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Effect of alpha-melanocyte stimulating hormone on lordosis : role of estrogen, progesterone, and serotonin Raible, Lyn Helene 1985

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EFFECT OF ALPHA-MELANOCYTE STIMULATING HORMONE ON LORDOSIS: ROLE OF ESTROGEN, PROGESTERONE,AND SEROTONIN by LYN HELENE RAIBLE B.A. San F r a n c i s c o S t a t e U n i v e r s i t y M.A. The U n i v e r s i t y of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of 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 as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA (c) Lyn Helene R a l b l e , 1985 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f P s y c h o l o g y  The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date August 15, 1985 i i A b s t r a c t The p r e s e n t s e r i e s of s t u d i e s was und e r t a k e n to det e r m i n e the e f f e c t s o f p e r i p h e r a l l y and c e n t r a l l y a d m i n i s t e r e d a l p h a -melanocyte s t i m u l a t i n g hormone (MSH) on l o r d o s i s and to i n v e s t i g a t e some of the mechanisms u n d e r l y i n g these e f f e c t s . The r e s u l t s of Experiments 1-5 ( S e c t i o n I) i n d i c a t e d t h a t , when c o n f o u n d i n g f a c t o r s were m i n i m i z e d , p e r i p h e r a l l y a d m i n i s t e r e d MSH f a c i l i t a t e d r e c e p t i v i t y . C e n t r a l l y a d m i n i s t e r e d MSH was found to produce b o t h a l o n g and a s h o r t term i n h i b i t o r y e f f e c t . Experiment 6 ( S e c t i o n I I ) t e s t e d the h y p o t h e s i s t h a t the f a c i l i t a t o r y a c t i o n of p e r i p h e r a l l y a d m i n i s t e r e d MSH was due t o an MSH-induced r e l e a s e of p r o g e s t e r o n e o r some o t h e r f a c i l i t a t o r y a d r e n a l s t e r o i d . R e s u l t s i n d i c a t e d t h a t , w h i l e a d r e n a l e c t o m y per se d i d not i n h i b i t l o r d o s i s , i t b l o c k e d the f a c i l i t a t o r y a c t i o n of MSH, s u p p o r t i n g the h y p o t h e s i s . I n Exp e r i m e n t s 7-9 ( S e c t i o n I I I ) , the r o l e of e s t r o g e n and p r o g e s t e r o n e i n the i n h i b i t o r y a c t i o n s of MSH was examined. The r e s u l t s of these s t u d i e s s u g g e s t e d t h a t both e s t r o g e n and p r o g e s t e r o n e are n e c e s s a r y f o r the s h o r t term i n h i b i t o r y a c t i o n of MSH. However, the l o n g term i n h i b i t o r y a c t i o n of MSH appears to be due, i n p a r t , t o an MSH-induced decrease i n the a v a i l a b i l i t y of c y t o p l a s m i c p r o g e s t i n r e c e p t o r s . In Exp e r i m e n t s 10-15 ( S e c t i o n I V ) , the r o l e o f s e r o t o n i n i n the p r o d u c t i o n of the i n h i b i t o r y a c t i o n s of MSH was examined. P a r a c h l o r o p h e n y l -a l a n i n e (PCPA), a s e r o t o n i n d e p l e t o r , was found t o p r e v e n t the lo n g term i n h b i t o r y a c t i o n of MSH. In a d d i t i o n , the i n h i b i t o r y e f f e c t s of PCPA or p i r e n p e r o n e , a s e r o t o n i n type I I r e c e p t o r i i i a n t a g o n i s t , d i d not summate w i t h the inh i b i tory. a c t i o n of MSH. T h i s s u g g e s t e d t h a t s e r o t o n i n type I I r e c e p t o r s were i n v o l v e d i n the p r o d u c t i o n of the i n h i b i t o r y a c t i o n s of MSH. In Experiment 12, q u i p a z i n e , a s e r o t o n i n type I I a g o n i s t , was found t o a t t e n u a t e f u l l y the s h o r t term I n h i b i t o r y a c t i o n of MSH. However, q u i p a z i n e d i d not f u l l y a t t e n u a t e the l o n g term i n h i b i t o r y a c t i o n of MSH, s u g g e s t i n g t h a t the s h o r t and l o n g term i n h i b i t o r y a c t i o n s o f MSH are mediated t h r o u g h d i f f e r e n t mechanisms. T h i s p o s s i b i l i t y was s u p p o r t e d by the r e s u l t s of Experiment 13, which i n d i c a t e d t h a t 20 ng MSH produced a l o n g term, but not a s h o r t term, i n h i b i t o r y e f f e c t . The r e s u l t s o f Experiment 14 i n d i c a t e d t h a t s u b t h r e s h o l d doses of p i r e n p e r o n e and of MSH, when a d m i n i s t e r e d t o g e t h e r , would i n h i b i t r e c e p t i v i t y . Experiment 15 i n d i c a t e d t h a t t h i s i n h i b i t i o n c o u l d be a t t e n u a t e d by q u i p a z i n e . Thus, the f o l l o w i n g c o n c l u s i o n s can be drawn: 1) the f a c i l i t a t o r y a c t i o n of p e r i p h e r a l l y a d m i n i s t e r e d MSH i s p r o b a b l y mediated by an MSH-induced r e l e a s e of p r o g e s t e r o n e from the a d r e n a l s , 2) the s h o r t term i n h i b i t o r y a c t i o n of MSH i s mediated, to a l a r g e e x t e n t , by an MSH-induced de c r e a s e i n s e r o t o n i n type I I a c t i v i t y , and 3) the l o n g term i n h i b i t o r y a c t i o n of MSH i s mediated, i n p a r t , by an MS H / s e r o t o n i n - i n d u c e d d e c r e a s e i n the a v a i l a b i l i t y of p r o g e s t i n r e c e p t o r s . In a d d i t i o n , i t was h y p o t h e s i z e d t h a t : 1) p r o g e s t e r o n e a c t s i n the MRF t o i n c r e a s e s e r o t o n i n type I I a c t i v i t y . Thus, MSH-induced d e c r e a s e s i n s e r o t o n i n type I I a c t i v i t y and i n p r o g e s t i n r e c e p t o r s p r o b a b l y o c c u r a t t h i s l o c a t i o n , and 2) e s t r o g e n a c t s i n the AH-POA to decrease s e r o t o n i n type I i v a c t i v i t y . T h e r e f o r e , any a c t i o n s of MSH on s e r o t o n i n type I a c t i v i t y o r on e s t r o g e n r e c e p t o r s i s l i k e l y t o o c c u r i n t h i s r e g i o n . F i n a l l y , i t was s u g g e s t e d t h a t MSH p l a y s a r o l e i n the i n d u c t i o n and maintenance of pseudopregnancy, t h e r e b y p r o v i d i n g MSH w i t h a f u n c t i o n a l r o l e i n the r e g u l a t i o n of r e c e p t i v e s t a t e s . V TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v l l LIST OF FIGURES vi i i ACKNOWLEDGEMENTS x INTRODUCTION 1 SECTION I - The Search for the MSH Effect 12 General Methods 12 Experiment 1 15 Experiment 2 22 Experiment 3 24 Experiment 4 31 Experiment 5 37 Discussion: Section I 38 SECTION II - MSH and Receptivity: Role of Adrenal Progesterone 43 Experiment 6 45 Discussion: Section II 46 SECTION III - MSH and Receptivity: Role of Estrogen and Progesterone 49 General Methods 56 Experiment 7 57 Experiment 8 63 Experiment 9 67 Discussion: Section III 72 SECTION IV - MSH and Receptivity: Role of Serotonin 76 General Methods 81 Experiment 10 81 v i Experiment 11 86 Experiment 12 91 Experiment 13 95 Experiment 14 102 Experiment 15 106 Discussion: Section IV 110 GENERAL DISCUSSION 115 REFERENCES 134 v i i LIST OF TABLES Table I. The temporal effects of subcutaneously administered MSH on receptivity 25 Table II. The temporal effects of centrally and peripherally administered MSH on receptivity 33 v i i i LIST OF FIGURES Figure 1. Pro-opiocortin and related peptides 3 Figure 2. A r t i f a c t u a l e f f e c t s of threshold estrogen and progesterone doses and repeated tests on r e c e p t i v i t y < 17 Figure 3. The e f f e c t s of c e n t r a l l y and peripherally administered MSH on r e c e p t i v i t y 20 Figure 4. The e f f e c t s of c e n t r a l l y and peripherally administered MSH on r e c e p t i v i t y 28 Figure 5. The e f f e c t s of c e n t r a l l y and peripherally administered MSH on r e c e p t i v i t y 34 Figure 6. The e f f e c t s of subcutaneously administered MSH on r e c e p t i v i t y in subjects displaying low levels of sexual responding 39 Figure 7. The e f f e c t s of subcutaneously administered MSH on r e c e p t i v i t y in ovariectomized and ovariectomized-adrenalectomized subjects 47 Figure 8. The e f f e c t s of MSH on r e c e p t i v i t y in subjects receiving estrogen alone 60 Figure 9. The e f f e c t s of estrogen and progesterone dose on the MSH-induced i n h i b i t i o n of r e c e p t i v i t y ... 65 Figure 10. The e f f e c t of increased progesterone dose on the long term inhibitory action of MSH on r e c e p t i v i t y 70 Figure 11. The e f f e c t s of MSH and parachloropheny1-alanine on r e c e p t i v i t y 84 Figure 12. The e f f e c t s of MSH and pirenperone on r e c e p t i v i t y 88 Figure 13. The e f f e c t s of MSH and quipazine on r e c e p t i v i t y 93 Figure 14. The dose-response e f f e c t s of pirenperone on r e c e p t i v i t y 97 Figure 15. The dose-response e f f e c t s of MSH on r e c e p t i v i t y 100 Figure 16. The e f f e c t s of subthreshold doses of MSH and pirenperone on r e c e p t i v i t y 104 ix Figure 17. The effects of quipazine on the inhibition of receptivity produced by subthreshold doses of MSH and pirenperone 108 Figure 18. Serotonergic and hormonal influences on receptivity 125 Figure 19. A model of MSH action 128 X ACKNOWLEDGEMENTS I would like to thank my supervisor. Dr. Boris Gorzalka, for his assistance, encouragement, honesty, and friendship throughout my tenure as a graduate student. I would also like to thank Dr. Rod Wong for his a b i l i t y to listen, his invaluable assistance in times of s t r i f e , and for his willingness to help me broaden my knowledge of ethology. To George 'HB' Renfrey, who hardly batted an eyelid when I threw half of my comprehensive reading material across the room and who was always there to provide emotional support, I give my thanks, my love, my comprehensive reading material, and the portion of cortex that I blew reading i t . In addition, I thank Dr. Phil Smith and Dr. Fred Valle for their suggestions with regards to this manuscript. Finally, I would like to thank two of my undergraduate instructors: Marc Russel, for imparting to me some of his enthusiasm for the fi e l d of psychology, and Dr. Richard Hunderfund, for renewing my enthusiasm for biology and zoology. This research was supported by a Natural Sciences and Engineering Research Council of Canada operating grant held by Dr. Boris B. Gorzalka. 1 Effect of alpha-Melanocyte Stimulating Hormone on Lordosis: Role of Estrogen, Progesterone, and Serotonin Since the discovery that gut and pituitary peptides are also found in the brain, the role of peptides In the regulation of behaviour has been a topic of intensive investigation. Of particular interest are the neuropeptides, those peptides that affect the nervous system and are present in neural tissue (de Wied & Jolles, 1982; de Wied, van Wimersma Greidanus, & Bohus, 1974). Peptides produced both by the brain and pituitary (e.g., adrenocorticotrophic hormone (ACTH), alpha-melanocyte stimulating hormone (MSH), beta-1ipotropin, endorphins, enkephalins) have particularly intrigued researchers since they appear to be involved in the mediation of learning and memory, analgesia, sleep, aggression, and sexual activity (de Wied & Jolles, 1982; Gray & Gorzalka, 1980; Krieger & Liotta, 1979; Thody, 1980). de Wied et a l . (1974) have suggested that the behavioural effects of these peptides result from a direct, rather than an indirect, action on the central nervous system. This contrasts with the effects of peptides such as luteinizing hormone (LH) that act through peripheral mechanisms. One example of the peripheral action of peptides can be seen in the regulation of gonadal hormone production and secretion. Gonadal steroids such as estrogen and testosterone are essential for the induction and maintenance of sexual activity. However, the production of these steroids is regulated, to a large extent, by peptides secreted by the pituitary. In rodents, the phases of the estrous cycle are controlled by the secretion of LH and 2 f o l l i c l e stimulating hormone (FSH) by the pituitary (Brown-Grant, 1971; Gorski, 1974, 1979). In the early stages of the estrous cycle, the pituitary secretes FSH, which stimulates f o l l i c u l a r growth and the secretion of estrogen by the granulosa cells of the f o l l i c l e (Dorrlngton, 1979). Estrogen from the granulosa cells appears to Initiate the 'ovulatory surge* of LH from the pituitary (Brown-Grant, 1971; Gorski, 1974). The LH surge causes the f o l l i c l e to rupture and release the ovum. The continued action of LH results in the transformation of the f o l l i c l e into the corpus luteum, which secretes progesterone (Dorrington, 1979). Estrogen and progesterone act on the brain to induce receptivity. Thus, both LH and FSH, via their regulation of estrogen and progesterone production, play an important role in the induction and maintenance of sexual behaviour. Other peptides may play a modulatory rather than a regulatory role in regards to sexual activity. For example, rather than directly affecting the production of gonadal steroids, they may modify or counteract the actions of gonadal steroids in the brain. Of particular interest is the pituitary peptide pro-opiocortin, which serves as a precursor to several neuropeptides, many of which alter sexual responding. Pro-opiocortln Is cleaved Into ACTH and beta-1lpotropic hormone (LPH; de Wied & Jolles, 1982). These peptides are further cleaved into MSH (from ACTH) and the . endorphins and enkephalins (from LPH; de Wied & Jolles, 1982; see Figure 1). Although the total number of studies examining the effects of these and other peptides on sexual activity Is relatively small, there is Increasing evidence that peptides act 3 Figure 1. Pro-opiocortin and related peptides. Pro-opiocortin contains the entire adrenocorticotrophic hormone <ACTH) and beta-1ipotrophic hormone C/^-LPH) molecules. Within the ACTH molecule is the amino acid sequence of alpha-melanocyte stimulating hormone (oC-MSH) as well as the ACTH fragment ACTH4-10. Within they«£?-LPH molecule are the amino acid sequences for yd?-MSH,/£?-endorphin, ACTH4-10, and met-enkephalin. The numbers listed reflect the location and sequence of the amino acids comprising each peptide within the pro-opiocortin molecule. PRO-OPIOCORTIN 13 «s*MSH 41 58 61 91 y£? MSH ^-ENDORPHIN 4 10 ACTH 4-10 47 53 61 65 ACTH, i n Met-ENKEPHALIN 4-10 5 both centrally and peripherally to alter sexual responding. Most research concerned with peptides and sexual behaviour has focussed on female sexual activity. However, a few studies have examined the effects of peptides on male sexual behaviour. Both beta-endorphin and an analogue of met-enkephalin have been reported to inhibit sexual activity in the male rat (Gessa, Paglietti, & Pellegrini Quarantotti, 1979? Meyerson & Terenius, 1977). On the other hand, central administration of ACTH has been reported to e l i c i t erection and ejaculation in male mammals (Bertolini, Gessa, & Ferrari, 1975). When examining the effects of peptides on sexual receptivity in female rats, researchers have tended to use the lordosis response as their index of receptivity. Lordosis is characterized by a concave arching of the back and dorsiflexion of the t a i l , which exposes the perineal region, f a c i l i t a t i n g penetration by the male. Studies in female rats indicate that centrally administered beta-endorphin inhibits sexual receptivity (SirinathsinghjI, 1984; SlrlnathslnghjI, Whittington, Audsley, & Fraser, 1983; Wiesner & Moss, 1984). Furthermore, the inhibitory action of beta-endorphin may be mediated through a beta-endorphin- induced decrease in gonadotropin-releasing hormone (GnRH; Drouva, Epelbaum, Tapia-Arancibia, Leplante, & Kordon, 1981; Sirinathsinghji, 1984). GnRH has been found to fac i l i t a t e receptivity (Moss & McCann, 1973; Pfaff, 1973; Risklnd & Moss, 1979; Thus, beta-endorphin may decrease receptivity by eliminating the stimulating effect of GnRH on sexual responding. Finally, the central administration of cortlcotropin-releasing 6 factor (CRF) strongly inhibits receptivity, apparently by releasing beta-endorphin (Sirinathsinghji, Rees, Rivier, & Vale, 1983). It is also conceivable that a CRF-induced release of ACTH may contribute to the inhibitory action of CRF. A large portion of the research examining the effects of peptides on female sexual behaviour has focussed on the effects of ACTH and its fragments. Although the central administration of ACTH appears to fa c i l i t a t e sexual receptivity in the female rabbit (Baldwin, Haun, & Sawyer, 1974), it is reported to inhibit (de Catanzaro, Gray, & Gorzalka, 1981) or have no effect on receptivity in the female rat (Dudley, Jamison, & Moss, 1982; Thody, Wilson, & Everard, 1981; Wilson, Thody, & Everard, 1979). Subcutaneous administration of ACTH facilitates receptivity in the estrogen-primed, ovariectomized but not ovariectomized-adrenalectomized female rat (de Catanzaro et a l . , 1981; de Catanzaro & Gorzalka, 1980). The rat adrenal gland releases progesterone in response to ACTH (Feder & Ruf, 1969) and, as progesterone acts synergistical1y with estrogen to fac i l i t a t e receptivity, it is likely that the ACTH-induced f a c i l i t a t i o n of receptivity is due to this action. Adrenalectomy would remove this source of progesterone and thus abolish the ACTH effect. The 13 amino acid sequence of MSH is identical to the i n i t i a l 13 amino acids of ACTH (Marks, Stern, & Kastin, 1976; Schwyzer & Eberle, 1977). This raises the possibility that the effects of ACTH on receptivity are produced by its MSH fragment. In support of this possibility is the finding that the peripheral administration of either MSH or the fourth through tenth amino 7 acid sequence of ACTH (ACTH4-10) facilitates receptivity (Wilson et a l . , 1979). Because peripheral administration of ACTH also faci l i t a t e s receptivity (de Catanzaro et a l . , 1981; de Catanzaro & Gorzalka, 1980), it is possible that the MSH fragment of ACTH is responsible for the effects of ACTH on receptivity. However, other data suggest that MSH and ACTH do not always exert similar effects on receptivity. Centrally administered MSH has been found to facil i t a t e receptivity in female rats displaying low levels of sexual responding without altering sexual activity In females displaying high levels of receptivity (Thody et a l . , 1981). In contrast, even in females displaying low levels of receptivity, centrally administered ACTH has been reported to inhibit receptivity (de Catanzaro et a l . , 1981). Furthermore, although peripherally administered MSH has been reported to faci l i t a t e receptivity in animals displaying low levels of sexual responding, an ACTH-like effect, it was found to inhibit sexual responding in females displaying high levels of sexual activity (Everard, Wilson, & Thody, 1977; Thody et a l . , 1981; Thody, Wilson, & Everard, 1979). Thody et a l . (1981) suggested that the stimulatory effect of MSH on receptivity is due to a potentiation by MSH of the effects of estrogen. They further suggest that, because the inhibitory action of MSH is observed after subcutaneous but not intracerebroventricular MSH administration, the effect must be peripherally mediated (Thody et a l . , 1981). However, the results of Thody and coworkers are questionable for reasons discussed below. Thody and coworkers (Everard et a l . , 1977; Thody et a l . . 8 1979, 1981) employ a design in which one half of the subjects receive f i r s t an MSH test and then, one week later, a saline test. The remaining subjects receive tests in the reverse order. Subjects are then assigned to a high or low receptivity group on the basis of the single saline score. In addition, the design used by Thody's group requires the use of threshold doses of estrogen and progesterone, i.e., identical doses that will produce both high levels of receptivity in some animals and low levels in other animals due to individual differences in hormone sensitivity. Observations in our laboratory indicate that threshold doses of estrogen and progesterone may produce both high and low levels of receptivity in the same animal tested once a week for two weeks. This in itself is not a major problem because animals displaying an Increase in receptivity one week will be balanced by other animals displaying a decrease in receptivity. For example, while one subject might drop from a high level of receptivity to a low level of receptivity over two weekly tests, another animal might go from a low to a high level of receptivity. The mean level of receptivity within the group would remain unchanged because the decrease displayed by one animal will be balanced by the increase displayed by another animal. This is no longer the case when the animals are divided into high and low receptivity subgroups, as was done by Thody's group. If Thody and coworkers had employed multiple control (saline) tests, they would have been able to identify animals with unstable scores between tests. However, the design used by Thody and coworkers precludes the identification of animals with 9 unstable scores and also prevents those animals showing an increase in receptivity from being balanced by those displaying a decrease since these animals now belong to separate groups. In a study to be discussed in more detail later, I found that this procedure, when used with pairs of saline scores, consistently produced a significant ' f a c i l i t a t i o n ' and, less consistently, an 'Inhibition' of receptivity, even though no drug was ever administered. Thus?, the results of Thody and coworkers on the effects of MSH may be due, to some degree, to experimental artifa c t . However, since Thody and coworkers obtained somewhat different results after the peripheral and central administration of MSH, it seems unlikely that their results were entirely artifactual. Thody et a l . (1981) found that, centrally administered MSH did not inhibit receptivity. This led them to suggest that the inhibitory action of subcutaneously administered MSH must be peripherally mediated. However, previous research in our laboratory (Parsons & Gorzalka, unpublished observations) has indicated that, in contrast to the results of Thody et a l . (1981), MSH does exert an inhibitory effect when administered centrally. In light of these findings plus the possibility that Thody et al.'s (1981) results may be due, in part, to experimental artifact, it remains possible that the inhibitory action of peripherally administered MSH on receptivity may be mediated via central mechanisms. Further experimentation is required to c l a r i f y the locus of the inhibitory action of MSH. Accumulating evidence Is consistent with the suggestion that 1 0 the inhibitory action of MSH may be mediated via central serotonergic mechanisms. For example, chronic MSH treatment has been shown to increase serotonin activity in the rat hypothalamus (Leonard, Kafoe, Thody, & Shuster, 1976). Conversely, depletion of serotonin has been found to reduce the concentration of MSH in the hypothalamus, thalamus, and brain stem (Fukata, Nakai, Impura, & Takeuchi, 1984). Although considerable contrary evidence exists, the majority of studies indicates that, in the female rat, increases in serotonin activity produce decreases in sexual receptivity (Everitt, Fuxe, & Hokfelt, 1975a; Everitt, Fuxe, Hokfelt, & Jonsson, 1975b; Sietnicks & Meyerson, 1980; Meyerson, 1964a,b, 1968; Ward, Crowley, Zemlan, & Margules, 1975). The recent work of Mendelson & Gorzalka (1985) may provide an explanation of the contradictory data. They found that serotonin type II receptor antagonists decreased receptivity and that this effect was reversed by serotonin type II agonists. Furthermore, i t appears that the majority of early research on the effects of serotonin agonists and antagonists employed drugs either low in specificity or with greater specificity for serotonin type I than type II receptors (Mendelson, 1985). This and other evidence led Mendelson & Gorzalka (1985) to hypothesize that increases in serotonin type I activity inhibit receptivity while increases in serotonin type II activity act to increase receptivity. It is possible that an inhibitory effect of MSH could be mediated via MSH-induced alterations in serotonin activity. Lending further plausibility to this suggestion is the recent 11 finding that serotonin receptors appear to possess a peptide binding site (Roth, Chuang, & Costa, 1984). Furthermore, MSH itsel f possesses a serotonin binding site (Root-Bernstein & Westall, 1984) that could be of behavioural significance. The findings that serotonin receptors possess a peptide binding site and that MSH possesses a serotonin binding site indicate that an MSH-serotonin interaction is possible and may even be required for the expression of certain MSH and serotonin effects. If Mendelson & Gorzalka's (1985) hypothesis is correct, MSH could inhibit receptivity by increasing serotonin type I activity and/or by decreasing serotonin type II activity. The present studies are designed to determine the effects of MSH on receptivity and to examine potential mechanisms mediating these effects. An attempt will then be made to integrate this information with a proposed model of receptivity in order to determine the potential functional significance of MSH in the regulation of receptivity in the rat. Since i n i t i a l studies in our laboratory (Experiments 1-5) indicated that MSH administered centrally exerted both a long and a short term inhibitory effect on receptivity while MSH administered peripherally facilitated receptivity, additional experiments were designed to examine potential mechanisms mediating these effects. Experiment 6 was designed to test the hypothesis that the facilitatory effect of peripherally administered MSH on receptivity is due to the release of progesterone from the adrenals. Experiments 7-9 were designed to examine the importance of estrogen and progesterone in the production of the short and long term inhibitory actions 12 of MSH. Experiments 7-8 were designed specifically to test the hypothesis that the long term inhibitory action of MSH is due to an MSH-induced decrease in cytoplasmic progestin receptors. Experiment 9 was designed to further c l a r i f y the role of progesterone in the expression of the long term inhibitory effect of MSH. Experiments 10-15 were designed to investigate the role of serotonin in the production of the long and short term inhibitory actions of centrally administered MSH. Experiments 12-15 were designed specifically to test the hypothesis that MSH inhibits receptivity, in part, by decreasing serotonin type II receptor activity. Section I - The Search for the MSH Effect As noted earlier, the results of Thody and coworkers may reflect a certain degree of experimental ar t i f a c t . Thus, my i n i t i a l studies were designed to determine the effect of centrally and peripherally administered MSH on receptivity in female rats. qenera.1 Methpcls, Subjects Female Sprague-Dawley rats, obtained from our breeding colony in the Department of Psychology at the University of British Columbia, were used as subjects. Animals were housed under a reversed 12:12 hr light:dark cycle with food and water available ad libitum. Procedure and Apparatus Surgery and Histology When subjects involved in experiments requiring central 13 a d m i n i s t r a t i o n of MSH were a p p r o x i m a t e l y 70 days of age, c h r o n i c guide c a n n u l a e were i m p l a n t 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 . Guide cannulae were c o n s t r u c t e d by g r i n d i n g s t a i n l e s s s t e e l 23 g i n j e c t i o n n e e d l e s t o the a p p r o p r i a t e l e n g t h (8 mm). To p r e v e n t p o s s i b l e i n f e c t i o n o r c o n t a m i n a t i o n of the c e r e b r o s p i n a l f l u i d , s t a i n l e s s s t e e l o b d u r a t o r s , made from 30 g n e e d l e s , were i n s e r t e d i n t o the g u i d e c a n n u l a e . C o o r d i n a t e s f o r c a n n u l a placement 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 (A-P: -0.2; M-L: 1.5; D-V: -2.8) were ta k e n from the a t l a s o f P e l l e g r i n o , P e l l e g r i n o , & Cushman (1979). S u r g e r y was performed w i t h s u b j e c t s under sodium p e n t o b a r b i t o l a n e s t h e s i a ( S o m n i t o l ) and c o n s i s t e d of a m i d s a g i t t a l s c a l p i n c i s i o n , r e t r a c t i o n of the s c a l p , i n s t a l l a t i o n of 4 s t a i n l e s s s t e e l anchor screws i n the s k u l l , i m p l a n t a t i o n of the guide c a n n u l a , and f i x a t i o n of the c a n n u l a assembly w i t h a c r y l i c cement ( F l a s h D e n t a l A c r y l i c ) . S e v e r a l weeks a f t e r c a n n u l a i m p l a n t a t i o n , a n i m a l 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 w h i l e under e t h e r a n e s t h e s i a . At a p p r o x i m a t e l y 80 days of age, s u b j e c t s i n v o l v e d i n e x p e r i m e n t s r e q u i r i n g o n l y p e r i p h e r a l a d m i n i s t r a t i o n o f MSH r e c e i v e d b i l a t e r a l o v a r i e c t o m i e s w h i l e under e t h e r a n e s t h e s i a . A f t e r s u r g e r y , a l l a n i m a l s were housed i n d i v i d u a l l y i n s t a n d a r d l a b o r a t o r y s i n g l e w i r e mesh cag e s . Because of the time i n v o l v e d i n c a n n u l a i m p l a n t a t i o n , a n i m a l s were used i n s e v e r a l e x p e r i m e n t s b e f o r e h i s t o l o g i c a l v e r i f i c a t i o n of the i m p l a n t a t i o n s i t e was c a r r i e d o u t . Between e x p e r i m e n t s , a c c u r a c y of c a n n u l a placement was a s s e s s e d by measuring the l a t e n c y t o d r i n k a f t e r the a d m i n i s t r a t i o n of a n g i o t e n s i n I I , a p o t e n t d l p s o g e n , v i a the c a n n u l a . Animals t h a t 14 f a i l e d to drink within 1 min af t e r being returned to t h e i r cage were eliminated from the experiment. Upon completion of t h e i r series of experiments, subjects were s a c r i f i c e d and 4 ul of India ink was infused into the ventricle via the cannula. The brain was removed and examined for traces of ink in the t h i r d ventricle at the level of the tuber cinereum and in the cerebral aqueduct, which was severed during brain removal. The appearance of ink at these locations was taken as confirmation of cannula placement into the l e f t l a t e r a l v e n t r i c l e . If India ink was not observed at these locations, the brain was fixed in formalin and then sectioned in order to v e r i f y cannula placement. Drug and Hormone Treatments Unless otherwise noted, animals received subcutaneous (SO injections of 2 jxg e s t r a d i o l benzoate (EB)/0.05 cc peanut o i l 50-53 hr p r i o r to testing and 200 >ig progesterone (P)/0.05 cc peanut o i l 4-7 hr pr i o r to testing. MSH was dissolved in s t e r i l e 0.9% (physiological) saline solution. Except where noted, MSH was administered 4 hr pr i o r to testing at a dose of 20 ^ ug/0.05 cc for SC injections and a dose of 200 ng/4 ,ul for intracerebroventric-ular (ICV) injections. Central injections were administered with an infusion pump at a rate of 1 / i l / 7 sec. Behavioural Testing The lordosis response was used as the primary indicator of r e c e p t i v i t y in the current studies. Tests of sexual r e c e p t i v i t y consisted of placing the female in a c y l i n d r i c a l Pyrex jar (29 cm diam) with a sexually vigorous male for a tota l of 10 mounts with p e l v i c thrusting. A lordosis quotient (LQ), the number of 15 lordosis responses displayed divided by the number of mounts by the male and multiplied by 100%, was calculated for each subject and s t a t i s t i c a l analyses performed on these scores. In experiments where subjects were divided into high and low LQ groups, an LQ of 50 or greater was considered to be high and an LQ of less than 50 was considered to be low. Animals were tested for sexual receptivity at seven day intervals unless otherwise noted. Analyses of variance (ANOVAs) were used to determine the st a t i s t i c a l significance of the data. Significant effects were then examined by using the Newman-Keul*s procedure with alpha set at 0.05. Experiment 1 Although Thody and coworkers (Everard et a l . , 1977; Thody et a l . , 1979, 1981) suggest that centrally and peripherally administered MSH increase LQs in subjects displaying low levels of receptivity (low LQ subjects) and that peripherally administered MSH also decreases LQs in subjects displaying high levels of receptivity (high LQ subjects), flaws in their experimental design make their results questionable. The major problem with the design used by Thody and coworkers is the use of threshold doses of EB and P combined with the post hoc division of subjects on the basis of a single control score. A pilot study was conducted to determine if this procedure could, in i t s e l f , produce results similar to those of Thody and coworkers. In this study, animals received two consecutive weekly tests of receptivity after saline administration. Animals were assigned to the high or low LQ group on the basis of one saline score 16 with the other saline score being treated as the "drug* score. An analysis of variance performed on these data revealed a significant group x "drug'' interaction, F< 1,28) = 21 .03, p=0.0001. A Newman-Keul's analysis indicated that, in low LQ subjects, saline 'drug* scores were significantly higher than saline scores while, for the high LQ subjects, saline 'drug' scores were significantly lower than saline scores (Figure 2a). This study was repeated using a second set of animals to reduce the probability that the results had occurred by chance. An analysis of variance performed on these data again indicated that, in low LQ animals, saline 'drug' scores were significantly higher than saline scores. However, the difference between the two saline scores in high LQ subjects was not significant (Figure 2b). These patterns of significance are reminiscent of the significant facilitatory and inhibitory effects observed by Thody and coworkers after the peripheral administration of MSH and of the significant facilitatory effect observed by Thody et a l . (1981) after the central administration of MSH. Thus, it is possible that the effects of MSH observed by Thody and coworkers were due, in part, to experimental artifact rather than to an effect of MSH administration. Because no other laboratory has produced data on the effects of MSH on sexual receptivity, this raises serious questions as to whether MSH is active in this respect. Experiment 1 was designed to determine the effects of both centrally and peripherally administered MSH on receptivity in high and low LQ animals. 17 Figure 2a and 2b. Artifactual effects of threshold estrogen and progesterone doses and repeated tests on receptivity. Subjects received 2 >ug estradiol benzoate 52 hr prior to testing and 200 u^g progesterone 4-7 hr before testing. Animals were tested for sexual receptivity twice at seven day intervals. Subjects were assigned to the high or low LQ (lordosis quotient; lordosis/mount x 100) group on the basis of their 'Saline 1' with the 'Saline 2' score being treated as the 'drug' score. 1 9 Methods Subjects served in each of four conditions: 1) central MSH administration; 2) central saline administration; 3) peripheral MSH administration; 4) peripheral saline administration. In order to replicate the procedure of Thody and coworkers as closely as possible, SC injections of MSH were administered 28 hr prior to testing while ICV injections of MSH were administered 4 hr prior to testing. The order in which subjects received each treatment was counterbalanced to control for possible effects of day and time of testing. Tests occurred once a week for four weeks. At the end of this period, subjects were assigned to either the high or the low LQ group on the basis of the two saline scores. Animals with unstable (one high and one low) saline scores were discarded from the analysis. Results After data from 9 animals with unstable levels of receptivity during the two saline tests and from 4 animals with poor cannula placements were discarded, a total of 25 subjects (10 high LQ and 15 low LQ) had served under each of the four treatment conditions. An examination of the data suggests that peripherally administered MSH both inhibited receptivity In high LQ subjects and facilitated receptivity in low LQ subjects while centrally administered MSH appeared to inhibit receptivity in high LQ subjects without affecting low LQ subjects (see Figure 3). A 2x2x2 (LQ x route of administration x MSH) ANOVA revealed a significant overall effect of drug, F(1,69)=9.47, p=0.003, with a Newman-Keul's analysis indicating that the mean LQ on MSH tests 20 Figure 3. The effects of centrally and peripherally administered MSH on receptivity. Subjects received 2 >ig estradiol benzoate 52 hr prior to testing and 200 >ig progesterone 4-7 hr before testing. A dose of 200 ng MSH was used for intracerebro-ventricular (ICV) injections while a dose of 20 ug was employed for subcutaneous (SO injections. For control tests, injections of the saline vehicle were employed. ICV injections were administered 4 hr prior to testing while SC injections were given 24 hr before testing. Subjects were assigned to the high or low LQ (lordosis quotient; lordosis/mount x 100) group on the basis of their control scores. 100 Legend • SALINE Q MSH 8CH 22 was significantly less than the mean LQ for saline tests. A significant LQ x drug interaction was also found, F<1,69)=79.07, p<0.0001. Further examination of this effect indicated that the mean LQs from the low LQ-saline test and the high LQ-MSH test did not d i f f e r from each other and were significantly lower than the mean LQ for the low LQ-MSH test which, in turn, was significantly lower than the mean LQ for the high LQ-saline test. The drug x administration route interaction was also significant, F< 1,69)=8.98, p=0.004, with a Newman-Keul's analysis indicating that the mean LQ obtained after central MSH administration was significantly less than for peripheral MSH administration or saline administration. Discussion The results of Experiment 1 indicate that the peripheral administration of MSH increases receptivity in animals with low LQs and decreases receptivity in subjects with high LQs. These results are similar to those obtained by Thody and coworkers (Everard et a l . , 1977; Thody et a l . , 1979, 1981). However, the finding that centrally administered MSH decreased receptivity in high LQ subjects without significantly altering receptivity in low LQ subjects is the opposite of the effect observed by Thody et a l . (1981). It seems likely that this difference can be accounted for by the differences in experimental design discussed earlier. However, further replication would help c l a r i f y the effects of MSH on receptivity. Experiments 2a-2c The results of Experiment 1 indicate that peripherally, but 23 not centrally administered MSH increases receptivity in subjects with low LQs. However, in order to replicate the procedures of Thody and coworkers as closely, as possible, testing occurred 24 hr after SC administration and 4 hr after ICV administration of MSH. It is possible that the differential effects observed are due to time dependent effects of MSH rather than to differences in peripheral versus central mechanisms of action. Experiments 2a-2c were designed to examine this partially by determining the temporal pattern of MSH effects on receptivity after SC administrat ion. Methods Subjects received MSH or physiological saline 1, 4, or 24 hr prior to testing. Half of the subjects received saline for their f i r s t test of sexual behaviour while the remainder received MSH. The following week, the saline subjects received MSH while the MSH subjects received saline. Receptivity data obtained 1, 4, and 24 hr after MSH administration failed to reveal any differential effects of time interval on MSH action. Therefore, one week after the last saline test of Experiment 2a, subjects were again administered MSH and tested either 15, 30, or 60 min after its administration (Experiment 2b). Because differential temporal effects were s t i l l not observed, subjects were given another MSH test one week after the last saline test of Experiment 2b. For this test, MSH was administered 1, 5, or 15 min prior to testing (Experiment 2c). Thus, testing was carried out over a total of six weeks. One half of the subjects received tests in the following pattern: Saline, MSH, Saline, MSH, Saline, 24 MSH. The other half of the subjects began with an MSH test and ended with a saline test. Results and Discussion It i n i t a l l y appeared that the peripheral administration of MSH facilitated receptivity in subjects with low LQs and inhibited receptivity in subjects with high LQs at virtually a l l of the times tested (Table I). However, as testing progressed, subjects seemed to display an increasing degree of instability. An examination of the data indicated that the number of animals displaying LQs of 0 was increasing for each saline test (from 15/38 to 17/38 to 22/38). This suggested that repeated administration of MSH might exert a slight, long term inhibitory effect on receptivity. It might be argued that repeated sexual testing i t s e l f produced an inhibition the following week. However, weekly sexual testing does not appear to have this effect (Raible & Gorzalka, 1981; Experiments 3, 5, 7-16). Because of the low number of subjects with stable scores and the possible long term action of repeated MSH administrations on receptivity, no analysis was performed on the data from Experiments 2a-2c. The results of this experiment do suggest, however, that the differential effects of MSH administered ICV and SC are not due to time-dependent effects of MSH action. Experiment 3 The results of Experiments 2a-2c failed to c l a r i f y the effects of MSH on receptivity. On the contrary, they served to further complicate the issue of the nature of the effect of MSH on sexual responding in the female rat. Indeed, the potential Time of High LQ Low LQ MSH NaCl _ MSH _NaCl __ MSH Experiment Administration X SE]v/f X S EM X X SE M 24 hr 86.7 8.8 36.7 31.8 10.0 4.5 24.0 13.0 2a 4 hr 73.3 7.1 56.7 12.6 3.3 3.3 21.7 14.0 1 hr 83.3 .8.0 63.3 20.1 ..5.7 5.7 47.1 17.3 60 min 82.0 7.9 54.0 22.7 10.0 5.7 38.8 14.1 2b 30 min 85.0 6.5 10.0 10.0 1.4 1.4 51.4 14.8 15 min 86.7 8.8 80.0 15.3 11.0 4.6 45.0 15.1 15 min 80.0 5.8 40.0 3.7 0.0 0.0 51.1 14.9 2c 5 min 72.0 7.3 36.0 20.6 10.0 4.9 57.1 12.2 1 min 100.0 0.0 55.0 18.9 0.0 0.0 17.8 7.9 Table I. The temporal e f f e c t s of subcutaneously administered MSH on r e c e p t i v i t y . Subjects received 2/lg e s t r a d i o l benzoate 52 hr p r i o r to testing and 200 )x% progesterone 4-7 hr before testing. MSH was administered subcutaneously at a dose of 20 jig. The number of high LQ subjects was as follows: 24 hr, n=3; 4 hr and 1 hr (Exp. 2a), n=6; 60 min (Exp. 2b), n=5; 30 min, n=4; 15 min (Exp. 2b and 2c) and 1 min, n=3; 5 min, n=5. The number of low LQ subjects was as follows: 24 hr, n=10; 4 hr, n=6; 1 hr (Exp. 2a), n=7; 60 min (Exp. 2b), n=8; 30 min, n=7; 15 min (Exp. 2b), n=10; 15 min (Exp. 2c) and 1 min, n=9; 5 min, n=7. 26 long terra effects of MSH also bring into question the results of Experiment 1. Because peptides are rapidly degraded and therefore do not ordinarily exert long term effects on behaviour, this result could not have been anticipated. Furthermore, if the effect is replicable, it may be valuable in the study of mechanisms of peptide action. Thus, the search for the MSH effect continued but now a third possible effect of MSH had been added, a long term effect. Experiment 3 was designed to 1) determine the effects of MSH administered SC and ICV on receptivity in high and low LQ subjects and 2) investigate the possibility that MSH might exert long term effects on recept i v i ty. Methods To insure maximal st a b i l i t y within subjects, a pilot study was carried out with the experimental animals to determine the doses of EB and P that would produce consistent LQs within subjects. On the basis of this study, subjects in the current experiment were administered 2 >ig EB either 50-53 hr or 50-53 and 26-29 hr prior to testing. A ll subjects received 100 /ig P 4-7 hr prior to testing. Sexual testing occurred once a week for four weeks, with subjects receiving control tests on Weeks 1, 3, and 4 and an MSH test on Week 2. As a pilot study had indicated that MSH administered either ICV or SC altered receptivity when administered 4 hr prior to testing, this time interval was used. Data from unstable subjects were discarded from the analysis. Results After data from 2 subjects with unstable levels of 27 receptivity were discarded, a total of 46 subjects (17 high LQ, ICV MSH; 14 high LQ SC MSH; 8 low LQ, ICV MSH; 7 low LQ SC MSH) had completed a l l 4 tests. An examination of the data (Figure 4) suggests that centrally administered MSH inhibits receptivity in females with high LQs. Peripherally administered MSH appears to f a c i l i t a t e receptivity in females with low LQs and may exert an inhibitory effect on receptivity in subjects with high LQs. Furthermore, it appears that MSH treatment might be exerting an effect on receptivity up to one week after MSH administration. A 2x2x4 (LQ x route of administration x test) ANOVA revealed significant effects of test, F(3,126)=2.70, p<0.05 and route of administration, F(1,42)=5.44, p=0.02. The analysis also revealed significant LQ x test, F(3,126)=13.42, p<0.0001, and route of administration x test, F(3,126)=3.12, p=0.03, interactions. A Newman-Keul's analysis was used to further examine the effects of route of administration. This analysis indicated that subjects receiving central injections of MSH displayed significantly lower LQs than those receiving peripheral MSH injections. Because of this, separate analyses were performed on the central MSH and the peripheral MSH data. An ANOVA on the data obtained from subjects receiving peripheral MSH revealed a significant LQ x test interaction, F(3,57)=6.87, p=0.0006. A Newman-Keul*s analysis indicated that the mean LQ for the MSH test was significantly higher than the mean LQs for the f i r s t and last control tests. However, the mean LQ obtained during the control test one week after MSH administration did not differ significantly from the mean LQ 28 Figure 4. The effects of centrally and peripherally administered MSH on receptivity. Subjects received 2 >ig estradiol benzoate 52 or 52 and 24 hr prior to testing and 100 >ig progesterone 4-7 hr before testing. Sexual testing occurred once a week for four weeks, with subjects receiving control tests on Weeks 1, 3, and 4 and an MSH test on Week 2. A dose of 200 ng MSH was used for intracerebroventr icular (ICV) injections while a dose of 20 ^ jug was used for subcutaneous (SC) injections.' MSH was administered 4 hr prior to testing. Subjects were assigned to the high or low LQ (lordosis quotient; lordosis/mount x 100) group on the basis of their control scores. Legend A HIGH LQ, SC MSH • LOW LQ, SC MSH O HIGH LQ, ICV MSH • LOW LQ, ICV MSH CONTROL CONTROL NJ 30 the mean LQs for the f i r s t and last control tests (Figure 4). For high LQ subjects, the mean LQ after MSH administration was significantly lower than the mean LQ for the f i r s t but not the last two control tests. Analysis of the data obtained for subjects receiving central MSH administration indicated a significant effect of drug, F(3,23)=4.84, p=0.004, with the mean LQ for the MSH test being significantly lower than the mean LQs for the f i r s t and last control tests. A significant drug x test interaction was also found, F(3,23)=6.81, p=0.0005. A Newman-Keul's analysis indicated that the mean LQs for low LQ subjects did not dif f e r over the four tests. For high LQ subjects, the mean LQ for the MSH test was significantly less than the mean LQs for the f i r s t and last control tests. However, the mean LQ for the control test occurring one week after MSH administration did not differ significantly from the mean LQ for the MSH test or the mean LQ from the f i r s t control test (Figure 4). D i scuss i on The results of this experiment indicate that peripherally administered MSH significantly facilitates receptivity in subjects with low LQs. There was also some indication that peripherally administered MSH might exert a long term effect on receptivity in these animals (Figure 4). However, the inhibitory effect of peripherally administered MSH on receptivity was rather weak. In contrast, centrally administered MSH exerted a strong inhibitory effect on receptivity in high LQ subjects but did not alter receptivity in animals with low LQs. Once again there was 31 a suggestion of a long term action of MSH on receptivity (Figure 4). The finding that peripheral MSH administration failed to strongly inhibit receptivity in subjects with high LQs Is In contrast to the results of Thody and coworkers (Everard et a l . , 1977; Thody et a l . , 1979, 1981) and to the results of Experiment 1.. However, the results of Thody and coworkers could be due to undetected wIthIn-subject variability, that is, an experimental art i f a c t . The significant inhibition observed after peripheral MSH administration in Experiment 1 may have been due, in part, to the prior central administration of MSH in some subjects, which appeared to produce a long term effect on receptivity. The design of the current experiment avoided that confounding factor. However, the possibility that the weak inhibitory effect observed in the current experiment resulted from some of the peripherally administered MSH reaching the brain can not be ruled out. Experiment 4 Because of the d i f f i c u l t i e s encountered previously with respect to the determination of the effects of MSH on receptivity, Experiment 4 was designed both to replicate the results of Experiment 3 and to examine the temporal parameters of MSH action. To determine the time of onset of MSH effects, MSH was administered at various times prior to testing. Methods Subjects received 2 >ig EB 50-53 hr prior to testing and 100 ,ug P 4-7 hr before testing. All subjects received control tests on Weeks 1, 3, and 4, with an MSH test occurring during Week 2. On the day of MSH testing, animals received either an SO 32 or an ICV injection of MSH 30 min, 1 hr, 2 hr, or 4 hr prior to testing. After eliminating subjects with unstable control scores, subjects were further divided into high and low LQ groups, creating a total of 16 groups (high or low LQ x SC or ICV MSH x 30 min, 1 hr, 2 hr, or 4 hr prior to testing). Results Upon elimination of 34 subjects with unstable control scores, some cells no longer contained sufficient subjects for an ANOVA. However, an examination of the data suggested that the inhibitory effects of ICV MSH occurred within 30 min while the facilitatory action of SC MSH occurred more than 1 hr after MSH administration (Table II). To determine the extent to which the current experiment replicated the basic findings of Experiment 3, data were collapsed into 4 groups (high or low LQ x SC or ICV administration). The following groups were eliminated from the analysis because of insufficient sample size: 1) high LQ, peripheral MSH administered 30 min, 1 hr, and 2 hr prior to testing; 2) high LQ, central MSH administered 30 min prior to testing; 3) low LQ, central MSH administered 30 min and I hr prior to testing. The data from the remaining groups were collapsed into four groups: high LQ, ICV MSH; high LQ, SC MSH; low LQ, ICV MSH; low LQ SC MSH. This resulted in 11 to 13 subjects in each of these groups. An examination of these data suggested that centrally administered MSH decreased receptivity in subjects with high LQs without altering receptivity in subjects with low LQs (Figure 5). Peripherally administered MSH appeared to increase receptivity in low LQ subjects without Route of 30 min 1 hr 2 hr 4 hr Adminis- . NaCl MSH NaCl MSH NaCl MSH NaCl MSH Group t r a t i o n X" SE M X SE^ X SE M X SE^ X SE^ X S EM X S E M ICV 68.0 2.2 32.0 14.6 70.0 7.1 20.0 12.6 62.0 5.8 50.0 18.7 63.0 3.3 10.0 5.8 High LO SC 81.4 8.1 74.3 7.8 . 83.6 8.9 83.6 9.3 . 97.5 1.3 80.8 8.1 100.0 0.0 87.3 8.9 ICV 10.0 10.0 12.5 12.5 .8.6 5.9 20.0 5.8 7.1 5.6 12.8 8.4 0.0 0.0 2.5 2.5 Low LQ SC 40.0 30.6 46.7 17.7 . 11.4 5.9 18.5 6.7 , 10.0 10.0 50.0 25.2 . 0.0 0.0 35.0 35.0 Table I I . The temporal e f f e c t s of c e n t r a l l y and pe r i p h e r a l l y administered MSH on r e c e p t i v i t y . Subjects received 2 /ig e s t r a d i o l benzoate 52 hr pr i o r to testing and 200 ^ ug progesterone 4-7 hr before t e s t i n g . MSH was administered subcutaneously at a dose of 20^g or i n t r a c e r e b r o v e n t r i c u l a r l y at a dose of 200 ng. The number of high LQ subjects per group was as follows: ICV-30 min, 1 hr, and 2 hr, n=5; ICV-4 hr, n=3; SC-30 min, n=14; SC-1 hr and 4 hr, n=ll; SC-2hr, n=12. The number of lowLQ subjects per group was as follows: ICV-30 min and 4 hr, n=4; ICV-1 hr and 2 hr, n=7; SC=30 min and 2 hr, n=3; SC-1 hr, n=7; SC-4 hr, n=2. 3 4 F i g u r e 5. The e f f e c t s of c e n t r a l l y and p e r i p h e r a l l y a d m i n i s t e r e d MSH on r e c e p t i v i t y . S u b j e c t s r e c e i v e d 2 jiq e s t r a d i o l benzoate 52 h r p r i o r t o t e s t i n g and 100 tag p r o g e s t e r o n e 4-7 h r b e f o r e t e s t i n g . T e s t s of s e x u a l r e c e p t i v i t y o c c u r r e d once a week f o r f o u r weeks w i t h c o n t r o l t e s t s o c c u r r i n g on Weeks 1, 3, and 4. S u b j e c t s were a s s i g n e d t o the h i g h o r low LQ ( l o r d o s i s q u o t i e n t ; l o r d o s i s / m o u n t x 100) group on the b a s i s of t h e i r c o n t r o l s c o r e s . MSH was a d m i n i s t e r e d p r i o r to t e s t i n g on Week 2. A dose of 200 ng MSH was employed f o r i n t r a c e r e b r o v e n t r i a c u l a r (ICV) i n j e c t i o n s w h i l e a dose of 20 jxq was used f o r subcutaneous (SC) i n j e c t i o n s . See methods s e c t i o n of Experiment 4 f o r tim e s of MSH a d m i n i s t r a t i o n . Legend A HIGH LQ, SC MSH • LOW LQ, SC MSH O HIGH LQ, ICV MSH • LOW LQ, ICV MSH . CONTROL CONTROL 36 altering receptivity in subjects with high LQs (Figure 5). An ANOVA performed on these data indicated a significant LQ x test interaction, F(3,t38>= 10.14, p<0.0001. A Newman-Keul*s analysis revealed that the overall mean LQ for low LQ subjects receiving MSH was significantly greater than the mean LQs for subjects during the control tests. It was also found that the overall mean LQ from high LQ subjects receiving MSH was significantly lower than the mean LQs for these subjects during the f i r s t and last control tests but did not differ from the mean LQ for these subjects during the control test occurring one week after MSH administration. Discuss ion The low subject numbers in some cells made it d i f f i c u l t to determine the onset of MSH effects. However, an examination of Table II suggests that the inhibitory effect of MSH administered ICV was observable 30 min after MSH administration while the facilitatory effect of MSH administered SC was not observable until over 2 hr after MSH administration. These results confirmed those of a pilot study, which indicated that the effects of MSH administered ICV and SC could be observed 4 hr after MSH administration. When collapsed over time, the results of the current experiment indicate that centrally administered MSH inhibits receptivity while peripherally administered MSH faci l i t a t e s receptivity. Furthermore, ICV administration of MSH appears to exert a long term inhibitory action on receptivity. These findings are consistent with those of Experiment 3 and make it less likely that these results are due to extraneous 37 variables. The d i f f i c u l t i e s in producing stable LQs within animals suggests that it would be desirable to perform separate experiments to examine the effects of MSH on receptivity in high and low LQ subjects. Experiment 5 Experiments 3 and 4 indicated that the peripheral administration of MSH facilitated receptivity in animals with low LQs. However, because of the d i f f i c u l t i e s encountered previously, an additional replication of this effect using a somewhat different design would lend further confirmation. Once one were certain that peripherally administered MSH alters receptivity, the possible mechanism behind this effect could then be examined. Thus, Experiment 5 was designed to replicate the finding that peripherally administered MSH increases receptivity in subjects displaying low LQs. Methods Twelve subjects were administered doses of EB and P that had previously been found to consistently produce LQs of less than 50 within each subject. Five combinations of EB and P doses were used: 2 >ig EB 50-53 hr prior to testing + 200 >ig P (2 subjects); 2 ;jg EB 50-53 hr prior to testing + 300 >ig P (2 subjects); 2 jxg EB 50-53 and 26-29 hr prior to testing + 200 >ig P (3 subjects); 2 /ig EB 50-53 and 26-29 hr prior to testing + 300 »jg P (3 subjects); and 4 ^ g EB 50-53 hr prior to testing + 200 jig P (2 subjects). In a l l cases, P was administered 4-7 hr prior to testing. Animals were tested once a week for 8 weeks, with control tests occurring on Weeks 1, 2, 4, 5, 6, and 8. MSH tests 38 occurred on Weeks 3 and 7. Results and Discussion An examination of the data suggests that peripherally administered MSH facilitates receptivity (Figure 6). An ANOVA performed on the data revealed a significant effect of test, F(7,77)=5.40, p=0.0001. A Newman-Keul's analysis indicated that the mean LQs for the MSH tests were significantly higher than the mean LQs for a l l control tests except for the control test following the f i r s t MSH test. However, the mean LQ for this control test did not differ from the mean LQs for the remaining control tests. These results are consistent with those obtained in Experiments 4 and 5 and indicate that the facilitatory action of peripherally administered MSH on receptivity in subjects with low LQs is a robust effect. DISCUSSION: SECTION I Experiments 1 - 3 indicated that flaws in experimental design and a long term effect of MSH on receptivity were problems that needed to be considered prior to the design of further experiments. Experimental designs were altered such that subjects received multiple control tests between MSH administrations. This allowed verification of sta b i l i t y within subjects and provided additional time for any long term effects of MSH to dissipate. Experiments 3-5 indicated that SC MSH administration produces a facilitatory effect on receptivity while Experiments 3 and 4 indicated that ICV MSH administration produces a short term inhibitory effect on receptivity. ICV MSH administration also 3 9 Figure 6. The effect of subcutaneously administered MSH on receptivity in subjects displaying low levels of sexual responding. Subjects were administered estradiol benzoate 52 hr prior to testing and progesterone 4-7 hr before testing. Subjects received doses of estrogen and progesterone that had previously been determined to produce stable levels of receptivity. Tests of sexual receptivity occurred once a week for eight weeks with control tests occurring on Weeks 1, 2, 4, 5, 6, and 8. MSH was administered subcutaneously at a dose of 20 jug on Weeks 3 and 7. 41 appears to produce a somewhat weaker long term inhibitory action on receptivity. Although the finding of a facilitatory effect of MSH administered SC on receptivity is consistent with the results of Thody and coworkers, the remaining results are not. Thody and coworkers observed an inhibitory effect of MSH administered SC on receptivity in subjects with high LQs but this result was not consistently obtained in the present experiments. Furthermore, Thody et a l . <1981) reported a significant facilitatory effect of MSH administered ICV in low LQ subjects and no effect on receptivity in high LQ subjects. This led Thody et a l . (1981) to suggest that the inhibitory action of MSH was peripherally mediated. However, the pilot studies discussed in the introduction to Experiment 1 indicate that s t a t i s t i c a l l y significant but artifactual inhibitory and facilitatory effects can be otained by using the design employed by Thody and coworkers. This brings the conclusions of Thody and coworkers into question. Experiments 3 and 4 indicated that MSH administered ICV inhibited receptivity in subjects with high LQs and did not alter receptivity in subjects with low LQs. This, plus the finding that MSH administered SC did not consistently inhibit receptivity in low LQ subjects suggests that the inhibitory action of MSH is mediated by a central mechanism. It is worth noting that, in Thody et al.'s (1981) paper examining the effect of ICV MSH administration, there was a strong trend towards an inhibition in high LQ subjects. The mean LQ for these subjects was 69.2 after saline administration but only 39.6 after 42 MSH administration. This difference apparently failed to reach s t a t i s t i c a l significance. Thus, Thody et al.'s (1981) finding that ICV MSH did not inhibit receptivity may simply be due to a large degree of variance between and within subjects. It could be argued that the inhibition of receptivity observed in the present experiments is due to a non-specific debilitating effect of MSH. However, the finding (Davis, Kastin, Beilstein, & Vento, 1980; Raible & Gorzalka, unpublished observations) that even relatively high doses of MSH do not alter locomotor activity argues against this possibility. The results of Experiments 3 and 4 also suggested that MSH, unlike other peptides examined to date, may produce long term behavioural effects. A clue to the potential significance of this effect may l i e in the recent finding that, in rats, pseudo-pregnancy induced by cervical stimulation leads to the release of MSH and adrenal progesterone (Volosin & Celis, 1979a, 1984). In addition, the chronic infusion of MSH has been found to produce physiological indications of pseudopregnancy (Volosin & Celis, 1984). Thus, it is possible that MSH serves a truly functional role in the regulation of receptivity in the rat. Cervical stimulation produced by copulation may increase MSH release which may i n i t i a l l y stimulate the release of adrenal progesterone, thus enhancing sexual responding. A long term inhibition could ensure that implantation and development of f e r t i l i z e d eggs can occur without the potentially disrupting effects of additional copulations. Although entirely speculative at this point, the current research Is consistent with the notion that MSH may play 4 3 a functional role in the regulation of receptive states. Section II - MSH and Receptivity: Rpjje of Adrenal Progesterone The finding in Section I that peripherally administered MSH facilitates receptivity Is of Interest In light of similar results obtained after ACTH administration <de Catanzaro et a l . , 1981; de Catanzaro & Gorzalka, 1980; Wilson et a l . , 1979). The 13 amino acid sequence of MSH is identical to the f i r s t 13 amino acids of ACTH (de Wied & Jolles, 1982; Marks et a l . , 1976; Schwyzer & Eberle, 1977), suggesting that MSH and ACTH may act via the same mechanism. Indeed, the MSH fragment of ACTH may be responsible for the behavioural effects of ACTH. As ACTH appears to faci l i t a t e receptivity by increasing the release of progesterone from the adrenals (de Catanzaro et a l . , 1981; de Catanzaro & Gorzalka, 1980), it is plausible that MSH also acts via this mechanism. Furthermore, other adrenal steroids, such as deoxycorticosterone (DOC), are also known to facil i t a t e receptivity (Gorzalka & Whalen, 1977). Thus, i t is possible that ACTH or MSH may facil i t a t e receptivity by f a c i l i t a t i n g the release of DOC or progesterone from the adrenals. As an alternative, Thody & coworkers have suggested that the stimulatory effect of peripherally administered MSH on receptivity was due to a potentiation by MSH of the effects of estrogen rather than to a release of adrenal progesterone. However, it is not clear that Thody and coworkers have performed the studies necessary to reach this conclusion. Thody et a l . (1979, 1981) appear to have excluded a possible MSH-induced release of adrenal progesterone for three reasons. First, they report (Thody & Wilson, 1983) that adrenalectomy does not eliminate the facilitatory action of MSH in subjects receiving estrogen plus progesterone. Second, they cite a study that indicates that MSH has l i t t l e or no adrenal stimulating activity in adult animals (Lowry, McMartin, & Peters, 1973). Third, they appear to conclude that, because the stimulatory effects of peripherally administered MSH are observed in ovariectomized animals given estrogen only, the presence of progesterone is not necessary for the effect. However, as discussed earlier, the design used by Thody's group may result in confounded data and erroneous conclusions. Thus, the results of their adrenalectomy experiment are subject to question. This is particularly true since their experiment employed doses of estrogen (10 ug) and progesterone (400 ug) that generally produce high levels of receptivity in a l l rats (Whalen, 1974). Curiously, in Thody & Wilson's (1983) experiment, this hormone regimen produced 35 subjects with low LQs and 34 subjects with high LQs. The reasons behind this low degree of receptivity are unclear, particularly since they obtained a more even division of LQs in earlier studies that employed doses of 2 ug EB and 200 ug P (e.g.* Everard et a l . , 1977; Thody et a l . , 1981; Wilson et a l . , 1979). In addition to these d i f f i c u l t i e s with Thody & Wilson's (1983) study, there is increasing evidence that indicates that MSH does stimulate adrenal activity (Page, Boyd, & Mulrow, 1974; Vinson, Whitehouse, Dell, Etienne, & Moris, 1980; Vinson, Whitehouse, & Thody, 1981; Volosin & Celis, 1984). Furthermore, the 4 5 demonstration that peripherally administered MSH facilitates receptivity in animals receiving only estrogen does not eliminate the possibility that it does so by stimulating the release of adrenal progesterone. Experiment 6 The results of Experiments 3-5 indicate that SC administration of MSH faci l i t a t e s receptivity. It is possible that, like ACTH, MSH increases receptivity by f a c i l i t a t i n g the release of progesterone from the adrenals. To determine the role of adrenal steroids in the mediation of the facilitatory action of MSH, ovariectomized-adrenalectomized animals receiving estrogen alone will be employed. If MSH facilitates receptivity by stimulating the release of adrenal progesterone or DOC, adrenalectomy will prevent this action and would thereby eliminate the facilitatory effect of MSH. This result would also suggest that the MSH sequence of the ACTH molecule mediates the ACTH effect. Experiment 6 is designed to determine the effect of adrenalectomy on the response to MSH in subjects with low levels of receptivity. Methods One half of the subjects were ovariectomized (ovx) and the remainder ovariectomized and adrenalectomized (ovx-adx) while under ether anesthesia. Ovx-adx subjects were provided with 0.9% saline instead of water for drinking to allow compensation for adrenalectomy-induced sodium deficiency. Subjects received daily injections of 0.8 >ig EB for the duration of the experiment. Previous research in our laboratory (Gorzalka & Raible, 1981) 46 indicates that this regimen can induce a moderate level of receptivity in ovx and ovx-adx rats. Testing began on Day 3 of EB administration and continued daily until the termination of the experiment. One half of the subjects in each surgical condition received SC injections of MSH 4 hr prior to testing on Day 5 of EB administration. MSH was administered on this day as the mean LQs on Day 4 of EB administration indicated a low level of receptivity had been reached. A total of four groups was created: 1) ovx-saline, n=12» 2)ovx-MSH, n=10; 3) ovx-adx-saline, n=tl> 4) ovx-adx-MSH, n=14. The MSH test constituted the final test of this experiment. Results An examination of the data suggested that MSH facilitated receptivity in ovx but not ovx-adx subjects (Figure 7). An ANOVA performed on the data revealed a significant surgery x MSH interaction, F(1,43)=4.11, p<0.05. A Newman-Keul's analysis indicated that ovx subjects receiving MSH were significantly more receptive than ovx subjects receiving saline. Ovx-adx subjects receiving MSH did not differ significantly from ovx-adx subjects receiving saline nor did they differ from ovx subjects receiving either MSH or saline. DISCUSSION: SECTION II The results of the current experiment indicate that MSH facilitated receptivity in ovx but not in ovx-adx subjects. Since the mean LQ of the ovx-adx groups was approximately 59.0, it is unlikely that the failure to observe a facilitatory action of MSH was due to a ceiling effect. These results suggest that. 47 F i g u r e 7. The e f f e c t s of s u b c u t a n e o u s l y a d m i n i s t e r e d MSH on r e c e p t i v i t y i n o v a r i e c t o m i z e d (OVX) and o v a r i e c t o m i z e d - a d r e n a l -e c t o m i z e d (OVX-ADX) s u b j e c t s . S u b j e c t s r e c e i v e d i n j e c t i o n s o f 0.8 >ig e s t r a d i o l benzoate 72, 48, and 24 hr p r i o r to t e s t i n g . 20 jig MSH or the s a l i n e v e h i c l e was 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 4 h r p r i o r to t e s t i n g . 100 80H o o X o CO CO o Q Cxi O 60H 40 20 OVX Legend CZ] SALINE a MSH 49 l ike ACTH, MSH may f a c i l i t a t e r e c e p t i v i t y by increasing the release of progesterone or other f a c i l i t a t o r y s tero ids from the adrenal . The current re su l t s f a i l to r e p l i c a t e Thody & Wilson's (1983) f inding that adrenalectomy does not e l iminate the f a c i l i t a t o r y act ion of MSH. However, due to the experimental design employed, Thody & Wilson's (1983) re su l t s are quest ionable . In a d d i t i o n , the suggestion by Thody and coworkers (1979, 1981) that p e r i p h e r a l l y administered MSH f a c i l i t a t e s r e c e p t i v i t y by potent ia t ing the e f fects of estrogen is not supported by the current r e s u l t s . If MSH did act by potent iat ing the e f fec ts of estrogen, then adrenalectomy should not have prevented the f a c i l i t a t o r y ac t ion of MSH. The f inding that adrenalectomy el iminated the f a c i l i t a t o r y ac t ion of MSH suggests that the e f fect is produced by progesterone or other s teroids of adrenal o r i g i n and not by enhanced estrogenic a c t i o n . Sect ion III - MSH and Recept iv i ty : Role of Estrogen and Progesterone The re su l t s of Experiments 3-5 in Section I indicate that MSH exerts both a short and a long term i n h i b i t o r y a c t i o n . It is poss ible that MSH produces both these e f fects by i n t e r f e r i n g with the f a c i l i t a t o r y act ions of estrogen and progesterone on r e c e p t i v i t y . A l t e r n a t i v e l y , the long and the short term i n h i b i t o r y act ions of MSH may represent two d i f f eren t mechanisms. For example, the i n i t i a l inh ib i tory e f fect of MSH may be the r e s u l t of immediate changes in serotonin (or other neurotransmitter) a c t i v i t y while the long term ef fec t may be due 50 to changes in estrogen, progesterone, or serotonin receptor concentrations. Changes in receptor concentration could be due to a direct action of MSH or could be mediated through MSH-induced changes in serotonin (or other neurotransmitter) activity. The current series of experiments was designed to examine the role of estrogen and progesterone in the production of the inhibitory actions of MSH. Of importance in any investigation of an effect on receptivity is a knowledge of the presumed sites and mechanisms of estrogen and progesterone action. Although a variety of models concerning the regulation of receptivity have been proposed (e.g., Clemens, 1978? Gorski, 1974, 1976, 1979; Kow, Malsbury, & Pfaff, 1974; Meyerson, Palis, & Sietnieks, 1979), few o f them (e.g., Gorski, 1974; Kow et a l . , 1974; Meyerson et a l . , 1979) have incorporated serotonergic mechanisms. Those that have often f a i l to incorporate hormonal factors (e.g., Kow et a l . , 1974). However, it is possible to integrate these models somewhat and arrive at a few basic conclusions about the regulation of receptivity in the female rat. Two brain areas thought crucial for the expression of lordosis are the anterior hypothalamus-preoptic area (AH-POA) and the mesencephalic reticular formation (MRF; Clemens, 1978; Gorski, 1974, 1976). It has long been accepted by many researchers (e.g., Clemens, 1978; Gorzalka and Mogenson, 1977; Pfaff, 1980) that the AH-POA exerts an excitatory influence on lordosis in the rat. This notion appears to be based more on wishful thinking than on direct evidence. As Gorski has pointed out (1974, 1976), much of the evidence in favor of this 51 hypothesis is contradictory: lesions of the AH-POA have been reported to decrease (Clark, 1942; Law & Meagher, 1958; Singer, 1968), increase (Dorner, Docke, & Hinz, 1969; Powers & Valenstein, 1972) or have no effect on receptivity (Law & Meagher, 1958; Raisman & Brown-Grant, 1977). Furthermore, most of these studies contain methodological flaws that make the interpretation of results questionable (see Gorski, 1974, for a review). However, although lesion studies provide l i t t l e consistent evidence that the AH-POA exerts a facilitatory action on lordosis, there is strong evidence that estrogen does in fact act in this area to facil i t a t e lordosis. The AH-POA contains neurons that retain radiolabelled estradiol (Pfaff, 1968a,b; Pfaff & Keiner, 1973). In addition, the application of estradiol to the AH-POA has been found to fac i l i t a t e or reinstate lordotic responding in ovariectomized females (Lisk, 1962; Ross, Claybaugh, Clemens,' & Gorski, 1971). Thus, i t would appear that the AH-POA plays an important role in the hormonal regulation of receptivity, only the manner in which i t does so remains in quest ion. Of interest is the finding that the hypothalamus contains a much greater proportion of serotonin type I receptors than of serotonin type II receptors (Leysen, Niemegeers, Van Nueten, & Laduron, 1982; Peroutka & Snyder, 1981). According to the hypothesis of Mendelson & Gorzalka (1985), increases in serotonin type I activity should inhibit receptivity while decreases in serotonin type I activity should faci l i t a t e receptivity. Although contrary to current opinion, it is possible that estrogen may act 5 2 to f a c i l i t a t e receptivity in the AH-POA by reducing serotonin type I receptors and/or activity, thereby disinhibiting lordosis. This idea is supported by the finding that: 1) electrical stimulation of neurons in the MPOA, which contains predominantly serotonin type I receptors, inhibits receptivity in female rats (Pfaff & Sakuma, 1979), 2> estrogen administration decreases serotonin type I receptor concentrations in the POA and hypothalamus (Biegon & McEwen, 1982), 3) estrus is associated with a decline in serotonin content in the AH-POA (Biegon, Bercovitz, & Samuel, 1980; Kueng, Wirz-Justice, Menzi, & Chappuis-Arndt, 1976), and 4) parachlorophenylalanine (PCPA), a serotonin synthesis inhibitor, facilitates lordosis in subjects receiving estrogen alone (Zemlan, Ward, Crowley, & Margules, 1973). As with other models, the lesion data present some d i f f i c u l t i e s . One would expect a POA lesion to f a c i l i t a t e receptivity as it would eliminate serotonin's inhibitory action. However, lesion data have failed to provide consistent results. It has been suggested (Clemens, 1978) that POA lesions increase the sensitivity of estrogen-sensitive regions not destroyed by the lesions. Thus, the effect obtained would depend on the extent and location of the lesion. At present, there is an insufficient number of methodologically sound studies to allow an evaluation of this idea. The role of the MRF in the expression of lordosis behaviour seems somewhat less controversial. Progesterone, when applied to the MRF, rapidly faci l i t a t e s lordosis (Luttge & Hughes, 1976; Ross et a l . , 1971). In addition, this procedure has also been 53 found to increase multi-unit activity and single-unit activity in the MRF (Gorski, 1976). Furthermore, high concentrations of radiolabelled progesterone are taken up in the mesencephalon (Whalen & Luttge, 1971a,b; Wade, Harding, & Feder, 1973). Finally, the raphe nuclei contain c e l l bodies of an ascending serotonergic pathway that courses through the MRF (Fuxe, Hokfelt, & Ungerstedt, 1970; Moore, 1981;Ungerstedt, 1971). This has led Gorski (1974) to suggest that, in the MRF, progesterone action may correlate with this ascending serotonergic system. Thus, progesterone may facil i t a t e lordosis by stimulating serotonergic activity in the MRF. According to the hypothesis of Mendelson & Gorzalka (1985), increasing serotonin type II activity should increase lordosis. Thus, progesterone may act specifically to increase the activity of serotonin type II-containing neurons in the MRF. The finding that serotonin type II receptors are found throughout the mesencephalon (Peroutka & Snyder, 198 1) is consistent with this notion. Furthermore, it may help explain an apparently contradictory finding: PCPA facilitates receptivity in subjects receiving estrogen alone but inhibits receptivity in subjects receiving estrogen plus progesterone. It has already been suggested that estrogen may facil i t a t e receptivity by decreasing the concentration of serotonin type I receptors in the AH-POA. PCPA may further decrease serotonin type I activity in this area, thus fa c i l i t a t i n g lordosis. Because progesterone acts synergistically with estrogen to fac i l i t a t e receptivity, a lower dose of estrogen is generally used in estrogen + progesterone studies than in studies employing estrogen alone. Thus, there 5 4 may be a smaller estrogen-induced reduction in serotonin type I receptors in the AH-POA in subjects receiving estrogen + progesterone. In addition, if progesterone acts in the MRF to f a c i l i t a t e lordosis by stimulating facilitatory serotonergic pathways, PCPA would reduce this activity, removing the facilitatory effect. As animals receiving estrogen alone were never dependent upon this facilitatory action, their behaviour would not reflect the elimination of this facilitatory action. Thus, the net result of the differences in hormonal treatment could be a f a c i l i t a t i o n in one case and an inhibition in the other. If so, increasing the dose of estrogen administered in conjunction with progesterone should attenuate PCPAs inhibitory action in rats treated with estrogen and progesterone. This hypothesis is being investigated in our laboratory. There are at least two mechanisms by which MSH could produce a short term inhibition of receptivity. F i r s t , MSH might interfere with the action of estrogen or progesterone on the release of serotonin or some other neurotransmitter. For example, MSH might alter the configuration of the estrogen or progesterone receptor, thereby decreasing the a f f i n i t y of the receptor for the hormone. This, in turn, would interfere with steroid-induced neurotransmitter release. Alternatively, MSH might activate neurons that synapse upon the axons of estrogen-or progesterone-activated neurons. This would decrease the electrical potential of these axons (presynaptic Inhibition) and reduce or eliminate the release of neurotransmitter from the terminal button. Second, MSH could exert a direct effect on 55 neurotransmitter release or binding and this effect could counteract the actions of estrogen or progesterone. For example, progesterone may fac i l i t a t e the release of serotonin in the MRF while MSH may Inhibit serotonin release in this region. Alternatively, MSH may alter the configuration of the serotonin receptor, reducing the af f i n i t y of the receptor for serotonin. If MSH produced the short term inhibition via the f i r s t mechanism, increasing the dose of estrogen or progesterone should attenuate the inhibitory action of MSH. However, i f MSH produced the short term effect by a direct action on neurotransmitter activity, increasing the hormone dose should be relatively ineffective in attenuating the short term effect. The long term inhibitory effect of MSH may be mediated to a large extent by MSH-induced decreases in serotonin type II activity, or a long term increase in serotonin type I activity. However, this seems an improbable explanation since it is unlikely that a peptide that is rapidly broken down could directly affect neurotransmitter activity one week after its administration. Rather, it is probable that the long term inhibitory action of MSH is produced by a long term change in the mechanism(s) mediating receptivity, e.g., estrogen, progesterone, or serotonin receptor concentrations. Several lines of research, when taken together, suggest that a reduction in the concentration of progestin receptors may be involved in the production of the long term inhibitory effect of MSH. Fir s t , pregnancy and pseudopregnancy (PSP) are characterized by a lack of receptvity and elevated serum progesterone levels (Smith, 56 F r e e m a n , & N e i 1 1 , 1975; V o l o s i n & C e l l s , 1984; W e l s c h e n , Osman, D u l l a a r t , De G r e e f , V i l e n b r o e k , & de J o n g , 1 9 7 5 ) . S e c o n d , p r o g e s t e r o n e h a s b e e n f o u n d t o d e c r e a s e t h e c o n c e n t r a t i o n o f c y t o p l a s m i c p r o g e s t i n r e c e p t o r s ( B l a u s t e i n , 1982a,b; B l a u s t e i n & F e d e r , 1979a,b; S c h w a r t z , B l a u s t e i n , & Wade, 1 9 7 9 ) . T h i s s u g g e s t s t h a t t h e i n d u c t i o n a n d m a i n t e n a n c e o f PSP i s d u e , i n p a r t , t o i n c r e a s e s i n s e r u m p r o g e s t e r o n e , w h i c h w o u l d d e c r e a s e t h e a v a i l a b i l i t y o f c y t o p l a s m i c p r o g e s t i n r e c e p t o r s . T h i r d , i t h a s r e c e n t l y b e e n f o u n d t h a t MSH l e v e l s a r e e l e v a t e d d u r i n g PSP a n d t h a t i n f u s i o n s o f MSH c a n i n d u c e PSP ( V o l o s i n & C e l i s , 1979a,b, 1 9 8 4 ) . T h i s s u g g e s t s t h a t MSH p l a y s a n i m p o r t a n t r o l e i n b o t h t h e i n d u c t i o n a n d m a i n t e n a n c e o f PSP. T a k e n t o g e t h e r , t h e a b o v e f i n d i n g s s u g g e s t t h a t MSH c o u l d c o n t r i b u t e t o t h e i n d u c t i o n a n d m a i n t e n a n c e o f PSP by f a c i 1 i t a t i n g t h e down-r e g u l a t i o n o f p r o g e s t i n r e c e p t o r s . The f i n d i n g t h a t s e r o t o n i n a n d p r o g e s t i n r e c e p t o r s a r e f o u n d i n t h e AH-POA a n d MRF i n d i c a t e s t h a t s u c h a n i n t e r a c t i o n i s p o s s i b l e . I t s h o u l d be n o t e d t h a t t h e l o n g t e r m i n h i b i t o r y e f f e c t o f MSH may a l s o i n v o l v e a l t e r a t i o n s i n s e n s i t i v i t y t o e s t r o g e n o r i n t h e a v a i l a b i l i t y o f e s t r o g e n r e c e p t o r s . A l t h o u g h I know o f no r e s e a r c h t h a t w o u l d c o n n e c t t h e i n h i b i t o r y a c t i o n o f MSH w i t h d e c r e a s e d r e s p o n s i v e n e s s t o e s t r o g e n , t h i s p o s s i b l i t y c a n n o t be e l i m i n a t e d . The e x p e r i m e n t s i n t h e p r e s e n t s e c t i o n a r e d e s i g n e d t o e x a m i n e t h e i m p o r t a n c e o f b o t h e s t r o g e n a n d p r o g e s t e r o n e on t h e M S H - i n d u c e d i n h i b i t i o n o f r e c e p t i v 1 t y . G e n e r a l M e t h o d s The g e n e r a l m ethods e m p l o y e d i n e x p e r i m e n t s i n t h i s s e c t i o n 57 will be virtually identical to those described in Section I except for one change in the experimental design. Because the facilitatory and inhibitory effects of MSH are no longer being examined in a single experiment, there is no need to divide the subjects into high and low LQ groups. Thus, subjects with unstable scores need no longer be discarded from the analysis. However, because MSH can exert long term effects, an index of stab i l i t y is s t i l l desirable. Unless otherwise noted, the following testing scheme will be used for each experiment: Week 1-control; Week 2-MSH; Week 3-control (post-MSH test); Week 4-control. In this manner, sta b i l i t y can be determined by examining the f i r s t and last control scores while any long term action of MSH can be determined by examining the second control score. Unless otherwise noted, ANOVAs will be performed on data collected through a l l four weeks of testing. There will be a minimum 21 day interval between MSH injections in a l l subjects. Experiment 7 In Section II, MSH administered ICV was observed to exert both a short and a long term inhibitory action. It was suggested that MSH might produce these effects via a direct action on hormone or neurotransmitter receptors or via an indirect action on hormone-induced changes in neurotransmitter activity. In addition, MSH might directly alter neurotransmitter activity. Because peptides are rapidly degraded, different mechanisms might be involved in the mediation of the long and short term inhibitory actions of MSH. It is possible that the short term action of MSH is produced by relatively immediate alterations in 58 serotonin activity while longer term effects are mediated via slower, longer lasting changes in serotonin, estrogen, or progestin receptor concentrations. To the best of my knowledge, the effects of alterations in serotonin activity on estrogen and progestin receptors have not been investigated. However, there is evidence that alterations in neurotransmitter activity can alter the concentration of estrogen receptors (Blaustein, in press) and progestin receptors (Blaustein, 1985; Nock, Blaustein, & Feder, 1981). Thus, the long term inhibitory action of MSH could be produced by MSH-induced alterations in neurotransmitter activity that, in turn, produce decreases in the concentration of progestin receptors. The short term inhibitory action of MSH could be produced by an MSH-induced alteration in neurotrans-mitter activity that counters that produced by progesterone. Experiment 7 is designed to determine the importance of progesterone for the expression of the inhibitory action of MSH. If both the short and long term effects are observed in the absence of progesterone, it would suggest that alterations in progestin receptor concentrations are not important for the expression of these effects. In addition, it would suggest that the short term action of MSH may be produced by direct alterations in neurotransmitter activity. If the short term, but not the long term, effect is observed in the absence of progesterone, it would suggest that progestin receptors are important in the expression of the long term effect but that alterations in serotonin (or other neurotransmitter) activity may be more important in the expression of the short term effect. If 5 9 the long term, but not the short term, effect is observed, it would suggest that alterations in progestin receptor concentrations are not responsible for the expression of the long term effect while progesterone-induced alterations in neurotransmitter activity may play a role in the expression of the short term effect. Finally, i f neither the short or the long term effect is observed in the absence of progesterone, it would suggest that alterations in progestin receptor activity and/or progesterone-induced alterations in neurotransmitter activity are required for the expression of both effects. Methods Subjects received 20 u^ig EB/0.05 cc o i l 52 hr prior to testing. Pilot studies in our laboratory have indicated that this dose of estrogen reliably e l i c i t s high levels of receptivity. Subjects were assigned to one of three groups <n=13/group): saline; MSH; or no treatment. The no treatment group was included to determine if saline administered ICV had any effect on receptivity. Results and Discussion An examination of the data suggests that, in animals receiving estrogen alone, MSH exerts only a very weak short term inhibition and no long term effect (Figure 8). In addition, the ICV administration of saline appears to have no effect on receptivity. An ANOVA performed on the data revealed no significant differences. These data are of interest for two reasons. First, they support the notion that the inhibitory action of MSH is not due 60 Figure 8. The effects of MSH on receptivity in subjects receiving estrogen alone. Subjects received 20 jjg estradiol benzoate 52 hr prior to testing. Tests occurred once a week for four weeks, with control tests occurring on Weeks 1, 3, and 4 and an MSH test occurring on Week 2. MSH was administered intracerebroventricular1y at a dose of 200 ng 4 hr prior to test ing. 100-1 CH , - , — CONTROL MSH Legend A NO TREATMENT • SAUNE O MSH CONTROL CONTROL 6 2 to a non-specific debilitation, since this would presumably affect subjects receiving estrogen or estrogen plus progesterone. Second, the present results suggest that both the short and the long term inhibitory action of MSH may require the presence of progesterone. This is particularly surprising in the case of the short term effect as it would suggest that an MSH-progesterone interaction occurs rather rapidly. It is possible that MSH-induced alterations in serotonin activity may counteract progesterone-induced changes in serotonin activity. For example, progesterone may act in the MRF to stimulate the release of serotonin, which may then bind to serotonin type II receptors. MSH could act in the MRF to block this effect by decreasing serotonin release or perhaps by altering the configuration of serotonin type II receptors thereby reducing the receptor's a f f i n i t y for serotonin. Alternatively, MSH may in some manner alter the configuration of the progestin receptor. However, it is also possible that the failure to observe the short term and/or the long term inhibitory action of MSH is due to the high dose of estrogen employed in the current experiment. Because estrogen alone is less effective than estrogen plus progesterone in e l i c i t i n g receptivity, the estrogen dose employed in the current experiment was greater than that used in prior experiments. If either the short or the long term inhibitory action of MSH were due to an interaction with estrogen, increasing the dose of estrogen administered could attenuate this effect. Thus, it would be premature to conclude that progesterone is required for the production and/or expression of the short and long term 63 inhibitory actions of MSH. Experiment 8 The results of Experiment 7 indicated that neither the short term nor the long term inhibitory action of MSH is observed in the absence of progesterone. However, a failure to observe the short and long term inhibitory effects of MSH in Experiment 13 could have been due to the absence of progesterone or to an increase in the dose of estrogen employed. The relative importance of estrogen and progesterone in the expression of the inhibitory effect of MSH could be c l a r i f i e d by administering different doses of estrogen with varying doses of progesterone. If subjects receiving different doses of estrogen but the same dose of progesterone display the same degree of inhibition, i t would suggest that progesterone is important in the expression of the MSH effect. However, if subjects receiving the same dose of estrogen but different doses of progesterone exhibited the same degree of inhibition, it would suggest that estrogen is important in the expression of the MSH effect. If a l l groups displayed differing degrees of receptivity, it would suggest that both estrogen and progesterone are important in the expression of the short term inhibitory effect of MSH. Experiment 14 is designed to test these p o s s i b i l i t i e s . Methods Fifty-two hr prior to testing, one half of the subjects received 2 / i g EB while the remainder received 0.8 ,ug EB. Four to seven hr before testing, one half of the subjects in each EB condition received 200 jig P while the remainder received 500 M^g 64 P. Thus, four groups were created: 2 >ig EB + 200 ,ug P, n=12» 2 }xg EB + 500 ,wg P, n=ll; 0.8 ^ ig EB + 200 >ig P, n=ll; and 0.8 ^ ug EB + 500 >ig P, n=l 1. Results and Discussion An examination of the data suggests that MSH inhibited receptivity only in subjects receiving 2 ^ ug EB. In addition, it appears that subjects receiving 2 jsq EB + 500 ;ug P displayed a less pronounced inhibition than those receiving 2 jig EB + 200 >ig P (Figure 9). An overall ANOVA performed on the data revealed a significant effect of test, F(3,126)=8.22, p=0.0001. A Newman-Keul's analysis revealed that the mean LQ for the MSH test was significantly lower than the mean LQ for the f i r s t and last control tests. The mean LQ for the post-MSH test did not differ significantly from the other three tests. These results suggest that MSH exerted both a short and a long term inhibitory action. However, different hormone treatments produced varying levels of receptivity. In addition, because a l l groups received MSH on the day of MSH administration, there were no vehicle control groups with which the control groups could be compared. Thus, differential effects of MSH in the different groups could not be evaluated in the overall analysis. To examine the possibility that MSH exerts effects that are dependent upon the estrogen and/or progesterone dose, difference scores were calculated. These scores were derived by subtracting the test score (either from the MSH test or from the post-test) from the mean of the f i r s t and last control scores. ANOVAs performed on data from the day of MSH administration and from the post-MSH test failed to 65 F i g u r e 9. The e f f e c t s of e s t r o g e n and p r o g e s t e r o n e dose on the MSH-induced i n h i b i t i o n of r e c e p t i v i t y . One h a l f of the s u b j e c t s r e c e i v e d 0.8 jug e s t r a d i o l benzoate (EB) w h i l e the remainder r e c e i v e d 2 jig EB 52 hr p r i o r to t e s t i n g . One h a l f of the s u b j e c t s i n each c o n d i t i o n then r e c e i v e d 200 >ig p r o g e s t e r o n e (P) 4-7 h r p r i o r to t e s t i n g w h i l e the remainder r e c e i v e d 500 ^ ug P. T e s t s o c c u r r e d once a week f o r f o u r weeks, w i t h c o n t r o l t e s t s o c c u r r i n g on Weeks 1, 3, and 4 and an MSH t e s t o c c u r r i n g on Week 2. MSH was a d m i n i s t e r e d i n t r a c e r e b r o v e n t r i c u l a r l y a t a dose of 200 ng 4 h r p r i o r t o t e s t i n g . i 100 o o X h-z: Z) o \ C O C O O O O 80 60-\ 40 20-1 0 J Legend A 2.0 ug EB + 200 ug P • 2.0 ug EB + 500 ug P O 0.8 ug EB + 200 ug P • 0.8 ug EB + 500 ug P CONTROL MSH CONTROL CONTROL TEST 67 reveal any significant differences. These results suggest that the dose of estrogen and progesterone employed did not influence the short or long term inhibitory action of MSH. However, an examination of Figure 9 suggests that animals receiving 0.8 jxg EB failed to exhibit either the short or the long term inhibition generally produced by MSH. In addition, the long term inhibitory action of MSH appeared less pronounced in subjects receiving 2 jag EB + 500 >ig P than in those receiving 2 jxg EB + 200 ,ug P. It is possible that the lack of a significant effect of hormone dose in the current experiment is due to the doses of estrogen and progesterone employed. However, the rapid action of MSH administered ICV and the d i f f i c u l t y in obtaining a significant effect of hormone dose on the short term action of MSH suggest that this effect may be mediated predominantly through a non-hormonal mechanism. On the other hand, the long term action of MSH may involve estrogen and progesterone more directly. The finding that increasing the dose of progesterone may attenuate the long term inhibitory action of MSH lends support to this notion and suggests that MSH may produce its long term action by decreasing the availability of cytoplasmic progestin receptors. Experiment 9 The results of Experiment 7 indicated that the long term inhibitory action of MSH is not observed in the absence of progesterone. In addition, the results of Experiment 8 suggest that increasing the dose of progesterone might attenuate the long term inhibitory action of MSH. Given that the long term 68 inhibitory action of MSH appears progesterone-dependent, the effect might be mediated, at least in part, by a reduction in the ava i l a b i l i t y of cytoplasmic progestin receptors. It has been found that estrogen increases while progesterone decreases the concentration of cytoplasmic progestin receptors (Blaustein, 1982a,b; Blaustein & Feder, 1979a,b; Schwartz et a l . , 1979). This reduction in cytoplasmic progestin receptors appears to be responsible for the lack of responsiveness to progesterone that is observed at the offset of receptivity (Blaustein & Feder, 1980; Brown & Blaustein, 1984; Reading & Blaustein, 1984; Schwartz et a l . , 1979). In addition, research indicates that decreases in the concentration of cytoplasmic progestin receptors are associated with decreases in receptivity (Blaustein, 1982b; Brown & Blaustein, 1984). Furthermore, this decline in receptivity can be attenuated by increasing the dose of progesterone administered (Blaustein, 1982b; Brown & Blaustein, 1984). The results of Blaustein (1982b) and Brown & Blaustein (1984) suggest that, if the long term inhibitory action of MSH is due to a decrease in the availability of cytoplasmic progestin receptors, the administration of a large dose of progesterone should attenuate this effect. The current experiment is designed to determine the effects of an increased dose of progesterone on the display of the long term inhibitory effect of MSH. Methods Subjects were assigned to one of four groups (n=11/group): saline + low P; saline + high P; MSH + low P; MSH + high P. MSH administration occurred 4 hr prior to testing during Week 2. For 69 th i s t e s t , the usual dose of 200 ,ug P was employed. For the test occurring one week l a t e r , a dose of 200 jag P was used as the low P dose while a dose of 500 jug P was employed as the high P dose. A dose of 200 jxg P was used for contro l tests during Weeks 1 and 4. Results and Discussion An examination of the data suggests that , although MSH produced a short term i n h i b i t o r y ef fect in subjects rece iv ing both 200 and 500 >ig P, the long term inh ib i tory e f fect was observed only in subjects rece iv ing the lower dose of progesterone (Figure 10). An overa l l ANOVA performed on the data revealed a s i g n i f i c a n t e f fect of t e s t , F(3,120)=4.73, p<0.0004. To further examine the e f fect of progesterone on the long term i n h i b i t o r y act ion of MSH, a one-way ANOVA was run on data from the post-MSH tes t . This analys i s revealed a s i g n i f i c a n t e f fect of group, F(3,40)=3.82, p<0.02. A Newman-Keul's analys i s indicated that subjects rece iv ing MSH + 200 ;ug P were s i g n i f i c a n t l y less receptive than those rece iv ing sa l ine + 200 jig P. Scores for subjects rece iv ing MSH + 500 jxg P d id not d i f f e r s i g n i f i c a n t l y from scores of the other three groups. In a d d i t i o n , scores of the two sa l ine groups d id not d i f f e r from each other. These r e s u l t s indicate that increasing the dose of progesterone attenuates the long term i n h i b i t o r y ac t ion of MSH, an e f fec t expected i f the long term i n h i b i t o r y act ion of MSH is mediated, in par t , by a decrease in the a v a i l a b i l i t y of cytoplasmic progest in receptors . It is poss ible that the f a i l u r e to observe the long term inh ib i tory ef fect of MSH in subjects 70 Figure 10. The ef f e c t of increased progesterone dose on the long term inhibitory action of MSH on r e c e p t i v i t y . Subjects received 2 jug e s t r a d i o l benzoate 52 hr p r i o r to testing during Weeks 1-4 and 200 >ig progesterone 4-7 hr before testing during Weeks 1, 2, and 4. During Week 3, one half of the subjects receiving MSH and one half of the subjects receiving saline during Week 2 received 200 jxg progesterone <P) while the remainder received 500 jug P. P was administered 4-7 hr before testing. MSH was administered at a dose of 200 ng 4 hr pr i o r to testing during Week 2. 100 0 J , . n — CONTROL MSH Legend A 200 ug P, SALINE • 500 ug P, SALINE O 200 ug P,_MSH . • 500 ug P, MSH INCREASE P CONTROL 72 receiving 500 jug P is due simply to the fact that increasing the dose of progesterone from 200 jug to 500 jug increases receptivity. However, the finding that subjects receiving MSH + 500 jug P displayed a level of receptivity intermediate between that observed in controls and in subjects receiving MSH + 200 jug P suggests that: 1) 500 jug progesterone does not fully attenuate the long term inhibitory aciton of MSH. If i t did, subjects receiving MSH + 500 /ig P should be significantly more receptive than those receiving MSH + 200 jug P. 2) The attenuation of the long term inhibitory action of MSH by 500 tug progesterone is not due entirely to the increase in receptivity produced by increasing the dose of progesterone. If so, subjects receiving MSH + 500 jug P should remain significantly less receptive than those receiving saline + 500 <ug P, as increasing the dose of progesterone would shift both means upwards by a similar degree. However, subjects receiving MSH + 500 ijg P are not significantly less receptive than those receiving saline + 500 jig P. These results, and those of Experiment 8 suggest that increasing the dose of progesterone attenuates the long term inhibitory action of MSH. This supports the notion that the long term inhibitory effect of MSH is produced, in part, by an MSH-induced decrease in the availability of cytoplasmic progestin receptors. As of yet, the exact mechanism by which MSH induces this change is undetermined. DISCUSSION: SECTION III The results of Experiments 7-9 suggest that both estrogen and progesterone play a role in the expression of the inhibitory 73 actions of MSH. Although it seems clear that progesterone is involved in the long term inhibitory action of MSH, the role of estrogen is less apparent. However, it seems that both steroids may be necessary for the short term action of MSH. The results of Experiment 7 indicated that neither the short term nor the long term inhibitory actions of MSH were observed in the absence of progesterone. However, it was suggested that the failure to observe a significant inhibitory effect could have been due to the high dose of estrogen used, rather than to the absence of progesterone. To investigate this possibility, subjects in Experiment 8 were administered various doses of estrogen and progesterone and the degree of inhibition produced by MSH was examined. Results indicated that there were no significant differences in the degree of inhibition observed in subjects receiving estrogen and progesterone at the administered doses. However, certain trends did seem apparent. MSH appeared to exert a stronger inhibitory action in subjects receiving 2 jjg EB than in subjects receiving 0.8 jig EB. In addition, the long term inhibitory action of MSH appeared to be greater in subjects receiving 200 ;jg P than in those receiving 500 tjg P. Experiment 9 was conducted to investigate further the possibility that increasing the dose of progesterone might attenuate the long term inhibitory action of MSH. Results indicated that increasing the dose of progesterone significantly attenuated the long term inhibitory action of MSH. The results of Experiments 7-9 suggest that : 1) progesterone is required for the production and/or expression of 74 the long term inhibitory action of MSH (Experiment 7), 2) estrogen does not play a major role in the mediation of the long term inhibitory action of MSH (Experiments 7, 8), and 3) both estrogen and progesterone may play a role in the mediation of the short term action of MSH (Experiments 7-9). It was hypothesized that the long term inhibitory action of MSH was the result of an MSH-induced decrease in the availability of cytoplasmic progestin receptors. This decrease in the availability of progestin receptors, by decreasing progesterone binding, would reduce the synergistic action of progesterone with estrogen. In turn, the level of receptivity would decline. Increasing the dose of progesterone administered should attenuate the long term effect of MSH as this treatment is known to overcome decreases in receptivity produced by decreases in the availability of progestin receptors (Blaustein, 1982b; Brown & Blaustein, 1984). Presumably, increasing the dose of progesterone raises the probability that remaining progestin receptors will be bound by progesterone. In subjects receiving estrogen alone, the degree of receptivity is determined solely by the action of estrogen. Thus, a reduction in the availability of cytoplasmic progestin receptors should not influence receptivity in subjects receiving estrogen alone. If a long term inhibitory effect of MSH were observed in these subjects, i t would indicate that estrogen receptors may be involved. Experiment 7 indicated that the long term inhbitory action of MSH is not observed in subjects receiving estrogen alone. In addition, Experiments 8 and 9 indicated that subjects receiving 500 >ig P displayed a less pronounced long terra inhibition than subjects receiving 200 jig P These results support the notion that the long term inhibitory action of MSH is produced, at least in part, by an MSH-induced decrease in the availability of cytoplasmic progestin receptors. At present, the importance of estrogen and progesterone for the production and/or expression of the short term inhibitory action of MSH remains unclear. The results of Experiment 7 suggested that progesterone was required for the expression of the short term action of MSH. Furthermore, although subjects receiving 0.8 jig EB appeared to be less sensitive to the inhibitory actions of MSH than those receiving 2 jig EB (Experiment 8), this difference was not significant. Although these rather ambiguous findings might be accounted for by the small range of estrogen and progesterone doses employed or by some other factor, it seems more reasonable to conclude that estrogen, and possibly progesterone, do not play a major role i the short term inhibitory action of MSH, Rather, some other mechanism may be primarily responsible for this effect. This possibility seems even more likely when the interval between the administration of MSH and the onset of behavioural effects is examined. The results of Experiment 3 suggest that the short term inhibitory action of MSH may be observed as soon as 30 minutes after ICV administration. This suggests that the short term inhibitory action of MSH is produced by relatively rapid alterations in the central nervous system. A likely mediator of this effect is serotonin, which is known to play an important role in the regulation of receptivity in the female rat. 76 Section IV - MSH and Receptivity; Role of Serotonin The results of Experiments 3-5 indicate that MSH administered centrally exerts both a long and a short term inhibitory action on receptivity. The results of Experiments 7-9 suggest that, although the long term inhibitory action of MSH may be mediated by progestin receptors, the short term inhibitory action of MSH is probably produced by a direct action of MSH on neurotransmitter activity. The short term effect could be mediated through several neurotransmitters. However, there is reason to believe that serotonin might be involved. Decreases in serotonin activity generally produce increases in receptivity and vice versa (Everitt et a l . , 1975a,b; Meyerson, 1974, 1968; Sietnieks & Meyerson, 1980; Ward et a l . , 1975). In addition, depletion of serotonin has been found to reduce the concentration of MSH in the brain (Fukata et a l . , 1984). Furthermore MSH has been found to increase serotonin activity (Leonard et a l . , 1976). Finally, MSH possesses a serotonin binding site (Root-Bernstein & Westall, 1984) and serotonin receptors possess a peptide binding site (Roth et a l . , 1984). Thus, serotonergic involvement in the MSH-induced inhibition of receptivity is clearly a possibility. Although several researchers (e.g., Everitt et a l , 1975a,b; Meyerson, 1964a,b, 1968; Ward et a l . , 1975) believe that serotonin plays a major role in the regulation of receptivity in the female rat, studies examining the effects of serotonin on receptivity are somewhat contradictory. For example, both stimulation and inhibition of serotonin receptor activity have been reported to increase, decrease, or have no effect on 77 receptivity (e.g., Clemens, 1978; Davis & Kohl, 1978; Espino, Sano, & Wade, 1975; Seitnieks & Meyerson, 1980, 1982; Ward et a l . , 1975; Zemlan et a l . , 1973). Furthermore, the depletion of serotonin has been reported to increase (Zemlan et a l . , 1973), decrease (Gorzalka & Whalen, 1975), or have no effect on lordosis (Meyerson & Lewander, 1970). More recent research may serve to c l a r i f y the effects of serotonin on receptivity. It is now known that there are serotonin type I and type II receptors (Peroutka, Lebovitz, & Snyder, 1981; Peroutka & Snyder, 1979, 1981). Serotonin type I receptors have a greater a f f i n i t y for serotonin than for spiroperidol while serotonin type II receptors have a greater a f f i n i t y for spiroperidol than for serotonin (Peroutka et a l . , 1981; Peroutka & Snyder, 1979, 1981, 1982a,b, 1983). Mendelson & Gorzalka (1985) have recently hypothesized that increased serotonin type I activity inhibits, while increased serotonin type II receptor activity f a c i l i t a t e s , sexual respond-ing in the female rat. In support of this hypothesis are findings that serotonin type I receptor agonists (Fuxe, Everitt, Agnati, Fredholm, & Jonsson, 1976) and serotonin type II receptor antag-nists (Mendelson & Gorzalka, 1985) inhibit receptivity. Further-more, the serotonin type II receptor agonist, quipazine, was found to attenuate the inhibitory effect of the serotonin type II receptor antagonist, pirenperone, on receptivity (Mendelson & Gorzalka, 1985). In further support of a differential role of serotonin receptors on receptivity are the findings that estrogen and progesterone both alter brain concentrations of serotonin receptors but that the type of effect depends on the serotonin 78 receptor examined (Biegon, Fischette, Rainbow, & McEwen, 1982; Biegon & McEwen, 1982; Biegon, Reches, Snyder, & McEwen, 1983). Chronic MSH treatment has been found to produce an overall increase in serotonin activity (Leonard et a l . , 1976). However, equivalent studies examining the effects of acute MSH treatment on serotonin activity have apparently not been published. In light of evidence indicating that MSH alters serotonin activity and that alterations in serotonin activity alter lordosis, it seems reasonable to suggest that at least a portion of the inhibitory action of MSH may be mediated through a serotonergic mechanism. Furthermore, the finding that ovarian hormones can differentially affect the activity of serotonin type I and type II receptors suggests two possible mechanisms by which MSH could exert this effect: 1) MSH could increase serotonin type I receptor activity and/or 2) MSH could decrease serotonin type II receptor activity. The neuroanatomical distribution of serotonin receptors and of MSH receptors lends further plausibility to this idea. Both the AH-POA and the MRF play an important role in the regulation of lordosis. As mentioned previously, radiolabe1led estrogen is preferentially taken up by neurons in the AH-POA (Pfaff, 1968a,b; Pfaff & Keiner, 1973). Radiolabelled progesterone is preferentially taken up in the mesencephalon (Wade et a l . , 1973; Whalen & Luttge, 1971a,b). In addition, estrogen implanted into the AH-POA (Lisk, 1962; Ross et a l . , 1971) and progesterone implanted into the MRF (Gorski, 1976; Luttge & Hughes, 1976; Ross et a l . , 1971) have been found to 7 9 f a c i l i t a t e receptivity. In addition, both of these areas are innervated by serotonergic pathways (Dahlstrom & Fuxe, 1965; Fuxe, 1965; Moore, 1981; Ungerstedt, 1971), supporting the notion that serotonin may be involved in the regulation of sexual behaviour. Furthermore, the infusion of serotonin into the medial preoptic area <MPOA) or into the arcuate-ventromedial area (ARC-VM) has been found to inhibit receptivity while infusion of methysergide, a serotonin receptor blocker, to these areas has been found to increase receptivity (Foreman & Moss, 1978). Methysergide binds preferentially to serotonin type II receptors but also binds to serotonin type I receptors (Peroutka et a l . , 1981). If Mendelson & Gorzalka's (1985) hypothesis is correct, one might expect methysergide to inhibit receptivity as it would block the facilitatory actions of serotonin type II activity. However, the AH-POA, MPOA, and ARC-VM are a l l areas with relatively high concentrations of serotonin type I receptors and relatively low concentrations of serotonin type II receptors (Leysen et a l . , 1982; Peroutka & Snyder, 1981). Due to the paucity of serotonin type II receptors in these areas, it is likely that, methysergide binds predominantly to serotonin type I receptors when infused into these regions. An inhibition of sexual receptivity after the infusion of serotonin into these areas and the fa c i l i t a t i o n of receptivity after the infusion of methysergide into these areas is thus consistent with the hypothesis that serotonin type I receptors inhibit receptivity. There is some evidence that, under conditions that may produce blockade of serotonin type II (vs type I) receptors, methysergide 80 may inhibit receptivity (Clemens, 1978; Meyerson & Eliasson, 1977; Sietnieks, 1985), lending support to the notion that serotonin type II receptors exert a facilitatory action on receptivity. These findings suggest that the effects of serotonin in the hypothalamic area may be the result of this area being populated predominantly with serotonin type I receptors. MSH is produced in the arcuate nucleus of the hypothalamus and stored in vesicles that are then transported to other brain regions by axonal flow (O'Donohue, Miller, & Jacobowitz, 1979; Thody, 1980). In addition, immunohistochemical investigations indicate that MSH-positive fibres are found near the ependymal cells of the lateral and third ventricles (Dube, Cote, & Pelletier, 1979). These findings led Dube et a l . (1979) to suggest that these MSH-positive fibres might be the source of MSH in the CSF. Thus, these fibres would also provide another means by which MSH is distributed throughout the brain. Of particular importance to the current discussion is the finding that high concentrations of MSH are found in the POA, hypothalamus, and mesencephalon (Eskay, Giraud, Oliver, & Brownstein, 1979; O'Donohue & Chappell, 1982; O'Donohue et a l . , 1979; Oliver & Porter, 1978; Thody, 1980). The results of Leonard et a l . (1976) suggest that MSH may increase serotonin activity in the hypothalamus. As this area contains predominantly serotonin type I receptors, MSH may exert its inhibitory action on receptivity via an increase in serotonin type I receptor binding in the hypothalamus. However, MSH may also exert its inhibitory action by altering serotonin type II activity. The mesencephalon 81 contains both serotonin type I and type II receptors. However, the concentration of serotonin type II receptors is somewhat higher in the mesencephalon than in the hypothalamus (Peroutka & Snyder, 1981). Thus, MSH could inhibit receptivity by decreasing serotonin type II activity in the hypothalamus and/or mesencephalon. Experiments 10-15 were designed to examine the role of serotonin type II activity in the production of the inhibitory action of MSH on receptivity. General Methods The general methods employed in this section will be similar to those employed in Section III. Unless otherwise noted, a 4x4 repeated measures design will be used such that there are four groups on the day of MSH testing: saline + saline; MSH + saline; saline + drug; MSH + drug. The presence of one group that receives saline on the day of MSH testing will provide a further index of s t a b i l i t y . Because the experiments in this section are preliminary, MSH will be placed into the ventricle rather than into the AH-POA or MRF. Experiment 10 If the short term inhibitory action of MSH is due to an alteration in serotonin activity, the depletion of serotonin should prevent the effect. Furthermore, if the long term inhibitory effect of MSH is dependent upon an MSH-serotonin interaction, serotonin depletion should also prevent this effect as it would prevent the interaction. PCPA-induced depletions of serotonin have been found to decrease sexual responding in females primed with estrogen and progesterone but not those 82 primed with estrogen alone (Gorzalka & Whalen, 1975). However, if MSH acted on a neurotransmitter other than serotonin, one might expect the inhibitory effects of MSH and the inhibitory effects of a serotonin depletor to summate to produce an inhibition greater than that seen with either administered alone. On the other hand, i f the two acted upon the same neurotransmitter, one should not necessarily observe a significant additive effect. Of course, a failure to observe a significant additive effect does not necessarily indicate that MSH acts via a serotonin depletion. Nonetheless, the administration of PCPA will provide useful information concerning the involvement of serotonin versus other neurotransmitters in the mediation of the inhibitory effects of MSH. Thus, the present experiment was designed to determine the effects of PCPA treatment on the long and short term inhibitory actions of MSH. Methods Subjects were assigned to one of four groups: saline + saline, n=16; MSH + saline, n=ll; saline + PCPA, n=16; or MSH + PCPA, n=ll. PCPA (150 mg/kg; concentration, 150 mg/1.6 cc) was administered intraperitoneally (IP) 72 hr prior to testing as research indicates that the effects on catecholamines are minimal at this time while the effects on serotonin levels are maximal (Ahlenius, Engel, Eriksson, & Sodersten, 1972; Everitt et a l . , 1975a,b; Koe & Weissman,1966). Results and Discussion An examination of the data suggests that the administration of MSH and PCPA, alone and in combination, produced an Inhibitory 83 e f f e c t o n r e c e p t i v i t y on t h e d a y o f MSH t e s t i n g (Week 2 ) . F u r t h e r m o r e , a l t h o u g h MSH a d m i n i s t e r e d a l o n e a p p e a r s t o h a v e e x e r t e d a l o n g t e r m i n h i b i t o r y a c t i o n on r e c e p t i v i t y , MSH + PCPA d i d n o t e x e r t t h i s e f f e c t ( F i g u r e 1 1 ) . An a n a l y s i s o f v a r i a n c e p e r f o r m e d on t h e d a t a r e v e a l e d a s i g n i f i c a n t g r o u p s x t e s t i n t e r a c t i o n , F ( 9 , 1 5 0 ) = 4 . 5 7 , p=0.0001. A Newman-Keul's a n a l y s i s i n d i c a t e d t h a t s u b j e c t s r e c e i v i n g MSH, PCPA, o r MSH + PCPA d u r i n g Week 2 o f t e s t i n g were s i g n i f i c a n t l y l e s s r e c e p t i v e t h a n s u b j e c t s r e c e i v i n g s a l i n e i n j e c t i o n s b u t d i d n o t d i f f e r f r o m e a c h o t h e r . T h i s i n d i c a t e s t h a t a l t h o u g h b o t h MSH a n d PCPA i n h i b i t r e c e p t i v i t y , t h e i r e f f e c t s a r e n o t a d d i t i v e a n d t h e r e f o r e may be p r o d u c e d by t h e same m e c h a n i s m . A l t e r n a t i v e l y , t h e low d e g r e e o f r e c e p t i v i t y p r o d u c e d by e i t h e r s u b s t a n c e may h a v e p r e v e n t e d t h e o b s e r v a t i o n o f a n a d d i t i v e e f f e c t when t h e two were a d m i n i s t e r e d t o g e t h e r ( a f l o o r e f f e c t ) . H owever, a n e x a m i n a t i o n o f F i g u r e 9 s u g g e s t s t h a t t h e d e g r e e o f i n h i b i t i o n p r o d u c e d by MSH, PCPA, o r MSH + PCPA was v i r t u a l l y i d e n t i c a l . I n a d d i t i o n , t h e mean LQ f o r s u b j e c t s r e c e i v i n g MSH + PCPA was a p p r o x i m a t e l y 20. T h u s , I t w o u l d h a v e b e e n p o s s i b l e t o o b s e r v e a t l e a s t a t r e n d t o w a r d s a n a d d i t i v e e f f e c t i n t h e s e s u b j e c t s . The f a i l u r e t o o b s e r v e s u c h a t r e n d , w h i l e n o t c o n c l u s i v e e v i d e n c e a g a i n s t t h e p o s s i b i l i t y o f a n a d d i t i v e e f f e c t , a r g u e s a g a i n s t t h i s e x p l a n a t i o n . S u b j e c t s r e c e i v i n g MSH d u r i n g Week 2 d i s p l a y e d a s i g n i f i c a n t l y l o w e r mean LQ d u r i n g Week 3 t h a n s u b j e c t s r e c e i v i n g MSH + PCPA. T h i s s u g g e s t s t h a t PCPA c a n p r e v e n t t h e l o n g t e r m i n h i b i t o r y a c t i o n o f MSH. T h u s , b o t h t h e l o n g a n d t h e s h o r t t e r m 84 Figure 11. The effects of MSH and PCPA on receptivity. Subjects received 2 jig estradiol benzoate 52 hr prior to testing and 200 u^g progesterone 4-7 hr before testing. Testing occurred once a week for four weeks, with control tests occurring on Weeks 1, 3, and 4 and an MSH/drug test occurring on Week 2. MSH was administered intracerebroventricularly at a dose of 200 ng 4-7 hr prior to testing. Parachlorophenylalanine (PCPA) was administered intraperitoneal1y at a dose of 150 mg/kg 72 hr prior to testing. 100-1 I— 1— CONTROL DRUG Legend A S A U N E + S A L I N E • SALINE+PCPA O MSH+SALINE • MSH+PCPA CONTROL CONTROL 86 effects of MSH may be mediated through a serotonergic mechanism. However, the finding that PCPA prevents the long term, but not the short term, effects of MSH suggests that two different mechanisms may be involved. Other effects found to be significant were groups, F<3,50)= 4.68, p=0.006 and test, F(3,150)=30.87, p<0.000l. Experiment 11 The results of Experiment 10 suggest that serotonin is involved in the mediation of both the long and the short term inhibitory actions of MSH. However, because PCPA is a serotonin synthesis inhibitor, i t is not possible to determine the importance of serotonin type I versus type II receptors in the production of this effect. The work of Mendelson & Gorzalka (1985) suggests that serotonin type II receptors play an important role in mediating sexual responding. MSH may exert its inhibitory effect, in part, by decreasing the activity of serotonin type II receptors. If so, the administration of a serotonin type II receptor antagonist in combination with MSH should produce no greater inhibitory effect than i f either were administered alone. However, if serotonin type II receptors are not responsible for the inhibitory action of MSH-, the administration of MSH in conjunction with a serotonin type II antagonist might produce an inhibition greater than that observed with either administered alone. The present experiment is designed to investigate the effects of pirenperone, a relatively specific serotonin type II antagonist (Colpaert & Janssen, 1983; Green, 0*shaughnessy, Hammond, Schachter, & Grahame-Smith, 1983; 87 Janssen, 1983), on receptivity in subjects receiving MSH. A non-additive effect would suggest that MSH is exerting a portion of its inhibitory action via serotonin type II receptors. An additive effect could be taken as an indication of potential serotonin type I receptor involvement in the MSH effect. Alternatively, an additive effect may reflect the involvement of other neurotransmitters in the production of the inhibitory action of MSH. Methods Subjects were assigned to one of four groups: saline + saline, n=13l saline + pirenperone, n=13» MSH + saline, n=14» MSH + pirenperone, n=14. Pirenperone was administered IP at a dose of 100 jjg/kg (concentration, 30 jig/0. 1 cc) 1 hr prior to testing (3 hr after MSH). This procedure has previously been found to inhibit receptivity in female rats (Mendelson & Gorzalka, 1985). Results and Discussion An examination of the data suggested that, although both MSH and pirenperone inhibited receptivity, their effects were not additive (Figure 12). An ANOVA performed on the data revealed a significant effect of test, F(3,150)=20.88, p<0.0001, and a significant group x test, F(9,150)=5.22, p<0.0001, interaction. A Newman-Keul's analysis indicated that subjects receiving MSH or pirenperone + MSH were significantly less receptive than those receiving saline on the day of MSH administration. In addition, although subjects receiving MSH + pirenperone were significantly less receptive than those receiving pirenperone, they did not 88 Figure 12. The effects of MSH and pirenperone on receptivity. Subjects received 2 jug estradiol benzoate 52 hr prior to testing and 200 jug progesterone 4-7 hr before testing. Testing occurred once a week for four weeks, with control tests occurring on Weeks 1, 3, and 4 and an MSH/drug test occurring during Week 2. MSH was administered intracerebroventricularly at a dose of 200 ng 4 hr prior to testing. Pirenperone was administered intraperitoneal1y at a dose of 100 >ug/kg 1 hr before testing. 100-1 CONTROL CONTROL TEST CO 90 differ significantly from those receiving MSH. Finally, subjects receiving MSH did not differ significantly from those receiving pirenperone. The analysis also indicated that, when tested one week later, subjects receiving MSH, but not subjects receiving pirenperone, were significantly less receptive than their respective controls. These results indicate that, although both MSH and pirenperone inhibit receptivity, their effects do not appear to be additive. In addition, MSH, but not pirenperone, exerted a long term inhibitory effect on receptivity. These findings suggest that MSH may inhibit receptivity by decreasing serotonin type II activity, as is the case with pirenperone. In addition, it is clear that temporarily decreasing serotonin type II activity is not sufficient to produce a long term inhibition of receptivity as pirenperone did not produce this effect. Thus, it seems likely that the long term inhibitory effect of MSH is not the direct result of an acute decrease in serotonin type II activity. This opens up the possibility that the long term inhibitory action of MSH may be mediated by serotonin type I receptors, by some other neurotransmitter, or by an MSH-serotonin interaction of some type. The finding that PCPA prevents the long term inhibitory action of MSH suggests that serotonin type I receptors or a serotonin-MSH interaction may mediate the effect. It should be noted that the level of receptivity displayed by MSH + pirenperone subjects was extrememly low. Thus, i t is possible that a 'floor' effect prevented the observation of an additive effect. Although the current results cannot eliminate this 91 possibility, additional experiments could help c l a r i f y the importance of the serotonin type II receptor in the production of the inhibitory action of MSH. Experiment 12 The results of Experiment 11 suggest that serotonin type II receptors may play an important role in the mediation of the short term inhibitory effect of MSH. However, the role of serotonin type II receptors in the mediation of this effect remains unclear. Quipazine is a relatively specific serotonin type II agonist (Green et a l . , 1983; Mendelson & Gorzalka, 1985). If the inhibitory action of MSH is mediated to some extent by a decrease in serotonin type II receptor activity, quipazine should attenuate this effect. The present experiment is designed to investigate the effects of quipazine administration on the long and short term inhibitory action of MSH. Methods Subjects were assigned to one of four groups: saline + saline, n=14; saline + quipazine, n=14» MSH + saline, n=13» or MSH + quipazine, n=13. Quipazine was administered IP at a dose of 3 mg/kg (concentration, 3 mg/0.4 cc) 1 hr prior to testing (3 hr after MSH) on the day of MSH testing (Week 2). This dose of quipazine has previously been found effective in attenuating the effects of pirenperone (Mendelson & Gorzalka, 1985). Results and Discussion An examination of the data suggests that MSH exerted both a short and a long term inhibitory effect. In addition, although quipazine alone had no effect on receptivity, quipazine 92 administered with MSH appeared to attenuate the short term inhibitory action of MSH while only partially attenuating the long term inhibitory effect (Figure 13). An ANOVA performed on the data indicated a significant effect of group F(3,150)=4.97, p<0.004 and a significant group x test interaction, F(9,150)=5.58, p<0.0001. A Newman-Keul's test performed on the group x test interaction indicated that the mean LQs for subjects receiving MSH on the day of MSH administration and one week after MSH administration were significantly lower than a l l other mean LQs. Mean LQs for subjects receiving saline, quipazine, or MSH + quipazine did not differ from each other. These results indicate that quipazine attenuates the short term inhibitory effect of MSH. In addition, quipazine did not appear to attenuate fully the long term inhibitory effect of MSH. However, this effect did not reach significance: subjects receiving MSH + quipazine did not differ significantly from those receiving saline when tested one week after MSH administration. The current findings confirm those of Experiments 10 and 11 in that they strongly suggest a role for serotonin in the inhibitory action of MSH. In addition, the finding that quipazine reversed the short term inhibitory action of MSH makes it unlikely that the failure to observe an additive effect of MSH with pirenperone was due solely to a 'floor' effect. Nonetheless, the possibility that quipazine is less effective in attenuating the long term (vs short term) action of MSH suggests that the two effects may be mediated via somewhat different mechanisms. The current results suggest that alterations in 93 Figure 13. The effects of MSH and quipazine on receptivity. Subjects received 2 tug estradiol benzoate 52 hr prior to testing and 200 jug progesterone 4-7 hr prior to testing. Tests occurred once a week for four weeks with control tests occurring on Weeks 1, 3, and 4 and an MSH/drug test occurring on Week 2. MSH was administered intracerebroventricularly at a dose of 200 ng 4 hr prior to testing. Quipazine was administered intraperitoneal1y at a dose of 3 mg/kg 1 hr prior to testing. » 100 - i 0 i 1— CONTROL DRUG • MSH+QUIPAZINE CONTROL CONTROL TEST 95 serotonin type II activity at the time of MSH administration may be involved in the production of the long term inhibitory effect of MSH. That PCPA prevents the long term effect also supports this notion. However, the finding that pirenperone does not produce a long term inhibition indicates that an alteration in serotonin type II activity is not the only factor Involved in the production of the long term effect. Experiment 13 The results of Experiments 11 and 12 suggest an important role for serotonin type II receptors in the mediation of the inhibitory action of MSH. A further verification of the importance of serotonin type II receptors in this effect could be gained by the demonstration of an additive effect of a subthreshold dose of MSH administered with a subthreshold dose of a serotonin type II antagonist. In preparation for such an experiment, the present experiment is designed to determine doses of both MSH and pirenperone that are subthreshold with respect to the inhibition of lordosis. Methods P irenperone Subjects were assigned to one of four groups: 100 >ig/kg pirenperone; 50 jug/kg pirenperone; 25 jjg/kg pirenperone; or saline (n=7/group). The pirenperone doses selected are based upon a previous dose response where animals were treated with estrogen alone (Mendelson & Gorzalka, 1985). As these doses a l l proved to be above threshold, additional subjects were run after being injected with either 2, 5, or 15 ug/kg pirenperone Cn=6/group). 96 All pirenperone doses were administered IP in concentrations such that the average volume per subject was 0.12 cc (a volume similar to that used in Experiment 8). MSH Subjects were assigned to one of 3 groups: 100 ng MSH; 50 ng MSH; or 20 ng MSH (n=8/group). Because these subjects were tested at the same time as the pirenperone-treated subjects, the subjects receiving saline during the pirenperone dose response study were also used for the saline control group in the MSH dose response study. All doses of MSH were administered in a volume of 4 y U l . Results and Discussion Pirenperone An examination of the data from subjects receiving different pirenperone doses suggested that 2 and 5 ^ ug/kg pirenperone had no effect on receptivity. However, 15 jxg/kg pirenperone appeared to have a moderate inhibitory effect on receptivity while 25, 50, and 100 jxg/kg pirenperone appeared to exert a strong inhibitory effect on receptivity (Figure 14). A one-way ANOVA performed on the data revealed a significant effect of dose, F(6,39)=8.44, p<0.0001. A Newman-Keul's analysis indicated that 25, 50, and 100 jkg/kg pirenperone produced a significantly greater inhibition than 0, 2, or 5 ^ ug/kg pirenperone. The mean LQ for the 15 jig/kg pirenperone group did not d i f f e r significantly from the mean LQs for any other group. These results indicate that doses of pirenperone above 15 ug/kg inhibit receptivity while doses of pirenperone below this 9 7 Figure 14. The dose-response effects of pirenperone on receptivity. Subjects received 2 jug estradiol benzoate 52 hr prior to testing and 200 jug progesterone 4-7 hr before testing. Pirenperone was administered intraperitoneally 1 hr prior to test ing. 100 80-60-40-20-0 5 15 2 5 PIRENPERONE DOSE j j g / k g 99 have no effect. This suggests that a dose of pirenperone between 5 and 15 jug/kg would represent an optimal threshold dose one that has some biological activity but is not sufficient to produce a behavioural effect. These findings are comparable to those obtained by Mendelson & Gorzalka (1985) in subjects receiving estrogen alone. MSH An examination of the data from subjects receiving various doses of MSH suggests that 50 and 100 ng MSH, but not 20 ng MSH, were sufficient to produce a short-term inhibitory effect (Figure 15). However, a l l three doses appeared to produce a long term inhibition. An ANOVA performed on this data revealed a significant effect of test, F(3,81)=20.25, p<0.0001, and a significant dose x test interaction, F(9,81)=4.84, p<0.0001. A Newman-Keul's analysis performed on the dose x test interaction revealed that subjects receiving 50 or 100 ng MSH were significantly less receptive than subjects receiving 20 ng MSH or saline on the day of MSH administration. However, when tested one week later, a l l three MSH groups were significantly less receptive than the saline group. These results indicate that 50 and 100 ng MSH produce both a long and short term inhibitory effect while 20 ng MSH produces only the long term effect. Thus, 20 ng MSH is a threshold dose for the short term, but not the long term, inhibitory action of MSH. This finding, in conjunction with the finding that PCPA blocked the long term inhibitory action of MSH (Experiment 10) and that quipazine did not fully attenuate the long term 1 0 0 F i g u r e 15. The dose-response e f f e c t s of MSH on r e c e p t i v i t y . S u b j e c t s r e c e i v e d 2 jug e s t r a d i o l benzoate 52 h r p r i o r to t e s t i n g and 200 jug p r o g e s t e r o n e 4-7 h r b e f o r e t e s t i n g . T e s t i n g o c c u r r e d once a week f o r f o u r weeks, w i t h c o n t r o l t e s t s o c c u r r i n g d u r i n g Weeks 1, 3, and 4 and an MSH t e s t o c c u r r i n g on Week 2. MSH was a d m i n i s t e r e d i n t r a c e r e b r o v e n t r i c u l a r l y 4-7 hr p r i o r t o t e s t i n g . 1 0 0 - 1 -a' Legend A 0 ng MSH • 20 ng MSH O 50 ng MSH_ • 100ng_MSH CC0NTR0L CONTROL 102 inhibitory action of MSH (Experiment 12) suggests that the short term and the long term inhibitory actions of MSH are mediated by different mechanisms. Furthermore, these 2 mechanisms would appear to be differentially sensitive to the actions of MSH. It is possible that the short term inhibitory action of MSH is dependent primarily upon MSH-induced alterations in serotonin type II activity while the long term inhibitory action is due to MSH/serotonin-induced alterations in estrogen, progestin, or serotonin receptor avai l a b i l i t y . Furthermore, relatively large concentrations of MSH may be required to decrease serotonin type II activity while smaller concentrations may be sufficient to alter receptor avai l a b i l i t y . Experiment 14 The current experiment is designed to determine the effects of subthreshold doses of MSH and pirenperone, administered alone and in combination, on receptivity. If an inhibition is observed with MSH + pirenperone but not with either alone, i t would be consistent with the idea that both manipulations are acting through the same mechanism, an inhibition of serotonin type II receptor activity. Methods Animals were assigned to one of four groups: saline + saline, n=14» saline + pirenperone, n=14» MSH + saline, n=12; and MSH + pirenperone, n=12. Doses of 10 jig/kg pirenperone and 20 ng MSH were used as the results of Experiment 10 suggested that these would be appropriate subthreshold doses. MSH and pirenperone were administered to subjects prior to testing during 103 Week 2 at the times indicated in Experiment 11. Results and Discussion An examination of the data suggests that subthreshold doses of MSH and of pirenperone did not inhibit receptivity when administered alone but did inhibit receptivity when administered in combination (Figure 16). An overall ANOVA performed on the data revealed a significant effect of test, F(3,144)=6.36, p=0.005, and a significant group x test interaction, F(9,144)= 4.65, p<0.0001. A Newman-Keul's analysis of the group x test interaction indicated that subjects receiving MSH + pirenperone were significantly less receptive than subjects receiving MSH, pirenperone, or saline on the day of MSH testing. In addition, subjects receiving MSH or pirenperone did not differ from those receiving saline on the day of MSH testing. When tested one week later, subjects receiving MSH or MSH + pirenperone did not differ from each other but were were significantly less receptive than subjects receiving pirenperone or saline. These findings indicate that doses of MSH and pirenperone that produce no behavioural effect when administered alone can inhibit receptivity when administered in combination. As pirenperone is a serotonin type II antagonist, this suggests that MSH may also act to antagonize serotonin type II activity. Thus, although the decrease in serotonin type II activity produced by a subthreshold dose of MSH or pirenperone is not sufficient to decrease receptivity, the decrease in serotonin type II activity produced by the two together is. The possibility remains that MSH produces Its effects by some 104 F i g u r e 16. The e f f e c t s of s u b t h r e s h o l d doses of MSH and p i r e n p e r o n e on r e c e p t i v i t y . S u b j e c t s r e c e i v e d 2 jug e s t r a d i o l benzoate 52 h r p r i o r t o t e s t i n g and 200 jug p r o g e s t e r o n e 4-7 h r b e f o r e t e s t i n g . T e s t s o c c u r r e d once a week f o r f o u r weeks, w i t h c o n t r o l t e s t s o c c u r r i n g on Weeks 1, 3, and 4 and an MSH/drug t e s t o c c u r r i n g on Week 2. MSH was a d m i n i s t e r e d i n t r a c e r b r o v e n t r i c u -l a r l y a t a dose of 20 ng 4 hr p r i o r t o t e s t i n g . P i r e n p e r o n e was a d m i n i s t e r e d i n t r a p e r i t o n e a l 1 y a t a dose of 10 jug/kg 1 h r b e f o r e t e s t i n g . o o X r -z O 01 o Q C£ O 100-1 80H 60H 40 H 20 H 0-" Legend A SALINE+SALINE • SALINE+PIRENPERONC O MSH + SALINE • MSH + PIRENPERONE CONTROL DRUG CONTROL CONTROL TEST o Ln 106 mechanism other than serotonin type II activity and that this effect summated with the effect produced by pirenperone. However, the finding (Experiment 12) that quipazine reverses the inhibitory action of MSH makes this possibility unlikely. Thus, it would appear that serotonin type II activity plays an important role in the mediation of the short term inhibitory effect of MSH. The current results also confirmed those of Experiment 13, in which 20 ng MSH was observed to produce a long term, but not a short term, inhibitory action on receptivity. Since pirenperone alone did not exert a long term inhibitory action on receptivity, it seems likely that the long term effect observed in subjects receiving MSH + pirenperone was due to an action of MSH. The current findings lend further support to the notion that the short and long term actions of MSH are mediated by different mechan i sms. Experiment 15 The results of Experiment 14 indicated a significant inhibition of receptivity in subjects administered MSH + pirenperone but not MSH or pirenperone alone. This suggests that the short term inhibitory effect of MSH is mediated, at least in part, by a decrease in serotonin type II receptor activity. Further support for this hypothesis would be gained i f it could be demonstrated that a serotonin type II agonist could reverse this effect. The present experiment is designed to determine the effectiveness of quipazine in reversing the inhibitory effect produced when a subthreshold dose of MSH is administered in 107 conjunction with a subthreshold dose of pirenperone. Mefrhod,s Subjects were assigned to one of four groups: saline + saline, n=14; saline + pirenperone + MSH, n=ll; quipazine + saline, n=l3; and quipazine + pirenperone + MSH, n=12. Doses and times of MSH and drug administration followed those used in Experiments 12, 13, and 14. Results and Discussion An examination of the data suggests that quipazine reversed the short term inhibitory action of subthreshold doses of pirenperone + MSH (Figure 17). In addition, quipazine appeared to have facilitated receptivity in subjects receiving quipazine alone. An ANOVA performed on the data revealed a significant group x test interaction, F(9,138)=5.34, p<0.0001. A Newman-Keul 's analysis revealed that, on the day of drug administration, subjects receiving quipazine were significantly more receptive than subjects receiving saline or quipazine + pirenperone + MSH, which were significantly more receptive than subjects receiving pirenperone + MSH. When tested one week later, subjects receiving pirenperone + MSH and quipazine + pirenperone + MSH were found to be significantly less receptive than subjects receiving saline or qu ipaz ine. These results indicate that quipazine, a serotonin type II agonist, can reverse the short term inhibitory action of subthreshold doses of MSH + pirenperone on receptivity. This further supports the notion that MSH acts, at least in part, by decreasing serotonin type II activity. The results also indicate 108 Figure 17. The effects of quipazine on the inhibition of receptivity produced by subthreshold doses of MSH and pirenperone. Subjects received 2 >ig estradiol benzoate 52 hr prior to testing and 200 >ug progesterone 4-7 hr before testing. Testing occurred once a week for four weeks, with control tests occurring during Weeks 1, 3, and 4 and an MSH test occurring during Week 2. MSH was administered intracerebroventricularly at a dose of 20 ng 4 hr prior to testing. Pirenperone (10 >ig/kg) and quipazine (3 mg/kg) were administered intraperitoneal1y 1 hr before testing. Legend 100 80 O O X 60 oo O 40 Q oc o 20 0 J CONTROL DRUG A SALINE*SALINC*S*LWE • S*UNC+SALINC+OUIPAZ'NC _ O USH4PIRENPER0NE + SMJNC • MSH+PIRCNPCR0NC+0UIP4ZIUC CONTROL CONTROL TEST O 110 that quipazine , when administered alone, f a c i l i t a t e s r e c e p t i v i t y . This f ind ing is consistent with the f indings of Hunter, Hole, & Wilson (1985) but not with the f indings of Experiment 12. However, subjects in Experiment 12 displayed a higher degree of r e c e p t i v i t y than those in the current experiment. Thus, i t is poss ible that a c e i l i n g e f fect prevented the observation of a f a c i l i t a t i o n in Experiment 12. Also in contrast to Experiment 12 was the f ind ing that quipazine d id not attenuate the long term 1 i n h i b i t o r y ac t ion of MSH. However, an examination of the data from Experiment 12 (Figure 13) suggests that quipazine d id not f u l l y attenuate the long term inh ib i tory ef fect of MSH. This f i n d i n g , plus those of Experiments 10 and 12 suggest that the long term inh ib i tory ac t ion of MSH is mediated by something other than simple changes in serotonin type II a c t i v i t y . Indeed, the f inding that PCPA adminis trat ion prevents the long term i n h i b i t i o n and that quipazine does not f u l l y reverse the e f fec t suggests that both MSH and serotonin are required to produce the long term e f f e c t . DISCUSSION: SECTION IV O v e r a l l , the resu l t s of Experiments 10-15 support the notion that MSH produces i t s i n h i b i t o r y e f f e c t , at least in p a r t , by a l t e r i n g serotonin a c t i v i t y . The resu l t s of Experiment 10 indicated that , although both MSH and PCPA exerted an inh ib i tory e f fect on r e c e p t i v i t y , t h e i r e f fects were not a d d i t i v e . This suggested that the short term inh ib i tory ac t ion of MSH was mediated, in part , by changes in serotonin a c t i v i t y . The re su l t s of Experiment 10 also indicated that the long term I l l inhibitory effect of MSH was prevented by PCPA administration. This suggests that an MSH-serotonin interaction is required for the production of the long term effect. In Experiment 11, pirenperone was administered to determine if alterations in serotonin type II receptor activity were involved in the inhibitory action of MSH. It was found that, although both MSH and pirenperone produced a short term inhibitory action on receptivity, their effects were not additive. Furthermore, MSH, but not pirenperone, produced a long term inhibition of receptivity. These findings suggest that the short term inhibitory action of MSH is due, in part, to a reduction in serotonin type II activity. Furthermore, it would appear that an acute reduction in serotonin type II activity cannot by i t s e l f account for the long term inhibitory action of MSH. If an acute decrease in serotonin type II activity were entirely responsible for the long term action of MSH, then pirenperone should also have produced this effect. Thus, the long term inhibitory action of MSH must involve other mechanisms as well. That an acute reduction in serotonin type II activity is involved to some degree is indicated by the finding that PCPA prevents the long term effect of MSH. In Experiment 12, quipazine was administered in conjunction with MSH to determine if quipazine could reverse the inhibitory action of MSH. Quipazine fu l l y attenuated the short term action of MSH and partially attenuated the long term action of MSH. This further supports the notion that MSH produces its short term inhibitory effect on receptivity via a reduction in serotonin 112 type II activity. The finding that quipazine partially attenuated the long term inhibitory effect of MSH also suggests a role for serotonin type II receptors in the mediation of this effect. Experiment 13 was conducted to determine the subthreshold doses of MSH and of pirenperone to be used in Experiment 14. One interesting finding was that the subthreshold dose of MSH for the short term inhibition was above threshold for the long term inhibit ion. In Experiment 14, results indicated that subthreshold doses of MSH and of pirenperone, when administered together, would summate to produce a short term inhibitory effect. This lent further support to the notion that MSH acts, at least in part, by decreasing serotonin type II activity. As the final test of this notion, Experiment 15 was performed to determine if quipazine would reverse the inhibition produced by subthreshold doses of MSH + pirenperone. Results indicated that quipazine did indeed attenuate the short term inhibition produced by the coadministration of subthreshold doses of MSH and pirenperone. However, as suggested by the results of Experiment 12, quipazine did not fully attenuate the long term inhibitory action of MSH. These experiments strongly suggest that, while the short term inhibitory action of MSH is mediated, to a large extent, by an acute reduction in serotonin type II activity, the long term inhibitory effect of MSH is not. The finding that the short term inhibitory effect of MSH may be produced by an MSH-induced decrease in serotonin type II 113 activity is of great interest in light of studies demonstrating the importance of serotonergic mechanisms in the regulation of receptivity. A large number of studies indicate that increases in serotonin activity produce decreases in sexual receptivity (Everitt et a l . , 1975b; Meyerson, 1964a,b, 1968; Meyerson & Lewander, 1970; Sietnieks & Meyerson, 1980, 1982; Ward et a l . , 1975; Zemlan et a l . , 1973). This led to the general hypothesis that serotonin exerts a tonic inhibitory effect on receptivity, an idea somewhat contrary to the results of this section. However, the hypothesis has recently been revised and evidence suggests that the inhibitory action of serotonin may be mediated by serotonin type I receptors while a facilitatory action of serotonin may be mediated by serotonin type II receptors (Mendelson & Gorzalka, 1985; Mendelson, 1985). The present findings that pirenperone inhibits receptivity (Experiment 11) while quipazine facilitates receptivity (Experiment 15) supports 0 this notion. The results of Experiments 10-15 indicate that serotonin type II receptors may also be involved in the mediation of the inhibitory actions of MSH. The results of Experiments 11-15 indicate that MSH, like the serotonin type II antagonist pirenperone, inhibits receptivity and that this inhibition can be reversed by the administration of the serotonin type II agonist quipazine. In fact, the attenuation of the inhibitory action of MSH by quipazine was so complete that the potential involvement of serotonin type I receptors seems questionable. As 114 the hypothalamus contains predominantly serotonin type I receptors, it seems unlikely that MSH-serotonin type II interactions occur at this site. However, it is possible that MSH may act in some fashion to decrease serotonin type II binding in the hypothalamus or perhaps to decrease the number of serotonin type II receptors per se, thereby producing an inhibitory effect. Alternatively, MSH may act to inhibit serotonin type II activity in mesencephalic areas. Although the mesencephalon contains both serotonin type I and type II receptors, the concentration of serotonin type II receptors is somewhat higher than that observed in the hypothalamus (Peroutka & Snyder, 1981). Thus, it is possible that the effects of substances that alter receptivity via changes in serotonin type II activity are mediated by mesencephalic structures. The long term inhibitory effect of MSH also appears to be mediated, in part, by serotonin activity. The results of Experiment 10 indicated that PCPA prevented the long term action of MSH, suggesting that an MSH-serotonin interaction is required for the production of this effect. However, the results of Experiment 11 indicated that an acute decrease in serotonin type II activity would not, in i t s e l f , produce a long term inhibition of receptivity, suggesting that the long term action of MSH is produced by a more complex interaction. It is possible that MSH acts in some manner to increase serotonin type I receptor concentrations or to decrease serotonin type II receptor concentrations. The results of Experiments 12 and 15 indicate that quipazine, when administered with MSH, attenuates, but does 115 not entirely eliminate, the long term inhibitory action of MSH. This suggests that serotonin type II receptors may be involved. The results of Experiments 7-9 in Section III suggest that the long term inhibitory action of MSH is due, in part, to a decrease in the availability of progestin receptors. When combined, these findings suggest that the long term Inhibitory action of MSH may be produced by an MSH-serotonin interaction that leads to a decrease in the concentration of progestin receptors. In summary, the results of Experiments 10-15 indicated that the short term inhibitory action of MSH may be due to an MSH-induced decrease in serotonin type II activity. Although many brain areas could be involved in the mediation of this effect, it was suggested that the mesencephalon, rather than the AH-POA might be of particular importance. The long term inhibitory effect of MSH appears to require an MSH-serotonin interaction. More specifically, alterations in serotonin receptor activity may be required for the manifestation of the long term effect. Serotonin type II receptors may play a modulatory role, providing MSH with a means by which it can alter an animals sensitivity to estrogen, or progesterone. GENERAL DISCUSSION The current series of studies was conducted to determine the effects of peripherally and centrally administered MSH on lordosis and to examine some potential mechanisms mediating these effect. Results from experiments in Section I indicated that, when contaminating or confounding factors were minimized, MSH administered SC facilitated receptivity while MSH administered 116 ICV inhibited receptivity. The inhibitory action of MSH administered ICV was also observed one week after MSH administration. It is tempting to speculate that the inhibitory action of MSH is nothing more than a non-specific debilitation. However, several lines of evidence suggest that this is unlikely. Fi r s t , Davis et a l . (1980) failed to observe an effect of MSH on locomotor activity, even when relatively high doses of MSH were administered, indicating that motor responding was not affected. Second, the results of Experiment 7 indicated that MSH did not inhibit receptivity in animals receiving estrogen alone. If the inhibitory action of MSH on receptivity is due to a non-specific debilitation, i t should have been observable in subjects receiving estrogen or estrogen plus progesterone. Finally, the finding that quipazine reverses the short term action of MSH suggests that the effect is due more specifically to a reduction in serotonin type II activity. It might also be suggested that the long term inhibitory effect of MSH is due to a response carry-over from the previous tests. However, the finding (Experiments 13 and 14) that subjects receiving 20 ng MSH show a long term, but not a short term, inhibition argues against this possibi1ity. In Section II, i t was hypothesized that the release of progesterone or other facilitatory steroids from the adrenals was the mechanism by which MSH administered SC facilitated receptivity in estrogen-primed animals. Results indicated that this may indeed be the case: adrenalectomized animals failed to exhibit an MSH-induced f a c i l i t a t i o n of receptivity. Sections III 117 and IV addressed questions concerning the mechanisms behind the short and long term inhibitory actions of MSH. It was suggested that the short term inhibitory action of MSH might be mediated through MSH-induced alterations in serotonin activity while the long term inhibitory action might be mediated through changes in estrogen or progestin receptor availability. The results of Section III indicate that, although estrogen and progesterone may play some role in the short term action of MSH, they are unlikely to be of major importance in the production of this effect. However, it does appear that MSH-induced decreases in cytoplasmic progestin receptors may play a major role in the production of the long term inhibitory action of MSH. The results of Section IV suggest that the short term inhibitory action of MSH is mediated, to a large extent, by changes in serotonin type II receptor activity. The results of Section I reveal that confounding factors may often remain undetected, even when an experimental design has been used repeatedly in the behavioural endocrinology literature. For example', regression towards the mean and instability within subjects may remain undetectable in experiments employing a single control test. Experiments employing multiple control tests are thus preferable. Furthermore, rather than performing a single experiment to examine high and low levels of a response, it may be wise to perform two separate experiments: one to examine the effects of a substance in low response subjects, the other to examine the effects of a substance in high response subjects. The experiments in Section I also indicate that the 118 standard procedure of counterbalancing is not always desirable. Counterbalancing was employed by Thody and coworkers and in Experiments 1 and 2 of this thesis. However, during the course of Experiment 2, it became clear that MSH was exerting a long term inhibitory action on lordosis. This means that subjects receiving an MSH test f i r s t and a saline test second would display a lower degree of receptivity during the saline test than those receiving tests in the reverse order. This a r t i f i c i a l lowering of control scores could give rise to false conclusions about the effects of MSH. As an example, assume that MSH reduced the mean LQ of the control group from 75 to 50. Experimental Group 1 displays a mean LQ of 75 after MSH treatment while experimental Group 2 displays a mean LQ of 50. The conclusion from this hypothetical experiment would be that treatment 1 fac i l i t a t e s receptivity while treatment 2 has no effect. However, the opposite conclusion would have been reached had the control scores not been confounded by the long term effects of MSH. Presumably, this should be detected when investigators test for order effects. However, these tests are not always run. In addition, the inhibitory action of a substance may summate over tests. Thus, although tests performed after a single administration of the substance may f a i l to reveal an order effect, tests after several administrations may reveal an order effect. Thus, greater caution should be exercized in experiments employing the repeated administration of a substance. It is worth noting that there are few studies that have investigated the possibility that a substance may exert a long term Influence on 119 receptivity. This may be due, in part, to the experimenal designs employed in studies of receptivity. The finding that MSH exerts a long term inhibitory action suggests that It may well be worth the extra time required to determine if a substance does exert a long term effect. This possibility requires careful consideration in future experiments. The results of Section II suggest that MSH, administered SC, may f a c i l i t a t e receptivity by f a c i l i t a t i n g the release of progesterone from the adrenals. Alternatively, MSH may induce the release of DOC from the adrenals. However, a potential role for an MSH-induced release of progesterone is suggested by research indicating that cervical stimulation can lead to the release of MSH (Volosin & Celis, 1979a, 1984). Cervical stimulation is also known to increase receptivity (see Komisaruk, 1974, for a review). It is possible that MSH released in response to cervical stimulation may serve i n i t i a l l y to stimulate the release of progesterone by the adrenals. This progesterone may then synergize with estrogen to further enhance sexual responding. Thus, one purpose of MSH may be to enhance the probability of pregnancy. The inhibition of receptivity produced by MSH administered ICV would appear to conflict with this role. However, a closer examination of the effects of MSH suggests that the inhibitory action of this peptide may actually be consonant with enhancing the probability of successful pregnancy. The studies by Volosin's group (Volosin & Celis, 1979a,b, 1984) examined serum MSH levels but not brain MSH levels. Therefore, it is not clear that cervical stimulation actually 120 increases brain MSH concentrations. However, it is possible that the neural systems f a c i l i t a t i n g the release of MSH from the pituitary also fac i l i t a t e the transport of MSH from the arcuate nucleus to other regions of the brain, where i t is then released. In addition, research suggests that it Is possible for pituitary hormones to enter the brain via retrograde transport from the pituitary to the portal blood vessels (Mezey, Palkovitz, De Kloet, Verhoef, & de Wied, 1979; Oliver, Mical, & Porter, 1977). Thus, it Is conceivable that cervical stimulation also results In the release of MSH into various brain areas where it may then act to inhibit receptivity. This inhibition of receptivity may occur several hours after cervical stimulation, at a time when further copulation might interfere with f e r t i l i z a t i o n and implantation. It is also possible that the. amount of MSH released after cervical stimulation is not sufficient to produce a short term inhibition but is sufficient to produce a long term inhibition. The prolonged inhibitory effect of MSH may ensure that the implantation and development of the f e r t i l i z e d eggs proceeds without the potentially dispruptlng effects of further copulations. It is interesting to note that progesterone also has a biphasic action on receptivity. The administration of progesterone in estrogen-primed animals i n i t i a l l y facilitates receptivity. However, this facilitatory action is followed by a period of insensitivity to progesterone (Blaustein & Feder, 1979c; Lisk, 1969) and thus a very low degree of sexual responding. Furthermore, as with MSH, this decrease in sensitivity to progesterone appears to be due to a decrease in the concentration of progestin receptors (Blaustein, 1982a; Blaustein & Feder, 1979a, 1980; Brown & Blaustein, 1984). Thus, MSH and progesterone may act in concert to inhibit receptivity after copulation. The results of the experiments in Section IV shed considerable light upon one potential mechanism mediating the short term inhibitory effect of MSH. These studies strongly suggest that an MSH-induced reduction in serotonin type II activity is responsible, possibly to a great extent, for the short term inhibitory action of MSH. However, the possibility that MSH also increases serotonin type I activity has not been eliminated and should be considered in any model of MSH action. It was suggested that MSH may exert its short term inhibitory effect by decreasing serotonin type II activity in the mesencephalon. More specifically, MSH may serve to decrease progesterone-induced increases in serotonin type II activity in this area. If this were the only mechanism underlying the short term action of MSH, then subjects receiving estrogen alone should not show the inhibition. The results of Experiment 7 suggested that MSH did not significantly alter receptivity in subjects receiving estrogen alone. However, the results of Experiment 9 suggested that the dose of estrogen employed may influence the degree of inhibition produced by MSH. It was suggested that estrogen may increase receptivity by decreasing serotonin type I activity in the AH-POA. The results of Leonard et a l . (1976) suggest that MSH does increase serotonin activity in the hypothalamus. As this area contains predominantly serotonin type 122 I receptors (Leysen et a l . , 1982; Peroutka & Snyder, 1981), an MSH-induced increase in serotonin activity in this area should result more specifically in an increase in serotonin type I activity. Thus, MSH, by increasing serotonin type I activity in the AH-POA, could counter the effects of estrogen on receptivity. However, the lack of a strong inhibition in the absence of progesterone suggests that the short term inhibitory action of MSH may be mediated predominantly by MSH-induced decreases in serotonin type II activity. The findings of Section IV also suggest that MSH-induced alterations in serotonin type II activity may play a role in the long term action of MSH. The depletion of serotonin by PCPA was found to prevent the long term inhibitory action of MSH. Furthermore, quipazine, when administered on the day of MSH administration, was found to partially attenuate the long term effect of MSH. Thus, i t would appear that alterations in serotonin activity produced by MSH may induce long term changes in receptivity. Because it is unlikely that MSH continues to alter serotonin activity one week after its administration, it was suggested that the long term effect was produced by some other mechanism. For example, the short term alterations in serotonin activity produced by MSH may result in long term alterations in the availability of estrogen, progesterone, or serotonin receptors. The results of the experiments in Section III further supported this notion. The results of Experiment 7 indicated that the long term inhibitory action of MSH is not observed in subjects receiving estrogen alone. In Experiment 9, 123 i t was f o u n d t h a t i n c r e a s i n g t h e d o s e o f p r o g e s t e r o n e p a r t i a l l y a t t e n u a t e d t h e l o n g t e r m i n h i b i t o r y a c t i o n o f MSH. T h e s e f i n d i n g s s u g g e s t t h a t a d e c r e a s e i n t h e a v a i l a b i l i t y o f p r o g e s t i n r e c e p t o r s i s one o f t h e m e c h a n i s m s u n d e r l y i n g t h e l o n g t e r m i n h i b i t o r y a c t i o n o f MSH. The f i n d i n g t h a t PCPA o r q u i p a z i n e c a n a t t e n u a t e t h e l o n g t e r m i n h i b i t o r y a c t i o n o f MSH s u g g e s t s t h a t M S H - i n d u c e d a l t e r a t i o n s i n s e r o t o n i n a c t i v i t y may be r e s p o n s i b l e f o r t h e d e c r e a s e i n p r o g e s t i n r e c e p t o r a v a i l a b i l i t y . A l t h o u g h t h e e f f e c t o f s e r o t o n i n r e c e p t o r a c t i v i t y on e s t r o g e n a n d p r o g e s t i n r e c e p t o r s h a s n o t b e e n e x a m i n e d , t h e r e i s e v i d e n c e t h a t a l t e r a t i o n s i n n e u r o t r a n s m i t t e r a c t i v i t y c a n a l t e r t h e c o n c e n t r a t i o n o f t h e s e hormone r e c e p t o r s ( B l a u s t e i n , 1985 & i n p r e s s , Nock e t a l . , 1 9 8 1 ) . T h u s , i t i s c e r t a i n l y p o s s i b l e t h a t M S H - i n d u c e d a l t e r a t i o n s i n s e r o t o n i n a c t i v i t y m e d i a t e M S H - i n d u c e d a l t e r a t i o n s i n p r o g e s t i n r e c e p t o r a v a i l a b i 1 i t y . T h u s f a r , t h e f o l l o w i n g m e c h a n i s m s u n d e r l y i n g t h e a c t i o n o f MSH on r e c e p t i v i t y h a v e b e e n p r o p o s e d : 1) t h e f a c i l i t a t o r y e f f e c t o f MSH a d m i n i s t e r e d SC i s m e d i a t e d by t h e r e l e a s e o f p r o g e s t e r o n e o r o t h e r f a c i l i t a t o r y s t e r o i d s f r o m t h e a d r e n a l s , 2) t h e s h o r t t e r m i n h i b i t o r y e f f e c t o f MSH a d m i n i s t e r e d ICV i s m e d i a t e d , t o a l a r g e e x t e n t , by d e c r e a s e s i n s e r o t o n i n t y p e I I a c t i v i t y , a n d 3) t h e l o n g t e r m i n h i b i t o r y a c t i o n o f MSH a d m i n i s t e r e d ICV i s m e d i a t e d , i n p a r t , by a d e c r e a s e i n t h e a v a i l a b i l i t y o f c y t o p l a s m i c p r o g e s t i n r e c e p t o r s . I n a d d i t i o n , i t h a s b e e n s u g g e s t e d t h a t : 1) e s t r o g e n f a c i l i t a t e s r e c e p t i v i t y by d i s i n h i b i t i n g t h e i n h i b i t o r y a c t i o n s o f s e r o t o n i n t y p e I 124 receptors in the AH-POA and 2) progesterone facilitates receptivity by increasing the activity of facilitatory serotonin type II fibres in the MRF. To this should be added a potential means by which progesterone can act synergistically with estrogen to fa c i l i t a t e receptivity while exerting no action on receptivity in the absence of estrogen. One possibility is that progesterone acts to facilitate receptivity by enhancing the a b i l i t y of estrogen to decrease the activity of serotonin type I activity. This could be achieved in several ways. For example, progesterone, by increasing the number of estrogen receptors, could decrease serotonin type I activity. Alternatively, progesterone may act via presynaptic inhibition: it may activate neurons that synapse upon the axons of estrogen-activated neurons. When the progesterone-activated neurons f i r e , they would effectively reduce the electrical potential of the axon upon which they synapse. This reduction in the electrical potential of the axon would reduce or eliminate the release of serotonin from the terminal buttons of the estrogen-activated neurons. Whatever the mechanism, it is important to remember that, although progesterone interacts synergistically with estrogen, progesterone alone can not induce receptivity. Thus, the facilitatory action of progesterone is due to an interaction between estrogen and progesterone and not to a direct facilitatory action of progesterone. This model of receptivity is presented in Figure 18. How do the effects of MSH on lordosis f i t into the proposed model of the regulation of lordosis behaviour and what might be 125 F i g u r e 18. S e r o t o n e r g i c and hormonal i n f l u e n c e s on r e c e p t i v i t y . E s t r o g e n b i n d s to e s t r o g e n r e c e p t o r s i n the a n t e r i o r h y p o t h a l a m u s - p r e o p t i c a r e a (AH-POA). T h i s , i n t u r n , may i n h i b i t the a c t i v i t y of s e r o t o n i n type I r e c e p t o r s , t h e r e b y i n c r e a s i n g l o r d o s i s . P r o g e s t e r o n e b i n d s t o p r o g e s t i n r e c e p t o r s i n the me s e n c e p h a l i c r e t i c u l a r f o r m a t i o n (MRF). T h i s may a c t i v a t e s e r o t o n i n type I I r e c e p t o r s . I n c r e a s e d s e r o t o n i n type I I r e c e p t o r a c t i v i t y may i n c r e a s e the c o n c e n t r a t i o n of p r o g e s t i n r e c e p t o r s . S e r o t o n i n type I I f i b r e s may f a c i l i t a t e the i n h i b i t o r y a c t i o n of e s t r o g e n on s e r o t o n i n type I a c t i v i t y , t h e r e b y i n c r e a s i n g l o r d o t i c r e s p o n d i n g . 126 LORDOSIS 127 the functional importance of MSH? Figure 19 illustrates the potential mechanisms mediating the effects of MSH and how they might influence receptivity. In this model, we find that MSH administered SC stimulates the release of progesterone from the adrenals. Presumably, this progesterone is carried in the blood to the brain where it binds to progestin receptors in a variety of regions, including the MRF. In the MRF, the progesterone-receptor complex activates facilitatory serotonin type II fibres which might then act to fac i l i t a t e the action of estrogen on serotonin type I activity in the AH-POA. This would further reduce serotonin type I activity and thereby fa c i l i t a t e lordosis responding. The most likely natural source of peripheral MSH would be that released from the pituitary. This factor has also been incorporated in the model of MSH action presented in Figure 19. Although there is some evidence that pituitary MSH may reach the brain via retrograde transport, the majority of MSH released by the pituitary remains outside of the central nervous system (Eskay et a l . , 1979; Pelletier, Leclerc, Saavedra, Brownstein, Vaudry, Ferland, & Labrie, 1980; Thody, 1980). Thus, like MSH administered SC, MSH from the pituitary can act upon the adrenal to stimulate the release of progesterone, or other facilitatory steroids, which could then fa c i l i t a t e sexual responding. Although not indicated in Figure 19, it seems likely that one function of the MSH released upon cervical stimulation is to initiate the above chain of events. The model of MSH action presented in Figure 19 also indicates the various mechanisms that may mediate the short term 128 Figure 19. A model of MSH action. MSH reaches the anterior hypothalamus-preoptic area (AH-POA) and the mesencephalic reticular formation (MRF) via the cerebrospinal fluid (CSF) and via fibres ascending from the arcuate nucleus. In addition, MSH may reach the hypothalamic area through retrograde transport via the portal blood system of the pituitary stalk. In the AH-POA, MSH may stimulate serotonin type I receptor activity, thereby inhibiting lordosis. In the MRF, MSH may act to decrease progestin receptor availability, producing the long term inhibitory action of MSH. MSH may also decrease serotonin type II receptor activity, producing the short term inhibitory action of MSH. 129 LORDOSIS 130 inhibitory action of MSH. Recall that a progesterone-induced increase in serotonin type II activity in the MRF was hypothesized to facilitate receptivity. Therefore, as indicated in Figure 19, the short term inhibitory action of MSH could be produced by an MSH-induced inhibition or blockade of this action of progesterone in the MRF. It was also suggested that estrogen facilitated receptivity by decreasing the inhibitory effect of serotonin type I activity in the AH-POA. Thus, MSH, by enhancing serotonin type I activity in this area, may negate the disinhibitory effects of estrogen on receptivity. Finally, it was suggested that progesterone functions to enhance the action of estrogen. If this action of progesterone is produced by the inhibition of serotonin type I activity by serotonin type II fibres, MSH could block this action by decreasing serotonin type II activity. The long term inhibitory effect of MSH appears to be mediated, in part, by a decrease in the availability of cytoplasmic progestin receptors. As progesterone is hypothesized to exert its action on receptivity via the MRF, it is assumed that this is the region in which a reduction in progestin receptors would take place. Although the process by which this reduction is achieved remains to be determined, it was suggested that MSH-induced decreases in serotonin type II activity may be involved. Alternatively, MSH may directly decrease the availability of progestin receptors. Both of these possiblities are indicated in Figure 19. As can be seen upon examination of the model, a reduction in progestin receptor 131 a v a i l a b i l i t y would r e s u l t in a lower degree of binding and therefore a reduction in serotonin type II a c t i v i t y . T h i s , in turn , would decrease the strength of the s y n e r g i s t i c act ion of progesterone with estrogen, r e s u l t i n g in a lower level of r e c e p t i v i t y . The natural corre la te of MSH administered ICV is MSH produced by the arcuate nucleus and transported to various brain reg ions . In Figure 19, MSH is shown to reach the AH-POA and the MRF v ia three d i f f eren t mechanisms. F i r s t , MSH-containing f ibres from the arcuate nucleus project to the AH-POA and the MRF. Second, MSH containing f ibres from the arcuate nucleus project to the ependymal tanacytes near the t h i r d and l a t e r a l v e n t r i c l e s . MSH released from these f ibres apparently enters the cerebrospinal f l u i d and can then be taken up by a var ie ty of brain regions . T h i r d , small amounts of MSH may reach the bra in v ia retrograde transport from the p i t u i t a r y to the hypothalamus. At th i s po int , several cautionary factors and l i m i t a t i o n s of th i s model should be pointed out. F i r s t , there are several bra in areas that influence lordos i s behaviour but are not included in the model. For example, both the cortex and the septum appear to exert an inh ib i tory influence on lordos i s (Gorsk i , 1976). However, the cortex and septum appear to serve as mediators between the environment and the animal and are not primary regulators of lordos i s behaviour (Gorsk i , 1976). Second, MSH is known to a l t e r dopamine a c t i v i t y as well as serotonin a c t i v i t y (Leonard et a l . , 1976). In a d d i t i o n , i t is l i k e l y that future research w i l l reveal that an even greater number of 1 3 2 neurotransmitters are influenced by MSH. The current research explored only one aspect of the a c t i v i t y of MSH in the central nervous system. Thus, the model based on thi s research is limited in scope. Furthermore, although a variety of control procedures were employed to eliminate potential confounds, r e p l i c a t i o n of the current studies would add strength to the proposed model of the action of MSH. F i n a l l y , conclusions based upon studies using serotonin agonists and antagonists should be v e r i f i e d through physiological means. Although drugs such as quipazine and pirenperone are r e l a t i v e l y s p e c i f i c to serotonin type II receptors, they also show some a c t i v i t y at serotonin type I receptors. In addition, they may also be active at the receptors for other neurotransmitters. Thus, physiological v e r i f i c a t i o n of an MSH-induced decrease in serotonin type II a c t i v i t y is des i r a b l e . Despite these l i m i t a t i o n s , the model proposed here also has it s strengths. It incorporates a wide range of experimental data derived from lesion and stimulation studies, estrogen and progesterone binding studies, and serotonin agonist and antagonist studies. It then attempts to lay upon t h i s framework the r e s u l t s of the MSH research in an attempt to explain how MSH might exert i t s e f f e c t s on l o r d o s i s . The model is also s p e c i f i c enough to allow several hypotheses to be generated, tested, and thei r accuracy determined. Despite i t s s p c i f i c i t y , the model is also f l e x i b l e . Thus, i f new data indicate that some aspect of the model is incorrect, t h i s portion of the model can be altered without discarding the entire model. F i n a l l y , the proposed model 133 provides a framework upon which the effects of substances other than MSH can be added and their potential modes of action determined. The current model suggests several future experiments that would greatly enhance our understanding of the regulation of lordosis behaviour. For example, an examination of neurophysiological changes after the direct application of estrogen to cells in the AH-POA would provide valuable information in regards to the mechanisms underlying the facilitatory action of estrogen on lordosis. Furthermore, after the effects of estrogen on c e l l activity were observed, estrogen could be removed and specific serotonin agonists and antagonists applied to determine if similar changes in ce l l activity are observed. In this manner, the role of estrogen-serotonin interactions in the regulation of lordosis could be examined. Similar experiments employing progesterone and serotonergic drugs could also be performed on cel l s in the MRF. In addition, studies in which MSH was administered directly to the AH-POA and MRF would help c l a r i f y the role these regions play in the production of the inhibitory actions of MSH. Finally experiments examining the effects of MSH on the concentrations of estrogen, progesterone, and serotonin receptors in the AH-POA and MRF would provide valuable confirmation of hypotheses generated from behavioural observations. 134 Re f e r e n c e s A h l e n i u s , S., E n g e l , J . , E r i k s s o n , H., & S o d e r s t e n , P. 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