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The role of opioid receptors in lordosis behaviour Pfaus, James George 1986

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THE ROLE OF OPIOID RECEPTORS IN LORDOSIS BEHAVIOUR By JAMES GEORGE PFAUS B . A . , The American U n i v e r s i t y , Washington , D . C . , 1983 A THESIS SUBMITTED IN THE REQUIREMENTS MASTER PARTIAL FULFILLMENT OF FOR THE DEGREE OF OF ARTS i n THE FACULTY OF GRADUATE STUDIES (Department of Psycho logy) We a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1986 © James George P f a u s , 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Psychology  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 3 October 1986 i i A b s t r a c t The R o l e of O p i o i d R e c e p t o r s i n L o r d o s i s Behav iour by James George Pfaus O p i o i d a g o n i s t s such as h e r o i n , morphine , methadone, M e t 5 -e n k e p h a l i n a m i d e , and ^ - e n d o r p h i n have been shown to i n h i b i t s e x u a l b e h a v i o u r i n a v a r i e t y of mammalian s p e c i e s . A l t h o u g h an e x t e n s i v e l i t e r a t u r e e x i s t s on the n e u r o e n d o c r i n o l o g y of o p i o i d a d m i n i s t r a t i o n , l i t t l e i s known about the r o l e of i n d i v i d u a l o p i o i d r e c e p t o r s i n the i n h i b i t i o n of s e x u a l b e h a v i o u r . T h i s l a c k of i n f o r m a t i o n i s due , i n l a r g e p a r t , to the l a c k of o p i o i d r e c e p t o r s e l e c t i v i t y of the drugs used to s tudy s e x u a l b e h a v i o u r . A c c o r d i n g l y , the p r e s e n t exper iments a s s e s s e d the d o s e -response and t i m e - c o u r s e e f f e c t s of s e v e r a l s e l e c t i v e o p i o i d p e p t i d e s , 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 ( i c v ) , on the l o r d o s i s b e h a v i o u r of o v a r i e c t o m i z e d r a t s r e n d e r e d s e x u a l l y r e c e p t i v e by e s t r o g e n and p r o g e s t e r o n e . In Exper iments 1 through 4, l o r d o s i s behav iour was a s s e s s e d i n each female r a t 15, 30, 60, 90, and 120 min a f t e r the i n f u s i o n of d i f f e r e n t doses of a s e l e c t i v e p e p t i d e . In Exper iment 5, l o r d o s i s behav iour was measured 60 min a f t e r the i n f u s i o n of e f f e c t i v e doses of each of the p e p t i d e s s t u d i e d in the p r e c e d i n g e x p e r i m e n t s . In Exper iment 1, the e f f e c t of u/S r e c e p t o r a c t i v a t i o n was a s s e s s e d u s i n g /3 -endorphin . /3-endorphin produced a dose -dependent d u a l e f f e c t on l o r d o s i s b e h a v i o u r ; a low dose (200 ng) s i g n i f i c a n t l y i n h i b i t e d l o r d o s i s , whereas a higher dose (2000 ng) produced a s i g n i f i c a n t f a c i l i t a t i o n . Experiment 2 examined the e f f e c t of the s e l e c t i v e u receptor agonist, mbrphiceptin, on lordosis behaviour. Morphiceptin also produced a dose-dependent dual e f f e c t in which the lowest dose (20 ng) s i g n i f i c a n t l y i n h i b i t e d , whereas the two higher doses ( 2 0 0 , 2000 ng) s i g n i f i c a n t l y f a c i l i t a t e d l o r d o s i s . In Experiment 3, the selective 6 receptor agonist, 6-receptor peptide, s i g n i f i c a n t l y f a c i l i t a t e d lordosis behaviour at a l l doses. In contrast, in Experiment 4 the s e l e c t i v e K receptor agonist, dynorphin 1-9, had no s i g n i f i c a n t e f f e c t on l o r d o s i s behaviour. F i n a l l y , in Experiment 5, the a b i l i t y of the opioid receptor antagonist naloxone to reverse the e f f e c t s of ^-endorphin, morphiceptin, and 6-receptor peptide observed in the f i r s t four experiments was determined. The e f f e c t s of each peptide were re p l i c a t e d and were reversed by naloxone, indicating that the e f f e c t s observed in the f i r s t four experiments were r e l i a b l e and that they were produced by the a c t i v a t i o n of opioid receptors. Naloxone alone, however, had no e f f e c t on lordosis behaviour. These results indicate that the selective a c t i v a t i o n of opioid receptors d i f f e r e n t i a l l y a f f e c t s lordosis behaviour in female r a t s . It appears that binding to h i g h - a f f i n i t y Mi receptors exerts an i n h i b i t o r y influence on l o r d o s i s , whereas binding to l o w - a f f i n i t y n2 receptors or 6 receptors exerts a f a c i l i t a t o r y influence. Binding to K receptors does not appear to a f f e c t lordosis behaviour. The f a i l u r e of naloxone alone to affect lordosis behaviour suggests that endogenous opioid systems do not exert a tonic i n h i b i t o r y or f a c i l i t a t o r y i v i n f l u e n c e i n o v a r i e c t o m i z e d r a t s pr imed w i t h e s t r o g e n and p r o g e s t e r o n e . The p r e s e n t exper iments demonstrate the importance of u s i n g h i g h l y s e l e c t i v e o p i o i d r e c e p t o r l i g a n d s i n the study of the b e h a v i o u r a l e f f e c t s of o p i o i d d r u g s . B o r i s B . G o r z a l k a , P h . D . Content s I n t r o d u c t i o n 1 Pharmacology and B i o c h e m i s t r y of O p i o i d s 3 H i s t o r y 3 C h e m i s t r y 5 O p i o i d R e c e p t o r s 5 C e n t r a l and P e r i p h e r a l E f f e c t s 11 O p i o i d s and Sexua l Behav iour 12 R a t i o n a l e 20 G e n e r a l Methods 24 Animal s and Surgery 24 Drug P r o c e d u r e s 25 B e h a v i o u r a l T e s t i n g 26 H i s t o l o g i c a l A n a l y s e s 27 Exper iment 1: E f f e c t of M/6 Receptor A c t i v a t i o n wi th /3-endorphin 28 Method 29 R e s u l t s and D i s c u s s i o n 29 Exper iment 2: E f f e c t of n Receptor A c t i v a t i o n w i t h M o r p h i c e p t i n 35 Method 35 R e s u l t s and D i s c u s s i o n 36 Exper iment 3: E f f e c t of 6 Receptor A c t i v a t i o n w i t h 5 -Receptor P e p t i d e . . . 4 1 Method 41 R e s u l t s and D i s c u s s i o n 42 v i Exper iment 4: E f f e c t of K Receptor A c t i v a t i o n w i t h Dynorphin 1-9 47 Method 47 R e s u l t s and D i s c u s s i o n 48 Exper iment 5: R e p l i c a t i o n and N a l o x o n e - R e v e r s i b i l i t y 53 Method 53 R e s u l t s and D i s c u s s i o n 54 G e n e r a l D i s c u s s i o n 63 Re ferences 70 v i i L i s t of Figures Figure 1. Dose- and time-response curves for the ef f e c t of 0-endorphin on lordosis behaviour in Experiment 1 31 Figure 2. Dose- and time-response curves for the e f f e c t of morphiceptin on lordosis behaviour in Experiment 2 38 Figure 3. Dose- and time-response curves for the eff e c t of 6-receptor peptide on lor d o s i s behaviour in Experiment 3 ..44 Figure 4. Dose- and time-response curves for the e f f e c t of dynorphin 1-9 on lor d o s i s behaviour in Experiment 4 50 Figure 5. E f f e c t of naloxone (10 mg/kg) or saline on the display of lordosis following e f f e c t i v e doses of 0-endorphin in Experiment 5 56 Figure 6. E f f e c t of naloxone (10 mg/kg) or saline on the display of lordosis following e f f e c t i v e doses of morphiceptin in Experiment 5 58 Figure 7. Ef f e c t of naloxone (10 mg/kg) or saline on the display of lordosis following e f f e c t i v e doses of 8-receptor peptide in Experiment 5 60 Acknowledgements I am deeply indebted to Dr. Boris Gorzalka for his support and guidance throughout the execution of these experiments and during the preparation of t h i s t h e s i s . I am also grat e f u l to the other members of my Thesis Committee, Dr. John Pinel and Dr. Wolfgang Linden, for their comments and recommendations which contributed to the f i n a l form of t h i s manuscript. I would also l i k e to thank Scott Mendelson for teaching me how to evaluate sexual r e c e p t i v i t y and procep t i v i t y in female rat s . F i n a l l y , I would l i k e to thank my fiance Denise Stapelman for her patience and encouragement, and for occasionally nudging me back into the real world. The research reported in t h i s thesis was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to Dr. Boris Gorzalka. I dedicate t h i s thesis to my parents. Foreshadow Dr. Strauss: It seems to be strange that the a l i e n i s interested in heroin. But there could be a l o t of reasons for that. We know now, for example, because of the research of a few American s c i e n t i s t s in the late 1970s, that there are special receptors, opiate receptors, in the human brain.... Ms. Goldman: "Well,- what are a l l these opiate receptors doing s i t t i n g around in the human brain? Waiting for someone to come along and... give them heroin?" Dr. Strauss: "Some physicians think that there i s a naturally-occurring molecule in the human body with nearly the same molecular structure as opiates." Ms. Goldman: "You mean to say that opium occurs naturally in the human body?" Dr. Strauss: "Not opium; I said nearly the same molecule with nearly the same properties. Opium users have said that the drug creates a similar f e e l i n g to what people experience during orgasm. It could be that t h i s molecule i s released into the brain during orgasm." Ms. Goldman: "During orgasm? Well... that's very i n t e r e s t i n g . " From Liquid Sky (1983), written and directed by Slava Tzukerman. Reprinted with permission. 1 I n t r o d u c t i o n For c e n t u r i e s ' o p i o i d s have p r o v i d e d a re fuge from p a i n and s t r e s s ; t h e i r use as a n a l g e s i c s , a n x i o l y t i c s , and n a r c o t i c s has been we l l -documented ( K r u e g e r , Eddy , & Sumwalt, 1941,1943; S t i m m e l , 1975; W i c k l e r , 1980). O p i o i d s have a l s o en joyed use s o l e l y f o r p l e a s u r e or to enhance a r t i s t i c c r e a t i v i t y ( C h e s s i c k , 1960; DeQuincey , 1821; K o l b , 1925; L a r n e r , 1967; Rado, 1933). In the absence of p h y s i c a l p a i n , a common f e a t u r e of a c u t e o p i o i d use has been a b r i e f , but very i n t e n s e e u p h o r i a f o l l o w e d by a p r o l o n g e d p e r i o d of r e l a x a t i o n , s e d a t i o n , and sense of w e l l -be ing ( M i r i n , Meyer , Mende l son , & E l l i n g b o e , 1980). Throughout h i s t o r y , o p i o i d u s e r s have d e s c r i b e d these e f f e c t s i n s e x u a l t erms , o f t e n e q u a t i n g them w i t h s e x u a l orgasm ( B e c k e r , 1903; DeQuincey , 1821; J o n e s , 1700; Jones & J o n e s , 1977; K a a n , 1891). In c o n t r a s t to d e s c r i p t i o n s of an o r g a s m - l i k e e u p h o r i a wi th acute o p i o i d a d m i n i s t r a t i o n , c h r o n i c o p i o i d use has l o n g been a s s o c i a t e d w i t h a p r o g r e s s i v e and i n s i d i o u s d e t e r i o r a t i o n of s e x u a l f u n c t i o n i n g (Ashworth , 1914; Bloom & B u t c h e r , 1970; Cushman, 1972; C o l t m a n , 1890; DeLeon & W e x l e r , 1973; G a r b u t t & G o l d s t e i n , 1972; G u e r r a , 1974; H a p p e l , 1892; J o n e s , 1700; Jones & J o n e s , 1977; L a r n e r , 1967; M a t h i s , 1970; N o i r o t , 1902; Pace , 1892; Passower, 1893; Wholey, 1912; W i e l u n d & Yunger , 1970). The l o n g - t e r m use of o p i o i d s by male a d d i c t s has been a s s o c i a t e d wi th the e l i m i n a t i o n of s e x u a l dreams, d e l a y e d e j a c u l a t i o n , d e c r e a s e d e j a c u l a t e volume, anorgasmia , i n h i b i t e d s e x u a l d e s i r e , and i n some cases i n f e r t i l i t y . S i m i l a r l y , the l o n g - t e r m use of o p i o i d s by female a d d i c t s has been a s s o c i a t e d . w i t h the e l i m i n a t i o n of s e x u a l dreams, amenorrhea, anorgasmia , i n h i b i t e d 2 sexual desire, and in some cases i n f e r t i l i t y . Eventually, female and male addicts experience a severely diminished capacity for sexual arousal; for them, the use of opioids seems to "replace sex" (Jones & Jones, 1977; Rado, 1933). Subsequent withdrawal from opioids, either through drug abstinence or challenge with opioid antagonists, i s characterized by a gradual restoration of sexual interest and functioning. Although the central and peripheral mechanisms underlying the c l a s s i c analgesic effect of opioids have been described in d e t a i l ( A k i l & Watson, 1979; Basbaum & F i e l d s , 1984; Watkins & Meyer, 1982; Wickler, 1980), i t i s not yet known how opioids produce an orgasm-like euphoria in the absence of pain, or whether the analgesic or euphoric actions of opioids are related to the i n h i b i t i o n of sexual behaviour. In fact, i t i s only within the l a s t decade that the e f f e c t of opioids on sexual behaviour has received extensive experimental attention. However, such research has not kept pace with the ever-expanding knowledge of opioid pharmacology and biochemistry (cf. Pfaus & Gorzalka, 1986). The experiments reported in t h i s thesis are a f i r s t attempt at bridging t h i s gap in information by addressing the fundamental question of whether opioid receptors are d i f f e r e n t i a l l y involved in the i n h i b i t i o n of sexual behaviour. This question i s addressed in the present experiments by testing the e f f e c t of several r e l a t i v e l y s e l e c t i v e opioid receptor ligands on lordosis behaviour in female rats. In order to provide a broad background for the rationale and discussion of the present series of experiments, i t i s necessary to b r i e f l y review the pharmacology and biochemistry of opioids. 3 The Pharmacology and Biochemistry of Opioids History. Opium, from the Greek opos meaning " j u i c e , " i s the dried, powdered form of the milky exudate obtained from the seed capsules of the poppy plant. It contains over 20 a l k a l o i d s , some of which are powerfully psychoactive. Before the i s o l a t i o n and production of these p a r t i c u l a r a l k a l o i d s , opium and i t s extracts, eg., tincture of opium or laudanum, were used widely as analgesics, sedatives, narcotics, and aphrodisiacs. In 1805, Serturner in Germany produced morphine, named in honour of Morpheus, the god of dreams in Ovid's play Metamorphoses, from the a l k a l i n e base obtained after separating meconic acid from opium. His procedure, which generally involved the separation of acid constituents from opium, was used by Robiquet in France, who isolated narcotine in 1817 and subsequently codeine in 1832 (Krueger et a l . , 1941). Heroin (diacetylmorphine) was f i r s t synthesized in 1874 by Wright in England, who boiled morphine in acetic acid to acetylate the hydroxyl groups in the molecule and f a c i l i t a t e l i p i d permeability. It was not u n t i l 1897, however, that heroin was marketed to the public by the Beyer Pharmaceutical Company in E l b e r s f e l d , Germany. The effectiveness of heroin as an analgesic was so great that in 1906 the American Medical Association o f f i c i a l l y approved i t s general use and recommended i t over morphine for patients suffering from chronic pain. Although heroin was o f f i c i a l l y banished from medical use in the United States during the 1920s and in Canada in the 1940s, s p e c i f i c a l l y because of i t s powerful abuse po t e n t i a l , i t has remained the opioid of choice for recreational use. 4 Great advances were made in understanding the b i o l o g i c a l d i s p o s i t i o n and the s t r u c t u r e / a c t i v i t y relationships of various opioid a l k a l o i d s over the past 40 years. These advances provided the c r i t i c a l information needed to synthesize analgesics with diminished abuse p o t e n t i a l . Some of the analgesic compounds synthesized from morphine or thebaine during the 1950s and 1960s included the benzomorphans, eg., pentazocine; the phenylpiperidines, eg., meperidine; the diphenylamines, eg., methadone; the morphans, eg., levorphanol; and the 6,14-endo-ethynyloripavines, eg., etorphine. In rats, many of these compounds could be discriminated from morphine and heroin at very low doses, yet were as e f f e c t i v e as morphine in attenuating withdrawal symptoms in morphine-dependent animals (Way, 1979). More importantly, many of these compounds could not serve as reinforcers in self-administration paradigms, indicating both a diminished abuse potential and a possible d i s s o c i a t i o n between analgesia and euphoria. The early 1960s saw the c l i n i c a l a p p l i c a t i o n of t h i s work. Methadone and some of the more active benzomorphans, eg., cyclazocine, were introduced as drug maintenance therapy for heroin addiction. The r e l a t i v e analgesic properties of synthetic opioids were assessed in humans. In the l a t e 1960s, the f i r s t "pure" opioid antagonist, N-allylnoroxymorphone (naloxone) was introduced. The biochemical basis of opioid tolerance and dependence was examined, as was the mediating role of other putative neurotransmitter systems, eg., dopamine or serotonin. Most importantly, central and peripheral s i t e s of the a c t i v i t y of opioids were i d e n t i f i e d in the late 1960s and early 1970s, 5 which led several investigators to search for "endogenous opioids" and drug-specific receptor proteins. Chemistry. The term opiate refers s p e c i f i c a l l y to the al k a l o i d s obtained d i r e c t l y from the juice of the poppy. The generic term opioid refers to any compound with opiate-like a c t i v i t y that i s reverse*d by a general opioid antagonist such as naloxone (Goldstein, 1975). Opioids may also possess nonopioid actions which, by d e f i n i t i o n , cannot be reversed with a general antagonist. Opioid peptides are s p e c i f i c amino acid sequences that possess o p i a t e - l i k e pharmacological a c t i v i t y which i s reversed with naloxone. A l l endogenous opioid peptides are cleaved by p r o t e o l y t i c enzymes, eg., aminopeptidase or enkephalinase, from larger precursory hormones (see DeWied & J o l l e s , 1982; Marx, 1983; O'Donohue & Dorsa, 1982, for reviews). B r i e f l y , 0-endorphin and methionine-enkephalin (Met 5-enkephalin) are derived from Proopiomelanocortin, a 30 kD protein from which i s also cleaved the ACTH and MSH peptide sequences. Met 5-enkephalin is also derived from Proenkephalin, a 28 kD protein from which leucine-enkephalin (Leu s-enkephalin) i s derived. F i n a l l y , the dynorphin sequences, eg., dynorphin 1-17, dynorphin 1-9, leumorphin, are cleaved from Prodynorphin, a 29 kD protein. Endogenous opioid peptides are generally produced in the anterior lobe and pars intermedia of the p i t u i t a r y and are released into the portal vasculature. A r e l a t i v e l y small but important proportion of the t o t a l endogenous opioid peptide production occurs within perikarya and on c e l l membranes in several other brain areas (see below). 6 Opioid Receptors. Endogenous opioid receptors were discovered concurrently in the myenteric plexus of the guinea-pig ileum ( K o s t e r l i t z , Lord, & Watt, 1973) and in the brain tissue of monkeys (Pert & Snyder, 1973). Subsequently, several brain-borne peptides were i s o l a t e d and c l a s s i f i e d as possible endogenous ligands for these receptors. Among the early candidates were Met 5-enkephalin and Leu 5-enkephalin, two pentapeptides with potent, but short acting opioid a c t i v i t y (Hughes, Smith, K o s t e r l i t z , F o t h e r g i l l , Morgan, & Morris, 1975). /3-endorphin, a 31-amino acid peptide representing positions 61-91 of the j3-lipotropin molecule, was found to possess opiate-l i k e a c t i v i t y very similar to that of morphine (Hughes, 1975; Pasternak, Goodman, & Snyder, 1977; Teschmacher, Opheim, Cox, & Goldstein, 1975). Several studies followed that l o c a l i z e d opioid receptors in various brain areas using autoradiographic techniques (Pert, Kuhar, & Snyder, 1976; Simantov, Childers, & Snyder, 1978). It soon became apparent, however, that more than one opioid receptor existed. K o s t e r l i t z and associates discovered that some endogenous peptides bound to peripheral tissue preparations d i f f e r e n t l y . The enkephalins, for example, were found to bind with higher s e l e c t i v i t y , i e . , at lower concentrations, to receptors in the mouse vas deferens than to receptors in either the rat vas deferens (se l e c t i v e for /3-endorphin) or the guinea-pig ileum (selective for morphine). Other opioids,' such as the benzomorphan drugs ketazocine and ketocyclazocine, were found to bind with high a f f i n i t y to receptors in the rabbit vas deferens but not at a l l to receptors in the mouse vas deferens 7 ( K o s t e r l i t z et a l . , 1973). In 1976, Martin and associates proposed that three functional classes of opioid receptor existed and named them after prototypic ligands: u receptors (after morphine); K receptors (after ketazocine); and a receptors (after SKF-10047 or N-allylnormetazocine) (Martin, Eades, Thompson, Huppler, & G i l b e r t , 1976; G i l b e r t & Martin, 1976). K o s t e r l i t z added a fourth c l a s s , the 5 receptor (after mouse vas deferens), to explain the p r e f e r e n t i a l binding of the enkephalins (Paterson et a l . , 1983). The dynorphin peptide sequences were discovered in the late 1970s and were subsequently shown to be endogenous ligands for K receptors (Corbett, Paterson, McNight, Magnan, & K o s t e r l i t z , 1982; Oko, Negishi, Suda, Sawa, Fujino, & Wakimasu, 1982; Paterson, Robson, & K o s t e r l i t z , 1983). In the early 1980s, i t was also proposed that ^-endorphin bound to a macromolecular receptor complex, referred to as the epsilon receptor (after endorphin), consisting of a protein-bound 8 and a lipid-bound M receptor (Smith, Lee, & Loh, 1983; Zuckin & Zuckin, 1984). Generally, the discovery and c l a s s i f i c a t i o n of opioid receptor subtypes has been hastened by the development of agonists and antagonists have r e l a t i v e l y s e l e c t i v e a f f i n i t i e s . The receptors described above were i n i t i a l l y characterized using both _iri v i t r o binding assays and in vivo bioassay techniques, eg., measures of analgesia such as the t a i l - f l i c k t e s t . The presence of heterogeneous populations of opioid receptors was l a t e r confirmed in various brain areas using _in v i t r o selective protection techniques, in which the binding of a drug to a p a r t i c u l a r receptor i s examined following the saturation of 8 other receptor types _in s i t u with other r e l a t i v e l y s e l e c t i v e drugs. More recent evidence, gathered with the use of more sele c t i v e opioid receptor ligands, has suggested further subdivisions of opioid receptors. There are currently believed to be two subclasses of n receptor, a h i g h - a f f i n i t y ( M I ) s i t e and a l o w - a f f i n i t y ( A I 2 ) s i t e . The u2 s i t e corresponds to the morphine-selective receptor o r i g i n a l l y described by K o s t e r l i t z et a l . (1973) in the guinea-pig ileum. The n\ s i t e , o r i g i n a l l y referred to as the " h i g h - a f f i n i t y opiate binding s i t e " by Pasternak and Snyder (1975), was found to be s e l e c t i v e l y blocked in several studies by the long-acting antagonist naloxazone, a hydrazone derivative of naloxone (Hazum, Chang, Cuatrecasas, & Pasternak, 1981; Ling, Spiegel, N i s h i m i r i , & Pasternak, 1982; Pasternak, Childers, & Snyder, 1980a,b; Wolozin & Pasternak, 1981). Naloxazone was also shown to block the analgesia produced by low doses of morphine without a f f e c t i n g the respiratory depression produced by higher doses (Pasternak et a l . , 1980a). Naloxazone also d i f f e r e n t i a l l y displaces r a d i o l a b e l l e d D-Ala 2-Met s-enkephalinamide and dihydromorphine binding in various regions of rat brain (Zhang & Pasternak, 1980). Brain areas r i c h in y, receptors, eg., the hypothalamus and spinal cord, were most se n s i t i v e to the e f f e c t of naloxazone, whereas areas containing both uy and nz receptors, eg., the striatum and thalamus, were less affected. Brain areas r i c h in 6 receptors, eg., the cortex and midbrain, were markedly in s e n s i t i v e to naloxazone. Although the functional role of n receptor subtypes has not yet been established, i t has been suggested that y, receptors may mediate c l a s s i c opioid e f f e c t s such as analgesia, 9 whereas M2 receptors may mediate ef f e c t s s i m i l a r to those produced by 6 receptor a c t i v a t i o n ( C h a i l l e t , Couland, Zajac, Fournie-Zaluski, Constantin, & Roques, 1984; Pasternak, G i n t z l e r , Houghten, Ling, Goodman, et a l . , 1983; Pazos & Florez, 1984). In fact, recent biochemical evidence indicates that the M 2 receptor i s s t r u c t u r a l l y similar to the 6 receptor (Rothman, Danks, Herkenham, Jacobson, Burke, & Rice, 1985). Although the a receptor was o r i g i n a l l y c l a s s i f i e d by G i l b e r t and Martin (1976) as an opioid receptor, the drugs that s e l e c t i v e l y bind to i t , eg., SKF-10047 or phencyclidine, do not possess the pharmacological p r o f i l e of other opioids. Instead of analgesia, these drugs produce hallucinations and sedation, both of which may involve an action on other neurotransmitter systems, eg., serotonin. Furthermore, i t has recently been shown using s e l e c t i v e protection techniques _in vivo that the sedative e f f e c t of SKF-10047 i s mediated through p a r t i a l agonist a c t i v i t y at K receptors (Khazan, Young, El-Fakany, Hong, & Ca l l i a g r o , 1984). The status of the a receptor as a "true" opioid receptor, therefore, remains in question. The central d i s t r i b u t i o n of opioid receptors has also been of great i n t e r e s t . Opioid receptor subtypes have been found to occur together on the same membrane in some brain regions and separately in others. In mammals, opioid receptors have been l o c a l i z e d in the olfactory bulbs; the projection areas of the vomeronasal organ, a chemosensory system implicated in the processing of pheromonal s t i m u l i ; the cortex; ventral septal n u c l e i ; nucleus accumbens; pe r i v e n t r i c u l a r nucleus of the thalamus; striatum; preoptic area; mediobasal hypothalamus; 10 median eminence; p i t u i t a r y ; amygdala; mesencephalic central grey area (MCG); ventral tegmentum; locus coeruleus; r e t i c u l a r formation, eg., the dorsal raphe and the nucleus r e t i c u l a r i s g i g a n t o c e l l u l a r i s ; cerebellum; the spinal nucleus of the trigeminal nerve; and d i f f u s e l y throughout spinal dorsal horn nuclei (see A k i l & Watson, 1979; Bloom, 1983; Goodman & Snyder, 1982; Khachaturian, Lewis, Schafer, & Watson, 1985; Robson, Foote, Maurer, & K o s t e r l i t z , 1984; Szara, 1982, for reviews). In the cortex, K receptors are found largely in laminae I, V, and VI, whereas a predominance of 6 and some M receptors are found in laminae I I , I I I , IV, and V (Foote & Maurer, 1982; Goodman, Snyder, Kuhar, & Young, 1980). Opioid receptors that have been implicated in the regulation of p i t u i t a r y function are located in both the arcuate nucleus of the hypothalamus and in the median eminence and appear to be of the u and 6 type or the epsilon complex because /3-endorphin, Met 5-enkephalin, and morphine a l l bind to these s i t e s . K receptors have not been found in the mediobasal hypothalamus. Peripherally, opioid receptors have been found in the myenteric plexus of the ileum, the vas deferens, the heart, kidneys, and gut (Jaffe & Martin, 1980). However, the opioid receptor subtypes found in these areas, e s p e c i a l l y in the ileum and vas deferens, d i f f e r among species (Paterson et a l . , 1983). Such differences have made the functional role of peripheral opioid receptors d i f f i c u l t to assess. The d i f f u s e central d i s t r i b u t i o n of opioid receptors has also made i t d i f f i c u l t to i d e n t i f y s p e c i f i c central mechanisms through which opioids influence behaviour. Nevertheless, as 11 s p e c i f i c receptor pathways become apparent, the a b i l i t y to "map" opioid e f f e c t s with selective ligands increases. Central and Peripheral E f f e c t s . Although opioids are best known for th e i r analgesic, narcotic, and euphoric actions, they produce several other notable e f f e c t s in the central and peripheral nervous systems. Pu p i l l a r y c o n s t r i c t i o n i s a prominent e f f e c t in humans and forms a diagnostic c r i t e r i o n of opioid overdose in conjunction with respiratory depression. Respiratory depression produced by opioids i s a result of the i n h i b i t i o n of medullary s i t e s responsible for the breathing r e f l e x . In subanalgesic doses, morphine and other opiates act as cough suppressants (Jaffe & Martin, 1980). Opioids decrease glandular secretions and contract smooth muscle f i b r e s . Both of these e f f e c t s , along with decreased i n t e s t i n a l p e r i s t a l s i s , result in constipation, a common side e f f e c t of opioid administration. Opioids also produce vestibular stimulation, which results in nausea and vomiting in some users. Hypotension and decreased blood pressure are other prominent e f f e c t s along with hypothermia (Jaffe & Martin, 1980). Opioids also disrupt neuroendocrine function. For example, heroin and methadone both decrease serum l u t e i n i z i n g hormone (LH) and testosterone l e v e l s , although the i n h i b i t o r y effect of methadone on testosterone secretion occurs at high doses only ( A z i z i , Vaginakis, Longcope, Ingbar, & Braverman, 1973; Cicero, Schmoeker, Meyer, M i l l e r , B e l l , et a l . , 1986; Mendelson, Ellingboe, Kuenhle, & Mello, 1980). Endogenous opioid systems have been implicated as putative neurotransmitter or neuromodulatory hormonal substrates of a 12 wide variety of psychologically relevant phenomena: Pain mediation, tolerance and dependence to exogenously administered opioid drugs, regulation of neuroendocrine function and dysfunction, sedation, sleep, euphoria, reinforcement mechanisms, consummatory behaviour, sexual orgasm, sexual dysfunction, anxiety, learning and memory, and assorted psychopathological states (see Cooper & Martin, 1982; Olson, Olson, & Kastin, 1984; Pfaus & Gorzalka, 1986; Reid & S i v i y , 1983; Rossier & Bloom, 1979; Szara, 1982, for reviews). Opioids and Sexual Behaviour The impetus to study the ef f e c t of opioids on sexual behviour comes from a long history of anecdotes and c l i n i c a l accounts (Pfaus & Gorzalka, 1986). As mentioned previously, acute administration of heroin i s reported by users to produce an immediate, orgasm-like "rush" of euphoria that gradually tapers off into feelings of general relaxation and contentment. Although long-term opioid use has not been reported to diminish t h i s subjective experience, provided an e f f e c t i v e dose i s maintained, the actual sexual motivation and functioning of the user diminishes. A large part of the psyc h i a t r i c maladjustment of heroin addicts i s based on the i r i n a b i l i t y to maintain intimate and supportive relationships. The progressive decline, f i r s t in the capacity to enjoy, and then to maintain, sexual a c t i v i t y i s an important factor. Furthermore, the sexual g r a t i f i c a t i o n experienced by many addicts following intravenous heroin administration appears to play a major role in the maintenance of opioid use (Chessick, 1960; Rado, 1933). 13 As the d e b i l i t a t i n g e f f e c t s of long-term opioid use in humans were f i r s t examined c l i n i c a l l y during the early part of t h i s century, comparative animal models of those e f f e c t s were sought in order to examine more c l o s e l y the mechanisms of action of opioid drugs (Kraus, 1918). The' acute and chronic e f f e c t s of opiates were o r i g i n a l l y studied in dogs, mice, rats, rabbits, monkeys, and chimpanzees, and were shown to mimic many of the symptoms observed in humans. Likewise, many of the symptoms of opiate withdrawal, eg., restlessness, diarrhea, respiratory a l t e r a t i o n s , muscular twitching, and r i g i d i t y , were evident in a l l of the species examined. Sexual behaviour was usually given only a passing mention in those studies. Single i n j e c t i o n s of low doses of morphine or heroin (usually 2 to 5 mg/kg) were generally reported to have no e f f e c t upon sexual behaviour. Long-term administration of higher doses of morphine or heroin (up to 70 mg/kg) invariably resulted in an i n h i b i t i o n of sexual a c t i v i t y in female and male dogs (Eddy & Reid, 1939; Plant & Pierce, 1928), female mice (Ko, 1935), female and male monkeys (Seevers, 1936; Tatum, Seevers, & C o l l i n s , 1929), chimpanzees (Spragg, 1940), and also the elimination of sexual odourants in the vaginal secretions of intact dogs (Hashimoto, 1938). During subsequent drug abstinence, a restoration of sexual a c t i v i t y was observed in a l l species. Interestingly, the restored interest in copulation and masturbation observed in male monkeys during drug abstinence was reportedly preceded by a period of spontaneous ejaculations not dependent upon genital stimulation (Tatum et a l . , 1929). This ef f e c t was i d e n t i c a l to that l a t e r reported in human males 14 during heroin withdrawal (Greenberg, 1985). Reports of an i n h i b i t o r y e f f e c t of long-term opioid administration on female sexual behaviour, however, was controversial in the early l i t e r a t u r e . Although overt s o l i c i t a t i o n was not generally observed in female dogs or rats during long-term morphine treatment, a number of these animals reportedly became pregnant and bore healthy l i t t e r s during treatment (Myers & Flynn, 1928; Plant & Pierce, 1928). Myers (1931) tested the e f f e c t of long-term morphine administration (50 to 70 mg/kg/day) in intact female albino rats for a period of 5 months, beginning 6 days after weaning. No evidence was found supporting the predictions of retarded growth, i n h i b i t i o n of the estrous cycle, or an i n a b i l i t y to mate and subsequently bear healthy l i t t e r s . Although i t could be argued that tolerance to the i n h i b i t o r y e f f e c t s of morphine on ovulation or sexual behaviour might have developed long before the f i f t h month of drug treatment, those data coincided with reports in the human c l i n i c a l l i t e r a t u r e that pregnancy did indeed occur in some female heroin addicts, who presumably adjusted their doses to avoid tolerance e f f e c t s (McGuigan, 1929; Wholey, 1912). Unfortunately, the e f f e c t s of opioids on sexual behaviour in laboratory animals did not receive further attention u n t i l the early 1970s, when Cushman, and l a t e r Mendelson and associates, published detailed analyses of endocrine dysfunction in human heroin addicts (Cushman, 1972; Mendelson, Mendelson, & Patch, 1975; Mendelson, Meyer, Ellingboe, Mirin, & McDougle, 1975). Those reports were followed by studies demonstrating a similar e f f e c t of opioids in rats, eg., decreases in serum LH or 15 testosterone l e v e l s ( C i c e r o , Wilcox, B e l l , & Meyer, 1976; Tokunaga, Muraki, & Hosoya, 1977). Because of these s i m i l a r i t i e s , r a t s , and subsequently hamsters, became the animal models of choice i n the modern l i t e r a t u r e , although nonhuman primates have been used o c c a s i o n a l l y . In the l a t e 1970s and e a r l y 1980s, parametric s t u d i e s of dose, time course, route of a d m i n i s t r a t i o n , i n a d d i t i o n to the acute and long-term i n h i b i t o r y e f f e c t of morphine on copulatory behaviour i n male r a t s began to appear i n the l i t e r a t u r e (Hetta, 1977; Kumar, Mumford, & T e i x e i r a , 1977; Mcintosh, V a l l a n o , & B a r f i e l d , 1980; Meyerson, 1981; Mumford & Kumar, 1979). I n h i b i t o r y e f f e c t s of morphine were a l s o reported on the l a t e r a l displacement of the t a i l during c o p u l a t i o n i n female golden hamsters (Ostrowski, S t a p l e t o n , Noble, & Reid, 1979), and on l o r d o s i s , the concave arching of the back and l i f t i n g of the a n o g e n i t a l region to f a c i l i t a t e p e n i l e i n t r o m i s s i o n , i n female r a t s (Pfaus & Gorzalka, 1986; W i e s e n f e l d - H a l l i n £ Sodersten, 1984). I n h i b i t o r y e f f e c t s of methadone were a l s o reported on autosexual behaviour i n male macaques (Crowley, Stynes, Hydinger, & Kaufman, 1974) and on c o p u l a t i o r y behaviour i n male golden hamsters (Murphy, 1981). Although o p i o i d peptides such as ^-endorphin or D-Ala 2-Met 5-enkephalinamide have been reported to i n h i b i t copulatory behaviour c o n s i s t e n t l y i n male r a t s (Mcintosh, et a l . , 1981; Meyerson, 1981; Meyerson & Terenius, 1977; P e l l e g r i n i -Q u a r a n t o t t i , Corda, P a g l i e t t i , B i g g i o , & Gessa, 1979), the e f f e c t s of o p i o i d peptides on l o r d o s i s behaviour i n female r a t s are more d i f f i c u l t to i n t e r p r e t . 16 Low doses of 0-endorphin have been reported to i n h i b i t the display of lordosis in ovariectomized (OVX), steroid-primed rats when infused into the MCG ( S i r i n a t h s i n g h j i , 1984; S i r i n a t h s i n g h j i , Whittington, Audsley, & Fraser, 1983). S i r i n a t h s i n g h j i and associates demonstrated t h i s e f f e c t in females that received both estrogen and progesterone or demonstrated t h i s e f f e c t in females that received both estrogen and progesterone or estrogen alone. When ^-endorphin (250 ng) was infused into the MCG, mean lordosis quotients, that i s , the mean lordosis/mount r a t i o s , were s i g n i f i c a n t l y reduced in both groups of rats within 30 min. In females that received estrogen treatment alone, lordosis behaviour was abolished within 2 hr and remained so for 7 hr. In females that received both estrogen and progesterone .treatment, l o r d o s i s behaviour was s i g n i f i c a n t l y , but not completely i n h i b i t e d for 4 hr. During t h i s time, females s i g n i f i c a n t l y increased the frequency of active rejections of the stimulus male, eg., displayed boxing stances, crouching, etc., when compared with control females that received saline infusions. The inhib i t o r y e f f e c t of |3-endorphin in both s t e r o i d treatment groups was reversed with naloxone (2.5 mg/kg, i p ) , administered 15 min before 0-endorphin. Wiesner and Moss (1984) described a similar i n h i b i t i o n of lordosis behaviour in OVX rats primed with estrogen and progesterone when a very low dose of j3-endorphin (100 ng) was infused into the t h i r d v e n t r i c l e . Lordosis quotients were s i g n i f i c a n t l y reduced in contrast to those of saline treated control rats 15 and 45 min after infusion. At 75 and 135 min, 17 however, lordosis quotients had risen to control l e v e l s . The i n h i b i t i o n of lordosis at 15 and 45 min was reversed by naloxone (2 mg/kg, sc) administered concurrently with ^-endorphin. The difference in the duration of i n h i b i t i o n between these data and those reported by S i r i n a t h s i n g h j i (1984) and S i r i n a t h s i n g h j i et a l . (1983) could r e f l e c t differences in dose, however, a more l i k e l y explanation i s that ^-endorphin was administered to d i f f e r e n t parts of the brain. It i s well-known, for example, that the cerebral v e n t r i c l e s contain a high concentration of various p r o t e o l y t i c enzymes. Although hypothalamic structures adjacent to the t h i r d v e n t r i c l e in rats also contain opioid receptors (Bugnan, Bloch, Lenys, Goudet, & Fellman, 1979; Grandison, F r a t t a , & Guidotti, 1980), peptides infused into the v e n t r i c l e s might be degraded more rapidly than those infused into discrete brain areas such as the MCG. Hence i t might be expected that the behavioural e f f e c t s of peptides infused into the v e n t r i c l e s would be of comparatively short duration. It should not be construed from the explanation presented above that dose parameters are unimportant. In fact, they may be c r i t i c a l in determining whether ^-endorphin i n h i b i t s or f a c i l i t a t e s l o r d o s i s in female rats. Preliminary experiments conducted for t h i s thesis demonstrated that 2 nq of 0-endorphin f a c i l i t a t e s l o r dosis 60 min aft e r infusion into the l a t e r a l v e n t r i c l e s of OVX rats primed with estrogen and progesterone. This confirmed e a r l i e r unpublished observations from our laboratory that 2 uq of 0-endorphin f a c i l i t a t e s lordosis in OVX rats primed with estrogen alone. These results raise several 18 inter e s t i n g questions. Because /3-endorphin interacts with both u and 5 receptors i t could be that the dual e f f e c t of /3-endorphin on lordosis behaviour r e f l e c t s a d i f f e r e n t i a l a c t i v a t i o n of these receptors. Another p o s s i b i l i t y i s that /3-endorphin f a c i l i t a t e s lordosis by a c t i v a t i n g an anatomically d i s t i n c t population of opioid receptors " near the l a t e r a l v e n t r i c l e s . Answers to either of these questions would have important implications, not only for the understanding of the opioid receptor mechanisms involved in the modulation of l o r d o s i s , but for a more general understanding of opioid receptor interactions. Although no d e f i n i t i v e answers e x i s t , several hints have been presented. In addition to /3-endorphin, S i r i n a t h s i n g h j i ( 1984) tested the e f f e c t of Met 5-enkephalin (0.5 or 10 ug) or dynorphin 1-17 (0.25 or 10 ug) on lordosis in OVX rats primed with estrogen alone. Although Met 5-enkephalin binds to both u and 5 receptors, i t has an a f f i n i t y 10 times greater for 6 than M receptors (Paterson et a l . , 1983) and i t s duration of action at both receptors i s r e l a t i v e l y short (Hughes, 1983). Dynorphin 1-17 is rapidly cleaved into dynorphin 1-9 or dynorphin 1-8, both of which possess very potent, but r e l a t i v e l y short action on K receptors ( G r i f f i t h s & McDermott, 1984; Paterson et a l . , 1983). S i r i n a t h s i n g h j i found no ef f e c t of either peptide on lordosis 2 hr a f t e r infusion into the MCG. Thus S i r i n a t h s i n g h j i concluded that only u receptor ac t i v a t i o n in the MCG may be necessary for the i n h i b i t i o n of lordosis in female rats by /3-endorphin. Although t h i s i s a tempting hypothesis, a potential flaw existed in the design of those experiments that casts some doubt 19 on the notion that 6 or K receptors have no role in the modulation of lordosis behaviour. Because Met 5-enkephalin and dynorphin 1-17 are r e l a t i v e l y short-acting peptides, i t i s not clear whether r e l a t i v e l y selective 6 or K receptor a c t i v a t i o n actually had no effect on lordosis or whether both peptides had been cleaved into inactive metabolites long before l o r d o s i s was tested. Time-response analyses of the e f f e c t s of short-acting peptides may require shorter observation i n t e r v a l s . Indeed, another r e l a t i v e l y selective K ligand, leumorphin, has been reported to produce a short-term f a c i l i t a t i o n of lor d o s i s in OVX, estrogen-primed rats (Imura, 1985; Suda, Nakao, Sakamoto, Morii, Sugawara, & Imura, 1986). In addition, peripheral administrations of estrogen are known to increase endogenous Met 5-enkephalin levels in the MCG (DuPont, Borden, Cusan, Merand, LaBrie, & Vaudry, 1980) and to increase the synthesis of Proenkephalin in the ventromedial hypothalamus (D. Pfa f f , 1986, personal communication). Both of these areas have been implicated in the central hormonal regulation of lordosis behaviour in female rats (Pfaff, 1980). Thus, i t may be that s e l e c t i v e 6 or K receptor activation serves to potentiate, rather than diminish, the display of l o r d o s i s . Administration of Met 5-enkephalin to the MCG, however, had no effect on lordosis in OVX rats primed with estrogen and progesterone in a single test 4 hr a f t e r the concurrent administration of Met 5-enkephalin and progesterone ( S i r i n a t h s i n g h j i , 1984). It is not clear why S i r i n a t h s i n g h j i chose to administer Met 5-enkephalin at the same time as progesterone priming and not 3 to 4 hr after progesterone, when female, rats normally begin to display 20 consistent patterns of lordosis behaviour. Once again, the p o s s i b i l i t y that Met 5-enkephalin had been cleaved into an inactive metabolite during the 4-hr period between infusion and lordosis testing cannot be discounted. To summarize, a major focus of research concerning the e f f e c t of opioids on sexual behaviour has been the categorization of the e f f e c t of various opioid agonists on patterns of sexual behaviour in laboratory animals. The promise of such research has been to i d e n t i f y the physiological or pharmacological mechanisms through which opioids i n h i b i t sexual behaviour. Although d i s t i n c t pharmacological classes of opioid drugs have been shown to i n h i b i t sexual behaviour in a variety of mammalian species, our knowledge of the mechanisms through which opioids exert such an i n h i b i t o r y action remains poor. Differences in experimental procedure, most notably in dose, time course, route of administration, sex of species, and dependent measure, further confuse matters. However, such differences have provided the necessary inconsistencies to spark new questions and new research designs. Rationale Our i n a b i l i t y to treat the sexual dysfunctions commonly associated with long-term opioid use stems in part from a general lack of knowledge concerning the central or peripheral mechanisms that underly the i n h i b i t i o n of sexual behaviour produced by opioid drugs. For example, i t i s currently impossible to determine the s t r u c t u r e / a c t i v i t y relationship of opioid agonists to sexual behaviour. Although agonists such as 21 hero in , morphine, methadone, Met 5 -enkephalinamide, and 0-endorphin i n h i b i t sexual behaviour, they d i f f e r widely in chemical s t r u c t u r e . S i m i l a r l y , i t i s d i f f i c u l t to re la te the i n h i b i t i o n of sexual behaviour produced by op io id agonists to the a c t i v a t i o n of s p e c i f i c op io id receptors . This d i f f i c u l t y is due, in large par t , to a lack of receptor s e l e c t i v i t y in the opioids l i s t e d above. Morphine, for example, i s 50 times more potent at u receptors than at 6 receptors , whereas ^-endorphin has an approximately equal a f f i n i t y for n and 6 receptors (Paterson et a l . , 1983; Smith et a l . , 1983). C l e a r l y , a systematic ana lys is of the op io id receptors that may be involved in the regulat ion of sexual behaviour i s not poss ib le using these drugs a lone. However, the i s o l a t i o n and c l a s s i f i c a t i o n of peptides that act as endogenous l igands for op io id receptors has hastened the development of synthet ic peptide sequences that possess a r e l a t i v e l y high a f f i n i t y for d i f f e ren t op io id receptors . A systematic ana lys is of the ro le of op io id receptors in the i n h i b i t i o n of sexual behaviour using highly s e l e c t i v e receptor l igands could provide important c lues for the s t r u c t u r e / a c t i v i t y r e l a t i o n s h i p of op io id receptor l igands to sexual behaviour, in addi t ion to a more comprehensive understanding of the regional d i s t r i b u t i o n of op io id receptors involved in the i n h i b i t i o n of sexual behaviour. The l o r d o s i s posture assumed by sexual ly recept ive female rats which f a c i l i t a t e s peni le in t romission by sexual ly act ive males i s i d e a l l y sui ted for an ana lys is of the e f f e c t s of s e l e c t i v e op io id receptor l i g a n d s . Lordosis quotients can be determined in a manner of minutes, depending upon the sexual 22 vigor of the stimulus male. This makes lordosis quotients an excellent dependent measure in time course analyses, es p e c i a l l y of peptides that may possess r e l a t i v e l y short action. In addition, the neuroanatomic pathways involved in the central and peripheral expression of lordosis behaviour have been studied in d e t a i l (Pfaff, 1980). Many of the central s i t e s involved in the hormonal regulation of lordosis behaviour, e s p e c i a l l y the MCG and ventromedial hypothalamus, in addition to brainstem areas such as the l a t e r a l vestibular nucleus and the nucleus g i g a n t o c e l l u l a r i s , contain immunoreactive opioid peptide-containing neurons and are r i c h in opioid receptors (Atweh & Kuhar, 1977; Bugnan et a l . , 1979; Finley, Lindstrom, & Petrusz, 1981; Finley, Maderdrut, & Petrusz, 1981; H o l l t , Haarman, Boverman, T e r l i c z , & Herz, 1980; Khachaturian et a l . , 1985). This suggests that endogenous opioid peptides may be naturally involved in the central regulation of lordosis behaviour. However, as mentioned e a r l i e r , although the e f f e c t s of opioid peptides such as /3-endorphin, Met 5-enkephalin, dynorphin 1-17, and leumorphin have been studied on lordosis behaviour, those e f f e c t s remain c o n t r o v e r s i a l . A systematic analysis of the involvement of opioid receptors in lordosis behaviour, using highly s e l e c t i v e receptor ligands, would c l e a r l y help to resolve the controversy. j Accordingly, in the present series of experiments, opioid peptides that are highly s e l e c t i v e ligands for u receptors (morphiceptin), 5 receptors (6-receptor peptide), K receptors (dynorphin 1-9), and the M/6 receptor complex (/3-endorphin) were administered to the l a t e r a l v e n t r i c l e s of OVX, steroid-primed 23 r a t s i n an e f f o r t to determine which o p i o i d r e c e p t o r s are i n v o l v e d i n the c e n t r a l r e g u l a t i o n of l o r d o s i s behaviour. In Experiments 1 through 4, dose-response and time course analyses were conducted on the e f f e c t s of each p e p t i d e on l o r d o s i s behaviour. In Experiment 5, the a b i l i t y of the o p i o i d a n t a g o n i s t naloxone to r e v e r s e the e f f e c t s of each p e p t i d e was t e s t e d . The r e s u l t s of these experiments demonstrated c l e a r l y t h a t u, 5, and K o p i o i d r e c e p t o r s are d i f f e r e n t i a l l y i n v o l v e d i n the c e n t r a l r e g u l a t i o n of l o r d o s i s behaviour. 24 General Methods Animals and Surgery The female Sprague-Dawley rats that served as subjects were obtained from Charles River Canada, Inc., Montreal. At approximately 100 days of age, a l l subjects were b i l a t e r a l l y OVX under sodium pentobarbital anesthesia (Somnital, 40 mg/kg, ip) and implanted with 23-gauge, s t a i n l e s s - s t e e l guide, cannulae aimed at the right l a t e r a l v e n t r i c l e s . B r i e f l y , the surgical procedure for guide cannula implantation involved placing each female rat in a stereotaxic apparatus with the i n c i s o r bar set 5 mm above the interaural l i n e . The scalp was cut, retracted, and 4 s t a i n l e s s - s t e e l anchor screws were i n s t a l l e d into the top of the s k u l l . A burr hole was d r i l l e d into the s k u l l through which the guide cannula was lowered into place. Bach guide cannula was positioned according to the a t l a s of Pellegrino, Pellegrino, and Cushman (1979), with the t i p of the cannula protruding approximately 0.5 mm into the right l a t e r a l v e n t r i c l e (A-P: -0.20 mm; M-L: 1.75 mm; D-V: -3.00 mm from the surface of the brain). Each guide cannula was fixed into place with a c r y l i c cement. In order to protect the guide cannula when not in use, a 30-gauge, s t a i n l e s s - s t e e l syringe needle, crimped at one end and s l i g h t l y longer than the guide cannula, was inserted into the guide cannula. Following surgery, females were housed i n d i v i d u a l l y in standard wire-mesh cages in a colony room maintained on a reversed 12 hr light/12 hr dark cycle at approximately 21°C. Females were allowed free access to food and water. The placement of each guide cannula was tested 1 week before each 25 experiment by i n f u s i n g 2 uq of a n g i o t e n s i n II i n t o the r i g h t l a t e r a l v e n t r i c l e of each r a t . Because i c v i n f u s i o n s of a n g i o t e n s i n II r e l i a b l y induce t h i r s t ( E p s t e i n , F i t z s i m o n s , & R o l l s , 1970; L i n d & Johnson, 1982), on l y those females that d i s p l a y e d v i g o r o u s d r i n k i n g w i t h i n 5 min of i n f u s i o n served as s u b j e c t s . S e x u a l l y a c t i v e , a d u l t Long-Evans hooded male r a t s , bred i n our colony from stock o r i g i n a l l y o b t a i n e d from C h a r l e s R i v e r Canada, Inc., Montreal, were used as st i m u l u s males. A l l of these males were housed i n groups of s i x i n standard wire mesh group cages and maintained under colony c o n d i t i o n s i d e n t i c a l to those of the females. Drug Procedures E s t r a d i o l benzoate (EB) and progesterone (P) ( S t e r a l o i d s ) were d i s s o l v e d i n peanut o i l and i n j e c t e d sc i n 0.1 ml of s o l v e n t . A n g i o t e n s i n II (Bachem) was d i s s o l v e d i n a p h y s i o l o g i c a l s a l i n e s o l u t i o n at a c o n c e n t r a t i o n of 1 uq/ul. 0-endorphin (Sigma), morphiceptin (Bachem), 6-receptor p e p t i d e (Bachem), and dynorphin 1-9 (Peninsula) were d i s s o l v e d i n a p h y s i o l o g i c a l s a l i n e s o l u t i o n to o b t a i n c o n c e n t r a t i o n s of 10, 100, or 1000 nq/ul of s o l v e n t . A l l doses of these p e p t i d e s were i n f u s e d i n 2 M1 of s o l v e n t . Under c o n t r o l c o n d i t i o n s , r a t s r e c e i v e d an eguivolume i n f u s i o n of the p h y s i o l o g i c a l s a l i n e s o l u t i o n . A l l c e n t r a l i n f u s i o n s were made with an e l e c t r i c a l l y d r i v e n i n f u s i o n pump (Sage Instruments Model 3H1A) at a r a t e of 5 Ml/min. Naloxone h y d r o c h l o r i d e (a generous g i f t from Dr. V . N i c h o l s o n of duPont Pharmaceuticals) was d i s s o l v e d i n a p h y s i o l o g i c a l s a l i n e s o l u t i o n and adm i n i s t e r e d sc • at a concentration of 10 mg/ml. Behavioural Testing Lordosis testing involved the presentation of each experimental female to a sexually vigorous stimulus male in a 29 x 45 cm Plexiglas testing chamber. The floor of the chamber was covered with 5 cm of San-i-cel bedding. Each female was placed with a male u n t i l 20 mounts with pelvic thrusting had occurred. Lordosis quotients were calculated as the percentage of mounts with pelvic thrusting that resulted in a lordosis posture. A moderate degree of sexual r e c e p t i v i t y was induced in a l l experimental females by subcutaneous (sc) administration of 10 Mg EB 48 hr and 250 ng P 4 hr before each t e s t . A l l testing occurred during the middle t h i r d of the dark cycle in a room dimly l i t by red l i g h t s . In Experiments 1 through 4, the f i r s t group of experimental females (N=16) received infusions of either 0, 20, 200, or 2000 ng of each peptide at weekly in t e r v a l s in a l a t i n i z e d fashion, such that a l l females were administered each dose of the peptide over a 4-week period in each experiment. Within each experiment, lor d o s i s quotients were determined for each female 15, 30, 60, 90, and 120 min afte r infusion. In Experiment 5, the second group of experimental females (N=72) was assigned randomly to an e f f e c t i v e dose of each peptide and received sc injections of either naloxone (10- mg/kg) or physiological saline 10 min before peptide infusion in a l a t i n i z e d fashion at weekly i n t e r v a l s . A l l experiments were separated by a 2-week period, during which an intervening angiotensin II test was conducted. Any female not displaying vigorous drinking was removed from the study and 27 replaced with a new experimental female. In order to assess the behavioural s p e c i f i c i t y of the ef f e c t of each peptide on l o r d o s i s , other behavioural measures, such as the frequency of ambulation, grooming, and the degree of r e a c t i v i t y to a pencil touching the lumbar region of the spine, were recorded during the 5-min period before each lo r d o s i s t e s t . H i s t o l o g i c a l Analyses Guide cannula placements were v e r i f i e d for the group of experimental females used in the f i r s t four experiments and for those used in Experiment 5, by infusing a 5 til quantity of black India ink into the right l a t e r a l v e n t r i c l e of each rat once testing was complete. Rats were asphyxiated with C0 2 20 min after ink infusion and were then perfused i n t r a c a r d i a l l y with saline followed by a 10 percent formalin solution. The brains were removed and dissected so that the l a t e r a l v e n t r i c l e s could be examined. Di f f u s i o n of ink throughout the v e n t r i c l e s was taken as v e r i f i c a t i o n of the correct placement of cannulae. Only the data from females with accurate cannula placements were subjected to s t a t i s t i c a l analyses. 28 Experiment 1: Effec t of M/6 Receptor Activation with 0-endorphin /3-endorphin appears to produce a dose-dependent dual e f f e c t on lordosis behaviour in female rats. Although the central infusion of low doses of /3-endorphin, eg., 100 to 250 ng, has been reported to i n h i b i t l o r dosis in OVX, steroid-primed rats ( S i r i n a t h s i n g h j i , 1984; S i r i n a t h s i n g h j i et a l . , 1983; Wiesner & Moss, 1984; I986a,b), preliminary experiments in our laboratory demonstrated that a higher dose of 2 uq f a c i l i t a t e d lordosis behaviour in OVX, estrogen-primed rats 60 min after infusion into the l a t e r a l v e n t r i c l e s . However, several factors make i t d i f f i c u l t to compare these r e s u l t s . For example, due to differences in route of administration, i t i s premature to conclude that the dual e f f e c t of /3-endorphin represents the act i v a t i o n of d i f f e r e n t populations of opioid receptors that exert either an inh i b i t o r y or f a c i l i t a t o r y action on lordosis behaviour. Differences in the steroid-priming regimens used could also contribute to the dual e f f e c t of /3-endorphin on lo r d o s i s . For example, a steroid-priming regimen that induces maximal lordosis quotients would be expected to mask any f a c i l i t a t o r y e f f e c t of /3-endorphin. F i n a l l y , because /3-endorphin binds to u and 6 receptors with r e l a t i v e l y equal a f f i n i t y , i t i s impossible to determine whether the dual e f f e c t represents dose-or time-dependent a c t i v i t y at either of these receptors. A dose-response and time course analysis of the effect of /3-endorphin on lo r d o s i s , using a common route of administration and an i d e n t i c a l steroid-priming regimen for a l l animals in every dose condition, could provide a systematic way of 29 addressing whether the dual effect of /3-endorphin i s a function of one or more of the factors l i s t e d above. If route of administration and steroid-priming regimen can be ruled out as important variables in either the i n h i b i t i o n or f a c i l i t a t i o n of lordosis behaviour by /3-endorphin, then an hypothesis concerning possible dose- or time-dependent a c t i v i t y at d i f f e r e n t opioid receptors would be strengthened considerably. Accordingly, in Experiment 1, dose-response and time course analyses were conducted for the e f f e c t of /3-endorphin on lordosis behaviour. Method In a l a t i n i z e d , repeated measures design, 16 experimental females received weekly treatments of 10 uq of EB followed 48 hr la t e r by 250 uq of P. The administration of P was followed in 4 hr by icv infusions of either 0, 20, 200, or 2000 ng of /3-endorphin. Lordosis behaviour was tested in each animal 15, 30, 60, 90, and 120 min aft e r infusion. Data were evaluated using a 4x5x4 repeated measures analysis of variance to assess the main ef f e c t s of dose, time course, order of presentation, and a l l interactions. For a l l s t a t i s t i c a l l y s i g n i f i c a n t main e f f e c t s and interactions, post-hoc comparisons were conducted using the Newman-Keuls method, p^.05. Results and Discussion /3-endorphin produced a dose dependent dual e f f e c t on lordosis behaviour as shown in Figure 1. The analysis of variance detected s i g n i f i c a n t main effects for dose of /3-endorphin, F(3,36) = 4.83, p<.006, and for time course, F(4,48) = 25.75, p<.000l. No s i g n i f i c a n t main eff e c t was found for order 30 Figure 1. Dose- and time-response curves for the e f f e c t of 0-endorphin on lordosis behaviour in Experiment 1. Values represent mean lordosis quotients ± standard errors. BETA-ENDORPHIN DOSE/TIME RESPONSE L e g e n d • 0 ng X 20 ng • 200 ng B) 2000 ng ~r- 1 —i 1 r , — , , . . 4 0 5 0 6 0 70 80 90 100 110 120 130 MINUTES A F T E R INJECTION of presentation. Post-hoc comparisons for the main e f f e c t of dose revealed that the 200 ng dose of /3-endorphin s i g n i f i c a n t l y reduced lo r d o s i s quotients o v e r a l l in contrast to the other three doses. Post-hoc comparisons for the main eff e c t of time course revealed s i g n i f i c a n t differences in o v e r a l l lordosis quotients at 15, 30, and 60 min, but not at 90 or 120 min. The analysis of variance also detected a s i g n i f i c a n t interaction between dose and time course, F(12,144) = 4.36, P<.0001. Subsequent post-hoc comparisons revealed that the 200 ng dose of /3-endorphin s i g n i f i c a n t l y reduced lordosis quotients 15, 30, and 60 min after infusion, but not at 90 or 120 min. The 2000 ng dose of /3-endorphin s i g n i f i c a n t l y f a c i l i t a t e d lordosis quotients at 60 min, but did not produce s i g n i f i c a n t differences from control values at any other time. The progressive r i s e in lordosis quotients throughout the testing period in animals that received control infusions of saline i s a common e f f e c t of repeated l o r d o s i s testing (Larsson, Feder, & Komisaruk, 1974). However, t h i s r i s e in lordosis quotients throughout the testing period did not reach s t a t i s t i c a l s i g n i f i c a n c e . A l l other interactions between dose and time course were also nonsignificant. F i n a l l y , none of the treatments with |3-endorphin produced any noticeable changes in other behavioural measures such as r e a c t i v i t y , ambulation, or grooming. This suggests that the suppression or f a c i l i t a t i o n of lordosis behaviour by 200 or 2000 ng of (3-endorphin respectively i s not due to a general eff e c t on motor a c t i v i t y . The re s u l t s of Experiment 1 are consistent with previous reports of an inhi b i t o r y e f f e c t of /3-endorphin on lordosis 33 behaviour ( S i r i n a t h s i n g h j i , 1984; S i r i n a t h s i n g h j i et a l . , 1983; Wiesner & Moss, 1984; I986a,b). The time course of the i n h i b i t o r y e f f e c t of 200 ng in Experiment 1 i s si m i l a r to that reported by Wiesner and Moss (1984) following the infusion of 100 ng into the t h i r d v e n t r i c l e . This suggests that the i n h i b i t i o n of lordosis produced by infusions of /3-endorphin into either the l a t e r a l or t h i r d v e n t r i c l e s r e f l e c t s a common interaction with opioid receptors. The f a c i l i t a t o r y e f f e c t of 2000 ng of /3-endorphin 60 min a f t e r infusion in Experiment 1 i s also consistent with a previous observation in our laboratory that t h i s dose f a c i l i t a t e s l o r dosis behaviour in OVX, estrogen-primed rats. However, t h i s e f f e c t i s not consistent with recent data by Wiesner and Moss (1986b) showing an i n h i b i t o r y e f f e c t of 2 uq of /3-endorphin on lordosis behaviour. In that study, lordosis was tested 30 min after infusion of /3-endorphin into the t h i r d v e n t r i c l e s of OVX rats primed with high doses of EB and P. Although the high degree of sexual r e c e p t i v i t y induced by that steroid-priming regimen obviously precluded the a b i l i t y to detect a f a c i l i t a t o r y e f f e c t of /3-endorphin in that study, i t i s more d i f f i c u l t to explain why Wiesner and Moss found an i n h i b i t i o n of lordosis with 2 uq of /3-endorphin whereas the results of Experiment 1 c l e a r l y demonstrate a f a c i l i t a t i o n with thi s dose. Therefore, procedural differences between these studies need to be considered. Comparable doses of /3-endorphin or morphine, for example, produce a time dependent dual e f f e c t on continuous motor a c t i v i t y in an open f i e l d eg., ambulation and exploration, consisting of a short-term decrease followed by a prolonged 34 increase in a c t i v i t y (Babbini & Davis, 1972; Browne & Segal, 1980). It could be that higher doses of /3-endorphin f a c i l i t a t e l ordosis only in animals given repeated lordosis t e s t s . /3-endorphin may also interact with higher doses of P to i n h i b i t l o r d o s i s . Such an interaction would be reminiscent of the i n h i b i t o r y e f f e c t of p-Chlorophenylalanine (Gorzalka & Whalen, 1975) or cholecystokinin (Mendelson & Gorzalka, 1984) on lordosis behaviour. It may also be the case that progesterone a l t e r s the a f f i n i t y or number of opioid receptors in the brain. It would be interesting to examine the e f f e c t of d i f f e r e n t steroid-priming regimens on opioid binding. It i s also impossible to rule out route of administration as a contributing factor. Although the i n h i b i t i o n of l o r d o s i s in the present experiment i s consistent with previous reports from other investigators, who u t i l i z e d d i f f e r e n t routes of administration, the f a c i l i t a t o r y e f f e c t of 2 vg of /3-endorphin may r e f l e c t a dose-dependent act i v a t i o n of opioid receptors near the l a t e r a l v e n t r i c l e s that serve to f a c i l i t a t e rather than i n h i b i t l o r dosis behaviour. It should be noted, however, that Wiesner and Moss (1986b) f a i l e d to r e p l i c a t e the i n h i b i t i o n of lordosis reported by S i r i n a t h s i n g h j i (1984) following infusions of /3-endorphin into the MCG. Thus, a precise determination of the factors that may contribute to these discrepant findings awaits further parametric research. 35 Experiment 2; Effect of n Receptor Activation with Morphiceptin Experiment 1 demonstrated a dose-dependent dual ef f e c t of /3-endorphin on lordosis behaviour. However, because /3-endorphin binds to n and 6 receptors with approximately equal a f f i n i t y , i t i s impossible on the basis of Experiment 1 to determine the role of either of these opioid receptors in the i n h i b i t i o n or f a c i l i t a t i o n of lordosis by /3-endorphin. A more appropriate analysis of opioid receptor mechanisms in l o r d o s i s behaviour requires the use of highly s e l e c t i v e receptor ligands. Morphiceptin (NH2-'Tyr-Pro-Phe-Pro-CONH2) i s an opioid tetrapeptide derived from the milk protein /3-casein. It has an a f f i n i t y over 100 times greater for n than 6 or K opioid receptors (Chang, Cuatrecasas, Wei, & Chang, 1982; Chang, K i l l i a n , Hazum, Cuatrecasas, & Chang, 1981) and produced a long-term, naloxone-reversible analgesia in mice following central administrations (Chang et a l . , 1982; Pasternak, Childers, & Snyder, I980a,b; Zhang, Chang, & Pasternak, 1981). In order to assess the role of u receptor a c t i v a t i o n in lordosis behaviour, dose-response and time course analyses of the ef f e c t of morphiceptin were conducted. Method In a l a t i n i z e d , repeated-measures design, the 16 experimental females used in Experiment 1 again received weekly treatments of 10 nq EB followed in 48 hr by 250 nq of P. The administration of P was followed in 4 hr by icv infusions of either 0, 20, 200, or 2000 ng of morphiceptin. As in Experiment 1, the lordosis behaviour of each female was assessed 15, 30, 36 60, 90, and 120 min after infusion. Data were evaluated s t a t i s t i c a l l y as in Experiment 1. Results and Discussion Morphiceptin produced a dose-dependent dual e f f e c t on lordosis behaviour as shown in Figure 2. The analysis of variance detected s i g n i f i c a n t main ef f e c t s of dose of morphiceptin, F(3,36) = 29.15, p<.000l, and of time course, F(4,48) = 19.17, p< .0001. As in Experiment 1, no s i g n i f i c a n t main e f f e c t was found for order of presentation. Post-hoc comparisons for the main e f f e c t of dose revealed that the 20 ng dose of morphiceptin s i g n i f i c a n t l y reduced lordosis quotients overal l in contrast to the other three doses. However, the two higher doses of 200 and 2000 ng s i g n i f i c a n t l y increased lordosis quotients o v e r a l l in contrast to control infusions of s a l i n e . Although mean lordosis quotients at each time i n t e r v a l were higher in animals receiving 2000 ng than in those receiving 200 ng, t h i s difference was not s t a t i s t i c a l l y s i g n i f i c a n t . Post-hoc comparisons for the main e f f e c t of time course revealed a s i g n i f i c a n t difference in overa l l l o r d o s i s quotients at 15 min but not at any other time. Overall lordosis quotients were s i g n i f i c a n t l y lower at 15 min in contrast to lordosis quotients at the other testing times. The analysis of variance also detected a s i g n i f i c a n t interaction between dose, and time course, F(12,144) = 6.14, P<.0001. Subsequent post-hoc comparisons revealed that the 20 ng dose of morphiceptin s i g n i f i c a n t l y reduced lordosis quotients for the duration of the experiment in contrast to animals receiving saline infusions. Mean' lordosis quotients for animals 37 Figure 2. Dose- and time-response curves for the e f fec t of morphiceptin on l o r d o s i s behaviour in Experiment 2. Values represent mean l o r d o s i s quot ients ± standard e r r o r s . MORPHICEPTIN DOSE/TIME RESPONSE 100-1 to - r — 20 — T -30 l 40 l l 1 1— 50 60 70 80 90 MINUTES AFTER INJECTION — i — 100 — I — 110 I 120 130 L e g e n d • On a. X 20 ng • 200 ng El 2000 ng CO 39 receiving 20 ng did not d i f f e r s i g n i f i c a n t l y at 15, 30, or 60 min. However, at 90 and 120 min lordosis quotients had risen s i g n i f i c a n t l y from the previous three testing times, although lordosis quotients at 90 or 120 min were s i g n i f i c a n t l y lower than those of control animals. The 200 and 2000 ng doses of morphiceptin s i g n i f i c a n t l y f a c i l i t a t e d lordosis quotients for the duration of the experiment in contrast to control animals. S i g n i f i c a n t differences in the degree of f a c i l i t a t i o n by these two doses were also detected at 15 and 30 min, but not at any other testing time. As in Experiment 1, the progressive r i s e in the mean lordosis quotients of saline treated control animals throughout the testing period did not reach s t a t i s t i c a l s i g n i f i c a n c e . A l l other interactions between dose and time course were nonsignificant. F i n a l l y , none of the treatments with morphiceptin produced any noticeable changes in the other behavioural measures, eg., r e a c t i v i t y , ambulation, or grooming. This suggests that the dual ef f e c t of morphiceptin i s not due to a general e f f e c t of the peptide on motor a c t i v i t y . It i s d i f f i c u l t to explain the dual effect of morphiceptin in terms of i t s action on opioid receptors. Given the high degree of s e l e c t i v i t y for u receptors, a li n e a r dose-response function would be the most l o g i c a l prediction and, indeed, the easiest to explain. The dual ef f e c t raises several p o s s i b i l i t i e s . Morphiceptin has been shown to interact with both the high- (MO and low- (u2) a f f i n i t y configurations of the u receptor (Chang et a l . , 1982; V i l l i g e r , 1984; Zhang et a l . , 1981). Thus the i n h i b i t i o n of lordosis by 20 ng of morphiceptin could be due to the r e l a t i v e l y selective a c t i v a t i o n of M. 40 receptors whereas the f a c i l i t a t o r y e f f e c t of the 2 higher doses could be due to the activation of u2 receptors or other opioid receptors. Although t h i s hypothesis i s tentative, there i s evidence to suggest that M I and n2 receptors mediate d i f f e r e n t , and in some cases opposite, e f f e c t s of opioid drugs (Pasternak et a l . , 1983; Yonehara & Clouet, 1984). Systemic injections of morphine, for example, result in analgesia, hypothermia, respiratory depression, bradycardia, sedation, catalepsy, increased release of p r o l a c t i n and growth hormone, and a dual e f f e c t on dopamine turnover (Broderick, 1985; Pasternak et a l . , 1983; Yonehara & Clouet, 1984). Through the use of selective opioid receptor antagonists, Pasternak et a l . have determined that supraspinal analgesia, p r o l a c t i n release, hypothermia, catalepsy, and the decreased turnover of s t r i a t a l dopamine, are produced by the agonist a c t i v a t i o n of Mi receptors. Other e f f e c t s of morphine, such as respiratory depression, bradycardia, and the increased turnover of s t r i a t a l dopamine appear to involve either *z2 o r 6 receptors. Consistent with t h i s , the analgesic, but not the respiratory depressant, ef f e c t of morphine has been reversed by the ^ i antagonist naloxazone (Zhang et a l . , 1981). In order to determine whether the dual e f f e c t of morphiceptin on lordosis behaviour r e f l e c t s a dose-dependent a c t i v a t i o n of M receptor subtypes, i t i s necessary f i r s t to rule out the p o s s i b i l i t y that morphiceptin produces either of these e f f e c t s by binding to other opioid receptors. If the r e l a t i v e l y s e l e c t i v e a c t i v a t i o n of 6 or K receptors has no e f f e c t on lordosis behaviour, then t h i s hypothesis would be strengthened considerably. 41 Experiment 3; E f f e c t of 6 Receptor Activation with 5-Receptor Peptide Experiments 1 and 2 demonstrated a dose dependent dual eff e c t on lordosis behaviour of /3-endorphin and morphiceptin, respectively. The results of Experiment 2, however, have made i t d i f f i c u l t to interpret the dual e f f e c t of /3-endorphin as a function of the d i f f e r e n t i a l a c t i v a t i o n of n and 6 receptors. In l i g h t of the dual ef f e c t of morphiceptin, the e f f e c t s of /3-endorphin could r e f l e c t the d i f f e r e n t i a l a c t i v a t i o n of Mi and u2 receptors. It i s therefore necessary to test the e f f e c t of a r e l a t i v e l y s e l e c t i v e 6 receptor agonist on lordosis behaviour. 5-receptor peptide (NH2-D-Tyr-Ser-Gly-Phe-Leu-Thr-CONH2) i s a synthetic hexapeptide over 620 times more potent at 6 receptors than at a receptors, with no a c t i v i t y whatsoever at K receptors (Gacel, Fournie-Zaluski, & Roques, 1980). Its r e l a t i v e potency and long duration of action at 6 receptors occurs because of the novel arrangement of amino acids in the peptide, which greatly retards i t s degradation. The substitution of a D-Serine in the second position of the peptide i n h i b i t s the action of aminopeptidase. The addition of a Threonine residue at the C-terminal prevents the normally rapid cleavage of the Phe*-Leu 5 bond by enkephalinase. In order to assess the role of 6-receptor a c t i v a t i o n in lordosis behaviour, dose-response and time course analyses were conducted for the e f f e c t of 5-receptor peptide. Method In a l a t i n i z e d , repeated-measures design, the 16 experimental females used in Experiments 1 and 2 again received weekly treatments of 10 nq EB followed in 48 hr by 250 nq of P. 42 The administration of P was followed in 4 hr by icv infusions of either 0, 20, 200, or 2000 ng of 6-receptor peptide. Lordosis behaviour was tested in each animal 15, 30, 60, 90, and 120 min afte r infusion. Data were evaluated s t a t i s t i c a l l y as in Experiment 1. Results and Discussion 6-receptor peptide produced a dose-dependent f a c i l i t a t i o n of lordosis behaviour as shown in Figure 3. The analysis of variance detected s i g n i f i c a n t main e f f e c t s for dose of 6-receptor peptide, F(3,36) = 25.16, p<.000l, and for time course, F(4,48) = 4.46, p<.004. As in Experiments 1 and 2, no s i g n i f i c a n t main eff e c t was found for order of presentation. Post-hoc comparisons for the main e f f e c t of dose revealed that 20, 200, and 2000 ng of 6-receptor peptide s i g n i f i c a n t l y increased mean lordosis quotients o v e r a l l in contrast to control infusions of s a l i n e . No s i g n i f i c a n t differences were detected among the three doses of 6-receptor peptide on o v e r a l l lordosis quotients. The analysis of variance also detected a s i g n i f i c a n t i nteraction between dose and time course, F(12,144) = 7.56, p<.0001. Subsequent post-hoc comparisons revealed no differences among doses of 5-receptor peptide u n t i l 90 min, when lordosis quotients produced by 20 ng were s i g n i f i c a n t l y lower than those produced by 2000 ng. The increases in lordosis quotients produced by a l l three doses of 6-receptor peptide were s i g n i f i c a n t l y d i f f e r e n t from control values at 15, 30, 60, and 90 min. At 120 min, however, lordosis quotients of animals that received 20 or 200 ng were not s i g n i f i c a n t l y d i f f e r e n t from 43 Figure 3. Dose- and time-response curves for the e f fec t of 5-receptor peptide on l o r d o s i s behaviour in Experiment 3. Values represent mean l o r d o s i s quot ients ± standard e r r o r s . DELTA-RECEPTOR PEPTIDE DOSE/TIME RESPONSE 100 T 90 80 70-^ O 60 O in </) 50 o Q Qt O 40-3 30 20 A 10 A 10 20 I )— 30 40 1— 50 - r -60 70 -r— 80 —j— 90 — I — 100 110 MINUTES AFTER INJECTION — i — 120 L e g e n d A Ong X 2 0 ng • 2 0 0 ng (3 2 0 0 0 ng —i 130 45 those of control animals. Although the 2000 ng dose produced a s i g n i f i c a n t increase in lordosis quotients at 120 min, in contrast to the lordosis quotients of control animals, the magnitude of t h i s e f f e c t was not s i g n i f i c a n t l y d i f f e r e n t from that of the two lower doses. Unlike the trends observed in Experiments 1 and 2, the progressive r i s e in the lordosis quotients of control animals throughout the testing period did reach s t a t i s t i c a l s i g n i f i c a n c e in Experiment 3. Lordosis quotients were s i g n i f i c a n t l y lower for control animals at 15 min than at any of the other te s t i n g times. A l l other interactions between dose and time course were nonsignificant. F i n a l l y , none of the treatments with 6-receptor peptide produced any noticeable changes in the other behavioural measures, eg., r e a c t i v i t y , ambulation, or grooming. This suggests that the f a c i l i t a t o r y e f f e c t of 6-receptor peptide on lordosis behaviour is not due to a general e f f e c t of the peptide on motor a c t i v i t y . The re s u l t s of Experiment 3 indicate that the r e l a t i v e l y s e l e c t i v e a c t i v a t i o n of 5 receptors with 5-receptor peptide serves to f a c i l i t a t e l o r d osis behaviour. In l i g h t of t h i s , the f a c i l i t a t o r y e f f e c t of /3-endorphin or morphiceptin on lordosis cannot be a t t r i b u t e d conclusively to agonist a c t i v i t y at u2 receptors. Higher doses of /3-endorphin or morphiceptin could conceivably f a c i l i t a t e l o r d osis by an action on 6 receptors. Resolution of t h i s question awaits further research with se l e c t i v e u and 6 receptor antagonists. It i s interesting to speculate, however, on the role of endogenous act i v a t i o n of 8 receptors in lordosis behaviour. DuPont et a l . (1980) have shown that a single i n j e c t i o n of 46 e s t r a d i o l increases endogenous Met 5-enkephalin l e v e l s in the MCG. S i m i l a r l y , Pfaff (1986, personal communication) has reported an increase in the synthesis of Proenkephalin in opioid neurons of the ventromedial hypothalamus following chronic e s t r a d i o l administration. As mentioned e a r l i e r , lordosis behaviour i s dependent upon the presence of estrogen, and chronic estrogen regimens f a c i l i t a t e lordosis in OVX ra t s . Thus, i t i s tempting to suggest that increases in endogenous enkephalin synthesis and release may d i s i n h i b i t or f a c i l i t a t e l o r d o sis behaviour. Although S i r i n a t h s i n g h j i (1984) reported that Met 5-enkephalin f a i l e d to influence lordosis behaviour 4 hr after infusion into the MCG of OVX rats primed with estrogen and progesterone, the results of Experiment 3, using a more se l e c t i v e and longer acting 6 receptor agonist, support t h i s suggestion. 47 Experiment 4: E f f e c t of K Receptor Activation with Dynorphin 1-9 The role of K receptors in lo r d o s i s behaviour has been co n t r o v e r s i a l . Imura (1984) and Suda et a l . (1986) reported a short-term f a c i l i t a t o r y e f f e c t of leumorphin following icv infusions to OVX, estrogen-primed rats, whereas S i r i n a t h s i n g h j i (1984) reported no e f f e c t of dynorphin 1-17 4 hr aft e r infusion to the MCG of OVX, estrogen-primed rat s . However, because dynorphin 1-17 i s short-acting, the results reported by S i r i n a t h s i n g h j i might have d i f f e r e d had lordosis t e s t i n g been conducted closer to the time of administration. Dynorphin 1-17 expresses i t s agonist action at K receptors through i t s f i r s t metabolite, dynorphin 1-9 (NH 2-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-CONH 2). Dynorphin 1-9 i s 16 times more potent at K receptors than at 5 receptors and over 20 times more potent at *c receptors than at u receptors (Paterson et a l . , 1983). Because of i t s r e l a t i v e potency at K receptors, and i t s diffuse l o c a l i z a t i o n throughout the p i t u i t a r y , striatum, cortex, and cerebellum, dynorphin 1-9 has been c l a s s i f i e d as an endogenous ligand for K receptors (Corbett et a l . , 1982). In order to assess the role of r e l a t i v e l y selective K receptor ac t i v a t i o n on lordosis behaviour, dose-response and time course analyses were conducted for the e f f e c t of dynorphin 1-9. Method In a l a t i n i z e d , repeated-measures design, the 16 experimental females used in Experiments 1, 2, and 3 again received weekly treatments of 10 jig EB followed in 48 hr by 250 ng of P. The administration of P was followed in 4 hr by icv 48 infusions of either 0, 20, 200, or 2000 ng of dynorphin 1-9. Lordosis behaviour was tested in each animal 15, 30, 60, 90, and 120 min aft e r infusion. Data were analysed as in Experiment 1. Results and Discussion Dynorphin 1-9 did not appear to aff e c t l o rdosis behaviour, although the lordosis quotients of animals treated with dynorphin 1-9 appeared higher than those of control animals at 30 min (Figure 4). The analysis of variance detected a s i g n i f i c a n t main e f f e c t for time course, F(4,48) = 8.53, p<.05, but not for dose of dynorphin 1-9 or order of presentation. Post-hoc comparisons for the main effect of time course revealed that the o v e r a l l mean lordosis quotient at 15 min was s i g n i f i c a n t l y lower than the o v e r a l l mean lordosis quotients at 90 and 120 min. However, no differences in o v e r a l l mean lordosis quotients were detected between 15 and 30 min, nor between those at 30, 60, 90, or 120 min. The analysis of variance also detected a s i g n i f i c a n t i nteraction of dose and time course F(12,144) = 3.82, p<.01. However, subsequent post-hoc comparisons revealed no s i g n i f i c a n t differences among doses of dynorphin 1-9 at any testing time. Although a l l three doses of dynorphin 1-9 appeared to f a c i l i t a t e l o r d o s i s at 30 min, in contrast to saline-treated control animals, the magnitude of t h i s effect was not s t a t i s t i c a l l y s i g n i f i c a n t . However, the f a c i l i t a t o r y e ffect of the 2000 ng dose almost reached s t a t i s t i c a l significance in contrast to the e f f e c t of saline (p<.l0). The progressive r i s e in the lordosis quotients of control animals throughout the testing period did reach s t a t i s t i c a l significance in this experiment. For control 49 Figure 4. Dose- and time-response curves for the e f f e c t of dynorphin 1-9 on lordosis behaviour in Experiment 4. Values represent mean lordosis quotients ± standard er r o r s . MEAN LORDOSIS QUOTIENT % OS 51 animals, mean lordosis quotients at 15 and 30 min d i f f e r e d s i g n i f i c a n t l y from those at 60, 90, and 120 min. F i n a l l y , none of the treatments with dynorphin 1-9 produced any s i g n i f i c a n t changes in the other behavioural measures. In contrast to the robust f a c i l i t a t i o n produced by 6-receptor peptide or the two higher doses of morphiceptin, the f a c i l i t a t o r y effect of dynorphin 1-9 was small and, more importantly, not s t a t i s t i c a l l y s i g n i f i c a n t . Although i t would be s t a t i s t i c a l l y correct to argue that dynorphin 1-9 had no effect on l o r d o s i s behaviour in Experiment 4, i t would be premature to suggest that K receptors play no role in the expression of lordosis behaviour based solely on the magnitude of the effect of dynorphin 1-9 in t h i s experiment. The trend toward a •significant f a c i l i t a t i o n of lordosis by the highest dose must be taken into consideration, es p e c i a l l y in l i g h t of data showing a f a c i l i t a t o r y e f f e c t of K receptor a c t i v a t i o n with leumorphin (Imura, 1984; Suda et a l . , 1986). The time course of the f a c i l i t a t o r y e f f e c t of dynorphin 1-9, however, provides ad d i t i o n a l , a l b e i t speculative evidence against a role of K receptors in lor d o s i s behaviour. Although dynorphin 1-9 acts as a pr e f e r e n t i a l agonist at K receptors, i t displays a r e l a t i v e l y lower a f f i n i t y for 6 receptors. Moreover, dynorphin 1-9 i s transformed by the action of aminopeptidase into Leu 5-enkephalin following cleavage of the Leu 5-Arg 6 bond ( G r i f f i t h s & McDermott, 1984). Thus, the r e l a t i v e s p e c i f i c i t y of dynorphin 1-9 i s shif t e d over time from K to 5 receptors. Given the r e s u l t s of Experiment 3, i t i s tempting to suggest that the delayed f a c i l i t a t o r y e f f e c t of dynorphin 1-9 r e f l e c t s a weak 52 action on 6 receptors rather than on *c receptors. It i s interesting to note that leumorphin i s also transformed into Leu s-enkephalin by aminopeptidase. The f a c i l i t a t o r y action of dynorphin 1-9 or leumorphin, therefore, may r e f l e c t the action of Leu 5-enkephalin at 6 receptors. It i s not clear whether a larger dose of dynorphin 1-9 might s i g n i f i c a n t l y f a c i l i t a t e lordosis behaviour. Resolution of the question of receptor s p e c i f i c i t y awaits the use of either a s e l e c t i v e 6 or K receptor antagonist in conjunction with dynorphin 1-9 or leumorphin. 53 Experiment 5: Replication and Naloxone-Reversibility In any study of the behavioural e f f e c t s of opioid agonists, i t i s necessary to test whether those e f f e c t s are reversible with an opioid antagonist. R e v e r s i b i l i t y indicates that the ef f e c t of a p a r t i c u l a r agonist r e f l e c t s the s p e c i f i c a c t i v a t i o n of opioid receptors. When conducting experiments with novel compounds, or when acquiring re s u l t s that are contrary to prediction, eg., a f a c i l i t a t o r y e f f e c t of morphiceptin or 6-receptor peptide on lordosis behaviour, i t i s also advisable to test whether those results are r e l i a b l e . Therefore, in Experiment 5, naloxone or saline was administered in conjunction with icv infusions of /3-endorphin, morphiceptin, or 5-receptor peptide to a new group of experimental females. The e f f e c t i v e doses and testing time were chosen on the basis of results from Experiments 1 through 3. Method New experimental females were randomly assigned to one of the three peptide treatment groups (N = 24/group). Within each peptide treatment group, females were randomly assigned to a p a r t i c u l a r dose of the peptide in a between-subjects design. Females assigned to /3-endorphin received either 0, 200, or 2000 ng of the peptide (n = 8/dose). Females assigned to morphiceptin received either 0, 20, or 2000 ng of the peptide (n = 8/dose). Females assigned to 6-receptor peptide received either 0 or 200 ng of the peptide (n = 12/group). Within each dose group, females received naloxone (10 mg/kg) or the saline vehicle at weekly inter v a l s in a l a t i n i z e d , repeated-measures design. 54 Naloxone or saline was administered 10 min before peptide infusions. Lordosis testing of the subjects in a l l groups commenced 60 min after peptide infusion. The data were analysed using mixed-design analyses of variance for each peptide treatment group to assess the main e f f e c t s of peptide dose, antagonist treatment, and the interaction of peptide dose with antagonist treatment. For a l l s t a t i s t i c a l l y s i g n i f i c a n t main ef f e c t s and interactions, the si g n i f i c a n c e of pairwise comparisons was determined by the Newman-Keuls method, p£.05. Results and Discussion In saline-treated animals, /3-endorphin and morphiceptin produced a dose- dependent dual e f f e c t on lordosis behaviour that was reversed by naloxone (Figures 5 and 6). 5-receptor peptide f a c i l i t a t e d lordosis in saline-treated animals and t h i s e f f e c t was also reversed with naloxone (Figure 7). The analysis of variance detected s i g n i f i c a n t main e f f e c t s for dose of 0-endorphin, F(2,21) = 4.63, p<.02; dose of morphiceptin, F(2,21) = 12.62, p<.005; and dose of 5-receptor peptide, F(1,22) = 5.74, p<.02. Post-hoc comparisons for the main e f f e c t of /3-endorphin revealed that the 200 ng dose s i g n i f i c a n t l y reduced the o v e r a l l mean lor d o s i s quotient compared to that of control animals. Although the o v e r a l l mean lordosis quotient produced by the 2000 ng dose was higher than that produced by saline infusions, t h i s difference was not s t a t i s t i c a l l y s i g n i f i c a n t . Post-hoc comparisons for the main e f f e c t of morphiceptin revealed that the 20 ng dose s i g n i f i c a n t l y reduced, whereas the 2000 ng dose s i g n i f i c a n t l y increased, the o v e r a l l mean lordosis quotient in contrast to that of control animals. Post-hoc comparisons for 55 Figure 5. Ef f e c t of naloxone (10 mg/kg) or saline on the display of lordosis behaviour following e f f e c t i v e doses of 0-endorphin in Experiment 5. Values represent mean lord o s i s quotients ± standard errors. Open bars: Saline; hatched bars: Naloxone. 57 Figure 6. Ef f e c t of naloxone (10 mg/kg) or of lordosis behaviour following morphiceptin in Experiment 5. lordosis quotients ± standard Saline; hatched bars: Naloxone. saline on the display e f f e c t i v e doses of Values represent mean errors. Open bars: 100 90-0 20 2000 DOSE OF MORPHICEPTIN (ng) 59 Figure 7. E f f e c t of naloxone (10 mg/kg) or saline on the display of lordosis behaviour following e f f e c t i v e doses of 6-receptor peptide in Experiment 5. Values represent mean lordosis quotients ± standard errors. Open bars: Saline; hatched bars: Naloxone 100-j 90-0 200 DOSE OF DELTA RECEPTOR PEPTIDE (ng) 61 the main e f f e c t of 6-receptor peptide revealed that the 200 ng dose s i g n i f i c a n t l y increased the o v e r a l l mean lordosis quotient in contrast to that of control animals. A s i g n i f i c a n t main eff e c t of antagonist administration was detected only for 5-receptor peptide, F(1,22) = 21.11, p<.0002. Post-hoc comparisons revealed that animals receiving saline administrations, regardless of the dose of 5-receptor peptide, had an o v e r a l l mean lordosis quotient s i g n i f i c a n t l y higher than animals receiving naloxone. S i g n i f i c a n t interactions between peptide dose and antagonist administration were detected for /3-endorphin, F(2,21) = 24.09, p<.000l; morphiceptin, F(2,21) = 17.80, p<.000l; and 6-receptor peptide, F(1,22) = 8.04, p<.009. Subsequent post-hoc comparisons revealed that administrations of naloxone s i g n i f i c a n t l y reversed the i n h i b i t i o n of lordosis produced by 200 ng of /3-endorphin and by 20 ng of morphiceptin. Naloxone administration also s i g n i f i c a n t l y reversed the f a c i l i t a t i o n of lordosis produced by 2000 ng of morphiceptin and by 200 ng cf 6-receptor peptide. Although animals that received the 2000 ng dose of /3-endorphin displayed lordosis quotients s i g n i f i c a n t l y lower in conjunction with naloxone than with sal i n e , the saline scores of these animals were not s i g n i f i c a n t l y d i f f e r e n t from those of control animals that received either naloxone or s a l i n e . Naloxone had no ef f e c t in animals that received control infusions of saline within each treatment group. The results of Experiment 5 have replicated and extended the results of Experiments 1, 2, and 3 by demonstrating that the in h i b i t o r y or f a c i l i t a t o r y e f f e c t of each peptide i s reversible 62 with naloxone. These results strongly support the conclusion that the i n h i b i t o r y or f a c i l i t a t o r y e f f e c t of /3-endorphin, morphiceptin, or 6-receptor peptide r e f l e c t s the s p e c i f i c a c t i v a t i o n of opioid receptors. It i s not clear why the highest dose of /3-endorphin f a i l e d to f a c i l i t a t e l o r d o s i s behaviour s i g n i f i c a n t l y in contrast to saline-treated control animals in t h i s experiment. However, the a b i l i t y of naloxone to s i g n i f i c a n t l y reduce lo r d o s i s quotients in animals that received the highest dose of /3-endorphin suggests that the 2000 ng dose may have exerted a f a c i l i t a t o r y e f f e c t in those animals. The fact that the magnitude of th i s e f f e c t was not s i g n i f i c a n t l y d i f f e r e n t from the mean lordosis quotients of control animals may be due to d i f f e r e n t i a l baselines of lordosis behaviour within each dose group. F i n a l l y , although infusions of naloxone to the MCG or the spinal cord have been reported to f a c i l i t a t e lordosis behaviour in OVX, steroid-primed rats ( S i r i n a t h s i n g h j i , 1984; Wiesenfeld-H a l l i n & Sodersten, 1984), the i n a b i l i t y of naloxone to a f f e c t the l o r d o s i s behaviour of control rats in Experiment 5 suggests that endogenous opioid systems do not exert a tonic i n h i b i t o r y or f a c i l i t a t o r y influence on lordosis in OVX rats primed with estrogen and progesterone. This finding i s consistent with previous reports that peripheral administration of naloxone has no e f f e c t on lordosis behaviour in OVX, estrogen-primed rats (Wiesner & Moss, 1984). 63 General Discussion The r e s u l t s of the present series of experiments have demonstrated that the r e l a t i v e l y s e l e c t i v e a c t i v a t i o n of central opioid receptors d i f f e r e n t i a l l y a f f e c t s l o r d o s i s behaviour in OVX, steroid-primed rats. Activation of the u/6 receptor complex with /3-endorphin in Experiment 1 resulted in a dose-dependent dual e f f e c t in which a low dose (200 ng) i n h i b i t e d whereas a higher dose (2000 ng) f a c i l i t a t e d lordosis behaviour. S i m i l a r l y , a c t i v a t i o n of u receptors with morphiceptin in Experiment 2 produced a dose dependent dual e f f e c t in which the lowest dose (20 ng) i n h i b i t e d whereas the two higher doses (200, 2000 ng) f a c i l i t a t e d l o r d o s i s behaviour. Activation of 6 receptors with 6-receptor peptide in Experiment 3 dose-dependently f a c i l i t a t e d lordosis behaviour. Activation of K receptors with dynorphin 1-9 in Experiment 4, however, did not have a s i g n i f i c a n t e f f e c t on lor d o s i s , although a nonsignificant trend toward f a c i l i t a t i o n appeared at the highest dose (2000 ng) 30 min after infusion. In Experiment 5, the dual e f f e c t s of ^-endorphin and morphiceptin, and the f a c i l i t a t o r y e f f e c t of 6-receptor peptide, were replicated in a d i f f e r e n t group of.rats and were reversed with naloxone. This indicates that these e f f e c t s are r e l i a b l e and s p e c i f i c to the ac t i v a t i o n of opioid receptors in the present paradigm. The f a i l u r e of naloxone alone to a f f e c t lordosis suggests that endogenous opioid systems do not exert a tonic action on lordosis behaviour. Some of the results of the present experiments are contrary to e a r l i e r findings. For example, Wiesner and Moss (l986a,b) reported an i n h i b i t i o n of l o r d o s i s behaviour with 1, 2, and 4 ug 64 of /3-endorphin. The results of Experiments 1 and 5 in the present thesis show a f a c i l i t a t i o n of lordosis with 2 ng of /3-endorphin. Although the f a c i l i t a t i o n produced by t h i s dose did not reach s t a t i s t i c a l significance in Experiment 5, the lack of an i n h i b i t o r y e f f e c t i s consistent with the observation in Experiment 1. Several methodological factors may have contributed to the d i f f e r e n t e f f e c t s reported for higher doses of /3-endorphin. Wiesner and Moss infused /3-endorphin into the t h i r d v e n t r i c l e s of OVX rats primed with estrogen and a high dose of progesterone. In the present experiments, /3-endorphin was infused into the l a t e r a l v e n t r i c l e s in OVX rats primed with estrogen and a low dose of progesterone. Although an interaction of /3-endorphin with progesterone cannot be ruled out as a contributing factor, i t seems rather unlike l y given that the time course of the i n h i b i t o r y e f f e c t of 200 ng in Experiment 1 was almost i d e n t i c a l to that reported by Wiesner and Moss (1984) following the infusion of 100 ng into the t h i r d v e n t r i c l e . This suggests that /3-endorphin may i n h i b i t lordosis in either of these paradigms by a common interaction with opioid receptors whereas the f a c i l i t a t o r y or d i s i n h i b i t o r y e f f e c t observed in Experiments 1 and 5 may r e f l e c t the interaction of higher doses of /3-endorphin with a population of opioid receptors near the l a t e r a l v e n t r i c l e s which serves to f a c i l i t a t e lordosis behaviour. It i s inter e s t i n g to note that differences in the ef f e c t of serotonin on lordosis have been reported following l a t e r a l or t h i r d v e n t r i c u l a r infusions (Wilson & Hunter, 1985). Another p o s s i b i l i t y i s that higher doses of /3-endorphin may f a c i l i t a t e lordosis in animals given repeated lordosis t e s t s . In 65 the recent experiments by Wiesner and Moss (I986a,b), the ef f e c t of higher doses of /3-endorphin was tested once, 30 min after infusion. Further parametric research i s required to determine the precise factors that may contribute to the inconsistent findings for higher doses of /3-endorphin. The present experiments have also demonstrated a f a c i l i t a t o r y e f f e c t of 6 receptor a c t i v a t i o n on lordosis behaviour. However, t h i s e f f e c t i s not consistent with the lack of e f f e c t reported by S i r i n a t h s i n g h j i (1984) following infusion of the 6 receptor agonist Met 5-enkephalin to the MCG. Unlike 8-receptor peptide, Met s-enkephalin i s a r e l a t i v e l y short-acting peptide. Instead of te s t i n g the effect of Met 5-enkephalin on lordosis soon after infusion into the MCG, S i r i n a t h s i n g h j i tested i t s e f f e c t 4 hr a f t e r infusion. The p o s s i b i l i t y that Met s-enkephalin had been cleaved by aminopeptidase into an inactive metabolite long before lordosis was tested in that experiment cannot be discounted. In contrast, the ef f e c t of 8-receptor peptide on lordosis was tested in Experiments 3 and 5 much sooner a f t e r infusion. Further research i s required to determine whether the f a c i l i t a t o r y e f f e c t of 6-receptor peptide is s p e c i f i c to i t s infusion into the l a t e r a l v e n t r i c l e s . Although S i r i n a t h s i n g h j i (1984) also reported no ef f e c t of the K receptor agonist dynorphin 1-17 on lordosis behaviour, Imura (1984) and Suda et a l . (1986) have reported a short term f a c i l i t a t o r y e f f e c t of leumorphin on l o r d o s i s . Both dynorphin 1-17 and leumorphin are r e l a t i v e l y short acting K agonists and both peptides contain the Leu s-enkephalin sequence at the N-terminus. Separating the Leu 5-enkephalin sequence from either of 66 these peptides s h i f t s their r e l a t i v e a f f i n i t y from K to 6 receptors. In Experiment 4, a trend toward a s i g n i f i c a n t f a c i l i t a t i o n of lordosis by dynorphin 1-9 was observed 30 min after infusion of the highest dose (2000 ng). In l i g h t of the f a c i l i t a t o r y e f f e c t observed after 6 receptor a c t i v a t i o n in Experiments 3 and 5, i t i s tempting to speculate that the f a c i l i t a t o r y e f f e c t of either dynorphin 1-9 or leumorphin may r e f l e c t the agonist action of the Leu 5-enkephalin metabolite at 8 receptors. Taken as a whole, the re s u l t s of the present experiments suggest several p o s s i b i l i t i e s about the role of opioid receptors in the central regulation of l o r d o s i s behaviour. As noted in the discussion of Experiment 2, morphiceptin has been shown to interact with both Mi and u2 receptor conformations. The dual e f f e c t of morphiceptin on lordosis may r e f l e c t the d i f f e r e n t i a l a c t i v a t i o n of these s i t e s , such that binding to jii receptors i n h i b i t s lordosis whereas binding to u2 receptors f a c i l i t a t e s t h i s behaviour. In a recent study, Pfaus, Pendleton, and Gorzalka (1986) showed that the s e l e c t i v e , long acting M I receptor antagonist naloxazone reversed the i n h i b i t o r y but not the f a c i l i t a t o r y e f f e c t of morphiceptin on l o r d o s i s . Thus, i t appears that jx, receptors may play an exclusively i n h i b i t o r y role in lordosis behaviour. The dual ef f e c t of /3-endorphin may be a result of the d i f f e r e n t i a l a c t i v a t i o n of n and 6 receptors. Recent evidence indicates that n and 6 receptors may be a l l o s t e r i c a l l y coupled in a mutually i n h i b i t o r y manner, such that binding to one receptor i n h i b i t s the conformational expression of the other 67 (Rothman et a l . , 1985; Rothman & Westfall, 1982). Thus, the i n h i b i t i o n of lordosis produced by low doses of /3-endorphin may be the res u l t of the a c t i v a t i o n of m receptors whereas the f a c i l i t a t i o n or d i s i n h i b i t i o n observed with the 2 uq dose may r e f l e c t increased a c t i v i t y at 6 receptors which a l l o s t e r i c a l l y i n h i b i t s the binding of /3-endorphin to M i receptors. If t h i s hypothesis proves correct, then s p e c i f i c brain areas r i c h in u receptors, eg., hypothalamus, thalamus, striatum, and brainstem, or 6 receptors, eg., f r o n t a l cortex, midbrain, and brainstem (Khachaturian et a l . , 1985; Zhang & Pasternak, 1980) would be implicated in the central regulation of lordosis by opioid drugs. It i s important to r e i t e r a t e that the ac t i v a t i o n of u2 receptors or 5 receptors with highly s e l e c t i v e ligands produces several s i m i l a r e f f e c t s , eg., respiratory depression (Pasternak et a l . , 1983; Pasternak & Wood, 1986; Rothman et a l . , 1985). In fact, Rothman et a l . (1985) have suggested on the basis of binding data that u2 and 6 receptors may be the same receptor protein. Thus, agonist a c t i v i t y at u2 or 6 receptors may serve a similar f a c i l i t a t o r y or d i s i n h i b i t o r y role in lordosis and may suppress the i n h i b i t i o n of lordosis produced by agonist a c t i v i t y at M i receptors through an a l l o s t e r i c mechanism. The use of se l e c t i v e opioid receptor antagonists in conjunction with central infusions of /3-endorphin, morphiceptin, or 5-receptor peptide should provide a useful method of testing t h i s Yiypothesis. /) The re s u l t s of the present experiments also allow speculation concerning the role of endogenous opioids in 68 lordosis beahviour. Hormonal treatments that f a c i l i t a t e lordosis behaviour, eg., chronic peripheral administrations of estrogen, are known to increase endogenous enkephalin l e v e l s in the midbrain and ventromedial hypothalamus (DuPont et a l . , 1980; P f a f f , 1986, personal communication). Treatment with estrogen and progesterone also depletes immunoreactive /3-endorphin levels in hypothalamic tissue homogenates along with concurrent elevations of /3-endorphin l e v e l s in plasma and p i t u i t a r y tissue homogenates (Hulse & Coleman, 1984). Thus, endogenous opioids may act to i n h i b i t or f a c i l i t a t e l o r d osis behaviour depending upon the brain area, receptor type, or hormonal state of the animal. However, i t should be remembered that the f a i l u r e of naloxone alone to a f f e c t l o r d o s i s behaviour in Experiment 5 strongly suggests that endogenous opioid systems were not t o n i c a l l y active during those t e s t s . Like any f i r s t step, the data c o l l e c t e d in t h i s thesis generate more questions than they answer. Certainly the e f f e c t s reported for the opioid peptides used in the present experiments are s p e c i f i c to lordosis behaviour in female rats and cannot be used in a more general sense to make inferences about their e f f e c t s in male rats or in other species. Likewise, the speculation concerning the role of opioid receptors presented above i s s p e c i f i c to lordosis behaviour in female r a t s . The real value of the present experiments l i e s in the introduction of a new methodology. Cle a r l y , the use of highly s e l e c t i v e , long acting opioid receptor ligands provides a valuable tool for determining the function of opioid receptors in any behavioural ana l y s i s . Conducting a systematic analysis of the sexual effects 69 of s e l e c t i v e ligands in discrete brain areas such as the MCG or the mediobasal hypothalamus in female rats could more thoroughly elucidate the role of opioid receptors in lordosis behaviour and provide important data on opioid receptor control of neuroendocrine function in female rat s . A similar approach might be taken to investigate the role of opioid receptors in the sexual behaviour of male rats or other rodents. A major goal of such research i s to provide animal models of opioid e f f e c t s on sexual behaviour from which c l i n i c a l l y - r e l e v a n t information can be derived for the treatment of opioid induced sexual dysfunction. Although the data in the present thesis constitute no more than a f i r s t step, they contribute to an already expansive knowledge of opioid receptors and their role in various aspects of behaviour. 70 References A k i l , H. & Watson, S. (1979). 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(1980). u~ and 6-opiate receptors: Correlations with high- and low-affinity opiate binding s i t e s . Eur. J. Pharmacol., 67, 323-324. Zhang, A., Chang, J . , S> Pasternak, G. (1981). The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2-Tyr-Pro-Phe-Pro-CONH2), a sele c t i v e mu-receptor ligand. L i f e S c i . , 28, 2829-2836. Zuckin, R. & Zuckin, S. (1984). The case for multiple opiate receptors. Trends in Neurosciences, 7, 160-164. PUBLICATION RECORD Nanai Pfaus, James G. Department: Psychology Faculty: Arts A. Publications 1. Published Papers Pfaus, J.G. (1981). Everyman's guide to the diagnosis and treatment of testicular cancer. Planned Parenthood Publication #237-H, Planned Parenthood of Metropolitan Washington, DC. 23 pp. Pfaus, J.G. (1982). An analysis of gas warfare: Historical perspectives and pharmacological considerations. Arch. Amer. Univ., 25, 1-103. Pfaus, J.G. (1983). Opioid peptide and receptor distribution in hypothalamic and pituitary nuclei: The role of opioids in the control of hormonal activity. Arch. Amer. Univ., 26, 1-30. Pfaus, J.G. 4 Gorzalka, B.B. (1986). Opioids and sexual behavior. Neurosci. Biobehav. Rev., in press. Pfaus, J.G. 4 Gorzalka, B.B. (1986). Selective activation of opioid receptors differentially affects lordosis behavior in female rats. Peptides, in press. Pfaus, J.G., Myronuk, L.D.S., & Jacobs, W.J. (1986). Soundtrack contents and depicted sexual violence. Arch. Sex. Behav., 15, 231-237. Pfaus, J.G., Pendleton, N., 4 Gorzalka, B.B. (1986). Dual effect of morphiceptin on lordosis behavior: Possible mediation by different opioid receptor subtypes. Pharmacol. Biochem. Behav., 24, 1461-1464. Jacobs, W.J., Blackburn, J.R., Buttrick, M., Harpur, T., Kennedy, D., Mana, M., MacDonald, M., McPherson, L., Paul, D., & Pfaus, J.G. Observations. Submitted to Behav. Brain Sciences, 78 pp. 2. Published Abstracts Pfaus, J.G. 4 Phillips, A.G. (1986). Apomorphine reverses the facilitation of ejaculation by cholecystoklnin in male rats. Soc. Neurosci. Abst., 12. in press. Pfaus, J.G. 4 Gorzalka, B.B. (1985). Dual effect of morphiceptin on lordosis behavior In female rats. Canadian Psychology/Psychologie  Canadienne, 26(3), 457. Pfaua/2 Pfaus, J.G., Jacobs, W.J., & Wong, R. (1986). Conditional olfactory cues facilitate the acquisition of copulatory behavior and influence sexual selection in male rats. Canadian Psychology/  Psychologie Canadienne, 27(2), 470. Pfaus, J.G., Mastropaolo, J.P:, 4 Riley, A.L. (1983). Conditioned taste aversions to delayed-onset toxins. Proceedings and Abstracts  of the 54th Annual Meeting of the Eastern Psychological Association, Philadelphia, PA., p. 115. Riley, A.L., Mastropaolo, J.P., k Pfaus, J.6. (1982). Conditioned taste aversions to slow onset toxins: A behavioral index of toxicity. Soc. Neurosci. Abst., _, 357. 

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