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The central administration of oxytocin and oxytocin analogs to steroid-primed female rats : an investigation… Schulze, H. Georg 1991

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THE CENTRAL ADMINISTRATION OF OXYTOCIN AND OXYTOCIN ANALOGS TO STEROID-PRIMED FEMALE RATS: AN INVESTIGATION OF THE EFFECTS OF BRAIN S I T E , DOSE, RECEPTOR TYPE, AND TIME PARAMETERS ON THE GENERATION OF LORDOSIS BEHAVIOR b y H. GEORG SCHULZE B.S c . E n g . (Chem)., The U n i v e r s i t y o f P r e t o r i a , 1980 A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n THE FACULTY OF GRADUATE STUDIES D e p a r t m e n t o f P s y c h o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A u g u s t 1991 ©Hans G e o r g S c h u l z e In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) i i Abstract Oxytocin plays an important r o l e i n the orchestration of many behaviors, including reproductive behaviors, i n the female r a t . Although i t i s known to influence sexual r e c e p t i v i t y , r e l a t i v e l y l i t t l e i s known about the central s i t e s of action of oxytocin and how these contribute to the generation of lor d o s i s . Furthermore, i t i s not cle a r what the mechanisms of action of t h i s peptide at these s i t e s may be, and how temporal factors influence oxytocinergic a c t i v i t y . The purpose of t h i s thesis i s to investigate the ro l e of oxytocin i n the generation of lordosis behavior i n the female r a t from a variety of perspectives i n order to gain a broader understanding of t h i s behavior. The administration of low doses of oxytocin to the l a t e r a l v e n t r i c l e of female rats treated with 5 jug e s t r a d i o l benzoate and 150 ng progesterone i n h i b i t e d lordosis behavior while higher doses had no e f f e c t . This contrasted with the administration of sim i l a r oxytocin doses to the medial preoptic area where low doses had l i t t l e e f f e c t and higher doses tended to elevate primarily lordosis quotients. Higher oxytocin doses administered to the ventromedial hypothalamus extended lordosis durations but had a smaller e f f e c t on lordosis quotients. In the central nucleus of the amygdala, the same high doses also extended lordosis durations but not lordosis quotients. i i i These r e s u l t s i m p l y t h a t d i f f e r e n t b r a i n s i t e s may c o n t r i b u t e t o d i f f e r e n t a s p e c t s o f t h e l o r d o s i s b e h a v i o r : t h e m e d i a l p r e o p t i c a r e a appears t o be r e s p o n s i b l e f o r l o r d o s i s f r e q u e n c y (as measured by t h e l o r d o s i s q u o t i e n t ) , t h e amygdala f o r l o r d o s i s d u r a t i o n , and t h e hypothalamus perhaps f o r l o r d o s i s i n i t i a t i o n . The i n f u s i o n o f an o x y t o c i n a g o n i s t and a n t a g o n i s t i n t o t h e l a t e r a l v e n t r i c l e s o f s t e r o i d - p r i m e d female r a t s r e d u c e d l o r d o s i s q u o t i e n t s . The same agents i n f u s e d i n t o t h e m e d i a l p r e o p t i c a r e a and t h e v e n t r o m e d i a l hypothalamus d i d not produce any s i g n i f i c a n t e f f e c t s . These r e s u l t s a r e d i f f i c u l t t o i n t e r p r e t but appear t o be congruent w i t h t h e n o t i o n t h a t b o t h t h e v a s o p r e s s i n e r g i c system and t h e o x y t o c i n e r g i c system e x e r c i s e c o n t r o l o v er l o r d o s i s b e h a v i o r . The former system i s i n h i b i t o r y w h i l e t h e l a t t e r system appears t o f a c i l i t a t e s e x u a l b e h a v i o r o n l y when a c e r t a i n t h r e s h o l d o f n e u r a l a c t i v i t y has been reached. I t f u r t h e r m o r e appears l i k e l y t h a t b o t h p e p t i d e s can a c t on t h e r e c e p t o r s o f b o t h systems. When i n f u s e d i n t o t h e l e f t l a t e r a l v e n t r i c l e s o f s t e r o i d -t r e a t e d female r a t s , t h e o x y t o c i n a g o n i s t employed i n t h e s e s t u d i e s f a c i l i t a t e d l o r d o s i s b e h a v i o r 32 h a f t e r e s t r a d i o l a d m i n i s t r a t i o n but not 48 h l a t e r when most r e p o r t e d i n v e s t i g a t i o n s have been c a r r i e d o u t . T h i s i m p l i e s t h a t s u f f i c i e n t ' f u n c t i o n a l o x y t o c i n r e c e p t o r s a r e i n d u c e d w i t h i n i v 32 h a f t e r estrogen treatment. I t also r a i s e s the p o s s i b i l i t y that receptor induction and decay at d i f f e r e n t central s i t e s may follow d i f f e r e n t time courses. Taken together, these studies indicate that several brain s i t e s contribute to the generation of lordosis and that both the vasopressinergic and oxytocinergic systems may be involved. Furthermore, these systems may be d i f f e r e n t i a t e d by temporal patterns of a c t i v a t i o n . V PREFACE Some of the material contained i n t h i s thesis have been published, submitted for publication, or are being considered for submission. In p a r t i c u l a r , the r e s u l t s of Experiments 2 and 4 have been published. The relevant reference i s the following: Schulze, H. G.; Gorzalka, B. B. 1991. Oxytocin e f f e c t s on lordosis frequency and lordosis duration following infusion into the medial preoptic area and ventromedial hypothalamus of female rats. Neuropeptides 18: 99-106. The r e s u l t s of Experiments 1 and 3 have been submitted: Schulze, H. G.; Gorzalka, B. B. Low concentrations of oxytocin suppress lordosis when infused into the l a t e r a l v e n t r i c l e of female r a t s . Endocrine Regulations: manuscript submitted. The r e s u l t s from Experiments 7 and 8 are being considered for publication and may be submitted a f t e r completion of t h i s t h e s i s . The submitted work i s expected to be a duplication of Section IV and w i l l be reported by the same authors i n the same order. A l l the material published, submitted for publication and considered for publication i s the work of the f i r s t author. The second author was responsible for ca r e f u l revisions of the material and for input regarding s t y l e , c l a r i t y , and presentation. In addition, a l l ' o f the work was ca r r i e d out i n the laboratory of and with the funds available to the second author. Signed: B.B.GORZALKA Signed: H.G.SCHULZE v i i TABLE OF CONTENTS ABSTRACT i i PREFACE V LIST OF TABLES v i i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x INTRODUCTION 1 GENERAL METHODS. . . . 6 SECTION I (The a d m i n i s t r a t i o n o f o x y t o c i n to the c e r e b r a l v e n t r i c l e s ) 9 Exper iment 1 10 Exper iment 2 15 Exper iment 3 20 SECTION I I (The a d m i n i s t r a t i o n o f o x y t o c i n t o b r a i n r e g i o n s c o n t a i n i n g e s t r o g e n - i n d u c e d o x y t o c i n r e c e p t o r s ) 31 Exper iment 4 35 Exper iment 5 47 SECTION I I I (The r e c e p t o r types m e d i a t i n g the e f f e c t s o f o x y t o c i n on l o r d o s i s ) 51 Exper iment 6 53 SECTION IV (Some t e m p o r a l f a c t o r s i n the e f f e c t s o f c e n t r a l l y a d m i n i s t e r e d o x y t o c i n on l o r d o s i s ) 61 Exper iment 7 62 Exper iment 8 68 GENERAL DISCUSSION 84 REFERENCES 94 v i i i LIST OF TABLES 1. A c t i v i t i e s a t v a r i o u s r e c e p t o r s o f t h e o x y t o c i n a g o n i s t and a n t a g o n i s t .52 2. R e c e p t o r a c t i v i t i e s and b e h a v i o r a l e f f e c t s o f some o x y t o c i n a n a l o g s a t o x y t o c i n and v a s o p r e s s i n r e c e p t o r s . ..87 i x L I S T OF FIGURES 1. The e f f e c t on l o r d o s i s o f o x y t o c i n a d m i n i s t e r e d t o t h e l a t e r a l a n d t h i r d v e n t r i c l e s 14 2. L o r d o s i s q u o t i e n t s a n d l o r d o s i s d u r a t i o n s a s a f u n c t i o n o f p r o g e s t e r o n e t r e a t m e n t i n e s t r o g e n - p r i m e d r a t s 19 3. The d o s e - r e s p o n s e c u r v e o f t h e e f f e c t s o f o x y t o c i n i n f u s i o n s i n t o t h e l e f t l a t e r a l v e n t r i c l e 23 4. The e f f e c t s o f v a r i o u s d o s e s o f o x y t o c i n a d m i n i s t e r e d t o t h e MPOA, VMH, a n d MCG 4 0 5. The e f f e c t s o f v a r i o u s d o s e s o f o x y t o c i n a d m i n i s t e r e d t o t h e c e n t r a l n u c l e u s o f t h e a m y g d a l a 50 6. The e f f e c t s o n l o r d o s i s o f o x y t o c i n a g o n i s t a n d a n t a g o n i s t a f t e r i n f u s i o n s i n t o t h e LV, VMH, a n d MPOA. ...58 7. The e f f e c t s o f a n o x y t o c i n a g o n i s t a n d a n t a g o n i s t o n l o r d o s i s o v e r a 24 h p e r i o d s t a r t i n g 28 h a f t e r e s t r o g e n a d m i n i s t r a t i o n 67 8. A c o m p a r i s o n o f t h e e f f e c t s on l o r d o s i s o f t h e o x y t o c i n a g o n i s t a t t i m e s lOhOO a nd 22h00 72 9. The e f f e c t s o f a n o x y t o c i n a g o n i s t a n d a n t a g o n i s t on l o r d o s i s o v e r a 24 h p e r i o d s t a r t i n g 28 h a f t e r e s t r o g e n a d m i n i s t r a t i o n ( d a t a f r o m e x p e r i m e n t s 7 & 8) 75 X ACKNOWLEDGEMENTS I would l i k e t o thank my t h e s i s s u p e r v i s o r , Dr. B o r i s G o r z a l k a , f o r h i s g r e a t p a t i e n c e and support and h i s c o n s i d e r a b l e courage i n a l l o w i n g an unknown e n t i t y i n t o h i s l a b o r a t o r y . I a l s o wish t o express my a p p r e c i a t i o n t o the o t h e r members of my committee, Dr. C a t h a r i n e Rankin and Dr. Lawrence Ward, f o r t h e i r support and encouragement. Dr. Ward deserves a f u r t h e r word of thanks f o r a t t e n d i n g t o my t h e s i s concerns w h i l e on s a b b a t i c a l . F i n a l l y , a word of g r a t i t u d e aimed a t another Psychology f a c u l t y member, Dr. John P i n e l , whose h e l p f u l and e n t h u s i a s t i c c o u n s e l l i n g t u r n e d a n a i v e and u n c e r t a i n student i n t o one headed f o r Graduate School i n Biopsychology. T h i s r e s e a r c h was funded by a N a t u r a l S c i e n c e s 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 Oxy t o c i n (OT), which i n 1953 became the f i r s t p e p t i d e t o be s y n t h e s i z e d (du Vignaud e t a l . 1953), i s known t o be p h y s i o l o g i c a l l y important i n p a r t u r i t i o n and l a c t a t i o n and has a l s o been shown t o e x e r t b e h a v i o r a l e f f e c t s , namely i n the e x p r e s s i o n of maternal behavior, l e a r n i n g and memory, drug t o l e r a n c e and a r o u s a l (see reviews by Kovacs 1986, A r g i o l a s and Gessa 1991) . T h i s p e p t i d e i s s y n t h e s i z e d p e r i p h e r a l l y i n the o v a r i e s ( I v e l l and R i c h t e r 1984, see a l s o I v e l l 1986), i s s e c r e t e d i n t o the systemic c i r c u l a t i o n by the p o s t e r i o r p i t u i t a r y (Hashimoto e t a l . 1988), i s r e l e a s e d i n phase wi t h progesterone ( F l i n t and S h e l d r i c k 1985; Schams e t a l . 1985) and i t s r e c e p t o r s are t o be found i n the smooth muscle of the female r e p r o d u c t i v e t r a c t , i n mammary m y o e p i t h e l i a l c e l l s (see S o l o f f 1985) and a t some c e n t r a l s i t e s (de K l o e t et a l . 1986). C e n t r a l l y OT i s s y n t h e s i z e d mainly i n the s u p r a o p t i c and p a r a v e n t r i c u l a r n u c l e i (see Robinson 1983, B u i j s e t a l . 1985). Moreover, t h e r e are o x y t o c i n e r g i c p r o j e c t i o n s t o the midbrain c e n t r a l grey (MCG), v e n t r a l tegmental area, d o r s a l v a g a l complex and s p i n a l c o r d ( B u i j s e t a l . 1985). OT i s t r a n s p o r t e d t o the s p i n a l c o r d from the s u p r a o p t i c and p a r a v e n t r i c u l a r n u c l e i (Camier e t a l . 1985) v i a f a s t axonal t r a n s p o r t (White e t a l . 1986) which may imply a n e u r o t r a n s m i t t e r r o l e f o r t h i s p e p t i d e i n the s p i n a l c o r d . In the s p i n a l c o r d of newborn r a t s , i o n t o p h o r e t i c a p p l i c a t i o n of OT induces a marked 2 d e p o l a r i z a t i o n of motorneurons (Suzue e t a l . 1981). OT i s furthermore p r e s e n t i n sensory neurons i n the r a t s p i n a l c o r d ( K a i - K a i e t a l . 1985). The presence of OT i n motor and sensory systems may al l o w the p e p t i d e t o p l a y a r o l e i n the c o n t r o l o f l o r d o s i s by i n f l u e n c i n g or modulating the behavior a t s p i n a l l e v e l s . In a d d i t i o n , OT l e v e l s i n the p o r t a l b l o o d (Sarkar and Gibbs 1984) and the p o s t e r i o r p i t u i t a r y (Crowley e t a l . 1978) vary a c r o s s the e s t r o u s c y c l e - l e v e l s being h i g h e s t j u s t b e f o r e the onset of b e h a v i o r a l e s t r u s . The importance of OT i n r e p r o d u c t i v e f u n c t i o n s and behaviors has k i n d l e d i n t e r e s t i n t h i s n e u r o a c t i v e substance as p u t a t i v e l y being i n v o l v e d i n se x u a l behavior. Three components of sexual behavior i n female mammals have been d e s c r i b e d : a t t r a c t i v i t y , p r o c e p t i v i t y and r e c e p t i v i t y (Beach 1976). A major aspect of r e c e p t i v i t y i n female r a t s i s the l o r d o s i s r e f l e x which c o n s i s t s of a concave a r c h i n g of the back, e l e v a t i o n of the head and l a t e r a l d e f l e x i o n of the t a i l , executed i n response t o a mount by a male r a t . The l o r d o s i s q u o t i e n t (LQ) i s wi d e l y used as an index of r e c e p t i v i t y and i s expressed as a percentage of the number of l o r d o s e s shown i n response t o a s e t number (e.g. 10) of mounts (Hardy and DeBold 1972). L o r d o s i s d u r a t i o n i s another index of sexual r e c e p t i v i t y and i t i s e s p e c i a l l y u s e f u l f o r measuring r e c e p t i v i t y i n hamsters (e.g. L e s t e r and Gorzalka 1988). Although i t has 3 been measured at l e a s t once i n the female r a t (see Madlafousek and H l i n a k 1977), l o r d o s i s d u r a t i o n i s seldom used as an index f o r r e c e p t i v i t y i n t h i s animal and has not been used t o i n v e s t i g a t e the e f f e c t s of o x y t o c i n on r e c e p t i v i t y . Whether OT p l a y s a r o l e i n the a t t r a c t i v i t y or p r o c e p t i v i t y components of s e x u a l behavior i n female r a t s i s not known, but i t has been e s t a b l i s h e d t h a t l o r d o s i s i s f a c i l i t a t e d by the a d m i n i s t r a t i o n of exogenous OT (e.g. A r l e t t i and B e r t o l i n i 1985, Gorzalka and L e s t e r 1987). Rec e n t l y , i t has been demonstrated t h a t OT i s indeed i n v o l v e d i n the e x p r e s s i o n of sexual behaviors i n the r a t ( A r l e t t i and B e r t o l i n i 1985, C a l d w e l l e t a l . 1986, G o r z a l k a and L e s t e r 1987, Hughes e t a l . 1987, C a l d w e l l e t a l . 1989). However, a t t h i s stage r e l a t i v e l y l i t t l e i s known about the c e n t r a l s i t e s of a c t i o n of t h i s p e p t i d e and i n p a r t i c u l a r about the mechanisms or modes of a c t i o n at these s i t e s . For i n s t a n c e , i t i s l i k e l y t h a t the a c t i o n of OT on s e x u a l behavior, l i k e the a c t i o n on maternal behavior, i s e s t r o g e n -dependent (see Fahrbach et a l . 1985 f o r r e v i e w ) . S e v e r a l b r a i n r e g i o n s , p a r t i c u l a r l y hypothalamic r e g i o n s , have been shown t o c o n c e n t r a t e r a d i o l a b e l l e d e s t r a d i o l ( P f a f f 1968, P f a f f and K e i n e r 1973). Estrogen a l s o i n c r e a s e s the number of OT r e c e p t o r s i n the VMH when c h r o n i c a l l y a d m i n i s t e r e d (de K l o e t e t a l . 1986, Johnson e t a l . 1989), s t i m u l a t e s the r e l e a s e of OT i n t o the p e r i p h e r a l c i r c u l a t i o n (Yamaguchi e t a l . 1979), i n f l u e n c e s the e l e c t r i c a l a c t i v i t y of OT neurons 4 (Negoro e t a l . 1973, Negoro e t a l . 1983), m o d i f i e s the p a t t e r n of c e n t r a l OT i n n e r v a t i o n ( C a l d w e l l e t a l . 1988, J i r i k o w s k i e t a l . 1988) and may cause i n c r e a s e d l e v e l s of OT mRNA ( M i l l e r e t a l . 1989). OT l e v e l s a l s o vary i n the p a r a v e n t r i c u l a r nucleus d u r i n g the r a t e s t r o u s c y c l e (Greer e t a l . 1986). C u r i o u s l y enough, d e s p i t e the involvement of OT i n female r e p r o d u c t i v e f u n c t i o n s , OT i n n e r v a t i o n of the b r a i n does not appear t o be s e x u a l l y dimorphic as i s t h a t of a r g i n i n e v a s o p r e s s i n ( B u i j s e t a l . 1985). The above evidence suggests a s t r o n g dependence of OT on the m e t a b o l i c e f f e c t s of e s t r o g e n . However, the p r e c i s e nature of t h i s dependence remains t o be determined. The second important o v a r i a n hormone r e q u i r e d f o r the f u l l e x p r e s s i o n of sexual behavior i n the female r a t i s progesterone ( B o l i n g and Blandau 1939, see Clemens and Weaver 1985 f o r a d i s c u s s i o n of the r o l e of gonadal hormones i n s e x u a l b e h a v i o r ) . The a c t i o n s of OT on l o r d o s i s are dependent on the presence of progesterone a t some s i t e s , e. g. the v e n t r o m e d i a l hypothalamus (VMH) (Schumacher e t a l . 1989, Schumacher e t a l . 1990) and the l a t e r a l v e n t r i c l e (Gorzalka and L e s t e r 1987), but may not be a t o t h e r s , e.g. the medial p r e o p t i c area ( C a l d w e l l e t a l . 1989). In the VMH progesterone appears t o f a c i l i t a t e l o r d o s i s by i n c r e a s i n g the area of d i s p e r s i o n of OT r e c e p t o r s (Schumacher e t a l . 1989, Schumacher e t a l . 1990). I n v e s t i g a t i n g the a c t i o n s of progesterone on o x y t o c i n e f f e c t s on l o r d o s i s i s complicated 5 by the s y n e r g i s t i c e f f e c t s of progesterone and estrogen on sexual behavior. For example, estrogen modulates p r o g e s t i n r e c e p t o r s i n some b r a i n areas but not i n others (MacLusky and McEwen 1978) and can have p r o g e s t e r o n e - l i k e e f f e c t s on rep r o d u c t i v e behavior (Parsons et a l . 1984). Progesterone can a l s o i n h i b i t o x y t o c i n r e l e a s e during v a g i n a l d i s t e n s i o n (Roberts and Share 1970). At t h i s stage, the r o l e of progesterone i n the expression of OT-mediated l o r d o s i s at other c e n t r a l s i t e s i s undetermined. Ongoing i n v e s t i g a t i o n s of the r o l e of OT i n sexual behavior focus on the c e n t r a l s i t e s of a c t i o n of OT (Caldw e l l et a l . 1989) and the modes and mechanisms of a c t i o n of OT at some of these s i t e s (Caldwell et a l . 1989, Schumacher et a l . 1989, Ca l d w e l l et a l . 1990, Schumacher et a l . 1990). These issues a l s o form the main o b j e c t i v e s of the present set of experiments. In p a r t i c u l a r , the e f f e c t s on l o r d o s i s of the a d m i n i s t r a t i o n of var i o u s concentrations of OT i n t o a few s e l e c t e d estrogen-concentrating b r a i n regions, known t o induce OT receptor s , w i l l be i n v e s t i g a t e d i n animals t r e a t e d w i t h both the ovarian hormones estrogen and progesterone. In a d d i t i o n , the s p e c i f i c c o n t r i b u t i o n of a p a r t i c u l a r s i t e i n the generation of the l o r d o s i s r e f l e x w i l l be f u r t h e r i n v e s t i g a t e d through the a d m i n i s t r a t i o n of an OT agonist and antagonist. F i n a l l y , the r e s u l t s obtained w i l l be cont r a s t e d t o attempt a synt h e s i s where the s i t e s of a c t i o n of OT, some of the modes and mechanisms of a c t i o n of 6 OT at these s i t e s and the contribution of these s i t e s i n generating the lordosis r e f l e x w i l l be related. GENERAL METHODS Animals and Surgery Female Sprague-Dawley rats, from Charles River Canada, Inc., Montreal, were b i l a t e r a l l y ovariectomized through lumbar i n c i s i o n s while under sodium pentobarbital (Somnotol) anesthesia at about 70 days of age. Following surgeries, a l l animals were i n d i v i d u a l l y housed i n standard laboratory wire mesh cages i n a colony room maintained on a reversed 12h light/dark cycle and at 50% r e l a t i v e humidity, 21 C with free access to food and water. Approximately one week af t e r the ovariectomies, animals receiving central manipulations were anesthetized as before, placed i n a stereotaxic instrument, and provided with a chronic i n t r a c r a n i a l cannula assembly using the stereotaxic a t l a s of Pellegrino et a l . (1979) as guide. These procedures are described f u l l y by Gray and Gorzalka (1979) and., except . for placements i n the l a t e r a l v e n t r i c l e , were modified s l i g h t l y , using a method outlined by E l l i o t t (1986) to accommodate b i l a t e r a l or double placements. Following surgeries, animals were returned to t h e i r home cages. 7 I n t h e c a s e o f i n t r a v e n t r i c u l a r c a n n u l a e , p l a c e m e n t s w e r e t e s t e d a b o u t one week a f t e r s u r g e r y by i n f u s i o n o f 2 /ug o f a n g i o t e n s i n I I (1 ixq/ixl) i n t o t h e l a t e r a l v e n t r i c l e o f e a c h a n i m a l . O n l y a n i m a l s t h a t showed a p r o n o u n c e d d r i n k i n g r e s p o n s e t o t h i s t r e a t m e n t w e r e r e t a i n e d f o r e x p e r i m e n t a t i o n . The a c c u r a c y o f a l l o t h e r p l a c e m e n t s was v e r i f i e d b y t r i a l s u r g e r i e s f o l l o w e d by t h e a p p r o p r i a t e h i s t o l o g i e s . Once t h e h i s t o l o g i e s i n d i c a t e d c o r r e c t p l a c e m e n t o f t h e c a n n u l a e , t h e r e m a i n i n g a n i m a l s w e r e s u b j e c t e d t o s u r g e r y u s i n g t h e p l a c e m e n t c o o r d i n a t e s d e t e r m i n e d i n t h e t r i a l s u r g e r i e s . I n t h i s manner a b o u t 20 a n i m a l s p e r p l a c e m e n t g r o u p w e r e p r e p a r e d . S e x u a l l y v i g o r o u s S p r a g u e - D a w l e y m a l e r a t s s e r v e d a s s t u d s . T h e s e w e r e g r o u p h o u s e d , s i x t o a c a g e , u n d e r c o n d i t i o n s i d e n t i c a l t o t h o s e o f t h e f e m a l e s . D r u g P r o c e d u r e s E s t r a d i o l b e n z o a t e (EB) and p r o g e s t e r o n e ( b o t h f r o m Sigma) w e r e d i s s o l v e d i n warm p e a n u t o i l a n d a d m i n i s t e r e d s u b c u t a n e o u s l y ( s c ) i n O . l c c o f v e h i c l e . A l l OT i n f u s i o n s w e r e made u s i n g a n i n f u s i o n pump (Sage i n s t r u m e n t s , m o d e l 34IA) d e l i v e r i n g a t a r a t e o f 4 j i i l / m i n u t e . 8 B e h a v i o r a l T e s t i n g B e h a v i o r a l t e s t i n g i n v o l v e d the p r e s e n t a t i o n of an experimental female t o a s e x u a l l y v i g o r o u s male, p r e v i o u s l y h a b i t u a t e d t o the t e s t i n g arena and g i v e n b r i e f access t o a f u l l y r e c e p t i v e s t i m u l u s female, i n a c i r c u l a r g l a s s chamber measuring 29 cm i n diameter, 45 cm i n h e i g h t and f i l l e d w i t h about 4 cm of bedding m a t e r i a l (Corncob Granules, Coeval Inc., S t . Joseph, 111.). T e s t i n g was conducted d u r i n g the middle p e r i o d of the dark c y c l e i n a t e s t i n g room where a computer was used as an event r e c o r d e r . Every experimental female was g i v e n 10 mounts wi t h p e l v i c t h r u s t i n g by a stud male. I f a male d i d not mount a f t e r 2 minutes, the female was presented t o another s t u d male u n t i l she had r e c e i v e d 10 mounts or u n t i l 15 minutes had elapsed i n which case the l o r d o s i s q u o t i e n t was c a l c u l a t e d on the number of mounts a c t u a l l y r e c e i v e d by the female. L o r d o s i s q u o t i e n t s were c a l c u l a t e d as a percentage of the number of mounts t h a t r e s u l t e d i n the l o r d o s i s p o s t u r e . L o r d o s i s postures were dichotomized and o n l y p o s t u r e s w i t h moderate or f u l l d o r s i f l e x i o n of the sp i n e c o r r e s p o n d i n g t o the 2-point and 3-point l o r d o s i s s c o r e s of Hardy and DeBold (1972) were c o n s i d e r e d p o s i t i v e . 9 SECTION I The a d m i n i s t r a t i o n of o x y t o c i n t o the c e r e b r a l v e n t r i c l e s . Although c e n t r a l OT r e c e p t o r s have been i d e n t i f i e d ( B r i n t o n e t a l . 1984), t h e r e i s a remarkable mismatch between s i t e s of OT i n n e r v a t i o n and OT r e c e p t o r s ( B u i j s e t a l . 1985). T h i s may i n d i c a t e t h a t r e l e a s e of OT i n t o the c e r e b r o s p i n a l f l u i d (CSF) p l a y s an important r o l e i n the t r a n s m i s s i o n of o x y t o c i n e r g i c s i g n a l s . OT i s known t o f a c i l i t a t e s e x u a l r e c e p t i v i t y i n female r a t s when i n f u s e d i n t o the l a t e r a l c e r e b r a l v e n t r i c l e ( A r l e t t i and B e r t o l i n i 1985, C a l d w e l l e t a l . 1986, Gorzalka and L e s t e r 1987, C a l d w e l l e t a l . 1989). To date, no l i t e r a t u r e e x i s t s on the e f f e c t s of OT i n f u s i o n s i n t o the t h i r d v e n t r i c l e . I t i s known t h a t the i n f u s i o n of b e h a v i o r a l l y a c t i v e substances i n t o the l a t e r a l versus t h i r d v e n t r i c l e s can have d i f f e r e n t i a l e f f e c t s . For example, Wilson and Hunter (1985) found t h a t the i n f u s i o n of s e r o t o n i n i n t o the l a t e r a l v e n t r i c l e of female r a t s primed with low l e v e l s of estrogen and progesterone s i g n i f i c a n t l y f a c i l i t a t e d l o r d o s i s but t h a t the same dose i n f u s e d i n t o the t h i r d v e n t r i c l e d i d not. Given the p r o x i m i t y of the t h i r d v e n t r i c l e t o the ven t r o m e d i a l hypothalamus (VMH) and the medial p r e o p t i c area (MPOA), s i t e s which are known t o be e f f e c t i v e f o r the o x y t o c i n e r g i c f a c i l i t a t i o n of l o r d o s i s (Schulze and Gorzalka 1991; C a l d w e l l e t a l . 1989; Schumacher et a l . 1989), i t seems reas o n a b l e t o expect t h a t t h i s r e g i o n would be more 10 s e n s i t i v e t h a n t h e l a t e r a l v e n t r i c l e t o i n f u s i o n . M o r e o v e r , l e a k a g e o f OT f r o m i n f u s i o n s i t e s s u c h a s t h e VMH a n d MPOA i n t o t h e n e a r b y v e n t r i c l e c a n n o t a l w a y s be r u l e d o u t . I t r e m a i n s t o be d e t e r m i n e d w h e t h e r i n f u s i o n s o f OT i n t o t h e t h i r d v e n t r i c l e w o u l d h a v e t h e same e f f e c t a s i n f u s i o n s i n t o t h e VMH o r MPOA. G i v e n a d i f f e r e n t i a l e f f e c t b e t w e e n i n f u s i o n s i t e e m p l o y e d a n d a d j a c e n t v e n t r i c l e , one c a n be more c o n f i d e n t a b o u t t h e r e s u l t i n g e f f e c t b e i n g due t o t h e a c t i o n o f OT a t t h e i n f u s i o n s i t e r a t h e r t h a n due t o l e a k a g e i n t o t h e v e n t r i c l e a n d s u b s e q u e n t a c t i o n a t a r e m o t e s i t e . The p r e s e n t s e r i e s o f e x p e r i m e n t s w e r e d e s i g n e d t o e x a m i n e t h e e f f e c t s o n l o r d o s i s o f t h i r d a n d l a t e r a l v e n t r i c u l a r OT a d m i n i s t r a t i o n . E x p e r i m e n t 1 E x p e r i m e n t 1 was d e s i g n e d t o com p a r e t h e e f f e c t o n l o r d o s i s o f OT i n f u s e d i n t o t h e l a t e r a l v e n t r i c l e w i t h a n i d e n t i c a l d o s e i n f u s e d i n t o t h e t h i r d v e n t r i c l e . The d o s e s e l e c t e d was r e l a t i v e l y l o w , b e c a u s e h i g h e r d o s e s w o u l d be l e s s l i k e l y t o r e v e a l d i f f e r e n t i a t i o n b e t w e e n t h e v e n t r i c l e s . W i t h h i g h e r d o s e s t h e p r o b a b i l i t y a n d e x t e n t o f d i f f u s i o n o r c i r c u l a t i o n o f i n f u s e d OT w o u l d be g r e a t e r . 11 M a t e r i a l s and methods Animals and surgery Twenty female Sprague-Dawley r a t s were b i l a t e r a l l y o v a r i e c t o m i z e d and r e c e i v e d i n t r a c r a n i a l cannulae aimed a t the t h i r d and l e f t l a t e r a l v e n t r i c l e s a c c o r d i n g t o procedures d e s c r i b e d i n the General Methods s e c t i o n . For the l a t e r a l v e n t r i c l e the c o o r d i n a t e s used f o r surgery were: 0.2 mm p o s t e r i o r , 1.8 mm l a t e r a l and 2.9 mm v e n t r a l ; and f o r the t h i r d v e n t r i c l e : 0.0 mm a n t e r i o r , 0.0 mm medial and 9.0 mm v e n t r a l . Placement t e s t i n g r e s u l t e d i n the e x c l u s i o n of data from 11 animals from s t a t i s t i c a l a n a l y s i s . Drug procedures E s t r a d i o l benzoate (5 /xg) and progesterone (150 jug) were prepared and ad m i n i s t e r e d as d e s c r i b e d under General Methods. E s t r a d i o l benzoate was g i v e n 4 8 h and progesterone 4 - 6 h b e f o r e b e h a v i o r a l t e s t i n g . OT and a n g i o t e n s i n I I (both from Sigma) were 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 . Each animal r e c e i v e d a t o t a l of 0.34 ng OT d e l i v e r e d i n 4/zl of v e h i c l e or v e h i c l e o n l y a d m i n i s t e r e d 20-30 minutes b e f o r e b e h a v i o r a l t e s t i n g commenced. The t e s t animals were randomly assigned t o one of f o u r groups of f i v e each. Each group r e c e i v e d , i n a 12 counterbalanced f a s h i o n , e i t h e r OT or s a l i n e t o e i t h e r the l e f t l a t e r a l v e n t r i c l e or the t h i r d v e n t r i c l e i n such a way t h a t each female r e c e i v e d each treatment over the d u r a t i o n of the experiment. A l l groups were t e s t e d on the same day of the week a t weekly i n t e r v a l s . B e h a v i o r a l t e s t i n g B e h a v i o r a l t e s t i n g was conducted as d e s c r i b e d under General Methods. L o r d o s i s d u r a t i o n s were not recorded. R e s u l t s The r e s u l t s , i l l u s t r a t e d i n F i g u r e 1, i n d i c a t e an i n h i b i t i o n of LQ when OT i s i n f u s e d i n t o the v e n t r i c l e s . A r epeated measures 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 s i g n i f i c a n t e f f e c t s (F(3,32) = 4.69, Huynh-Feldt p < 0.05). Tukey's post-hoc comparisons r e v e a l e d a s i g n i f i c a n t i n h i b i t i o n of l o r d o s i s i n response t o OT as compared t o v e h i c l e i n f u s i o n i n t o the l a t e r a l v e n t r i c l e s (p < 0.05). No s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on LQ r e s u l t e d from an OT i n f u s i o n i n t o the t h i r d v e n t r i c l e . D i s c u s s i o n An OT dose of 0.34 ng admi n i s t e r e d t o the l e f t l a t e r a l v e n t r i c l e s i g n i f i c a n t l y reduced the l o r d o s i s q u o t i e n t of 13 F i g u r e 1. The mean l o r d o s i s q u o t i e n t s ± S.E.M. f o r animals i n f u s e d w i t h 0.34 ng of o x y t o c i n and s a l i n e v e h i c l e i n t o the l a t e r a l and t h i r d v e n t r i c l e are shown. A l l animals were primed w i t h 5 jug e s t r a d i o l benzoate and 150 jug progesterone and r e c e i v 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 i n f u s i o n s 20-30 minutes b e f o r e b e h a v i o r a l t e s t i n g . 100.0-1 80.0 FIGURE 1 E^Saline CXI Oxytocin UJ I— O O GO CO O a cc o 60.0 40.0-20.0-0.0 Lateral Third VENTRICULAR INFUSION SITE f e m a l e " r a t s t r e a t e d w i t h e s t r a d i o l b e n z o a t e a n d p r o g e s t e r o n e . A d m i n i s t r a t i o n o f t h e same d o s e t o t h e t h i r d v e n t r i c l e h a d no e f f e c t . The i n h i b i t i o n was t o t a l l y u n e x p e c t e d i n l i g h t o f p r e v i o u s r e s e a r c h f r o m t h i s a n d o t h e r l a b o r a t o r i e s i n d i c a t i n g t h a t OT i n f u s i o n i n t o t h e l a t e r a l v e n t r i c l e f a c i l i t a t e d l o r d o s i s p e r f o r m a n c e i n f e m a l e r a t s ( A r l e t t i a n d B e r t o l i n i 1 985, C a l d w e l l e t a l . 1986, G o r z a l k a a n d L e s t e r 1987, C a l d w e l l e t a l . 1 9 8 9 ) . I n o r d e r t o d e m o n s t r a t e t h a t t h i s i n h i b i t i o n was n o t s i m p l y a f u n c t i o n o f t h e s p e c i f i c d o s e s o f e s t r o g e n a n d p r o g e s t e r o n e , t h e p r e s e n t r e s u l t s w e r e c o n f i r m e d i n 6 a n i m a l s p r i m e d w i t h l o w e r d o s e s o f s t e r o i d s (2 jug e s t r a d i o l b e n z o a t e a n d 100 jug;' p r o g e s t e r o n e ) . The i n h i b i t o r y e f f e c t was s t i l l p r e s e n t : LQ means ± S.E.M. w e r e 2 8 % ± 6.6 f o r t h e 0.34 ng OT i n f u s i o n a n d 6 0 % ± 17.0 f o r t h e s a l i n e c o n t r o l g r o u p ; d e p e n d e n t t -t e s t : t = 2.795, d f = 5, p < 0.035. I n o r d e r t o f u r t h e r e x a m i n e t h i s s u r p r i s i n g f i n d i n g , we d e s i g n e d E x p e r i m e n t 3 t o i n v e s t i g a t e t h e e f f e c t s on l o r d o s i s b e h a v i o r o f v a r i o u s d o s e s o f OT, when i n f u s e d i n t o t h e l e f t l a t e r a l v e n t r i c l e o f f e m a l e r a t s . E x p e r i m e n t 2 I n p r e v i o u s s t u d i e s o f t h e e f f e c t s o f o x y t o c i n on s e x u a l r e c e p t i v i t y , l o r d o s i s f r e q u e n c y was u s e d a s a d e p e n d e n t m e a s u r e ( e . g . A r l e t t i a n d B e r t o l i n i 1 985, G o r z a l k a a n d L e s t e r 1987, C a l d w e l l e t a l . 1989, S c h u m a c h e r e t a l . 1 9 8 9 ) . 16 To d a t e , t h e d u r a t i o n o f t h e l o r d o s i s p o s t u r e h a s n o t b e e n e m p l o y e d , n o r p r o p o s e d , a s a m e a s u r e i n s u c h s t u d i e s . D u r a t i o n may p r o v i d e a more s e n s i t i v e m e a s u r e o f r e c e p t i v i t y t h a n t h e more f r e q u e n t l y u s e d LQ o r may p r o v i d e i n f o r m a t i o n a b o u t s e x u a l b e h a v i o r i n t h e f e m a l e r a t n o t a t t a i n a b l e t h r o u g h t h e u s e o f t h e LQ s c o r e . I n o r d e r t o v a l i d a t e t h e u s e o f l o r d o s i s d u r a t i o n ( L D ) , we d e s i g n e d E x p e r i m e n t 2 t o i n v e s t i g a t e t h e e f f e c t o f d i f f e r e n t d o s e s o f p r o g e s t e r o n e on LD a n d t o c o n t r a s t t h a t w i t h LQ f o r t h e same a n i m a l s . M a t e r i a l s a n d m e t h o d s A n i m a l s a n d s u r g e r y F o r t y f e m a l e S p r a g u e - D a w l e y r a t s w e r e b i l a t e r a l l y o v a r i e c t o m i z e d a s d e s c r i b e d i n t h e G e n e r a l M e t h o d s s e c t i o n . D r u g p r o c e d u r e s E s t r a d i o l b e n z o a t e a n d p r o g e s t e r o n e w e r e d i s s o l v e d i n warm p e a n u t o i l a n d a d m i n i s t e r e d s u b c u t a n e o u s l y i n 0.1 c c o f v e h i c l e . The e x p e r i m e n t a l a n i m a l s w e r e t h e n r a n d o m l y a s s i g n e d t o f o u r g r o u p s o f t e n a n i m a l s e a c h . A l l g r o u p s w e r e t e s t e d on t h e same d a y . A l l a n i m a l s r e c e i v e d 10 /xg e s t r a d i o l b e n z o a t e 48 h b e f o r e b e h a v i o r a l t e s t i n g a n d e i t h e r o i l v e h i c l e , 100 300 jxg o r 500 /xg p r o g e s t e r o n e 4-6 h b e f o r e t e s t i n g . 17 B e h a v i o r a l t e s t i n g B e h a v i o r a l t e s t i n g was conducted as d e s c r i b e d i n the General Methods s e c t i o n . In a d d i t i o n t o l o r d o s i s frequency, the d u r a t i o n of every l o r d o s i s was a l s o recorded and a LD c a l c u l a t e d f o r each animal c o r r e s p o n d i n g t o the average d u r a t i o n . LD was c o n s i d e r e d t o be 0 when a female r a t d i d not show any l o r d o s e s . R e s u l t s and d i s c u s s i o n S t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n s were obtained between progesterone dose and LQ (r = 0.687, B o n f e r r o n i a d j u s t e d p < 0.001), progesterone dose and LD (r = 0.486, B o n f e r r o n i p < 0.01) as w e l l as LQ and LD (r = 0.813, B o n f e r r o n i p < 0.001). F i g u r e 2 d i s p l a y s the LQ and LD v a l u e s f o r the f o u r groups t e s t e d . The h i g h c o r r e l a t i o n between LQ and LD suggests t h a t LD may be as v a l i d a measure of sexual r e c e p t i v i t y i n the female r a t as LQ. However, i t appears t o be somewhat l e s s s e n s i t i v e than LQ t o the e f f e c t s of progesterone, s u g g e s t i n g t h a t i t i s not simply a f u n c t i o n of the LQ measure. The p o s s i b i l i t y e x i s t s t h a t f o r some phar m a c o l o g i c a l treatments, 18 F i g u r e 2. Mean l o r d o s i s f r a c t i o n (LQ/100) ± S.E.M: and mean l o r d o s i s d u r a t i o n ± S.E.M. as a f u n c t i o n of progesterone dose a d m i n i s t e r e d subcutaneously 4-6 h bef o r e t e s t i n g t o female r a t s primed w i t h 10 /xg e s t r a d i o l benzoate 48 h be f o r e t e s t i n g . BEHAVIORAL SCORE 20 LD c o u l d be a more s e n s i t i v e measure than LQ. We t h e r e f o r e d e c i d e d t o g e n e r a l l y assess both measures of female s e x u a l r e c e p t i v i t y . Experiment 3 Experiment 3 was conducted as a dose-response study t o i n v e s t i g a t e the e f f e c t s on l o r d o s i s of v a r i o u s doses of OT when i n f u s e d 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 . M a t e r i a l s and methods Animals and surgery F o r t y animals were o v a r i e c t o m i z e d and cannulated as d e s c r i b e d f o r Experiment 1. Instead of r e c e i v i n g two cannulae, each animal r e c e i v e d a s i n g l e cannula p l a c e d i n the l e f t l a t e r a l v e n t r i c l e . Drug procedures Each animal was primed with 5 /Ltg e s t r a d i o l benzoate and 150 jug progesterone as d e s c r i b e d f o r Experiment 1 and r e c e i v e d an i n f u s i o n of one of the f o l l o w i n g - s a l i n e v e h i c l e , 0.34, 3.4 or 34 ng of OT. I n f u s i o n volumes and r a t e s were as d e s c r i b e d i n Experiment 1. Before every t e s t i n g s e s s i o n , animals were randomly assi g n e d t o one of 21 the f o u r drug treatment groups, i n a manner which r e s u l t e d i n approximately equal groups. B e h a v i o r a l t e s t i n g B e h a v i o r a l t e s t i n g was conducted b l i n d a t weekly i n t e r v a l s a c c o r d i n g t o procedures d e s c r i b e d i n Experiment 1. A microcomputer w i t h custom w r i t t e n software was employed as an event r e c o r d e r and both the l o r d o s i s frequency and the d u r a t i o n of each i n d i v i d u a l l o r d o s i s response were recorded. LQ and LD s c o r e s were c a l c u l a t e d as b e f o r e . Based on procedures used t o c o n f i r m v e n t r i c l e placements d e s c r i b e d i n Experiment 1, the data from some animals were e l i m i n a t e d . T h i s r e s u l t e d i n group s i z e s of 10, 11, 11, and 11 f o r the 0 - 34 ng OT doses, r e s p e c t i v e l y . R e s u l t s I n s p e c t i o n of the data presented i n F i g u r e 3 suggests t h a t OT, when i n f u s e d i n t o the l a t e r a l v e n t r i c l e , had an i n h i b i t o r y e f f e c t on LQ. An a n a l y s i s of v a r i a n c e confirmed t h a t t h e r e was a s i g n i f i c a n t treatment e f f e c t (F(3,39) = 3.96, p < 0.05). A s i g n i f i c a n t i n h i b i t i o n (Dunnett, p < 0.05) of LQ o c c u r r e d i n response t o 0.34 ng OT but not i n response t o 3.4 or 34 ng OT. None of the doses t e s t e d had 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 e f f e c t on LD (means ± S.E.M. were 22 F i g u r e 3. D o s e - r e s p o n s e c u r v e o f t h e e f f e c t s on l o r d o s i s o f o x y t o c i n i n f u s i o n s i n t o t h e l e f t l a t e r a l v e n t r i c l e o f f e m a l e r a t s p r i m e d w i t h 5 jug e s t r a d i o l b e n z o a t e a n d 150 jug p r o g e s t e r o n e i s i l l u s t r a t e d . I n f u s i o n s w e r e p e r f o r m e d 20-30 m i n u t e s b e f o r e b e h a v i o r a l t e s t i n g . Shown a r e t h e mean l o r d o s i s q u o t i e n t s ± S.E.M. FIGURE 3. 24 1.28 ± 0.08, 1.12 ± 0.14, 1.19 ± 0.16 and 1.21 ± 0.11 f o r the 0 - 34 ng doses, r e s p e c t i v e l y ) . D i s c u s s i o n An OT dose of 0.34 ng i n f u s e d i n t o the l a t e r a l v e n t r i c l e s i g n i f i c a n t l y i n h i b i t e d the frequency of l o r d o s i s . T h i s confirmed the r e s u l t s o b t ained i n Experiment 1. There was, however, no i n d i c a t i o n of a f a c i l i t a t i o n . T h i s may have been due t o the r e l a t i v e l y low OT doses used i n Experiment 3 (0.34, 3.4 and 34 ng) as opposed t o those employed by o t h e r s (e.g. 100 and 500 ng) (Ca l d w e l l e t a l . 1989). Although 500 ng OT has been shown t o f a c i l i t a t e l o r d o s i s when i n f u s e d i n t o the l a t e r a l v e n t r i c l e , 100 ng OT produced a non-s i g n i f i c a n t f a c i l i t a t i o n i n the same study ( C a l d w e l l e t a l . 1989). In a f u r t h e r attempt t o demonstrate a f a c i l i t a t o r y OT e f f e c t , we i n f u s e d a h i g h e r dose of OT (340 ng) i n t o the l a t e r a l v e n t r i c l e s of 9 female r a t s primed w i t h 3 /ug e s t r a d i o l - b e n z o a t e and 150 /xg progesterone. T h i s r e s u l t e d i n a LQ of 38% ± 13.0 as compared t o a score f o r the s a l i n e treatment of 42% ± 10.4. These d i f f e r e n c e s 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 . General d i s c u s s i o n The i n f u s i o n of a r e l a t i v e l y low dose of OT (0.34 ng) 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 of female r a t s s i g n i f i c a n t l y 25 a t t e n u a t e d t h e f r e q u e n c y o f l o r d o s i s . The same d o s e when a p p l i e d t o t h e t h i r d v e n t r i c l e was i n e f f e c t i v e . Of a l l t h e OT d o s e s (0.34 - 340 ng) e m p l o y e d , none f a c i l i t a t e d l o r d o s i s when a d m i n i s t e r e d t o t h e l a t e r a l v e n t r i c l e o f e s t r o g e n a n d p r o g e s t e r o n e t r e a t e d a n i m a l s . T h e r e a r e s e v e r a l r e a s o n s why OT i n f u s i o n s i n t o t h e l a t e r a l a n d t h e t h i r d v e n t r i c l e m i g h t be e x p e c t e d t o d i f f e r i n t h e i r e f f e c t s on l o r d o s i s . A l t h o u g h t h e v e n t r i c u l a r s y s t e m o f t h e r a t i s i n t e r c o n n e c t e d a n d c e r e b r o s p i n a l f l u i d i s a c t i v e l y c i r c u l a t e d t h r o u g h t h e s y s t e m , t h e p r o x i m i t i e s o f t h e d i f f e r e n t v e n t r i c l e s t o s p e c i f i c b r a i n s t r u c t u r e s v a r y . I n p a r t i c u l a r , t h e t h i r d v e n t r i c l e i s much c l o s e r t h a n t h e l a t e r a l v e n t r i c l e s t o t h e h y p o t h a l a m u s a n d t h e m e d i a l p r e o p t i c a r e a . The t h i r d v e n t r i c l e i s b o r d e r e d by t h e p a r a v e n t r i c u l a r a n d s u p r a o p t i c n u c l e i w h i c h c o n t a i n OT ( A r m s t r o n g 1 9 8 5 ) . D e n d r i t e s o f p a r a v e n t r i c u l a r n e u r o n s p a s s a l o n g t h i s v e n t r i c l e i n t h e mouse ( C a s t e l a n d M o r r i s 1988) a n d i m m u n o r e a c t i v e v a s o p r e s s i n a n d OT a x o n s p r o j e c t i n t o t h e t h i r d v e n t r i c l e u n d e r some c o n d i t i o n s o f s a l t l o a d i n g ( D e l l m a n n e t a l . 1 9 8 8 ) . The a b o v e c o n s i d e r a t i o n s w o u l d a l s o l e a d one t o e x p e c t i n f u s i o n s o f OT i n t o t h e t h i r d v e n t r i c l e t o be more l i k e l y t o p r o d u c e a n e f f e c t on l o r d o s i s t h a n a s i m i l a r t r e a t m e n t o f t h e l a t e r a l v e n t r i c l e . H o wever, t h e r e s u l t s o f E x p e r i m e n t 1 p r o v e d c o n t r a r y t o t h i s e x p e c t a t i o n . On t h e o t h e r h a n d , d i f f e r e n t i a l v e n t r i c u l a r e f f e c t s o f OT o n l o r d o s i s a r e c o n s i s t e n t w i t h t h e l i t e r a t u r e o h s e r o t o n i n a n d 6-endorphin. I n f u s i o n of 10 jug s e r o t o n i n i n t o the l a t e r a l , but not the t h i r d , v e n t r i c l e of female r a t s f a c i l i t a t e d l o r d o s i s , whereas 100 nq s e r o t o n i n was i n e f f e c t i v e i n e i t h e r v e n t r i c l e (Wilson and Hunter 1985) . A dose of 2 jxg 6-endorphin i n h i b i t e d l o r d o s i s when admi n i s t e r e d i n t o the t h i r d (Wiesner and Moss 1986) but not the l a t e r a l v e n t r i c l e (Pfaus and Gorz a l k a 1987). I t may be t h a t when i n f u s e d i n t o t h e l a t e r a l v e n t r i c l e , OT more r e a d i l y g ains access t o undetermined c e n t r a l s i t e s a s s o c i a t e d with the i n h i b i t i o n of l o r d o s i s . Because known c e n t r a l s i t e s of a c t i o n of OT on l o r d o s i s are f a c i l i t a t o r y ( C a l d w e l l e t a l . 1989, Schumacher e t a l . 1989, Schulze and Gorzalka 1991), t h i s appears t o be the f i r s t evidence s u g g e s t i v e of an i n h i b i t o r y s i t e . I t i s s u r p r i s i n g t h a t OT i n f u s i o n s i n t o the l a t e r a l v e n t r i c l e s hould produce a f a c i l i t a t i o n of l o r d o s i s ( A r l e t t i and B e r t o l i n i 1985, C a l d w e l l e t a l . 1986, Gorzalka and L e s t e r 1987, C a l d w e l l e t a l . 1989) as w e l l as an i n h i b i t i o n (Experiments 1 and 2) depending on the OT dose a d m i n i s t e r e d . T h i s f i n d i n g i s r e m i n i s c e n t of the i n h i b i t o r y e f f e c t on l o r d o s i s of a lower dose of G-endorphin and the f a c i l i t a t o r y e f f e c t of a h i g h e r dose (Pfaus and Gorzalka 1987). There are f o u r p o s s i b i l i t i e s t h a t we wish t o address concerning the o p p o s i t e e f f e c t s of OT on female sexual r e c e p t i v i t y : ( i ) t h a t an i n h i b i t o r y s i t e e x i s t s ; ( i i ) t h a t an OT a u t o r e c e p t o r e x i s t s ; ( i i i ) t h a t OT a t some c o n c e n t r a t i o n s may modulate oth e r p e p t i d e r g i c systems a f f e c t i n g l o r d o s i s and (iv ) t h a t 27 t h e e f f e c t s o f OT o n l o r d o s i s may be d e p e n d e n t on t h e p r o g e s t e r o n e a n d e s t r o g e n d o s e . A s p o i n t e d o u t a b o v e , known c e n t r a l s i t e s o f a c t i o n o f OT on l o r d o s i s a r e f a c i l i t a t o r y ( C a l d w e l l e t a l . 1989, S c h u m a c h e r e t a l . 1989, S c h u l z e a n d G o r z a l k a 1991) a n d t h e i n h i b i t i o n r e p o r t e d h e r e s u g g e s t s t h a t a n i n h i b i t o r y s i t e e x i s t s . A t r e l a t i v e l y l o w d o s e s o f OT, t h e i n h i b i t o r y s i t e may be a c t i v a t e d , w h i l e a t h i g h e r d o s e s OT d i f f u s e s t o f a c i l i t a t o r y s i t e s , t h u s m a s k i n g t h e i n h i b i t i o n . I t seems l i k e l y t h a t t h i s p u t a t i v e i n h i b i t o r y s i t e i s n o t i n t h e i m m e d i a t e v i c i n i t y o f t h e t h i r d v e n t r i c l e . The e x i s t e n c e o f an OT a u t o r e c e p t o r m i g h t e x p l a i n b o t h t h e i n h i b i t i o n a n d t h e f a c i l i t a t i o n s e e n i n r e s p o n s e t o i n f u s i o n s i n t o t h e l a t e r a l v e n t r i c l e s . S u p p o s e t h a t OT f u n c t i o n s t o f a c i l i t a t e s e x u a l r e c e p t i v i t y i n f e m a l e r a t s . A t l o w c o n c e n t r a t i o n s o f OT t h e a u t o r e c e p t o r may be a c t i v a t e d , i n h i b i t i n g t h e f a c i l i t a t o r y e f f e c t s o f OT on l o r d o s i s . I t a p p e a r s t h a t a c t i v a t i o n o f V 2 r e c e p t o r s o n o x y t o c i n e r g i c c e l l s d o w n - r e g u l a t e s OT s e c r e t i o n (Cheng a n d N o r t h 1 9 8 9 ) . F u r t h e r m o r e , OT may b i n d t o t h e s e V 2 r e c e p t o r s ( H r u b y a n d S m i t h 1 9 8 7 ) . I f h i g h l e v e l s o f a c t i v i t y o f OT n e u r o n s a r e n e c e s s a r y f o r t h e e x p r e s s i o n o f l o r d o s i s , t h i s i n h i b i t i o n o f OT s e c r e t i o n w i l l t h e n i n h i b i t l o r d o s i s . A t h i g h e r d o s e s o f OT s u c h a c t i v a t i o n d o e s n o t o c c u r a n d no i n h i b i t i o n w o u l d be s e e n . T h i s i s s u p p o r t e d by e v i d e n c e 28 i n d i c a t i n g t h a t h i g h l e v e l s of a c t i v i t y of OT c e l l s , or the i n f u s i o n of OT i n t o the t h i r d v e n t r i c l e , cause the r e c r u i t m e n t of other c e l l s ( B e l i n and Moos 1986) p o s s i b l y through the l o c a l r e l e a s e of OT (Mason e t a l . 1986). T h e r e f o r e , h i g h doses of OT may t r i g g e r such r e c r u i t m e n t r e s u l t i n g i n a f a c i l i t a t i o n of l o r d o s i s . Thus v a s o p r e s s i n V2 r e c e p t o r s l o c a t e d on OT neurons may f u n c t i o n as OT a u t o r e c e p t o r s . A l t e r n a t i v e l y , i t i s q u i t e p o s s i b l e t h a t OT normally i n h i b i t s mating i n the female r a t and t h a t lower (and more p h y s i o l o g i c a l ) doses of OT when a p p l i e d c e n t r a l l y r e f l e c t t h i s . At h i g h e r doses of OT, the a u t o r e c e p t o r s would become a c t i v a t e d and shut the i n h i b i t o r y system down, r e s u l t i n g i n a f a c i l i t a t i o n of sexual r e c e p t i v i t y . The a c t i v a t i o n of other p e p t i d e r g i c systems p r o v i d e s another e x p l a n a t i o n f o r the dua l e f f e c t of OT. Although a t t h i s stage o n l y a s i n g l e c e n t r a l OT r e c e p t o r - t y p e i s known t o e x i s t , OT a l s o binds t o v a s o p r e s s i n V3 r e c e p t o r s (Antoni 1987) and oth e r v a s o p r e s s i n r e c e p t o r s (Hruby and Smith 1987). T h i s r a i s e s the p o s s i b i l i t y t h a t OT may bin d t o y e t othe r types of r e c e p t o r s . The d i f f e r e n t e f f e c t s of OT on l o r d o s i s may be mediated through a c t i v a t i o n of d i f f e r e n t types of r e c e p t o r s i n a manner analogous t o the i n h i b i t o r y and e x c i t a t o r y s e r o t o n e r g i c r e c e p t o r subtypes r e g u l a t i n g male and female s e x u a l behavior (Gorzalka e t a l . 1990). I t i s c o n c e i v a b l e , f o r example, t h a t the p r o l y l - l e u c y l -g l y c i n a m i d e fragment of the o x y t o c i n molecule a c t i v a t e s a-29 f a c i l i t a t o r y s i t e (Gorzalka et a l . 1991) whereas another fragment of the o x y t o c i n molecule might, depending on the b r a i n s i t e and s t e r o i d m i l i e u , a c t i v a t e an i n h i b i t o r y r e c e p t o r . Moreover, a t c e r t a i n c o n c e n t r a t i o n s , OT may be a c t i n g on the r e c e p t o r s of other p e p t i d e s t o i n v o l v e these p e p t i d e r g i c systems i n the e x p r e s s i o n of l o r d o s i s . Recent work by C a l d w e l l e t a l . (1990) i n v o l v i n g the v a s o p r e s s i n a n t a g o n i s t [d(CH2)5 Tyr(Me)]-AVP i n d i c a t e s t h a t b l o c k i n g a c t i v i t y a t v a s o p r e s s i n V]_ r e c e p t o r s may f a c i l i t a t e l o r d o s i s through the l i f t i n g of a t o n i c i n h i b i t i o n . However, the same a n t a g o n i s t used by these authors has been shown t o a c t as a p a r t i a l V3 a g o n i s t (Antoni 1987). T h e r e f o r e i t remains p o s s i b l e t h a t the e f f e c t s of t h i s a n t a g o n i s t on l o r d o s i s behavior are due t o i t s a c t i o n a t V3 r e c e p t o r s . P i t u i t a r y V3 r e c e p t o r s mediate the r e l e a s e of a d r e n o c o r t i c o t r o p i c hormone which, when p e r i p h e r a l l y administered, i n c r e a s e s l o r d o s i s (deCatanzaro e t a l . 1981). The f a c i l i t a t i o n seen w i t h OT i n some s t u d i e s may p a r t i a l l y r e f l e c t the e f f e c t s of a d r e n o c o r t i c a l a c t i v a t i o n . The e f f e c t s of OT on other p e p t i d e r g i c systems i m p l i c a t e d i n l o r d o s i s behavior may i n v o l v e , i n a d d i t i o n t o the a c t i v a t i o n of d i f f e r e n t r e c e p t o r s , other mechanisms as w e l l . For i n s t a n c e , OT i s c o n s i d e r e d t o be a s t r e s s hormone and i s r e l e a s e d i n response t o v a r i o u s s t r e s s o r s : h y p e r o s m o l a l i t y ( F o r s l i n g and Brimble 1985), immersion i n water (Lang e t a l . 1983), and i m m o b i l i z a t i o n (Lang e t a l . 1983, Gibbs 1984). In 30 agreement w i t h t h i s view, i t has been shown t h a t OT and v a s o p r e s s i n f u n c t i o n as c o r t i c o t r o p i c r e l e a s i n g f a c t o r s (CRF) (Antoni 1987), r e s u l t i n g i n i n c r e a s e d ACTH l e v e l s . Moreover, i n some b r a i n areas, CRF and OT are c o l o c a l i z e d (see Sawchenko and Swanson 1985 f o r d i s c u s s i o n ) and one may expect them t o be r e l e a s e d c o ncomitantly. Both CRF ( S i r i n a t h s i n g h j i 1985) and ACTH (deCatanzaro e t a l . 1981), however, c e n t r a l l y i n h i b i t l o r d o s i s i n female r a t s . In a d d i t i o n , OT r e l e a s e s p r o l a c t i n c e n t r a l l y (Arey and Freeman 1989) and p r o l a c t i n i n t u r n r e l e a s e s OT (Sarkar 1989). C h r o n i c h y p e r p r o l a c t i n e m i a and the i n t r a v e n t r i c u l a r i n f u s i o n of p r o l a c t i n i n h i b i t l o r d o s i s behavior i n female r a t s (Dudley e t a l . 1982). T h i s r a i s e s the p o s s i b i l i t y t h a t OT f a c i l i t a t e s the d i r e c t a c t i v a t i o n of f u n c t i o n a l o x y t o c i n e r g i c b r a i n s i t e s (e. g. C a l d w e l l e t a l . 1989, Schumacher et a l . 1989, Schulze and Gorzalka 1991), but may i n h i b i t l o r d o s i s behavior i n d i r e c t l y through the a c t i v a t i o n of o t h e r p e p t i d e r g i c systems. The i n d i r e c t e f f e c t s of OT may be m a n i f e s t o n l y a t r e l a t i v e l y low c o n c e n t r a t i o n s s i n c e h i g h c o n c e n t r a t i o n s , r e s u l t i n g i n the a c t i v a t i o n of f a c i l i t a t o r y o x y t o c i n e r g i c systems, may o v e r r i d e them. Other p o s s i b l e f a c t o r s c o n t r i b u t i n g t o an i n h i b i t o r y e f f e c t of o x y t o c i n i n f u s i o n s i n t o the l a t e r a l v e n t r i c l e are the dose and d u r a t i o n of estrogen treatment and the presence or absence of progesterone. In p r iming regimens where an acute dose of estrogen was f o l l o w e d by an acute dose of 31 progesterone, OT f a c i l i t a t e d l o r d o s i s when the EB dose was 10 ng ( A r l e t t i and B e r t o l i n i 1985, G orzalka and L e s t e r 1987) but i n h i b i t e d l o r d o s i s when the EB dose was 2-5 /xg (Experiments 1 and 3). Moreover, i n a p r i m i n g regimen where progesterone was absent and even lower doses of EB were a d m i n i s t e r e d c h r o n i c a l l y f o r t h r e e days, OT was f a c i l i t a t o r y ( C a l d w e l l e t a l . 1986, C a l d w e l l e t a l . 1989). T h e r e f o r e , the o b s e r v a t i o n of both a f a c i l i t a t i o n and an i n h i b i t i o n i n animals primed with i d e n t i c a l s t e r o i d doses may be u n l i k e l y . T h i s c o u l d e x p l a i n our f a i l u r e t o see a f a c i l i t a t i o n of l o r d o s i s w i t h 340 ng OT i n f u s e d i n t o the l a t e r a l v e n t r i c l e s of female r a t s . Now t h a t an i n h i b i t o r y e f f e c t of a low dose of OT has been demonstrated, f u r t h e r i n v e s t i g a t i o n s are needed t o r e v e a l the p o s s i b l e i n t e r a c t i o n s between OT dose, e s t r o g e n dose and progesterone. SECTION II The a d m i n i s t r a t i o n of o x y t o c i n t o b r a i n r e g i o n s t h a t show i n c r e a s e s i n the number of o x y t o c i n r e c e p t o r s i n response t o estrogen treatment. The MPOA ( L i s k e t a l . 1972, Bast 1987, C a l d w e l l e t a l . 1989, C a l d w e l l e t a l . 1990), VMH ( L i s k 1962, Doerner e t a l . 1968, L i s k e t a l . 1972, N a p o l i e t a l . 1972, Mathews and Edwards 1977, P f a f f and Sakuma 1979a, P f a f f and Sakuma 1979b, G l a s e r e t a l . 1987, Schumacher et a l . 1989, 32 Schumacher e t a l . 1990), mesencephalic c e n t r a l grey (MCG) (Sakuma and P f a f f 1979a, Sakuma and P f a f f 1979b, Arendash and G o r s k i 1983, Harl a n e t a l . 1983, R o t h f e l d e t a l . 1986) and amygdala ( L i s k and B a r f i e l d 1975) are a l l s i t e s t h a t have been shown t o conce n t r a t e r a d i o l a b e l l e d e s t r a d i o l and t o e x e r t c o n t r o l over l o r d o s i s , the major index of se x u a l r e c e p t i v i t y . Some of the e f f e c t s of estrogen on l o r d o s i s may i n v o l v e i n t e r a c t i o n w i t h OT. For i n s t a n c e , e s t r a d i o l i n f l u e n c e s OT immunoreactivity i n the MPOA (Ca l d w e l l et a l . 1988), OT immunoreactive l e v e l s i n the MPOA of r a t s t r e a t e d w i t h e s t r a d i o l and progesterone are i n c r e a s e d as a r e s u l t of mounting and the number of c e l l bodies s t a i n e d f o r OT are decreased i n the same animals ( C a l d w e l l e t a l . 1989) -perhaps i n d i c a t i n g i n c r e a s e d OT r e l e a s e . A l s o , OT when ad m i n i s t e r e d t o t h i s nucleus f a c i l i t a t e s l o r d o s i s i n female r a t s t r e a t e d c h r o n i c a l l y with estrogen ( C a l d w e l l e t a l . 1989). In the VMH e s t r a d i o l i n c r e a s e s the number of OT r e c e p t o r s when c h r o n i c a l l y administered (de K l o e t e t a l . 1986, Johnson e t a l . 1989) and i n c r e a s e s l e v e l s of OT mRNA ( M i l l e r e t a l . 1989). Estrogen treatment a l s o m o d i f i e s the p a t t e r n of c e n t r a l OT i n n e r v a t i o n (Rhodes e t a l . 1981, J i r i k o w s k i e t a l . 1988, C a l d w e l l e t a l . 1988) and furthermore, OT l e v e l s vary i n the p a r a v e n t r i c u l a r nucleus as a f u n c t i o n of the es t r o u s c y c l e (Greer e t a l . 1986). The MCG i s an area t h a t may be a f f e c t e d by changes i n the OT 33 i n n e r v a t i o n i n response t o the presence of estrogen. I t i s known t o r e c e i v e p r o j e c t i o n s from the VMH (see And r e z i k and B e i t z 1985) and OT f i b e r s from the p a r a v e n t r i c u l a r nucleus ( B u i j s e t a l . 1985). Although progesterone i n t e r a c t s s y n e r g i s t i c a l l y w i t h e s t r o g e n f o r the f u l l e x p r e s s i o n of sexual behavior ( B o l i n g and Blandau 1939), some c o n t r o v e r s y e x i s t s as t o whether the OT f a c i l i t a t i o n of l o r d o s i s behavior i s progesterone dependent. Supporting evidence f o r dependence i n d i c a t e s t h a t progesterone a d m i n i s t r a t i o n i n c r e a s e s l e v e l s of immunoreactive OT i n the MPOA and i n f i b e r s l a t e r a l t o the VMH and decreases them elsewhere ( C a l d w e l l e t a l . 1986, C a l d w e l l e t a l . 1988). In agreement with t h i s evidence, dependence has been demonstrated i n s e v e r a l s t u d i e s of sex u a l r e c e p t i v i t y ( A r l e t t i and B e r t o l i n i 1985, Gorz a l k a and L e s t e r 1987). Such s t u d i e s suggest t h a t the mechanism of a c t i o n o f OT l i k e l y i n v o l v e s more than mimicking the e f f e c t s of progesterone. In a r e c e n t study C a l d w e l l et a l . (1989) found t h a t OT i n f u s i o n s i n t o the MPOA and l a t e r a l v e n t r i c l e s (LV), but not i n t o the VMH, MCG, and v e n t r a l tegmental area (VTA), f a c i l i t a t e d l o r d o s i s i n female r a t s t r e a t e d c h r o n i c a l l y w i t h e s t r a d i o l benzoate. However, i n another study p u b l i s h e d a t the same time, i t was found t h a t progesterone i n c r e a s e d the d i s p e r s i o n of OT r e c e p t o r s i n the VMH and t h a t OT i n f u s i o n s 34 i n t o t he VMH enhanced the l o r d o s i s q u a l i t y , but appeared t o have l e s s of an e f f e c t on the l o r d o s i s q u o t i e n t (LQ), a measure o f l o r d o s i s frequency, i n r a t s t r e a t e d w i t h e s t r a d i o l benzoate and progesterone (Schumacher et a l . 1989). S i n c e n a t u r a l e s t r u s i n v o l v e s both of the o v a r i a n s t e r o i d s , and C a l d w e l l e t a l ' s (1989) i n v e s t i g a t i o n of OT a d m i n i s t r a t i o n t o the MPOA, VMH, LV, VTA and MCG employed r a t s not t r e a t e d with progesterone, we i n v e s t i g a t e d the/" e f f e c t s of OT i n f u s i o n s i n t o the MPOA, VMH and MCG i n animals t r e a t e d with both e s t r a d i o l benzoate and progesterone. T h i s r a t i o n a l e i s a l l the more co m p e l l i n g g i v e n t h a t progesterone can s t r o n g l y i n f l u e n c e the e f f e c t s of o t h e r b e h a v i o r a l l y a c t i v e substances. For example, some s e l e c t i v e s e r o t o n i n r e c e p t o r a g o n i s t s are f a c i l i t a t o r y i n the presence of estrogen and i n h i b i t o r y i n the presence of both e s t r o g e n and progesterone (Gorzalka e t a l . 1990). In the s t u d i e s by C a l d w e l l e t a l . (1989) and Schumacher e t a l . (1989), r e l a t i v e l y h i g h doses of OT were i n f u s e d c e n t r a l l y . In l i g h t of the prec e d i n g d i s c u s s i o n , we expected progesterone t o have a pe r m i s s i v e or f a c i l i t a t i v e a c t i o n on the e f f e c t of c e n t r a l OT i n f u s i o n s on l o r d o s i s and consequently examined the e f f e c t s of lower OT doses c o v e r i n g f o u r o r d e r s of magnitude. F i n a l l y , the two c i t e d s t u d i e s ( C a l d w e l l e t a l . 1989, Schumacher e t a l . 1989), taken t o g e t h e r , suggest t h a t o x y t o c i n e r g i c s t i m u l a t i o n of the MPOA may have p r i m a r i l y an e f f e c t on l o r d o s i s frequency w h i l e the VMH may be i n t e g r a l t o other aspects of the l o r d o s i s 35 p o s t u r e . In order t o f u r t h e r i n v e s t i g a t e t h i s p o s s i b i l i t y , we measured both l o r d o s i s d u r a t i o n (LD) and l o r d o s i s frequency i n the pr e s e n t study. Experiment 4 T h i s study was designed t o measure the e f f e c t s on the LQ and LD of v a r i o u s doses of OT admi n i s t e r e d t o the MPOA, VMH and MCG of female r a t s primed with e s t r a d i o l benzoate and progesterone. M a t e r i a l s and methods Animals and surgery Female Sprague-Dawley r a t s were o v a r i e c t o m i z e d and cannul a t e d as d e s c r i b e d under General Methods. F o l l o w i n g s u r g e r i e s , animals were r e t u r n e d t o t h e i r home cages. As d e s c r i b e d above, female r a t s were o v a r i e c t o m i z e d and r e c e i v e d cannulae b i l a t e r a l l y aimed a t the medial p r e o p t i c area (n = 2 0 ) , ventromedial hypothalamus (n = 20) and mesencephalic c e n t r a l grey (n = 2 0 ) . Each group was then randomly s u b d i v i d e d i n t o f i v e groups of f o u r animals each. A l l subgroups w i t h implants i n the same area were t e s t e d on the same day a t weekly i n t e r v a l s u s i n g a counterbalanced d e s i g n where one of fo u r d i f f e r e n t c o n c e n t r a t i o n s of OT or s a l i n e v e h i c l e was i n f u s e d i n t r a c r a n i a l l y . The accuracy of a l l p l a c e m e n t s was v e r i f i e d t h r o u g h t h e u s e o f h i s t o l o g i c a l p r o c e d u r e s a n d o n l y t h e d a t a f r o m a n i m a l s w i t h c o r r e c t c a n n u l a p l a c e m e n t s w e r e r e t a i n e d f o r s t a t i s t i c a l a n a l y s e s . D r u g p r o c e d u r e s A l l e x p e r i m e n t a l a n i m a l s w e r e g i v e n 3 jug o f EB a n d 150 ixg o f p r o g e s t e r o n e d i s s o l v e d i n p e a n u t o i l v e h i c l e a s d e s c r i b e d e a r l i e r . OT (Sigma) 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 i n f o u r d i f f e r e n t c o n c e n t r a t i o n s , 0.085, 0.85, 8.5 a n d 85 n g / j u l . A l l OT i n f u s i o n s w e r e a d m i n i s t e r e d a s d e s c r i b e d u n d e r G e n e r a l M e t h o d s . The MPOA, VMH a n d MCG i m p l a n t e d a n i m a l s w e r e b i l a t e r a l l y i n f u s e d w i t h a t o t a l o f 1 / i l o f s o l u t i o n p e r s i d e p e r t e s t i n g s e s s i o n . I n f u s i o n s w e r e p e r f o r m e d 3 0 m i n u t e s b e f o r e t e s t i n g s t a r t e d . B e h a v i o r a l t e s t i n g L o r d o s i s b e h a v i o r was r e c o r d e d a s d e s c r i b e d i n E x p e r i m e n t 2 a n d t h e LQ a n d LD a c c o r d i n g l y c a l c u l a t e d f o r t h e MPOA a n d VMH. L o r d o s i s f r e q u e n c y s c o r e s f o r t h e MCG w e r e t a k e n m a n u a l l y a n d t h e LQ was c a l c u l a t e d a s d e s c r i b e d i n E x p e r i m e n t 2. A l l t e s t i n g was c o n d u c t e d b l i n d . 37 R e s u l t s MPOA R e l a t i v e t o s a l i n e , an OT dose of 8.5 ng s i g n i f i c a n t l y (p < 0.01) f a c i l i t a t e d l o r d o s i s behavior as expressed by LQ sco r e s when i n f u s e d i n t o the MPOA (Wilcoxon s i g n e d ranks t e s t , z = 2.923). The other OT doses d i d not produce a s i g n i f i c a n t e f f e c t when compared t o the c o n t r o l c o n d i t i o n . r The same OT dose of 8.5 ng i n f u s e d i n t o the MPOA of EB and progesterone primed female r a t s s i g n i f i c a n t l y (p < 0.05) extended the d u r a t i o n of l o r d o s i s (Wilcoxon s i g n e d ranks t e s t , z = -2.261) compared t o the s a l i n e c o n t r o l v a l u e s . No othe r OT dose produced a s i g n i f i c a n t e f f e c t when compared t o the c o n t r o l c o n d i t i o n . VMH None of the OT doses caused a s i g n i f i c a n t i n c r e a s e i n LQ sc o r e s v i s - a - v i s the c o n t r o l i n f u s i o n s u s i n g the Wilcoxon s i g n e d ranks t e s t . However, a l l d u r a t i o n s were s i g n i f i c a n t l y extended: the 0.085 ng (z = 2.380, p < 0.05), the 0.85 ng (z = 1.960, p < 0.05), the 8.5 ng (z = 2.033, p < 0.05) and the 85 ng (z = 2.240, p < 0.05) OT doses produced i n c r e a s e s i n l o r d o s i s d u r a t i o n compared t o the c o n t r o l i n f u s i o n s . 38 MCG None of the OT doses i n f u s e d i n t o the MCG proved t o have a s i g n i f i c a n t e f f e c t on LQs. These r e s u l t s are re p r e s e n t e d i n F i g u r e 4. VMH ver s u s MPOA I t i s e v i d e n t from the pre c e d i n g r e s u l t s t h a t the e f f e c t of OT i n f u s i o n s i n t o the VMH on LD i s more dramatic than when i n f u s e d i n t o the MPOA. When combining a l l the d u r a t i o n s c o r e s o f a l l the OT treatments f o r the VMH and the MPOA, the mean l o r d o s i s d u r a t i o n + S.D. sco r e s f o r the two s i t e s were 0.928 + 0.238 and 0.752 + 0.356 r e s p e c t i v e l y . A Mann-Whitney U t e s t i n d i c a t e d t h a t the i n f u s i o n of OT i n t o the VMH was s i g n i f i c a n t l y (p < 0.05) more e f f e c t i v e i n i n c r e a s i n g LDs than OT i n f u s i o n s i n t o the MPOA. In c o n t r a s t , t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n LQ v a l u e s f o r the VMH ( a l l doses) and the MPOA ( a l l doses). General d i s c u s s i o n I n f u s i o n s of o x y t o c i n i n t o the VMH and MPOA were e f f e c t i v e i n i n c r e a s i n g the l o r d o s i s q u o t i e n t and the l o r d o s i s d u r a t i o n of female r a t s t r e a t e d w i t h both e s t r a d i o l benzoate and progesterone. None of the o x y t o c i n doses used 39 F i g u r e 4. The top two panels show the mean l o r d o s i s f r a c t i o n ( l o r d o s i s quotient/100) ± S.E.M. and mean l o r d o s i s d u r a t i o n ± S.E.M. as a f u n c t i o n of o x y t o c i n dose i n f u s e d i n 1 jul of v e h i c l e i n t o the medial p r e o p t i c area (MPOA - top panel) and the v e n t r o m e d i a l hypothalamus (VMH - middle panel) of female r a t s primed 48 h bef o r e t e s t i n g with 3 ug e s t r a d i o l benzoate and 4-6 h b e f o r e t e s t i n g with 150 jug progesterone. The bottom pan e l shows the mean l o r d o s i s f r a c t i o n ± S.E.M. f o l l o w i n g o x y t o c i n i n f u s i o n i n t o the mesencepahlic c e n t r a l grey (MCG). 40 41 had an e f f e c t on LQ when i n f u s e d i n t o the MCG. Furthermore, t h e VMH was more s e n s i t i v e t o the e f f e c t s of o x y t o c i n on LD than the MPOA. These r e s u l t s were o b t a i n e d u s i n g lower OT doses (0.085 - 85 ng) than those used by C a l d w e l l e t a l . (1989) (100 and 500 ng) and Schumacher e t a l . (1989) (100 ng) . C o n s i s t e n t w i t h the f i n d i n g s of C a l d w e l l e t a l . (1989), we observed t h a t OT i n f u s i o n i n t o the MPOA f a c i l i t a t e d s e x u a l r e c e p t i v i t y i n the female r a t and t h a t a lower dose of OT was more e f f e c t i v e than the h i g h e s t OT dose used. Our f i n d i n g s r e v e a l a f a c i l i t a t i o n a t a r a t h e r lower OT dose (8.5 ng) than t h e i r lowest dose (100 ng). In view of the f a c t t h a t i n our experiment the 85 ng OT dose f a i l e d t o produce an e f f e c t w h i l e t h e i r 100 ng dose d i d , i t appears t h a t one of the a c t i o n s of progesterone may be t o s h i f t the dose response curve toward the lower c o n c e n t r a t i o n s . Perhaps progesterone i n c r e a s e s the s e n s i t i v i t y of the MPOA o x y t o c i n system p e r m i t t i n g a b e h a v i o r a l e f f e c t of lower doses of OT. We a l s o found an e f f e c t of OT when ad m i n i s t e r e d t o the VMH i n EB and progesterone primed female r a t s , a r e s u l t which d i f f e r s w i t h t h a t of C a l d w e l l e t a l . (1989) - perhaps due t o the f a c t t h a t t h e i r animals r e c e i v e d no progesterone. Examination of F i g u r e 4 suggests t h a t a t the h i g h e r doses, OT f a c i l i t a t e d l o r d o s i s when i n f u s e d i n t o the VMH. Because t h e 8.5 and 85 ng groups d i d not d i f f e r s i g n i f i c a n t l y from 42 each ot h e r ( p a i r e d samples t - t e s t , t = 0.084, df = 7, p = 0.935) these two groups were combined. T h e i r combined mean was s i g n i f i c a n t l y e l e v a t e d with r e s p e c t t o the c o n t r o l i n f u s i o n ( o n e - t a i l e d independent t - t e s t : pooled v a r i a n c e s t = -2.011, df = 22, p < 0.05; separate v a r i a n c e s t = -1.858, df = 11.6, p < 0.05) i n d i c a t i n g a f a c i l i t a t o r y e f f e c t of OT i n the VMH on the LQ. Examination of F i g u r e 4 f u r t h e r r e v e a l s t h a t a t the h i g h e r doses OT seemed t o e s t a b l i s h a p l a t e a u by e l e v a t i n g LQs by about 27%. I t i s i n t e r e s t i n g t o s p e c u l a t e why t h i s s hould happen a t submaximal l e v e l s . One p o s s i b i l i t y i s t h a t the spread of OT r e c e p t o r s induced by progesterone i n t h i s b r a i n area (Schumacher e t a l . 1989) i s p r o p o r t i o n a l t o the amount of progesterone admi n i s t e r e d . I f one f u r t h e r p o s t u l a t e s t h a t the LQ e x h i b i t e d by a p a r t i c u l a r animal i s a l s o p r o p o r t i o n a l t o the spread of OT r e c e p t o r s induced (which c o u l d make more OT r e c e p t o r s a c c e s s i b l e t o m o l e c u l e s ) , a p o s s i b l e mechanism t o account f o r t h i s e f f e c t can be e s t a b l i s h e d b e a r i n g i n mind t h a t a submaximal dose of progesterone (150 /ig/ animal) was g i v e n . I t i s a l s o p o s s i b l e t h a t the s m a l l EB dose (3 /zg/ animal) l e a d t o the s y n t h e s i s of a submaximal number of OT r e c e p t o r s and t h a t they were f u l l y spread when g i v e n the c u r r e n t progesterone dose. The c e i l i n g e f f e c t thus may r e f l e c t the s m a l l e r number of a c c e s s i b l e or a v a i l a b l e OT r e c e p t o r s . I t would t h e r e f o r e 43 seem worthwhile t o examine the e f f e c t s of i n t r a c r a n i a l OT a t a v a r i e t y of estrogen and progesterone doses. In t he MCG none of the OT doses t e s t e d had an e f f e c t on the LQ. T h i s i s c o n s i s t e n t w i t h the f i n d i n g s i n animals t r e a t e d w i t h estrogen o n l y ( C a l d w e l l e t a l . 1989). I t seems l i k e l y t h a t the r o l e of the MCG i n l o r d o s i s i s independent of an OT mechanism. OT, when i n f u s e d i n t o the VMH and MPOA, i n c r e a s e d the d u r a t i o n of l o r d o s i s r e l a t i v e t o v e h i c l e i n f u s i o n s . Of these two s i t e s , the VMH appeared t o be the most s e n s i t i v e t o the e f f e c t s of OT i n f u s i o n on l o r d o s i s d u r a t i o n . I f the LD i s i n some way r e l a t e d t o l o r d o s i s q u a l i t y , then an i n c r e a s e i n LD due t o i n f u s i o n s i n t o the VMH supports the f i n d i n g of Schumacher e t a l . (1989). Schumacher e t a l . (1989) found a s i g n i f i c a n t e f f e c t of OT on l o r d o s i s q u a l i t y but d i d not r e p o r t a s i g n i f i c a n t i n c r e a s e i n LQ f o l l o w i n g i n f u s i o n i n t o the VMH. Furthermore, OT i n f u s i o n s a t the s i n g l e most e f f e c t i v e dose p r e c i p i t a t e d a much more robust e f f e c t on LQ sc o r e s i n the MPOA (p < 0.01) than i n the VMH (p < 0.1). T h i s supports the p o s i t i o n of C a l d w e l l e t a l . (1989) t h a t the MPOA i s the most s e n s i t i v e b r a i n area f o r the f a c i l i t a t o r y e f f e c t s of OT on LQ. We suggest, i n the l i g h t of the f i n d i n g s of Schumacher et a l . (1989), C a l d w e l l e t a l . (1989) and those presented here, t h a t the MPOA and VMH d i f f e r e n t i a l l y c o n t r o l a spects of the l o r d o s i s p o s t u r e -44 more s p e c i f i c a l l y - t h a t the MPOA may predominantly c o n t r o l the frequency of l o r d o s i s e x p r e s s i o n w h i l e the VMH c o n t r o l s l o r d o s i s q u a l i t y and d u r a t i o n . An examination of the data i n F i g u r e 4 suggests t h a t a c t i v i t y i n the MPOA a s s o c i a t e d with i n c r e a s e d l o r d o s i s frequency i s more l i k e l y t o be p h a s i c , t h a t i s , t o occur when a s p e c i f i c ' s t a t e ' of OT neu r o t r a n s m i s s i o n (here r e f l e c t e d by the OT dose) has been reached. In c o n t r a s t , i t appears t h a t the VMH may p r o v i d e a more t o n i c s i g n a l , t h a t i s , once t h i s nucleus i s a c t i v a t e d t o cause an i n c r e a s e i n l o r d o s i s d u r a t i o n , i t tends t o remain a c t i v a t e d . The h i e r a r c h i c a l r e l a t i o n s h i p s between these two n u c l e i i n e x e r t i n g c o n t r o l over l o r d o s i s are not c l e a r a t t h i s stage. Given the f a c t t h a t LD i s not merely l i n e a r l y r e l a t e d t o LQ, t h e r e are a t l e a s t t h r e e b a s i c p o s s i b l e r e l a t i o n s h i p s between the MPOA and the VMH. These are: (i) t h a t both independently p r o v i d e s i g n a l s t o a t h i r d s i t e , e.g. the MCG, where the incoming s i g n a l s are i n t e g r a t e d ; ( i i ) t h a t a s i g n a l o r i g i n a t e s i n the MPOA, i s passed on t o the VMH where d u r a t i o n i n f o r m a t i o n i s i n c o r p o r a t e d i n t o the s i g n a l b e f o r e b e i n g r e l a y e d f u r t h e r and ( i i i ) t h a t a s i g n a l o r i g i n a t e s i n the VMH c o n t a i n i n g d u r a t i o n i n f o r m a t i o n , i s passed along t o the MPOA which gates or superimposes frequency i n f o r m a t i o n on the s i g n a l b e f o r e being r e l a y e d downstream. These r e l a t i o n s h i p s remain t o be t e s t e d . 45 I t appears t h a t OT when a p p l i e d t o the MPOA i n c r e a s e s LQ sc o r e s i n e s t r o g e n - t r e a t e d r a t s whether progesterone i s pr e s e n t (Experiment 4) or not (Ca l d w e l l e t a l . 1989). By c o n t r a s t , i t appears t h a t the e f f e c t s of OT i n the VMH vary w i t h progesterone treatment. In the absence of progesterone, OT f a i l s t o i n c r e a s e LQ (C a l d w e l l e t a l . 1989, Schumacher e t a l . 1989) or l o r d o s i s q u a l i t y (Schumacher e t a l . 1989). In the presence of progesterone, OT i n f u s e d i n t o the VMH i n c r e a s e s LQ (Experiment 4), LD (Experiment 4) and l o r d o s i s q u a l i t y s c o r e s (Schumacher e t a l . 1989). Although C a l d w e l l e t a l . (1989) observed a f a c i l i t a t i o n of l o r d o s i s i n the absence of exogenous progesterone f o l l o w i n g i n f u s i o n of OT i n t o the MPOA and l a t e r a l v e n t r i c l e s , a s i g n i f i c a n t e f f e c t was f i r s t observed on the f o u r t h repeated t e s t , the f o u r t h t e s t o c c u r r i n g approximately 90 minutes a f t e r the f i r s t . I t should be noted t h a t repeated t e s t s o c c u r r i n g a t s h o r t i n t e r v a l s are l i k e l y t o i n c r e a s e the r e l e a s e of adren a l s t e r o i d s , i n c l u d i n g progesterone. In ovar i e c t o m i z e d , e s t r o g e n - t r e a t e d animals, progesterone o r i g i n a t i n g i n the adre n a l s may have b e h a v i o r a l consequences (Whalen e t a l . 1975). Furthermore, repeated t e s t i n g causes an i n c r e a s e i n r e c e p t i v i t y which appears t o be a d r e n a l l y mediated (Larsson e t a l . 1974). C o n s i s t e n t with t h i s i n t e r p r e t a t i o n are the f i n d i n g s of Gorz a l k a and L e s t e r (1987) i n d i c a t i n g t h a t i n the absence of repeated t e s t s , OT 46 i n f u s e d i n t o the l a t e r a l v e n t r i c l e s of estrogen-primed r a t s was i n e f f e c t i v e i n f a c i l i t a t i n g s e x u a l r e c e p t i v i t y , except when progesterone was p r e s e n t . I t would t h e r e f o r e seem reasonable, i n s t u d i e s employing a repeated t e s t paradigm, t o c o n s i d e r a p o s s i b l e r o l e f o r a d r e n a l s t e r o i d s . i The amygdala i s a l s o an e s t r o g e n - c o n c e n t r a t i n g r e g i o n ( P f a f f and K e i n e r 1973), s e x u a l l y dimorphic (Meany e t a l . 1985), responds d i f f e r e n t l y t o t e s t o s t e r o n e p r o p i o n a t e i n male and female neonatal r a t s (Takeshita e t a l . 1987), shows e s t r o u s c y c l e v a r i a t i o n s i n p e p t i d e content ( F r a n k f u r t e t a l . 1986) and has been shown t o a f f e c t l o r d o s i s ( L i s k and B a r f i e l d 1975, McGinnis e t a l . 1985). In response t o estrogen a d m i n i s t r a t i o n , a t w o - f o l d i n c r e a s e i n OT r e c e p t o r s are seen i n the c e n t r a l nucleus of the amygdala (ACE) (de K l o e t e t a l . 1986). Furthermore, OT f i b e r s p r o j e c t t o the amygdala ( B u i j s e t a l . 1985). I t i s p o s s i b l e then t h a t the amygdala c o n t r i b u t e s t o l o r d o s i s b ehavior v i a a c t i v a t i o n of the o x y t o c i n e r g i c system. The e f f e c t s on l o r d o s i s of v a r i o u s doses of OT i n f u s e d i n t o the ACE of e s t r o g e n and progesterone primed r a t s were i n v e s t i g a t e d i n Experiment 5. 47 Experiment 5 The e f f e c t s on l o r d o s i s behavior of the a d m i n i s t r a t i o n of v a r i o u s doses of OT i n t o the c e n t r a l nucleus of the amygdala i n female r a t s primed with estrogen and progesterone. M a t e r i a l s and methods Another s e t of 2 0 Sprague-Dawley female r a t s were used i n Experiment 5. A l l s u r g i c a l and drug procedures were e x a c t l y as d e s c r i b e d f o r Experiment 4, except t h a t cannulae were aimed a t the c e n t r a l nucleus of the amygdala and t h a t females were primed w i t h 10 jug of e s t r a d i o l benzoate and 100 jug progesterone. In a l l other r e s p e c t s , animals were a s s i g n e d t o groups, t e s t e d and the placements v e r i f i e d i n a manner i d e n t i c a l t o t h a t d e s c r i b e d f o r experiment 4. H i s t o l o g i c a l procedures r e s u l t e d i n the r e t e n t i o n of data from 7 animals f o r s t a t i s t i c a l a n a l y s i s . R e s u l t s and d i s c u s s i o n None of the OT doses i n f u s e d i n t o the ACE had a s i g n i f i c a n t e f f e c t on LQ scores when compared with the c o n t r o l i n f u s i o n s u s i n g the Wilcoxon signed ranks t e s t . However, the i n f u s i o n of 8.5 ng OT d i d s i g n i f i c a n t l y extend l o r d o s i s d u r a t i o n when compared with the c o n t r o l i n f u s i o n (z = 2.113, p < 0.05). A s i g n i f i c a n t c o r r e l a t i o n was o b t a i n e d between OT dose and LD (Pearson r = .343, df = 33, p < 0.05) but not between OT dose and LQ (Pearson r = .071, df = 33). These r e s u l t s are shown i n F i g u r e 5. The r e s u l t s from Experiment 5 s t r o n g l y suggest t h a t LQ and LD are independently r e g u l a t e d . OT i n f u s i o n s i n t o the ACE have no e f f e c t on LQ but do s i g n i f i c a n t l y extend LD. T h i s appears t o be f u r t h e r c o n f i r m a t i o n of the g e n e r a l h y p o t h e s i s t h a t d i f f e r e n t b r a i n s i t e s may c o n t r o l d i f f e r e n t a s p e c t s of l o r d o s i s . R e s u l t s from Experiment 4 i n d i c a t e t h a t the MPOA may p r i m a r i l y r e g u l a t e l o r d o s i s frequency while the VMH may be r e s p o n s i b l e f o r other aspects of l o r d o s i s . I t may now be p o s s i b l e t o f u r t h e r r e f i n e t h i s h y p o t h e s i s : i n the l i g h t o f Experiments 4 and 5, i t appears t h a t the VMH serves as g e n e r a t o r of t h i s behavior, t h a t the ACE c o n t r o l s l o r d o s i s d u r a t i o n , and t h a t the MPOA c o n t r o l s l o r d o s i s frequency. T h i s l o r d o s i s maintenance f u n c t i o n of the ACE i s c o n s i s t e n t w i t h the f i n d i n g s of Grossmann (1962) concerning the r o l e of the amygdala i n the maintenance of e a t i n g and d r i n k i n g b e h a v i o r s . Anatomical c o n s i d e r a t i o n s may p r o v i d e some a d d i t i o n a l support f o r t h i s h y p o t h e s i s . I t has been shown wit h t r a c i n g s t u d i e s t h a t the v e n t r o l a t e r a l VMH p r o j e c t s t o the ACE and the dorsomedial VMH t o the MPOA (Saper e t a l . 1976), but the ACE does not p r o j e c t t o the MPOA or d i r e c t l y t o the VMH (Kre t t e k and P r i c e 1978), which suggests t h a t l o r d o s i s F i g u r e 5. The f i g u r e shows the mean l o r d o s i s f r a c t i o n ( l o r d o s i s quotient/100) ± S. E. M. and the mean l o r d o s i s d u r a t i o n ± S. E. M. f o r v a r i o u s doses of o x y t o c i n i n f u s e d i n t o the c e n t r a l nucleus of the amygdala i n female r a t s t r e a t e d subcutaneously w i t h 3/Ltg e s t r a d i o l benzoate 48 h be f o r e t e s t i n g and 150 ng progesterone 4-6 h b e f o r e t e s t i n g . BEHAVIORAL SCORE > O ! I >. O 51. r e l a t e d s i g n a l s may o r i g i n a t e i n the VMH. The hypothalamus i s c o n s i d e r e d t o be a neurohumoral t r a n s d u c e r t h a t c o n v e r t s n e u r a l s i g n a l s i n t o hormonal s i g n a l s (Scharrer and S c h a r r e r 1954). I t may be e q u a l l y u s e f u l t o c o n s i d e r i t a humoroneural t r a n s d u c e r responding t o humoral c o n d i t i o n s w i t h n e u r a l output. In p a r t i c u l a r , the VMH may respond t o humoral events f o l l o w i n g estrogen and progesterone treatment w i t h the g e n e r a t i o n of n e u r a l s i g n a l s t h a t r e s u l t i n l o r d o s i s b ehavior. These s i g n a l s are then r e l a y e d t o the MPOA and ACE f o r the i n c o r p o r a t i o n of the r e l e v a n t frequency and d u r a t i o n i n f o r m a t i o n , r e s p e c t i v e l y . SECTION I I I I n v e s t i g a t i o n s of the r e c e p t o r types i n v o l v e d i n the g e n e r a t i o n of l o r d o s i s a t c e n t r a l s i t e s t h a t c o n c e n t r a t e r a d i o l a b e l l e d e s t r a d i o l and respond t o o x y t o c i n a d m i n i s t r a t i o n . Although t h e r e appears t o be o n l y one OT r e c e p t o r , OT can a l s o i n t e r a c t w i t h the primary v a s o p r e s s i n r e c e p t o r s due t o t h e s t r u c t u r a l s i m i l a r i t i e s between o x y t o c i n and v a s o p r e s s i n (Antoni 1987, Cheng and North 1989, Manning and Sawyer 1989). There are two primary v a s o p r e s s i n r e c e p t o r subtypes t o which OT can b i n d : V]_ r e c e p t o r s mediate the v a s o p r e s s o r e f f e c t s of v a s o p r e s s i n wtiile V2 r e c e p t o r s mediate the a n t i d i u r e t i c e f f e c t s of v a s o p r e s s i n . 52 I t i s t h e r e f o r e i m p o r t a n t t o i n v e s t i g a t e w h i c h r e c e p t o r s may be r e s p o n s i b l e f o r t h e e f f e c t s o f c e n t r a l OT i n f u s i o n s o n l o r d o s i s , e s p e c i a l l y g i v e n t h e r e s u l t s o f E x p e r i m e n t s 1 -4 w h e r e i t became a p p a r e n t t h a t OT h a d b o t h a n i n h i b i t o r y e f f e c t ( E x p e r i m e n t s 1 and 2) and a f a c i l i t a t o r y e f f e c t ( E x p e r i m e n t 4 ) . F u r t h e r m o r e , l o r d o s i s c a n be m e d i a t e d b y d i f f e r e n t r e c e p t o r s a t d i f f e r e n t s i t e s . The OT a g o n i s t [ T h r 4 , G l y 7 ] - o x y t o c i n a n d a n t a g o n i s t [d(CH2)5, T y r ( 0 M e ) 2 , 0 r n 8 ] - v a s o t o c i n h a v e a c t i v i t i e s a t t h e v a r i o u s r e c e p t o r s a s i n d i c a t e d i n t h e t a b l e b e l o w ( a d a p t e d f r o m 4 1 , 4 3 ) . T a b l e 1. r e c e p t o r t y p e o x y t o c i c v a s o p r e s s o r (V^) a n t i d i u r e t i c (V2) a g o n i s t ( m o d e r a t e +) ( v e r y weak +) ( v e r y weak +) a n t a g o n i s t ( p o t e n t -) ( p o t e n t -) ( v e r y weak +) (+) a g o n i s t i c (-) a n t a g o n i s t i c I t s h o u l d be c l e a r f r o m t h e t a b l e t h a t t h e u s e o f t h e s e t w o compounds may g i v e some i n d i c a t i o n o f w h i c h r e c e p t o r m e d i a t e s l o r d o s i s a t a p a r t i c u l a r c e n t r a l s i t e . I f V2 r e c e p t o r s o n l y a r e r e s p o n s i b l e f o r l o r d o s i s , t h e n n e i t h e r a g o n i s t n o r a n t a g o n i s t s h o u l d h a v e a n e f f e c t on l o r d o s i s . I f V I r e c e p t o r s e x c l u s i v e l y m e d i a t e l o r d o s i s , t h e n t h e 53 antagonist should have an e f f e c t but the agonist not. I f the oxytocin receptor was so l e l y responsible for the induction of l o r d o s i s , then the agonist and antagonist should have opposite e f f e c t s on lor d o s i s . However, there i s some in d i c a t i o n that both OT and VI receptors are involved i n lord o s i s behavior (Caldwell et a l . 1990). In p a r t i c u l a r , i t appears that i n the MPOA of female rats c h r o n i c a l l y treated with estrogen, a uterotonic antagonist (OT receptor antagonist) i n h i b i t s lordosis while a VI antagonist f a c i l i t a t e s l o r d o s i s . I t i s not known what the time course of OT and VI receptor a c t i v a t i o n by the antagonist i s and i t i s d i f f i c u l t to make a prediction about r e s u l t s to be obtained from the concurrent blocking of OT and VI receptors. Experiment 6 The e f f e c t s of the OT agonist [Thr4, Gly7] - oxytocin and antagonist [d(CH2)5, Tyr(OMe)2, 0rn8] - vasotocin on lordosis when infused into the LV, MPOA and VMH of female rats treated with estrogen and progesterone. Infusion of OT into the l a t e r a l v e n t r i c l e s of female rats treated with estrogen and progesterone can either f a c i l i t a t e ( A r l e t t i and B e r t o l i n i 1985, Gorzalka and Lester 1987, Caldwell et a l . 1989) or i n h i b i t lordosis (Experiments 1 and 2), an e f f e c t that may be dependent on OT concentration, 54 s t e r o i d reg imen or r e c e p t o r type a c t i v a t e d . F o r i n s t a n c e , a t low c o n c e n t r a t i o n s OT may e i t h e r i n a c t i v a t e OT r e c e p t o r s o r p r i m a r i l y b i n d t o an i n h i b i t o r y r e c e p t o r , perhaps the V2 r e c e p t o r . In the MPOA, a u t e r o t o n i c a n t a g o n i s t b l o c k e d the f a c i l i t a t o r y e f f e c t s o f OT i n f u s i o n s i n female r a t s t r e a t e d c h r o n i c a l l y w i t h e s t r o g e n ( C a l d w e l l e t a l . 1990) . S i n c e the p r e s e n c e o r absence o f p r o g e s t e r o n e can have an e f f e c t on OT r e c e p t o r s (Schumacher e t a l . 1989, Schumacher e t a l . 1990) and O T - i n d u c e d l o r d o s i s ( G o r z a l k a and L e s t e r 1987, Schumacher e t a l . 1989), i t i s i m p o r t a n t to c o n f i r m the r e s u l t s o b t a i n e d by C a l d w e l l and co -workers ( C a l d w e l l e t a l . 1990) i n an imal s t r e a t e d a l s o w i t h p r o g e s t e r o n e . Whether a u t e r o t o n i c r e c e p t o r i s r e s p o n s i b l e f o r the i n d u c t i o n o f l o r d o s i s upon the i n f u s i o n o f OT i n t o the VMH i n a manner analogous t o t h a t i n the MPOA ( C a l d w e l l e t a l . 1990) i s not known, a l t h o u g h i t i s known t h a t e s t r o g e n induces these r e c e p t o r s i n the VMH (de K l o e t e t a l . 1986, Johnson e t a l . 1989) and t h a t they a r e d i s p e r s e d i n response t o p r o g e s t e r o n e (Schumacher e t a l . 1989, Schumacher e t a l . 1990) . The r e c e p t o r type r e s p o n s i b l e f o r l o r d o s i s b e h a v i o r i n re sponse t o i n f u s i o n s o f OT i n t o the L V , MPOA, and VMH of s t e r o i d - p r i m e d female r a t s was i n v e s t i g a t e d i n Exper iment 6. M a t e r i a l s and methods Twenty Sprague-Dawley female r a t s were b i l a t e r a l l y o v a r i e c t o m i z e d and p r o v i d e d w i t h c h r o n i c i n t r a c e r e b r a l 55 cannulae directed at the LV, MPOA and VMH according to procedures described i n the General Method section. The oxytocin agonist [Thr 4, Gly 7] - oxytocin and antagonist [d(CH2)5, Tyr(OMe) 2, Orn 8] - vasotocin were obtained from Peninsula Laboratories Inc., Belmont, CA. These peptide analogues were dissolved i n physiological saline a few hours p r i o r to use and administered 30 minutes before behavioral t e s t i n g of each animal. Two concentrations of agonist (100 ng and 500 ng) and two concentrations of antagonist (100 ng and 500 ng) as well as a saline control group were employed instead of the various oxytocin doses used i n Experiment 4. Cannula placements were h i s t o l o g i c a l l y v e r i f i e d and only the data from animals with correct i n t r a c r a n i a l cannula placements were analyzed. Due to a t t r i t i o n and the fact that the LV and VMH tests were conducted for three weeks only, the data from these groups were analyzed using the Mann-Whitney U-test while data from the MPOA tests were analyzed with the Wilcoxon signed ranks procedure. In a l l other respects, animals were assigned to groups and tested i n a manner i d e n t i c a l to that described for Experiment 4. Results LV Relative to the saline control infusions, infusions of both the agonist and antagonist i n h i b i t e d l ordosis. An infusion of 100 ng agonist s i g n i f i c a n t l y i n h i b i t e d LQ scores 56 (Mann-Whitney U = 13; n l f n 2 = 8; p < 0.05) and an i n f u s i o n of 500 ng a n t a g o n i s t a l s o s i g n i f i c a n t l y i n h i b i t e d LQs (Mann-Whitney U = 11.5; n! = 8, n 2 = 9; p < 0.05). The i n f u s i o n of 500 ng a g o n i s t and 100 ng a n t a g o n i s t d i d not have a s i g n i f i c a n t e f f e c t on l o r d o s i s q u o t i e n t s . L o r d o s i s d u r a t i o n s c o r e s were not s i g n i f i c a n t l y a f f e c t e d by any a g o n i s t or a n t a g o n i s t dose. MPOA No s i g n i f i c a n t e f f e c t s of the i n f u s i o n of a g o n i s t or a n t a g o n i s t i n t o the MPOA of estrogen and progesterone primed female r a t s were obtained. VMH No s i g n i f i c a n t e f f e c t s on l o r d o s i s q u o t i e n t and l o r d o s i s d u r a t i o n were observed i n response t o the i n f u s i o n of any dose of a g o n i s t or a n t a g o n i s t . / A l l the r e s u l t s are gi v e n i n F i g u r e 6. General d i s c u s s i o n Both the a g o n i s t and a n t a g o n i s t attenuated l o r d o s i s frequency when i n f u s e d i n t o the LV of female r a t s t r e a t e d w i t h e s t r o g e n and progesterone. Although the e f f e c t on LQ of an i n f u s i o n of 500 ng a g o n i s t i n t o the LV was a p p a r e n t l y s i m i l a r t o t h a t of a 100 ng i n f u s i o n , the e f f e c t of the F i g u r e 6. The mean l o r d o s i s q u o t i e n t s ± S. E. M. f o r the o x y t o c i n a g o n i s t , s a l i n e c o n t r o l , and o x y t o c i n a n t a g o n i s t i n f u s i o n s i n t o the LV (top p a n e l ) , VMH (middle p a n e l ) , and MPOA (bottom panel) are shown. A l l animals were t r e a t e d subcutaneously w i t h 3 jug e s t r a d i o l benzoate 48 h b e f o r e t e s t i n g and 150 jug progesterone 4-6 h b e f o r e t e s t i n g . 00 ID FIGURE 6. o o o 1.0n 0.8-0.6-0.4-0.2-0.0 D 1 1 ' • D . 500 100' Agonist, ng/rat 0 Control 0 — O LV • •D 1 a A - - A VMH • • • • • MPOA 100 500 Antagonist, ng/rat 59 former was not s t a t i s t i c a l l y s i g n i f i c a n t due t o gre a t e r v a r i a n c e . The i n f u s i o n of 100 ng antagonist had l e s s of an e f f e c t on LQ than the 500 ng dose. LD was not a f f e c t e d by these manipulations. In the VMH, n e i t h e r agonist nor antagonist had any s i g n i f i c a n t e f f e c t on LQ or LD. This i s l i k e l y due t o the small sample s i z e s (n = 4-6) si n c e the p a t t e r n of r e s u l t s g e n e r a l l y mirrored those obtained i n the LV. Data generated from i n f u s i o n s of agonist and antagonist i n t o the MPOA are of poor q u a l i t y and l i t t l e use but do show t h a t the agonist tended t o elevate and the antagonist t o suppress LQ scores. Although LD scores i n the VMH were much higher than those i n the MPOA, a comparison between these two s i t e s was not done f o r s e v e r a l reasons. C o n t r o l LD l e v e l s f o r the VMH were s i g n i f i c a n t l y higher than c o n t r o l MPOA l e v e l s (independent t = 4.013, df = 8, p < 0.01). Since c o n t r o l LQ scores f o r the VMH (85 ± 8.66) were a l s o much higher than c o n t r o l LQ scores f o r the MPOA (20 ± 14.37), the p o s s i b i l i t y remains t h a t s t e r o i d manipulations of the two groups may not have been eq u i v a l e n t i n s p i t e of the f a c t t h a t the same regimen was foll o w e d . I t i s a l s o p o s s i b l e t h a t r a t i n g d i s c r e p a n c i e s between two d i f f e r e n t experimenters may have produced t h i s e f f e c t . F i n a l l y , the agonist and antagonist tended t o have d i f f e r e n t trends i n the two s i t e s which renders a comparison of LQ and LD values between them d i f f i c u l t . 60 The i n h i b i t i o n p r o d u c e d by b o t h t h e a g o n i s t a n d a n t a g o n i s t when a d m i n i s t e r e d t o t h e LV was u n e x p e c t e d . T h e s e d a t a a r e d i f f i c u l t t o i n t e r p r e t . I t h a s b e e n shown t h a t a n t a g o n i s m o f V^ r e c e p t o r s r e s u l t s i n t h e e l e v a t i o n o f LQ s c o r e s ( C a l d w e l l e t a l . 1 9 9 0 ) . I f o x y t o c i n r e c e p t o r s a r e l o c a t e d a t p o s t s y n a p t i c s i t e s a n d v a s o p r e s s i n r e c e p t o r s a t p r e s y n a p t i c s i t e s , t h e n t h e a n t a g o n i s m o f b o t h V I a n d OT r e c e p t o r s s h o u l d r e s u l t i n t h e d e p r e s s i o n o f LQ s c o r e s . T h i s h a p p e n s b e c a u s e p o s t s y n a p t i c e v e n t s a r e more 'downstream' i n t h e n e u r o t r a n s m i s s i o n p a t h w a y a n d b l o c k i n g n e u r o t r a n s m i s s i o n d o w n s t r e a m w i l l i n h i b i t s e x u a l b e h a v i o r i r r e s p e c t i v e o f a n u p s t r e a m f a c i l i t a t i o n o f n e u r o t r a n s m i s s i o n . The i n h i b i t i o n shown b y t h e 500 ng d o s e o f a n t a g o n i s t i s c o m p a t i b l e w i t h t h e v i e w t h a t V I r e c e p t o r s a r e l o c a t e d on p r e s y n a p t i c s i t e s . The i n h i b i t i o n f o u n d i n E x p e r i m e n t s 1 and 3 c o u l d be c o n s i d e r e d t o b e , amongst o t h e r p o s s i b i l i t i e s , due t o a c t i v a t i o n o f V2 r e c e p t o r s l o c a t e d on o x y t o c i n n e u r o n s . A c t i v a t i o n o f t h e s e r e c e p t o r s a t t e n u a t e s OT n e u r o t r a n s m i s s i o n a n d t h e s e r e c e p t o r s may c o n s e q u e n t l y a c t a s OT a u t o r e c e p t o r s . The a g o n i s t u s e d i n E x p e r i m e n t 6 h a s o n l y a t h i r d t h e a c t i v i t y o f t h e OT p e p t i d e a t OT r e c e p t o r s ( L o w b r i d g e e t a l . 1 9 7 6 ) . T h i s t r a n s l a t e s i n t o a n e q u i v a l e n t OT d o s e o f a b o u t 33 ng i n r e s p o n s e t o a 100 ng a g o n i s t i n f u s i o n . However, i n E x p e r i m e n t 3, a c o m p a r a b l e d o s e o f OT h a d no e f f e c t on LQ. A c t i v i t y o f t h e a g o n i s t a t V2 r e c e p t o r s a r e a l s o 1 0 0 0 - f o l d w e a k e r t h a n t h a t o f OT a t t h e s e 61 r e c e p t o r s . I t i s t h e r e f o r e d i f f i c u l t t o r e c o n c i l e the i n h i b i t i o n of LQ by the i n f u s i o n of 100 ng of OT a g o n i s t w i t h the h y p o t h e s i s t h a t the a c t i v a t i o n of V2 r e c e p t o r s l o c a t e d on OT neurons are r e s p o n s i b l e f o r such i n h i b i t i o n . Consequently, one of the other a l t e r n a t i v e s , e s p e c i a l l y the e x i s t e n c e of an i n h i b i t o r y s i t e or the a c t i v a t i o n of oth e r p e p t i d e r g i c systems should r e c e i v e more credence. SECTION IV Some temporal f a c t o r s i n the e f f e c t s of c e n t r a l l y a d m i n i s t e r e d o x y t o c i n on l o r d o s i s . I n v e s t i g a t i o n s of the o x y t o c i n e r g i c pathways i n v o l v e d i n l o r d o s i s behavior f r e q u e n t l y i n v o l v e the c e n t r a l a d m i n i s t r a t i o n N o f OT or i t s analogues ( A r l e t t i and B e r t o l i n i 1985, G o r z a l k a and L e s t e r 1987, Schumacher et a l . 1989, C a l d w e l l e t a l . 1990). I t would t h e r e f o r e seem reasonable t o apply these substances at a time when the maximum number of OT r e c e p t o r s are a v a i l a b l e . T h i s appears t o be a t about 24 h a f t e r acute subcutaneous estrogen a d m i n i s t r a t i o n (Johnson e t a l . 1989). However, many s t u d i e s employing the c e n t r a l a d m i n i s t r a t i o n of OT or i t s analogues have been conducted approximately 48 h a f t e r e s t r a d i o l benzoate a d m i n i s t r a t i o n (e.g. A r l e t t i and B e r t o l i n i 1985, Gorzalka and L e s t e r 1987, Schumacher e t a l . 1989, Schulze and Gorzalka 1991) when l e v e l s of OT r e c e p t o r s may have d e c l i n e d a p p r e c i a b l y (Johnson e t a l . 1989) although they may s t i l l be e l e v a t e d 62 r e l a t i v e t o c o n t r o l l e v e l s . The hypothesis t h a t f u n c t i o n a l OT r e c e p t o r s a l r e a d y e x i s t t h i s soon ( c i r c a 24 h) a f t e r e s t r o g e n treatment has not been t e s t e d . Experiment 7 Sin c e the p e p t i d e o x y t o c i n p l a y s an important p h y s i o l o g i c a l r o l e i n p a r t u r i t i o n (Fuchs 1985) and l a c t a t i o n (Robinson 1986), i s employed c l i n i c a l l y (Dawood 1985) and has e f f e c t s on many behaviors, i n c l u d i n g l o r d o s i s (reviews by A r g i o l a s and Gessa 1991, Kovacs 1986), an understanding of i t s time dependency i s e s s e n t i a l . We consequently designed Experiment 7 t o i n v e s t i g a t e the e f f e c t s of the time s i n c e e s t r o g e n a d m i n i s t r a t i o n on the e f f e c t s of o x y t o c i n on l o r d o s i s behavior i n female r a t s employing the same OT a g o n i s t and a n t a g o n i s t used i n Experiment 6. M a t e r i a l s and methods Animals and surgery T h i r t y - t h r e e female Sprague-Dawley r a t s , from C h a r l e s R i v e r Canada, Inc., Montreal, were b i l a t e r a l l y o v a r i e c t o m i z e d and cannulated as d e s c r i b e d i n the General Method s e c t i o n . The c o o r d i n a t e s used f o r c a n n u l a t i o n i n the l e f t l a t e r a l v e n t r i c l e were: 0.2 mm p o s t e r i o r , 1.8 mm l a t e r a l and 2.9 mm v e n t r a l . Cannula placements were v e r i f i e d 63 as d e s c r i b e d b e f o r e and on l y data from animals w i t h c o r r e c t placements were r e t a i n e d f o r s t a t i s t i c a l a n a l y s e s . Placement t e s t i n g r e s u l t e d i n the e x c l u s i o n of data from 14 animals from s t a t i s t i c a l a n a l y s i s . F o l l o w i n g surgery, a l l animals were i n d i v i d u a l l y housed i n s t a n d a r d l a b o r a t o r y wire mesh cages i n a colony room maintained on a r e v e r s e d 12 h l i g h t / d a r k c y c l e ( l i g h t s o f f a t 06h30) and a t 50% r e l a t i v e humidity, 21°C with f r e e access t o food and water. Drug procedures E s t r a d i o l benzoate and progesterone (both from Sigma) were d i s s o l v e d i n warm peanut o i l and ad m i n i s t e r e d subcutaneously. A l l animals r e c e i v e d 3 izg e s t r a d i o l benzoate i n 0.05 cc of v e h i c l e a t 14h00 and 28 h be f o r e the b e h a v i o r a l t e s t of the f i r s t group ( t h i s means t h a t group one r e c e i v e d e s t r a d i o l benzoate 28 h before t e s t i n g , but t h a t group s i x r e c e i v e d the s t e r o i d 48 h be f o r e t e s t i n g ) and 150 ng progesterone i n 0.1 cc of v e h i c l e 4 h be f o r e each group was t e s t e d . The o x y t o c i n a g o n i s t [Thr4, Gly7] -o x y t o c i n and a n t a g o n i s t [d(CH2)5, Tyr(0Me)2, Orn8] -v a s o t o c i n were ob t a i n e d from P e n i n s u l a L a b o r a t o r i e s Inc., Belmont, CA. These p e p t i d e analogues were 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 a few hours p r i o r t o use and ad m i n i s t e r e d 30 minutes be f o r e b e h a v i o r a l t e s t i n g of each 64 animal. Both a g o n i s t and a n t a g o n i s t c o n c e n t r a t i o n s were 12 5 ng/ / i l and a t o t a l dose of 500 ng per animal was i n f u s e d . A n g i o t e n s i n I I (Sigma) 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 t o a c o n c e n t r a t i o n of 1 /ig/ / i l and g i v e n a t a dose of 1 /ig per animal. A l l i n f u s i o n s were performed a t a flow r a t e of 4 / i l / minute and the i n f u s i o n needle was l e f t i n the cannula f o r an a d d i t i o n a l 30 s t o a l l o w d i s p e r s i o n of the p e p t i d e away from the i n f u s i o n s i t e . B e h a v i o r a l t e s t i n g L o r d o s i s t e s t i n g was conducted as d e s c r i b e d i n the General Method s e c t i o n . Only l o r d o s i s frequency was measured. The experimental animals were randomly assig n e d t o f i v e groups of s i x animals each as w e l l as a s i x t h group w i t h o n l y t h r e e animals. A l l groups were t e s t e d by a b l i n d o bserver o n l y once d u r i n g the same 24 h p e r i o d . The f i r s t group was t e s t e d a t 18h00 and 28 h a f t e r e s t r a d i o l benzoate a d m i n i s t r a t i o n . T h e r e a f t e r , every f o u r hours another group was t e s t e d u n t i l group s i x was t e s t e d a t 14h00 and 48 h a f t e r e s t r a d i o l benzoate a d m i n i s t r a t i o n . E x a c t l y two weeks l a t e r , the animals were randomly r e a s s i g n e d t o s i x groups and the t e s t i n g procedure was repeated as d e s c r i b e d above. R e s u l t s and d i s c u s s i o n The r e s u l t s are i l l u s t r a t e d i n F i g u r e 7. No s t a t i s t i c a l a n a l y s e s were done due t o - t h e s m a l l number of s u b j e c t s per group. An examination of F i g u r e 7 s t r o n g l y suggests rhythmic a c t i v i t y i n the e f f e c t s of o x y t o c i n on l o r d o s i s . I n t e r e s t i n g l y , a c i r c a d i a n f l u c t u a t i o n i s a l s o m a n i f e s t i n the c o n t r o l s c o r e s . However, c a u t i o n needs t o be e x e r c i s e d i n the i n t e r p r e t a t i o n of these data. Due t o the e l i m i n a t i o n of data from s e v e r a l animals, some of the data p o i n t s i n F i g u r e 7 r e p r e s e n t s i n g l e t e s t s c o r e s . P a u c i t y of data i s p a r t i c u l a r l y p r o b l e m a t i c a t the 22h00, lOhOO and 14h00 time p o i n t s . U n f o r t u n a t e l y , these p o i n t s are the ones of p a r t i c u l a r i n t e r e s t . At 22h00, i t appears as i f the a g o n i s t s t r o n g l y f a c i l i t a t e s l o r d o s i s w h ile the a n t a g o n i s t and c o n t r o l s c o r e s are s i m i l a r t o each other. I f such a f a c i l i t a t i o n indeed e x i s t s , a f u l l 16 h bef o r e those g e n e r a l l y r e p o r t e d i n the l i t e r a t u r e ( A r l e t t i and B e r t o l i n i 1985, G o r z a l k a and L e s t e r 1987, Schumacher e t a l . 1989, Schulze and G o r z a l k a 1991), i t would be of i n t e r e s t because i t may r e q u i r e r e s e a r c h e r s t o modify t h e i r experimental procedures and/or t h e o r e t i c a l p o s i t i o n s r e g a r d i n g the e f f e c t s of o x y t o c i n on sexual behavior i n female r a t s . For s i m i l a r reasons the g e n e r a l d e c l i n e i n l o r d o s i s s c o r e s from h i g h v a l u e s a t 06h00 t o lower v a l u e s a t lOhOO may a l s o be of i n t e r e s t . C o n t r o l v a l u e s seem t o i n c r e a s e again a f t e r lOhOO w h i l e a g o n i s t and a n t a g o n i s t v a l u e s continue t o d e c l i n e . I f F i g u r e 7. Mean l o r d o s i s q u o t i e n t s f o r the o x y t o c i n a g o n i s t , o x y t o c i n a n t a g o n i s t , and s a l i n e c o n t r o l groups over a 24 h p e r i o d s t a r t i n g 28 h a f t e r the subcutaneous a d m i n i s t r a t i o n of 3 jug e s t r a d i o l benzoate. A l l groups r e c e i v e d 150 jug progesterone 4 h and drug i n f u s i o n s 30 minutes b e f o r e t e s t i n g . I n d i c a t e d on the same graph i s the l i g h t - d a r k regimen f o l l o w e d . TIME OF DAY 68 these f i n d i n g s c o u l d be r e p l i c a t e d , i t would suggest t h a t i n v e s t i g a t i o n s of o x y t o c i n e r g i c a c t i o n s on s e x u a l behavior ought t o be r e c o n s i d e r e d . A follow-up experiment was undertaken t o f u r t h e r i n v e s t i g a t e these f i n d i n g s . Experiment 8 Experiment 8 was designed t o t e s t the e f f e c t s of the OT a g o n i s t and a n t a g o n i s t a t 22h00 and 14h00 and t o t e s t the e f f e c t s of on l y the a g o n i s t a t lOhOO. These times were chosen because Experiment 7 y i e l d e d l i t t l e data a t these times and they were of p a r t i c u l a r i n t e r e s t as d i s c u s s e d above. M a t e r i a l s and methods Animals and surgery The same animals used i n Experiment 7 were a l s o used i n Experiment 8. Because some animals were e l i m i n a t e d from the s t a t i s t i c a l a n a l y s i s as d e s c r i b e d i n Experiment 7, an a d d i t i o n a l group of 13 animals were o v a r i e c t o m i z e d , cann u l a t e d and housed as d e s c r i b e d i n Experiment 7. 69 Drugs Drug procedures were i d e n t i c a l t o those i n Experiment 7. Animals i n the 22h00 t e s t group r e c e i v e d e s t r a d i o l benzoate 32 h b e f o r e t e s t i n g , those i n the lOhOO t e s t group r e c e i v e d the s t e r o i d 44 h be f o r e t e s t i n g and those i n the 14h00 group r e c e i v e d e s t r a d i o l benzoate 48 h b e f o r e t e s t i n g . Progesterone and the o x y t o c i n e r g i c drugs were a d m i n i s t e r e d 4 h b e f o r e and 30 minutes before t e s t i n g , r e s p e c t i v e l y . Animals i n the 22h00 and 14h00 groups r e c e i v e d e i t h e r the OT a n t a g o n i s t , a g o n i s t or s a l i n e v e h i c l e and those i n the lOhOO group r e c e i v e d e i t h e r the a g o n i s t or s a l i n e v e h i c l e . B e h a v i o r a l t e s t i n g B e h a v i o r a l t e s t i n g was i d e n t i c a l t o t h a t d e s c r i b e d i n Experiment 7 wi t h the e x c e p t i o n t h a t animals were t e s t e d a t o n l y t h r e e time p o i n t s . One group of animals was t e s t e d a t 22h00 and another a t 14h00. The t e s t a t 22h00 was repeated a few weeks l a t e r with an a d d i t i o n a l group of animals t o i n c r e a s e the sample s i z e s . T h i s same group was a l s o used a week l a t e r f o r t e s t i n g a t lOhOO. R e s u l t s 22h00. The mean LQ ± S.E.M. f o r the a g o n i s t , a n t a g o n i s t and s a l i n e groups was, r e s p e c t i v e l y : 76.7% ± 5.58; 21.7% ± 14.24; and 23.3% ± 8.43. These r e s u l t s suggest a f a c i l i t a t i o n of l o r d o s i s by the a g o n i s t when compared t o the c o n t r o l group. An a n a l y s i s of v a r i a n c e i n d i c a t e d a s i g n i f i c a n t treatment e f f e c t (F(2,15) = 9.627, p <0.01) and a f o l l o w - u p (Tukey, p <0.01) confirmed t h a t the f a c i l i t a t i o n o f l o r d o s i s by the a g o n i s t was s i g n i f i c a n t . lOhOO. L o r d o s i s q u o t i e n t s (mean ± S.E.M.) f o r the a g o n i s t and s a l i n e v e h i c l e c o n t r o l groups were, r e s p e c t i v e l y , 35.0% ± 9.06 and 32.5% ± 10.65. These d i f f e r e n c e s 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 . 14h00. No s i g n i f i c a n t d i f f e r e n c e s were found between the a g o n i s t , a n t a g o n i s t and s a l i n e groups a t t h i s time. LQs ± S.E.M. were, r e s p e c t i v e l y : 65.0% ± 14.78; 55.0% ± 16.48; and 62.0% ± 19.08. 22h00 v s. lOhOO. The a g o n i s t induced a s i g n i f i c a n t l y h i g h e r LQ a t 22h00 than i t d i d a t lOhOO (independent samples, t = 3.593, df = 12, p < 0.01). As i n d i c a t e d above, the means ± S.E.M. were 76.7% ± 5.58 and 35.0% ± 9.06, r e s p e c t i v e l y . The va l u e s f o r the s a l i n e c o n t r o l groups a t these times were 23.3% ± 8.43 and 32.5% ± 10.65, r e s p e c t i v e l y , and d i d not d i f f e r s i g n i f i c a n t l y . These r e s u l t s are i l l u s t r a t e d i n F i g u r e 8. 71 F i g u r e 8. The mean l o r d o s i s q u o t i e n t ± S.E.M. i s i n d i c a t e d f o r the o x y t o c i n a g o n i s t and s a l i n e groups a t 22h00 and lOhOO. The o x y t o c i n a g o n i s t induced a s i g n i f i c a n t l y h i g h e r LQ a t 22h00 than i t d i d a t lOhOO (p < 0.01). The o x y t o c i n a g o n i s t - i n d u c e d l e v e l s of l o r d o s i s a t 22h00 were a l s o s i g n i f i c a n t l y h i g h e r than those of the c o n t r o l group a t the same time (p < 0.01). 72 LORDOSIS QUOTIENT N O > cn oo o o o o o o o b b b b b b i i i i i i 22h00 vs 06h00 vs lOhOO. C o n t r o l s cores a t 06h00, 80.0% ± 7.07, were s i g n i f i c a n t l y h i g h e r than both the c o n t r o l s c o r e s a t 22h00 (23.3% ± 8.43, independent t = 4.753, df = 8, p < 0.01) and those a t lOhOO (32.5% ± 10.65, independent t = 2.94, df = 10, p < 0.02). D i s c u s s i o n The r e s u l t s of Experiment 8 confirmed the t r e n d towards a f a c i l i t a t i o n of l o r d o s i s a t 22h00 by the a g o n i s t as seen i n Experiment 7. A l s o confirmed were the g e n e r a l l y low l e v e l s of l o r d o s i s seen a t lOhOO i n response t o the a g o n i s t and i n the c o n t r o l group. At 14h00, however, the c o n t r o l s c o r e s were lower and the a g o n i s t scores h i g h e r than those observed i n Experiment 7. The combined r e s u l t s of Experiments 7 and 8 are shown i n F i g u r e 9. General d i s c u s s i o n The a g o n i s t used i n t h i s study f a c i l i t a t e d l o r d o s i s i n female r a t s as e a r l y as 32 h a f t e r p r iming w i t h e s t r a d i o l benzoate. T h i s f i n d i n g p r o v i d e s support f o r the h y p o t h e s i s t h a t f u n c t i o n a l OT r e c e p t o r s a l r e a d y e x i s t c i r c a 24 h a f t e r subcutaneous estrogen a d m i n i s t r a t i o n . At 44 h and 48 h a f t e r e s t r a d i o l benzoate i n j e c t i o n s , no f a c i l i t a t i o n was seen. The a n t a g o n i s t used i n t h i s study d i d not i n h i b i t l o r d o s i s a t any of the time p o i n t s t e s t e d . In a d d i t i o n t o the l e n g t h of 74 F i g u r e 9. The combined data from Experiments 7 and 8 showing the mean l o r d o s i s q u o t i e n t s f o r the o x y t o c i n a g o n i s t , the o c x y t o c i n a n t a g o n i s t , and s a l i n e c o n t r o l groups over a 24 h p e r i o d s t a r t i n g 2 8 h a f t e r the subcutaneous a d m i n i s t r a t i o n of 3 jug e s t r a d i o l benzoate. The 22h00 time p o i n t shows the mean l o r d o s i s q u o t i e n t ± S.E.M. A l l groups r e c e i v e d 150 jLtg progesterone 4 h and drug i n f u s i o n s 3 0 minutes be f o r e t e s t i n g . A l s o i n d i c a t e d on the same graph i s the l i g h t - d a r k regimen f o l l o w e d . 75 LORDOSIS QUOTIENT O o l > o o o o 3 r-t-3 > > S l 76 time since e s t r a d i o l benzoate injections, the time of day also appeared to a f f e c t the l e v e l s of sexual r e c e p t i v i t y expressed by female rats as was mainly evident i n the control group. A s i g n i f i c a n t increase i n hypothalamic OT receptors occurs as early as 24 h a f t e r subcutaneous ( s i l a s t i c capsule implantation) estrogen administration to female rats (Johnson et a l . 1989). If the a c t i v a t i o n of central OT receptors causes an increase i n lordosis of female rats as suggested i n the l i t e r a t u r e ( A r l e t t i and B e r t o l i n i 1985, Gorzalka and Lester 1987, Schumacher et a l . 1989, Caldwell et a l . 1990, Schulze and Gorzalka 1991), then a f a c i l i t a t i o n of lordosis may be seen as early as 24 h a f t e r subcutaneous t estrogen administration. This hypothesis i s given support by the finding of a s i g n i f i c a n t f a c i l i t a t i o n of lordosis i n response to the i n t r a v e n t r i c u l a r infusion of an oxytocin agonist at 22h00 or 32 h a f t e r acute subcutaneous administration of e s t r a d i o l benzoate. Since i t appears that functional OT receptors e x i s t c e n t r a l l y at t h i s time, i t i s reasonable to conclude that the rate l i m i t i n g step i n the a c t i v a t i o n of the oxytocinergic pathway contributing to lordosis behavior i s that of OT peptide synthesis. Submaximal l e v e l s of OT (levels below that required for the f u l l a c t i v a t i o n of a l l possible OT receptors contributing to lordosis behavior) can be augmented from exogenous sources and should r e s u l t i n a f a c i l i t a t i o n of lordosis behavior. 77 Maximal l e v e l s of OT, on the other hand, would a l r e a d y take f u l l advantage of a l l OT r e c e p t o r s and the a d m i n i s t r a t i o n of exogenous p e p t i d e w i l l r e p r e s e n t no g a i n t o the system and hence no f a c i l i t a t i o n of l o r d o s i s would occur. L o r d o s i s l e v e l s a t 02h00 and 06h00 are very h i g h f o r a l l groups and may r e f l e c t the g r e a t e r a v a i l a b i l i t y of endogenous o x y t o c i n a t these times. Since OT r e c e p t o r s are a l r e a d y a v a i l a b l e a t 22h00 but h i g h l e v e l s of l o r d o s i s are not seen a t t h i s time without the a d m i n i s t r a t i o n of exogenous hormone, i t would seem t h a t the r a t e d e t e r m i n i n g s t e p i n the a c t i v a t i o n of o x y t o c i n e r g i c pathways i n v o l v e d i n the e x p r e s s i o n of l o r d o s i s a t t h i s time p o i n t i s the r a t e of p e p t i d e s y n t h e s i s . However, an o x y t o c i n e r g i c pathway may not be the ' l a s t common pathway 1 and the i n c r e a s e i n d i u r n a l s e x u a l r e c e p t i v i t y may r e f l e c t the a c t i v a t i o n of another pathway c o n t r i b u t i n g t o l o r d o s i s behavior. A prime candidate f o r such a pathway i s t h a t mediated by s e r o t o n i n . I t i s known t h a t s e r o t o n i n type 2 r e c e p t o r s (5HT 2) f a c i l i t a t e l o r d o s i s i n female r a t s (Gorzalka e t a l . 1990). Furthermore, 5HT 2 r e c e p t o r s show a c i r c a d i a n rhythm wi t h peak numbers being expressed d u r i n g the l i g h t phase (Moser and Redfern 1985) and thus c o i n c i d e n t with the h i g h l e v e l s of l o r d o s i s seen i n Experiment 7 a t 02h00 and 06h00. The g e n e r a l d e c l i n e i n r e c e p t i v i t y l e v e l s a f t e r 06h00 may r e f l e c t a d e c l i n e i n OT r e c e p t o r l e v e l s as d i s c u s s e d 78 e a r l i e r . T h i s may imply t h a t , i n c o n t r a s t t o the s i t u a t i o n a t 22h00, OT r e c e p t o r l e v e l s are now the r a t e l i m i t i n g f a c t o r and t h a t , p r o v i d e d adequate l e v e l s of OT p e p t i d e e x i s t , exogenous p e p t i d e or analogues would not e l e v a t e l o r d o s i s q u o t i e n t s . T h i s would account f o r the p r e s e n t f i n d i n g s where l i t t l e d i f f e r e n c e was observed between a g o n i s t and s a l i n e c o n t r o l scores a t lOhOO and 14h00 i n Experiment 8, but would c o n f l i c t w i t h s e v e r a l s t u d i e s r e p o r t i n g a f a c i l i t a t i o n of l o r d o s i s by i n t r a v e n t r i c u l a r OT a d m i n i s t r a t i o n a t 14h00 (48 h a f t e r estrogen a d m i n i s t r a t i o n ) . I f , p r i o r t o the d e c l i n e of the OT r e c e p t o r , t h e r e i s a conformation change of the r e c e p t o r from a h i g h t o a low a f f i n i t y s t a t e a c c e s s i b l e t o p e p t i d e molecules but not t o the OT a g o n i s t employed i n t h i s study, then the p r e s e n t f i n d i n g s may be r e c o n c i l e d w i t h e a r l i e r o + work. For i n s t a n c e , i t i s known t h a t the presence of Mg can i n f l u e n c e the a f f i n i t y of the OT r e c e p t o r f o r OT (Antoni and Chadio 1989) as w e l l as f o r the a g o n i s t (Lowbridge e t a l . 1976). I t i s not c l e a r why t h e r e i s almost a d o u b l i n g of LQ s c o r e s from lOhOO t o 14h00. T h i s i n c r e a s e i s 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 , perhaps due t o the r e l a t i v e l y s m a l l sample s i z e s used i n our study. However, i f a t t h i s time t h e r e i s indeed a d e c l i n e i n OT r e c e p t o r l e v e l s as suggested by other r e s e a r c h (Johnson et a l . 1989), the apparent i n c r e a s e i n sexual r e c e p t i v i t y seen t o occur 79 between lOhOO and 14h00 may be due t o the a c t i v a t i o n of a f a c i l i t a t o r y or i n a c t i v a t i o n of an i n h i b i t o r y non-o x y t o c i n e r g i c pathway. For i n s t a n c e , i t i s known t h a t a c t i v i t y i n the v a s o p r e s s i n e r g i c system i n h i b i t s l o r d o s i s b e havior (Sodersten e t a l . 1983) and t h a t a pronounced d a i l y rhythm e x i s t s i n female, r a t s with estrogen implants which i s o p p o s i t e t o t h a t of the d a i l y v a s o p r e s s i n rhythm c o n t r o l l e d by the s u p r a c h i a s m a t i c nucleus (Hansen e t a l . 1979). T h i s d e c l i n e i n r e c e p t o r l e v e l s would imply t h a t s t u d i e s t h a t i n v e s t i g a t e d the e f f e c t s of OT on l o r d o s i s behavior 48 h a f t e r e s t r o g e n a d m i n i s t r a t i o n d i d so a t a time t h a t was not op t i m a l f o r t h i s type of i n v e s t i g a t i o n . Furthermore, when t h i s d e c l i n e i n c e n t r a l OT r e c e p t o r l e v e l s i s coupled w i t h h i g h endogenous OT l e v e l s , i t would render the i n t e r p r e t a t i o n of much of the e x i s t i n g l i t e r a t u r e on the e f f e c t s of OT on l o r d o s i s p r o b l e m a t i c . S t u d i e s i n v e s t i g a t i n g the e f f e c t s of o x y t o c i n on l o r d o s i s behavior i n the female r a t have f r e q u e n t l y ignored the p o s s i b l e i n f l u e n c e s of c i r c a d i a n rhythms (e.g. A r l e t t i and B e r t o l i n i 1985, Gorzalka and L e s t e r 1987, Schumacher e t a l . 1989, C a l d w e l l e t a l . 1989, C a l d w e l l e t a l . 1990, Schulze and Gorz a l k a 1991). Since many f a c t o r s a s s o c i a t e d w i t h r e p r o d u c t i v e behaviors i n the r a t c l e a r l y show c i r c a d i a n f l u c t u a t i o n s , e.g the onset and t e r m i n a t i o n of e s t r u s (Kuehn and Beach 1963), l o r d o s i s behavior i n female r a t s w i t h estrogen implants (Hansen e t a l . 1979), the 0 80 e x p r e s s i o n o f s e r o t o n i n r e c e p t o r s ( A k i y o s h i e t a l . 1989), s e r o t o n i n b i n d i n g (Wesemann e t a l . 1986) and s e r o t o n i n t u r n o v e r (Cohen and Wise 1988), and t h e r e l e a s e o f l u t e i n i z i n g hormone r e l e a s i n g hormone (Ching 1982), t h e e f f e c t s o f OT on l o r d o s i s may be c i r c a d i a l l y modulated. E v i d e n c e t o i n d i c a t e t h a t c i r c a d i a n f a c t o r s may be a t work i n t h e e x p r e s s i o n o f l o r d o s i s i s s u g g e s t e d p r i m a r i l y by t h e c o n t r o l groups o f E x p e r i m e n t s 7 and 8 (due t o l a r g e r samples a t t h e t i m e p o i n t s compared). Here i t was found t h a t l o r d o s i s s c o r e s d u r i n g t h e l i g h t phase t e n d e d t o be h i g h e r t h a n t h o s e o f t h e d a r k phase, i n p a r t i c u l a r , l o r d o s i s l e v e l s a t 06h00 (40 h a f t e r e s t r a d i o l benzoate a d m i n i s t r a t i o n ) were s i g n i f i c a n t l y e l e v a t e d w i t h r e s p e c t t o t h e l e v e l s a t 18h00 (28 h a f t e r e s t r a d i o l benzoate a d m i n i s t r a t i o n ) and lOhOO (44 h a f t e r e s t r a d i o l benzoate a d m i n i s t r a t i o n ) . These r e s u l t s c o n t r a s t w i t h t h o s e o f Hansen e t a l . (1979) who found a pronounced c i r c a d i a n rhythm, w i t h peak l o r d o s i s a c t i v i t y i n t h e d a r k phase, i n female r a t s w i t h e s t r o g e n i m p l a n t s . I t s h o u l d be n o t e d t h a t t h e r e s u l t s r e p o r t e d h e r e may r e f l e c t a t r a n s i t i o n phenomenon s i n c e t h e rhythm o b s e r v e d appears t o s h i f t from peak a c t i v i t y i n t h e l i g h t phase toward i n c r e a s e d a c t i v i t y i n t h e d a r k phase. As p o i n t e d out b e f o r e , t h i s r i s e and d e c l i n e i n l e v e l s o f r e c e p t i v i t y may r e f l e c t t h e a v a i l a b i l i t y o f OT r e c e p t o r s o r t h o s e o f s e r o t o n i n r e c e p t o r s . 81 However, i t cannot be concluded from the p r e s e n t data t h a t the a c t i v i t y of the o x y t o c i n e r g i c system or systems i n v o l v e d i n l o r d o s i s i s dependent on the time of day. S e v e r a l l i n e s of r e s e a r c h i n d i c a t e t h a t the c e n t r a l o x y t o c i n e r g i c systems may not show d a i l y rhythmic a c t i v i t y . For example, although t h e r e i s a d a i l y f l u c t u a t i o n i n the OT l e v e l s of c e r e b r o s p i n a l f l u i d (CSF) i n monkeys (Perlow 1982), such v a r i a t i o n s do not occur i n the r a t (Mens et a l . 1982) and furthermore, no d i u r n a l v a r i a t i o n occurs i n the l e v e l s of o x y t o c i n messenger RNA i n the s u p r a o p t i c and p a r a v e n t r i c u l a r n u c l e i of the hypothalamus (Burbach e t a l . 1988) . In a d d i t i o n , the c o n t r o l of p r o l a c t i n r e l e a s e by OT i s not dependent on the time of day (Arey and Freeman 1989). On the o t h e r hand, t h e r e i s evidence t h a t the a d m i n i s t r a t i o n of o x y t o c i n i n c r e a s e s s e r o t o n i n l e v e l s ( P f i s t e r and Muir 1989) . T h i s may then t r a n s l a t e i n t o c i r c a d i a n e f f e c t s of OT on l o r d o s i s because of the mentioned c i r c a d i a n rhythm expressed by 5-HT2 r e c e p t o r s (Moser and Redfern 1985). Thus, although the a c t i v a t i o n of the c e n t r a l o x y t o c i n e r g i c system(s) i n v o l v e d i n l o r d o s i s behavior show no d i r e c t c i r c a d i a n v a r i a t i o n s , they may a c t i v a t e systems t h a t are c i r c a d i a l l y rhythmic and i n such an i n d i r e c t manner cause l o r d o s i s b ehavior t o show rhythmic e f f e c t s . We b e l i e v e , t h e r e f o r e , t h a t s t u d i e s i n v e s t i g a t i n g the s e x u a l behavior of female r a t s should take i n t o account the p o s s i b l e o p e r a t i o n of c i r c a d i a n f a c t o r s i n t h e i r i n t e r p r e t a t i o n of the r e s u l t s . 82 Taken t o g e t h e r , l i k e the phenomenon of postpartum e s t r u s , the o x y t o c i n e r g i c f a c i l i t a t i o n of l o r d o s i s may be dependent on both the time of day and the l e n g t h of time s i n c e e s t r o g e n a d m i n i s t r a t i o n . I t i s w e l l known t h a t OT i s important d u r i n g p a r t u r i t i o n and t h a t plasma l e v e l s of t h i s hormone are e l e v a t e d d u r i n g and up t o one hour a f t e r p a r t u r i t i o n (Evans e t a l . 1989). However, postpartum e s t r u s does not occur u n t i l about 10 h a f t e r p a r t u r i t i o n or l a t e r , depending on the time of day of p a r t u r i t i o n ( G i l b e r t e t a l . 1985). I t appears t h a t by t h i s time plasma l e v e l s of OT had r e t u r n e d t o normal (Evans et a l . 1989). I f OT d i d f a c i l i t a t e l o r d o s i s behavior and was a l s o c e n t r a l l y a v a i l a b l e c o n c o m i t a n t l y w i t h p e r i p h e r a l r e l e a s e , one would have expected peak l e v e l s of postpartum mating t o occur w i t h i n a s h o r t p e r i o d a f t e r p a r t u r i t i o n . I t i s p o s s i b l e t h a t no r e c e p t i v i t y i s seen s h o r t l y a f t e r p a r t u r i t i o n because c e n t r a l OT r e c e p t o r s , which r e q u i r e estrogen f o r i n d u c t i o n (de K l o e t e t a l . 1986), may not be p r e s e n t . Estrogen l e v e l s are low d u r i n g pregnancy i n the r a t and s t a r t t o r i s e s h o r t l y b e f o r e p a r t u r i t i o n ( S l o t n i c k 1975). E s t r a d i o l , when ad m i n i s t e r e d by way of s i l a s t i c c apsule i m p l a n t a t i o n , causes i n c r e a s e d OT r e c e p t o r b i n d i n g i n the ventromedial hypothalamus w i t h i n 2 4 h and withdrawal r e s u l t s i n a s i g n i f i c a n t r e d u c t i o n i n OT r e c e p t o r b i n d i n g w i t h i n 24 h (Johnson e t a l . 1989). Furthermore, post-partum i n c r e a s e s i n o x y t o c i n r e c e p t o r s occur i n d i s c r e t e b r a i n r e g i o n s (Gelhard e t a l . 1986). T h i s supports the suggestion t h a t mating i s 83 seen o n l y a f t e r the i n d u c t i o n of c e n t r a l OT r e c e p t o r s and t h a t a c e r t a i n time i n t e r v a l i s r e q u i r e d f o r t h e i r i n d u c t i o n . In a d d i t i o n , postpartum mating does not occur t i l l r o u g h l y the beginning of the dark phase, a phenomenon t h a t i s c l e a r l y c i r c a d i a n . Whether t h i s e f f e c t i s due t o a c t i v i t y of the s e r o t o n i n system i s u n c l e a r g i v e n the f a c t t h a t 5-HT2 r e c e p t o r s are p r i m a r i l y expressed d u r i n g the l i g h t phase (Moser and Redfern 1985) and t h a t e s t r a d i o l can change the c i r c a d i a n rhythms of s e r o t o n i n t u r n o v e r expressed i n c e r t a i n b r a i n areas (Cohen and Wise 1988). In the l i g h t of the e x i s t i n g l i t e r a t u r e and those r e s u l t s r e p o r t e d i n t h i s paper, we t e n t a t i v e l y suggest t h a t sexual r e c e p t i v i t y i n female r a t s i s d e s c r i b e d by a model i n which an i n t e r v a l t i m e r i s coupled t o a c i r c a d i a n g a t i n g mechanism i n a manner analogous t o t h a t of postpartum mating ( G i l b e r t e t a l . 1985). In p a r t i c u l a r , we propose t h a t the i n t e r v a l timer c o n s t i t u t e s the process of o x y t o c i n r e c e p t o r (and perhaps peptide) s y n t h e s i s a c t i v a t e d by estrogen a d m i n i s t r a t i o n and t h a t the c i r c a d i a n g a t i n g mechanism i s another n e u r o t r a n s m i t t e r system ( p o s s i b l y the s e r o t o n i n or v a s o p r e s s i n system) t h a t shows c i r c a d i a n r h y t h m i c i t y and i s coupled t o the o x y t o c i n system. The p r e s e n t f i n d i n g s p o i n t t o a s e r i o u s need t o i n v e s t i g a t e the time course of OT r e c e p t o r i n d u c t i o n and OT p e p t i d e s y n t h e s i s i n response t o acute subcutaneous estrogen a d m i n i s t r a t i o n . For i n s t a n c e , t h e r e e x i s t s the p o s s i b i l i t y 84 t h a t the time course of OT r e c e p t o r i n d u c t i o n and/or OT p e p t i d e s y n t h e s i s may vary depending on the c e n t r a l s i t e i n v e s t i g a t e d . In a r e c e n t paper, C a l d w e l l e t a l . (1989) found a f a c i l i t a t i o n of l o r d o s i s i n response t o OT a d m i n i s t r a t i o n t o the medial p r e o p t i c area but not t o the vent r o m e d i a l hypothalamus. T h i s f i n d i n g , although s u b j e c t t o othe r i n t e r p r e t a t i o n s (Schulze and Gorzalka 1991), may a l s o be due t o a d i f f e r e n c e i n the time course of OT r e c e p t o r decay a t these two s i t e s . F i n a l l y , such responses of the o x y t o c i n e r g i c system a t p a r t i c u l a r c e n t r a l s i t e s t o estrogen a d m i n i s t r a t i o n may depend on both the time of day of t e s t i n g and the time e l a p s e d s i n c e estrogen a d m i n i s t r a t i o n as proposed i n the p r e c e d i n g paragraph. Unless these i s s u e s are c l a r i f i e d , the i n t e r p r e t a t i o n of s t u d i e s i n v e s t i g a t i n g the r o l e of the o x y t o c i n e r g i c system i n the c o n t r o l of l o r d o s i s w i l l be d i f f i c u l t . One would t h e r e f o r e suggest t h a t s t u d i e s i n v e s t i g a t i n g the e f f e c t s of OT and i t s analogues on l o r d o s i s behavior should c o n s i d e r both the time of day and the time s i n c e estrogen a d m i n i s t r a t i o n . GENERAL DISCUSSION The p r e s e n t s e r i e s of experiments concerning the c e n t r a l e f f e c t s of o x y t o c i n on l o r d o s i s has y i e l d e d s e v e r a l f i n d i n g s . O x y t ocin i n low c o n c e n t r a t i o n s i n h i b i t s l o r d o s i s when a d m i n i s t e r e d t o the l a t e r a l v e n t r i c l e , but not t h i r d v e n t r i c l e , of female r a t s primed with both estrogen and 85 progesterone. In s i m i l a r l y s t e r o i d - p r i m e d animals, o x y t o c i n i n f u s i o n s i n t o the MPOA enhance p r i m a r i l y l o r d o s i s frequency, i n f u s i o n s i n t o the VMH i n c r e a s e p r i m a r i l y l o r d o s i s d u r a t i o n w h i l e i n f u s i o n s i n t o the ACE extend l o r d o s i s d u r a t i o n but have no e f f e c t on l o r d o s i s frequency. The a d m i n i s t r a t i o n of a moderate o x y t o c i c a g o n i s t t o the l e f t l a t e r a l v e n t r i c l e i n h i b i t s LQ wh i l e a compound wit h potent a n t i - o x y t o c i c e f f e c t s and potent a n t i - v a s o p r e s s o r e f f e c t s a l s o i n h i b i t s LQ. F i n a l l y , the same o x y t o c i n a g o n i s t a d m i n i s t e r e d t o the l e f t l a t e r a l v e n t r i c l e s of female r a t s i n c r e a s e d l o r d o s i s frequency as e a r l y as 32 h a f t e r estrogen treatment b u t not 48 h l a t e r . The f i n d i n g s obtained from the i n f u s i o n of s e v e r a l d i f f e r e n t doses of o x y t o c i n i n t o v a r i o u s c e n t r a l s i t e s p o i n t t o the f a c t t h a t no s i n g l e b r a i n s i t e ' c o n t r o l s ' l o r d o s i s b ehavior, but t h a t s e v e r a l s i t e s c o n t r i b u t e toward the ge n e r a t i o n of d i f f e r e n t aspects of t h i s behavior. In t h i s r e gard, I have p o i n t e d out t h a t the VMH may be the s i t e where l o r d o s i s i s pe r m i t t e d or i n i t i a t e d , the MPOA c o n t r i b u t e s frequency i n f o r m a t i o n t o t h i s "behavior w h i l e the ACE may be r e s p o n s i b l e f o r the maintenance of the l o r d o s i s p o s t u r e once i n i t i a t e d . T h i s i n t e r p r e t a t i o n a l s o suggests a h i e r a r c h y of the s i t e s i n v o l v e d : i t i s l i k e l y t h a t t a c t i l e s t i m u l i a r i s i n g from c o n t a c t with the male induce the VMH t o produce ' l o r d o s i s s i g n a l s ' which i t then r e l a y s t o the MPOA and ACE. These two n u c l e i then r e l a y i n f o r m a t i o n t o the MCG 86 where a l l incoming s i g n a l s are i n t e g r a t e d t o produce the l o r d o s i s p o s t u r e . The r e s u l t s from Experiments 3 and 4 suggest t h a t o x y t o c i n may have e f f e c t s a t more than one c e n t r a l r e c e p t o r type. In a d d i t i o n t o the o x y t o c i n r e c e p t o r , o x y t o c i n i s known t o b i n d t o v a s o p r e s s i n and V 2 r e c e p t o r s (Lowbridge e t a l . 1976, Manning and Sawyer 1989). As d i s c u s s e d b e f o r e , a c t i v i t y a t V 2 - l i k e r e c e p t o r s l o c a t e d on o x y t o c i n neurons i n h i b i t o x y t o c i n r e l e a s e (Cheng and North 1989) and may i n h i b i t l o r d o s i s . Conversely, v a s o p r e s s i n binds t o the o x y t o c i n r e c e p t o r w i t h a pharmacologic a c t i v i t y about 3% t h a t of o x y t o c i n . T h e r e f o r e , the a d m i n i s t r a t i o n of a moderately h i g h dose of v a s o p r e s s i n can be c o n s i d e r e d t o be e q u i v a l e n t t o t h a t of a low dose of o x y t o c i n . V a s o p r e s s i n i n h i b i t s l o r d o s i s (Sodersten e t a l . 1983) and may consequently do so by a c t i n g i n the manner of a low dose of o x y t o c i n (Experiment 3). A f u r t h e r c o m p l i c a t i o n i s the f a c t t h a t i n the presence of Mg the a f f i n i t y of the OT r e c e p t o r f o r OT and some OT a g o n i s t s i s i n c r e a s e d (Antoni and Chadio 1989). I t has a l s o been e s t a b l i s h e d t h a t i n the presence of L i + the o x y t o c i n molecule changes i t s conformation t o resemble t h a t of v a s o p r e s s i n (Rholam et a l . 1985). I t i s t h e r e f o r e p o s s i b l e t h a t M g 2 + may cause a c o n f o r m a t i o n a l change i n the p e p t i d e and not the r e c e p t o r . T h i s may account f o r the f a c t t h a t a r g i n i n e v a s o p r e s s i n i s a more potent a g o n i s t a t OT r e c e p t o r s than o x y t o c i n i t s e l f i n the absence 87 of Mg but l e s s powerful i n the presence of Mg (Antoni and Chadio 1989). U n f o r t u n a t e l y , the r e s u l t s from Experiment 6 do not a l l o w an unambiguous i n t e r p r e t a t i o n of the r e s u l t s o b t a i n e d i n Experiments 3 and 4. What does become c l e a r from the r e s u l t s of experiments r e p o r t e d here and elsewhere, i s t h a t t h e r e may be a c o n s i d e r a b l e amount of ' c r o s s t a l k ' between the o x y t o c i n and v a s o p r e s s i n systems. Some r e s u l t s , presented i n Table 2, s t r o n g l y imply t h a t both the v a s o p r e s s i n and o x y t o c i n systems p l a y a r o l e i n the c o n t r o l of l o r d o s i s behavior. T a b l e 2 E f f e c t a t r e c e p t o r B e h a v i o r a l e f f e c t Reference Agent V l v 2 OT LQ OT 4 u/mg 4 u/mg 520 u/mg depr e s s e d / e l e v a t e d a/b AVP 3 69 u/mg 32 3 u/mg 14 u/mg depressed c GT-OT .01 u/mg .002 u/mg 166 u/mg depressed d VT pA =-7.96 ."01 u/mg pA =-8.52 depressed d PM-OT an t a g o n i s t depressed e dTM pA = -8.62 .31 u/mg pA =-6.62 e l e v a t e d e OT = o x y t o c i n , AVP = a r g i n i n e v a s o p r e s s i n , GT-to = [Thr4, Gly7] - o x y t o c i n , VT = [d(CH2)5, Tyr(OMe)2, 0rn8] -v a s o t o c i n , PM-to = [Penl, pMePhe2, Thr4, Orn8] - o x y t o c i n , dTM = [d(CH2)5, Tyr(Me)] - AVP; a = Experiment 3, b = 88 G o r z a l k a and L e s t e r 1987, c = Sodersten e t a l . 1983, d = Experiment 6, e = C a l d w e l l e t a l . 1990. I f o n l y one of the OT or r e c e p t o r types mediated l o r d o s i s behavior, one would have expected t o see s i m i l a r e f f e c t s on LQ upon t h e i r a d m i n i s t r a t i o n . Given the f a c t t h a t o p p o s i t e e f f e c t s are obtained, one i s l e a d t o conclude t h a t at l e a s t two systems with o p p o s i t e a c t i o n s mediate l o r d o s i s . A p o s s i b l e way i n which these two p e p t i d e r g i c systems may i n t e r a c t i s the f o l l o w i n g . Oxytocin may a c t as an a g o n i s t a t o x y t o c i n r e c e p t o r s and as a p a r t i a l a g o n i s t a t v a s o p r e s s i n r e c e p t o r s (e.g. r e c e p t o r s ) , w h i l e v a s o p r e s s i n may a c t as an a g o n i s t a t v a s o p r e s s i n r e c e p t o r s and as a p a r t i a l a g o n i s t at o x y t o c i n r e c e p t o r s . Low c o n c e n t r a t i o n s of o x y t o c i n may then i n h i b i t l o r d o s i s because t h e r e may be l i t t l e e f f e c t a t o x y t o c i n r e c e p t o r s w h i l e the t o n i c i n h i b i t i o n of v a s o p r e s s i n would be enhanced, e s p e c i a l l y i f o x y t o c i n a t low c o n c e n t r a t i o n s i s more l i k e l y t o b i n d a t v a s o p r e s s i n r e c e p t o r s . High c o n c e n t r a t i o n s of o x y t o c i n would a c t i v a t e the l o r d o s i s f a c i l i t a t i n g o x y t o c i n system and tend t o d i s r u p t the l o r d o s i s i n h i b i t i n g v a s o p r e s s i n system. Low c o n c e n t r a t i o n s of exogenous v a s o p r e s s i n would augment t o n i c v a s o p r e s s i n i n h i b i t i o n and have l i t t l e e f f e c t a t the OT system, w h i l e h i g h c o n c e n t r a t i o n s of v a s o p r e s s i n would f u r t h e r a c t i v a t e the i n h i b i t o r y v a s o p r e s s i n system and tend t o d i s r u p t the f a c i l i t a t o r y o x y t o c i n system. The net e f f e c t 89 o f such a system would be t h a t l o r d o s i s o n l y o c c u r s when h i g h l e v e l s o f o x y t o c i n a r e p r e s e n t . F i n a l l y , t h e r e s u l t s from E x p e r i m e n t s 7 and 8 i n d i c a t e t h a t t e m p o r a l f a c t o r s s h o u l d be t a k e n i n t o c o n s i d e r a t i o n when e v a l u a t i n g t h e e f f e c t s o f o x y t o c i n on l o r d o s i s . The c i r c a d i a n rhythms e x p r e s s e d by t h e v a s o p r e s s i n e r g i c and s e r o t o n e r g i c systems (Mens e t a l . 1982) and t h e t i m e c o u r s e o f a c t i v a t i o n o f t h e o x y t o c i n e r g i c system (Experiment 8) may be i m p o r t a n t f a c t o r s i n t h e r e g u l a t i o n o f l o r d o s i s b e h a v i o r g i v e n t h a t i n t e r a c t i o n s between t h e s e two systems can o c c u r (see S e c t i o n I I I ) . S i n c e t e m p o r a l f a c t o r s a r e known t o be i m p o r t a n t i n t h e s e r o t o n e r g i c and v a s o p r e s s i n e r g i c systems, t h e y c o u l d a l s o e x t e n d t o o t h e r n e u r o t r a n s m i t t e r systems i n v o l v e d i n l o r d o s i s . When a d m i n i s t e r e d i n t o t h e LV, t h e o x y t o c i n a g o n i s t employed e l e v a t e d LQ s c o r e s w i t h r e s p e c t t o c o n t r o l i n f u s i o n s as e a r l y as 32 h a f t e r e s t r o g e n i n j e c t i o n . U n f o r t u n a t e l y , l o r d o s i s d u r a t i o n s c o r e s have n o t been o b t a i n e d and i t i s not c e r t a i n whether LD would a l s o show i n c r e a s e s a t t h i s t i m e . I t would be i n t e r e s t i n g t o e s t a b l i s h whether t h e t i m e c o u r s e o f f u n c t i o n a l o x y t o c i n r e c e p t o r i n d u c t i o n by e s t r o g e n a d m i n i s t r a t i o n f o l l o w s t h e same p a t t e r n i n t h e MPOA, t h e VMH and t h e ACE. Whether t h e e f f e c t s o f OT on l o r d o s i s a r e medi a t e d by o t h e r n e u r o t r a n s m i t t e r systems, e.g. t h e s e r o t o n i n system, o r whether OT mediates t h e e f f e c t s on l o r d o s i s o f o t h e r 90 n e u r o t r a n s m i t t e r systems i s not c u r r e n t l y known. These e x p e r i m e n t s ( r e p o r t e d i n S e c t i o n s I I I and IV) s u g g e s t t h a t i n t e r a c t i o n s do o c c u r w i t h o t h e r n e u r o t r a n s m i t t e r systems and t h e o x y t o c i n e r g i c system may be o n l y one o f s e v e r a l n e u r o t r a n s m i t t e r systems m e d i a t i n g l o r d o s i s . I t i s a l s o p o s s i b l e t h a t a s p e c i f i c a s p e c t o f l o r d o s i s b e h a v i o r , such as d o r s i f l e x i o n , i s mediated by a s p e c i f i c n e u r o t r a n s m i t t e r system, w h i l e a d i f f e r e n t a s p e c t o f l o r d o s i s , such as t h e maintenance o f t h e l o r d o s i s p o s t u r e , i s med i a t e d by a n o t h e r n e u r o t r a n s m i t t e r system. A s i d e from t h e c l i n i c a l a p p l i c a t i o n o f OT d u r i n g l a b o r and l a c t a t i o n , OT may have o t h e r p o t e n t i a l c l i n i c a l a p p l i c a t i o n s as w e l l . I t i s known t h a t OT p l a y s a r o l e i n t h e development o f drug t o l e r a n c e (see Kovacs 1986 and A r g i o l a s and Gessa 1991 f o r r e v i e w s ) and t h a t e s t r o g e n s t i m u l a t e d n e u r o p h y s i n s ( p r e c u r s o r m o l e c u l e s o f o x y t o c i n and v a s o p r e s s i n ) a r e i n c r e a s e d more t h a n 100% i n a l l p s y c h i a t r i c p a t i e n t s who r e c o v e r s a t i s f a c t o r i l y from e l e c t r o c o n v u l s i v e t h e r a p y ( S c o t t e t a l . 1986). As f a r as s e x u a l b e h a v i o r i s con c e r n e d , OT c o n c e n t r a t i o n s i n CSF r i s e s h o r t l y a f t e r e j a c u l a t i o n i n male r a t s (Hughes e t a l . 1987), w h i l e i n t r a c e r e b r o v e n t r i c u l a r i n f u s i o n s o f OT i n male r a t s produce r e p e a t e d e p i s o d e s o f p e n i l e e r e c t i o n s and yawning ( M e l i s e t a l . 1989). I n human f e m a l e s , t h o s e t a k i n g o r a l c o n t r a c e p t i v e s showed i n c r e a s e d o x y t o c i n plasma l e v e l s d u r i n g t h e m e n s t r u a l c y c l e compared t o t h o s e n o t t a k i n g o r a l 91 c o n t r a c e p t i v e s (Uvnas-Moberg e t a l . 1989). Given the involvement of OT i n reinforcement (see Kovacs 1986) and se x u a l f u n c t i o n , and the p o s s i b l e involvement i n emotional d y s f u n c t i o n ( S c o t t e t a l . 1986), one c o u l d s p e c u l a t e t h a t therapy aimed a t the o x y t o c i n e r g i c system may address problems a s s o c i a t e d with both emotional and se x u a l d y s f u n c t i o n . I t i s furthermore tempting t o c o n j e c t u r e t h a t the e f f e c t i v e n e s s of l i t h i u m i n the treatment of manic and b i p o l a r depressed p a t i e n t s (Cooper e t a l . 1986) can be a s c r i b e d t o i t s e f f e c t s on the conformation of the OT molecule (Rholam e t a l . 1985) r e s u l t i n g i n a modulation of o x y t o c i n e r g i c f u n c t i o n . Whether any of the f i n d i n g s r e p o r t e d here w i l l have c l i n i c a l r e l e v a n c e , remains t o be determined. I t i s expected though t h a t the e f f e c t of OT dose on se x u a l b e h a v i o r ( S e c t i o n I) and the temporal parameters of OT a c t i o n ( S e c t i o n IV) may prove u s e f u l i n any a p p l i c a t i o n of OT-based therapy. Oxytocin has been shown t o have m a n i f o l d b e h a v i o r a l e f f e c t s (see Kovacs 1986 and A r g i o l a s and Gessa 1991 f o r reviews) and the r e s u l t s o b tained i n the experiments r e p o r t e d here need t o be i n t e r p r e t e d i n the l a r g e r c o n t e x t of o x y t o c i n ' s v a r i e t y of b e h a v i o r a l e f f e c t s . To put i t more f o r c e f u l l y , i f the c e n t r a l i n f u s i o n of o x y t o c i n can l e a d t o one of s e v e r a l d i f f e r e n t b e haviors, some of which may be mutu a l l y incompatible, how i s a p a r t i c u l a r behavior s e l e c t e d f o r execution? 92 There i s evidence i n d i c a t i n g t h a t independent o x y t o c i n e r g i c systems may e x i s t , f o r i n s t a n c e , OT i s 0 r e l e a s e d i n t o the c i r c u l a t i o n from the p o s t e r i o r p i t u i t a r y by p a r a v e n t r i c u l a r and s u p r a o p t i c n e u r o s e c r e t o r y c e l l s t h a t do not p r o j e c t t o other b r a i n areas ( B u i j s e t a l . 1985) and i t has been shown t h a t l e v e l s of OT i n the CSF and i n blood are independently r e g u l a t e d (Robinson 1983). I t i s f u r t h e r supported by the f a c t t h a t immunostaining of p e r i v a s c u l a r neurons i n the p r e o p t i c r e g i o n disappear by day two postpartum and t h a t another p o p u l a t i o n of o x y t o c i n e r g i c neurons, not a s s o c i a t e d with blood v e s s e l s , appear i n these r e g i o n s ( J i r i k o w s k i e t a l . 1989). Furthermore, on l y 30% of OT neurons i n the VMH conc e n t r a t e r a d i o l a b e l l e d estrogen ( M o r r e l l and P f a f f 1982) and i n some b r a i n r e g i o n s , but not i n o t h e r s , OT r e c e p t o r s are modulated by estrogen (de K l o e t e t a l . 1986). Separate o x y t o c i n e r g i c systems may c o n t r o l separate b e h a v i o r s and some of these behaviors may be estrogen dependent w h i l e o t h e r s may not be. I t has been demonstrated t h a t OT induces maternal behaviors such as pup r e t r i e v a l , pup l i c k i n g and nest b u i l d i n g (see Mena e t a l . 1985). S e v e r a l s t u d i e s a l s o p o i n t t o the p o s s i b i l i t y t h a t maternal a g g r e s s i o n may be r e g u l a t e d by OT. Mayer and R o s e n b l a t t (1987) found t h a t treatments known to reduce s e n s i t i z a t i o n l a t e n c i e s t o pups a l s o l e a d t o i n c r e a s e d a g g r e s s i v e n e s s of 93 the female toward a novel male. Furthermore, s u c k l i n g s t i m u l a t i o n by the pups, which r e l e a s e s OT (Mena e t a l . 1985), i s e s s e n t i a l f o r the i n i t i a t i o n of m a l e - d i r e c t e d a g g r e s s i o n i n mice d u r i n g the time immediately post partum (Garland and Svare 1988). In r a t s , l e s i o n s of the p e r i p e d u n c u l a r area of the l a t e r a l midbrain, a p a r t of the ascending m i l k - e j e c t i o n pathway, d i m i n i s h e d maternal a g g r e s s i o n (Hansen and F e r r e i r a 1986). Although no d i r e c t evidence e x i s t s t o i n d i c a t e c o n t r o l of maternal a g g r e s s i o n by OT, the a v a i l a b l e data are too p r o v o c a t i v e t o i g n o r e . E x a c t l y t h i s c r e a t e s a dilemma: i t i s not c l e a r how maternal a g g r e s s i o n toward a novel male i s compatible w i t h c o p u l a t i o n w i t h a n o v e l male - g i v e n t h a t both behaviors may be e l i c i t e d by OT. Oxy t o c i n appears t o have e f f e c t s on many be h a v i o r s . Some of these behavio r s and some components of some of these b e h a v i o r s may be incompatible. T h i s n e c e s s i t a t e s c o o r d i n a t i o n among the v a r i o u s o x y t o c i n systems. 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