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Effects of serotonin type 3 receptor activity on receptive and proceptive behaviours in female rats Tanco, Sheryl Anne 1991

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EFFECTS OF SEROTONIN TYPE 3 RECEPTOR ACTIVITY ON RECEPTIVE AND PROCEPTIVE BEHAVIOURS IN FEMALE RATS by SHERYL ANNE TANCO B.A. (Psych)., The U n i v e r s i t y of B r i t i s h Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n THE FACULTY OF GRADUATE STUDIES Department of Psychology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1991 © S h e r y l Anne Tanco, 1991 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 /~tsrycAo/^g c / The University of British Columbia Vancouver, Canada Date &Csf~ 9. /99/  DE-6 (2/88) i i ABSTRACT The 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 ( 5-HT) i n f l u e n c e s many c e n t r a l l y - m e d i a t e d behaviours. In female r a t s , 5-HT a f f e c t s the e x p r e s s i o n of l o r d o s i s , a b e h a v i o u r a l index of sexual r e c e p t i v i t y . C o n f l i c t i n g e m p i r i c a l evidence r e g a r d i n g the r o l e of 5-HT, with r e s p e c t t o l o r d o s i s , and i d e n t i f i c a t i o n of subtypes of c e n t r a l 5-HT r e c e p t o r s l e d to f o r m u l a t i o n of the h y p o t h e s i s t h a t 5-HT p l a y s a dual r o l e with r e s p e c t to female sexual behaviour. Evidence suggests t h a t 5-HT 1 A r e c e p t o r s i n h i b i t , while 5-HT 2 r e c e p t o r s f a c i l i t a t e l o r d o s i s . The r e c e n t l y i d e n t i f i e d c e n t r a l 5-HT 3 r e c e p t o r a f f e c t s the r e l e a s e of n e u r o t r a n s m i t t e r s such as dopamine, n o r e p i n e p h r i n e and a c e t y l c h o l i n e ; modulates the e f f e c t s of o p i a t e s , amphetamine and n i c o t i n e ; and i n f l u e n c e s anxiety, l e a r n i n g , n o c i c e p t i o n , nausea and v o m i t i n g . I t remains to be determined whether 5-HT 3 r e c e p t o r s a l s o i n f l u e n c e r e p r o d u c t i v e a c t i v i t y . In female r a t s s exual a c t i v i t y i s comprised of two types of behaviours: r e c e p t i v e ( l o r d o s i s ) and p r o c e p t i v e (ear w i g g l i n g , hopping and d a r t i n g ) . In the c u r r e n t l i t e r a t u r e , l o r d o s i s stands as the primary measure; p r o c e p t i v e behaviours are seldom r e p o r t e d . The purpose of t h i s t h e s i s i s to f u r t h e r c h a r a c t e r i z e the 5 - H T 3 r e c e p t o r with r e s p e c t to female r e p r o d u c t i v e a c t i v i t y and to i n c r e a s e the understanding of neurochemical f a c t o r s which i n f l u e n c e r e c e p t i v e and p r o c e p t i v e behaviours i n female r a t s . The c u r r e n t s e r i e s of experiments i n v e s t i g a t e d the e f f e c t s of 5 -HT3 a g o n i s t s and a n t a g o n i s t s , a d m i n i s t e r e d alone and i n c o n j u n c t i o n with morphine and apomorphine, on sexual behaviours i n o v a r i e c t o m i z e d , s t e r o i d - p r i m e d female r a t s . The 5 - H T 3 a n t a g o n i s t s MDL 72222, ondansetron and ICS 205-930 f a i l e d t o a f f e c t r e c e p t i v e or p r o c e p t i v e behaviours, although a t r e n d towards a f a c i l i t a t o r y e f f e c t was ev i d e n t i n the case of ondansetron. S i m i l a r l y , the 5-HT3 a g o n i s t s 1-p h e n y l b i g u a n i d e and 2- m e t h y l - s e r o t o n i n , a d m i n i s t e r e d i n t r a c e r e b r o v e n t r i c u l a r l y , d i d not s i g n i f i c a n t l y i n f l u e n c e female c o p u l a t o r y behaviours. While low doses of morphine s i g n i f i c a n t l y i n h i b i t e d sexual a c t i v i t y , t h i s i n h i b i t i o n was not att e n u a t e d by any of the 5-HT3 a n t a g o n i s t s . Likewise, apomorphine p r o f o u n d l y i n h i b i t e d both r e c e p t i v e and p r o c e p t i v e behaviours, but these e f f e c t s were not antagonized by ICS 205-930. Although these data do not support the i d e a t h a t 5-HT3 r e c e p t o r s are i n f l u e n t i a l i n the modulation of female r e p r o d u c t i v e b e h a v i o u r s , c o n s i d e r a t i o n of v a r i o u s p h a r m a c o l o g i c a l and methodological f a c t o r s c a u t i o n a g a i n s t a premature c o n c l u s i o n i n t h i s regard. i v TABLE OF CONTENTS /ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 Components of Female Sexual Behaviour 1 5-HT Receptor Subtypes and Sexual Behaviour 3 5-HT3 Receptors 5 Ob j e c t i v e s 10 GENERAL METHODS 11 PART I - ADMINISTRATION OF 5~HT3 ANTAGONISTS 14 Experiment 1 14 Experiment 2 20 Experiment 3 28 PART II - ADMINISTRATION OF 5-HT3 AGONISTS 31 Experiment 4 31 Experiment 5 38 PART I I I - ADMINISTRATION OF MORPHINE AND 5-HT3 ANTAGONISTS. . .41 Experiment 6 44 Experiment 7 46 Experiment 8 50 PART IV - ADMINISTRATION OF APOMORPHINE AND ICS 205-930 56 Experiment 9 56 GENERAL DISCUSSION 62 REFERENCES 7 6 V L I S T OF T A B L E S 1 . E f f e c t s o f MDL 7 2 2 2 2 o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s 18 2 . E f f e c t s o f O n d a n s e t r o n o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s 2 5 3 . E f f e c t s o f I C S 2 0 5 - 9 3 0 o n S e x u a l B e h a v i o u r i n E B - p r i m e d F e m a l e R a t s 33 4 . E f f e c t s o f 1 - P h e n y l b i g u a n i d e o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s 37 5 . E f f e c t s o f 2 - M e t h y l - S e r o t o n i n o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s 4 3 6 . E f f e c t s o f M o r p h i n e a n d MDL 7 2 2 2 2 o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s 4 8 7 . E f f e c t s o f M o r p h i n e a n d O n d a n s e t r o n o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s 5 2 8 . E f f e c t s o f M o r p h i n e a n d I C S 2 0 5 - 9 3 0 o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s ' 5 5 9 . E f f e c t s o f A p o m o r p h i n e a n d I C S 2 0 5 - 9 3 0 o n S e x u a l B e h a v i o u r i n E B - a n d P - p r i m e d F e m a l e R a t s 60 LIST OF FIGURES v i 1. E f f e c t of Ondansetron on L o r d o s i s Quotient i n EB- and P-primed Female Rats 27 v i i ACKNOWLEDGEMENTS I would l i k e t o express s i n c e r e a p p r e c i a t i o n t o my r e s e a r c h s u p e r v i s o r , Dr. B o r i s Gorzalka and t o members of my t h e s i s committee, Dr. Wolfgang Linden and Dr. Roderick Wong. In p a r t i c u l a r , I am indebted t o Dr. Gorzalka f o r h i s support d u r i n g the p r e p a r a t i o n of t h i s t h e s i s . I would a l s o l i k e t o thank two f e l l o w graduate students: Georg Schulze, who p a t i e n t l y i n s t r u c t e d me i n the techniques of s t e r e o t a x i c surgery and N e i l Watson, who was a constant source of he l p and encouragement. F i n a l l y , I would l i k e t o thank my husband, Dean f o r h i s p a t i e n c e , support and l o v e . Ondansetron was a k i n d g i f t of Dr. B.M. Bain, Glaxo Group Research L i m i t e d , Middlesex, U.K. and ICS 205-930 was a generous g i f t of Dr. K. G a l l a n t , Sandoz Canada Inc. Thi s r e s e a r c h was funded by a N a t u r a l Sciences and E n g i n e e r i n g Research C o u n c i l grant t o Dr. B o r i s G o r z a l k a . 1 INTRODUCTION The 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 (5-hydroxytryptamine, 5-HT) i s a c t i v e both c e n t r a l l y and p e r i p h e r a l l y . In the c e n t r a l nervous system (CNS) , 5-HT r e c e p t o r s mediate many d i v e r s e behaviours, p l a y i n g a r o l e i n a p p e t i t i v e and compulsive behaviours, a n x i e t y and depression, n o c i c e p t i o n and r e p r o d u c t i o n (Glennon, 1990). With r e s p e c t to r e g u l a t i o n of female r e p r o d u c t i v e a c t i v i t y , the i n f l u e n c e of s e r o t o n i n was o r i g i n a l l y h y p o t h e s i z e d t o be i n h i b i t o r y (Meyerson, 1964). Today however, s e r o t o n e r g i c e f f e c t s on sexual behaviours are known to be f a r more complex (e.g. Gorzalka, Mendelson & Watson, 1990). Components of Female Sexual Behaviour Female r e p r o d u c t i v e behaviour has been c h a r a c t e r i z e d i n terms of t h r e e components: r e c e p t i v i t y , p r o c e p t i v i t y and a t t r a c t i v i t y (Beach, 1976). In female r a t s , sexual r e c e p t i v i t y i s i n d i c a t e d by the d i s p l a y of l o r d o s i s i n response t o mounting by a male. The l o r d o t i c response i s c h a r a c t e r i z e d by d o r s i f l e x i o n of the v e r t e b r a l column, lowering of the thorax and e l e v a t i o n of the neck, rump and t a i l b a s e ( P f a f f , 1980) . In r e c e p t i v e females, l o r d o s i s can be e l i c i t e d by cutaneous s t i m u l a t i o n of the f l a n k s f o l l o w e d by p r e s s u r e on the rump, t a i l b a s e and perineum ( P f a f f , 1980) . During c o p u l a t i o n , such s t i m u l i are p r o v i d e d by the male. However, i t i s i n i t i a l rump 2 e l e v a t i o n by the female which permits the male t o apply s t i m u l i , s u f f i c i e n t t o evoke those female responses necessary f o r s u c c e s s f u l c o p u l a t i o n . Thus, rump e l e v a t i o n i s r e q u i r e d e a r l y i n c o p u l a t i o n , p r i o r t o p e n i l e i n t r o m i s s i o n ( P f a f f , 1980) . H i g h l y r e c e p t i v e females f r e q u e n t l y h o l d the l o r d o t i c p o s t u r e a f t e r the male has dismounted. This suggests l o r d o s i s i s the r e s u l t , at l e a s t i n p a r t , of female muscular a c t i v i t y , v i s a v i s the p a s s i v e consequence of male mounting and t h r u s t i n g ( P f a f f , 1980). From the above d e s c r i p t i o n i t i s apparent t h a t while the female p l a y s an a c t i v e r o l e i n c o p u l a t i o n , her responses are a l s o , i n p a r t , a f u n c t i o n of male-provided s t i m u l i . L o r d o s i s i s a b o l i s h e d by ovariectomy. In o v a r i e c t o m i z e d r a t s , a d m i n i s t r a t i o n of s u f f i c i e n t estrogen or estrogen and progesterone w i l l r e s t o r e l o r d o s i s ; however, a d m i n i s t r a t i o n of progesterone alone i s not s u f f i c i e n t t o promote l o r d o s i s ( P f a f f , 1980) . L o r d o s i s performance may be enhanced by i n c r e a s i n g estrogen, stimulus p r e s s u r e or the t o t a l area s t i m u l a t e d ( P f a f f , 1980). In a d d i t i o n t o l o r d o s i s , e s t r o u s females f r e q u e n t l y d i s p l a y p r o c e p t i v e b e haviours. In the female r a t , p r o c e p t i v e behaviours i n c l u d e ear w i g g l i n g and hopping and d a r t i n g ( P f a f f , 1980) . Moreover, f o r c o n s p e c i f i c males, female a t t r a c t i v i t y , c h a r a c t e r i z e d by o l f a c t o r y and v i s u a l cues, i s h i g h e s t d u r i n g e s t r u s (Beach, 1976). Such enhanced a t t r a c t i v e n e s s and p r o c e p t i v i t y i n c r e a s e the p r o b a b i l i t y of s u c c e s s f u l c o p u l a t i o n o c c u r r i n g d u r i n g e s t r u s when the female i s f e r t i l e . T h i s i s 3 f u r t h e r evidence t h a t females p l a y an a c t i v e r o l e i n sexual behaviour, a f a c t o f t e n obscured by a focus i n the l i t e r a t u r e on the r e l a t i v e l y " p a s s i v e " l o r d o s i s response (Beach, 1976) . Female a t t r a c t i v i t y must be i n f e r r e d v i a o b s e r v a t i o n of the male, i . e . how f r e q u e n t l y he approaches, mounts, e j a c u l a t e s , e t c . (Beach, 1976). Thus, assessment of female a t t r a c t i v i t y i s confounded by c h a r a c t e r i s t i c s of the male, making d i r e c t e v a l u a t i o n of female a t t r a c t i v i t y very d i f f i c u l t . P r o c e p t i v e behaviours can be observed d i r e c t l y and are t h e r e f o r e l e s s a f u n c t i o n of the male i n v o l v e d . Nonetheless, c h a r a c t e r i s t i c s of the male, e.g. h i s a t t r a c t i v e n e s s , v i g o u r , e t c . may a f f e c t the female's d i s p l a y of p r o c e p t i v e behaviours (Beach, 1976) . 5-HT Receptor Subtypes and Sexual Behaviour In r a t b r a i n , [3H] 5-HT and [3H] spiperone were demonstrated t o b i n d t o two d i s t i n c t s e r o t o n e r g i c s i t e s , d e s i g n a t e d 5-HT} and 5-HT2 r e c e p t o r s r e s p e c t i v e l y (Peroutka & Snyder, 1979); subsequently each was determined to comprise a f a m i l y of r e l a t e d subtypes (e.g. 5-HT1A_D, 5-HT 2 < l c; Peroutka, 1990) . These f i n d i n g s were f o l l o w e d by the i d e n t i f i c a t i o n of a t h i r d c e n t r a l b i n d i n g s i t e , 5-HT3, a s i t e b e l i e v e d i d e n t i c a l t o the so c a l l e d "M-r e c e p t o r " known t o be r e s p o n s i b l e f o r 5-HT-induced, n e u r o n a l l y -mediated, c o n t r a c t i o n s of gu i n e a - p i g ileum ( K i l p a t r i c k , Jones & Tyers, 1987) . Today i t i s r e c o g n i z e d t h a t the s e r o t o n e r g i c system i s a d i f f u s e l y o r g a n i z e d p r o j e c t i o n system of 4 m o r p h o l o g i c a l l y d i s t i n c t neurons i n n e r v a t i n g v i r t u a l l y the e n t i r e CNS (Tork, 1990) . Moreover, pharmacologic and b e h a v i o u r a l d i s t i n c t n e s s of c e n t r a l 5-HT r e c e p t o r subtypes has been demonstrated (Bradley et a l . , 1 9 8 6 ) . C h a r a c t e r i z a t i o n of subtypes of c e n t r a l s e r o t o n i n r e c e p t o r s , t o g e t h e r with c o n t r a d i c t o r y f i n d i n g s i n the l i t e r a t u r e r e g a r d i n g the i n f l u e n c e of 5-HT on sexual a c t i v i t y , prompted the hypothesis by Mendelson and Gorzalka (1985) t h a t 5 -HT p l a y s a dual r o l e i n the r e g u l a t i o n of female sexual behaviour. Indeed, t h e r e i s now evidence t h a t s e r o t o n e r g i c i n f l u e n c e upon r e p r o d u c t i v e behaviour i s a f u n c t i o n both of sex and the subtype of r e c e p t o r a c t i v a t e d (Gorzalka et a l . , 1990) . In female r a t s , 5-HT1A r e c e p t o r a c t i v i t y appears r e s p o n s i b l e f o r the l o r d o s i s - i n h i b i t i n g e f f e c t s of 5-HT while 5-HT 1 B and 5-HT2 r e c e p t o r s mediate the l o r d o s i s - e n h a n c i n g e f f e c t s of 5-HT (Mendelson & Gorzalka, 1989) . Data r e g a r d i n g the e f f e c t s of 5-HT3 a c t i v i t y on r e p r o d u c t i v e behaviour are l i m i t e d and r e s u l t s c o n t r a d i c t o r y . In male r a t s , both the 5-HT3 a g o n i s t , 2-methyl - 5-hydroxytryptamine (2-Me-5-HT) and the 5-HT3 a n t a g o n i s t , BRL 43694 ( g r a n i s i t r o n ) appear t o f a c i l i t a t e c o p u l a t o r y behaviour (N.V. Watson, p e r s o n a l communication, 1991) . Although no complete p u b l i s h e d s t u d i e s r e g a r d i n g the i n f l u e n c e of 5-HT3 a c t i v i t y on female r e p r o d u c t i v e behaviour appear t o e x i s t , p r e l i m i n a r y data are mentioned i n two review c h a p t e r s . In estrogen-primed female r a t s , the s e l e c t i v e 5-HT3 a n t a g o n i s t s , ICS 205-930 (5mg/kg) and MDL 72222 (5mg/kg) 5 r e p o r t e d l y f a c i l i t a t e and f a i l t o a f f e c t l o r d o s i s , r e s p e c t i v e l y (Mendelson & Gorzalka, 1989) . Furthermore, James and c o l l e a g u e s (1989) r e p o r t f a c i l i t a t i o n of l o r d o s i s i n non r e c e p t i v e , but not i n r e c e p t i v e , females by the 5-HT3 a n t a g o n i s t s MDL 72222, g r a n i s i t r o n and GR 38032F (ondansetron) at dosages of both 0.2 and 0.5 mg/kg. Together with the known i n f l u e n c e of other 5-HT r e c e p t o r subtypes on sexual behaviour and the e x i s t e n c e of 5-HT3 b i n d i n g s i t e s i n hypothalamic and s p i n a l c o r d areas fundamental t o o r c h e s t r a t i o n of sexual behaviour, these p r e l i m i n a r y r e s u l t s suggest t h a t 5-HT3 r e c e p t o r a c t i v i t y may indeed i n f l u e n c e r e p r o d u c t i v e behaviour. 5-HT3 Receptors By d e f i n i t i o n , 5-HT3 r e c e p t o r s are s u s c e p t i b l e t o antagonism by nanomolar c o n c e n t r a t i o n s of MDL 72222 (Fozard, 1984) and ICS 205-930 (Richardson, Engel, Donatsch & S t a d l e r , 1985) , r e s i s t a n t t o antagonism by the 5-HT: and 5-HT2 a n t a g o n i s t s methiothepin, methysergide and k e t a n s e r i n , and s e n s i t i v e t o the s e r o t o n i n a g o n i s t 2-Me-5-HT (Bradley et a l . , 19 8 6 ) . 5-HT3 r e c e p t o r s are l o c a t e d on neurons of both the p e r i p h e r a l and c e n t r a l nervous systems; t h e i r a c t i v a t i o n induces a q u i c k l y d e s e n s i t i z i n g , r a p i d membrane d e p o l a r i z a t i o n (Tyers, 1990) . U n l i k e 5-HTx and 5-HT2 r e c e p t o r s , which modulate a c e l l ' s a c t i v i t y v i a GTP-binding p r o t e i n s , 5-HT3 r e c e p t o r s are l i g a n d -gated, membrane i o n channels (Derkach, Suprenant & North, 1989) . 6 T r i t i a t e d l i g a n d s have enabled i d e n t i f i c a t i o n of 5-HT3 b i n d i n g s i t e s i n p e r i p h e r a l and c e n t r a l t i s s u e s of s e v e r a l mammalian species i n c l u d i n g humans and r a t s . 5-HT3 b i n d i n g s i t e s have been i d e n t i f i e d on sensory, e n t e r i c , sympathetic and parasympathetic nerves (Bradley et a l . , 1986) . In the r a t , c e n t r a l 5-HT3 b i n d i n g has been demonstrated to occur i n the e n t o r h i n a l , r e t r o s p l e n i c , f r o n t a l , c i n g u l a t e , temporal, o c c i p i t a l and p a r i e t a l c o r t i c e s , as w e l l as i n the amygdala, hippocampus, nucleus accumbens, septum, thalamus, hypothalamus and s t r i a t u m ( K i l p a t r i c k , et a l . , 1987) ; the highest 5-HT3 b i n d i n g d e n s i t i e s have been found i n d i s c r e t e brainstem regions, i n c l u d i n g the nucleus t r a c t u s s o l i t a r i u s , the d o r s a l nucleus of the vagus nerve, the su b s t a n t i a g e l a t i n o s a of the s p i n a l t r i g e m i n a l nucleus, the area postrema and i n the s u b s t a n t i a g e l a t i n o s a i n the s p i n a l cord ( P a l a c i o s , Waeber, Hoyer & Mengod, 1 9 9 0 ) . I t i s necessary to supplement r e s u l t s from l i g a n d - b i n d i n g s t u d i e s w i t h p h y s i o l o g i c a l and behavioural data to e s t a b l i s h the presence of f u n c t i o n a l receptor s i t e s . Receptors may be defined as those b i n d i n g s i t e s on which a c e l l u l a r t r a n s f o r m a t i o n or b i o l o g i c a l e f f e c t i s i n i t i a t e d ( P l i s k a , 1991) . Guinea-pig ileum, r a b b i t and r a t heart and vagal t i s s u e and su p e r i o r c e r v i c a l ganglion preparations have been employed as f u n c t i o n a l bioassays f o r assessment of 5-HT3 receptor a c t i v i t y (Tyers, 1990) . Anaesthetized r a t s d i s p l a y a vagally-mediated r e f l e x bradycardia i n response to bolus i n j e c t i o n of 5-HT i n t o the ju g u l a r v e i n . This response, known as the von B e z o l d - J a r i s c h r e f l e x , i s 7 mediated v i a 5-HT3 r e c e p t o r s (Richardson et a l . , 1985) and has a l s o been used e x t e n s i v e l y i n the c h a r a c t e r i z a t i o n of 5-HT3 a g o n i s t s and a n t a g o n i s t s . Receptor s i t e s f o r which 5-HT3 a g o n i s t s and a n t a g o n i s t s show a f f i n i t y may not be homogeneous. That i s , t h e r e may be more than one s p e c i e s of 5-HT3 r e c e p t o r . 5-HT3 a n t a g o n i s t s show s u b s t a n t i a l v a r i a t i o n i n s e n s i t i v i t y f o r 5-HT3 s i t e s on d i f f e r e n t b i o a s s a y t i s s u e s , s u g g e s t i n g t h a t as c u r r e n t l y d e f i n e d , 5-HT3 b i n d i n g s i t e s may be comprised of d i s t i n c t subtypes (Richardson et a l . , 1985) . However, others (e.g. Tyers, 1990) have argued these d i s c r e p a n c i e s i n s e n s i t i v i t y t o be a r e f l e c t i o n of s p e c i e s v a r i a t i o n , r a t h e r than evidence of w i t h i n -s p e c i e s 5-HT3 r e c e p t o r h e t e r o g e n e i t y . F u r t h e r c o m p l i c a t i n g t h i s i s s u e are recent r e p o r t s of a novel 5-HT r e c e p t o r s e n s i t i v e t o drugs showing a f f i n i t y f o r 5-HT3 r e c e p t o r s . A r e c e p t o r s i t e , having pharmacologic p r o p e r t i e s s i m i l a r t o t h a t d e s c r i b e d f o r the p u t a t i v e 5-HT4 r e c e p t o r found i n mouse c o l l i c u l i neurons (Dumuis, Sebben & Bockaert, 1 9 8 9 ) , has r e c e n t l y been i d e n t i f i e d on guinea p i g ileum ( C r a i g & C l a r k , 1990) . I n t e r e s t i n g l y , ICS 2 0 5 - 9 3 0 i s a c t i v e at t h i s r e c e p t o r , though with lower a f f i n i t y than at 5-HT3 r e c e p t o r s i t e s (Craig, Eglen, Walsh, P e r k i n s , Whiting & C l a r k , 1 9 9 0 ). Furthermore, 2-Me-5-HT and 5-HT induce i n h i b i t o r y responses i n r a t sympathetic neurons which appear to be mediated v i a a 5-HT s i t e other than the 5-HT!, 5-HT2 or 5-HT3 r e c e p t o r (Lewis & Coote, 1990) . Although f u r t h e r evidence i s necessary t o c l a r i f y such f i n d i n g s , the e x i s t e n c e of 8 he t e r o g e n e i t y w i t h i n c e n t r a l 5-HT3 b i n d i n g s i t e s / r e c e p t o r s remains a p o s s i b i l i t y . Many of the f u n c t i o n s of 5-HT3 r e c e p t o r s i n v o l v e modulation of the r e l e a s e of other n e u r o t r a n s m i t t e r s . Evidence i n d i c a t e s t h a t 5-HT3 r e c e p t o r s modulate d i s t u r b a n c e s i n , but not b a s a l l e v e l s of, mesolimbic dopamine (DA; C o s t a l l et a l . , 1990b). Furthermore, 5-HT3 r e c e p t o r s a l s o i n f l u e n c e the r e l e a s e of nor e p i n e p h r i n e (NE) and a c e t y l c h o l i n e (ACh). The 5-HT3 a g o n i s t , 2-Me-5-HT, i n h i b i t s NE r e l e a s e i n r a t hypothalamic s l i c e s (Blandina, G o l d f a r b , Walcott, & Green, 1991) and b l o c k s r e l e a s e of ACh i n i s o l a t e d r a t e n t o r h i n a l cortex (Barnes, Barnes, C o s t a l l , Naylor & Tyers, 1989). As w e l l , 2-Me-5-HT has been demonstrated t o i n h i b i t ACh r e l e a s e i n the c e r e b r a l c o r t i c e s of unanaesthetized, f r e e l y - m o v i n g guinea p i g s ( B i a n c h i , S i n i s c a l c h i & Beani, 1990). Moreover, 2-Me-5-HT-induced i n h i b i t i o n of NE and ACh r e l e a s e i s b l o c k e d by treatment with 5-HT3 a n t a g o n i s t s (Barnes et a l . , 1989/ B i a n c h i et a l . , 1990/ Bl a n d i n a et a l . , 1991) . Therefore, 5-HT 3-active compounds may, i n p a r t , exert t h e i r b e h a v i o u r a l and pharm a c o l o g i c a l e f f e c t s by modulating r e l e a s e of DA, ACh and NE. Although i t remains to be confirmed whether 5-HT3 r e c e p t o r s modulate sexual a c t i v i t y , other b e h a v i o u r a l e f f e c t s of 5-HT3 a c t i v a t i o n are reasonably w e l l - e s t a b l i s h e d . 5-HT3 a n t a g o n i s t s r e p o r t e d l y improve performance on ob j e c t d i s c r i m i n a t i o n and r e v e r s a l l e a r n i n g t a s k s (Barnes et a l . , 1990b), d i s p l a y a n x i o l y t i c p r o p e r t i e s (e.g. C o s t a l l , 1988a; C u t l e r , 1990/ Jones 9 et a l . , 1988; Papp, 1988; Rodgers, Shepherd & R a n d a l l , 1990), antagonize the a n t i n o c i c e p t i v e e f f e c t s of 5-HT (Glaum, P r o u d f i t & Anderson, 1988), i n h i b i t nausea and vomitin g (e.g. Higgins, K i l p a t r i c k , Bunce, Jones & Tyers, 1989) and attenuate c e r t a i n symptoms of withdrawal from benzodiazepines ( C o s t a l l et a l . , 1989; Goudie & Leat h l e y , 1990), e t h a n o l , n i c o t i n e and cocaine ( C o s t a l l , Jones, K e l l y , Naylor, O n a i v i & Tyers, 1990a) . As noted p r e v i o u s l y , the i n f l u e n c e of 5-HT3 r e c e p t o r a c t i v i t y on mating behaviour i s u n c l e a r . Furthermore, the mechanism whereby 5-HT3 r e c e p t o r s may modulate such behaviours has yet t o be determined. P o t e n t i a l e f f e c t s on sexual behaviour may be due e i t h e r t o d i r e c t s e r o t o n e r g i c a c t i o n or t o modulation of other n e u r o t r a n s m i t t e r s . Furthermore, the r e l a t i o n s h i p between e f f e c t s on l o r d o s i s frequency and e f f e c t s on l o r d o s i s i n t e n s i t y and p r o c e p t i v e behaviours i s not c l e a r . No drug demonstrates complete s e l e c t i v i t y f o r a s i n g l e type of s i t e ; moreover, c u r r e n t l y u n i d e n t i f i e d s i t e s may e x i s t f o r which seemingly s e l e c t i v e compounds show a f f i n i t y . Thus, use of m u l t i p l e a g o n i s t s and a n t a g o n i s t s i s necessary i n the c h a r a c t e r i z a t i o n of a r e c e p t o r . Convergent evidence from s t u d i e s u s i n g d i f f e r e n t drugs with v a r y i n g s e l e c t i v i t i e s i s needed t o i n c r e a s e c o n f i d e n c e . i n the r e s u l t s o btained. Many n e u r a l and hormonal mechanisms i n t e r a c t i n the e x p r e s s i o n of female sexual behaviour. In the presence of estrogen, s e r o t o n e r g i c (Gorzalka, et a l . , 1990), dopaminergic (Fernandez-Guasti, A h l e n i u s , H j o r t h , & Larsson, 1987), o p i o i d 10 (Pfaus & Gorzalka, 1987a) , c h o l i n e r g i c (Richmond & Clemens, 1986) and a d r e n e r g i c a c t i v i t y (Mendelson & Gorzalka, 1988) a l l c o n t r i b u t e t o r e g u l a t i o n of female r e p r o d u c t i v e a c t i v i t y . O p iates, f o r example, may f a c i l i t a t e or i n h i b i t female c o p u l a t o r y behaviour, depending on b r a i n s i t e and subtype of r e c e p t o r a c t i v a t e d (Pfaus & Gorzalka, 1987b). Systemic a d m i n i s t r a t i o n of the L i - o p i o i d a g o n i s t morphine i n h i b i t s l o r d o s i s (Pfaus & Gorzalka, 1987a) . Dopamine a l s o a f f e c t s c o p u l a t o r y behaviour, i n gen e r a l p l a y i n g an i n h i b i t o r y r o l e i n females (Crowley & Zemlan, 1981). O b j e c t i v e s The f o l l o w i n g s t u d i e s were conducted with the aim of f u r t h e r c h a r a c t e r i z i n g the 5-HT3 r e c e p t o r and i n c r e a s i n g understanding of the neurochemical c o n t r o l of female r e p r o d u c t i v e behaviour. The e f f e c t s of 5-HT3 a n t a g o n i s t s and a g o n i s t s on b a s a l sexual a c t i v i t y i n the female r a t were i n v e s t i g a t e d i n P a r t s I and I I , r e s p e c t i v e l y . O p i o i d s and dopamine i n f l u e n c e female r e p r o d u c t i v e behaviours and 5-HT3 a n t a g o n i s t s modulate e f f e c t s produced by o p i o i d s and dopamine. For example, 5-HT3 a n t a g o n i s t s b l o c k morphine-induced p l a c e p r e f e r e n c e (Carboni, Acquas, Leone, & Di C h i a r a , 1989) and dopamine-induced h y p e r a c t i v i t y ( C o s t a l l et a l . , 1990b). I t i s t h e r e f o r e p l a u s i b l e t h a t s e r o t o n i n may i n t e r a c t with these n e u r o t r a n s m i t t e r s i n order t o r e g u l a t e sexual a c t i v i t y . Thus, i n 11 P a r t I I I , the a b i l i t y of 5-HT3 a n t a g o n i s t s to b l o c k morphine-induced i n h i b i t i o n of r e p r o d u c t i v e behaviour was examined. Part IV was conducted with an aim towards examining the i n t e r a c t i v e e f f e c t s of dopamine agonism and 5-HT3 blockade on female r a t sexual behaviour. GENERAL METHODS Animals and Surgery Female Long-Evans r a t s d e r i v e d from stock o r i g i n a l l y o b t a i n e d from Charles R i v e r , Quebec served as s t i m u l u s and experimental s u b j e c t s . At a minimum of 60 days of age, animals underwent b i l a t e r a l ovariectomy v i a lumbar i n c i s i o n while under g e n e r a l a n a e s t h e s i a (sodium p e n t o b a r b i t a l 45 mg/kg and ketamine 40 mg/kg). F o l l o w i n g surgery, which o c c u r r e d at l e a s t one week p r i o r t o any b e h a v i o u r a l t e s t i n g , animals were housed i n sta n d a r d l a b o r a t o r y cages i n an e x c l u s i v e l y female colony maintained on a r e v e r s e 12 h l i g h t / 1 2 h dark c y c l e at 21+1 °C with food and water a v a i l a b l e ad l i b i t u m . Animals used i n Experiments 4 and 5 had guide cannulae implanted s t e r e o t a x i c a l l y under the g e n e r a l a n a e s t h e s i a regime d e s c r i b e d above. Cannula placement was estimated u s i n g the s t e r e o t a x i c a t l a s of P e l l e g r i n o , P e l l e g r i n o and Cushman (1979). S t a i n l e s s s t e e l guide cannulae were i n s e r t e d v i a b u r r h o l e s and cemented i n p l a c e with d e n t a l a c r y l i c t o j e w e l l e r ' s screws 12 i n s e r t e d i n t o the s k u l l . The s c a l p was then s u t u r e d around the s k u l l cap and an obdurator i n s e r t e d i n t o the guide cannula. F o l l o w i n g surgery, animals were s i n g l y housed and given two weeks t o re c o v e r p r i o r t o any b e h a v i o u r a l t e s t i n g . Cannulae placements were assessed approximately one week f o l l o w i n g surgery by i n f u s i o n of 2 Lig a n g i o t e n s i n II i n a 2 | i l volume i n t o the l a t e r a l v e n t r i c l e . Only animals demonstrating a s i g n i f i c a n t d r i n k i n g response were r e t a i n e d . Male Long-Evans r a t s s e l e c t e d f o r t h e i r c o p u l a t o r y v i g o u r served as studs. Drug Procedures Females were primed with subcutaneous (SC) e s t r a d i o l benzoate (EB) and progesterone (P) ( S t e r a l o i d s ) d i s s o l v e d i n 0.1 cc of peanut o i l , a d m i n i s t e r e d 48 h and 4 h p r i o r t o t e s t i n g , r e s p e c t i v e l y . As females show i d i o s y n c r a t i c responses t o these hormones, dosages necessary t o induce a p p r o p r i a t e l e v e l s of r e c e p t i v i t y were e s t a b l i s h e d on the b a s i s of p r e t e s t i n g with each group of animals. In P a r t s I and I I , s t e r o i d doses which induced moderate l e v e l s of r e c e p t i v i t y were employed i n order t o permit o b s e r v a t i o n of e i t h e r a f a c i l i t a t o r y or i n h i b i t o r y e f f e c t . In P a r t s I I I and IV, s t e r o i d doses which produced h i g h e r l e v e l s of r e c e p t i v i t y were used t o f a c i l i t a t e o b s e r v a t i o n of i n h i b i t o r y e f f e c t s . Nonexperimental stimulus females were primed with 10 ug EB and 500 ug P SC. 13 B e h a v i o u r a l T e s t i n g T e s t i n g took p l a c e at 7 day i n t e r v a l s d u r i n g the dark phase of the l i g h t c y c l e . T e s t i n g o c c u r r e d i n a c y l i n d r i c a l p l e x i g l a s s chamber, 45 cm i n height and 29 cm i n diameter, l i n e d with san-i - c e l l bedding m a t e r i a l . Subsequent to a 10 minute h a b i t u a t i o n p e r i o d and p r i o r t o i n t r o d u c t i o n of the experimental female, males were b r i e f l y exposed to a f u l l y r e c e p t i v e s t i m u l u s female. B e h a v i o u r a l s c o r i n g was conducted by an observer b l i n d t o the animals' treatment c o n d i t i o n s . Hardy and DeBold (1972) d e s c r i b e t h r e e i n t e n s i t i e s of l o r d o s e s : marginal ( s l i g h t f l e x i o n of the spine, s l i g h t l y r a i s e d head, h i p s with t a i l base e l e v a t e d from f l o o r ) , normal (moderate s p i n a l f l e x i o n , head at a 30° angle, f r o n t paws s l i g h t l y forward, h i n d l e g s s t r a i g h t and s t i f f ) and exaggerated (pronounced s p i n a l f l e x i o n , head at a minimum 45° a n g l e ) . As t e s t e r s were not able to a c c u r a t e l y d i s c r i m i n a t e between these t h r e e i n t e n s i t i e s , only two l e v e l s of l o r d o s e s , f u l l and p a r t i a l , were recorded. To q u a l i f y as a f u l l l o r d o s i s (corresponding to Hardy and DeBold's "exaggerated" c a t e g o r y ) , a response had to i n v o l v e f u l l d o r s i f l e x i o n of the back, displacement of the t a i l and hyperextension of the neck to an angle 45° or g r e a t e r above the h o r i z o n t a l . P a r t i a l l o r d o s e s i n c l u d e d those responses c o n t a i n i n g some but not a l l of the f u l l complement of behaviours l i s t e d above, or a t t e n u a t e d v e r s i o n s t h e r e o f . The number of f u l l and p a r t i a l l o r d o s e s observed i n 14 r e s p o n s e t o 10 m o u n t s b y a m a l e a n d t h e p r e s e n c e o r a b s e n c e o f e a r w i g g l i n g (EW) a n d h o p p i n g / d a r t i n g - (HD) w e r e r e c o r d e d . A n i m a l s w e r e t e s t e d t w o a t a t i m e i n s e p a r a t e c y l i n d e r s ; t e s t i n g c o n t i n u e d u n t i l t h e f e m a l e w a s m o u n t e d 10 t i m e s o r f o r a t o t a l o f 10 m i n u t e s . I f a m a l e e j a c u l a t e d o r f a i l e d t o m o u n t , h e w a s r e p l a c e d w i t h a n e w s t u d . D a t a A n a l y s i s F u l l a n d p a r t i a l l o r d o t i c r e s p o n s e s w e r e s c o r e d a s 2 a n d 1 r e s p e c t i v e l y . L Q a n d m e a n l o r d o s i s i n t e n s i t y ( L I ) w e r e c a l c u l a t e d f o r e a c h s u b j e c t a s [ ( # l o r d o s e s / # m o u n t s ) X 100%] a n d [ t o t a l n u m b e r o f p o i n t s / t o t a l n u m b e r o f l o r d o t i c r e s p o n s e s ] r e s p e c t i v e l y ( H a r d y & D e B o l d , 1972) . U n l e s s o t h e r w i s e s t a t e d , n o n p a r a m e t r i c t e s t s w e r e e m p l o y e d : L Q a n d L I d a t a w e r e a n a l y z e d u s i n g F r i e d m a n t w o - w a y a n a l y s i s o f v a r i a n c e ( A N O V A ) , f o l l o w e d , w h e n a p p r o p r i a t e , b y W i l c o x o n t e s t s f o r p a i r w i s e c o m p a r i s o n s ; p r o p o r t i o n s o f a n i m a l s d i s p l a y i n g EW a n d HD w e r e a n a l y z e d v i a C o c h r a n ' s Q t e s t , f o l l o w e d b y M c N e m a r ' s t e s t f o r p a i r w i s e c o m p a r i s o n s . P a r t I - A d m i n i s t r a t i o n o f 5 - H T 3 A n t a g o n i s t s E x p e r i m e n t 1 MDL 72222 ( l o c H , 3 a , 5 a H - t r o p a n - 3 - y l - 3 , 5 - d i c h l o r o b e n z o a t e ) , a 15 s t r u c t u r a l analogue of cocaine, i s a potent 5-HT3 a n t a g o n i s t (Fozard, 1984; Fortune & I r e l a n d , 1984). I t i s s e l e c t i v e f o r 5-HT3 s i t e s over other subtypes of 5-HT r e c e p t o r s and mu s c a r i n i c a c e t y l c h o l i n e r g i c r e c e p t o r s (Fozard, 1984) . Re c e n t l y however, MDL 72222 has been found t o be equipotent i n b l o c k i n g 5-HT3 and n i c o t i n i c r e c e p t o r s (Vanner & Suprenant, 1990), making i t a l e s s s e l e c t i v e a n t a g o n i s t than o r i g i n a l l y thought. MDL 72222 has been observed t o i n f l u e n c e c e n t r a l l y mediated behaviours f o l l o w i n g p e r i p h e r a l a d m i n i s t r a t i o n . In mice, SC a d m i n i s t r a t i o n of MDL 72222 produces dose-dependent a n a l g e s i a of chemical, but not thermal or mechanical p a i n (Giordano & Dyche, 1989) . In f e r r e t s , MDL 72222 admi n i s t e r e d e i t h e r SC or d i r e c t l y i n t o the area postrema i n h i b i t s c i s p l a t i n - i n d u c e d emesis (Higgins et a l . , 1989) and, i n r a t s r e c e i v i n g an i s o l e u c i n e - i m b a l a n c e d d i e t , o r a l a d m i n i s t r a t i o n of MDL 72222 attenuates the observed decrease i n food i n t a k e (Hammer, Gietzen, Beverly, & Rogers, 1990). As w e l l , MDL 72222 has been observed t o have a n x i o l y t i c e f f e c t s i n both rodents and primates (Tyers et a l . , 1987) . In a d d i t i o n t o these e f f e c t s , MDL 72222 has a l s o been p u r p o r t e d t o i n f l u e n c e sexual behaviour i n r a t s . In p a r t i c u l a r , MDL 72222 has been r e p o r t e d both to exert no i n f l u e n c e on l o r d o s i s i n EB-primed r a t s (Mendelson & Gorzalka, 1989) and to f a c i l i t a t e (James, Lane, Hole & Wilson, 1989) l o r d o s i s i n no n r e c e p t i v e but not i n r e c e p t i v e r a t s . I t i s p o s s i b l e however t h a t the r e p o r t e d f a c i l i t a t i o n simply r e f l e c t s a s t a t i s t i c a l r e g r e s s i o n toward the mean as even s a l i n e treatment can i n h i b i t 16 or f a c i l i t a t e l o r d o s i s when animals are d i v i d e d i n t o r e c e p t i v e and n o n r e c e p t i v e groups p r i o r t o s t a t i s t i c a l a n a l y s i s (Raible & Gorzalka, 1986). The present study was t h e r e f o r e designed t o he l p c l a r i f y these c o n f l i c t i n g f i n d i n g s . Method F i f t e e n o v a r i e c t o m i z e d female Long-Evans r a t s , approximately 6 months of age and weighing between 300 and 385 grams served as s u b j e c t s . Animals were primed with 5 fig EB and 100 (ig P, dosages which had r e s u l t e d i n a b a s e l i n e LQ of 72% d u r i n g p r e t e s t i n g . MDL 72222 (Research B i o c h e m i c a l s Inc.) was d i s s o l v e d i n a drop of d i l u t e g l a c i a l a c e t i c a c i d and made up to volume with d i s t i l l e d H20. In a counterbalanced, repeated measures design, animals r e c e i v e d SC i n j e c t i o n s of 0 ( i . e . v e h i c l e ) , 0.05, 0.5 or 5 mg/ml/kg MDL 72222 30 minutes p r i o r t o t e s t i n g , such t h a t each animal r e c e i v e d a l l drug dosages. R e s u l t s and D i s c u s s i o n One animal f a i l e d t o d i s p l a y l o r d o s i s under c o n t r o l c o n d i t i o n s and one d i e d p r i o r to completion of the experiment, r e s u l t i n g i n a sample s i z e of 13. Examination of Table 1 suggests t h a t MDL 72222 d i d not e f f e c t sexual a c t i v i t y . S t a t i s t i c a l a n a l y s i s confirmed t h i s : i n EB- and P-primed animals, MDL 72222 f a i l e d t o modify LQ (X 2=3.9462, £=.2673), LI 17 Table 1. E f f e c t s of MDL 72222 on l o r d o s i s q u o t i e n t (LQ) , l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g ear w i g g l i n g (EW) and h o p p i n g / d a r t i n g (HD) i n animals primed with 5 fig e s t r a d i o l benzoate (EB) and 100 |lg progesterone (P) . Values r e p r e s e n t means +_ S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW and HD). E f f e c t s o f MDL 72222 on S e x u a l B e h a v i o u r i n EB- a n d P - p r i m e d F e m a l e R a t s DOSE LQ L I EW HD (mg/kg) 0.00 88.08 ± 6.29 1.80 ± .06 0.92 0.38 0.05 82.31 ± 6.22 1.78 ± .07 0.92 0.23 0.50 73.85 ± 7.38 1.73 ± .08 0.85 0.23 5.00 80.77 ± 6.15 1.79 ± .06 0.77 0.31 19 ( X 2 = 1 . 1 7 5 0 , £ = . 7 5 9 0 ) , EW (Q= 4 . 0 0 0 0 , p_=.2615) or HD (0=1.0645, p_=.7856) . The f a i l u r e of MDL 72222 t o modify b a s a l l e v e l s of r e c e p t i v e or p r o c e p t i v e behaviours i s c o n s i s t e n t with i t s l a c k of e f f e c t on other b e h a v i o u r a l parameters when ad m i n i s t e r e d alone (e.g. Carboni et a l . , 1989) . Moreover, the l a c k of e f f e c t with r e s p e c t to female sexual behaviour i s c o n s i s t e n t with r e s u l t s o b t a i n e d by James et a l . , (1989) i n r e c e p t i v e animals, and with f i n d i n g s r e p o r t e d by Mendelson and Gorzalka (1989) . However, i t should be noted t h a t o b s e r v a t i o n of a f a c i l i t a t o r y e f f e c t would have been h i n d e r e d by the h i g h LQ of the c o n t r o l group (88%) . Nonetheless, the c u r r e n t f i n d i n g s suggest t h a t , i n female EB- and P-primed r a t s , MDL 7 2 2 2 2 does not i n f l u e n c e sexual behaviour. MDL 7 2 2 2 2 ' s l a c k of s p e c i f i c i t y may have c o n t r i b u t e d to the c u r r e n t f i n d i n g s . That MDL 72222 has been found equipotent at b l o c k i n g 5-HT3 s i t e s and n i c o t i n i c c h o l i n o c e p t o r s (Vanner & Suprenant, 1990) suggests the p o s s i b i l i t y of c h o l i n e r g i c a t t e n u a t a t i o n of a s e r o t o n e r g i c e f f e c t . For example, e f f e c t i v e blockade of n i c o t i n i c c h o l i n o c e p t o r s at neuromuscular j u n c t i o n s may have prevented b e h a v i o u r a l e x p r e s s i o n of l o r d o s i s . However, t h i s seems u n l i k e l y : no a l t e r a t i o n s i n motor behaviour were noted. Furthermore, i n E B - and P-primed females, a d m i n i s t r a t i o n of the n i c o t i n i c r e c e p t o r a n t a g o n i s t , mecamylamine, f a i l s to e f f e c t r e c e p t i v i t y (Weaver & Clemens, 1984) . The present r e s u l t s may a l s o be r e l a t e d t o MDL 7 2 2 2 2 ' s 20 mechanism of a c t i o n . MDL 72222 does not appear t o act as a simple, c o m p e t i t i v e 5-HT3 a n t a g o n i s t . Although i t i s a potent a n t a g o n i s t of 5-HT-induced d e p o l a r i z a t i o n of r a t vagus nerve, MDL 72222 produces a concentration-dependent s u p p r e s s i o n of the maximum response t o 5-HT, a mechanism of a c t i o n not c o n s i s t e n t with t h a t of a simple r e v e r s i b l e c o m p e t i t i v e a n t a g o n i s t ( I r e l a n d and Tyers, 1987). C o n s i s t e n t with t h i s f i n d i n g , Fozard et a l . , (1985) note t h a t while 1 hour exposure t o MDL 72222 produces c o m p e t i t i v e blockage, longer exposure r e s u l t s i n a f u r t h e r insurmountable blockade. Moreover, at guinea p i g ileum, MDL 72222 a c t s only weakly and as a n o n s e l e c t i v e a n t a g o n i s t of 5 - H T 3 receptor-mediated c o n t r a c t i o n s (Fozard, 1984) . Thus, while MDL 72222 appears t o act as a 5-HT3 a n t a g o n i s t at c e r t a i n p e r i p h e r a l r e c e p t o r s and i n the modulation of some c e n t r a l l y - m e d i a t e d behaviours, i t s mechanism of a c t i o n , as w e l l as reasons f o r i n a c t i v i t y at 5-HT3 s i t e s on guinea p i g ileum remain u n c l e a r . MDL 72222's l a c k of e f f e c t with regard t o sexual behaviour may be unique; the c u r r e n t f i n d i n g s do not u n e q u i v o c a l l y r e s o l v e whether 5-HT3 r e c e p t o r a c t i v i t y i s i n v o l v e d i n modulation of female r e p r o d u c t i v e a c t i v i t y . Experiment 2 Ondansetron (GR38032F; 1,2,3,9-tetrahydro-9-methyl-3-[2 -methyl-lH-imidazol-l-yl)-methyl]-4H-carbazol-4-one,HCL- 2H20) i s a potent and s e l e c t i v e 5-HT3 r e c e p t o r a n t a g o n i s t ( B u t l e r , H i l l , I r e l a n d , Jordan & Tyers, 1988) . In c o n t r a s t t o MDL 72222, ondansetron i s a c t i v e at g u i n e a - p i g ileum, although i t does f a i l t o antagonize a p o r t i o n of the b i p h a s i c smooth muscle response t o 5-HT seen i n t h i s b i o a s s a y ( B u t l e r et a l . , 19 8 8 ) . Furthermore, i t s a c t i o n s on r a b b i t heart t i s s u e are not c o n s i s t e n t with t h a t of a simple, r e v e r s i b l e c o m p e t i t i v e a n t a g o n i s t ( B u t l e r et a l . , 1988) . Ondansetron has a s e l e c t i v i t y r a t i o g r e a t e r than 1000 f o r 5-HT3 s i t e s over 5-HTx or 5-HT2 s i t e s and d i s p l a y s n e g l i g i b l e a c t i v i t y at non5-HT s i t e s ( B u t l e r et a l . , 1988). Moreover, u n l i k e MDL 72222, ondansetron i s 1 0 0 - f o l d more s e l e c t i v e f o r 5-HT3 s i t e s than n i c o t i n i c c h o l i n e r g i c s i t e s (Vanner & Suprenant, 1990) . Ondansetron i s a b e h a v i o u r a l l y a c t i v e compound. I t has potent a n x i o l y t i c e f f e c t s i n both rodents and primates with no s e d a t i v e , a n t i c o n v u l s a n t or hypnotic e f f e c t s (e.g. Jones et a l . , 1988), and i s e f f e c t i v e i n modifying i n c r e a s e s i n mesolimbic dopamine f u n c t i o n (Hagan, Jones, Jordan & Tyers, 1990) and the r e s u l t a n t h y p e r a c t i v i t y ( C o s t a l l et a l , 1990b). Ondansetron produces dose-dependent a n a l g e s i a i n chemical p a i n t e s t s i n mice (Giordano & Dyche, 1989) and i n h i b i t s c i s p l a t i n - i n d u c e d emesis i n the f e r r e t (Higgins, et a l . , 1989) . In t e s t s of c o g n i t i v e f u n c t i o n , such as o b j e c t d i s c r i m i n a t i o n and r e v e r s a l l e a r n i n g t a s k s , ondansetron improves b a s a l performance and attenuates decrements i n performance due to scopolamine- or l e s i o n - i n d u c e d c h o l i n e r g i c d e f i c i t s (Barnes et a l . , 1990b) . In marmosets, ondansetron reduces a l c o h o l i n t a k e f o l l o w i n g e t h a n o l withdrawal 22 and re-exposure and attenuates behaviours induced by withdrawal (Oakley et a l . , 1988) . As ondansetron i s e f f e c t i v e i n a l t e r i n g c e r t a i n b a s e l i n e behaviours, i t i s c o n c e i v a b l e t h a t i t may e f f e c t b a s a l female r e p r o d u c t i v e a c t i v i t y . Such an e f f e c t may be unique from t h a t of MDL 72222 as these two compounds show somewhat d i f f e r e n t b e h a v i o u r a l e f f e c t s and pharmacodynamic p r o f i l e s . The purpose of present study was to e s t a b l i s h whether the f a c i l i t a t o r y e f f e c t of ondansetron, demonstrated i n nonreceptive females (James et a l . , 1989), c o u l d be extended t o i n c l u d e r e c e p t i v e females. Thus, t h i s experiment i n v e s t i g a t e d the e f f e c t s of v a r y i n g doses of ondansetron on p r o c e p t i v e and r e c e p t i v e behaviours i n EB- and P-primed, o v a r i e c t o m i z e d r a t s . Method Ei g h t e e n o variectomized, female Long-Evans r a t s 60 days of age, weighing 250 to 315 grams served as s u b j e c t s . Animals were primed with 3 (ig EB and 50 (ig P. These dosages had r e s u l t e d i n a mean LQ of 69% d u r i n g p r e t e s t i n g . Ondansetron (Glaxo) was d i s s o l v e d i n d i s t i l l e d H20. In a counterbalanced, repeated measures design, animals r e c e i v e d SC i n j e c t i o n s of 0 ( i . e . v e h i c l e ) , .001, .01, 0.1, 1.0, or 10 mg/ml/kg ondansetron, 45 minutes p r i o r t o t e s t i n g , such t h a t each animal r e c e i v e d a l l drug dosages. 23 R e s u l t s and D i s c u s s i o n R e s u l t s of Experiment 2 are presented i n Table 2. Ondansetron f a i l e d t o s i g n i f i c a n t l y e f f e c t LQ (X 2=7.6349, p_=.1775), LI (X 2=2.5143, p_=.7743), EW (Q=2.6238, p_=.7578), or HD (Q=8.4146, p_=.1348). Examination of F i g u r e 1 suggests a f a c i l i t a t o r y t r e n d of ondansetron on LQ at doses of 0.001, 0.01, 0.10 and 1.00 mg/kg. S i m i l a r l y , more animals d i s p l a y e d ear w i g g l i n g and h o p p i n g / d a r t i n g under the drug c o n d i t i o n s of 0.001, 0.01 and 0 . 1 mg/kg than under c o n t r o l c o n d i t i o n s . Although i t i s not evident from Table 2 and F i g u r e 1, the data o b t a i n e d suggest the i n f l u e n c e of an unknown v a r i a b l e r e l a t e d t o week of t e s t i n g . The mean LQ of c o n t r o l group animals decreased from 63 and 67% i n weeks 1 and 2, r e s p e c t i v e l y , t o 20, 33, 20 and 13% i n the subsequent 4 weeks. Moreover, at week 6, 11/18 animals f a i l e d t o d i s p l a y l o r d o s i s . P r i o r t o t h i s time, the maximum number of animals f a i l i n g t o d i s p l a y l o r d o s i s i n a s i n g l e s e s s i o n was 5. A subsequent t e s t i n g s e s s i o n on week 7 r e v e a l e d l o r d o t i c responses were absent i n 14/18 animals. The impact of any unknown i n f l u e n c e should be e q u a l l y d i s t r i b u t e d a c r o s s c o n d i t i o n s . Nonetheless, while the f a c i l i t a t o r y t r e n d of ondansetron, at doses other than 10 mg/kg, was manifest weekly, the apparent presence of an i n t e r v e n i n g v a r i a b l e makes i n t e r p r e t a t i o n d i f f i c u l t . I d e n t i f i c a t i o n of such a v a r i a b l e i s v e x a t i o u s at b e s t . Although o v a r i e c t o m i z e d animals are known to become l e s s 24 Table 2. E f f e c t s of ondansetron on l o r d o s i s q u o t i e n t (LQ) , mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g ear w i g g l i n g (EW) and h o p p i n g / d a r t i n g (HD) i n animals primed with 3 (ig e s t r a d i o l benzoate (EB) and 50 ug progesterone (P) . Values r e p r e s e n t means +, S.E.M.s (LQ and L I ) ; p r o p o r t i o n s (EW, HD). 25 E f f e c t s of Ondansetron on Sexual Behaviour i n EB- and P-primed Female Rats DOSE LQ LI EW HD (mg/kg) 0.000 36.11 + 8.93 1.63 + .09 0.27 0.00 0.001 60.56 + 8.69 1.55 + .09 0.39 0.11 0.010 63.33 + 9.04 1.66 + .08 0.39 0.06 0.100 57.22 + 9.10 1.55 + .08 0.50 0.22 1.000 56.11 + 7.63 1.60 + .08 0.44 0.00 10.000 44.44 + 10.67 1.66 + .09 0.39 0.11 26 F i g u r e 1. E f f e c t s of ondansetron on l o r d o s i s q u o t i e n t i n animals primed with 3 |ig e s t r a d i o l benzoate and 50 \xg progesterone. CN 28 r e s p o n s i v e t o given dosages of hormones with i n c r e a s i n g age and extended estrogen a d m i n i s t r a t i o n or d e p r i v a t i o n , the c u r r e n t s u b j e c t s were r e l a t i v e l y young, had undergone gonadectomy only 1 week p r i o r t o t e s t i n g and had r e c e i v e d minimal amounts of est r o g e n . Thus, the reasons u n d e r l y i n g observed a l t e r a t i o n s i n su b j e c t response remain u n c l e a r . The presence of a f a c i l i t a t o r y t r e n d i s c o n s i s t e n t with the r e s u l t s of James et a l . , (1989) i n non r e c e p t i v e animals. I t i s worth n o t i n g the low c o n t r o l group LQ (36%) observed i n the present study, which suggests c o m p a r a b i l i t y with the no n r e c e p t i v e animals of James et a l . The c u r r e n t r e s u l t s are a l s o c o n s i s t e n t with unpublished o b s e r v a t i o n s from our l a b o r a t o r y wherein the tendency of ondansetron t o f a c i l i t a t e c e r t a i n components of male r a t sexual behaviour was noted (N.V. Watson, p e r s o n a l communication, 1991). However, c l e a r e l u c i d a t i o n of the e f f e c t of ondansetron on female sexual behaviour r e q u i r e s r e p l i c a t i o n of t h i s experiment. Experiment 3 ICS 205-930 ( ( 2 - t r o p a n y l ) - l H - i n d o l e - 3 - c a r b o x y l i c a c i d e s t e r ) i s a potent and s e l e c t i v e 5-HT3 a n t a g o n i s t , a c t i v e on guinea p i g ileum, where, u n l i k e MDL 72222, i t behaves as a t r u e c o m p e t i t i v e a n t a g o n i s t (Donatsch, Engel, Richardson & S t a d l e r , 1984). ICS 205-930 i s 300 f o l d more s e l e c t i v e i n b l o c k i n g 5-HT3 than n i c o t i n i c c h o l i n o c e p t o r s whereas ondansetron i s approximately 100 f o l d more s e l e c t i v e and MDL 72222 i s equipotent (Vanner & Surprenant, 1990). Furthermore, the potency of ICS 205-930 i n i n h i b i t i n g the von B e z o l d - J a r i s c h e f f e c t i n v i v o i s 100 times g r e a t e r than t h a t of MDL 72222 (Donatsch, Engel, Richardson & S t a d l e r , 1984) . ICS 205-930 i n f l u e n c e s many behaviours and p h a r m a c o l o g i c a l e f f e c t s . In mice, ICS 205-930 produces dose-dependent a n a l g e s i a of chemical p a i n (Giodano & Dyche, 1989) and b l o c k s the a n o r e c t i c response of r a t s f e d an amino acid-imbalanced d i e t (Hammer et a l . , 1990). I n d i c a t i v e of a n x i o l y t i c p r o p e r t i e s , ICS 205-930 i n c r e a s e s elements of s o c i a l behaviours i n g e r b i l s under a v e r s i v e environmental c o n d i t i o n s ( C u t l e r , 1990) and i n r a t s , b l o c k s shock-induced c o n d i t i o n e d p l a c e a v e r s i o n (Papp, 1988). Moreover, ICS 205-930 p o t e n t l y attenuates h y p e r a c t i v i t y induced by r a i s e d mesolimbic dopamine ( C o s t a l l , et a l . , 1987c). ICS 205-930 i s e f f e c t i v e under c e r t a i n c o n d i t i o n s where other 5-HT3 a n t a g o n i s t s d i s p l a y l i t t l e or no i n f l u e n c e . In p a r t , t h i s i s l i k e l y a r e f l e c t i o n of i t s a c t i v i t y at a novel r e c e p t o r s i t e , the p u t a t i v e 5-HT4 r e c e p t o r (e.g. C r a i g et a l . , 1990) . ICS 205-930 i s a more potent a n t a g o n i s t of 5-HT-induced a n t i n o c i c e p t i o n than i s MDL 72222 (Glaum, P r o u d f i t & Anderson, 1990) and h i g h c o n c e n t r a t i o n s of ICS 205-930, but not MDL 72222 or ondansetron, b l o c k 5-HT produced r e l a x a t i o n of smooth muscle i n r a t esophagus (Reeves, Bunce, & Humphrey, 1991) . F u r t h e r evidence of ICS 205-930's unique pharmacodynamic p r o f i l e i s suggested by f i n d i n g s t h a t , i n r a t s , decreases i n d i a s t o l i c and 30 i n t r a g a s t r i c p r e s s u r e s , evoked by i n c r e a s e d duodenal p r e s s u r e are a t t e n u a t e d by ICS 205-930, but not by ondansetron (Moss & Sanger, 1990) . Furthermore, antagonism by ICS 205-930 of the von B e z o l d - J a r i s c h r e f l e x i s of longer d u r a t i o n than t h a t produced by ondansetron (Cohen, Bloomquist, Gidda & L a c e f i e l d , 1989) . As noted p r e v i o u s l y , p r e l i m i n a r y evidence suggests t h a t ICS 205-930 p l a y s a f a c i l i t a t o r y r o l e i n the modulation of female sexual behaviour (Mendelson & Gorzalka, 1989) . Moreover, d i s c r e p a n c i e s between pharmacodynamic and b e h a v i o u r a l e f f e c t s of ICS 205-930 and other 5-HT3 a n t a g o n i s t s suggest t h a t the i n f l u e n c e of ICS 205-930 on female r e p r o d u c t i v e behaviour may be unique from t h a t of MDL 72222 or ondansetron. The f o l l o w i n g study i s aimed at r e p l i c a t i n g p r e v i o u s f i n d i n g s r e g a r d i n g the i n f l u e n c e of ICS 205-930, with r e s p e c t t o female r a t sexual behaviour. Method T h i r t e e n female Long-Evans r a t s , approximately 12 months of age, weighing 270 to 410 grams served as s u b j e c t s . In order to r e p l i c a t e the hormonal paradigm of Mendelson and Gorzalka (1989), animals were primed with EB (7.5 Lig) o n l y . ICS 205-930 (Sandoz) was d i s s o l v e d i n a drop of d i l u t e g l a c i a l a c e t i c a c i d and made up to volume with d i s t i l l e d H20. In a counterbalanced, repeated measures design, animals r e c e i v e d SC i n j e c t i o n s of 0 ( i . e . v e h i c l e ) , 0.005, 0.05, 0.5 or 5.0 mg/kg/ml ICS 205-930 40 31 minutes p r i o r to t e s t i n g , such that each animal r e c e i v e d a l l drug dosages. Re s u l t s and Di s c u s s i o n Re s u l t s are presented i n Table 3 . ICS 2 0 5 - 9 3 0 ( 0 . 0 0 5 t o 5 . 0 mg/kg) f a i l e d t o s i g n i f i c a n t l y a f f e c t LQ ( X 2 = 2 . 4 6 1 5 , p_=.6515), LI ( X 2 = 1 . 2 1 5 4 , p_=.8756), EW (Q=5.8667, p_=.2093) or HD (Q=.091, p_=.9233) i n EB-primed r a t s . These r e s u l t s c o n t r a s t with those of Mendelson and Gorzalka, (1989). While reasons f o r t h i s discrepancy are not c l e a r , i t must be noted t h a t a s i n g l e drug dose was u t i l i z e d i n the previous experiment while the current i n v e s t i g a t i o n employed 4 d i f f e r e n t doses, a l l of which f a i l e d to produce evidence of f a c i l i t a t i o n . P art I I - A d m i n i s t r a t i o n of 5-HT3 Agonists Experiment 4 1-Phenylbiguanide (PBG) , a chemical s t r u c t u r a l l y u n r e l a t e d to 5-HT, i s a s e l e c t i v e 5-HT3 receptor agonist ( I r e l a n d & Tyers, 1 9 8 7 ). On r a t i s o l a t e d vagus nerve, PBG evokes 5-HT 3-mediated d e p o l a r i z a t i o n s , mimicking those produced by 5-HT (Ir e l a n d & Tyers, 1 9 8 7 ). As w e l l , i n j e c t i o n s of PBG i n t o r a b b i t c a r d i a c a t r i a produce decreases i n heart r a t e and a t r i a l pressure and t r a n s i e n t hypopnea, e f f e c t s a l s o mediated by 5-HT3 receptors 32 Table 3 . E f f e c t s of ICS 205-930 on l o r d o s i s q u o t i e n t (LQ), mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g ear w i g g l i n g (EW) and h o p p i n g / d a r t i n g (HD) i n animals primed with 7 .5 pig e s t r a d i o l benzoate (EB) . Values re p r e s e n t means ± S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW, HD). 33 E f f e c t s o f ICS 205-930 on S e x u a l B e h a v i o u r i n E B - p r i m e d F e m a l e R a t s DOSE LQ L I EW HD (mg/kg) 0.000 86.92 + 5.59 1.63 + .09 0.92 0.39 0.005 77.69 + 9.21 1.69 + .09 0.62 0.38 0.050 83.85 + 7.21 1.72 + .08 0.78 0.46 0.500 78.46 + 9.12 1.68 + .10 0.69 0.31 5.000 76.92 + 6.24 1.68 + .10 0.78 0.39 34 (Evans, Ludbrook & M i c h a l i c e k , 1990) . Due to the poor CNS p e n e t r a t i o n of PBG (Watling, 1989), i n v e s t i g a t i o n s concerning c e n t r a l l y - m e d i a t e d (e.g. behavioural) e f f e c t s r e q u i r e i t s c e n t r a l a d m i n i s t r a t i o n . Furthermore, PBG i s known to induce n o n s e r o t o n e r g i c c a r r i e r - m e d i a t e d r e l e a s e of [ 3H]dopamine (Schmidt & Black, 1989), making i n t e r p r e t a t i o n of b e h a v i o u r a l e f f e c t s d i f f i c u l t . Together, these encumbrances have r e s u l t e d i n a p a u c i t y of b e h a v i o u r a l i n v e s t i g a t i o n s . PBG may e f f e c t female c o p u l a t o r y a c t i v i t y by a d i r e c t s e r o t o n e r g i c mechanism. P e r i p h e r a l l y , PBG mimics the e f f e c t s of 5-HT (e.g. I r e l a n d & Tyers, 1987) . I f i t a l s o mimics the e f f e c t s of 5-HT when ad m i n i s t e r e d i n t o the l a t e r a l v e n t r i c l e , a f a c i l i t a t i o n of l o r d o s i s may be a n t i c i p a t e d , although i n the past t h i s e f f e c t has been a t t r i b u t e d t o 5-HT2 r e c e p t o r a c t i v i t y (Wilson & Hunter, 1985) . Conversely, i t i s p o s s i b l e t h a t by s t i m u l a t i n g dopamine r e l e a s e , PBG may i n f l u e n c e mating behaviour v i a a n o n s e r o t o n e r g i c mechanism. Dopamine a g o n i s t s decrease l o r d o s i s (e.g. Fernandez-Guasti et a l . , 1 9 8 7 ) ; t h e r e f o r e , i f dopaminergic e f f e c t s predominate, treatment with PBG can be expected t o i n h i b i t l o r d o s i s . The f o l l o w i n g study was designed to examine the i n f l u e n c e of v a r y i n g doses of c e n t r a l l y a d m i n i s t e r e d PBG on female r a t sexual behaviours. Method Six month o l d female Long-Evans r a t s weighing 250 to 350 35 grams were o v a r i e c t o m i z e d and had cannulae implanted i n t o the l e f t l a t e r a l v e n t r i c l e at the f o l l o w i n g c o o r d i n a t e s r e l a t i v e t o bregma: p o s t e r i o r 0 mm; l a t e r a l 1.7 mm; v e n t r a l 3.6 mm. Subjects were primed with 5 |lg EB and 100 ug P, dosages which produced a mean LQ of 82% d u r i n g p r e t e s t i n g . PBG (Research B i o c h e m i c a l s Inc.) was d i s s o l v e d i n s t e r i l e s a l i n e . A Sage Instruments (Orion Research Inc.) e l e c t r o n i c i n f u s i o n pump and 50 m i c r o l i t r e Hamilton s y r i n g e were used to d e l i v e r v e h i c l e and drug i n f u s i o n s . A l l animals r e c e i v e d 1 | ! l volumes d e l i v e r e d over 15 seconds; the i n f u s i o n needle was l e f t i n p l a c e f o r a f u r t h e r 45 seconds t o ensure t h a t a l l f l u i d had d i f f u s e d away from the t i p of the needle. In a counterbalanced, repeated measures design, s u b j e c t s r e c e i v e d 0 ( i . e . v e h i c l e ) , 0.4, 2.0, 10.0 and 50.0 pig doses of PBG such t h a t each animal r e c e i v e d a l l doses. I n f u s i o n s o c c u r r e d 30 minutes p r i o r t o b e h a v i o u r a l t e s t i n g . R e s u l t s and D i s c u s s i o n R e s u l t s are p r e s e n t e d i n Table 4. Of 2 6 animals d r i n k i n g v i g o r o u s l y i n response to a n g i o t e n s i n II i n f u s i o n , 1 was s a c r i f i c e d mid-experiment due to i l l n e s s , and a f u r t h e r 11 d i d not undergo a l l c o n d i t i o n s due to b l o c k e d or m i s s i n g cannulae, r e s u l t i n g i n a sample s i z e of 14. S t a t i s t i c a l a n a l y s i s confirmed t h a t PBG d i d not e f f e c t LQ (X 2=1.9286, p_=.7489), LI (X 2=2.1385, p_=.7103), EW (Q=8.2353, p_=.0833) or HD (Q=0.7692, £=.9425). The l a c k of i n h i b i t i o n suggests t h a t b e h a v i o u r a l l y , PBG-36 Table 4. E f f e c t s of 1-phenylbiguanide on l o r d o s i s q u o t i e n t (LQ), mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g ear w i g g l i n g (EW) and ho p p i n g / d a r t i n g (HD) i n animals primed with 5 |lg e s t r a d i o l benzoate (EB) and 100 |lg progesterone (P) . Values represent means ± S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW, HD). 37 Effe c t s of 1-Phenylbiguanide on Sexual Behaviour in EB- and P-primed Female Rats DOSE LQ LI EW HD (l^g) 0.0 95.71 + 4 .29 1.86 + .04 1 . 00 0 .71 0.4 89.29 + 5 .39 1.79 + .06 0 . 93 0 .71 2 . 0 92.86 + 4 . 96 1.79 + . 05 0 .71 0 .71 10 .0 80 .00 + 9 .38 1.74 + .09 0 .71 0 . 64 50.0 96.43 + 2 .25 1.88 + .05 0 . 93 0 .79 38 evoked dopamine e f f e c t s do not predominate. S i m i l a r l y , l a c k of f a c i l i t a t i o n suggests t h a t PBG does not mimic 5-HT with r e s p e c t t o female sexual behaviour. I t i s however p o s s i b l e t h a t both systems, s t i m u l a t e d simultaneously, a c t i v a t e d opposing mechanisms thus p r e v e n t i n g any net b e h a v i o u r a l e f f e c t . Experiment 5 L i k e PBG, 2-Me-5-HT (3-(2-Aminoethyl)-2-methyl-lH-indol-5-o l maleate) i s a s e l e c t i v e 5-HT3 a g o n i s t ( C r a i g et a l . , 1990; Is m a i e l , T i t e l e r , M i l l e r , Smith & Glennon, 1990), a c t i n g e q u i p o t e n t l y at guinea p i g ileum and r a b b i t h eart and vagus nerve (Richardson et a l . , 1985) . At guinea p i g ileum, i t i n h i b i t s the response t o 5-HT mediated v i a 5-HT3 r e c e p t o r s but does not b l o c k t h a t induced v i a the p u t a t i v e 5-HT4 r e c e p t o r s ( C r a i g et a l . , 1990). 2-Me-5-HT suppresses f i r i n g of medial p r e f r o n t a l c o r t i c a l c e l l s (Ashby, Edwards, Harkins, & Wang, 1989). U n l i k e PBG, 2-Me-5-HT does not induce n o n s e r o t o n e r g i c , c a r r i e r - m e d i a t e d r e l e a s e of dopamine (Schmidt & Black, 1989); i t does however, act v i a 5-HT3 r e c e p t o r s t o modulate r e l e a s e of no n s e r o t o n e r g i c n e u r o t r a n s m i t t e r s . 2-Me-5-HT i n c r e a s e s spontaneous r e l e a s e of dopamine i n s t r i a t a l s l i c e s (Blandina, G o l d f a r b , Craddock-Royal & Green, 1989) and i n the nucleus accumbens (Jiang, Ashby, Kasser & Wang, 1990), decreases n o r e p i n e p h r i n e r e l e a s e i n r a t hypothalamic s l i c e s (Blandina et 39 a l . , 1991) and decreases c o r t i c a l a c e t y l c h o l i n e r e l e a s e i n v i t r o (Barnes et a l . , 1989) and i n v i v o (Bianchi et a l . , 1990). On r a t sympathetic p r e g a n g l i o n i c neurons, 2-Me-5-HT evokes both i n h i b i t o r y and b i p h a s i c e f f e c t s , although evidence suggests the i n h i b i t o r y e f f e c t i s mediated v i a an, as yet u n i d e n t i f i e d , non5-HT3 r e c e p t o r (Lewis & Coote, 1990) . 2-Me-5-HT i s a b e h a v i o u r a l l y a c t i v e compound. Ad m i n i s t e r e d i n t r a t h e c a l l y , i t i n c r e a s e s t a i l f l i c k l a t e n c y , s u g g e s t i n g i t has a n t i n o c i c e p t i v e p r o p e r t i e s (Glaum et a l . , 1990). Infused d i r e c t l y i n t o the area postrema, 2-Me-5-HT produces sig n s of "nausea": s a l i v a t i o n , s e a r c h i n g , l i p l i c k i n g , r e t c h i n g , but not emesis (Higgins et a l . , 1989). I n j e c t e d i n t o g u i n e a - p i g hypothalami, i t decreases g a s t r i c emptying ( C o s t a l l , K e l l y , Naylor, Tan & T a t t e r s a l l , 1986) and adm i n i s t e r e d b i l a t e r a l l y i n t o r a t nucleus accumbens, 2-methyl-5-HT f a c i l i t a t e s amphetamine-induced h y p e r a c t i v i t y ( C o s t a l l et a l , 1987a). As 2-Me-5-HT mediates v a r i o u s b a s a l pharmacologic and b e h a v i o u r a l e f f e c t s and does not evoke c a r r i e r - m e d i a t e d dopamine r e l e a s e , i t may be e f f e c t i v e where PBG was not. The f o l l o w i n g experiment was t h e r e f o r e intended t o i n v e s t i g a t e the e f f e c t s of v a r y i n g doses of 2-Me-5-HT, admin i s t e r e d i n t o the l a t e r a l v e n t r i c l e , on female c o p u l a t o r y a c t i v i t y . Method Six month o l d female Long-Evans r a t s weighing 250 to 350 40 grams were o v a r i e c t o m i z e d and had cannulae implanted i n t o the l e f t l a t e r a l v e n t r i c l e at the f o l l o w i n g c o o r d i n a t e s r e l a t i v e t o bregma: p o s t e r i o r 0 mm; l a t e r a l 1.7 mm; v e n t r a l 4.1 mm. R e s u l t s from Experiment 4 i n d i c a t e t h a t PBG does not i n h i b i t r e c e p t i v e or p r o c e p t i v e behaviour, suggesting t h a t 5-HT3 a c t i v i t y i s not i n h i b i t o r y . Were the e f f e c t of 5-HT3 agonism t o be one of f a c i l i t a t i o n , i t may have been obscured by the h i g h LQ of the c o n t r o l group. Therefore, i n the c u r r e n t study the EB dose was decreased t o enable o b s e r v a t i o n of e i t h e r a f a c i l i t a t o r y or i n h i b i t o r y e f f e c t . Thus, s u b j e c t s were primed with 3 |ig EB and 100 (ig P, dosages which produced a mean LQ of 73% d u r i n g p r e t e s t i n g . 2-Me-5-HT (Research Biochemicals Inc.) was d i s s o l v e d i n s t e r i l e H20. A Sage Instruments (Orion Research Inc.) i n f u s i o n pump and 50 m i c r o l i t r e Hamilton s y r i n g e were used t o d e l i v e r drugs and v e h i c l e . A l l animals r e c e i v e d 2 ( i l volumes d e l i v e r e d over 30 seconds; the i n f u s i o n needle was l e f t i n p l a c e f o r a f u r t h e r 30 seconds t o ensure f l u i d d i s p o s i t i o n . In a counterbalanced, repeated measures design, s u b j e c t s r e c e i v e d 0 ( i . e . v e h i c l e ) , 1.5, 4.5, 13.5 and 40.5 u.g doses of 2-Me-5-HT such t h a t each animal r e c e i v e d a l l doses. I n f u s i o n s o c c u r r e d 15 minutes p r i o r t o b e h a v i o u r a l t e s t i n g . R e s u l t s and D i s c u s s i o n R e s u l t s are pre s e n t e d i n Table 5. Of the 15 animals d r i n k i n g i n response t o a n g i o t e n s i n II t e s t i n g , 1 d i e d and 8 had o b s t r u c t e d or m i s s i n g cannulae r e s u l t i n g i n a f i n a l sample s i z e of 6 . S t a t i s t i c a l a n a l y s i s r e v e a l e d t h a t 2-Me-5-HT d i d not i n f l u e n c e LQ ( X 2 = 2 . 8 6 6 7 , p_= .5804) , LI ( X 2 = 5 . 6 0 0 0 , p_= .2311) , EW (Q= 4 . 5 7 1 4 , p_=.3342) or HD (Q= 8 . 0 0 0 0 , £ = . 0 9 1 6 ) . In c o n t r a s t to the f a c i l i t a t o r y e f f e c t s observed i n males (N.V. Watson, p e r s o n a l communication, 1 9 9 1 ), the c u r r e n t r e s u l t s suggest t h a t 5-HT 3 r e c e p t o r agonism does not a f f e c t b a s a l l e v e l s of female c o p u l a t o r y behaviour. However, the l a c k of s t a t i s t i c a l power, due to the s m a l l number of s u b j e c t s , makes a d e f i n i t i v e c o n c l u s i o n premature. P a r t I I I - A d m i n i s t r a t i o n of Morphine and 5-HT3 A n t a g o n i s t s The a b i l i t y of o p i o i d s to modulate sexual f u n c t i o n i n r a t s , humans and other s p e c i e s i s w e l l documented (f o r review see Pfaus & Gorzalka, 1987a). Moreover, t h e r e i s i n c r e a s i n g evidence t h a t endogenous o p i o i d s p l a y a r o l e i n r e p r o d u c t i v e behaviour. Endogenous o p i o i d s are found i n brainstem and hypothalamic regions i m p l i c a t e d i n the r e g u l a t i o n of r e p r o d u c t i v e behaviour (Khachaturian, Lewis, Schafer & Watson, 1985) . Female r e c e p t i v i t y r e q u i r e s estrogen-induced p r o t e i n s y n t h e s i s w i t h i n the hypothalamus and estrogen treatment has been found to i n c r e a s e proenkephalin s y n t h e s i s i n the v e n t r o m e d i a l hypothalamus (VMH; Romano, Harlan, S h i v e r s , Howells & P f a f f , 1986). Furthermore, i n the v e n t r o l a t e r a l VMH, e s t r o g e n -accumulating c e l l s i n which endogenous o p i o i d p e p t i d e s (EOP) are 42 Table 5. E f f e c t s of 2-methyl-serotonin on l o r d o s i s q u o t i e n t (LQ) , mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals ear w i g g l i n g (EW) and h o p p i n g / d a r t i n g (HD) i n animals primed with 3 |lg e s t r a d i o l benzoate (EB) and 100 fig progesterone (P) . Values r e p r e s e n t means ± S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW, HD). 43 E f f e c t s of 2-Methyl-Serotonin on Sexual Behaviour i n EB- and P-primed Female Rats DOSE LQ LI EW HD (ng) 0.0 60.00 + 16 . 93 1 .68 + .07 0.50 0 .33 1.5 68 .33 + 17 . 40 1 .80 + .11 0 . 67 0 .50 4.5 83.33 + 13. 08 1 .83 + .09 0 . 67 0 . 17 13 .5 66.67 + 19. 61 1 .50 + .19 0 .33 0 .00 40.5 76 . 67 + 16. 06 1 .84 + .07 0 .50 0 .50 c o l o c a l i z e d , have been i d e n t i f i e d , thus s u g g e s t i n g t h a t e strogen a c t s d i r e c t l y t o induce EOP s y n t h e s i s (Akesson & Micevych, 1 9 9 1 ) . In female r a t s , s e c r e t i o n of l u t e i n i z i n g hormone (LH), which promotes o v u l a t i o n , i s t o n i c a l l y i n h i b i t e d by endogenous o p i o i d s a c t i n g at the hypothalamus (Wiesner, Koenig, K r u l i c h & Moss, 1984) . Moreover, t h i s o p i o i d i n f l u e n c e on LH s e c r e t i o n i s s e r o t o n e r g i c a l l y mediated (Gopalan, Gilmore & Brown, 1989) . At doses which do not a l t e r gross motoric responses, p e r i p h e r a l a d m i n i s t r a t i o n of morphine, a s p e c i f i c Li-opioid r e c e p t o r a g o n i s t , i n h i b i t s l o r d o s i s i n s t e r o i d - p r i m e d female r a t s (Pfaus & Gorzalka, 1987a) . Morphine i s a l s o known to i n c r e a s e r a t hypothalamic 5-HT a c t i v i t y (Gopalan et a l . , 1989) and t o p r e f e r e n t i a l l y i n c r e a s e s y n a p t i c dopamine c o n c e n t r a t i o n s i n r a t nucleus accumbens (Di C h i a r a & Imperato, 1988) . The e x i s t e n c e of hypothalamic 5-HT3 r e c e p t o r s ( K i l p a t r i c k et a l . , 1987) and the f a c t t h a t 5-HT3 a n t a g o n i s t s decrease r a i s e d l e v e l s of mesolimbic dopamine and the accompanying h y p e r a c t i v i t y ( C o s t a l l et a l . , 1990b), suggests t h a t such compounds may a l t e r the e f f e c t s of o p i o i d s on sexual behaviour. Experiment 6 R e s u l t s from Part I suggest t h a t 5-HT3 r e c e p t o r a n t a g o n i s t s have l i t t l e e f f e c t on b a s a l female sexual b e h a v i o u r s . That 5-HT 3 a n t a g o n i s t s modify d r u g - a l t e r e d behaviours i n i n s t a n c e s where they are i n e f f e c t i v e i n a l t e r i n g 4 5 b a s a l a c t i v i t y , suggests t h a t they may e f f e c t i v e l y modulate c h e m i c a l l y - i n h i b i t e d sexual a c t i v i t y . Morphine (1.5 mg/kg) i n h i b i t s l o r d o s i s 60 minutes f o l l o w i n g i n j e c t i o n (Gorzalka, Luck & Tanco, 1991) . A d m i n i s t e r e d alone, naloxone, a n o n s p e c i f i c o p i a t e a n t a g o n i s t , i s i n e f f e c t i v e i n a l t e r i n g l o r d o s i s ; however, given i n c o n j u n c t i o n with o p i o i d s , naloxone attenuates the e f f e c t s of P-endorphin, m o r p h i c e p t i n and 8 - r e c e p t o r p e p t i d e on l o r d o s i s (Pfaus & Gorzalka, 1987b). I n t e r e s t i n g l y , MDL 72222 (0.03 mg/kg) a b o l i s h e s p l a c e a v e r s i o n induced by naloxone (Acquas, Carboni, Garau & Di C h i a r a , 1990) and antagonizes morphine-induced p l a c e - p r e f e r e n c e c o n d i t i o n i n g (Carboni, Acquas, Leone, P e r e z z a n i , & Di C h i a r a , 1988). Thus, independent of i t s e f f e c t when adm i n i s t e r e d i n i s o l a t i o n , MDL 72222 may f a c i l i t a t e c o p u l a t o r y behaviour by a t t e n u a t i n g morphine-induced sexual i n h i b i t i o n . Method F i f t e e n o v ariectomized, Long-Evans females primed with 5 ug EB and 100 Lig P served as s u b j e c t s . Subjects were 6 months of age and weighed 280 to 380 grams. Morphine (BDH Chemicals) was d i s s o l v e d i n d i s t i l l e d H20 and MDL 72222 (Research Biochemicals Inc.) was d i s s o l v e d i n a drop of d i l u t e g l a c i a l a c e t i c a c i d and made up to volume with d i s t i l l e d H20. In a counterbalanced, repeated measures design, animals r e c e i v e d SC i n j e c t i o n s of v e h i c l e alone, MDL 72222 (0.05 mg/ml/kg) and v e h i c l e , morphine 4 6 (1.5 mg/ml/kg) and v e h i c l e , and MDL 72222 and morphine, such t h a t each animal r e c e i v e d a l l treatment c o n d i t i o n s . MDL 72222 and morphine were ad m i n i s t e r e d 30 and 60 minutes p r i o r t o t e s t i n g , r e s p e c t i v e l y . R e s u l t s and D i s c u s s i o n R e s u l t s are p r e s e n t e d i n Table 6. MDL 72222 f a i l e d t o modify LQ, (X2= 2.58, p_=.4610), LI (X2=1.300, p_=.7291), EW (Q=4.000 p_=.2615), or HD (Q=2.4545 p_=.4836) i n EB- and P-primed animals. Although morphine 1.5 mg/kg f a i l e d t o i n h i b i t l o r d o s i s i n a s t a t i s t i c a l l y s i g n i f i c a n t manner, both groups r e c e i v i n g morphine demonstrated approximately a 20% r e d u c t i o n i n LQ. As w e l l , p r o p o r t i o n s of animals demonstrating EW and HD were decreased, a l b e i t n o n s i g n i f i c a n t l y , i n both morphine c o n d i t i o n s r e l a t i v e t o c o n t r o l . Experiment 7 Ondansetron moderates e f f e c t s produced by other compounds. Systemic a d m i n i s t r a t i o n of ondansetron a t t e n u a t e s h y p e r a c t i v i t y induced by b i l a t e r a l i n f u s i o n of dopamine i n t o the nucleus accumbens ( C o s t a l l 1987b). Furthermore, ondansetron attenuates b e h a v i o u r a l symptoms induced by withdrawal from c h l o r d i a z e p o x i d e (Goudie & L e a t h l e y , 1990), e t h a n o l , n i c o t i n e and cocaine ( C o s t a l l et a l . , 1990a), drugs which, l i k e morphine, i n c r e a s e 47 Table 6. E f f e c t s of morphine (MOR) and MDL 72222 (MDL) on l o r d o s i s q u o t i e n t (LQ), mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g ear w i g g l i n g (EW) and h o p p i n g / d a r t i n g (HD) i n animals primed with 5 Jig e s t r a d i o l benzoate (EB) and 100 |lg progesterone (P) . Values r e p r e s e n t means ± S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW, HD). 48 E f f e c t s o f M o r p h i n e a n d MDL 72222 on S e x u a l B e h a v i o u r i n EB- a n d P - p r i m e d F e m a l e R a t s DOSE LQ L I EW HD MDL / MOR (mg/kg) 0.00 / 0.00 0.05 / 0.00 0.00 / 1.50 0 . 05 / 1.50 89.33 + 1.53 83.33 + 5.40 74.00 + 6.75 71 . 33 + 8.50 1.85 + .15 1.79 + .06 1.79 + .05 1.74 + .09 0.80 0.53 0.87 0.33 0.60 0.33 0.67 0.33 4 9 dopamine l e v e l s i n the nucleus accumbens (Di C h i a r a & Imperato, 1988) . The a b i l i t y of ondansetron t o e f f e c t i v e l y modify c h e m i c a l l y - i n d u c e d behaviours suggests t h a t i t may attenuate o p i o i d - i n d u c e d i n h i b i t i o n of sexual behaviour. Therefore, the purpose of the f o l l o w i n g study was to i n v e s t i g a t e the a b i l i t y of v a r y i n g doses of ondansetron to b l o c k morphine-induced sexual i n h i b i t i o n i n EB- and P-primed r a t s . Method Ei g h t e e n Long-Evans female r a t s , 70 days o l d and weighing between 190 and 240 grams were used as s u b j e c t s ; each was primed with 3 |lg EB and 100 fig P 48 and 4 h p r i o r t o t e s t i n g , r e s p e c t i v e l y . Morphine (BDH Chemicals) and ondansetron (Glaxo) were d i s s o l v e d i n d i s t i l l e d H20. As 3.0 mg/kg morphine does not i n h i b i t motor f u n c t i o n i n female r a t s (Pfaus & Gorzalka, 1987a), the morphine dose was i n c r e a s e d s l i g h t l y t o produce g r e a t e r sexual i n h i b i t i o n . In a counterbalanced, repeated measures design s u b j e c t s r e c e i v e d SC i n j e c t i o n s of v e h i c l e alone, morphine (2.0 mg/ml/kg) and v e h i c l e or ondansetron 4.0, 0.8, 0.16, 0.032 mg/ml/kg i n combination with morphine (2.0 mg/ml/kg), such t h a t each animal r e c e i v e d a l l treatment c o n d i t i o n s over 6 weeks of t e s t i n g . Ondansetron and morphine were a d m i n i s t e r e d 60 and 30 minutes p r i o r t o t e s t i n g r e s p e c t i v e l y . 50 R e s u l t s and D i s c u s s i o n R e s u l t s are p r e s e n t e d i n Table 7. S t a t i s t i c a l analyses r e v e a l e d s i g n i f i c a n t o v e r a l l treatment e f f e c t s f o r LQ (X2=23.54 p_=.0003), EW (Q=21.89, p_=.0006) and HD (Q=11.43 p_=.0435), but not f o r LI (X2=2.69, p_=.7483); subsequent analyses i n d i c a t e d these e f f e c t s were due to morphine. Morphine c o n s i s t e n t l y decreased both LQ (p_<.002) and the p r o p o r t i o n of animals d i s p l a y i n g EW (p_<.004). Ondansetron, 0.032 to 4.0 mg/kg, however, f a i l e d t o attenuate these e f f e c t s . Experiment 8 ICS 205-930 i n h i b i t s morphine-induced s t i m u l a t i o n of dopamine r e l e a s e i n the nucleus accumbens (Carboni et a l . , 1989) , b l o c k s p l a c e a v e r s i o n induced by naloxone (Acquas et a l . , 1990) and antagonizes morphine- as w e l l as n i c o t i n e - i n d u c e d p l a c e - p r e f e r e n c e c o n d i t i o n i n g (Carboni et a l . , 1988) . ICS 205-930 i s e f f e c t i v e at s i t e s and i n i n s t a n c e s where MDL 72222 and ondansetron are not. Thus, i t s a b i l i t y to antagonize o p i o i d -induced sexual i n h i b i t i o n may d i f f e r from those of MDL 72222 and ondansetron. Method Subjects were twelve, 12 month o l d female Long-Evans r a t s 51 Table 7. E f f e c t s of morphine (MOR) and ondansetron (OND) on l o r d o s i s q u o t i e n t (LQ), mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g , ear w i g g l i n g (EW) and ho p p i n g / d a r t i n g (HD) i n animals primed with 5 (ig e s t r a d i o l benzoate (EB) and 100 |lg progesterone (P) . Values represent means +_ S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW, HD). 52 E f f e c t s of Morphine and Ondansetron on Sexual Behaviour i n EB- and P-primed Female Rats DOSE L Q LI EW HD OND / MOR (mg/kg) 0.000 / 0.0 95.33 + 2.15 1.79 + .04 1.00 0.60 0.000 / 2.0 35.33 + 8.99 1.54 + .13 0.40 0.27 0.032 / 2.0 41.33 + 10.14 1.62 + .12 0.53 0.13 0.160 / 2.0 40.00 + 10.56 1.50 + .12 0.47 0.20 0.800 / 2.0 26.67 + 10.81 1.48 + .18 0.27 0.20 4.000 / 2.0 30.67 + 8.86 1.44 + .13 0.33 0.20 53 weighing between 330 and 430 grams; each was primed with 7.5 fig EB and 100 fig P 48 and 4 h p r i o r t o t e s t i n g , r e s p e c t i v e l y . Morphine (BDH Chemicals) was d i s s o l v e d i n s t e r i l e H20. ICS 205-930 (Sandoz) was d i s s o l v e d i n a drop of d i l u t e g l a c i a l a c e t i c a c i d and made up to volume with s t e r i l e H20. In a counterbalanced, repeated measures design, animals r e c e i v e d SC i n j e c t i o n s of v e h i c l e alone, morphine (2.0 mg/ml/kg) and v e h i c l e , or 0.005, 0.05, 0.5 or 5.0 mg/ml/kg ICS 205-930 i n combination with morphine (2.0 mg/kg/ml), such t h a t each animal r e c e i v e d a l l treatment c o n d i t i o n s over 6 weeks of t e s t i n g . ICS 205-930 and morphine were admi n i s t e r e d 45 and 30 minutes p r i o r t o t e s t i n g , r e s p e c t i v e l y . R e s u l t s and D i s c u s s i o n R e s u l t s are presented i n Table 8. S t a t i s t i c a l a n a l y s i s r e v e a l e d a s i g n i f i c a n t e f f e c t of treatment on LQ (X2=15.83, p_=.0073), which follow-up t e s t i n g r e v e a l e d was due to morphine i n h i b i t i o n . LQ's of a l l treatment c o n d i t i o n s were s i g n i f i c a n t l y a t t e n u a t e d r e l a t i v e t o the c o n t r o l group (p_<.01) . However, ICS 205-930 f a i l e d t o antagonize any of these e f f e c t s . Although the e f f e c t s of morphine on LI, EW and HD were 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 , an i n h i b i t o r y t r e n d e x i s t e d with fewer morphine-t r e a t e d than c o n t r o l animals d i s p l a y i n g EW or HD. The f a i l u r e of ICS 205-930 to modify morphine-induced sexual i n h i b i t i o n i s c o n s i s t e n t with the f i n d i n g s of Hasegawa and c o l l e a g u e s 54 Table 8. E f f e c t s of morphine (MOR) and ICS 205-930 (ICS) on l o r d o s i s q u o t i e n t (LQ), mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g ear w i g g l i n g (EW and h o p p i n g / d a r t i n g (HD) i n animals primed with 7 .5 [ig e s t r a d i o l benzoate (EB) and 100 |lg progesterone (P) . Values r e p r e s e n t means ± S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW, HD). 55 E f f e c t s o f M o r p h i n e a n d ICS 205-930 on S e x u a l B e h a v i o u r i n EB- a n d P - p r i m e d F e m a l e R a t s DOSE LQ L I EW HD ICS / MOR (mg/kg) 0.000 / 0 .0 95 . 83 + 2 .88 1 . 66 + .09 0 . 67 0 . 42 0 .000 / 2 .0 69 . 17 + 10 . 69 1.38 + .07 0 .50 0 . 08 0.005 / 2 .0 68 .33 + 6 .83 1.57 + .09 0 .58 0 . 17 0.050 / 2 .0 54 .17 + 10 . 97 1.35 + .08 0 .42 0 .33 0 .500 / 2 . 0 63 .33 + 10 .89 1.36 + .09 0 .25 0 . 17 5.000 / 2 . 0 67 .50 + 8 .71 1.49 + . 11 0 .42 0 . 08 (1990) who r e p o r t t h a t ICS 205-930 d i d not antagonize morphine-induced Straub t a i l . I n t e r e s t i n g l y , ICS 205-930 b l o c k e d the e f f e c t of U-50, 488H, a K - o p i o i d a g o n i s t , which a l s o a c t s as a f l -o p i o i d a n t a g o n i s t (Hasegawa, Kurachi, Okuyama, A r a k i , & Otomo, 1990) . P a r t IV - A d m i n i s t r a t i o n of Apomorphine and ICS 205-930 Experiment 9 Although r e s u l t s from the p r e v i o u s experiments suggest t h a t 5-HT3 r e c e p t o r s and o p i a t e s do not i n t e r a c t i n the n e u r a l c o n t r o l of female sexual behaviour, t h i s does not p r e c l u d e the p o s s i b i l i t y of i n t e r a c t i o n between 5-HT3 r e c e p t o r s and other n o n s e r o t o n e r g i c n e u r o t r a n s m i t t e r s . That 5-HT3 r e c e p t o r s are d i s t r i b u t e d i n d o p a m i n e r g i c - r i c h b r a i n areas, i . e . e n t o r h i n a l cor t e x , amygdala, nucleus accumbens and tuberculum o l f a c t o r i u m , ( K i l p a t r i c k et a l . , 1987) suggests the p o s s i b i l i t y of such an i n t e r a c t i o n . Indeed, i n i s o l a t e d r a t s t r i a t a l t i s s u e , 5-HT3 a c t i v i t y i s a s s o c i a t e d with i n c r e a s e d dopamine r e l e a s e (Blandina, G o l d f a r b & Green, 1988/ B l a n d i n a et a l . , 1989). In v i v o , 5-HT3 a n t a g o n i s t s attenuate r a i s e d mesolimbic dopamine a c t i v i t y and the accompanying hypermotor a c t i v i t y (e.g. Hagan, B u t l e r , H i l l , Jordan, I r e l a n d & Tyers, 1987/ C o s t a l l , Domeney, K e l l y , Naylor & Tyers, 1987c/ C o s t a l l , Domeney, Naylor & Tyers, 1987a, 1987b, 1988b). However, they f a i l t o a l t e r s i m i l a r behaviour t h a t i s the r e s u l t of i n c r e a s e d s t r i a t a l dopamine a c t i v i t y ( C o s t a l l , et a l . , 1987a). 2-Me-5-HT p o t e n t i a t e s amphetamine-induced h y p e r a c t i v i t y , but does not a l t e r b a s a l motor a c t i v i t y ( C o s t a l l , et a l . , 1987a). S i m i l a r l y , i n drug-t r e a t e d animals, 5-HT3 a n t a g o n i s t s reduce n e i t h e r motor a c t i v i t y nor l i m b i c dopaminergic a c t i v i t y t o l e v e l s below normal ( C o s t a l l , Naylor & Tyers, 1990b). Together, these f i n d i n g s suggest t h a t 5-HT3 r e c e p t o r s modulate d i s t u r b a n c e s i n , but not b a s a l l e v e l s of, mesolimbic dopamine ( C o s t a l l et a l . , 1990b). The dopamine D1/D2 ag o n i s t , apomorphine (0.4 mg/kg) i s known t o p r o f o u n d l y i n h i b i t l o r d o s i s (Fernandez-Guasti et a l . , 1987) . Moreover, ICS 205-930 attenuates e t h a n o l - i n d u c e d i n c r e a s e s of dopamine i n the nucleus accumbens and corpus s t r i a t u m (Wozniak, P e r t , & L i n n o i l a , 1990) . Therefore, i t i s c o n c e i v a b l e t h a t ICS 205-930 may f a c i l i t a t e female sexual behaviour by a t t e n u a t i n g the dopaminergic i n h i b i t i o n of l o r d o s i s . The f o l l o w i n g study i n v e s t i g a t e d the a b i l i t y of v a r y i n g doses of ICS 205-930 t o attenuate apomorphine-induced sexual i n h i b i t i o n . Method Subjects were 35 female Long-Evans r a t s , approximately 4 months of age and weighing between 240 and 340 grams. A l l were primed with 5 ug EB and 100 ug P. Any animal showing an LQ l e s s 58 than 70% d u r i n g b a s e l i n e t e s t i n g with the above hormone regime was e l i m i n a t e d from f u r t h e r t e s t i n g . Thus, 34 s u b j e c t s were randomly a s s i g n e d t o one of th r e e treatment c a t e g o r i e s : v e h i c l e (lml/kg), v e h i c l e and apomorphine ( 0 . 4 mg/ml/kg) or ICS 2 0 5 - 9 3 0 ( 0 . 0 3 mg/ml/kg) and apomorphine ( 0 .4 mg/ml/kg). A l l drugs were ad m i n i s t e r e d SC. Apomorphine (BDH Chemicals) was d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e and admi n i s t e r e d 5 minutes p r i o r t o t e s t i n g as peak b e h a v i o u r a l e f f e c t s on l o r d o s i s have been demonstrated to occur 5 minutes p o s t - i n j e c t i o n (Fernandez-Guasti, et a l . , 1987) . ICS 2 0 5 - 9 3 0 (Sandoz) was d i s s o l v e d i n a drop of d i l u t e g l a c i a l a c e t i c a c i d , made up to volume with p h y s i o l o g i c a l s a l i n e and a d m i n i s t e r e d 45 minutes p r i o r to i n j e c t i o n of apomorphine or s a l i n e . R e s u l t s and D i s c u s s i o n R e s u l t s are pres e n t e d i n Table 9 . K r u s k a l - W a l l i s a n a l y s i s of v a r i a n c e r e v e a l e d an o v e r a l l treatment e f f e c t f o r LQ ( X 2 = 1 5 . 5 2 , p_=.0004) and LI ( X 2 = 1 0 . 2 2 , p_=.0060); s i m i l a r l y , c h i square analyses r e v e a l e d s i g n i f i c a n t e f f e c t s of treatment on EW ( X 2 = 1 7 . 7 6 p_=.0001) and HD ( X 2 = 2 1 . 1 2 , p_=.0001) . Follow-up t e s t i n g u s i n g Mann Whitney U (LQ and LI) and F i s h e r ' s Exact Test (EW and HD) i n d i c a t e d t h a t while apomorphine s i g n i f i c a n t l y i n h i b i t e d LQ, LI, EW and HD, (p_<.001), ICS 2 0 5 - 9 3 0 f a i l e d t o attenuate these e f f e c t s . C o ntrary t o our hypothesis, ICS 2 0 5 - 9 3 0 d i d not attenuate 59 Table 9. E f f e c t s of 0.4 mg/kg apomorphine (APO) and 0.03 mg/kg ICS 205-930 (ICS) on l o r d o s i s q u o t i e n t (LQ), mean l o r d o s i s i n t e n s i t y (LI) and p r o p o r t i o n of animals d i s p l a y i n g ear w i g g l i n g (EW) and h o p p i n g / d a r t i n g (HD) i n animals primed with 5 Jig e s t r a d i o l benzoate (EB) and 100 jig progesterone (P) . Values r e p r e s e n t means + S.E.M.s (LQ and LI) and p r o p o r t i o n s (EW, HD). 60 E f f e c t s o f A p o m o r p h i n e a n d ICS 205-930 on S e x u a l B e h a v i o u r i n EB- a n d P - p r i m e d F e m a l e R a t s DOSE LQ L I EW HD ICS/APO (mg/kg) 0.00 / 0.0 82.05 + 11.37 1.66 + .08 0.64 0.73 0.00 / 0.4 25.29 + 7.87 1.27 + .11 0.00 0.00 0.03 / 0.4 14.82 + 7.12 1.18 + .18 0.00 0.00 61 apomorphine-induced i n h i b i t i o n of sexual behaviour. C o n s i s t e n t with t h i s i s the f i n d i n g t h a t 5-HT3 a n t a g o n i s t s f a i l t o b l o c k p l a c e - p r e f e r e n c e c o n d i t i o n i n g (Carboni et a l . , 1989) and hyperlocomotion ( C o s t a l l et a l . , 1990b) induced by p a r e n t e r a l amphetamine, the a d m i n i s t r a t i o n of which r e s u l t s i n i n c r e a s e d s t r i a t a l dopamine a c t i v i t y . The f a i l u r e of ICS 205-930 t o attenuate apomorphine-induced sexual i n h i b i t i o n may be r e l a t e d t o b i n d i n g s i t e l o c a t i o n and indeed may p r o v i d e an i n d i c a t i o n as t o the c e l l u l a r l o c a l i z a t i o n of 5-HT3 r e c e p t o r s i t e s . Although c e l l u l a r l o c a l i z a t i o n of 5-HT3 b i n d i n g s i t e s has yet to be f u l l y e s t a b l i s h e d , t h e r e i s p r e l i m i n a r y evidence s u g g e s t i v e of a p r e s y n a p t i c l o c a t i o n , at l e a s t i n the s p i n a l cord. S p e c i f i c a l l y , s e l e c t i v e d e s t r u c t i o n of a f f e r e n t neurons by neonatal c a p s a i c i n a d m i n i s t r a t i o n , decreases both o p i a t e l i g a n d b i n d i n g and 5-HT3 b i n d i n g i n the d o r s a l horn of r a t s p i n a l cord; as o p i a t e r e c e p t o r s on s p i n a l c a p s a i c i n - s e n s i t i v e f i b e r s are l o c a t e d p r e s y n a p t i c a l l y , t h i s suggests a p r e s y n a p t i c l o c a t i o n f o r s p i n a l 5-HT3 r e c e p t o r s as w e l l (Hamon, G a l l i s s o t , Menard, Gozlan, Bourgoin & Verge, 1989). I f 5-HT3 s i t e s mediating sexual behavior are l o c a t e d p r e s y n a p t i c a l l y , the i n e f f e c t i v e n e s s of ICS 205-930 i n the present paradigm may be due to apomorphine's p o s t s y n a p t i c dopaminergic a c t i v i t y . Thus, i t i s tempting t o s p e c u l a t e t h a t perhaps 5-HT3 a n t a g o n i s t s would attenuate e f f e c t s induced by a p r e s y n a p t i c dopamine ag o n i s t such as co c a i n e . 62 GENERAL DISCUSSION In P a r t I, 5-HT3 r e c e p t o r a n t a g o n i s t s f a i l e d t o a l t e r b a s a l l o r d o s i s or p r o c e p t i v i t y i n EB- (Experiment 3) or EB- and P-(Experiments 1 and 2) primed r a t s . Likewise the 5-HT3 a g o n i s t s , 2-Me-5-HT and PBG a l s o f a i l e d t o modulate female r e p r o d u c t i v e b e h a v i o u r s . In Part I I I , morphine decreased r e c e p t i v e and p r o c e p t i v e behaviours, but the 5-HT3 a n t a g o n i s t s , MDL 72222, ondansetron, and ICS 205-930 f a i l e d t o attenuate these e f f e c t s . F i n a l l y , i n Part IV, apomorphine s i g n i f i c a n t l y i n h i b i t e d both LQ and LI as w e l l as EW and HD; ICS 205-930 however d i d not b l o c k t h i s i n h i b i t i o n . Taken together, these r e s u l t s i n d i c a t e t h a t i n s t e r o i d - p r i m e d female r a t s , 5-HT3 a c t i v i t y does not a l t e r b e h a v i o u r a l r e c e p t i v i t y or p r o c e p t i v i t y . Present f i n d i n g s are congruent with aspects of p r e v i o u s l y r e p o r t e d r e s u l t s . S p e c i f i c a l l y , our data are s u p p o r t i v e of f i n d i n g s r e g a r d i n g the i n e f f e c t i v e n e s s of MDL 72222 (Mendelson & Gorzalka, 1989) and r e s u l t s i n d i c a t i n g t h a t 5-HT3 a n t a g o n i s t s f a i l t o e f f e c t sexual behaviour i n r e c e p t i v e females (James et a l . , 1989) . C o n s i s t e n t with these r e s u l t s , the c u r r e n t f i n d i n g s suggest t h a t 5-HT3 r e c e p t o r a c t i v i t y may not p l a y a r o l e i n female r e p r o d u c t i v e behaviour. Moreover, the l a c k of e f f e c t i n r e c e p t i v e animals supports the s u p p o s i t i o n t h a t i n no n r e c e p t i v e animals, f a c i l i t a t o r y e f f e c t s induced by 5-HT3 r e c e p t o r a n t a g o n i s t s may be s t a t i s t i c a l a r t i f a c t . Although i t i s p o s s i b l e t h a t such f a c i l i t a t i o n r e f l e c t s the r o l e of 5-HT3 63 r e c e p t o r a c t i v i t y i n r e g u l a t i n g f u n c t i o n a l d i s t u r b a n c e s , r a t h e r than normal b a s a l a c t i v i t y , the c u r r e n t f i n d i n g s do not support the i d e a t h a t 5 - H T 3 - r e l a t e d f a c i l i t a t i o n was due t o modulation of dopamine or L i - o p i o i d r e c e p t o r d i s t u r b a n c e . N e v e r t h e l e s s , i t i s p o s s i b l e t h a t the observed f a c i l i t a t i o n of sexual behaviour i n n o n r e c e p t i v e females f o l l o w i n g 5-HT3 r e c e p t o r blockade (James et a l . , 1989) i n v o l v e s a l t e r a t i o n s i n other n e u r o t r a n s m i t t e r systems. The c u r r e n t r e s u l t s c o n t r a s t with the f a c i l i t a t o r y e f f e c t of ICS 205-930 observed by Mendelson and Gorzalka ( 1 9 8 9 ) ; reasons f o r t h i s d i s c r e p a n c y are not apparent. The i n e f f e c t i v e n e s s of 5-HT 3 a g o n i s t s and a n t a g o n i s t s i n females c o n t r a s t s with the f a c i l i t a t o r y e f f e c t s observed i n males. T h i s may be a sex d i f f e r e n c e s i m i l a r t o t h a t observed with 5-HTx r e c e p t o r a c t i v i t y . In c o n t r a s t t o i t s i n h i b i t o r y e f f e c t i n females, 5-HT 1 A a c t i v i t y f a c i l i t a t e s c o p u l a t o r y behaviour i n males; c o n v e r s l y , 5-HT 1 B a c t i v i t y f a c i l i t a t e s female, but i n h i b i t s male sexual behaviour (Gorzalka et a l . , 1 9 9 0 ) . Reasons f o r such d i f f e r e n c e s may be r e l a t e d t o s e x u a l l y dimorphic b r a i n s t r u c t u r e s and hormone p h y s i o l o g y . For example, t h a t t e s t o s t e r o n e decreases 5-HT 3 b i n d i n g i n male r a t amygdala (Mendelson & McEwen, 1990) suggests the p o s s i b i l i t y t h a t g r e a t e r 5-HT 3 r e c e p t o r a c t i v i t y may occur n a t u r a l l y i n females due to lower androgen l e v e l s . Therefore, i f 5-HT3 r e c e p t o r agonism f a c i l i t a t e s c o p u l a t o r y behaviour i n females, as i t may i n males, i t i s p o s s i b l e t h a t , i n females, g r e a t e r b a s a l r e c e p t o r a c t i v i t y may have prevented o b s e r v a t i o n of such an e f f e c t . 6 4 D i f f e n c e s i n b e h a v i o u r a l s t r a t e g i e s may a l s o c o n t r i b u t e t o the sex d i f f e r e n c e observed to occur i n response t o a d m i n i s t r a t i o n of 5-HT 3-active compounds. For females, motoric i n h i b i t i o n i s a necessary component of c o p u l a t i o n . Females cease gross motor a c t i v i t y t o enable male mounting and p e n e t r a t i o n ; moreover, d u r i n g i n t r o m i s s i o n , females assuming the l o r d o t i c p o s t u r e are observed t o " f r e e z e " . Indeed, the s i m i l a r i t y between the l o r d o t i c p osture and a k i n e s i a and limb r i g i d i t y produced by n i g r o s t r i a t a l l e s i o n s i s no t a b l e (Crowley & Zemlan, 1981). In c o n t r a s t , male c o p u l a t o r y behavior r e q u i r e s p e r s i s t e n t motor a c t i v i t y . Perhaps motoric a c t i v i t y , heightened by sexual a r o u s a l , was f u r t h e r augmented by a d m i n s t r a t i o n of 2-Me-5-HT. In males, such an i n c r e a s e i n motor a c t i v i t y c o u l d c o n c e i v a b l y f a c i l i t a t e sexual a c t i v i t y ; i n c o n t r a s t , such an i n c r e a s e i n motor a c t i v i t y would not be expected to f a c i l i t a t e female c o p u l a t o r y b e h a v i o r . In support of such c o n j e c t u r e are f i n d i n g s t h a t dopamine agonism f a c i l i t a t e s sexual behaviour i n males but not i n females (Crowley and Zemlan, 1981) . The observed i n a b i l i t y of 5-HT3 a n t a g o n i s t s t o bl o c k o morphine-induced i n h i b i t i o n of sexual behaviour c o n t r a s t s with r e s u l t s from p l a c e - p r e f e r e n c e s t u d i e s (e.g. Carboni et a l . , 1988) . In these s t u d i e s , 5-HT3 a n t a g o n i s t s were observed t o b l o c k both morphine- and n i c o t i n e - i n d u c e d p l a c e - p r e f e r e n c e c o n d i t i o n i n g . That naloxone antagonized the e f f e c t s of morphine but was i n e f f e c t i v e i n b l o c k i n g n i c o t i n e - i n d u c e d p l a c e -p r e f e r e n c e suggests p o s s i b l e involvement of a n o n - o p i o i d 65 receptor (Aquas et a l . , 1990) . Thus, while 5-HT3 antagonists may not block e f f e c t s due to [ l - o p i o i d receptor s t i m u l a t i o n , the p o s s i b i l i t y remains that 5-HT3 a c t i v i t y may modulate e f f e c t s on sexual behaviour induced by a c t i v a t i o n of non-(i o p i o i d r e c e p t o r s . I t has been suggested that various components of female sexual behaviour may respond d i f f e r e n t i a l l y to treatment (Crowley & Zemlan, 1981) . However, l i m i t e d data regarding p r o c e p t i v e behaviours and l o r d o t i c i n t e n s i t y and du r a t i o n e x i s t . Therefore, i t i s i n t e r e s t i n g to note t h a t , i n general, decreases i n LQ produced by morphine and apomorphine, were accompanied by decreases i n proceptive behaviours. Although l o r d o s i s i n t e n s i t y was only s i g n i f i c a n t l y i n h i b i t e d by apomorphine, i t i s worth n o t i n g t h a t the mean L i s d i s p l a y e d by animals r e c e i v i n g morphine i n Experiments 7 and 8 were lower than L i s d i s p l a y e d by animals r e c e i v i n g only v e h i c l e i n j e c t i o n s . Nonetheless, i t remains conceivable t h a t l o r d o t i c i n t e n s i t y may be a d i s c r e t e component of female sexual a c t i v i t y , and as such be r e l a t i v e l y independent of l o r d o t i c frequency ( i . e . LQ). In support of t h i s s u p p o s i t i o n are recent f i n d i n g s that oxytocin-induced e f f e c t s on l o r d o s i s d u r a t i o n d i f f e r from e f f e c t s on LQ (Schulze & Gorzalka, 1 9 9 1 ) . The current r e s u l t s do not support the hypothesis of 5-HT3 receptor involvement i n the modulation of female sexual behaviour. Moreover, the p a u c i t y of 5-HT3 e f f e c t s are not s o l e l y a t t r i b u t a b l e to methodological concerns such as inaccurate s c o r i n g . Indeed, r e p l i c a t i o n of the w e l l e s t a b l i s h e d i n h i b i t o r y e f f e c t s of morphine and apomorphine supports t h i s c o n t e n t i o n . Nonetheless, experimental c o n d i t i o n s c e r t a i n l y c o n t r i b u t e d to the r e s u l t s . S e v e r a l p o s s i b l e e x p l a n a t i o n s r e g a r d i n g the l a c k of observed e f f e c t s e x i s t . I t i s p o s s i b l e t h a t s t r e s s a s s o c i a t e d with experimental procedures may have i n h i b i t e d sexual a c t i v i t y . Gopalan and c o l l e a g u e s (1989) note t h a t i n t h e i r work, c o n t r o l animals r e c e i v i n g SC i n j e c t i o n s under l i g h t e t h e r a n a e s t h e s i a , show hi g h e r hypothalamic 5-HT l e v e l s than animals r e c e i v i n g IP i n j e c t i o n s . The authors a t t r i b u t e t h i s t o e t h e r - r e l a t e d s t r e s s . As s t r e s s a s s o c i a t e d with r e s t r a i n t i s known to i n c r e a s e hypothalamic 5-HT (Mueller, Twohy, Chen, Advis & Meites, 1976), i t i s p l a u s i b l e t h a t s t r e s s induced by h a n d l i n g and i n j e c t i o n may have i n c r e a s e d hypothalamic 5-HT l e v e l s . Moreover, a d m i n i s t r a t i o n of 5-HT i n t o the hypothalamus has been found to i n h i b i t l o r d o s i s i n EB- and P-primed animals (Clemens, 1978) . During the a c q u i s i t i o n of b a s e l i n e data, animals d i d not r e c e i v e i n j e c t i o n s . I t i s t h e r e f o r e worth n o t i n g t h a t , under c o n t r o l c o n d i t i o n s , the LQ of animals i n Experiment 2 decreased, r e l a t i v e t o b a s e l i n e c o n d i t i o n s , by n e a r l y 50%. Conversely, i t i s a l s o p o s s i b l e t h a t experimental procedures may have i n c r e a s e d LQs. The LQs of animals under c o n t r o l c o n d i t i o n s i n Experiments 1 and 3, but not the LQ of c o n t r o l animals i n Experiment 2, i n c r e a s e d s u b s t a n t i a l l y over b a s e l i n e l e v e l s . S t r e s s a s s o c i a t e d with r e s t r a i n t and i n j e c t i o n may have i n c r e a s e d adrenal hormone output. As a d r e n a l hormones 67 such as progesterone and desoxycorticosterone can f a c i l i t a t e l ordosis (Gorzalka & Whalen, 1977), i t i s plausible that stress-induced hormone secretion may have resulted i n higher LQs under control conditions r e l a t i v e to baseline conditions. Interestingly, i n Experiment 2, where ondansetron appeared to produce a consistent f a c i l i t a t o r y .trend, increasing both receptive and proceptive behaviours, the mean LQ (36%) of control animals was substantially lower than that of control animals i n Experiments 1 (88%) and 3 (87%) . Thus, regardless of the mechanism involved, i t i s plausible that f a c i l i t a t o r y e f f e c t s were obscured by the high LQs present under control conditions i n Experiments 1 and 3. That the d i r e c t i o n of serotonergic e f f e c t s on sexual behaviour i s , i n some instances, hormone-dependent suggests hormonal environment may have contributed to the ineffectiveness of 5-HT 3-active drugs i n the current study. To date, the only s p e c i f i c evidence regarding interaction between 5-HT3 receptors and hormones i s based on work with male rat s . In castrated rats, testosterone has been demonstrated to decrease 5-HT3 ligand binding in the amygdala (Mendelson & McEwen, 1990) , an area involved i n lordosis modulation. Such hormonally-induced a l t e r a t i o n in 5-HT3 binding i n males suggests that ovarian hormones may be i n f l u e n t i a l i n females. Although the role of progesterone has yet to be f u l l y established, evidence suggests the capacity to a l t e r the di r e c t i o n of serotonergic e f f e c t s . For example, moderate dosages 68 of the 5-HT1A agonists, ipsapirone and gepirone f a c i l i t a t e lordosis i n EB-primed animals; however, i n EB- and P-primed females, these drugs i n h i b i t lordosis (Mendelson & Gorzalka, 1986) . Also, Gorzalka and Lester (1987) found that oxytocin f a c i l i t a t e d lordosis i n EB- and P-primed females, but not in females primed with EB alone, although control LQ scores were sim i l a r for both. These findings suggest the role of P i s not l i m i t e d to establishing an environment conducive to observation of i n h i b i t o r y e f f e c t s ; i . e . P does more than merely create high leve l s of r e c e p t i v i t y . Administration of P decreases 5-HT accumulation i n the ventromedial hypothalamic nucleus and midbrain central gray, but f a i l s to a l t e r 5-HT accumulation i n other hypothalamic s i t e s or in the dorsal raphe nucleus (Renner, Krey & Luine, 1987) . While the actions of P are c e r t a i n l y more complex than simple blockade of i n h i b i t o r y serotonergic pathways, as was o r i g i n a l l y suggested (Pfaff, 1 9 8 0 ) , the i n t e r a c t i v e e f f e c t s of progesterone with the 5-HT system have yet to be f u l l y elucidated. Interestingly, quipazine, a 5-HT2 agonist/ 5-HT3 antagonist f a c i l i t a t e s lordosis i n EB-primed animals, but i s i n e f f e c t i v e i n EB- and P-primed animals (Gorzalka et a l . , 1 9 9 0 ) ; however, given the j o i n t s i t e s of action, clear interpretation of these data i s d i f f i c u l t . Animals i n the current experiments were primed with estrogen and progesterone, the exception being animals i n Experiment 3 who were primed with EB alone. While i t i s possible that progesterone i n h i b i t e d observation of a serotonergic e f f e c t , 69 t h i s seems u n l i k e l y as ICS 205-930, the 5-HT3 a n t a g o n i s t having the broadest pharmacologic spectrum, was ad m i n i s t e r e d t o animals primed only with EB and s t i l l produced no s i g n i f i c a n t e f f e c t s . However, the h i g h c o n t r o l scores may have prevented o b s e r v a t i o n of a f a c i l i t a t o r y e f f e c t with ICS 205-930. Lack of r e c e p t o r s e l e c t i v i t y i s always p r o b l e m a t i c i n i n v e s t i g a t i o n s of t h i s nature. Even compounds c o n s i d e r e d t o be " s e l e c t i v e " show a f f i n i t y f o r more than one type of r e c e p t o r . Although Meyerson proposed t h a t low doses of 5-HT a g o n i s t s , observed t o f a c i l i t a t e l o r d o s i s , d i d so v i a a c t i v a t i o n of i n h i b i t o r y p r e s y n a p t i c r e c e p t o r s , i t now appears more probable t h a t such e f f e c t s are the r e s u l t of 5-HT2 r e c e p t o r a c t i v a t i o n (Gorzalka et a l . , 1990) . S i m i l a r l y , the p o s s i b i l i t y remains t h a t drugs employed f o r t h e i r 5-HT3 a c t i v i t y a l s o a c t i v a t e d other 5-HT or even non5-HT r e c e p t o r s . As noted above, q u i p a z i n e , o r i g i n a l l y c o n s i d e r e d a 5-HT2 a g o n i s t a l s o a c t s as a 5-HT3 a n t a g o n i s t (Peroutka & Hamik, 1988). Based on the present experiment, i t i s u n l i k e l y t h a t q u i p a z i n e a f f e c t s l o r d o s i s v i a a 5-HT3 mechanism. The p o s s i b i l i t y a l s o e x i s t s t h a t some 5-HT3 r e c e p t o r a n t a g o n i s t s may act as p a r t i a l a g o n i s t s . For example, zacopr i d e , a 5-HT3 a n t a g o n i s t and potent a n t i e m e t i c , has been found t o induce v o m i t i n g ; t h i s e f f e c t was subsequently d i s c o v e r e d t o be a r e s u l t of use of the S enantiomer. U n l i k e the racemic mixture or R isomer, both of which act as 5-HT3 a n t a g o n i s t s , S-zacopride a c t s as a p a r t i a l a g o n i s t ( M i d d l e f e l l & P r i c e , 1991) . 70 I t i s a l s o p o s s i b l e t h a t systemic a d m i n i s t r a t i o n c o n t r i b u t e d to the l a c k of e f f e c t s produced by 5-HT3 a n t a g o n i s t s . That drugs a d m i n i s t e r e d SC l i k e l y reached a l l areas of the b r a i n , suggests the p o s s i b i l i t y t h a t r e c e p t o r s i n b r a i n regions having opposing f u n c t i o n s may have been a c t i v a t e d . Indeed, 5-HT i t s e l f can have opposing e f f e c t s depending on s i t e of a d m i n i s t r a t i o n : l a t e r a l v e n t r i c u l a r a d m i n i s t r a t i o n f a c i l i t a t e s (Wilson & Hunter, 1985) whereas hypothalamic a d m i n i s t r a t i o n i n h i b i t s (Foreman & Moss, 1978) l o r d o s i s . I n t e r e s t i n g l y , i n s t u d i e s where low doses of ICS 205-930 have been e f f e c t i v e , h i g h e r doses have sometimes f a i l e d t o produce an e f f e c t (e.g. C o s t a l l et a l . , 1987c, 1988b). Moreover, evidence i n d i c a t e s t h a t ICS 205-930 i s a c t i v e at a non5-HT 3 s i t e (e.g. C r a i g & C l a r k e , 1990). Together, these f i n d i n g s suggest t h a t h i g h doses of ICS 205-930 may a c t i v a t e a low a f f i n i t y r e c e p t o r , having e f f e c t s i n o p p o s i t i o n to 5-HT3 r e c e p t o r s . Given the r a t h e r d i f f u s e d i s t r i b u t i o n of 5-HT3 s i t e s , and the evidence t h a t some 5-HT3- a c t i v e compounds show a f f i n i t y f o r non5-HT 3 s i t e s , a d m i n i s t r a t i o n to d i s c r e t e n u c l e i may prove to be more f r u i t f u l than systemic a d m i n i s t r a t i o n . A 5-HT3 r e c e p t o r mechanism may be i n v o l v e d i n the e f f e c t s on r e p r o d u c t i v e behaviour produced by a d r e n e r g i c a g o n i s t s and a n t a g o n i s t s . 5-HT3 r e c e p t o r a c t i v i t y i n f l u e n c e s r e l e a s e of NE, endogenous s e c r e t i o n of which r e s u l t s i n a c t i v a t i o n of both a-and [3-adrenoceptors (Hornykiewicz & F l a t t e r y , 1980) . Furthermore, evidence suggests an i n t e r a c t i o n between 5-HT and NE i n the c o n t r o l of r e p r o d u c t i v e behaviour. I t i s known f o r 7 1 example, t h a t s e r o t o n e r g i c c o n t r o l of LH r e l e a s e i s , i n p a r t , dependent upon n o r a d r e n e r g i c n e u r o t r a n s m i s s i o n (Gopalan et a l . , 1 9 8 9 ) . Furthermore, ( - ) p i n d o l o l , a [3-adrenergic a n t a g o n i s t and p a r t i a l a g o n i s t , attenuates i n h i b i t i o n of l o r d o s i s produced by 8 - h y d r o x y - 2 - ( d i - n - p r o p y l a m i n o ) t e t r a l i n , a 5-HT 1 A a g o n i s t (Fernandez-Guasti et a l . , 1 9 8 7 ) . As p e r i p h e r a l a d m i n i s t r a t i o n of ( - ) p i n d o l o l alone i n h i b i t s l o r d o s i s (Mendelson & Gorzalka, 1 9 8 8 ) , i t i s l i k e l y t h a t ( - ) p i n d o l o l - i n d u c e d a t t e n u a t i o n of s e r o t o n e r g i c i n h i b i t i o n occurs v i a i n t e r a c t i o n with the s e r o t o n e r g i c system. In male r a t s , e f f e c t s induced by l i s u r i d e (a dopamine/5-HT agonist) on sexual behaviour are a t t e n u a t e d by n o r a d r e n e r g i c l e s i o n s (Fernandez-Guasti, Hansen, Archer & Jonsson, 1 9 8 6 ) . I n t e r e s t i n g l y , i n females, l i s u r i d e i n h i b i t s l o r d o s i s v i a a mechanism demonstrated to be s e r o t o n e r g i c (Fernandez-Guasti et a l . , 1 9 8 7 ) . Data r e g a r d i n g the e f f e c t s of n o r a d r e n e r g i c l e s i o n s on l o r d o s i s are l a c k i n g . However, as noted p r e v i o u s l y , the 5-HT 3 a g o n i s t 2-Me -5-HT i n h i b i t s r e l e a s e of NE, an e f f e c t b l o c k e d by 5-HT 3 a n t a g o n i s t s . Thus, i t i s tempting to s p e c u l a t e t h a t i f n o r a d r e n e r g i c l e s i o n s do a f f e c t female r e p r o d u c t i v e behaviour, they may do so v i a i n t e r a c t i o n with 5 -HT3 r e c e p t o r s . Although a c t i v a t i o n of (3-adrenoceptors has been demonstrated to i n h i b i t l o r d o s i s , c o n t r o v e r s y remains r e g a r d i n g the r o l e of cc-adrenoceptors with r e s p e c t to female sexual behaviour (Mendelson & Gorzalka, 1 9 8 8 ) / r e g a r d l e s s , i t i s p o s s i b l e t h a t 5-HT 3 r e c e p t o r agonism may attenuate a d r e n e r g i c -induced e f f e c t s on female sexual behaviour. 72 5-HT3 r e c e p t o r s may mediate a l i n k between sexual a c t i v i t y and modulation of p a i n , at l e a s t i n the male. Acute o p i o i d use i s a s s o c i a t e d both with a n a l g e s i a and orgasm-like euphoria; moreover, o p i o i d s i n f l u e n c e sexual f u n c t i o n (Pfaus & Gorzalka, 1987a). Morphine and ICS 205-930 both b l o c k r e f l e x i v e p a i n responses induced by duodenal d i s t e n t i o n , v i a a mechanism l o c a t e d o u t s i d e of the gut (Moss & Sanger, 1 9 9 0 ) . Given the documented involvement of 5-HT3 r e c e p t o r s i n n o c i c e p t i v e modulation (e.g. Glaum et a l . , 1988, 1990), i t i s i n t e r e s t i n g t h a t other i n v e s t i g a t o r s (James, et a l . , 1989; Mendelson & Gorzalka, 1989, N.V. Watson, p e r s o n a l communication) have found t h a t modulation of 5-HT3 r e c e p t o r a c t i v i t y i n f l u e n c e s sexual behaviour. Thus, i t i s p o s s i b l e t h a t 5-HT3 r e c e p t o r s may mediate the r e l a t i o n s h i p between n o c i c e p t i o n and sexual a c t i v i t y . Evidence suggests t h a t 5-HT i n f l u e n c e s sexual behaviour i n humans as w e l l as i n rodents. I n t e r e s t i n g l y , i n humans, a v a r i e t y of p a r a p h i l i a s are documented t o respond t o treatment with s e r o t o n i n - e n h a n c i n g psychopharmacological agents (Kafka & Coleman, 1991). Buspirone has been r e p o r t e d t o attenuate t r a n s v e s t i c f e t i s h i s m (Fedoroff, 1988); l i t h i u m carbonate and imipramine r e p o r t e d l y attenuate compulsive masturbation, voyeurism and e x h i b i t i o n i s m (Kafka, 1991a). F l u o x e t i n e has been used s u c c e s s f u l l y i n the treatment of n o n p a r a p h i l i a c sexual a d d i c t i o n s (Kafka, 1991a) and i n treatment of p a r a p h i l i a c c o e r c i v e d i s o r d e r ( i . e . a r a p i s t ) (Kafka, 1991b). The authors of these r e p o r t s i n t e r p r e t t h e i r f i n d i n g s w i t h i n the context of 73 compulsive d i s o r d e r s or d r i v e d y s r e g u l a t i o n syndromes. However, t h a t enhancement of s e r o t o n i n n e u r o t r a n s m i s s i o n a t t e n u a t e d these s e x u a l d i s o r d e r s which had not responded to behaviour therapy suggests t h a t s e r o t o n i n may p l a y a p i v o t a l r o l e i n modulating such b e h a v i o u r s . I n v e s t i g a t i o n of 5-HT 3 a c t i v e compounds has now been extended to humans. I d e n t i f i c a t i o n of 5-HT 3 r e c e p t o r s i n human b r a i n t i s s u e (Barnes et a l . , 1 9 9 0 ) , t o g e t h e r with f i n d i n g s t h a t c l i n i c a l l y - a c t i v e compounds (e.g. metoclopramide) were a c t i v e at 5-HT 3 s i t e s (Hamik and Peroutka, 1989) spurred examination of 5-H T 3 - a c t i v e compounds i n humans. In c l i n i c a l t r i a l s 5-HT 3 a n t a g o n i s t s have been found to be potent i n h i b i t o r s of chemotherapy-induced emesis (e.g. Cunningham et a l . , 1987) and to e f f e c t g a s t r i c emptying (Stacher et a l . , 1990) and c o l o n i c a c t i v i t y (Stacher et a l , 1 9 8 9 ) . ICS 205-930 has been found to a t t e n u a t e p e r s i s t e n t d i a r r h e a due to c a r c i n o i d syndrome (Anderson, Coupe, M o r r i s , Hodgson & Bloom, 1987) . Moreover, Richardson (1990) notes the p o t e n t i a l t h e r a p e u t i c value of 5-HT 3 a n t a g o n i s t s i n terms of t h e i r a n a l g e s i c a b i l i t i e s , e s p e c i a l l y i n c o n d i t i o n s such as myocardial i n f a r c t i o n where p a i n i s the r e s u l t of s e r o t o n i n r e l e a s e . C o n s i s t e n t with f i n d i n g s i n d i c a t i n g t h a t 5-HT3 r e c e p t o r a n t a g o n i s t s attenuate 5-HT-induced v a s o d i l a t i o n i n the human forearm, (Blauw, van Brummelen & van Zwieten, 1 9 8 8 ) , MDL 72222 has been found e f f e c t i v e i n the treatment of migraine headaches (Loisy, B e orchia, Centonze, Fozard, Schechter & T e l l , 1 9 8 7 ) . 74 The a b i l i t y of 5-HT3 a n t a g o n i s t s t o modulate mesolimbic dopamine a c t i v i t y , suggests t h a t they may be e f f e c t i v e a n t i p s y c h o t i c s (e.g. T r i c k e l b a n k , 1989). In nonhuman models, 5-HT3 a n t a g o n i s t s , u n l i k e t r a d i t i o n a l n e u r o l e p t i c s , decrease n e i t h e r motor a c t i v i t y nor l i m b i c dopaminergic a c t i v i t y t o below normal l e v e l s ( C o s t a l l et a l . , 1990b); thus, such drugs may be f r e e of e x t r a p y r a m i d a l s i d e e f f e c t s i n humans. Indeed, even f o l l o w i n g c h r o n i c a d m i n i s t r a t i o n of 5-HT3 a n t a g o n i s t s , no a l t e r a t i o n s i n dopamine or s e r o t o n i n metabolism i n mesolimbic or n i g r o s t r i a t a l areas are found (Koulu, Lappalainen, H i e t a l a & Sjoholm, 1990). In humans, o r a l a d m i n i s t r a t i o n of ondansetron f o r 2.5 days produces no impairment of psychomotor f u n c t i o n ( H a l l & Ceuppens, 1991). I n t e r e s t i n g l y , s e v e r a l a t y p i c a l n e u r o l e p t i c s (e.g. c l o z a p i n e ) , which possess l i m i t e d s i d e e f f e c t s , have demonstrable a f f i n i t y f o r 5-HT3 s i t e s (Bolanos et a l . , 1990) . The nonsedating a n x y i o l y t i c e f f e c t s produced by 5-HT3 a n t a g o n i s t s i n rodents (Jones et a l . , 1988), suggest t h a t 5-HT3 a n t a g o n i s t s a l s o have p o t e n t i a l as a n x i o l y t i c agents i n humans. Furthermore, given t h e i r a b i l i t y t o b l o c k the b e h a v i o u r a l consequences of drug withdrawal i n nonhumans (Goudie & L e a t h l e y , 1990; C o s t a l l et a l . , 1990a), 5-HT3 a n t a g o n i s t s may a l s o prove e f f e c t i v e i n a t t e n u a t i n g symptoms of drug withdrawal i n humans. Although 5-HT3 r e c e p t o r a c t i v i t y has been i m p l i c a t e d i n the modulation of v a r i o u s b e h a v i o u r a l e f f e c t s , the present r e s u l t s do not support the c o n t e n t i o n of 5-HT3 involvement i n female r a t 75 sexual b e h a v i o u r s . Nonetheless, the p o s s i b i l i t y t h a t 5-HT3 r e c e p t o r s may modulate female r e p r o d u c t i v e behaviour v i a an i n t e r a c t i o n with a c e t y l c h o l i n e or n o r e p i n e p h r i n e awaits f u t u r e r e s e a r c h . 76 R E F E R E N C E S Acquas, E., Carboni, L., Garau, L., & Di C h i a r a , G. (1990) . 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