may account for 'the observed attenuation of morphine-produced analgesia. Although the main e f f e c t for pirenperone was s i g n i f i c a n t (F(1,40)=17.76, p<.005), the morphine x pirenperone interactions was not (F(1,40)=1.49 p>.05). Neither the main e f f e c t for pirenperone nor the morphine x pirenperone i n t e r a c t i o n was s i g n i f i c a n t i n the spinal transection condition (both p>.05). Discussion In Experiment 4, the s e l e c t i v e S2 receptor blocker, pirenperone attenuated the analgesic e f f e c t of morphine i n 60 c o n t r o l r a t s b u t n o t i n t h o s e w i t h t r a n s e c t e d s p i n a l c o r d s . M o r e o v e r , i n c o n t r a s t t o t h e h y p e r a l g e s i c e f f e c t o f p i r e n p e r o n e s e e n i n r a t s i n t h e s h a m - s u r g e r y c o n d i t i o n , i n j e c t i o n o f p i r e n p e r o n e a n d t h e v e h i c l e d i d n o t a f f e c t n o c i c e p t i o n i n t h e t r a n s e c t i o n c o n d i t i o n . T h e f i n d i n g s i n t h e s h a m - s u r g e r y c o n d i t i o n c o n f i r m t h o s e o f E x p e r i m e n t 1 . B e c a u s e t r a n s e c t i o n o f t h e s p i n a l c o r d i s o l a t e s t h e o p i o i d a n a l g e s i a c i r c u i t r y o f t h e s p i n a l c o r d f r o m t h e i n f l u e n c e o f d e s c e n d i n g i n h i b i t i o n o r e x c i t a t i o n o r i g i n a t i n g i n t h e b r a i n , t h e s e r e s u l t s p r o v i d e f u r t h e r e v i d e n c e t h a t S2 r e c e p t o r b l o c k a d e d o e s n o t p r o d u c e i t s a n t i - a n a l g e s i c a n d h y p e r a l g e s i c e f f e c t s v i a s p i n a l c o r d r e c e p t o r s . C o n s i d e r i n g t h e l a c k o f S2 r e c e p t o r s i n t h e s p i n a l c o r d ( M o n r o e & S m i t h , 1 9 8 2 ; P a z o s e t a l . , 1 9 8 5 ) t h e s e r e s u l t s a r e n o t s u r p r i s i n g . T h e y d o , h o w e v e r , r u n c o u n t e r t o t h e p r e v a i l i n g t h e o r y t h a t s e r o t o n i n m e d i a t e s m o r p h i n e - p r o d u c e d a n a l g e s i a a n d m o d u l a t e s n o c i c e p t i o n v i a s p i n a l c o r d s e r o t o n e r g i c r e c e p t o r s . T h e r e i s , h o w e v e r , a n o t h e r p o s s i b l e i n t e r p r e t a t i o n f o r t h i s f i n d i n g . T h e r a t s w i t h t r a n s e c t e d s p i n a l c o r d s h a d s u b s t a n t i a l l y s h o r t e r t a i l - f l i c k l a t e n c i e s , a n d t h e s e s h o r t b a s e l i n e s may h a v e b e e n l e s s s e n s i t i v e t o a n t i - a n a l g e s i c e f f e c t s . T h i s p o s s i b i l i t y c o u l d b e a s s e s s e d b y d e c r e a s i n g t h e t e m p e r a t u r e o f t h e w a t e r b a t h t o e s t a b l i s h h i g h e r b a s e l i n e s . 61 V I I . E X P E R I M E N T 5 : E F F E C T OF L Y 5 3 8 5 7 ON MORPHINE A N A L G E S I A T h e s e r o t o n i n a n t a g o n i s t s , p i r e n p e r o n e a n d k e t a n s e r i n a r e p o t e n t b l o c k e r s o f S2 r e c e p t o r s i t e s b u t h a v e v i r t u a l l y no a c t i v i t y a t S1 s i t e s . H o w e v e r , t h e s e t w o s e r o t o n i n a n t a g o n i s t s h a v e b e e n r e c e n t l y f o u n d t o b e m o d e r a t e l y p o t e n t b l o c k e r s o f a l p h a - a d r e n e r g i c r e c e p t o r s ( J a n s s e n , 1 9 8 3 ) . A p p r o x i m a t e l y 10% o f t h e b i n d i n g o f p i r e n p e r o n e a n d k e t a n s e r i n i s a t a l p h a - r e c e p t o r s . M o r e o v e r , some o f t h e e f f e c t s o f k e t a n s e r i n h a v e b e e n a t t r i b u t e d t o t h i s d r u g ' s a c t i v i t y a s a b l o c k e r o f a l p h a - r e c e p t o r s , r a t h e r t h a n t o i t s a c t i v i t y a t S2 r e c e p t o r s ( F o z a r d , 1 9 8 2 ; V a n h o u t t e e t a l . , 1 9 8 6 ) . I n E x p e r i m e n t s 1 , 2 , a n d 4 ( s e e a l s o P a u l & P h i l l i p s , 1 9 8 6 ) , p i r e n p e r o n e a n d k e t a n s e r i n b l o c k e d m o r p h i n e - p r o d u c e d a n a l g e s i a . H o w e v e r , i t i s n o t c l e a r w h e t h e r t h e s e e f f e c t s w e r e t h e r e s u l t o f t h e e f f e c t s o f t h e s e d r u g s o n S2 r e c e p t o r s o r t h e r e s u l t o f t h e i r r e c e n t l y d i s c o v e r e d e f f e c t s o n a l p h a - a d r e n e r g i c r e c e p t o r s ( N e a l & S p a r b e r , 1 9 8 6 ; P a u l & P h i l l i p s , 1 9 8 6 ) . A c c o r d i n g l y , E x p e r i m e n t 5 a s s e s s e d t h e e f f e c t o n m o r p h i n e - p r o d u c e d a n a l g e s i a o f L Y 5 3 8 5 7 , a s e l e c t i v e S2 a n t a g o n i s t w i t h a 3 0 0 , 0 0 0 - f o l d g r e a t e r a f f i n i t y f o r S2 r e c e p t o r s t h a n f o r a l p h a - a d r e n e r g i c r e c e p t o r s ( C o h e n , F u l l e r , & K u r t z , 1 9 8 3 ) . M e t h o d T h e 10 r a t s s e r v i n g a s s u b j e c t s r e c e i v e d 6 d a y s o f b a s e l i n e t e s t i n g . T h e n t h e y w e r e i n j e c t e d w i t h 10 m g / k g ( I P ) o f m o r p h i n e s u l p h a t e o r i t s s a l i n e v e h i c l e i n c o m b i n a t i o n w i t h 10 m g / k g ( I P ) o f L Y 5 3 8 5 7 o r i t s s a l i n e v e h i c l e . B e c a u s e o f t h e l o w s o l u b i l i t y o f L Y 5 3 8 5 7 , t h i s d r u g was d i s s o l v e d t o a c o n c e n t r a t i o n o f 10 rag/ml i n s a l i n e , a n d i n j e c t e d i n t r a p e r i t o n e a l l y i n a v o l u m e o f 1 m l / k g , r a t h e r t h a n t h e 0 . 2 m g / m l SC i n j e c t i o n s u s e d t h r o u g h o u t t h e s e e x p e r i m e n t s . R e s u l t s F i g u r e 5 i l l u s t r a t e s t h e r e s u l t s o f E x p e r i m e n t 5 . I n j e c t i o n o f 10 m g / k g o f m o r p h i n e s u l p h a t e b y i t s e l f p r o d u c e d p o t e n t a n a l g e s i a , a s i n d i c a t e d b y t h e s u b s t a n t i a l l y i n c r e a s e d mean t a i l -f l i c k l a t e n c i e s a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s i n t h e m o r p h i n e - v e h i c l e c o n d i t i o n i n c o m p a r i s o n t o t h o s e i n t h e v e h i c l e -v e h i c l e c o n d i t i o n . T h e m a j o r f i n d i n g o f t h i s s t u d y was t h a t when L Y 5 3 8 5 7 was i n j e c t e d w i t h t h e m o r p h i n e , t h e m o r p h i n e - p r o d u c e d a n a l g e s i a was a t t e n u a t e d . A s e c o n d i m p o r t a n t f i n d i n g was t h a t L Y 5 3 8 5 7 i n j e c t e d b y i t s e l f p r o d u c e d h y p e r a l g e s i a ; t h a t i s , t h e t a i l - f l i c k l a t e n c i e s i n t h e v e h i c l e - L Y 5 3 8 5 7 c o n d i t i o n a r e s i g n i f i c a n t l y s h o r t e r t h a n t h o s e i n t h e v e h i c l e - v e h i c l e c o n t r o l c o n d i t i o n . T h e o v e r a l l ANOVA r e v e a l e d a s i g n i f i c a n t m a i n e f f e c t f o r t r e a t m e n t ( F ( 3 , 2 7 ) = 1 4 . 7 , p < . 0 0 0 1 ) . T h e a n a l g e s i c e f f e c t o f m o r p h i n e was c o n f i r m e d b y t h e s i g n i f i c a n c e o f t h e o v e r a l l d i f f e r e n c e b e t w e e n t h e v e h i c l e - v e h i c l e t r e a t m e n t a n d t h e m o r p h i n e - v e h i c l e c o n d i t i o n ( N e w m a n - K e u l ' s p < . 0 5 ) . M o r p h i n e p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n mean t a i l - f l i c k l a t e n c y a t t h e 3 0 - , 6 0 - , a n d 9 0 - m i n t e s t i n t e r v a l s ( a l l t h r e e N e w m a n - K e u l ' s p < . 0 5 ) . A l t h o u g h t h e mean t a i l - f l i c k l a t e n c i e s o f r a t s i n j e c t e d w i t h F i g u r e 5 . T h e a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e c h a l l e n g e d w i t h 10 m g / k g o f L Y 5 3 8 5 7 . M e a n t a i l - f l i c k l a t e n c i e s (n=10) w e r e a s s e s s e d 0 , 30, 60, 90, a n d 120 m i n a f t e r i n j e c t i o n s i n t h e f o u r t r e a t m e n t c o n d i t i o n s . T h e a n a l g e s i c e f f e c t o f m o r p h i n e i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d M O R - V E H c o n d i t i o n s . T h e e f f e c t o f L Y 5 3 8 5 7 b y i t s e l f o n mean t a i l - f l i c k l a t e n c y i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e V E H - V E H a n d V E H - L Y 5 3 8 5 7 c o n d i t i o n s . T h e a t t e n u a t i o n o f m o r p h i n e -p r o d u c e d a n a l g e s i a b y L Y 5 3 8 5 7 i s i l l u s t r a t e d b y t h e d i f f e r e n c e b e t w e e n t h e M O R - V E H a n d M O R - L Y 5 3 8 5 7 c o n d i t i o n s . 64 TIME POST-INJECTION (min) m o r p h i n e a n d L Y 5 3 8 5 7 w e r e s h o r t e r a t t h e 6 0 - , 9 0 - , a n d 1 2 0 - m i n i n t e r v a l s t h a n when t h e y w e r e t r e a t e d w i t h m o r p h i n e a l o n e , t h i s d i f f e r e n c e r e a c h e d s i g n i f i c a n c e o n l y a t t h e 1 2 0 - m i n i n t e r v a l ( N e w m a n - K e u l ' s p < . 0 1 ) . T h e h y p e r a l g e s i c e f f e c t o f L Y 5 3 8 5 7 was c o n f i r m e d b y t h e s i g n i f i c a n c e o f t h e o v e r a l l d i f f e r e n c e b e t w e e n t h e v e h i c l e -v e h i c l e c o n d i t i o n a n d t h e v e h i c l e - L Y 5 3 8 5 7 c o n d i t i o n (Newman-K e u l ' s p < . 0 5 ) . H o w e v e r , o n l y t h e c o m p a r i s o n a t t h e 9 0 - m i n t e s t i n t e r v a l was s i g n i f i c a n t ( N e w m a n - K e u l ' s p < . 0 5 ) . D i s c u s s i o n I n E x p e r i m e n t 5 , L Y 5 3 8 5 7 p r o d u c e d a s i g n i f i c a n t o v e r a l l a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a , a n d i t p r o d u c e d a s i g n i f i c a n t o v e r a l l h y p e r a l g e s i a . B e c a u s e L Y 5 3 8 5 7 i s a n S2 r e c e p t o r b l o c k e r w i t h no a c t i v i t y a t a l p h a - a d r e n e r g i c r e c e p t o r s , t h i s p a t t e r n o f r e s u l t s p r o v i d e s e v i d e n c e t h a t t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y p i r e n p e r o n e , k e t a n s e r i n , a n d L Y 5 3 8 5 7 i s n o t d u e t o t h e e f f e c t o f t h e s e d r u g s o n a l p h a -a d r e n e r g i c r e c e p t o r s . L i k e w i s e , b e c a u s e L Y 5 3 8 5 7 b y i t s e l f p r o d u c e d h y p e r a l g e s i a , t h i s s t u d y p r o v i d e s e v i d e n c e t h a t t h e h y p e r a l g e s i a p r o d u c e d b y S2 r e c e p t o r b l o c k e r s i s n o t m e d i a t e d b y a l p h a - r e c e p t o r s . I t s eems t h a t t h e h y p e r a l g e s i c a n d a n t i -a n a l g e s i c e f f e c t s o f S2 r e c e p t o r b l o c k e r s a r e i n d e e d m e d i a t e d b y S2 r e c e p t o r s . L Y 5 3 8 5 7 p r o d u c e d a s i g n i f i c a n t o v e r a l l a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a a n d a s i g n i f i c a n t o v e r a l l h y p e r a l g e s i a , t h a t was s i g n i f i c a n t o n l y a t t h e 1 2 0 - m i n i n t e r v a l . 66 It appears, then, that t h i s drug may have a d i f f e r e n t time course than pirenperone and ketanserin. But i t i s also possible that the anti-analgesic e f f e c t of LY53857 was not observed u n t i l the l a t e r test i n t e r v a l s because t h i s drug was injected i n t r a p e r i t o n e a l l y , rather than subcutaneously. V I I I . D I S C U S S I O N T h e g e n e r a l g o a l o f t h i s d i s s e r t a t i o n was t o c l a r i f y t h e r o l e o f S2 r e c e p t o r s i n t h e p r o d u c t i o n o f m o r p h i n e a n a l g e s i a . F i v e e x p e r i m e n t s w e r e c o n d u c t e d t o a c h i e v e t h i s g o a l . The f i r s t f o u r e x p e r i m e n t s w e r e d e s i g n e d t o l o c a l i z e t h e S2 r e c e p t o r s t h a t a r e i n v o l v e d i n t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a ; w h e r e a s , t h e f i f t h was d e s i g n e d t o t e s t t h e p o s s i b i l i t y t h a t t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y S2 r e c e p t o r b l o c k e r s i s d u e t o t h e i r a c t i o n a t a l p h a - a d r e n e r g i c r e c e p t o r s . The g e n e r a l d i s c u s s i o n i s d i v i d e d i n t o f o u r s e c t i o n s . I n t h e f i r s t s e c t i o n , t h e r e s u l t s a r e s u m m a r i z e d a n d i n t e g r a t e d , a n d i t i s a r g u e d ' t h a t t o g e t h e r t h e y 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 m a j o r c o n c l u s i o n o f t h e d i s s e r t a t i o n : t h a t S2 r e c e p t o r s i n t h e b r a i n a r e i n v o l v e d i n m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e s e c o n d s e c t i o n d e s c r i b e s t h e r e s u l t s o f a r e c e n t l y c o m p l e t e d p i l o t s t u d y t h a t s u p p o r t s t h i s v i e w . T h e t h i r d c o n s i d e r s t h e i m p l i c a t i o n s o f t h i s c o n c l u s i o n f o r c u r r e n t t h e o r i e s o f n o c i c e p t i o n a n d a n a l g e s i a . A n d t h e f o u r t h c o n s i d e r s i t s g e n e r a l i m p l i c a t i o n s f o r f u t u r e d i r e c t i o n s o f p s y c h o p h a r m a c o l o g i c a l r e s e a r c h . 1 . Summary a n d G e n e r a l D i s c u s s i o n o f t h e E x p e r i m e n t s T h e e x p e r i m e n t s d e s c r i b e d i n t h i s d i s s e r t a t i o n h a d t w o g e n e r a l p u r p o s e s . T h e p u r p o s e o f t h e f i r s t f o u r e x p e r i m e n t s was t o d e t e r m i n e t h e l o c a t i o n o f t h e S2 r e c e p t o r s i n v o l v e d i n t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e p u r p o s e o f E x p e r i m e n t 5 was t o t e s t t h e h y p o t h e s i s t h a t t h e a n t i - a n a l g e s i c e f f e c t o f p i r e n p e r o n e a n d o t h e r S2 a n t a g o n i s t s i s d u e t o t h e i r 68 p u t a t i v e a c t i o n a t a l p h a - a d r e n e r g i c r e c e p t o r s ( J a n s s e n , 1983). A c c o r d i n g l y , t h e r e s u l t s o f t h e f i r s t f o u r e x p e r i m e n t s a n d t h e f i f t h e x p e r i m e n t a r e s e p a r a t e l y s u m m a r i z e d i n t h e f o l l o w i n g two s u b s e c t i o n s . A . G e n e r a l D i s c u s s i o n o f E x p e r i m e n t s 1, 2, 3 f a n d 4. E x p e r i m e n t 1 a s s e s s e d t h e e f f e c t o f t h e S 2 - s e l e c t i v e r e c e p t o r b l o c k e r , p i r e n p e r o n e o n m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e r e s u l t s o f t h i s e x p e r i m e n t c o n f i r m e d a n d e x t e n d e d t h e f i n d i n g o f P a u l a n d P h i l l i p s (1986) t h a t p i r e n p e r o n e a t t e n u a t e s t h e a n a l g e s i c e f f e c t o f m o r p h i n e . H o w e v e r , i n c o n t r a s t t o t h e f i n d i n g s o f P a u l a n d P h i l l i p s , p i r e n p e r o n e by i t s e l f was f o u n d t o p r o d u c e s i g n i f i c a n t h y p e r a l g e s i a . T h e r e a s o n f o r t h i s i n c o n s i s t e n c y i s n o t c l e a r , b u t i t c o u l d be a t t r i b u t a b l e t o m e t h o d o l o g i c a l d i f f e r e n c e s . P a u l a n d P h i l l i p s a s s e s s e d t h e e f f e c t o f p i r e n p e r o n e o n t a i l - f l i c k l a t e n c i e s i n a s i n g l e t e s t , 75 m i n a f t e r i n j e c t i o n , w h e r e a s i n E x p e r i m e n t 1 o f t h e p r e s e n t t h e s i s t h i s e f f e c t was a s s e s s e d f o u r t i m e s a t 30 m i n i n t e r v a l s . B e c a u s e p i r e n p e r o n e - p r o d u c e d h y p e r a l g e s i a was a l s o o b s e r v e d i n E x p e r i m e n t 4, t h e r e i s l i t t l e d o u b t t h a t t h i s i s a b o n a f i d e e f f e c t . B e c a u s e S2 r e c e p t o r s h a v e b e e n shown t o e x i s t i n t h e p e r i p h e r y ( V a n N u e t e n e t a l . , 1981) a s w e l l a s i n t h e b r a i n , i t was n o t c l e a r w h e t h e r t h e a n t i - a n a l g e s i c e f f e c t o f p i r e n p e r o n e was m e d i a t e d by c e n t r a l o r p e r i p h e r a l r e c e p t o r s . I n E x p e r i m e n t 2, t h e a n a l g e s i c e f f e c t o f m o r p h i n e was c h a l l e n g e d w i t h t h e S2 r e c e p t o r b l o c k e r , k e t a n s e r i n . T h i s d r u g i s p h a r m a c o l o g i c a l l y s i m i l a r t o p i r e n p e r o n e , h o w e v e r , i t d o e s n o t r e a d i l y e n t e r t h e 69 c e n t r a l n e r v o u s s y s t e m ( L a d u r o n e t a l . , 1 9 8 2 ) . B e c a u s e k e t a n s e r i n a t t e n u a t e d m o r p h i n e - p r o d u c e d a n a l g e s i a o n l y a t a v e r y h i g h d o s e (10 m g / k g ) , E x p e r i m e n t 2 p r o v i d e d e v i d e n c e t h a t t h e a n t i - a n a l g e s i c e f f e c t o f S2 r e c e p t o r b l o c k a d e i s m e d i a t e d b y r e c e p t o r s w i t h i n t h e c e n t r a l n e r v o u s s y s t e m . A l t h o u g h k e t a n s e r i n a d m i n i s t e r e d by i t s e l f s l i g h t l y r e d u c e d mean t a i l - f l i c k l a t e n c i e s a t a l l t e s t i n t e r v a l s a f t e r d r u g i n j e c t i o n , t h i s h y p e r a l g e s i c e f f e c t d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e . I t was n o t c l e a r f r o m t h e f i r s t t w o e x p e r i m e n t s w h e t h e r t h e a n t i - a n a l g e s i c e f f e c t o f S2 r e c e p t o r b l o c k e r s i s s p e c i f i c t o m o r p h i n e - p r o d u c e d a n a l g e s i a . C a n S2 r e c e p t o r b l o c k e r s a f f e c t a n a l g e s i a p r o d u c e d b y a g e n t s t h o u g h t t o a c t t h r o u g h d i f f e r e n t c i r c u i t s ? M o r p h i n e i s t h o u g h t t o p r o d u c e a t l e a s t p a r t o f i t s a n a l g e s i c e f f e c t t h r o u g h a P A G - t o - N R M - t o - s p i n a l c o r d d e s c e n d i n g i n h i b i t o r y s y s t e m ( F i e l d s & B a s b a u m , 1 9 7 8 ; B a s b a u m & F i e l d s , 1 9 7 8 , 1 9 8 4 ; M a y e r & P r i c e , 1 9 7 6 ) . I n c o n t r a s t , k e t a m i n e i s t h o u g h t t o p r o d u c e i t s a n a l g e s i c e f f e c t t h r o u g h a d i r e c t a c t i o n o n s p i n a l c o r d r e c e p t o r s ( O k u d a , 1 9 8 6 ; S m i t h , P e r r o t t i , M a n s e l l , & M o n r o e , 1 9 8 5 ) . A c c o r d i n g l y , I n E x p e r i m e n t 3 , k e t a m i n e - p r o d u c e d a n a l g e s i a was c h a l l e n g e d w i t h a d o s e o f k e t a n s e r i n t h a t h a d a t t e n u a t e d m o r p h i n e - p r o d u c e d a n a l g e s i a i n E x p e r i m e n t 2 (10 m g / k g ) . The f i n d i n g t h a t k e t a n s e r i n d i d n o t a t t e n u a t e k e t a m i n e -p r o d u c e d a n a l g e s i a e s t a b l i s h e d t h a t t h e a n t i - a n a l g e s i c e f f e c t S2 r e c e p t o r b l o c k a d e i s n o t g e n e r a l a n d t h a t i t may b e s p e c i f i c t o a n a l g e s i a p r o d u c e d b y m o r p h i n e o r o t h e r d r u g s t h a t a c t o n t h e same s y s t e m . A s i n E x p e r i m e n t 2 , k e t a n s e r i n , i n j e c t e d a l o n e , 70 produced a reduction i n mean t a i l - f l i c k latencies at a l l post-i n j e c t i o n test i n t e r v a l s , although again t h i s e f f e c t was not s t a t i s t i c a l l y s i g n i f i c a n t . Ketanserin has produced a — nonsignificant reduction i n t a i l - f l i c k latency on the eight occasions that I have assessed i t s e f f e c t s (the 3 doses of ketanserin i n Experiment 2, once i n Experiment 3, and i n 4 experiments i n Paul, Symons, and P i n e l , unpublished observations). This suggests that ketanserin has a s l i g h t hyperalgesic e f f e c t that may be d i s s o c i a b l e from i t s i n h i b i t o r y e f f e c t on morphine-produced analgesia. The finding of Experiment 2 that the anti-analgesic e f f e c t of S2 receptor blockers i s mediated by t h e i r e f f e c t i n the central nervous system, i n combination with the apparent lack of S2 receptors i n the spinal cord implicates brain S2 receptors i n the mediation of the anti-analgesic e f f e c t s of pirenperone and ketanserin. Experiment 4 addressed t h i s issue d i r e c t l y . In order to confirm that the blockade of morphine-produced analgesia by S2 recptor blockers i s mediated by S2 receptors i n the brain, Experiment 4 assessed the e f f e c t of pirenperone on morphine-produced analgesia i n rats with transected spinal cords. Transection of the spinal cord at the lower thoracic l e v e l i s o l a t e s the spinal c i r c u i t r y that mediates the t a i l - f l i c k r e f l e x from the influence of descending i n h i b i t i o n or e x c i t a t i o n o r i g i n a t i n g i n the brain. In Experiment 4, pirenperone attenuated morphine-produced analgesia i n sham-surgery control rats but not i n rats with transected spinal cords. This confirms 71 t h a t i t i s S2 r e c e p t o r s i n t h e b r a i n t h a t m e d i a t e t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y p i r e n p e r o n e . A l t h o u g h p i r e n p e r o n e p r o d u c e d h y p e r a l g e s i a i n b o t h E x p e r i m e n t s 1~and 4 i n i n t a c t r a t s , i t d i d n o t p r o d u c e h y p e r a l g e s i a i n t h e r a t s w i t h t r a n s e c t e d s p i n a l c o r d s i n E x p e r i m e n t 4 . T h i s r e s u l t s u g g e s t s t h a t S2 r e c e p t o r s i n t h e b r a i n a l s o m e d i a t e p i r e n p e r o n e - p r o d u c e d h y p e r a l g e s i a . I t i s p o s s i b l e , h o w e v e r , t h a t t h e r e d u c e d b a s e l i n e t a i l - f l i c k l a t e n c i e s o b s e r v e d i n r a t s w i t h t r a n s e c t e d s p i n a l c o r d s may h a v e d i s g u i s e d a h y p e r a l g e s i c e f f e c t o f p i r e n p e r o n e . I n s u m m a r y , when c o n s i d e r e d t o g e t h e r , t h e r e s u l t s o f E x p e r i m e n t s 1 , 2 , 3 , a n d 4 p r o v i d e e v i d e n c e t h a t t h e a n t i -a n a l g e s i c e f f e c t s o f t h e S2 r e c e p t o r b l o c k e r s , p i r e n p e r o n e a n d k e t a n s e r i n a r e m e d i a t e d b y S2 r e c e p t o r s i n t h e b r a i n . I t i s n o t c l e a r , h o w e v e r , i f t h e m i l d h y p e r a l g e s i a p r o d u c e d b y t h e s e S2 a n t a g o n i s t s i s m e d i a t e d b y t h e s e same s u p r a s p i n a l S2 r e c e p t o r s . B . G e n e r a l D i s c u s s i o n o f E x p e r i m e n t 5 . The s e r o t o n i n a n t a g o n i s t s , p i r e n p e r o n e a n d k e t a n s e r i n a r e s e l e c t i v e i n t h e s e n s e t h a t t h e y a r e p o t e n t b l o c k e r s o f S2 r e c e p t o r s i t e s b u t h a v e v i r t u a l l y no a c t i v i t y a t S1 s i t e s ( C o l p a e r t & J a n s s e n , 1 9 8 2 ; C o l p a e r t e t a l . , 1 9 8 3 ; L e y s e n e t a l . , 1 9 8 2 ) . H o w e v e r , r e c e n t l y b o t h p i r e n p e r o n e a n d k e t a n s e r i n h a v e b e e n f o u n d t o a l s o be m o d e r a t e l y p o t e n t b l o c k e r s o f a l p h a - a d r e n e r g i c r e c e p t o r s ( J a n s s e n , 1 9 8 3 ) . T h u s , i t i s n o t c l e a r w h e t h e r t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y p i r e n p e r o n e a n d k e t a n s e r i n o b s e r v e d i n E x p e r i m e n t s 1 , 2 , a n d 4 was m e d i a t e d b y t h e i r b l o c k a d e o f S2 r e c e p t o r s o r b y t h e i r a c t i o n a t a l p h a - a d r e n e r g i c 72 r e c e p t o r s . The r e c e n t l y s y n t h e s i z e d s e r o t o n i n r e c e p t o r b l o c k e r , L Y 5 3 8 5 7 b i n d s w i t h h i g h a f f i n i t y t o S2 r e c e p t o r s b u t h a s v i r t u a l l y no a c t i o n a t e i t h e r S1 r e c e p t o r s o r a t a l p h a - ~ a d r e n e r g i c r e c e p t o r s ( C o h e n e t a l . , 1 9 8 3 ) . A c c o r d i n g l y , t h e p u r p o s e o f E x p e r i m e n t 5 was t o t e s t t h e " a l p h a - a d r e n e r g i c " a l t e r n a t i v e h y p o t h e s i s b y a s s e s s i n g t h e e f f e c t o f L Y 5 3 8 5 7 o n n o c i c e p t i o n a n d m o r p h i n e - p r o d u c e d a n a l g e s i a . L i k e p i r e n p e r o n e a n d k e t a n s e r i n , L Y 5 3 8 5 7 a t t e n u a t e d m o r p h i n e - p r o d u c e d a n a l g e s i a , a n d i t p r o d u c e d s i g n i f i c a n t h y p e r a l g e s i a . T h e s e r e s u l t s i n d i c a t e t h a t b o t h t h e a n t i - a n a l g e s i c a n d h y p e r a l g e s i c e f f e c t s o f S2 r e c e p t o r b l o c k e r s a r e m e d i a t e d b y S2 r e c e p t o r s r a t h e r t h a n a l p h a - a d r e n e r g i c r e c e p t o r s . 2 . R e s u l t s o f a P i l o t E x p e r i m e n t t o T e s t D i r e c t l y W h e t h e r  C e n t r a l o r S p i n a l S2 R e c e p t o r s M e d i a t e t h e B l o c k a d e o f M o r p h i n e - P r o d u c e d A n a l g e s i a b y S2 R e c e p t o r B l o c k e r s E x p e r i m e n t 4 p r o v i d e d e v i d e n c e t h a t s p i n a l r e c e p t o r s d o n o t m e d i a t e t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y p i r e n p e r o n e , t h u s i m p l i c a t i n g b y e l i m i n a t i o n s u p r a s p i n a l S2 r e c e p t o r s i n t h i s e f f e c t . T h e r e l a t i v e c o n t r i b u t i o n o f b r a i n a n d s p i n a l c o r d r e c e p t o r s t o t h e h y p e r a l g e s i c a n d a n t i - a n a l g e s i c e f f e c t s o f S2 r e c e p t o r b l o c k a d e w a s a s s e s s e d i n a r e c e n t p i l o t s t u d y ( P a u l , P f a u s , & P i n e l , 1 9 8 7 ) . I n t h i s s t u d y , t h e a n a l g e s i c e f f e c t o f m o r p h i n e was c h a l l e n g e d w i t h k e t a n s e r i n i n j e c t e d i n t o t h e c e r e b r a l v e n t r i c l e s o r i n t o t h e s p i n a l s u b a r a c h n o i d s p a c e . I n t r a v e n t r i c u l a r i n j e c t i o n o f 5 u g o f k e t a n s e r i n a t t e n u a t e d a n a l g e s i c e f f e c t o f 10 m g / k g o f m o r p h i n e s u l p h a t e ( I P ) a t t h e 30 m i n t e s t i n t e r v a l . I n c o n t r a s t , 5 u g o f k e t a n s e r i n i n j e c t e d i n t r a t h e c a l l y e n h a n c e d m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e f i n d i n g t h a t i n t r a v e n t r i c u l a r i n j e c t i o n o f k e t a n s e r i n a t t e n u a t e s m o r p h i n e - p r o d u c e d a n a l g e s i a p r o v i d e s e v i d e n c e t h a t S2 r e c e p t o r s i n t h e b r a i n m e d i a t e t h e a n t i - a n a l g e s i c e f f e c t o f k e t a n s e r i n a n d p i r e n p e r o n e . B e c a u s e t h e r e i s no p r e c e d e n t f o r t h e o b s e r v e d e n h a n c e m e n t o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y i n t r a t h e c a l i n j e c t i o n o f k e t a n s e r i n t h i s e f f e c t r e q u i r e s r e p l i c a t i o n . N e i t h e r i n t r a v e n t r i c u l a r n o r i n t r a t h e c a l i n j e c t i o n o f k e t a n s e r i n b y i t s e l f h a d a n e f f e c t o n t a i l - f l i c k l a t e n c i e s . I f i t p r o v e s t o be r e p l i c a b l e , t h i s f i n d i n g w o u l d i m p l i c a t e p e r i p h e r a l r e c e p t o r s i n t h e m e d i a t i o n o f t h e h y p e r a l g e s i a p r o d u c e d b y S2 r e c e p t o r b l o c k e r s . 3 . I m p l i c a t i o n s o f t h e P r e s e n t F i n d i n g s f o r T h e o r i e s o f P a i n P e r c e p t i o n a n d A n a l g e s i a I t i s w i d e l y b e l i e v e d t h a t m o r p h i n e p r o d u c e s m o s t o f i t s a n a l g e s i c e f f e c t b y b i n d i n g t o o p i o i d r e c e p t o r s i n t h e P A G , w h i c h i n t u r n a c t i v a t e a n i n h i b i t o r y s y s t e m t h a t d e s c e n d s t o t h e s p i n a l c o r d v i a s y n a p s e s i n t h e NRM ( c f . S o j a & S i n c l a i r , 1 9 8 3 ) . T h i s s y s t e m i s p r e s u m e d t o i n h i b i t i n c o m i n g p a i n s i g n a l s c a r r i e d b y p r i m a r y s o m a t o s e n s o r y n e u r o n s i n t h e d o r s a l h o r n o f t h e s p i n a l c o r d . I t i s t h e s e d o r s a l h o r n i n h i b i t o r y s y n a p s e s t h a t a r e a s s u m e d t o be s e r o t o n e r g i c ( M a y e r & P r i c e , 1 9 7 6 ; F i e l d s & B a s b a u m , 1 9 7 8 ; B a s b a u m & F i e l d s , 1 9 8 4 ; M e s s i n g & L y t l e , 1 9 7 7 ) . 74 T h e v i e w t h a t s p i n a l c o r d s e r o t o n e r g i c s y n a p s e s m e d i a t e m o r p h i n e -p r o d u c e d a n a l g e s i a i s s u p p o r t e d b y t h e f a c t t h a t t h e NRM h a s b e e n i m p l i c a t e d i n t h e a n a l g e s i c e f f e c t o f m o r p h i n e a n d t h e " a x o n s o f s e r o t o n e r g i c NRM c e l l b o d i e s h a v e b e e n s h o w n t o p r o j e c t i n t o t h e d o r s a l h o r n ( B o w k e r , W e s t l u n d , & C o u l t e r , 1 9 8 1 ; D a h l s t r o m & F u x e , 1 9 6 5 ) . M o r e o v e r , i n t r a t h e c a l i n j e c t i o n s o f 5 , 6 -d i h y d r o x y t r y p t a m i n e o r 5 , 7 - d i h y d r o x y t r y p t a m i n e , w h i c h h a v e b e e n f o u n d t o s e l e c t i v e l y d e s t r o y s p i n a l c o r d s e r o t o n i n - c o n t a i n i n g n e u r o n s w i t h o u t a f f e c t i n g f o r e b r a i n n e u r o n s , h a v e b e e n s h o w n t o b l o c k t h e a n a l g e s i c e f f e c t o f m o r p h i n e ( D e a k i n & D o s t r o v s k y , 1 9 7 8 ; K u r a i s h i e t a l . , 1 9 8 3 ) . H o w e v e r , d e s p i t e t h e w i d e a c c e p t a n c e o f t h i s v i e w ( s e e C a r l s s o n , 1 9 8 1 ; K a n d e l & S c h w a r t z , 1 9 8 1 ) , t h e e v i d e n c e t h a t m o r p h i n e - p r o d u c e d a n a l g e s i a i s m e d i a t e d b y s e r o t o n e r g i c s y n a p s e s i n t h e d o r s a l h o r n r e m a i n s e q u i v o c a l ( B e r g e e t a l . , 1 9 8 3 ; R o b e r t s , 1 9 8 4 ; Z e m l a n , 1 9 8 3 ) . Some i n v e s t i g a t o r s h a v e f a i l e d t o c o n f i r m t h a t l e s i o n s o f s p i n a l c o r d s e r o t o n e r g i c n e u r o n s b y i n t r a t h e c a l i n j e c t i o n o f 5 , 6 -d i h y d r o x y t r y p t a m i n e r e s u l t i n a b l o c k a d e o f m o r p h i n e - p r o d u c e d a n a l g e s i a ( K u r a i s h i e t a l . , 1 9 8 3 ; P r o u d f i t & Y a k s h , 1 9 8 0 ) . A l t h o u g h i t h a s n o t r e c e i v e d w i d e r e c o g n i t i o n , s e v e r a l a u t h o r s ( B e r g e e t a l . , 1 9 8 3 ; f o r a r e v i e w s e e R o b e r t s , 1 9 8 4 ) h a v e p r o v i d e d e v i d e n c e t h a t t h e w e l l d o c u m e n t e d i n t e r a c t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a a n d s e r o t o n i n o c c u r s v i a r e c e p t o r s i n t h e b r a i n . F o r e x a m p l e , B e r g e e t a l . ( 1 9 8 3 ) f o u n d t h a t i n r a t s p r e t r e a t e d w i t h t h e s e r o t o n i n u p t a k e b l o c k e r z i m e l i d i n e , p a r a -c h l o r o a m p h e t a m i n e p r o d u c e d s e l e c t i v e l e s i o n s o f s e r o t o n i n -75 c o n t a i n i n g t e r m i n a l s i n t h e b r a i n s t e m . T h e s e s e l e c t i v e l e s i o n s a t t e n u a t e d t h e a n a l g e s i c e f f e c t o f m o r p h i n e , t h u s i m p l i c a t i n g b r a i n s t e m s e r o t o n i n r e c e p t o r s i n t h e m e d i a t i o n o f m o r p h i n e -p r o d u c e d a n a l g e s i a . T h e r e s u l t s o f t h e p r e s e n t e x p e r i m e n t s c o n s t i t u t e w h a t i s a r g u a b l y t h e s t r o n g e s t e v i d e n c e a g a i n s t t h e c u r r e n t v i e w t h a t t h e s e r o t o n e r g i c i n v o l v e m e n t i n m o r p h i n e - p r o d u c e d a n a l g e s i a i s p r i m a r i l y s p i n a l . B e c a u s e t h e r e a r e no S2 r e c e p t o r s i n t h e s p i n a l c o r d ( M o n r o e & S m i t h , 1 9 8 3 ; P a z o s e t a l . , 1 9 8 5 ) , t h e e v i d e n c e p r o v i d e d h e r e t h a t t h e s e r o t o n e r g i c m e d i a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a i n v o l v e s S2 r e c e p t o r s r u l e s o u t t h e p o s s i b i l i t y t h a t o n l y s p i n a l s e r o t o n i n r e c e p t o r s m e d i a t e m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e h y p o t h e s i s o f B e r g e e t a l . ( 1 9 8 3 ) t h a t b r a i n s t e m s e r o t o n i n s y n a p s e s a r e c r u c i a l f o r t h e m e d i a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a i s c o n s i s t e n t w i t h t h e d e n s e S2 b i n d i n g t h a t h a s b e e n f o u n d i n t h e d o r s a l r a p h e n u c l e i ( D e s a m u k h e t a l . , 1 9 8 3 ; L a d u r o n e t a l . , 1 9 8 2 ; M a r c i n k i e w i c z e t a l . , 1 9 8 4 ) . T h i s a r e a h a s b e e n i m p l i c a t e d i n t h e m e d i a t i o n c f m o r p h i n e - p r o d u c e d a n a l g e s i a . T h e f a c t t h a t many o f t h e n e u r o n s t h a t p r o j e c t f r o m t h e PAG t o r a p h e n u c l e i c o n t a i n s e r o t o n i n ( B e i t z , 1 9 8 2 b ) , p r o v i d e s f u r t h e r s u p p o r t f o r t h e h y p o t h e s i s t h a t m o r p h i n e - p r o d u c e d a n a l g e s i a i s p a r t i a l l y m e d i a t e d b y a P A G - t o -r a p h e s e r o t o n e r g i c s y s t e m t h a t s y n a p s e s o n S2 r e c e p t o r s i n t h e r a p h e n u c l e i . B e c a u s e t h e r e i s s t r o n g e v i d e n c e t h a t s e r o t o n e r g i c s y n a p s e s i n t h e s p i n a l c o r d m e d i a t e b o t h i n h i b i t o r y a n d e x c i t a t o r y m o d u l a t i o n o f n o c i c e p t i o n ( J o r d a n , K e n s h a l o , M a r t i n , H a b e r , & W i l l i s , 1 9 7 9 ; Y a k s h e t a l . , 1 9 7 6 ; Y a k s h & W i l s o n , 1 9 7 9 ; Z e m l a n e t a l . , 1 9 8 3 ) , I am n o t p r o p o s i n g t h a t t h e s e r o t o n e r g i c i n v o l v e m e n t i n m o r p h i n e - p r o d u c e d a n a l g e s i a i s e n t i r e l y c e r e b r a l ; my p o i n t i s t h a t t h e r e i s a m a j o r c e r e b r a l c o m p o n e n t . I n f a c t , e x i s t i n g e v i d e n c e s u g g e s t s t h a t t h e r e a r e t h r e e d i f f e r e n t s y s t e m s t h a t u s e s e r o t o n i n t o m o d u l a t e p a i n p e r c e p t i o n . One o f t h e s e s y s t e m s i s a s p i n a l s y s t e m t h a t f a c i l i t a t e s n o c i c e p t i v e r e f l e x e s . The e x i s t e n c e o f t h i s s y s t e m i s s u p p o r t e d b y t h e f i n d i n g s o f Z e m l a n e t a l . ( 1 9 8 3 ) t h a t s y s t e m i c i n j e c t i o n s o f s e r o t o n i n a g o n i s t s i n r a t s w i t h t r a n s e c t e d s p i n a l c o r d s r e d u c e s t a i l - f l i c k l a t e n c i e s . A s e c o n d s e r o t o n e r g i c s y s t e m i s t h o u g h t t o i n h i b i t a s c e n d i n g n o c i c e p t i v e n e u r o n s . E v i d e n c e f o r t h i s s y s t e m comes f r o m t h e f i n d i n g s t h a t i n t r a t h e c a l i n j e c t i o n o f s e r o t o n i n o r s e r o t o n i n a g o n i s t s s u p p r e s s e s w i t h d r a w a l r e f l e x e s t o p a i n f u l s t i m u l i ( W a n g , 1 9 7 7 ; Y a k s h & W i l s o n , 1 9 7 9 ) . B o t h o f t h e s e s y s t e m s a r e a s s u m e d t o i n v o l v e S1 r e c e p t o r s b e c a u s e t h e r e a r e no S2 r e c e p t o r s i n t h e s p i n a l c o r d . T h e r e s u l t s o f t h e p r e s e n t e x p e r i m e n t s s u g g e s t t h a t t h e r e i s a t h i r d , c e r e b r a l s e r o t o n e r g i c s y s t e m m e d i a t i n g t h e a n a l g e s i c e f f e c t o f m o r p h i n e , w h i c h i n v o l v e s S2 r e c e p t o r s . I t i s a l s o p o s s i b l e t h a t s u p r a s p i n a l S1 r e c e p t o r s a r e i n v o l v e d . I n d i r e c t e v i d e n c e s u g g e s t s t h a t t h i s s y s t e m i n v o l v e s s e r o t o n e r g i c n e u r o n s p r o j e c t i n g f r o m t h e PAG t o t h e r a p h e . C l e a r l y , a n i m p o r t a n t d i r e c t i o n f o r f u t u r e r e s e a r c h i s t o m o r e r i g i d l y d e f i n e t h e r o l e s o f s p i n a l a n d s u p r a s p i n a l S1 r e c e p t o r s i n n o c i c e p t i o n a n d m o r p h i n e - p r o d u c e d a n a l g e s i a . 77 4 . G e n e r a l I m p l i c a t i o n s a n d F u t u r e D i r e c t i o n s W i t h t h e d i s c o v e r y t h a t t h e r e a r e s e v e r a l t y p e s o f r e c e p t o r s f o r some n e u r o t r a n s m i t t e r s t h e r e h a s b e e n a c o n c e r t e d e f f o r t t o d e t e r m i n e t h e f u n c t i o n s o f t h e d i f f e r e n t r e c e p t o r t y p e s . T h e s y n t h e s i s o f d r u g s w i t h a p r e f e r e n t i a l a f f i n i t y f o r s p e c i f i c r e c e p t o r t y p e s h a s g r e a t l y f a c i l i t a t e d t h i s l i n e o f r e s e a r c h ( C a r l s s o n , 1 9 8 3 ) . I n v e s t i g a t i o n o f t h e f u n c t i o n a l r o l e o f r e c e p t o r t y p e s h a s b e e n p a r t i c u l a r l y i n t e n s e i n t h e c a s e o f o p i o i d r e c e p t o r s . F o u r t y p e s o f o p i o i d r e c e p t o r s h a v e b e e n i d e n t i f i e d : mu, d e l t a , k a p p a , a n d s i g m a . Many i n v e s t i g a t o r s h a v e a t t e m p t e d t o a t t r i b u t e s u b s e t s o f t h e m u l t i p l e e f f e c t s o f o p i a t e d r u g s t o e a c h o f t h e s e r e c e p t o r t y p e s b y a d m i n i s t e r i n g p e p t i d e s t h a t a r e s e l e c t i v e f o r a p a r t i c u l a r t y p e . For e x a m p l e , mu r e c e p t o r s a r e t h o u g h t t o m e d i a t e t h e c a t a l e p t i c e f f e c t o f o p i a t e s , w h e r e a s , d e l t a r e c e p t o r s a r e t h o u g h t t o m e d i a t e t h e r e s p i r a t o r y d e p r e s s a n t e f f e c t ( P a s t e r n a k e t a l . , 1 9 8 3 ) . R e c e n t l y , s i m i l a r f u n c t i o n a l i n v e s t i g a t i o n s h a v e b e e n c a r r i e d o u t f o r t h e s e r o t o n i n r e c e p t o r t y p e s . T h e d e v e l o p m e n t o f S1 r e c e p t o r a g o n i s t s a n d S2 r e c e p t o r a n t a g o n i s t s h a s g r e a t l y f a c i l i t a t e d t h i s l i n e o f r e s e a r c h . M e n d e l s o n a n d G o r z a l k a ' s ( 1 9 8 5 a , b ; 1 9 8 6 a , b ) a n a l y s i s o f t h e d i f f e r e n t i a l r o l e o f s e r o t o n i n r e c e p t o r t y p e s i n m a l e a n d f e m a l e s e x u a l b e h a v i o r h a s a c c o u n t e d f o r many o f t h e c o n t r a d i c t o r y d a t a i n t h i s a r e a o f r e s e a r c h . S i m i l a r l y , t h e l i n e o f r e s e a r c h o n s p e c i f i c s e r o t o n i n r e c e p t o r t y p e s a n d m o r p h i n e - p r o d u c e d a n a l g e s i a i n i t i a t e d h e r e may e v e n t u a l l y r e s o l v e some o f t h e c o n t r o v e r s i e s i n t h i s f i e l d . 78 T h e u s e o f r e c e p t o r - s e l e c t i v e c o m p o u n d s i n b e h a v i o r a l r e s e a r c h i s n o t , h o w e v e r , w i t h o u t i t s p r o b l e m s . T h e m a i n p r o b l e m i s t h a t t h e r e i s a l w a y s some q u e s t i o n a b o u t t h e s e l e c t i v i t y o f a n y c o m p o u n d . I t s eems t h a t t h e s e l e c t i v i t y o f many d r u g s h a s p r o v e n t o be i n v e r s e l y p r o p o r t i o n a l t o t h e e l a p s e d t i m e s i n c e i t s o r i g i n a l s y n t h e s i s . T h i s i s a s o m e w h a t f l i p p a n t way o f p o i n t i n g o u t t h a t t h e more a d r u g i s s t u d i e d , t h e g r e a t e r t h e n u m b e r o f e f f e c t s i t i s f o u n d t o h a v e . H o w e v e r , d r u g s d o n o t h a v e t o be t o t a l l y s e l e c t i v e t o be o f v a l u e i n p s y c h o p h a r m a c o l o g i c a l r e s e a r c h . P i r e n p e r o n e a n d k e t a n s e r i n a r e a c a s e i n p o i n t . T h e y w e r e o r i g i n a l l y r e p o r t e d t o b e h i g h l y s e l e c t i v e a n t a g o n i s t s o f S2 r e c e p t o r s , w i t h no a c t i v i t y a t S1 r e c e p t o r s ( C o l p a e r t & J a n s s e n , 1 9 8 2 ; C o l p a e r t e t a l . , 1 9 8 3 ; L e y s e n e t a l . , 1 9 8 1 ) . T h i s s e l e c t i v i t y f o r t h e S2 r e c e p t o r t y p e r e l a t i v e t o t h e S1 t y p e h a s h e l d , b u t r e c e n t l y t h e s e d r u g s h a v e b e e n r e p o r t e d t o a l s o b e m o d e r a t e l y p o t e n t b l o c k e r s o f a l p h a - a d r e n e r g i c r e c e p t o r s ( J a n s s e n , 1 9 8 3 ) . F o r t u n a t e l y , t h e m o r e s e l e c t i v e S2 r e c e p t o r b l o c k e r , L Y 5 3 8 5 7 was s y n t h e s i z e d a n d i t h a s t u r n e d o u t t o h a v e no a c t i v i t y a t a l p h a - a d r e n e r g i c r e c e p t o r s ( C o h e n , F u l l e r , & K u r t z , 1 9 8 3 ) . T h u s , b y c o m p a r i n g t h e e f f e c t s o n m o r p h i n e - p r o d u c e d a n a l g e s i a o f p i r e n p e r o n e , k e t a n s e r i n , a n d L Y 5 3 8 5 7 , t h e h y p o t h e s i s t h a t a n a c t i o n a t a l p h a - a d r e n e r g i c r e c e p t o r s was r e s p o n s i b l e f o r t h e a n t i - a n a l g e s i c e f f e c t o f p i r e n p e r o n e a n d k e t a n s e r i n c o u l d b e d i s c o u n t e d . A n o t h e r p r o b l e m e n c o u n t e r e d when u s i n g r e c e p t o r - s e l e c t i v e d r u g s i s t h e c o n t i n u a l d i s c o v e r y o f a d d i t i o n a l r e c e p t o r t y p e s . F o r e x a m p l e , t h e e x i s t e n c e o f a t h i r d s e r o t o n i n r e c e p t o r t y p e ( F o z a r d , 1 9 8 4 ; c f . F e u e r s t e i n & H e r t t i n g , 1 9 8 6 ) , k n o w n a s t h e M r e c e p t o r o r S3 r e c e p t o r , r a i s e s t h e p o s s i b i l i t y t h a t t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a b y p i r e n p e r o n e , k e t a n s e r i n , a n d L Y 5 3 8 5 7 may b e m e d i a t e d b y a n a c t i o n a t t h i s t h i r d s e r o t o n i n r e c e p t o r s i t e . H o w e v e r , t h i s h y p o t h e s i s i s u n l i k e l y f o r t w o r e a s o n s . F i r s t , t h e r e i s no f i r m e v i d e n c e t h a t S3 r e c e p t o r s e x i s t i n t h e c e n t r a l n e r v o u s s y s t e m . B e c a u s e t h e p r e s e n t e x p e r i m e n t s i m p l i c a t e c e n t r a l r e c e p t o r s i n t h e a n t i -a n a l g e s i c e f f e c t o f S2 r e c e p t o r b l o c k e r s , t h i s w o u l d a r g u e a g a i n s t a r o l e f o r S3 r e c e p t o r s . H o w e v e r , f i r m c o n c l u s i o n s m u s t a w a i t a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f S3 r e c e p t o r s . S e c o n d , F e u e r s t e i n a n d H e r t t i n g ( 1 9 8 6 ) r e c e n t l y f o u n d t h a t t h e s e l e c t i v e S3 r e c e p t o r b l o c k e r s MDL 7 2 2 2 2 a n d I C S 2 0 5 - 9 3 0 a t t e n u a t e d s e r o t o n i n - i n d u c e d t r a n s m i t t e r r e l e a s e , w h e r e a s k e t a n s e r i n d i d n o t . . T h i s s u g g e s t s t h a t k e t a n s e r i n d o e s n o t a c t a t S3 r e c e p t o r s . T h e p r e s e n t s t u d i e s a l s o t o o k a d v a n t a g e o f d i f f e r e n c e s i n t h e p h a r m a c o l o g i c a l p r o p e r t i e s o f t w o s i m i l a r d r u g s t o r o u g h l y l o c a l i z e S2 r e c e p t o r s t h a t m e d i a t e t h e a n t i - a n a l g e s i c e f f e c t o f S2 r e c e p t o r b l o c k a d e . P i r e n p e r o n e a n d k e t a n s e r i n a r e p h a r m a c o l o g i c a l l y s i m i l a r , e x c e p t t h a t p i r e n p e r o n e r e a d i l y e n t e r s t h e c e n t r a l n e r v o u s s y s t e m , w h e r e a s k e t a n s e r i n d o e s n o t ( L a d u r o n e t a l . , 1 9 8 2 ) . B y e x p l o i t i n g t h i s d i f f e r e n c e , i t was p o s s i b l e t o show t h a t t h e S2 r e c e p t o r s r e s p o n s i b l e f o r t h e a t t e n u a t i o n o f m o r p h i n e - p r o d u c e d a n a l g e s i a a r e i n t h e c e n t r a l n e r v o u s s y s t e m . T h i s s t r a t e g y i s n o t w i d e l y u s e d b e c a u s e i t i s r a r e f o r t w o d r u g s 80 o f h i g h l y s i m i l a r s t r u c t u r e t o d i f f e r o n l y i n t h e i r d i s p o s i t i o n . The g r e a t e r t h e d i f f e r e n c e s i n t h e t w o d r u g s , t h e g r e a t e r t h e n u m b e r o f p o s s i b l e i n t e r p r e t a t i o n s . ~ A r e c e n t t r e n d i n p s y c h o p h a r m a c o l o g y h a s b e e n t o c o m b i n e t h e u s e o f r e c e p t o r - s e l e c t i v e d r u g s w i t h n e u r o s u r g i c a l t e c h n i q u e s t o p r e c i s e l y l o c a l i z e r e c e p t o r p o p u l a t i o n s t h a t s u b s e r v e a p a r t i c u l a r d r u g e f f e c t . F o r e x a m p l e , Z e m l a n e t a l . ( 1 9 8 3 ) s y s t e m i c a l l y a d m i n i s t e r e d t h e s e l e c t i v e S1 a g o n i s t , 5 - m e t h o x y -N , N - d i m e t h y l t r y p t a m i n e t o r a t s w i t h t r a n s e c t e d s p i n a l c o r d s a n d o b s e r v e d a f a c i l i t a t i o n o f n o c i c e p t i o n . E x p e r i m e n t 4 o f t h e p r e s e n t t h e s i s , u s e d t h e t e c h n i q u e o f s p i n a l c o r d t r a n s e c t i o n t o a s s e s s t h e e f f e c t o f S2 r e c e p t o r b l o c k a d e o n t h e i s o l a t e d s p i n a l c o r d o p i a t e a n a l g e s i a s y s t e m a n d f o u n d t h a t p i r e n p e r o n e i n j e c t e d s y s t e m i c a l l y h a d no e f f e c t o n s p i n a l l y - m e d i a t e d m o r p h i n e - p r o d u c e d a n a l g e s i a t h u s i m p l i c a t i n g s u p r a s p i n a l S2 r e c e p t o r s i n t h e a n t i -a n a l g e s i c e f f e c t o f p i r e n p e r o n e . C e r e b r a l S2 r e c e p t o r s h a v e b e e n s h o w n t o b e p a r t i c u l a r l y a b u n d a n t i n t h e a c c u m b e n s , s t r i a t u m , o l f a c t o r y t u b e r c l e , d o r s a l r a p h e , a n d i n t h e f r o n t a l a n d o c c i p i t a l c o r t e x ( D e s a m u k h e t a l . , 1 9 8 3 ; L a d u r o n e t a l . , 1 9 8 2 ; M a r c i n k i e w i c z e t a l . , 1 9 8 4 ; P a z o s e t a l . , 1 9 8 5 ; P a z o s & P a l a c i o s , 1 9 8 5 ; S l a t e r & P a t e l , 1 9 8 3 ) . A t r a d i t i o n a l p h y s i o l o g i c a l m e t h o d t h a t c o u l d b e u s e d t o m o r e p r e c i s e l y l o c a l i z e t h e r e c e p t o r s t h a t m e d i a t e t h e a n t i - a n a l g e s i c e f f e c t o f S2 r e c e p t o r b l o c k e r s i s t h e " c e r v e a u i s o l e " p r e p a r a t i o n , i n w h i c h t r a n s e c t i o n o f t h e b r a i n b e t w e e n t h e s u p e r i o r a n d i n f e r i o r c o l l i c u l i i s o l a t e s t h e b r a i n s t e m f r o m t h e 81 midbrain and forebrain. If m i d c o l l i c u l a r transections eliminated the attenuation of morphine-produced analgesia by S2 receptor blockers, midbrain or forebrain S2 receptors would be implicated. Conversely, i f m i d c o l l i c u l a r transection f a i l e d to eliminate the attenuation of morphine-produced analgesia by S2 receptor blockers then brainstem S2 receptors would be implicated. Once the general areas mediating the anti-analgesic e f f e c t of S2 receptor blockers have been i d e n t i f i e d , d i r e c t i n j e c t i o n of S2 receptor blockers into brain regions that are r i c h i n S2 receptors may p r e c i s e l y l o c a l i z e the receptors that mediate the attenuation of morphine-produced analgesia by pirenperone, ketanserin, and LY53857. In summary, in t h i s series of experiments current psychopharmacological research strategies were adapted for the purpose of characterizing the nature and location of the serotonin receptors involved i n the mediation of morphine-produced analgesia. By challenging morphine-produced analgesia with s e l e c t i v e S2 receptor blockers, i t was fir m l y established that S2 receptors that mediate the analgesic e f f e c t of morphine. By e x p l o i t i n g the differences i n the three antagonists used i n these experiments, the attenuation of morphine-produced analgesia was l o c a l i z e d to the central nervous system, and the a l t e r n a t i v e hypothesis that t h i s e f f e c t was due to alpha-adrenergic receptor blockade was eliminated. By using the physiological technique of spinal cord transection, the mediation of the anti-analgesic e f f e c t of S2 receptor blockers was l o c a l i z e d to receptors i n the 82 brain. These conclusions stand i n marked contrast to current views of the involvement of serotonin i n morphine-produced analgesia. -I X . R E F E R E N C E S 83 A b b o t t , F . V . , G r i m e s , R . W . , & M e l z a c k , R . ( 1 9 8 4 ) . S i n g l e n e r v e c a p s a i c i n : E f f e c t s o n p a i n a n d m o r p h i n e a n a l g e s i a i n t h e f o r m a l i n a n d f o o t - f l i c k t e s t s . B r a i n R e s e a r c h , 2 9 5 , 7 7 - 8 4 . A b b o t t , F . V . , M e l z a c k , R . , & L e b e r , B . F . ( 1 9 8 2 ) . M o r p h i n e a n a l g e s i a a n d t o l e r a n c e i n t h e t a i l - f l i c k a n d f o r m a l i n t e s t s : D o s e r e s p o n s e r e l a t i o n s h i p s . P h a r m a c o l o g y ,  B i o c h e m i s t r y a n d B e h a v i o r , 1 7 , 1 2 1 3 - 1 2 1 9 . A b o l s , I . A . & B a s b a u m , A . I . ( 1 9 8 1 ) . A f f e r e n t c o n n e c t i o n s o f t h e r o s t r a l m e d u l l a o f t h e c a t : A n e u r a l s u b s t r a t e f o r t h e m i d b r a i n - m e d u l l a r y i n t e r a c t i o n s i n t h e m o d u l a t i o n o f p a i n . J o u r n a l o f C o m p a r a t i v e N e u r o l o g y , 2 0 1 , 2 8 5 - 2 9 7 . A k a i k e , A . , S h i b a t a , T . , S a t o h , M . , & T a k a g i , H . ( 1 9 7 8 ) . A n a l g e s i a i n d u c e d b y m i c r o i n j e c t i o n o f m o r p h i n e i n t o a n d e l e c t r i c a l s t i m u l a t i o n o f t h e n u c l e u s r e t i c u l a r i s p a r a g i g a n t o c e l l u l a r i s o f r a t m e d u l l a o b l o n g a t a . N e u r o p h a r m a c o l o g y , 1 7 , 7 7 5 - 7 7 8 . A r c h e r , T . , M i n o r , B . G . , & P o s t , C . ( 1 9 8 5 ) . B l o c k a d e a n d r e v e r s a l o f 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e - i n d u c e d a n a l g e s i a f o l l o w i n g n o r a d r e n a l i n e d e p l e t i o n . B r a i n  R e s e a r c h , 3 3 3 , 5 5 - 6 1 . A t w e h , S . F . & K u h a r , M . J . ( 1 9 7 7 ) . A u t o r a d i o g r a p h i c l o c a l i z a t i o n o f o p i a t e r e c e p t o r s i n r a t b r a i n . I . S p i n a l c o r d a n d l o w e r m e d u l l a . B r a i n R e s e a r c h , 1 2 4 , 5 3 - 6 7 . A t w e h , S . F . & K u h a r , M . J . ( 1 9 7 7 ) . A u t o r a d i o g r a p h i c l o c a l i z a t i o n o f o p i a t e r e c e p t o r s i n r a t b r a i n . I I . The b r a i n s t e m . B r a i n R e s e a r c h , 129, 1-12. A t w e h , S . F . & K u h a r , M . J . (1977). A u t o r a d i o g r a p h i c l o c a l i z a t i o n o f o p i a t e r e c e p t o r s i n r a t b r a i n . IITz The t e l e n c e p h a l o n . B r a i n R e s e a r c h , 134, 393-405. A t w e h , S . F . & K u h a r , M . J . (1983). D i s t r i b u t i o n a n d p h y s i o l o g i c a l s i g n i f i c a n c e o f o p i o i d r e c e p t o r s i n t h e b r a i n . B r i t i s h M e d i c a l B u l l e t i n , 39, 47-52. A z a m i , J . , L l e w e l y n , M . D . , & R o b e r t s , M . H . T . (1982). The c o n t r i b u t i o n o f t h e n u c l e u s r e t i c u l a r i s p a r a g i g a n t o c e l l u l a r i s a n d n u c l e u s r a p h e magnus t o t h e a n a l g e s i a p r o d u c e d b y s y s t e m i c a l l y a d m i n i s t e r e d m o r p h i n e , i n v e s t i g a t e d w i t h t h e m i c r o i n j e c t i o n t e c h n i q u e . P a i n , 12, 229-246. B a s b a u m , A . I . , C l a n t o n , C . H . , & F i e l d s , H . L . (1978). T h r e e b u l b o s p i n a l p a t h w a y s f r o m t h e r o s t r a l m e d u l l a o f t h e c a t : A n a u t o r a d i o g r a p h i c s t u d y o f p a i n m o d u l a t i n g s y s t e m s . J o u r n a l o f C o m p a r a t i v e N e u r o l o g y , 178, 209-224. B a s b a u m , A . I . & F i e l d s , H . L . (1978). E n d o g e n o u s p a i n c o n t r o l m e c h a n i s m s : R e v i e w a n d h y p o t h e s i s . A n n u a l o f N e u r o l o g y , 4, 451-462. B a s b a u m , A . I . & F i e l d s , H . L . (1979). T h e o r i g i n o f d e s c e n d i n g p a t h w a y s i n t h e d o r s o l a t e r a l f u n i c u l u s o f t h e c o r d o f t h e c a t a n d r a t : F u r t h e r s t u d i e s o n t h e a n a t o m y o f p a i n m o d u l a t i o n . J o u r n a l o f C o m p a r a t i v e N e u r o l o g y , 187, 513-532. B a s b a u m , A . I . & F i e l d s , H . L . (1984). E n d o g e n o u s p a i n c o n t r o l s y s t e m s : B r a i n s t e m s p i n a l p a t h w a y s a n d e n d o r p h i n c i r c u i t r y . A n n u a l R e v i e w o f N e u r o s c i e n c e , 7 , 3 0 9 - 3 3 8 . B a u m g a r t e n , H . G . , E v e t t s , K . D . , H o l m a n , R . B . , I v e r s e n , L . L . , V o g t , M . , & W i l s o n , G . ( 1 9 7 2 ) . E f f e c t s o f 5 , 6 -d i h y d r o x y t r y p t a n i i n e o n m o n o a m i n e r g i c n e u r o n s w i t h i n t h e c e n t r a l n e r v o u s s y s t e m o f t h e r a t . J o u r n a l o f  N e u r o c h e m i s t r y , 1 9 , 1 5 8 7 - 1 5 9 7 . B e a l , J . A . & B i c k n e l l , H . R . ( 1 9 8 1 ) . P r i m a r y a f f e r e n t d i s t r i b u t i o n p a t t e r n i n t h e m a r g i n a l z o n e ( l a m i n a I ) o f a d u l t monkey a n d c a t l u m b o s a c r a l s p i n a l c o r d . J o u r n a l o f  C o m p a r a t i v e N e u r o l o g y , 2 0 2 , 2 5 5 - 2 6 3 . B e h b e h a n i , M . M . & P o m e r o y , S. L . ( 1 9 7 8 ) . E f f e c t o f m o r p h i n e i n j e c t e d i n p e r i a q u e d u c t a l g r a y o n t h e a c t i v i t y o f s i n g l e u n i t s i n t h e n u c l e u s r a p h e magnus o f t h e r a t . B r a i n  R e s e a r c h , 1 4 9 , 2 6 6 - 2 6 9 . B e h b e h a n i , M . M . , P o m e r o y , S. L . , & M a c k , C. E . ( 1 9 8 1 ) . I n t e r a c t i o n b e t w e e n c e n t r a l g r a y a n d n u c l e u s r a p h e m a g n u s : R o l e o f n o r e p i n e p h r i n e . B r a i n R e s e a r c h , 1 4 9 , 2 6 6 - 2 6 9 . B e i t z , A . J . ( 1 9 8 2 a ) . T h e n u c l e i o f o r i g i n o f b r a i n s t e m e n k e p h a l i n a n d s u b s t a n c e P p r o j e c t i o n s t o t h e r o d e n t n u c l e u s r a p h e m a g n u s . N e u r o s c i e n c e , 7 , 2 7 5 3 - 2 7 6 8 . B e i t z , A . J . ( 1 9 8 2 b ) . T h e s i t e s o f o r i g i n o f b r a i n s t e m n e u r o t e n s i n a n d s e r o t o n i n p r o j e c t i o n s t o t h e n u c l e u s r a p h e m a g n u s . J o u r n a l o f N e u r o s c i e n c e , 2 , 8 2 9 - 8 4 2 . B e l c h e r , G . & R y a l l , R . W. ( 1 9 7 8 ) . D i f f e r e n t i a l e x c i t a t o r y a n d i n h i b i t o r y e f f e c t s o f o p i a t e s o n n o n - n o c i c e p t i v e a n d n o c i c e p t i v e n e u r o n e s i n t h e s p i n a l c o r d o f t h e c a t . B r a i n R e s e a r c h , 1 4 5 , 3 0 3 - 3 1 4 . B e r g e , O . - G . , F a s m e r , 0 . B . , & H o l e , K . ( 1 9 8 3 ) . S e r o t o n i n r e c e p t o r a n t a g o n i s t s i n d u c e h y p e r a l g e s i a w i t h o u t p r e v e n t i n g m o r p h i n e a n t i n o c i c e p t i o n . P h a r m a c o l o g y , B i o c h e m i s t r y a n d B e h a v i o r , 1 9 , 8 7 3 - 8 7 8 . B e r g e , O . - G . , H o l e , K . , & D a h l e , H . ( 1 9 8 0 ) . N o c i c e p t i o n i s e n h a n c e d a f t e r h i g h d o s e s o f t h e s e r o t o n i n r e c e p t o r a g o n i s t 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e . N e u r o s c i e n c e L e t t e r s , 1 9 , 2 1 9 - 2 2 3 . B e r g e , O . - G . , H o l e , K . , & O g r e n , S . - O . ( 1 9 8 3 ) . A t t e n u a t i o n o f m o r p h i n e - i n d u c e d a n a l g e s i a b y p - c h l o r o p h e n y l a l a n i n e a n d p -c h l o r o a m p h e t a m i n e : T e s t - d e p e n d e n t e f f e c t s a n d e v i d e n c e f o r b r a i n s t e m 5 - h y d r o x y t r y p t a m i n e i n v o l v e m e n t . B r a i n R e s e a r c h , 2 7 1 , 5 1 - 6 4 . B e s s o n , J . M . & L e B a r s , D . ( 1 9 7 8 ) . E f f e c t o f m o r p h i n e o n t h e t r a n s m i s s i o n o f p a i n f u l m e s s a g e s a t t h e s p i n a l l e v e l . I n M . L . A d l e r , L . M a n a r a , & R . S a m a n i n ( E d s . ) , F a c t o r s a f f e c t i n g  t h e a c t i o n o f n a r c o t i c s ( p p . 1 0 3 - 1 2 4 ) . New Y o r k : R a v e n P r e s s . B i e g o n , A . , R a i n b o w , T . C , & M c E w e n , B . S . ( 1 9 8 2 ) . Q u a n t i t a t i v e a u t o r a d i o g r a p h y o f s e r o t o n i n r e c e p t o r s i n t h e r a t b r a i n . B r a i n R e s e a r c h , 2 4 2 , 1 9 7 - 2 0 4 . B l a c k s h e a r , M . A . , S t e r a n k a , L . R . , & S a n d e r s - B u s h , E . ( 1 9 8 1 ) . M u l t i p l e s e r o t o n i n r e c e p t o r s : R e g i o n a l d i s t r i b u t i o n a n d e f f e c t o f r a p h e l e s i o n s . E u r o p e a n J o u r n a l o f P h a r m a c o l o g y , 7 6 , 3 2 5 - 3 3 4 . 87 B o w k e r , R . M . , W e s t l u n d , K . N . , & C o u l t e r , J . D . ( 1 9 8 1 ) . O r i g i n s o f s e r o t o n e r g i c p r o j e c t i o n s t o t h e s p i n a l c o r d i n r a t : A n i m m u n o c y t o c h e m i c a l r e t r o g r a d e t r a n s p o r t s t u d y . B r a i n  R e s e a r c h , 2 2 6 , 1 8 7 - 1 9 9 . B u x b a u r a , D . M . , Y a r b o r o u g h , G . G . & C a r t e r , M . E . ( 1 9 7 3 ) . B i o g e n i c a m i n e s a n d n a r c o t i c e f f e c t s . I . M o d i f i c a t i o n s o f m o r p h i n e - i n d u c e d a n a l g e s i a a n d m o t o r a c t i v i t y a f t e r a l t e r a t i o n o f c e r e b r a l a m i n e l e v e l s . J o u r n a l o f  P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 1 8 5 , 3 1 7 - 3 2 7 . C a l v i l l o , 0., H e n r y , J . L., & N e u m a n , R. S. ( 1 9 7 4 ) . E f f e c t s o f m o r p h i n e a n d n a l o x o n e o n d o r s a l h o r n n e u r o n e s i n t h e c a t . C a n a d i a n J o u r n a l o f P h y s i o l o g y a n d P h a r m a c o l o g y , 5 2 , 1 2 0 7 -1211 . C a r l s o n , N . R . ( 1 9 8 1 ) . P h y s i o l o g y o f B e h a v i o r . ( 2 n d e d . , p p . 5 7 1 - 5 7 8 ) . B o s t o n : A l l y n a n d B a c o n . C a r l s s o n , A . ( 1 9 8 3 ) . The r o l e o f b a s i c b i o m e d i c a l r e s e a r c h i n new d r u g d e v e l o p m e n t . I n F . 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A c t a P h y s i o l o g i a  S c a n d i a , 64 ( S u p p l . 247), 1-36. D ' A m o u r , F . E . & S m i t h , D . L. (1941). A m e t h o d f o r d e t e r m i n i n g l o s s o f p a i n s e n s a t i o n . J o u r n a l o f P h a r m a c o l o g y and E x p e r i m e n t a l T h e r a p e u t i c s , 7 2 , 74-79. D e a k i n , J . F . & D o s t r o v s k y , J . 0 . ( 1 9 7 8 ) . I n v o l v e m e n t o f t h e p e r i a q u e d u c t a l g r a y m a t t e r a n d s p i n a l 5 - _ h y d r o x y t r y p t a m i n e r g i c p a t h w a y s i n m o r p h i n e a n a l g e s i a : E f f e c t s o f l e s i o n s a n d 5 - h y d r o x y t r y p t a m i n e d e p l e t i o n . B r i t i s h J o u r n a l o f P h a r m a c o l o g y , 6 3 , 1 5 9 - 1 6 5 . D e n n i s , S. G. & M e l z a c k , R . ( 1 9 7 9 ) . C o m p a r i s o n o f p h a s i c a n d t o n i c p a i n i n a n i m a l s . I n J . J . B o n i c a , J . C . L i e b e s k i n d , & D . 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R o l e o f t h e n u c l e u s r a p h e magnus i n o p i a t e a n a l g e s i a a s s t u d i e d b y t h e m i c r o i n j e c t i o n t e c h n i q u e i n t h e r a t . B r a i n R e s e a r c h , 1 7 0 , 9 5 - 1 1 1 . D o s t r o v s k y , J . 0 . & P o m e r a n z , B . ( 1 9 7 6 ) . I n t e r a c t i o n o f i o n t o p h o r e t i c a l l y a p p l i e d m o r p h i n e w i t h r e s p o n s e s o f i n t e r n e u r o n s i n c a t s p i n a l c o r d . E x p e r i m e n t a l N e u r o l o g y , 5 2 , 3 2 5 - 3 3 8 . D u g g a n , A . W . , H a l l , J . G . , & H e a d l e y , P . M . ( 1 9 7 7 ) . S u p p r e s s i o n 90 o f t r a n s m i s s i o n o f n o c i c e p t i v e i m p u l s e s b y m o r p h i n e : S e l e c t i v e e f f e c t s o f m o r p h i n e a d m i n i s t e r e d i n t h e r e g i o n o f t h e s u b s t a n t i a g e l a t i n o s a . B r i t i s h J o u r n a l o f P h a r m a c o l o g y , 6 1 , 6 5 - 7 6 . D u g g a n , A . 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Afferents to brainstem nuclei (brainstem raphe, nucleus r e t i c u l a r i s pontis caudalis and nucleus g i g a n t o c e l l u l a r i s ) i n the rat as demonstrated by microiontophoretically applied horseradish peroxidase. Brain Research, 144, 257-276. Gardiner, T. H. & Eberhart, G. (1970). E f f e c t s of L-tryptophan and dl-5-hydroxytryptophan on narcotic analgesia i n mice. Federation Prodeedings, 29, 685abs. Genovese, E., Zonta, N., & Mategazza, P. (1973). Decreased antinociceptive a c t i v i t y of morphine i n rats pretreated i n t r a v e n t r i c u l a r l y with 5,6-dihydroxytryptamine, a long-l a s t i n g s e l e c t i v e d e p l e t o r o f b r a i n s e r o t o n i n . P s y c h o p h a r m a c o l o g i a ( B e r l i n ) , 3 2 , 3 5 9 - 3 6 4 . G o o d m a n , R . R . , S n y d e r , S . H . , K u h a r , M . J . , & Y o u n g , Wv S . I l l ( 1 9 8 0 ) . D i f f e r e n t i a t i o n o f d e l t a a n d mu o p i a t e r e c e p t o r l o c a l i z a t i o n s b y l i g h t m i c r o s c o p i c a u t o r a d i o g r a p h y . P r o c e e d i n g s o f t h e N a t i o n a l A c a d a m y o f S c i e n c e s , U . S . A . , 7 7 , 6 2 3 9 - 6 2 4 3 . G o r l i t z , B . - D . & F r e y , H . - H . ( 1 9 7 2 ) . C e n t r a l m o n o a m i n e s a n d a n t i n o c i c e p t i v e d r u g a c t i o n . E u r o p e a n J o u r n a l o f  P h a r m a c o l o g y , 2 0 , 1 7 1 - 1 8 0 . G r e e n , A. R . ( 1 9 8 4 ) . 5 - H T m e d i a t e d b e h a v i o u r . N e u r o p h a r m a c o l o g y , 2 3 , 1 5 2 1 - 1 5 2 8 . H a r v e y , J . A . , S c h l o s b e r g , A . J . , & Y u n g e r , L . M . ( 1 9 7 4 ) . E f f e c t o f p - c h l o r o p h e n y l a l a n i n e a n d b r a i n l e s i o n s o n p a i n s e n s i t i v i t y a n d m o r p h i n e a n a l g e s i a i n t h e r a t . . I n E . C o s t a , G . L . G e s s a , & M . S a n d l e r ( E d s . ) , A d v a n c e s i n B i o c h e m i c a l  P s y c h o p h a r m a c o l o g y : V o l 1 0 . S e r o t o n i n : New V i s t a s ( p p . 2 3 2 - 2 4 6 ) . New Y o r k : R a v e n P r e s s . H i l l a r , J . M . , P e a r s o n , J . , & S i m o n , E . J . ( 1 9 7 3 ) . D i s t r i b u t i o n o f s t e r e o s p e c i f i c b i n d i n g o f t h e p o t e n t n a r c o t i c a n a l g e s i c e t o r p h i n e i n t h e human b r a i n : P r e d o m i n a n c e i n t h e l i m b i c s y s t e m . R e s e a r c h C o m m u n i c a t i o n s i n C h e m i c a l P a t h o l o g y a n d  P h a r m a c o l o g y , 6 , 1 0 5 2 - 1 0 6 1 . H i l l a r , J . M . , S i m o n , E . J . , C r a i n , S . M . , & P e t e r s o n , E . R . ( 1 9 7 8 ) . O p i a t e r e c e p t o r s i n c u l t u r e o f f e t a l mouse d o r s a l r o o t g a n g l i a (DRG) a n d s p i n a l c o r d : P r e d o m i n a n c e i n DRG n e u r i t e s . B r a i n R e s e a r c h , 1 4 5 , 3 9 6 - 4 0 0 . I r w i n , S . , H o u d e , R . W . , B e n n e t t , D . R . , H e n d e r s h o t , L . C , & S e e v e r s , M . H . ( 1 9 5 1 ) . t h e e f f e c t s o f m o r p h i n e , m e t h a d o n e a n d m e p e r i d i n e o n some r e f l e x r e s p o n s e s o f s p i n a l a n i m a l s t o n o c i c e p t i v e s t i m u l a t i o n . J o u r n a l o f P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 1 0 1 , 1 3 2 - 1 4 3 . J a c o b s , B . L . ( 1 9 7 6 ) . A n a n i m a l b e h a v i o r m o d e l f o r s t u d y i n g c e n t r a l s e r o t o n e r g i c s y n a p s e s . L i f e S c i e n c e s , 1 9 , 7 7 7 - 7 8 6 . J a n s s e n , P. A . J . ( 1 9 8 3 ) . 5 - H T 2 r e c e p t o r b l o c k a d e t o s t u d y s e r o t o n i n - i n d u c e d p a t h o l o g y . T r e n d s i n P h a r m a c o l o g i c a l S c i e n c e 4 , 1 9 8 - 2 0 6 . J o r d a n , L . M . , K e n s h a l o , D . R . 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J o u r n a l o f  P h a r m a c y a n d P h a r m a c o l o g y , 3 5 , 3 2 4 - 3 2 5 . R e t h e l y i , M. & C a p o w s k i , J . J . ( 1 9 7 7 ) . T h e t e r m i n a l a r b o r i z a t i o n p a t t e r n o f p r i m a r y a f f e r e n t ' f i b e r s i n t h e s u b s t a n t i a g e l a t i n o s a o f t h e s p i n a l c o r d i n t h e c a t . J o u r n a l o f  P h y s i o l o g y , P a r i s , 7 3 , 2 6 9 - 2 7 7 . R i v o t , J . P . , C a l v i n o , B . , & B e s s o n , J . M. ( 1 9 8 7 ) . I s t h e r e a s e r o t o n e r g i c t o n i c d e s c e n d i n g i n h i b i t i o n o n t h e r e s p o n s e s o f d o r s a l h o r n c o n v e r g e n t n e u r o n s t o C - f i b e r i n p u t s ? B r a i n  R e s e a r c h , 4 0 3 , 1 4 2 - 1 4 6 . R o b e r t s , M. H . T . ( 1 9 8 4 ) . 5 - H y d r o x y t r y p t a m i n e a n d a n t i n o c i c e p t i o n . N e u r o p h a r m a c o l o g y , 2 3 , 1 5 2 9 - 1 5 3 6 . R o m a n d i n i , S . , E s p o s i t o , E . , & S a m a n i n , R . ( 1 9 8 6 ) . S t u d i e s o n t h e s e p a r a t e r o l e s o f f o r e b r a i n a n d s p i n a l s e r o t o n i n i n m o r p h i n e a n a l g e s i a . N a u n y n - S c h m i e d e b e r g ' s A r c h i v e s o f  P h a r m a c o l o g y , 3 3 2 , 2 0 0 - 2 1 2 . R u d a , M. A . ( 1 9 8 2 ) . O p i a t e s a n d p a i n p a t h w a y s : D e m o n s t r a t i o n o f e n k e p h a l i n s y n a p s e s o n d o r s a l h o r n p r o j e c t i o n n e u r o n s . S c i e n c e , 2 1 5 , 1 5 2 3 - 1 5 2 5 . S a m a n i n , R . , B e r n a s c o n i , S . , & Q u a t t r o n e , A . ( 1 9 7 6 ) . A n t i n o c i c e p t i v e a c t i o n o f q u i p a z i n e : R e l a t i o n t o c e n t r a l s e r o t o n e r g i c r e c e p t o r s t i m u l a t i o n . P s y c h o p h a r m a c o l o g i a  ( B e r l i n ) , 4 6 , 2 1 9 - 2 2 2 . S a m a n i n , R . , M i r a n d a , F . , & M e n n i n i , T . ( 1 9 7 8 ) . S e r o t o n e r g i c 1 02 m e c h a n i s m s o f n a r c o t i c a c t i o n . I n M . L . A d l e r , L . M a n a r a , & R . S a m a n i n ( E d s . ) f F a c t o r s a f f e c t i n g t h e a c t i o n o f n a r c o t i c s ( p p . 5 2 3 - 5 4 1 ) . New Y o r k : R a v e n P r e s s . _ S l a t e r , P . & P a t e l , S . ( 1 9 8 3 ) . A u t o r a d i o g r a p h i c d i s t r i b u t i o n o f s e r o t o n i n - 2 r e c e p t o r s i n r a t b r a i n . E u r o p e a n J o u r n a l o f P h a r m a c o l o g y , 9 2 , 2 9 7 - 2 9 8 . S o j a , P . J . & S i n c l a i r , J . (1 9 8 3 ) . S p i n a l v s . s u p r a s p i n a l a c t i o n s o f m o r p h i n e o n c a t s p i n a l c o r d m u l t i r e c e p t i v e n e u r o n s . B r a i n R e s e a r c h , 2 7 3 , 1 - 7 . S m i t h , D. J . , P e r r o t t i , J . M . , M a n s e l l , A . L . & M o n r o e , P . J . ( 1 9 8 5 ) . K e t a m i n e a n a l g e s i a i s n o t r e l a t e d t o a n o p i a t e a c t i o n i n t h e p e r i a q u e d u c t a l g r a y r e g i o n o f t h e r a t b r a i n . P a i n , 2 1 , 2 5 3 - 2 6 5 . S m i t h , D . L . , D ' A m o u r , M . C , & D ' A m o u r , F . E . ( 1 9 4 3 ) . T h e a n a l g e s i c p r o p e r t i e s o f c e r t a i n d r u g s a n d d r u g c o m b i n a t i o n s . J o u r n a l o f P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 7 8 , 1 8 4 - 1 9 3 . S t o n e , C . A . , W e n g e r , H . C , L u d d e n , C . T . , S t a v o r s k i , J . M . , & R o s s , C . A . ( 1 9 6 1 ) . A n t i s e r o t o n i n - a n t i h i s t a m i n i c p r o p e r t i e s o f c y p r o h e p t a d i n e . J o u r n a l o f P h a r m a c o l o g y a n d E x p e r i m e n t a l  T h e r a p e u t i c s , 1 3 1 , 7 3 - 8 4 . S u g r u e , M . F . ( 1 9 7 9 ) . E f f e c t o f d e p l e t i o n o f r a t b r a i n 5 -h y d r o x y t r y p t a m i n e o n m o r p h i n e - i n d u c e d a n t i n o c i c e p t i o n . J o u r n a l o f P h a r m a c y a n d P h a r m a c o l o g y , 3 1 , 2 5 3 - 2 5 5 . T a k a g i , H . , M a t s u m u r a , M . , Y a n a i , A . , & O g i u , K . ( 1 9 5 5 ) . T h e e f f e c t o f a n a l g e s i c s o n t h e s p i n a l r e f l e x a c t i v i t y o f t h e 1 03 c a t . J a p a n e s e J o u r n a l o f P h a r m a c o l o g y / 4 , 1 7 6 - 1 8 7 . T a k a g i , H . , T a k a s h i m i , T . , & K i m u r a , K . ( 1 9 6 4 ) . A n t a g o n i s m o f t h e a n a l g e t i c e f f e c t o f m o r p h i n e i n m i c e b y t e t r a b e n a z i n e a n d r e s e r p i n e . A r c h i v e s I n t e r n a t i o n a l e s d e P h a r m a c o d y n a m i e  e t T h e r a p i e , 1 f 4 8 4 - 4 9 2 . T a k e m o r i , F . C , T u l u n a y , A . E . , & Y a n o , I . ( 1 9 7 5 ) . D i f f e r e n t i a l e f f e c t s o n m o r p h i n e a n a l g e s i a a n d n a l o x o n e a n t a g o n i s m b y b i o g e n i c a m i n e m o d i f i e r s . L i f e S c i e n c e s / 1 7 , 2 1 - 2 8 . T e n n e n , S . S . ( 1 9 6 8 ) . A n t a g o n i s m o f t h e a n a l g e s i c e f f e c t o f m o r p h i n e a n d o t h e r d r u g s b y p - c h l o r o p h e n y l a l a n i n e , a s e r o t o n i n d e p l e t o r . P s y c h o p h a r m a c o l o g i a 1 2 , 2 7 8 - 2 8 5 . T i l s o n , H . A . & R e c h , R. H . ( 1 9 7 4 ) . T h e e f f e c t s o f p -c h l o r o p h e n y l a l a n i n e o n m o r p h i n e a n a l g e s i a , t o l e r a n c e a n d d e p e n d e n c e d e v e l o p m e n t i n t w o s t r a i n s o f r a t s . P s y c h o p h a r m a c o l o g i a ( B e r l i n ) , 3 5 , 4 5 - 6 0 . T s u o , K . & J a n g , C . S . ( 1 9 6 4 ) . S t u d i e s o n t h e s i t e o f a n a l g e s i c a c t i o n o f m o r p h i n e b y i n t r a c e r e b r a l m i c r o i n j e c t i o n . S c i e n t i a S i n i c a , 1 3 , 1 0 9 9 - 1 1 0 9 . T u l u n a y , F . C , Y a n o , I . , & T a k e m o r i , A . E . ( 1 9 7 6 ) . T h e e f f e c t o f b i o g e n i c a m i n e m o d i f i e r s o n m o r p h i n e a n a l g e s i a a n d i t s a n t a g o n i s m b y n a l o x o n e . V a n h o u t t e , P . M . , B a l l , S . G . , B e r d e a u x , A . , c o h e n , M . L . , H e n d e r , T . , M c C a l l , R . , R a m a g e , A . G . , R e i m a n n , I . H . , R i c h e r , C , S a x e n a , P . R . , S c h a l e k a m p , M . A . D . H . , S t r u y k e r - B o u d i e r , H . A . J . , S y m o e n s , J . , V a n N u e t e n , J . M . , & V a n Z w i e t e n , P . A . ( 1 9 8 6 ) . M e c h a n i s m o f a c t i o n o f 1 04 k e t a n s e r i n i n h y p e r t e n s i o n . T r e n d s i n P h a r m a c o l o g i c a l S c i e n c e , 7 , 5 8 - 5 9 . V a n N u e t e n , J . M . , J a n s s e n , P . A . J . , V a n B e c k , J . , X h O T i n e u x , R . , V e r b e u r e n , T . J . , & V a n h o u t t e , P . M . ( 1 9 8 1 ) . V a s c u l a r e f f e c t s o f k e t a n s e r i n (R 41 4 6 8 ) , a n o v e l a n t a g o n i s t o f 5 -HT2 s e r o t o n e r g i c r e c e p t o r s . J o u r n a l o f P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 2 1 8 , 2 1 7 - 2 3 0 . V o g t < M. ( 1 9 7 3 ) . The e f f e c t o f l o w e r i n g t h e 5 - h y d r o x y t r y p t a m i n e c o n t e n t o f t h e r a t s p i n a l c o r d o n a n a l g e s i a p r o d u c e d b y m o r p h i n e . J o u r n a l o f P h y s i o l o g y , 2 3 6 , 4 8 3 - 4 9 8 . W a n g , J . K . ( 1 9 7 7 ) . A n t i n o c i c e p t i v e e f f e c t o f i n t r a t h e c a l l y a d m i n i s t e r e d s e r o t o n i n . A n e s t h e s i o l o g y , 4 7 , 2 6 9 - 2 7 1 . W i l l i s , W. D . ( 1 9 8 5 ) . P a i n a n d H e a d a c h e . V o l . 8 . T h e P a i n S y s t e m : The n e u r a l b a s i s o f n o c i c e p t i v e t r a n s m i s s i o n . B a s e l : K a r g e r . W i l l i s , W. D . , H a b e r , H . , & M a r t i n , R . F . ( 1 9 7 7 ) . I n h i b i t i o n o f s p i n o t h a l a m i c t r a c t c e l l s a n d i n t e r n e u r o n s b y b r a i n s t e m s t i m u l a t i o n i n t h e m o n k e y . J o u r n a l o f N e u r o p h y s i o l o g y , 4 0 , 9 6 8 - 9 8 1 . W o o l f e , G . & M a c D o n a l d , A . D . ( 1 9 4 4 ) . T h e e v a l u a t i o n o f t h e a n a l g e s i c a c t i o n o f p e t h i d i n e h y d r o c h l o r i d e ( d e m e r o l ) . J o u r n a l o f P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 8 0 , 3 0 0 - 3 0 7 . Y a k s h , T . L . ( 1 9 7 9 ) . D i r e c t e v i d e n c e t h a t s p i n a l s e r o t o n i n a n d n o r a d r e n a l i n t e r m i n a l s m e d i a t e t h e s p i n a l a n t i n o c i c e p t i v e e f f e c t s o f m o r p h i n e i n t h e p e r i a q u e d u c t a l g r a y . B r a i n 105 R e s e a r c h , 1 6 0 , 1 8 0 - 1 8 5 . Y a k s h , T . L . ( 1 9 8 1 ) . S p i n a l o p i a t e a n a l g e s i a : C h a r a c t e r i s t i c s a n d p r i n c i p l e s o f a c t i o n . P a i n , 1 1 , 2 9 3 - 3 4 6 . _ Y a k s h , T . L . , D u C h a t e a u , J . C , & R u d y , T . A . ( 1 9 7 6 ) . A n t a g o n i s m b y m e t h y s e r g i d e a n d c i n a n s e r i n o f t h e a n t i n o c i c e p t i v e a c t i o n o f m o r p h i n e a d m i n i s t e r e d i n t o t h e p e r i a q u e d u c t a l g r a y . B r a i n R e s e a r c h , 1 0 4 , 3 6 7 - 3 7 2 . Y a k s h , T . L . & R e d d y , S . V . R . ( 1 9 8 1 ) . S t u d i e s i n t h e p r i m a t e o n t h e a n a l g e t i c e f f e c t s a s s o c i a t e d w i t h i n t r a t h e c a l a c t i o n s o f o p i a t e s , - a d r e n e r g i c a g o n i s t s a n d b a c l o f e n . A n e s t h e s i o l o g y , 5 4 , 4 5 1 - 4 6 7 . Y a k s h , T . L . & R u d y , T . A . ( 1 9 7 7 ) . S t u d i e s o n t h e d i r e c t s p i n a l a c t i o n o f n a r c o t i c s i n t h e p r o d u c t i o n o f a n a l g e s i a i n t h e r a t . J o u r n a l o f P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 2 0 2 , 4 1 1 - 4 2 8 . Y a k s h , T . L . & W i l s o n , P . R . ( 1 9 7 9 ) . S p i n a l s e r o t o n i n t e r m i n a l s y s t e m m e d i a t e s a n t i n o c i c e p t i o n . J o u r n a l o f P h a r m a c o l o g y  a n d E x p e r i m e n t a l T h e r a p e u t i c s , 2 0 8 , 4 4 6 - 4 5 3 . Y e u n g , J . C . & R u d y , T . ( 1 9 8 0 ) . S i t e s o f a n t i n o c i c e p t i v e a c t i o n o f s y s t e m i c a l l y i n j e c t e d m o r p h i n e : I n v o l v e m e n t o f s u p r a s p i n a l l o c i a s r e v e a l e d b y i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n o f n a l o x o n e . J o u r n a l o f P h a r m a c o l o g y a n d  E x p e r i m e n t a l T h e r a p e u t i c s , 2 1 5 , 6 2 6 - 6 3 2 . Y e z i e r s k i , R . P . , W i l c o x , T . K . , & W i l l i s , W. D . ( 1 9 8 2 ) . T h e e f f e c t o f s e r o t o n i n a n t a g o n i s t s i n t h e i n h i b i t i o n o f p r i m a t e s p i n o t h a l a m i c t r a c t c e l l s p r o d u c e d b y s t i m u l a t i o n i n n u c l e u s 1 06 r a p h e magnus o r p e r i a q u e d u c t a l g r a y . J o u r n a l o f  P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 2 2 0 , 2 6 6 - 2 7 7 . Y o u n g , W. S . & K u h a r , M . J . ( 1 9 8 0 ) . S e r o t o n i n r e c e p t o r -l o c a l i z a t i o n i n r a t b r a i n b y l i g h t m i c r o s c o p i c a u t o r a d i o g r a p h y . E u r o p e a n J o u r n a l o f P h a r m a c o l o g y , 6 2 , 2 3 7 -2 3 9 . Z e m l a n , F . P . , K o w , L . - M . & P f a f f , D . W. ( 1 9 8 3 ) . S p i n a l s e r o t o n i n ( 5 - H T ) r e c e p t o r s u b t y p e s a n d n o c i c e p t i o n . J o u r n a l  o f P h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s , 2 2 6 , 4 7 7 - 4 8 5 . Z i e g l g a n s b e r g e r , W. & B a y e r l , H . ( 1 9 7 6 ) . T h e m e c h a n i s m o f i n h i b i t i o n o f n e u r o n a l a c t i v i t y b y o p i a t e s i n t h e s p i n a l c o r d o f t h e c a t . B r a i n R e s e a r c h , 1 1 5 , 1 1 1 - 1 2 8 . 

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