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Reproductive behaviour in the male rat: importance of 5-HT2 receptor activity and relation to 5-HT2-dependent… Watson, Neil Verne 1994

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REPRODUCTIVE BEHAVIOUR IN THE MALE RAT: IMPORTANCE OF 5—HT2 RECEPTOR ACTIVITY AND RELATION TO 5-HT2-DEPENDENT SEROTONERGIC STEREOTYPY by NEIL VERNE WATSON B.A., M.A.,  The University of Western Ontario, The University of Western Ontario,  1985 1989  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Psychology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1993 © Neil Verne Watson,  1993  In  presenting  this  thesis  in  partial fulfilment of the  requirements  for an  advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  11  ABSTRACT  It is well established that the neurotransmitter serotonin participates in the control of sexual behaviour in the male rat. Recently, it has been found that serotonergic activity may either inhibit or facilitate sexual behaviour, depending on the subtypes of serotonin receptors involved. However, the participation of 5-HT2 receptors in the control of male rat copulation has received little experimental attention, and the published data are equivocal. In Experiments 1-4, it was established that the 5HT2/1C agonist DCI inhibits sexual behaviour in male rats; this inhibition is effectively reversed by the antagonists ritanserin, pirenperone, and ketanserin. Comparison of these effects  ,  with reference to the binding profiles of each  drug, provided strong evidence that 5-HT2/1C receptors mediate an inhibitory influence on sexual behaviour in male rats. In addition, a tentative claim may be made that the effects of these drugs may be more attributable to 5-HT2 activity than 5-UT1C activity. ‘Wet dog shake’ behaviour in rats is known to be 5-HT2dependent. Experiments 5—7  evaluated the novel proposition  that the incidence of spontaneous wet dog shaking (WDS) by male rats in mating tests may provide a behavioural assay of concurrent 5—HT2 activity. WDS was found to be associated  iii  with copulatory inhibition in noncopulating males, compared to normal copulators, and this relationship was specific to mating situations. Activating  5-HT2/1C receptors with DOl  simultaneously induced WDS and inhibited copulation. Thus, the incidence of spontaneous WDS in untreated males may reflect the function of a 5—HT2—mediated neural mechanism that tonically inhibits copulation in male rats. In Experiment 8, DOl microinjection in the nucleus raphe obscurus/inferior olivary complex also induced WDS and inhibited copulation. This suggests that the hypothesized 5flT2-dependent inhibitory mechanism is vested in the ventromedial brainstem. Recent anatomical findings support this suggestion: cells in this region have bifurcating axons, projecting collaterally to both the medial preoptic area (implicated in sexual behaviour) and to the ventral cervical spinal cord (implicated in WDS). Overall, the results of the eight experiments provide strong evidence that 5-HT2 receptors mediate some of the inhibitory effects of serotonin on male rat sexual behaviour.  iv  TABLE OF CONTENTS  .I.bstract Table of Contents  .  .  .  .  .  .  .  .  .  .  .  ii  .  iv  List of Tables  vi  List of Figures Ackno1edgeent  vii .  .  .  .  .  .  .  .  .  .  Introduction  ix 1  1. Serotonin: Multiple subtypes of receptors in the CNS  .  .  .  6  .  2. Serotonin receptor subtypes and maleratsexualbehaviour  12  3. Serotonin receptor subtypes and serotonin stereotypy  16  4. Objectives  19  Experiments General Method: Experiments 1—4 Experiment 1  .  .  .  .  .  .  .  .  .  .  .  22  .  .  .  .  .  .  .  Experiment 2  33  Experiment 3 Experiment 4  .  .  .  .  .  45  .  63  GeneralMethod:Experiments5-7 Experiment  5 .  Experinient 6  26  .  .  .  Experiment 7..  .  77 .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  80  .  87  .  95  V  Experiment 8  •  Discussion  100 113  1. General Discussion: Experiments 1-4  .  114  2. General Discussion: Experiments 5—8  ..  121  3. Conclusions, speculations, and implications for future research References  .  . . .  .  . .  126 .  134  vi  LIST OF TABLES  Table 1,  The effects of increasing doses of ritanserin on the copulatory behaviour of male Sprague—Dawley rats  Table 2.  40—41  The effectiveness of pirenperone in reversing DOl-induced inhibition of sexual behaviour in male Sprague— Dawley rats  Table 3.  57—58  The effectiveness of pirenperone in reversing DOl-induced inhibition of sexual behaviour in male Long-Evans rats  Table 4.  . . .  . . . .  59—60  . . . .  72—73  . .  74—75  The effectiveness of ketanserin in reversing DOI-induced inhibition of sexual behaviour in male Sprague— D awley rats  Table 5.  . .  .  The effectiveness of ketanserin in reversing DOl-induced inhibition of sexual behaviour in male Long—Evans rats  vii  LIST OF FIGURES  Figure 1. Proportion of males displaying mounts, intromissions, or ejaculations following treatment with DOI  31—32  Figure 2. Effectiveness of ritanserin in reversing DOl-induced inhibition of sexual behaviour inmale rats  42—43  Figure 3. Effectiveness of pirenperone in reversing DOl-induced inhibition of sexual behaviour in male Sprague—Dawley rats  50—51  Figure 4. Effectiveness of pirenperone in reversing DOl-induced inhibition of sexual behaviour in male Long—Evans rats  52—53  Figure 5. Effectiveness of ketanserin in reversing DOl-induced inhibition of sexual behaviour in male Sprague—Dawley rats  . . . . .  .  66—67  Figure 6. Effectiveness of ketanserin in reversing DOl-induced inhibition of sexual behaviour inmaleLong—Evansrats  68—69  .  Figure 7, Incidence of WDS in male rats of varying copulatory proficiency  .  ..  84—85  . .  91—92  Figure 8. Comparison of spontaneous WDS in male rats paired with differing types of partners  .  . . .  viii  Figure 9.  Effects of DOl treatment on concurrent WDS and ejaculations  97—98  Figure 10. Intracerebral microinjection of DOl into the region of the raphe obscurus and inferior olivary complex: Effects on WDS and ejaculatory behaviourinmalerats  ...110—111  ix  ACKNOWLEDGMENT I am indebted to my supervisor, Dr. Boris Gorzalka, for his advice, encouragement and friendship. Thanks also to the faculty of the Biopsychology area, particularly Drs. Dave Albert, Ariane Coury, and John Pinel. Dr. Doreen Kimura of introduced me to biopsychology, and has been my U.w.O. friend and mentor throughout my academic career. Fellow graduate students have been a source of support and stimulation in far too many ways to catalogue. This is especially true of (in no particular order!): Ingrid Moe, Bill Mah, Sheryl Tanco, Georg Schulze, Scott Mendelson, and Theresa Newlove. I particularly wish to single out David (Jimmeh) Carey, most recently of St. Andrews University, for my thanks his e-mail nagging, while we were writing our theses in parallel, kept me going at times when I thought the well was dry Lastly, I am fortunate to have family who have been tolerant of my somewhat mercurial behaviour my brother Pete, Ingrid & Nic Cuk and family, and most especially my parents, Kathleen and Dennis, to whom I owe so much. —-  ——  This thesis is dedicated to my wife, Maria, the love of my life, and the answer to an ancient question. S’tu ma cholil.  1  INTRODUCT ION  Havelock Ellis (1898) maintained that “Sex lies at the root of life, and we can never learn to reverence life until we know how to understand sex”  (preface, p. xxx). Although  one may dispute Ellis’ lofty ambitions for the pursuit, it is axiomatic that the study of sexual behaviour is the study of a fundamental life process of major adaptive and organizational significance across species. The sexual interaction of animals, and the exchange of genetic material that ensues, is the currency of natural selection and the basis of biological diversity. Comprehensive understanding of the control of sexual behaviour has remained elusive. Attempts to specify the physiological determinants of sexual behaviour have frequently focussed on the copulatory behaviour of the laboratory rat. Males and females of this species display highly stereotyped patterns of mating behaviour that are easily and reliably quantified and therefore are potentially sensitive to experimental manipulations. When a male rat encounters a sexually receptive female rat, copulation proceeds in a highly regular manner (Beach, 1956; Sachs & Barfield, 1976). The male grasps the female’s flanks, mounts her, and commences pelvic thrusting. In response to this tactile stimulation by the male, the female adopts the lordosis posture, arching  2  her back, elevating her rump and diverting her tail laterally. A typical copulatory bout consists of a series of such mounts, during some of which penile intromission of the vagina occurs. After a number of intromissions (usually between 6 and 10), the male ejaculates intravaginally. The copulatory bout is followed by a brief period of refractoriness in the male, following which a new copulatory bout is commenced. Normal male rats will usually complete between three and five copulatory bouts in 1 hour. The three male copulatory behaviours ejaculations  --  ——  mounts, intromissions, and  are readily and reliably discriminated from  one another, and they form the basis of a variety of measures of copulatory behaviour in the male rat (described in General Method: Experiment 1-4). Early work on the neurochemical bases of male rat sexual behaviour  particularly implicated the monoamine  neurotransmitters: the catecholamines dopamine, norepinephrine, and epinephrine, and the indoleamine, serotonin. Treatment of male rats with monoamine oxidase (MAO) inhibitors such as pargyline or nialamide was found to produce an overall inhibition of the sexual behaviour of male rats (e.g. Dewsbury, Davis, & Jansen, 1972; Maimnas & Meyerson, 1970). MAO inhibitors exert their effects by interfering with the enzyme that renders monoamine molecules inert, resulting in supranormal concentrations of these  3  transmitters in brain tissue. Consequently, the findings  that MAO inhibitors reduce male rat sexual behaviour were taken as evidence that increased monoaluinergic activity has a general inhibitory influence on copulation in male rats (Malmnas & Meyerson, 1970). The importance of serotonin (5-hydroxytryptamine; 5-HT) in the modulation of rat sexual behaviour became apparent with the development of methods for selectively altering 5HT concentrations in the brain. When rats are treated with the metabolic precursor of serotonin, 5-aT? (5-hydroxytryptophan), plus an enzyme inhibitor that prevents it from being metabolized peripherally, serotonin synthesis in the central nervous system (CNS) is accelerated and levels of 5-HT in the brain become elevated. This treatment has been repeatedly reported to inhibit copulatory behaviour in male rats  (e.g., Ahienius & Larsson,  Ahlenius, Larsson, & Svensson, Meyerson,  1984;  1980; Gessa, 1970; Maimnas &  1971; Tagliamonte, Fratta, Mercuro, Biggio, &  Cainba, 1972), suggesting a general inhibitory influence of serotonin on male rat sexual behaviour. Similarly, it was reported that the sexual behaviour of male rats was inhibited by treatment with PCA (p-chloroamphetamine), which induces serotonergic neurons to release larger than normal quantities of 5-HT (Sodersten, Berge, & Hole,  1978).  4  Serotonin reuptake inhibitors are drugs that prevent serotonergic neurons from reabsorbing serotonin after it has been released, resulting in supranormal serotonin concentrations in the brain. Treatment of male rats with drugs of this class, such as alaproclate (Ahienius, Heimann & Larsson,  1979; Ahlenius et al.,  1980), chiorimipramine  (Ahlenius et al., 1979), fluoxetine (Baum & Starr, 1980), and zimelidine (Ahienius et al.,  1979,  1980) has been  reported to inhibit mating behaviour in male rats. The drug PCPA (para-chiorophenylalanine) blocks the actions of the rate-limiting enzyme in the synthesis of serotonin; administration of a few large doses of PCPA produces rats whose brains are nearly devoid of serotonin (Koe & Weissman,  1966). Dozens of reports in the late 1960’s  and 1970’s, using a variety of experimental preparations, noted a facilitation of copulatory behaviour in male rats as a consequence of PCPA-induced 5-fiT depletion (e.g., Sheard, 1969,  1973; Shillito, 1969; Tagliatuonte, Tagliamonte, Gessa,  and Brodie,  1969; Ahienius, Eriksson, Larsson, Modigh, &  Sodersten, 1971; Malmnas & Meyerson, 1971; Salis & Dewsbury, 1971; Sodersten, Larsson, Ahienius, & Engel, 1980).  1976; Dahiof,  In some cases, however, PCPA was found to have little  or no effect, especially in sexually—experienced male rats (McIntosh & Barfield, 1984; Whalen & Luttge, 1970).  5  The availability of serotonin in the CNS may also be reduced by the administration of neurotoxins that selectively destroy serotonergic neurons. When injected into the lateral ventricles of the brain, which are filled with cerebrospinal fluid that circulates throughout the CNS, the serotonergic neurotoxin 5, 6-DHT (5 ,6-dihydroxytryptamine) was found to induce a general facilitation of male rat sexual behaviour (Da Prada, Carruba, O’Brien, Saner & Pletscher,  1972). A similar facilitation was reported with  the closely related neurotoxin 5,7-DHT, when injected into either the lateral ventricles  (Sodersten et al.,  1978;  McIntosh & Barfield, 1984) or directly into the region of dorsal and median raphe nuclei, which contain large numbers of serotonergic cells (Larsson, Fuxe, Everitt, Holmgren, & Sodersten, 1978; Rodriguez, Castro, Hernandez & Mas,  1984).  The direct application of 5-lIT into the raphe nuclei causes a decrease in serotonin release through a feedback mechanism, and it also facilitates copulatory behaviour in male rats (Hillegaart, Ahienius, & Larsson, 1989). Taken together, investigations in which brain-wide serotonin levels have been manipulated have led to the general conclusion that serotonergic activity in the CNS inhibits copulation in male rats. Male rats that received treatments that induced serotonin accumulation in the brain displayed deficient sexual behaviour, and males whose brains  6  were depleted of serotonin displayed facilitated copulation. Pharmacological and physiological discoveries since 1980, however, have revealed a remarkable degree of diversity within the serotonergic systems of the CNS, and it now appears that the general conclusion that serotonin inhibits sexual behaviour is too simple.  1. Serotonin: Multiple subtypes of receptors in the CNS Serotonin derives its name from its “serum tonic” vasoconstrictive effects; it was for these effects that 5-HT was first studied (for review: Sjoerdsma & Palfreyman, 1990; Zifa & Fillion,  1992). By the 1950s, 5-UT had been  demonstrated to be heterogeneously distributed in the CNS and to bind to at least two different kinds of receptors in peripheral tissues (Gaddum & Picarelli, 1957). During the period from the 195Os to the 1970s, studies using pharmacological tools that altered brain-wide serotonin concentrations revealed that serotonin plays a role in such diverse functions as sleep, memory, sexual behaviour, aggression, thermoregulation, feeding, and hallucinogenesis (Bevan, Cools, & Archer, 1989; Osborne & Hamon, 1988; Zifa & Fillion, 1992). Understanding of the complexity of serotonergic participation in the control of behaviour has begun to emerge, as multiple subtypes of receptors for 5-HT have been  7  described in the CNS and their functional significance investigated. Molecules of serotonin, which serve as chemical messengers in the CNS, may be released from the terminals or axonal varicosities of serotonergic neurons. Like other neurotransmitters, serotonins actions on the surface of an individual neuron alters its excitability. In order to do this, serotonin binds to membrane—bound proteins, or receptors, on the surface of the cell. These receptors have an electrochemical configuration that is complementary to that of the serotonin molecule; as a result, the receptor is particularly selective for serotonin, and is less likely to bind other types of molecules. When serotonin binds to the receptor, its electrochemical interaction with the receptor causes changes within the neuron that alter its excitability, that is, alter the probability that it will release its own neurotransmitter)  (for review: Kandel & Schwartz,  1992).  The first intimations that more than one type of serotonin receptor exists on CNS neurons came with the advent of radioligand binding analyses (see, e.g., Lyon & Titeler,  1988). A ligand’ is a substance that has affinity  for the type of receptor being investigated. In radioligand binding analysis, homogenized brain tissue is incubated with a radioactive form of a ligand for the type of receptors in question. In the case of serotonin receptors, this might be  8  radioactive serotonin ([ H]5-HT) or a radiolabelled form of 3 a drug that mimics serotonin. The labelled compound selectively binds to the serotonin receptors in the brain tissue, and any leftover radioactive material is rinsed away. In a binding competition analysis, which is the most common type, a nonradioactive compound that selectively binds to the same type of receptors as the labelled compound is added to the brain homogenate in increasing concentrations. If the unlabelled competitor has a high affinity for the same type of receptors as the labelled compound, it will effectively displace the labelled compound even at low concentrations. If the test compound’s affinity for the labelled receptors is low, high concentrations will be required to displace the labelled compound from the receptor sites. Mathematical analyses of the manner in which drugs compete for labelled receptors reveal two things: the affinity of the test compound for the type of receptor that is labelled, and the selectivity of the test compound across different types of receptors. Importantly, the pattern of these two characteristics may suggest the presence of more than one subtype of receptor that is labelled by the radioactive substance. An additional consideration with any new ligand is its intrinsic activity. When a drug binds to a receptor, its ability to produce the same effect as the  9  endogenous ligand (e.g., serotonin) reflects its intrinsic activity. If the drug  ‘turns on’ the receptor, thereby  altering the activity of the cell, it has high intrinsic activity and is called a receptor agonist. If, instead, the drug binds with high affinity but simply blocks the receptor, preventing any other ligand from activating it, the drug has low intrinsic activity and is called a receptor antagonist. Drugs that have moderate intrinsic activity produce partial activation of the receptors and are referred to as partial agonists (or partial antagonists) Kalant, Roschlau, & Sellers,  (see, e.g.,  1985).  In the early 1980 us, this type of radioligand binding analysis was applied to binding at serotonin receptors. By comparing the efficacy of unlabelled serotonin in displacing labelled serotonin and labelled spiperone, which binds to serotonin receptors, with the efficacy of unlabelled spiperone in the same preparation, Peroutka and his collaborators (Peroutka & Snyder,  1979; Peroutka, Lebovitz,  & Snyder, 1981) found that there existed two populations of receptors which, while differing in their pharmacological properties and anatomical distribution, were nevertheless subtypes of serotonin receptors. These they named the. 5-HT1 receptor (displaying high affinity for labelled 5-HT) and the 5-11T2 receptor (displaying high affinity for labelled spiperone).  10  In the last decade or so, further binding studies have revealed a remarkable profusion of different subtypes of serotonin binding sites in the CNS. Recent accounts suggest that there may be as many as 11 distinct subtypes of 5-HT binding sites, and evidence of new subtypes continues to accumulate (Bradley, Handley, Cooper, Key, Barnes, & Coote, 1992; Frazer, Maayani, & Wolfe, 1990; Glennon & Dukat, 1991; Zifa & Fillion,  1992). In many cases, however, these binding  sites have been characterized only pharmacologically, in isolated tissue preparations, and their functional significance remains unknown. Drugs that are truly selective for particular subtypes of serotonin receptors in the intact organism have yet to be developed, making it difficult to attribute particular functions to modulation of activity at particular 5-HT receptor subtypes (Glennon & Dukat, Zifa & Fillion,  1991;  1992). Nevertheless, ligands that are  moderately selective do exist, and they have been used for  in vivo studies of the functions of the six best-established 5-HT receptor subtypes: the 5-HT1A,  1B,  1C, and 10  receptors; 5-HT2 receptors; and 5-HT3 receptors (Bradley et al.,  1992; Glennon & Dukat, 1991; Zifa & Fillion, 1992).  Quantitative autoradiography has demonstrated that these subtypes of 5-HT receptors are differentially distributed throughout the brain (e.g., Palacios, Waeber, Hoyer, & Mengod,  1990); however, it is important to keep in mind that  11  the distribution of the various subtypes of serotonin receptors has provided little indication of their respective functions. It should be mentioned that pharmacological data suggest the existence of multiple subtypes of 5-HT2 binding sites, having in common a phosphoinositol hydrolysis second messenger system. In particular, both the classical 5—HT2 receptor and the 5-HT1C receptor belong to this class (e.g., Glennon & Dukat, 1991; Hoyer, 1988b,  1992). Indeed, cloning  studies have revealed an 80% homology between the 5-HT2 and 5-HT1C receptor subtypes  (Hartig, 1989; Glennon & Dukat,  1991). Given the great physical similarity of these two receptor subtypes, it is not surprising that ligands have yet to be developed that can potently differentiate between them (Hoyer, 1988b,  1992; Glennon & Dukat, 1991)  --  drugs  that bind to 5-HT2 receptors also tend to bind to 5-HT1C receptors with high affinity. However, because each serotonergic drug possesses its own unique profile of affinities for the various 5-HT receptor subtypes, comparing the behavioural effects of a spectrum of serotonergic drugs, that share affinity for a target subtype of receptor but differ in affinity for other types of receptors, does allow limited inferences about the behavioural roles of the target subtype. In particular, pharmacological probes have become available in recent years that provide a means of specifying  12  some behavioural effects of 5-HT2/1C receptor activity, and to a much more limited extent allow distinctions to be made between 5-HT2 and 5-HT1C-mediated functions (reviewed in Bradley et al.,  1992; Zifa & Fillion, 1992).  2. Serotonin receptor subtypes and male rat sexual behaviour. Although early work suggested a general inhibitory effect of serotonergic activity on the sexual behaviour of male rats, in the last 12 years or so research using receptor-selective ligands has suggested that 5-HT may inhibit or facilitate male rat copulation, depending on the subtypes of receptors involved (reviewed in Gorzalka, Mendelson,  & Watson,  1990; Zifa & Fillion,  1992).  The majority of this work has focussed on the roles of subtypes of 5—HT1 receptors in the control of male rat copulation. In particular, treatment of male rats with drugs that are agonists at the 5-HT1A site has been found to produce a net facilitation of sexual behaviour. Doses of the selective 5-HT1A agonist 8-OH-DPAT [8-hydroxy-2-(di-npropylamino)tetralin] ranging from 0.025 to 4.0 mg/kg are uniformly facilitatory, in some cases producing male rats that ejaculate on the first intromission of a copulatory bout (Ahlenius & Larson, 1984a, 1984b,  1985; Ahienius,  Larsson, Svensson, Hjorth, Carlsson, Lindberg, et al., 1981;  13  Dahiof, Ahlenius, & Larsson, 1986; Morali & Larsson, Davidson,  1988; Mendelson & Gorzalka,  1984; Schnur, Smith, Lee, Mas, &  1989). Administration of the related compound  8-OMe-DPAT is reported to produce a comparable facilitation (Ahlenius et al.,  1981). Treatment with 8-OH-DPAT has been  reported to restore copulatory function in castrates and in males that have undergone penile deaf ferentation (Dahlof et al.,  1988). Recent evidence suggests that the stimulatory  effects of 8-OH-DPAT are effectively reversed by the selective 5-HT1A receptor antagonist UH-301 Meyerson,  & Hacksell,  1991),  (Johansson,  supporting the conclusion that  5-HT1A receptors mediate a facilitation of male rat copulatory behaviour.  Interestingly, there also exists a  marked sex difference in the effects of 8-OH-DPAT on sexual behaviour; in female rats,  8-OH-DPAT is reportedly  inhibitory (e.g., Mendelson & Gorzalka,  1986).  In agreement  with the data collected using 8-OH-DPAT, facilitation of sexual behaviour has also been observed in male rats treated with the less selective 5-HT1A agonists lisuride (Ahienius, Larsson,  & Svensson,  & Jonsson,  1980; Fernandez—Guasti, Hansen, Archer,  1986) and RDS-127  Arneric, Long, & Davidson,  (Clark, Smith, Stefanick,  1982). The 5-HT1A partial  agonists buspirone (Mathes, Smith, Popa,  & Davidson,  1990)  and ipsapirone (Fernandez-Guasti, Escalante, & Agmo,  1989)  likewise appear to facilitate copulation in male rats, as  14  does the novel 5-HT1A agonist indorenate (Fernandez-Guasti, Escalante, Hong, & Agmo,  1990).  The 5-HT agonists TFMPP and mCPP display mixed affinities, but particularly bind with high affinity to 5-HT1C sites. Administration of these drugs to male rats has been reported to inhibit their sexual behaviour (Fernandez— Guasti et al.,  1989; Fernandez-Guasti & Rodriguez-Manzo,  1992; Mendelson & Gorzalka,  1990). The mixed 5-HT1A/1B  receptor agonist RU 24969 reportedly also produces an inhibition of copulation in male rats, presumably due to its activity at 5-HT1B receptors given that 5-HT1A receptor agonists are facilitatory (Fernandez-Guasti et al., Mendelson & Gorzalka,  1989;  1988).  Recent evidence suggests that brain 5—HT3 receptors play no role in regulating reproductive behaviour in rats. We have found that the highly selective 5-HT3 receptor antagonists ondansetron and granisetron are without effect in tests of male rat copulation (Tanco, Watson, & Gorzalka, 1993a). Similarly, the 5-HT3 receptor agonist 2-Me-5-HT (2—methylserotonin) does not influence copulation in male rats, and although the 5-HT3 agonist 1-PBG (1-phenylbiguanide) induces a slight facilitation in mating tests  (Watson, Tanco & Corzalka,  Gorzalka,  1991; Tanco, Watson, &  1993b), this effect may be attributable to 1-PBG-  induced dopamine release (Schmidt & Black,  1989).  15  The present thesis is concerned with some possible functional effects of 5-HT2 receptor activity. In contrast to the attention that has been directed at the roles of 5-HT1 receptors on the regulation of copulatory behaviour in the male rat, relatively few studies have directly investigated the effects of 5-HT2/1C-selective compounds. The data that have been collected thus far have been equivocal, and in some cases contradictory, and are discussed in greater detail in the introductory sections of Experiments 1-4. Briefly, the 5-HT2/1C receptor antagonists ketanserin and pirenperone have been reported to inhibit male rat sexual behaviour (Mendelson & Gorzalka, 1985), suggesting that 5-HT2/1C receptors mediate a facilitation of sexual behaviour in the male rat. Conversely, the 5-HT2/1C antagonists cyproheptadine and LY 53857 reportedly facilitate male rat copulation, suggesting that 5-HT2/1C activation is inhibitory (Abraham, Viesca, Plaza, & Mann, 1988; Foreman, Hall, & Love, 1989). This latter conclusion is supported by the report that the 5-HT2/1C receptor agonist DOl [1-(2 ,5-dimethoxy-4-iodophenyl)-2-aminopropane] inhibits copulation in male rats (Foreman et al.,  1989).  Given the contradictory conclusions of the published studies, therefore, one of the major objectives of the present dissertation is to attempt to clarify the role of 5-HT2 receptor activity in male rat sexual behaviour.  16  3. Serotonin receptor subtypes and serotonin stereotypy. It has been known for some time that a highly stereotyped “Serotonin Behavioural Syndrome” ensues when rats receive treatments that augment serotonergic activity in the brain (Come, Pickering, & Warner, 1963; Grahame Smith, 1971). This is most frequently accomplished by giving rats large doses of the metabolic precursor of serotonin, 5-HTP, plus an inhibitor of peripheral decarboxylation such as benserazide or carbidopa, or by giving the animals large amounts of the amino acid tryptophan plus an MAO inhibitor. The syndrome consists of a variety of bizarre motor patterns, including reciprocal forepaw treading, Straub tail (a stiff or “poker” tail), abnormal hindlimb abduction, flattened body posture, and ‘wet dog shaking’  (WDS), It is  presumed that these symptoms reflect supranormal or “spillover” activation of central serotonergic mechanisms (Green & Grahame-Smith, 1976; Green & Heal,  1985).  As has been discussed, significant advances in refining the pharmacology of the multiple subtypes of CNS receptors for serotonin have been made in the last decade, but the effects of activity at these receptor subtypes on overt behaviour remains largely uncharacterized. Nevertheless, evidence has accumulated that the different elements of the serotonin behavioural syndrome are mediated by different  17  5-HT receptor subtypes  particular,  (for review: Green & Heal,  1985).  In  it appears that WDS is primarily mediated by the  activation of 5-HT2 receptors (Green, 1985; Pranzatelli,  1990; Yap & Taylor,  1989; Green & Heal, 1983). WDS may be  defined as a paroxysmal, rapid rotational shudder of the head and shoulders; as its name suggests, the behaviour is reminiscent of a dog shaking water out of its coat. Individual episodes of WDS last between 500 and 1000 msec. WDS may be elicited in isolation from the other symptoms of the serotonin behavioural syndrome by administration of 5-fiT agonists which act at 5-HT2 receptors, such as the nonselective compounds LSD and quipazine (Green & Heal, 1985; Lucki & Minugh-Purvis,  1987) and the more highly  selective agonist DOl (Darmani, Martin, Pandey, & Glennon, 1990; Pranzatelli,  1990). Conversely, 5—HT antagonists such  as ritanserin, ketanserin, mianserin, methysergide, pirenperone, cinanserin, cyproheptadine, and pizotifen, all of which display some selectivity for 5-11T2 receptors, have generally been found to inhibit the production of WDS (Colpaert & Janssen, Eberle, Janssen,  1983; Darmani, et al.,  & Minugh-Purvis,  1990; Lucki,  1987; Meert, Niemegeers, Gelders, &  1989; Pranzatelli,  1990; Yamaguchi, Nabeshima,  Ishikawa, Yoshida, & Kameyama,  1987).  WDS has also been reported as a consequence of a variety of experimental treatments that are not direct manipulations  18  of 5-HT2 activity. For example, increased WDS has been reported following the administration of phencyclidine (Yamaguchi et al.,  1987; Nabeshima, Ishikawa, Yatuaguchi,  Furakawa, & Kameyama, 1987), the benzodiazepine clonazepam (Pranzatelli, 1989), the noradrenergic neurotoxin DSP4 (Eison, Yocca, & Gianutsos, 1988), insulin (Kleinrok & Juskiewicz,  1986) and the thyrotrophin releasing hormone  analogue CC 3509 (Fone, Johnson, Bennett, & Marsden,  1989).  Interestingly, large systemic injections of putrescine (a substance produced by decaying bodies!) have also been reported to induce WDS (Genendani, Bernardi, Tagliavini, Botticelli, & Bertolini, 1987). WDS has also been reported as a consequence of hippocampal stimulation (Araki & Aihara, 1986). Nevertheless, where it has been tested, modulation of central 5-HT activity has frequently been implicated in WOS produced by non5-HT manipulations (e.g., Pranzatelli, Fone et al., et al.,  1989; Kleinrok & Juskiewicz,  1987; Yameguchi, et al.  1989;  1986; Nabeshima,  1987). Overall, the evidence  generally supports the conclusion that WDS is mediated by 5—HT2 receptors, with other transmitters or mechanisms serving ancillary or permissive functions (Green & Heal, 1985). It is for this reason that pharmacologically induced WDS, in conjunction with the serotonin behavioural syndrome, has gained some acceptance as a model of neuronal serotonergic activity. Examination of the effectiveness of  19  novel psychoactive compounds in modulating the expression of WDS has gained acceptance as an index of central 5-HT2 receptor activity (e.g., Meert et al.,  1989; Pranzatelli,  1990; Yap & Taylor, 1983).  4. Obiectives As was discussed earlier, the existing data strongly suggest serotonergic participation in the control of male rat sexual behaviour, but the way in which subtypes of serotonin receptors contribute to this regulation is poorly understood. In particular, the effects of changes in activity at 5-HT2 receptors on the reproductive behaviour of male rats have received little attention. Therefore, the overall objective of the present dissertation was to attempt to elucidate the role of 5-HT2 activity in male rat sexual behaviour. This goal was formulated as three general objectives. a) The two published studies that had focussed on the contributions of 5-HT2 receptor activity in the control of the reproductive behaviour of male rats had led to contradictory conclusions. Accordingly, the first four experimental sections of this dissertation report the results of studies examining the effects of a spectrum of 5—HT2 receptor ligands on sexual behaviour in male rats. The agents employed in these studies were selected on the basis  20  of common affinity for 5-HT2  (and in some cases, 5-HT1C)  receptors, but different affinities for other types of receptors, in an effort to reconcile the conflicting data reported by others. b)  In Experiments 5-7, the issue of 5-HT2 receptor  mediation of male rat sexual behaviour was addressed in an entirely behavioural fashion by incorporating behavioural measures of central serotonergic activity with measures of male rat copulatory behaviour. Informal observations suggested to me that untreated normal rats spontaneously display WDS while engaging in other types of behaviour. In this set of experiments, I evaluated a novel approach in which WDS was employed as a behavioural index of 5-HT2 receptor activation during copulation. Given the established dependency of WDS on 5-HT2 receptor activity, it seemed reasonable to suppose that the occurrence of spontaneous WDS could constitutea “behavioural assay” of concurrent 5-HT2 activation that would converge on the pharmacological data. In Experiment 5-7, measures of .WDS by male rats were taken during various types of mating tests. c) Very little is known about the participation of brainstem mechanisms in the control of male rat sexual behaviour; most work has focussed on the critical importance of more rostral structures, such as the medial preoptic area and rostral midbrain (e.g., Rose,  1990). Likewise, the  21  neural substrate of WDS has yet to be established. In an initial attempt to identify neural sites involved in the control of WDS and sexual behaviour, measures of these behaviours were taken in male rats after direct manipulation of 5-HT2 receptor activity in the ventromedial brainstem, in Experiment 8. The degree to which WDS and male rat sexual behaviour rely on a common neural substrate was also evaluated.  22  GENERAL METHOD: EXPERIMENTS 1-4  Animals Male and female Sprague—Dawley and Long—Evans rats were bred from stock originally obtained from Charles River Canada Inc., Montreal. Rats were housed in our colonies, in groups of 6, in standard wire mesh cages, segregated by sex and strain. Free access to food and water was provided. Colony rooms were maintained on a reversed 12/12 hr light/dark cycle. At approximately 70 days of age,  females underwent  bilateral ovariectomy while under sodium pentobarbital anaesthesia (65 mg/kg). Males were between 90 and 180 days of age when tested. All males used in these experiments were sexually experienced, having been exposed to receptive females on at least three occasions prior to testing.  Steroid treatments Receptivity was induced in the ovariectomized females by subcutaneous injection of 10 [tg estradiol benzoate (Steraloids Inc.) 48 hr prior to testing, and 500 g progesterone (Steraloids Inc.) 4 hr prior to testing. Steroids were dissolved in 0.1 ml peanut oil.  23  Behavioural Testing With the aid of a microcomputer program (Holmes, Holmes, & Sachs,  1988), the copulatory behaviours of male rats were  recorded after they had been presented with a receptive female rat. Behavioural recording was performed by an observer blind to experimental hypotheses and treatment conditions. All testing was conducting during the middle 1/3 of the rats’ dark cycle, at which time rats are most active. Males were placed individually into clear Plexiglas testing arenas measuring 30 cm in width and length and 45 cm in height, and containing a layer of fresh San-i-cel bedding material. A period of 5 mm  was then allowed for the males  to habituate to their new surroundings. During each session, 4 or 5 males were scored simultaneously, in separate testing chambers. Following the habituation period, receptive females were introduced into the males’ testing chambers and recording of the males’ copulatory behaviours commenced. For each session, the proportion of males displaying mounts (%M), intromissions (%I), or ejaculations (%E) was quantified. In Experiments 3 and 4 the following additional measures of male rat copulatory behaviour were recorded: the number of mounts (M) and intromissions (I) preceding ejaculation; the latency from presentation of the stimulus female to the occurrence of the first mount (ML) or intromission (IL) by  24  the male; the latency from the first intromission to ejaculation (EL); and the postejaculatory interval (PEI) between ejaculation and the first intromission of the next copulatory bout. Scoring of the males  sexual behaviour was  discontinued either after the first intromission of the second copulatory bout, or after a maximum of 30 mm  had  elapsed. Stimulus females were rotated among the males every 10 mm  during the test session.  Under some experimental conditions, male rats fail to achieve an ejaculation, or exhibit no sexual behaviour whatsoever. In such cases, behaviour counts such as M, I, and E were set to zero. Missing latency scores (ML, IL, EL and PEI) were assigned the maximum value possible: 1800 sec. Additionally, because group variances of 0.0 grossly violate the assumption of normalcy in parametric tests, such as analysis of variance, the data were analysed using nonparametric statistics (reviewed in Siegel & Castellan, 1988). The statistical significance of the proportion data (%M, %I, and %E) was assessed with the Cochran Q statistic, which provides an overall test for differences in proportions across multiple groups. In the event of a statistically significant Cochran Q test, the significance of the differences between individual pairs of group  25  proportions was assessed with paired McNemar tests. All other behavioural measures were submitted to a Friedman nonparametric analysis of variance for k related samples. Significant Friedman tests were followed by Wilcoxon paired comparisons.  26  EXPERIMENTS  Experiment 1  Although many reports have appeared regarding the effects of potentiated overall serotonergic transmission on the sexual behaviour of male rats, few data have been collected using compounds that selectively bind to, and activate, 5-HT2 receptors. Agonists and antagonists for 5-HT2 receptors have been either characterized or synthesized only relatively recently, and even these compounds exhibit the problem, discussed earlier, of comparable binding to 5-HT2 and 5-HT1C receptors (Glennon & Dukat,  1991; Hoyer, 1988a,b).  The 5—fiT agonist quipazine has been reported to induce a net facilitation of male rat sexual behaviour (Mendelson & Gorzalka,  1985). This finding was attributed to 5-HT2  receptor activation, on the basis of functional analyses available at that time which suggested that quipazine exhibits preferential binding to 5-HT2 receptors (eg. Green, O’Shaugnessy, Hammon, Schacter & Grahame-Smith, 1983). However, it is no longer clear to what extent quipazine— induced facilitation of male rat copulation reflects 5-HT2 receptor activity, as more recent binding analyses have revealed that quipazine shows little selectivity between the  27  various 5-HT receptor subtypes Hoyer,  1988a; Peroutka & Hamik,  (Glennon & Dukat, 1988; Perry,  1991;  1990).  In fact,  H]quipazine is now used in radioligand binding studies of 3 [ 5-HT3 receptors, suggesting that,  if anything, it is  somewhat more selective for 5-HT3 receptors than for 5—HT2 receptors  (Peroutka & Hamik,  1988; Perry,  1990).  The 5-HT agonists DOM [1-(2,5-dimethoxy-4-methylphenyl)2—aminopropane], and to a lesser extent LSD [lysergic acid diethylamide], are now believed to exhibit a degree of selectivity for 5-HT2/1C binding sites & Jacobs,  1988; Wing, Tapson & Geyer,  (Glennon,  1990; Heym  1990; Zifa & Fillion,  1992). Administration of these compounds to male rats has been reported to have no effect on their copulatory behaviour (Ahienius et al.,  1981). However, it should be  noted that while LSD appears to bind with some selectivity to 5-HT2/1C receptors, its activity at those receptors is disputed. Although LSD has most frequently been reported to act as an agonist  ,  evidence also exists suggesting an  antagonistic action of LSD at 5-HT2 receptors Jacobs,  (eg. Heym &  1988).  More recently,  it has been reported that treatment with  the serotonin agonist DOl [1-(2 , 5-dimethoxy-4-iodophenyl)-2aiuinopropane] induces a net inhibition of copulatory behaviour in male rats  (Foreman et al.,  1989). DOl is a  prototypic 5-HT2/1C agonist (Dabiré, Chaouche-Teyara,  28  Cherqui, Fournier, Laubie, & Schmitt,  1989; Glennon & Dukat,  1991; Glennon, McKenney, Lyon, & Titeler, 1986), and thus exhibits high and selective affinity for 5-HT2 and 5-HT1C sites  (Hoyer,  1988a). When administered to male rats in  doses ranging from 0.1 mg/kg to 1.0 mg/kg, DOl has been reported to systematically reduce or eliminate copulatory behaviour (Foreman et al.,  1989).  In view of the  inconsistent findings regarding the effects of 5-HT2 receptor stimulation on male rat sexual behaviour, the objectives of Experiment 1 were to confirm the inhibitory action of DOl and to establish an effective dose of DOl for use in the subsequent experiments.  Method  Drugs DOl HC1 was obtained from Research Biochemicals,  Inc.,  Natick, MA, and stored at 5° C, protected from light. Fresh solutions of DOl were prepared before each test session, dissolved in sterile physiological saline and administered subcutaneously (SC) in a total volume of 1.0 ml per kg of body weight (mi/kg). DOl was administered 30 mm testing (Foreman et al.,  1989).  prior to  29  Procedures In Experiment 1, 20 sexually-experienced Sprague Dawley male rats were tested following administration of 0.0, 0.1, or 1.0 mg/kg DOl SC. Doses were administered in a random counterbalanced fashion over three consecutive sessions, such that by the end of the experiment the sexual behaviour of each male (% M, %I and %E) had been evaluated once at each dose. An interval of 1 week separated successive test sessions.  Results  The results of Experiment 1 are presented in Figure 1, in the form of dose—response curves for each behavioural measure. One rat failed to initiate normal copulatory behaviour under any treatment condition, and was excluded from analyses. Treatment with DOl inhibited the sexual behaviour of the male rats in a dose—dependent manner (Fig 1). Cochran Q analyses revealed a significant decrement in the proportions of males exhibiting mounts (Q(2) intromissions (Q(2) (Q(2)  =  13.0, p  =  =  16.9, p  =  =  21.0, p  <  0.0001),  0.0002), and ejaculations  0.0015). Subsequent McNemar tests revealed  that this inhibition was most pronounced in the 1.0 mg/kg DOl condition (see Fig 1 for group comparisons). Indeed,  30  after 1.0 mg/kg DCI, only one rat displayed mounting and intromitting behaviour, and no animal achieved ejaculation.  Discussion  The results of Experiment 1 confirm the previous finding (Foreman et al.,  1989) that DCI induces a profound  inhibition of copulatory behaviour in male rats, and informal observation suggested that this inhibition did not reflect a generalized motor deficit. To the extent that the effects of DCI on copulatory behaviour are attributable to 5-HT2 specific activation, the data provide preliminary evidence that 5-HT2 receptors mediate an inhibitory influence on copulation in male rats. Additionally, the results provide an empirical basis for selecting 1.0mg/kg DCI as an effective dose for use in the subsequent experiments of this series.  31  Fiaure 1. Proportion of male rats displaying mounts, intromissions or ejaculations following treatment with varying doses of DCI. *Differs from 1.0 mg/kg condition, p  <  .05  (McNemar test)  **Differs from both 0.1 and 1.0 mg/kg conditions, p (McNemar test).  <  .05  0  (I)  -o  ci)  0  >‘  C  ci 0 -c C.) Q a)  0 >  0•  20  40  60-  80-  100•  0.0  D.  **  *  *  Dose of DOl, mg/kg s.c.  0.1  *  *  1.0  —  Q  0—0  Ejaculations  Intromissions  Mounts  t’j  33  Experiment 2  In Experiment 1, the administration of DOl to male rats was found to profoundly inhibit their copulatory behaviour. The binding profile of DOl suggests that its inhibitory effect on male rat sexual behaviour is attributable to the high affinity of DOl for 5-HT2 and 5-HT1C receptors (Hoyer, 1988a). One method of substantiating this interpretation would be to examine the reversibility of the effect of DOl, using other pharmacological probes that share high affinity and selectivity for 5-HT2/1C receptors. This may be accomplished by administering DOI and coadministering a 5-HT2/1C antagonist, which blocks receptors rather than activating them. Like all receptor-selective drugs, these antagonists display unique affinity profiles across a variety of types of receptors, but nevertheless display high affinity for 5-11T2/1C receptors, in common with DOl (for review: Hoyer, 1988a, b; Jansson, 1991; Zifa & Fillion,  1983; Glennon & Dukat,  1992).  Previous work with 5-HT2 antagonists has yielded equivocal results. Drugs of this class bind to 5-HT2/1C receptors without activating them, and thereby reduce activity at these receptor populations to subnormal levels. Inhibition of sexual behaviour after treatment with a 5-HT2/1C antagonist would therefore imply that, in the  34  untreated male rat, 5-HT2/1C receptors mediate a facilitation of copulatory behaviour. Conversely, facilitation of sexual behaviour after such treatment would imply that 5-HT2/1C receptors mediate an inhibition of sexual behaviour in male rats. Although the 5-HT antagonist pirenperone was initially reported to be without effect on male rat sexual behaviour (Ahienius & Larsson, 1984),  later studies  have reported an  inhibition of sexual behaviour in male rats following pirenperone treatment (Mendelson & Gorzalka, 1985; Foreman et al.,  1989). A similar inhibition has been reported with  the structurally related compound ketanserin (Mendelson & Gorzalka,  1985). Pirenperone and ketanserin are serotonin  receptor antagonists displaying high affinity for 5-HT2/1C receptors and moderate selectivity (Janssen, 1983); the finding that they inhibit the sexual behaviour of male rats therefore suggests that 5-HT2/1C receptor activity facilitates male rat copulation. However, data collected using other antagonists have suggested the reverse. Cyproheptadine is a 5-HT antagonist that, while it is not very selective overall, displays a moderate preference for 5—HT2 and 5—HT1C receptors (e.g., Hoyer, 1988a). When administered to male rats, cyproheptadine reportedly facilitates their sexual behaviour (Abraham, Viesca, Plaza & Mann,  1988). The more highly  35  selective 5-11T2/1C antagonist LY 53857, and its congeners LY 237733 and LY 281067, has been reported to induce a similar facilitation of sexual behaviour in male rats (Foreman et al.,  1989). The results of these studies therefore suggest  that 5-HT2/1C receptor activity inhibits, rather than facilitates, the sexual behaviour of male rats. Studies employing a combination of agonists and antagonists have added to the confusion. As mentioned before, the agonist quipazine inhibits sexual behaviour in male rats when given alone (Ahlenius et al., Grabowska,  1981;  1975; Mendelson & Gorzalka, 1985). Paradoxically,  when coadministered with pirenperone, quipazine is reportedly effective in reversing the inhibitory effects of pirenperone (Mendelson & Gorzalka, 1985). In this case, then, quipazine acted in a facilitatory fashion. Furthermore, pirenperone has been reported to effectively reverse the inhibition of male rat copulatory behaviour induced by treatment with the precursor of serotonin, 5-HTP (Ahlenius & Larsson, 1984). Pirenperone thus exerted a facilitatory action in this study, which would be congruent with an inhibitory effect of 5-HT2/1C receptor activity. Taken together, the data from these studies are completely contradictory; both pirenperone and quipazine have displayed both inhibitory and facilitatory profiles in the various studies.  36  The conflicting results of previous studies are at least partly attributable to the relative lack of selectivity of the agonists employed. In the one previous study in which the selective 5-HT2/1C agonist DOl was employed in a competitive manner, it was found that DOl induced inhibition of male rat sexual behaviour was effectively blocked in rats that had been pretreated with the 5-HT2I1C antagonist LY 53857  (Foreman, et al.,  1989).  The 5-UT antagonist ritanserin displays very high affinity and relatively high selectivity for 5-HT2/1C receptors (Hoyer, Janssen,  1988a; Meert, Niemegeers, Gelders, &  1989). The effects of ritanserin on male rat sexual  behaviour have not been determined, but the unique binding profile of ritanserin suggests that it might be of value in assessing the particular contributions of 5-HT2/1C receptors to the control of male rat copulation.  In Experiment 2, the  effects of varying doses of ritanserin on male rat copulation were examined. In addition, the efficacy of ritanserin in reversing the inhibition of sexual behaviour induced by DOl was examined.  37  Methods  Drugs Ritanserin was obtained from Research Biochemicals, and stored at 5°C in dark conditions. Fresh solutions of rita.nserin and DCI were prepared before each test session. DCI was prepared as in Experiment 1. Ritanserin was initially dissolved in a minimal amount of lactic acid and diluted with sterile distilled water. Both drugs were administered SC in a total volume of 1.0 mi/kg; ritanserin was administered 90 mm  prior to testing (Meert et al.,  1989) and DCI was administered 30 mm  prior.  Procedures Experiment 2 was conducted in two parts. In the first part, Experiment 2a,  18 sexually experienced male Sprague  Dawley rats were observed following the administration of ritanserin alone, in the following doses: 0.0, 0.3, 0.6, or 1.2 mg/kg. The doses were administered in a random counterbalanced fashion over successive test sessions, such that by the end of the experiment each male rat had been evaluated (N, I, ML, IL, EL, PEI) once at each dose. Consecutive test sessions were separated by at least 1 week. In Experiment 2b, the effects of ritanserin on DOl induced inhibition were evaluated. The same male rats as had  38  been used in Experiment 2a received a pretreatment of ritanserin (0.0,  0.2, 1.0, or 5.0 mg/kg) plus 1.0 mg/kg DOl,  the dose of DOl that was found to be maximally effective in producing an inhibition of sexual behaviour in Experiment 1. For control purposes, an additional condition was included, in which the rats received only the drug vehicles. The male rats received each of the five treatments in counterbalanced fashion over the course of 5 weeks, and their copulatory behaviour was quantified (%M,  %I, %E) under each treatment  condition.  Results  The results of Experiment 2a are presented in Table 1. Friedman tests revealed that, when given alone, ritanserin did not significantly affect the sexual behaviour of the male rats, at any dose tested  2 (x  values ranging from 2.09 to  4.61, NS). To assess the possibility that male rat sexual behaviour may be affected only by a high dose of ritanserin, an additional follow—up study was performed in which the 18 male rats received 0.0 or 3.0 mg/kg of ritanserin, following the procedure of Experiment 2a. The 3.0 mg/kg dose did not significantly affect the male rats’ sexual behaviour, for any measure (Wilcoxon test; z values ranging from 0.2068 to 1.5148, NS).  39  The results of Experiment 2b are presented in Figure 2, in the form of a dose—response graph. In the absence of ritanserin, 1.0 mg/kg 001 completely abolished sexual behaviour in the male rats. Cochran Q tests revealed that pretreatment of the male rats with ritanserin potently blocked this inhibitory influence of DCI, in a dose dependent manner. This was reflected in systematic increases in %M (Q(3) and %E (Q(3)  =  31.2, p 16.1, p  < =  .0001), %I (Q(3)  =  31.4 p  <  .0001),  .003). Indeed, with ritanserin  pretreatments of 1.0 or 5.0 mg/kg, the copulatory behaviour of the male rats was maintained at baseline levels (Figure 2).  40  Table 1.  The effects of various doses of ritanserin on the  copulatory behaviour of male Sprague-Dawley rats.  Mounts  and intromissions are total counts, all other values are time in seconds. Data are presented as mean scores (S.E.M. in parentheses) after substitution of missing values. The numbers of rats that displayed each behaviour are presented in square brackets.  41  Effects of Ritanserin on Sexual Behaviour in Male Rats Ritanserin treatment, mg/kg Behavioural Measure  0.0  0.3  0.6  1.2  Mounts  10.3 (1.8) [20]  9.9 (1.6) [20]  9.8 (1.4) [20]  7.15 (0.9) [20]  Intromissions  10.0 (0.7) [20]  8.9 (0.6) [20]  8.7 (0.9) [19]  9.9 (0.9) [20]  Mount Latency  56.3 (19.0) [20]  38.3 (10.6) [20]  117.3 (89.2) [19]  47.4 (28.0) [20]  Intromission Latency  126.6 (42.5) [20]  62.9 (14.3) [20]  161.2 (88.0) [19]  67.4 (41.1) [20]  Ejaculation Latency  567.8 (99.1) [18]  441.9 (48.4) [20]  629.9 (117.2) [18]  428.7 (31.4) [20]  Postejaculatory 499.0 Interval (100.1) [18]  373.9 (16.0) [20]  572.4 (118.7) [17]  422.0 (74.1) [19]  42  Ficiure 2. Effectiveness of increasing doses of ritanserin in blocking the inhibition of male rat copulatory behaviour induced by DOl.  In all but the No-Drugs condition, rats  received a test dose of ritanserin 90 mm and 1.0 mg/kg DOl 30 mm  prior to testing  prior. In the No-Drugs condition,  animals received the drug vehicles 90 and 30 mm  prior to  testing. *Differs from 0.0 mg/kg condition, p<.05  (McNemar test).  **Differs from 0.0 and 0.2 iug/kg conditions, p<.O5 test).  (McNemar  U) 0  U)  0  U)  0  >‘  C  C-) 0 U)  U) 0  0 > 0  L  0  20  40  60  80  100  0.0  /  /  /  /  1.0  El.  *  S.C.  5.0  *  *  /*  Ritanserin pretreatment, mg/kg  0.2  *  -o  **  o  **  0  z  I  (I) 01  El  *  *  0  **  El-  —  Mounts  [1  Ejaculations  L tntromissjons  0—0  w  44  Discussion  Ritanserin effectively competes with 5-HT agonists for 5-HT2 and 5-HT1C binding sites Meert, Niemegeers, Gelders, experiment,  (eg. Hoyer,  & Janssen,  1988a,  1989).  1992;  In the present  ritanserin potently blocked the inhibiting  effects of DOl on the copulatory behaviour of male rats. Because both DOl and ritanserin exhibit high affinity for 5-HT2 and 5-HT1C receptors, but differ in their affinities for other types of receptors, the present data suggest that the efficacy of ritanserin in blocking D01-induced inhibition specifically reflects the displacement by ritanserin of DOl from 5-HT2/1C receptors. The present data therefore support the tentative conclusion,  from Experiment  1, that activation of 5-HT2/1C receptors mediates an inhibition of sexual behaviour in the male rat. It is somewhat curious that ritanserin was without effect when given alone. The 5-HT2/1C antagonists LY 53857, pirenperone,  and ketanserin have all been reported to  significantly influence the copulatory behaviour of male rats, albeit in opposing directions Mendelson & Gorzalka,  (Foreman et al.,  1989;  1985). One possibility is that these  drugs exert their effects through their interactions with other types of receptors, rather than through their effects at 5-HT2/1C binding sites.  45  Experiment 3  Although the results of Experiments 1 and 2 support the contention that 5-HT2/1C receptor activity mediates an inhibition of male rat copulatory behaviour, the finding that ritanserin did not affect sexual behaviour when given alone is not congruent with the previous reports in which 5-HT2/1C antagonists have been found to influence male rat copulation (Foreman et al.,  1989; Mendelson & Gorzalka,  1985). Two additional considerations may reconcile this apparent discrepancy. One possible explanation is related to the unique binding profiles of the different drugs that have been employed. Ritanserin and DOl both display high affinity and selectivity for 5-HT2 and 5-HT1C receptors relative to other types of receptors. However, neither drug displays appreciable selectivity between 5-HT2 and 5-HT1C sites (Moyer, 1988a; Glennon & Dukat, 1991). Pirenperone, by contrast, is less selective overall than is ritanserin, but it does display somewhat more selectivity for 5-HT2 sites than for 5-HT1C sites. That is, although pirenperone binds to a wider variety of non5-HT2/1C receptors than does ritanserin (Janssen,  1983), it does display slightly more  affinity for 5-HT2 than 5-UT1C receptors (Moyer, 1988a,b; Glennon & Dukat, 1991). Although the reported effects of  46  pirenperone on male rat copulation (Mendelson .& Gorzalka, 1985) may reflect the occupation of non5-HT2/1C sites at higher concentrations, low doses of pirenperone might selectively occupy 5-HT2 receptors, and to a lesser extent 5-HT1C receptors. This proposition is amenable to experimental evaluation, by examining the efficacy of pirenperone in reversing the inhibitory effects of DOl. Given the results of Experiments 1 and 2, it is probable that the smallest dose of pirenperone found to be effective in blocking DOl-induced inhibition would also be a dose at which pirenperone preferentially occupied 5-HT2 sites. The effects of this dose of pirenperone, in the absence of DOl, could then be evaluated for effects on male rat sexual behaviour. A second consideration is that the conflicting results of the previously published reports may be at least partly due to genetic differences between their respective experimental samples. Specifically, whereas Mendelson and Gorzalka (1985) used rats of the Long—Evans strain, Foreman et al.  (1989) employed Sprague-Dawley rats. Although strain  differences in the serotonergic control of sexual behaviour have not been established, such differences do exist between related species. For example, muroid species are known to differ in their response to serotonergic drugs; 8-OH-DPAT, a 5-HT1A agonist, reportedly facilitates the sexual behaviour  47  of male rats Gorzalka,  (Ahlenius & Larsson,  1984; Mendelson &  1986) but inhibits the sexual behaviour of male  mice (Svensson, Larsson, Ahlenius, Arvidsson,  & Carisson,  1987). In Experiment 3, the effects of pirenperone pretreatment on DOl—induced copulatory inhibition were assessed. Parallel experiments were run in the two genetic strains of rats, Long—Evans and Sprague—Dawley, in order to evaluate the hypothesis that the conflicting data reported by others may be a consequence of strain differences.  Method  Drugs Pirenperone was obtained from Janssen Pharmaceutica, Beerse, Belgium, and stored at 5° C in dark conditions. Fresh solutions of pirenperone and DCI were prepared before each test session; DCI was prepared as in Experiment 1. Pirenperone was initially dissolved in a. minimal quantity of warm 0.0007 M citrate solution, and diluted to final concentration with sterile physiological saline. Pirenperone was administered intraperitoneally (IP)  60 mm  testing, and DCI was administered SC 30 mm testing.  prior to  prior to  48  Procedures A total of 13 male Sprague-Dawley rats and 14 male LongEvans rats were tested as in Experiment 2b. All males were sexually experienced. Prior to each test session, the males received a pretreatment dose of pirenperone:  0.0, 20.0,  100.0, or 200.0 pg/kg, plus 1.0 mg/kg DCI. A control condition was also included in which animals were tested after administration of the drug vehicles alone. The treatments were administered in a random counterbalanced fashion, such that by the end of the experiment, male rats had been evaluated (%M, %I, M,  I, ML,  all the IL, EL, PEI)  once under each treatment condition, At the conclusion of the experiment, a follow—up session was conducted in which the males were tested after receiving a dose of pirenperone that corresponded to the smallest dose that significantly reversed DOl-induced inhibition.  In this session,  pirenperone was administered without DCI, in order to assess the effects of pirenperone on sexual behaviour in otherwise— untreated males.  Results  The effects of the pirenperone pretreatment on the proportion of Sprague—Dawley males showing each of the three copulatory behaviours  (%M,  %I, %E) are presented in  49  Figure 3. males.  Figure 4 presents the data for the Long-Evans  In both strains, pirenperone potently blocked the  inhibition of sexual behaviour induced by DCI. In neither strain was the effect of pirenperone dose dependent. Maximal reversal of the inhibitory effect of DCI occurred with the lowest dose of pirenperone (50.0 ig/kg).  50  Figure 3. Effectiveness of pirenperone in reversing DOl induced inhibition of copulatory behaviour in male Sprague Dawley rats. *Differs from 0.0 rig/kg condition, p < 0.05 **Differs from all other doses, p < 0.05  (McNemar test)  (McNemar test)  ci  ci)  Cl)  -o  Ci)  0  ci  >1  o a)  0  -c a)  0 > 0  0.0  20.0  40.0  60.0  80.0  100.0  /  50  U.  *  100  •D  Pirenperone pretreatment, pg/kg, 60 mm  0.0  /  /  0—  *  prior  200  U  *  U)  :1  Q)  Mounts  Q  Ejaculations  L Intromissions  0—0  U  (J1  52  Figure 4. Effectiveness of pirenperone in reversing DOl— induced inhibition of copulatory behaviour in male Long Evans rats. *Differs from 0.0 pg/kg condition, p  <  0.05  (McNemar test)  a) ci  (I)  -o  (I)  ci  >‘ 0  a)  C.) 0  0  -c a)  0 > 0  L.  0.0-  20.0  40.0-  60.0-  80.0-  100.0-  0.0  100  *  *  Pirenperone pretreatment, /Lg/kg  50  *  ‘)ñfl  *  *  z  0  1...  0)  U)  *  a-  L.  0  a)  C 0  —  Mounts  0  Ejaculations  L Intromissions  0—0  *  0  *  U’  54  The means and standard errors for the various parameters of male rat sexual behaviour are given in Table 2 for Sprague-Dawley males, and in Table 3 for Long—Evans males. For the Sprague—Dawley males, overall Friedman analyses revealed that pirenperone pretreatment had a significant effect for M 0.0148), ML p  =  ) 3 ( 2 (x ) 3 ( 2 (x  =  ) 3 ( 2 (x  15.3, p  =  0.0016), I  15.5, p  =  0.0014), and IL  =  10.5, p  ) 3 ( 2 (x  =  =  8.6,  0.0354). subsequent Wilcoxon pairwise comparisons  revealed that the 50.0,  100.0, and 200.0 pig/kg pirenperone  conditions significantly differed from the 0.0 pig/kg (i.e., DOl plus pirenperone vehicle) condition (Z ranging from 2.2 to 2.9, p  <  .02) for all four behavioural measures.  Pirenperone’s overall effect on EL and PEI was not statistically significant  ) 3 ( 2 (x  =  1.03, NS, and  ) 3 ( 2 x  =  0.6,  NS, respectively). In the Long-Evans rats, pirenperone had a pronounced effect on all measures of sexual behaviour in the DCI— treated male rats (Table 3). This was reflected in significant overall Friedman analyses for M p  <  p  <  p  =  ) 3 ( 2 (x  =  19.5,  (x = 19.8, p < 0.0002), ML (x ) 3 ( 2 ) = 20.25, 3 ( 2 0.0002), IL (x ) = 20.0, P < 0.0002), EL (x 3 ( 2 ) = 16.4, 3 ( 2 0.001), and PEI (x ) = 17.8, p = 0.0005). Subsequent 3 ( 2 0.0002), I  pairwise Wilcoxon tests revealed that the males displayed significantly facilitated sexual behaviour on all 6 of these measures, relative to the 0.0 fig/kg pirenperone condition,  55  when pretreated with 50.0,  100.0, or 200.0 FIg/kg pirenperone  (Z ranging from 2.5 to 3.2, P  <  0.01). Furthermore, a  •significant decrement in performance was apparent between the 50.0 and 200.0 i.g/kg pirenperone pretreatment conditions, for ML, IL, EL, and PEI (Z ranging from 2.0 to 2.4, p  <  0.05). Similarly, The 50.0 and 100.0 tg/kg doses  significantly differed on EL (Z  =  2.6, p  =  0.011). These  differences thereby statistically confirmed the trend, apparent in Figures 3 and 4, that pirenperone pretreatment was maximally efficacious in reversing the inhibiting effects of DCI at the low 50.0 p.g/kg dose. Behavioural measures of copulatory proficiency were significantly diminished with higher concentrations of pirenperone (100.0 and 200.0 pig/kg). In order to assess the effects of pirenperone on male rat sexual behaviour, when given in the absence of DCI, a follow—up session was conducted in which the male rats were tested following injections of 50.0 g/kg pirenperone 60 mm prior to testing, and physiological saline (DCI vehicle) 30 mm  prior. This dose of pirenperone did not significantly  affect the male rats’  sexual behaviour when given alone  (Figures 1 and 2), despite being effective in reversing DCI induced inhibition. The two genetic strains of rats displayed comparable responses to the drug treatments, although they differed  56  somewhat in scope and variability. Mann-Whitney U tests of the baseline data revealed Long—Evans males to be much more vigorous copulators than the Sprague—Dawley males across the various behavioural measures (p values ranging from 0.0427 to 0.0013). Nevertheless, it does not appear that strain differences account for the contradictory findings of Mendelson & Gorzalka (1985) and Foreman et al.  (1989), since  the pattern of results in the present study was in the same direction for both strains.  57  Table 2. The effectiveness of pirenperone in reversing DCI induced inhibition of sexual behaviour in male rats, of the Sprague-Dawley strain.  In all but the  1.0 mg/kg DCI was administered 30 mm Data are presented as mean scores  ‘No-Drugs’ condition, prior to testing.  (S.E.M. in parentheses);  the number of animals displaying each number are given in square brackets.  58  Effects of Pirenperone Pretreatment on DOl—Induced Inhibition of Male Rat Sexual Behaviour: Spracrue-Dawlev Strain. Pirenperone pretreatment, tg/kg Behavioural Measure  0.0  50.0  Mounts  0.0 (0.0) [0]  10.6 (2.3) [10]  7.3 (2.4) [10]  9.4 (2.7) [11]  20.4 (3.3) [13]  Intromissions  0.0 (0.0) [0]  3.9 (1.3) [9]  3.2 (1.4) [7]  3.6 (1.2) [6]  10.4 (0.8) [13]  Mount Latency  1800.0 (0.0) [0]  490.9 (207.4) [10]  783.3 (199.5) [10]  534.5 (170.7) [11]  62.9 (16.2) [13]  Intromission Latency  1800.0 (0.0) [0]  922.8 (209.7) [9]  1198.3 (205.2) [7]  1194.5 (208.0) [6]  179.7 (54.8) [13]  Ejaculation Latency  1800.0 (0.0) [0]  1668.0 (98.0) [2]  1551.4 (138.3) [3]  1583.4 (122.7) [3]  927.6 (144.0) [11]  Postejaculatory 1800.0 Interval (0.0) [0]  1700.3 (99.7) [1]  1578.7 (149.8) [2]  1574.9 (152.5) [2]  894.9 (206.7) [8]  100.0  200.0  No Drugs  59  Table 3. The effectiveness of pirenperone in reversing DOl induced inhibition of sexual behaviour in male rats, of the Long-Evans strain.  In all but the  mg/kg DCI was administered 30 mm presented as mean scores  ‘No-Drugs’ condition,  prior to testing. Data are  (S.E.M. in parentheses); the number  of animals displaying each number are given in square brackets.  1.0  60  Effects of Pirenperone Pretreatment on DOl—Induced Inhibition of Male Rat Sexual Behaviour: Long-Evans Strain. Pirenperone pretreatment, ig/kg Behavioural Measure  0.0  50.0  100.0  200.0  No Drugs  Mounts  0.1 (0.1) [1]  5.1 (1.1) [13)  8.9 (2.3) [12]  4.1 (1.2) [10]  9.7 (3.2) [13]  Intromissions  0.2 (0.2) [1]  8.1 (0.8) [13]  6.5 (1.2) [12]  5.0 (1.1) [9]  8.1 (1.2) [13]  Mount Latency  1680.7 (120.3) [1]  260.6 (147.4) [13]  475.4 (177.6) [12]  560.4 (217.9) [10]  149.9 (127.0) [13]  Introinission Latency  1720.8 (81.2) [1]  264.6 (147.0) [13]  517.4 (171.6) [12]  709.6 (226.2) [9]  164.1 (126.1) [13]  Ejaculation Latency  1677.5 (123.5) [1]  363.6 (113.4) [13]  634.6 (171.4) [11]  820.1 (203.5) [9]  378.7 (122.1) [13]  Postejaculatory 1800.0 Interval (0.0) [0]  404.2 (108.2) [13]  753.4 (184.6) [10]  860.3 (194.8) [9]  391.9 (110.1) [13]  61  Discussion Like ritanserin, pirenperone was found to effectively reverse the inhibition of male rat sexual behaviour induced by systemic administration of DCI. Because the effects of DCI are believed to be exerted via 5-HT2/1C receptor activation, and pirenperone’s main pharmacological effect is antagonism of these sites (Dabiré et al., 1989; Glennon & Dukat, 1991; Glennon et al.,  1986) this finding supports the  contention that 5-HT2/1C receptor activation inhibits male rat copulation. At a concentration of 50.0 tg/kg, pirenperone reversed the effects of DOl on male rat copulation. Because pirenperone and DCI share high affinity for 5-HT2/1C receptors, but differ in their affinity for other types of receptors (Dabiré et al., Tricklebank,  1989; Janssen,  1983; Middlemiss &  1992; McKenna & Peroutka, 1989), it may be  assumed that pirenperone’s behavioural effects were exerted through effective competion with DOl for binding at the receptor sites through which the inhibiting effects of DCI are realized (for review, e.g., Kalant et al.,  1985). Thus,  it may be assumed that when given in the absence of DCI, a 50.0 ig/kg dose of pirenperone saturates this same population of receptors. When given alone, 50.0 rig/kg of pirenperone was found to be without effect on the sexual behaviour of male rats in the present experiment. This is  62  congruent with the results of Experiment 2, in which doses of ritanserin also were without effect when given to otherwise untreated male rats. This pattern of results suggests that the previously reported facilitatory effects of pirenperone in otherwise untreated male rats (Mendelson & Gorzalka, 1985) may be attributable to binding of pirenperone to other non—5-HT2/1C receptor types when given in higher doses. Lastly, evidence exists that pirenperone exhibits a moderate degree of selectivity for 5-HT2 versus 5-HT1C receptors (Janssen, 1983; Hoyer, 1992), unlike ritanserin (foyer, 1992). This selectivity of pirenperone for 5-HT2 receptors, coupled with its efficacy in reversing the effects of DOl, provides preliminary evidence that the inhibitory effects of DOl on male rat sexual behaviour may be attributable to 5-HT2, rather than 5-HT1C, activation.  63  Experiment 4  In Experiments 2 and 3, the 5-HT2/1C antagonists ritanserin and pirenperone were found to effectively block DOl-induced inhibition of male rat sexual behaviour. The efficacy of pirenperone was evident at a dose that did not in itself affect male rat copulatory behaviour when given in the absence of DOl. Because this finding conflicts with the published data (Mendelson & Gorzalka, 1985), further investigation with an additional 5-HT2/1C antagonist was warranted. Comparing the data collected with a variety of antagonists, given their differing binding profiles, might clarify the unique contributions of 5-HT2/1C receptors to the control of male rat copulation, and may also permit some degree of discrimination between the effects of 5-HT2 and 5-HT1C activity. Ketanserin is considered to be a prototypic 5-11T2 receptor antagonist (Glennon & Dukat, 1991). Like pirenperone, it possesses moderately higher selectivity for 5-HT2 receptors, compared to 5-HT1C receptors, than does ritanserin (Hoyer, 1988a,  1988b,  1992; Middlemiss &  Tricklebank, 1992). Pirenperone and ketanserin differ in their affinity for other types of monoaminergic receptors, however. In particular, ketanserin displays less affinity for dopamine receptors than does pirenperone (Janssen,  64  1983). The efficacy of ketanserin in reversing the inhibition of male rat sexual behaviour induced by DOl was examined in Experiment 4. As in Experiment 3, the possibility of different responses between the Sprague Dawley and Long-Evans genetic strains was also evaluated,  Method  Drugs Ketanserin tartrate was obtained from Janssen Pharmaceutica, Beerse, Belgium, and stored at 5°C in dark conditions. Fresh solutions of ketanserin and DOl were prepared before each session; DOl was prepared as in the preceding experiments. Ketanserin was dissolved in warm physiological saline and injected IP in a total volume of 1.0 mi/kg, mm  60 mm  prior to testing. DOl was administered 30  prior to testing.  Procedures Experiment 4 was conducted in a fashion similar to Experiment 3. As before,  13 Sprague-Dawley and 14 Long-Evans  males were used; all were sexually experienced and of comparable age to the animals tested in Experiment 3. The copulatory behaviour of the male rats  (%M,  %I,  %E, M,  I, ML,  65  IL, EL, PEI) was evaluated after administration of 1.0 mg/kg DOl plus a test dose of ketanserin: 0.0, 0.2, 1.0, or 5.0 mg/kg  --  or after the administration of the drug vehicles  only. The treatment conditions were randomly counterbalanced over the course of the experiment, such that each animal was tested once under each treatment condition. Consecutive testing sessions were separated by a period of 1 week.  Results  Ketanserin potently blocked DOl-induced inhibition of male rat sexual behaviour, as was found with pirenperone in Experiment 3. In contrast to the data collected with pirenperone, however, the facilitatory effects of ketanserin were dose—dependent. The effects of ketanserin on the proportions of male rats displaying mounts, intromissions, and ejaculations are presented in Figures 5 and 6. Data on the effects of ketanserin pretreatment on DOl— induced inhibition of the other behavioural parameters are presented in Table 4 for Sprague-Dawley males, and in Table 5 for Long—Evans males. Baseline (‘No—Drugs’) data are included for comparison. In the Sprague—Dawley group, overall Friedman analyses revealed a significant treatment effect of ketanserin for M  ) 3 ( 2 (x  =  20.1, p  <  0.0002), I  ) 3 ( 2 (x  =  10.1, p  =  0.0181),  66  Figure 5. Effectiveness of ketanserin in reversing DOl— induced inhibition of copulatory behaviour in male Sprague Dawley rats. *Differs from 0.0 pjg/kg condition, p < 0.05  (McNemar test)  o  -  D  I  -  •5  20.0  40.0  60. 0  80.0  100.0  / ..  .  *  *  Ketanserin pretreatment, mg/kg  /  /  /  /,/* II  /  /-  0  *  //  /  /  *  *  *  *  z  0  *  g  Mounts  EjaculaUons  IS Intromissions  Q• •Q  —  0—0  *  E]  68  Figure 6. Effectiveness of ketanserin in reversing DOl— induced inhibition of copulatory behaviour in male Long Evans rats. *Differs from 0.0 tg/kg condition, p  <  0.05  (McNemar test)  -  U)  U)  *  C.)  fa)  o  0.0•  20.0  40.0  60.0  80.0  100.0  0.0  1.0  *  —  Ketanserin pretreatment, mg/kg  0.2  /  /  /  /“ 0  I.  *  L)  5.0  *-  *.  —  O  U)  *  U)  o  0w  C  Li  —  Mounts  Q  Ejaculations  L Intromissions  0—0  *  0•  70  ML  ) 3 ( 2 (x  =14.8, p  =  0.002), and IL  ) 3 ( 2 (x  =  11.2, p  =  0.0108). Subsequent Wilcoxon tests revealed that for these behavioural measures, scores for the 0.2,  1.0 and 5.0 mg/kg  ketanserin pretreatments all significantly differed from the 0.0 mg/kg (i.e. vehicle plus DCI) condition (Z ranging from 2.4 to 3.1, p  <  0.02).  In the Long—Evans males, overall Friedman tests revealed that ketanserin pretreatment significantly reversed DOl induced inhibition of male rat copulatory behaviour on all 6  (x ) 3 ( 2 ) = 3 ( 2 (x ).= 3 ( 2 (x  behavioural measures: M 19.1, p  =  0.0003), ML  24.7, p  <  0.0002), EL  ) 3 ( 2 (x  29.4, p  =  <  =  13.6, p  =  .0035), I  (2(3)  =  ) 3 ( 2 (x  =  25.8, p  <  0.0002), IL  12.8, p  =  0.0052), and PEI  0.0002). Subsequent Wilcoxon tests  indicated that, for each of these measures, the scores obtained for the 0.2,  1.0, and 5.0 mg/kg ketanserin  pretreatment groups significantly differed from the scores obtained with 0.0 mg/kg (i.e. vehicle plus DCI) ketanserin pretreatment (Z ranging from 2.4 to 3.3, p  <  0.02).  Moreover, dose-dependency was clearly evident in the pattern of results. Scores for ML and PEI were significantly improved (i.e. decreased) when the males received 1.0 mg/kg ketanserin compared to when they received 0.2 mg/kg (Z 2.43 and 1.96, p  =  =  0.015 and 0.049, respectively). A  significant facilitation of PEI was also evident for the 5.0 mg/kg ketanserin pretreatment condition, relative to the 1.0  71  mg/kg condition (Z  =  1.98, p  =  0.048). Lastly, ML, IL and EL  were all improved in the 5.0 mg/kg ketanserin pretreatment condition, when compared with the scores for 0.2 mg/kg of ketanserin  ( Z  =  2.98, 3.11, and 3.3; p  =  0.0029, 0.0019,  and 0.001, respectively). Taken together, these results indicate that the reversal by ketanserin of DOl—induced inhibition of sexual behaviour in the male rats was significantly greater with successively higher doses of ketanserin, As in Experiment 3, a follow-up session was conducted in which a minimal effective dose of ketanserin was administered to the male rats, in the absence of DOl, and their sexual behaviour was quantified. The low dose (0.2 mg/kg) of ketanserin was selected for this follow-up, because it had a significant effect on DOl-induced inhibition of male rat sexual behaviour in the present experiment. As is evident in Figures 5 and 6, treatment with this dose of ketanserin had no effect on the expression of sexual behaviour in otherwise untreated male rats. As was found in Experiment 3, Long—Evans males were more vigorous copulators than the Sprague—Dawley males (p values ranging from 0.022 to  <  0.0001), but the males of the two strains  responded in comparable fashion to the drug treatments.  72  Table 4. Effectiveness of ketanserin in reversing DOl— induced inhibition of sexual behaviour in male rats of the Sprague-Dawley strain.  In all but the No-Drugs condition,  1.0 mg/kg DCI was administered 30 mm are presented as mean scores  prior to testing. Data  (S.E.M. in parentheses); the  number of rats displaying each behaviour is given in square brackets.  73  Effects of Ketanserin Pretreatment on DOl-Induced Inhibition of Male Rat Sexual Behaviour: Sprague—Dawley Strain. Ketanserin pretreatment, mg/kg Behavioural Measure  0.0  0.2  1.0  5.0  No Drugs  Mounts  0.5 (0.5) [1]  9.9 (3.2) [8]  15.5 (4.4) [12]  10.8 (2.3) [13]  22.3 (4.5) [13]  Intromissions  0.0 (0.0) [0]  3.7 (1.2) [7]  4.9 (1.3) [9]  3.9 (1.0) [8]  6.6 (1.3) [11]  Mount Latency  1662.9 (137.1) [1)  829.3 (230.9) [8]  430.1 (174.8) [12]  547.2 (158.6) [13]  214.9 (113.2) [13]  Intromission Latency  1800.0 (0.0) [0]  916.5 (240.2) [7]  821.8 (228.1) [9]  902.9 (222.4) [8]  502.4 (178.6) [11]  Ejaculation Latency  1800.0 (0.0) [0]  1592.7 (141.1) [2]  1272.2 (195.9) [5]  1337.0 (163.1) [6]  1245.0 (168.7) [7]  Postejaculatory 1800.0 Interval (0.0) [0]  1578.5 (150.0) [2]  1471.4 (173.4) [3]  1454.3 (182.3) [3]  1245.2 (202.8) [5]  74  Table 5. Effectiveness of ketanserin in reversing DOl— induced inhibition of sexual behaviour in male rats of the Long-Evans strain.  In all but the No-Drugs condition,  mg/kg DOl was administered 30 mm presented as mean scores  1.0  prior to testing. Data are  (S.E.M. in parentheses); the number  of animals displaying each behaviour is given in square brackets.  75  Effects of Ketanserin Pretreatment on DOl-Induced Inhibition of Male Rat Sexual Behaviour: Long-Evans Strain. Ketanserin pretreatment, mg/kg Behavioural Measure  0.0  0.2  1.0  5.0  Mounts  0.8 (0.6) [2]  2.1 (0.6) [9]  6.2 (2.3) [12]  5.4 (1.6) [14]  6.1 (1.8) [14]  Intromissions  0.4 (0.4) [1]  4.9 (1.2) [9]  5.6 (1.0) [11]  7.5 (0.5) [14]  9.6 (1.0) [14]  Mount Latency  1594.0 (144.4) [2]  728.0 (225.9) [9]  311.4 (171.3) [12]  66.1 (37.6) [14]  41.1 (21.3) [14]  Intromission Latency  1676.4 (123.6) [1]  731.9 (224.9) [9]  471.9 (196.9) [11]  74.5 (37.4) [14]  50.9 (21.5) [14]  Ejaculation Latency  1679.2 (120.8) [1]  746.2 (218.4) [9]  564.0 (184.0) [11]  194.1 (21.6) [14]  229.8 (25.9) [14]  Postejaculatory 1706.9 Interval (93.1) [1]  904.6 (186.8) [9]  648.4 291.6 (168.2) (14.0) [11] [14]  No Drugs  308.2 (19.6) [14]  76  Discussion  In a similar manner to ritanserin and pirenperone, ketanserin effectively reversed the inhibitory influence of DOl on male rat sexual behaviour. That it did so at a concentration that did not in itself affect the males’ sexual behaviour suggests that previously reported effects of 5-HT2/1C antagonists, when given alone (Abraham et al., 1988; Mendelson & Gorzalka, 1985), may be attributable to nonselective activity at other types of receptors. Moreover, the data collected in Experiment 4 reinforce the notion that 5-HT2/1C receptors mediate an inhibition of male rat sexual behaviour. To the extent that ketanserin particularly shares affinity for 5-HT2 receptors with ritanserin and pirenperone, while differing from them in affinity for other receptor types, including 5-HT1C (Hoyer, 1992; Janssen, 1983), the present data allow a further limited inference: Specifically, the alterations in male rat sexual activity seen in Experiments 1 through 4 may be primarily attributable to modulation of a mechanism dependent on 5-HT2 receptor activity, rather than 5-HT1C receptor activity. The results of Experiments 1—4, taken together, are considered in greater detail in the General Discussion.  77  GENERAL METHOD: EXPERIMENTS 5-7  Animals Male and female Sprague-Dawley rats were bred from stock originally obtained from Charles River Canada, Inc., Montreal. Rats were housed in our colonies in groups of 6, segregated by sex and strain, in standard wire mesh laboratory cages. Free access to food and water was provided, and a reversed 12/12 hr light/dark cycle was maintained. At approximately 70 days of age, the female rats underwent bilateral ovariectomy while under sodium pentobarbital anaesthesia (65 mg/kg IP). Males were approximately 100 days old at the beginning of behavioural testing.  Steroid Treatments As in the preceding experiments, receptivity was induced in the ovariectomized female rats by subcutaneous injection of 10.0 pg estradiol benzoate (Steraloids) and 500.0 progesterone (Steraloids), 48 hr and 4 hr prior to testing, respectively. Steroids were dissolved in 0.1 ml peanut oil vehicle. The nonreceptive females employed in Experiment 6 were ovariectomized females that had not received steroid replacement for a minimum of 2 weeks prior to testing.  78  Behavioural Testing In order to evaluate the possibility that WDS and copulatory behaviour are systematically related in male rats, the coincidence of these behaviours was measured by an observer who was blind to the experimental hypotheses and unaware of individual treatment conditions. As before, all testing occurred during the middle 1/3 of the dark phase of the rats  light-dark cycle.  The experimental males were placed individually into clear Plexiglas testing arenas measuring 30 X 30 X 45 cm, and allowed 5 mm  to habituate to their new surroundings.  The floors of the testing arenas were covered with fresh San—i—Cel bedding material before each testing session, and the arenas were washed weekly. During each testing session, between 2 and 4 males were scored simultaneously in separate testing chambers. Following the 5-mm  habituation period, a stimulus rat  was introduced into each male’s testing chamber and behavioural recording commenced. During each 60-mm  testing  session, the occurrence of WDS was scored for each male, by means of a verbal recording on a voice—activated microcassette recorder (Sony model M-660V). In addition, the  79  incidence of mounts, intromissions, and ejaculations was recorded in Experiment 5 and 6, and the incidence of ejaculations was recorded in Experiment 7. These recordings were transcribed immediately following each testing session.  80  Experiment 5  The evidence discussed in the General Introduction suggests that the generation of WDS in rats is critically dependent on a 5-HT2-mediated mechanism. It is possible, therefore, that spontaneously occurring WDS could be used as a type of behavioural assay of concurrent 5—HT2 activation during the performance of other types of behaviour. Informal observation during the course of other experiments has suggested that males generate WDS during sexual behaviour. Consequently, it is reasonable to suppose that combining measures of WDS with measures of male rat copulatory behaviour might yield a behavioural index of 5—HT2 activity during copulation that converges on the pharmacological data collected in Experiments 1-4. In any group of male rats, a certain proportion reliably fails to initiate normal copulation when confronted with receptive females, even after numerous trials (Whalen, Beach, & Kuehn,  1961; Crowley, Popolow, & Ward,  1973). This  presented the opportunity to perform a natural experiment in drug-free animals, in which the sexual and WDS responses of noncopulators  (‘Duds’: Crowley et al.,  1973) were compared  with those of normal copulators (‘Studs’: Crowley et al., 1973). The extent to which WDS and male rat copulatory behaviours rely on a shared 5-HT2 mediated mechanism is  81  unknown. However, to the extent that such a mechanism underlies the WDS and copulatory behaviours, WDS frequency may be systematically related to copulatory proficiency and reflect concurrent 5-HT2 activation; specifically, based on the results of Experiments 1-4, it was hypothesized that increased WDS, reflecting augmented 5-HT2 activity, would be observed in males that failed to copulate.  Method  Procedures A total of 58 sexually inexperienced male rats were evaluated in Experiment 5. Previous observations suggested that this number would yield approximately 10 noncopulators. Following habituation to the observation chambers, receptive female rats were introduced into the chambers and behavioural recording commenced. During each 60-mm  session,  4 males were evaluated simultaneously for the incidence of WDS, mounts, intromissions, and ejaculations. The receptive female rats were rotated among the males every 15 mm.  Results  Upon completion of the experiment, the experimental males were divided into groups on the basis of their  82  measured sexual performance in the testing sessions. Inspection of the copulation data suggested that, in fact, three groups of males could be discriminated on the basis of their sexual proficiency. The levels of WDS recorded in these groups are presented in Figure 7. The incidence of spontaneously occurring WDS differed markedly between the Duds (males that did not display any sexual behaviour; n=8) and the Studs (males that ejaculated at least once; n=43), with Duds expressing approximately four times more WDS than the Studs. The third group, Slugs (males that mounted or intromitted at least once, but failed to achieve ejaculation; n=7) displayed an intermediate level of WDS. Slugs displayed very little copulatory behaviour, compared to Studs, and thus appeared to be a subset of the Duds category. Overall tests of the WDS scores across the three groups revealed significant differences, both by the nonparametric Kruskal—Wallis test  ) ( 2 (x  20.1, p  <  O.OOO1), and  parametric analysis of variance (F( ) 255 p  <  =  14.46,  0.00005). Subsequent comparisons of pairs of groups,  using the the Mann-whitney U test, revealed that Studs produced significantly fewer WDS than either Duds (U P  <  0.0004) or Slugs (U  =  44.5, p  <  =  33.5,  0.0004), and when tested  using the Newman—Keuls procedure, all paired comparisons were significant (p  <  0.05). Alternatively, the relation  83  between WDS and sexual behaviour was also evident in a simple correlation of E with WDS, collapsed across the groups (r (56)  =  —0.40., p  <  0.001).  84  Figure 7.  Incidence of WDS in male rats of varying  copulatory proficiency, during 60 mm receptive female rats  (Mean  ±  S.E.M.).  interactions with  LA  co  SPfl4S  s6ni  spn  0  z  .  9  9  0I.  m  U  (1)  86  Discussion  Total WDS scores for the three groups of males were all significantly different from one another. Both sexual behaviour and the incidence of WDS appeared to discriminate among the Duds,  Slugs,  and Studs groups. The results of  Experiment 5 thus suggest that normal copulatory behaviour is incompatible with the expression of WDS in the male rat. Given the established dependency of WDS on 5-HT2 receptor activity (e.g., Green & Heal,  1985), this pattern of results  provides preliminary, entirely behavioural, evidence that WDS and male rat sexual behaviour are directly related in some fashion, and therefore supports the proposition that a 5-HT2 receptor mediated mechanism exerts an inhibitory influence on sexual behaviour in male rats.  87  Experiment 6  The results of Experiment 5 suggested that the expression of WDS is incompatible with normal copulatory behaviour in male rats. However, the nature of this relation between the two types of behaviour remains unknown. In order to infer, with confidence, that the increased incidence of WDS in Duds during mating tests is attributable to activity of a 5-HT2-dependent mechanism that also contributes, at least in part, to the control of copulatory behaviour, it is necessary to examine the specificity of the effect. It is possible that the effect observed in Experiment 5 may be an artifact of some other mediating variable. For instance, it has been suggested that noncopulating males may exhibit a generalized syndrome characterized by diminished arousability (Pottier & Baran, 1973). In the context of the present experiments, a systematic difference between Duds and Studs in autonomic activity or general arousal levels might predispose certain males to fail to copulate and also to produce excessive WDS, without the two behaviours being directly related to one another. If this were true, one would expect the difference in WDS observed in Experiment 5 to obtain regardless of the type of stimulus rat presented. In Experiment 6, the expression of WDS was measured while the males were paired  88  with other types of stimulus rats, and the degree to which group differences in WDS emerge specifically during mating tests was assessed.  Method  Procedure The 58 male Sprague-Dawley rats used in Experiment 5 also served as subjects in Experiment 6. As in Experiment 5, an observer, blind to experimental condition, made verbal microcassette recordings of WDS and sexual behaviours exhibited by the male rats during 60 mm  test sessions. The  recorded data were transcribed at the end of each testing session. As in Experiment 5, the experimental males were evaluated following presentation of receptive females. Data were also collected while the experimental males were paired with another male, or with a nonreceptive female. Experimental males were paired with each of the three types of stimulus animals in a counterbalanced fashion, such that, over three consecutive test sessions, each experimental male had been tested once with each type of partner in the observation chaniber. Stimulus rats were rotated among the experimental males every 15 mm  during behavioural  89  observation. The experimental males were allowed at least 1 week between consecutive test sessions.  Results  The results of Experiment 6 are presented in Figure 8. The data collected in Experiment 5 suggested that Slugs are a subset of the Duds category, as evidenced by the marginally significant differences between Duds and Slugs on WDS incidence. Given their minimal and very deficient copulatory behavIour, Slugs were combined with Duds for the purposes of the present experiment, with Duds defined as those males (n=15) which failed to achieve ejaculation during 60 mm  mating tests with a receptive female (eg.  Zainble, Hadad, Mitchell, & Cutmore,  1985).  The mean incidence of WDS was significantly greater for Duds than for Studs, when the males were paired with receptive females (Mann-whitney U test: U  =  78.0, P  <  0.0001). In contrast, levels of WDS were comparable between Duds and Studs when paired either with nonreceptive females (U  =  266.0, NS) or with males (U  236.0, NS).  Interestingly, within-group Friedman tests revealed that while the mean WDS scores for the Duds group did not significantly differ across the three treatment conditions  ) ( 2 (x  =  0.433, NS), the types of partners which Studs were  90  paired with significantly affected their WDS scores 43.41,  p  <  ) ( 2 (x  =  0.0001). Subsequent pairwise comparisons using  the Wilcoxon test revealed that the Studs’ mean WDS scores significantly differed for each of the treatment conditions (p  <  0.0005 for all comparisons). Thus, not only did Studs  produce significantly more WDS when paired with a nonreceptive female than they did when paired with a receptive female, but they also displayed significantly more WDS when paired with a male than when paired with a nonreceptive female. The overall pattern of WDS production in Studs may thereby be characterized as minimal in the presence of receptive females, moderate in the presence of nonreceptive females, and maximal in the presence of males. Although not directly quantified, subjective observation suggested that the obtained results were not reducible to overall activity levels. Duds explored their cagemates quite actively throughout the test sessions, as did Studs. Similarly, although it might be argued that WDS appears to be related to grooming behaviour, group differences in the frequency of grooming do not appear to explain the present results, either. In fact, copulating males engage in considerably more grooming behaviour than do noncopulating males.  91  Figure 8. Comparison of spontaneous WOS production in male rats paired with differing types of partners in the testing chambers. Results are displayed as mean WDS scores  ±  S.E.M.  ci  0•  2  8  10  Female  Receptive  —  + Nonreceptive Female  +  Male  r iDuds  “Wet Dog Shake&’ under various conditions  eD 1%)  93  Discussion  The results of Experiment 6 support and extend the suggestion, from Experiment 5, that male rat copulation and the expression of WDS are almost mutually exclusive. Moreover, the present data indicate that the inverse relationship between copulation and WDS is highly specific to the copulatory milieu; Duds and Studs generate comparable levels of WDS when paired with either nonreceptive females or with males. This is the first demonstration of a behavioural correlate, that manifests specifically during mating tests,  of the copulatory failure phenomenon in male  rats. It is also important to note that Studs displayed significantly fewer WDS in the presence of nonreceptive females than in the presence of males. The Stud males were not engaging in copulation in either of these conditions. This suggests that the marked suppression of WDS production by Studs, measured when they were paired with receptive females, is not readily explicable on the basis of differential activity levels, or as an epiphenomenon of the copulatory behaviour itself. The present data therefore strengthen the argument that the in copula expression of spontaneous WDS, which is believed to be 5-HT2-mediated (Green & Heal,  1985; Zifa & Fillion, 1992), reflects levels  94  of activation of a 5-HT2--dependent neural mechanism that modulates both the WDS and copulatory behaviours.  95  Experiment 7  In light of the evidence implicating 5-HT2 receptor activation in the expression of WDS (e.g., Green, 1989; Green & Heal,  1985) the results of Experiments 5 and 6  support the idea that spontaneous WDS may reflect concurrent serotonergic activity, at least in mating tests. These data are also consonant with the pharmacological data collected in Experiments 1-4, which suggest that 5-HT2 receptor activity mediates an inhibition of copulatory behaviour in the male rat. Although Experiments 5 and 6 demonstrated a relation of WDS to copulatory proficiency that was specific to mating situations, and it was inferred that this stemmed from activity in a 5-HT2-mediated mechanism, it has yet to be directly established that the relation between these two behaviours is a reflection of 5-HT2 receptor activity. This issue could be addressed by assessing WDS and copulatory behaviours while using a pharmacological probe to modulate 5-HT2 receptor activity. Therefore, in Experiment 7, WDS and copulatory behavjours were evaluated in male rats that received varying doses of DCI.  96  Method  Drugs DOl was prepared in physiological saline, as in Experiments 1-4. All injections were given Sc, 30 mm  prior  to behavioural testing.  Procedures Sixty sexually experienced male rats served as experimental subjects. The males were randomly assigned to one of three treatment conditions; they received either 0.1 mg/kg DCI,  1.0 mg/kg DOl, or the saline vehicle in an  equivalent volume (approximately 0.4 ml). Each male rat was then paired with a receptive female rat, and the incidence of WDS and ejaculatory behaviour was scored for a period of 60 mm,  Results  The effects of DCI treatment on the sexual behaviour and simultaneous WDS of the male rats is depicted in Figure 9. Overall analyses of these measures were performed using the Kruskal-Wallis test for k independent samples. DOl had a significant effect on both the number of ejaculations achieved by the experimental males (x ) ( 2  =  21.02,  97  Figure 9. Effects of DOl treatment on concurrent WDS and ejaculations in male rats paired with receptive female rats for 60 mm. Data are presented as mean  ±  S.E.M.  o I  Mean WDS 0) I  /  hr  00  0  I  0  o  I  I  1  2 3  I \1  Co  I-.--’  I  I  CD C.,  .11  I  0  m C  C)  0  CM  Total Ejaculations  86  99  p  <  0.0001), and on the amount of WDS they produced  32.24, p  <  0.0001). Subsequent paired comparisons  ) ( 2 (x  =  (Mann  Whitney U test) revealed that WDS scores in each condition were significantly different from the scores in the other conditions  (p  <  0.0005): Minimal WDS was produced in the 0.0  mg/kg DCI condition, moderate levels were evident with 0.1 mg/kg DCI, and high levels of WDS occurred with the 1.0 mg/kg dose of DCI. Concurrently, ejaculatory behaviour was significantly suppressed in male rats treated with 1.0 mg/kg DCI relative to the other two treatments  (p  <  0.0001).  Discussion  The inverse relation of WDS and copulatory proficiency in male rats that was demonstrated in Experiments 5 and 6 was also evident in Experiment 7, this time induced using a pharmacological probe. The fact that DCI is a 5-HT2/1C agonist (Glennon et al.,  1986) extends the previous findings  by providing direct evidence that the relation between WDS and copulatory behaviour in the male rat is 5-HT2/1Cdependent.  It is noteworthy that the behaviour of the DOl  treated animals in the present experiment strongly resembled that of the untreated male rats in Experiments 5 and 6; in essence, the present data indicate that 5-HT2/1C activation causes Studs to become Duds.  100  Experiment 8  In Experiments 1-4, pharmacological manipulations of 5HT2/1C activity led to the conclusion that 5-HT2 receptor activation mediates an inhibition of male rat copulation. In Experiments 5 and 6, convergent behavioural evidence demonstrated that the incidence of spontaneous WDS discriminates between males of differing copulatory proficiency, and that the behavioural assay provided by concurrent WDS counts is quite specific to sexual behaviour testing, rather than being a component of a more generalized difference between subpopulations of male rats. The pharmacological data collected in Experiment 7 strongly suggest that differential WDS expression during mating tests does indeed reflect 5—HT2 receptor activation. It remains to be established, however, that the effects of 5-HT2 activity on WDS and copulatory behaviours are mediated by a unitary population of 5-HT2 receptors. No direct evidence has been collected demonstrating that the two behaviours rely on a shared neural substrate. It is possible, for example, that the relation between WDS and male rat sexual behaviour is somewhat coincidental; activation of one population of 5-HT2 receptors may inhibit sexual behaviour, while simultaneous activation of a different population of 5-HT2 receptors may induce WDS. If  101  this were so, the use of spontaneous WDS as a behavioural assay would not necessarily be invalidated, but the inference that WDS and male rat sexual behaviour are uniquely related would be weakened. The existence of overlap between neural structures that control the WDS and male rat copulatory behaviours may be assessed by employing a focal intracerebral manipulation, in which 5-HT2 activity is manipulated in only a circumscribed region of the brain. If WDS and male rat sexual behaviour rely on overlapping neural mechanisms, then a focal intracerebral manipulation that affects one behaviour should also affect the other. If the two behaviours are neurally independent, however, and artifactually correlated in tests using systemic drug injections, a dissociation of the behaviours should appear with discrete intracerebral treatments. The precise brain regions involved in the generation of WDS are unknown. However, research by others has narrowed the range of candidate regions through the process of elimination. The neurotoxin 5,7-dihydroxytryptantine (5,7DHT) destroys serotonergic neurons with reasonable selectivity. When injected intrathecally (i.e., into the fluid-containing space in the spine), 5,7-DHT treatment reportedly has no effect on the production of WDS (Fone, Johnson, Bennett, & Marsden,  1989). This suggests that WDS  102  is mediated by structures rostral to the spinal cord. The generation of WDS is also apparently unaffected by complete ablation of the frontal cortex (Lucki & Minugh-Purvis, 1987), an area which is rich in 5-HT2 receptors (Palacios, Mengod, Hoyer, Waeber, Pompeiano, Niclou, & Bruinvels, 1992; Palacios, Waeber, foyer, and Mengod,  1990). Furthermore,  Bedard & Pycock (1977) made a series of coronal transections of the brain rostral to the posterior coimuissure, none of which affected WDS production. Thus, it would appear from these studies that WDS must be mediated by a mechanism located in the brainstem. Very little is known about the possible involvement of the brainsteni in the control of copulatory behaviour in the male rat (Rose,  1990); most of the work on central nervous  system mechanisms contributing to mating behaviour in male rats has focussed on the critical role of the medial preoptic area (MPOA), which is an area that lies anterior to the hypothalamus, and thus also anterior to the posterior commissure. However, given the evidence suggesting that the neural substrate for WDS behaviour must be located in the brainstem, it follows that if WDS and male rat sexual behaviour are influenced by a common neural mechanism, that mechanism must be located in the brainstem. Experiment 8 was therefore intended to address several issues. It is of interest to attempt to identify neural  103  mechanisms mediating the effects of drug treatments on male rat copulation and simultaneously occurring WDS for at least two reasons. First, this would provide insights into the central mechanisms participating in the inhibition of male rat sexual behaviour observed after treatment with 5-HT2/1C agonists (Foreman et al.  1991; Watson & Gorzalka, 1990,  1991). Second, it would elucidate the neural basis of WDS  --  pharmacologically-induced WDS is increasingly employed in pharmacological research as an index of 5-HT2 activity, and the ability of new drugs to modulate 5-HT2 receptor activation is assessed on the basis of the ability of such drugs to elicit/inhibit WDS (e.g., Bedard & Pycock, Meert et al.,  1977;  1989). The utility of WDS as a pharmacological  screening tool would be improved by a more complete understanding of the neural mechanisms that contribute to the production of WDS. In previous pharmacological studies, for example, attempts have been made to correlate changes in WDS after drug treatment with changes in 5-HT2 receptor density in cortical tissues (Goodwin, Green, & Johnson, 1984). Inconsistent results have been obtained, presumably because the brain tissues being assayed are not from the structures that actually mediate the WDS behaviour (Lucki & Minugh-Purvis, 1987). In Experiment 8, copulatory behaviour and WDS were measured in male rats that received focal microinjections of  104  DOl in the brainstem, in the region of the nucleus raphe obscurus (ROb) and inferior olivary complex (ICC). This region was selected for two reasons. First, anatomical research has demonstrated that the neurons of this region project to the ventral horns of the spinal cord (Bowker, Westlund, Sullivan, & Coulter, 1982; Tork, 1985,  1990),  which contain the motor neurons that would ultimately be involved in the production of WDS. Second, it has been demonstrated that moderate densities of 5-HT2 receptors exist in the region of the Rob/Icc (Wieland, Krieder, Mcconigle, & Lucki, 1990), supporting the possibility of a role of cells in the Rob/Icc in the expression of WDS.  Method  Animals Twelve female and 24 male Long—Evans rats, derived from stock originally obtained from charles River Inc. (Montreal), were employed. Females were prepared as in the preceding experiments. Animals were housed in wire mesh cages, with free access to food and water, and were maintained on a reversed 12/12 hr light/dark cycle. At approximately 70 days of age, each male rat was anaesthetized (1.0 mg/kg atropine sulfate 12, as a preanaesthetic treatment to minimize respiratory secretions,  105  followed by sodium pentobarbital, ketamine hydrochloride,  45.0 mg/kg IP, plus  60.0 mg/kg IP) and placed in a Kopf  stereotaxic instrument. Using standard stereotaxic surgical procedures  (Cooley & Vanderwoif,  1978; Gray & Gorzalka,  1979), a single guide cannula constructed from 23 gauge stainless steel tubing was implanted in the Rob/bc. The tip of the guide cannula was aimed at a point on the midline, 12.6 mm posterior to bregma and 9.8 mm ventrally from the dural surface. Stereotaxic coordinates were derived from the atlas of Paxinos and Watson (1986). Following implantation, the cannula was affixed to the skull using dental acrylic and jeweller’s screws, and the rat was returned to its home cage for 1 week of postoperative recovery. All experimental male rats were housed individually following surgery.  Drugs Fresh drug solutions were prepared immediately prior to each testing session. Ritanserin (Research Biochemicals) was prepared as in Experiment 2. Doses of DOl nd  (Research  Biochemicals) were dissolved in physiological saline and injected in  a total volume of 1.0 tl, via the brainstem  cannula, using a 30 gauge injection needle. Each dose was delivered using a Sage Instruments infusion pump (Model 341A), at a flow rate of 4 p1/mm  for 15 s. The injection  needle was left in place for a further 45 s following  106  injection, to allow the drug solution to fully diffuse away from the needle tip. Ritanserin was administered 90 mm prior to testing; intracerebral injections of DOl were made 15 mm  prior to testing.  Procedure Following surgery, and prior to behavioural scoring, the experimental male rats were paired with receptive female rats for 45 mm, on at least three separate occasions, to allow screening of the male rats on the basis of copulatory proficiency. Males that failed to display normal copulatory behaviour (i.e. failed to ejaculate at least once in 45 mm) in at least one screening session were excluded. Drug treatments and behavioural scoring were conducted in five testing sessions, over the course of 5 consecutive weeks. All behavioural testing occurred during the middle 1/3 of the dark phase of the rats’  light-dark cycle. In a  repeated measures design, the incidence of WDS and sexual behaviour was recorded after the experimental males received intracerebral microinjection of 0.0 0.1,  (vehicle-only control),  1.0, or 10.0 tg of DOl, or after they had received 10.0  ig DOl intracerebrally plus a SC injection of 5.0 mg/kg ritanserin. The five drug treatments were administered to the male rats in random counterbalanced fashion, such that  107  by the conclusion of the experiment each animal had been tested once at each dose. Behavioural observations were conducted in much the same fashion as in Experiments 5-7. After the administration of drugs, the male rats were placed in Plexiglas observation chambers  (30 X 30 X 45 cm) and allowed 5 mm  to habituate to  their new surroundings. Receptive female rats were then introduced into the chambers for an observation period of 30 iuin. As in Experiments 5-7, an observer blind to experimental condition recorded the incidence of WDS and sexual behaviour on microcassette tape; the data were transcribed from tape at the end of each session. At the conclusion of the experiment, each male rat was deeply anaesthetized (sodium pentobarbital,  65 mg/kg IP),  and a 1 il volume of 1% Evans blue solution was injected via the brainstem cannula, to aid in histological verification of the injection site. Each male rat was then transcardially perfused with 10% formalin, and the brain was removed, sectioned (35 tm slices), and the slices were mounted on gelatin-coated microscope slides. Following staining of the slices with Cresyl violet, cannula placement was microscopically verified. Examination of the diffusion of Evans blue at the injection site revealed stain deposition throughout a spherical area 1-2 mm in diameter.  108  Results  A total of 7 experimental rats were excluded from the experiment, either on the basis of a persistent failure to initiate copulation in the pre-experimental screening trials (n=5), or due to inaccurate placement of the Rob/bc cannula. The effects of the various drug treatments on the WDS and copulatory behaviours of the male rats are depicted in Figure 10. The statistical significance of the treatment effects were assessed using overall Friedman tests, followed by Wilcoxon paired comparisons where appropriate. Microinjection of DOl into the region of the ROb/bC had a pronounced effect on both the sexual behaviour and WDS of the male rats. Thus, the overall test of the effect of intracerebral DOl on WDS was statistically significant  ) 4 ( 2 (x  =  32.04, p  <  0.001). With microinjection of 10.0  DOl, almost 400% more WDS was expressed than under the other treament conditions (p  <  0.001). Simultaneously, the  intracerebral microinjections of DOl induced a dose— dependent decrease in the mean number of ejaculations achieved during the 30-mm  test sessions  ) 4 ( 2 (x  =  36.25, p  <  0.001). Subsequent paired comparisons revealed that when the males received intra-ROb/IOC injections of DOl in doses of either 1.0 or 10.0 ig they displayed significantly less ejaculatory behaviour than they did when they received the  109  0.0 ig DOl control injection (p  <  0.03 and p  <  0.01,  respectively). Pretreatment of the male rats with a systemic injection of ritanserin effectively reversed the effects of intra ROb/bC microinjection of 10.0 tg DOl on WDS (p fact, the  <  0.001). In  ritanserin treatment almost completely abolished  the WDS response, lowering WDS to a level significantly lower than in the 0.0 tg DOl control condition (p  <  0.004).  Concurrently, ritanserin treatment significantly reversed the inhibitory influence of intra-ROb/IOC injection of 10.0 tg DCI on the copulatory behaviour of the male rats  (p  <  0.02), although baseline levels of ejaculatory behaviour were not re-established (p  <  0,001).  110  Figure 10. Effects on WDS and ejaculatory behaviour of male rats that received intracerebral microinjection of DOl into the region of the nucleus raphe obscurus and inferior olivary complex.  In the Ritanserin + DOl condition, the male  rats received a subcutaneous injection of ritanserin (5.0 mg/kg) in addition to an intra-ROb/IOC microinjection of 10 ig DOl. Results are presented as mean  ±  S.E.M.  C 0 0  U)  0  C  0  5  10  15  20  0.1  I—I WDS  0— -O EJAC  T 0  1.0  \  •.1.  .T  -  1 0.0  \  DOl j..tg/rat, brainstem injection  0.0  I  •  T  -p  Rltanserin + DOl tO jg/rat  -p  0  1  2  z  4  0  C 0  •  a  C.)  .2 -I a  C  U)  0  .  C  I-I  112  Discussion  In Experiment 7,  it was found that systemic injection of  DCI both inhibited sexual behaviour and stimulated WDS, presumably through D0Is agonistic actions at 5-HT2/1C receptors. These findings were mirrored in the present experiment. DCI microinjection into a discrete region of the ventromedial brainstem of male rats not only stimulated WDS but also inhibited their copulatory behaviour. That no dissociation of the effects of DOl emerged with the focal brainstem manipulation suggests that the two behaviours rely, to some degree, on a common neural substrate vested in the region of the nucleus raphe obscurus and inferior olivary complex. Furthermore, the efficacy of ritanserin in reversing the effects of intra Rob/ICC DCI supports the argument that the hypothesized brainstem mechanism is mediated by 5-HT2/1C receptors, given the known 5-HT2/1C selectivity of both DOl (Glennon, ritanserin (Meert et al.,  1989).  1986; Hoyer,  1992) and  113  DISCUSSION  Back to Havelock Ellis (1898) for a moment: He further noted that,  “I regard sex as the central problem of life”  (preface p.xxx). Fortunately, it would seem that this problem, at least in the academic sense, is tractable; some of the mysteries of the biochemical control of sexual behaviour are yielding to ever more detailed levels of analysis. In the present experiments, I have investigated the relationship between alterations of activity at 5-HT2 receptors (and, to some unknown extent, 5—HT1C receptors) and the control of reproductive behaviour in male rats. In order to do this, two different experimental approaches were employed.  In Experiments 1—4, a systemic pharmacological  approach was used to evaluate the sexual behaviour of male rats after the administration of various 5-HT2/1C receptor ligands. The drugs varied in their affinities, selectivity, and intrinsic activity. A novel behavioural approach was elaborated in Experiments 5-8: the incidence of WDS in male rats during mating tests was evaluated as a behavioural assay of concurrent 5—HT2 activity, and, furthermore, the neural basis of this relationship was explored. The results of the two sets of experiments that make up these approaches are reviewed and discussed separately in two following  114  sections. In the third section of the Discussion, the results of the two approaches are integrated, and it is argued that the data generally support the suggestion that 5—HT2 receptors mediate an inhibition of copulatory behaviour in the male rat. Implications for future research are also considered.  1. General Discussion: Experiments 1—4. In Experiment 1, the 5-HT2/1C receptor agonist DOl was found to inhibit the copulatory behaviour of male rats in a dose-dependent manner. This finding is consistent with that of Foreman and associates (1989), and it provided initial evidence that 5-HT2/1C receptor activation inhibited male rat sexual behaviour. In Experiments 2—4, the efficacy of the 5—HT2/1C receptor antagonists ritanserin, ketanserin, and pirenperone in reversing DOl-induced inhibition of copulation was assessed. All were highly effective in reversing this inhibition, and, interestingly, none had an effect on male rat sexual behaviour when administered alone at a dose that was effective in reversing DOl—induced inhibition. Inconsistencies in the literature regarding the effects of 5-HT2/1C antagonists on the sexual behaviour of male rats may be attributable to the use of excessively high doses, resulting in behavioural expression of nonselective binding to non5-HT2/1C receptors.  115  Like DOl, ritanserin has very high affinity for 5-HT2 and 5-HT1C receptors, but does not discriminate between the two subtypes (Hoyer, 1988b,  1992; Meert et al., 1989;  Middlemiss & Tricklebank, 1992; Zifa & Fillion,  1992;  Glennon & Dukat, 1991). Pirenperone and ketanserin both display moderate selectivity for 5-HT2 receptors over 5-HT1C receptors (Hoyer, 1988a, Janssen,  1988b, 1992; Glennon & Dukat, 1991;  1983; Zifa & Fillion, 1992). Basic pharmacological  principles  (e.g., Kalant, et al., 1985) suggest that these  three antagonists are effective in reversing 001-induced inhibition by competing with 001 for binding at the receptors through which 001’s effects are exerted. The binding data cited here thus suggest that it is the 5-HT2 receptor subtype that mediates the effects of these drugs on male rat sexual behaviour, because it is the subtype for which all four drugs have the highest affinity. However, as I have repeatedly noted, it is also important to keep in mind that all these drugs have high affinity for 5-HT1C receptors as well. An additional complication should be noted. Although pirenperone and ketanserin are moderately selective for 5-HT2 receptors, both drugs have appreciable affinity for other types of receptors, particularly histaminergic and alpha—adrenergic receptors (Janssen, 1983; Glennon & Dukat, 1991). Pirenperone also displays affinity for dopamine  116  receptors, but ketanserin lacks this activity, suggesting that the reversal of DOl-induced inhibition by the three antagonists is not attributable to dopamine receptor blockade. Furthermore, the 5-HT/1C antagonist LY 53857, which has also been reported to reverse DOl—induced inhibition of copulatory behaviour in male rats, reportedly lacks appreciable activity at alpha-adrenergic receptors (Foreman et al.,  1989). This suggests that the effects  observed in Experiments 1—4 are not mediated by alpha— adrenergic receptors. In Experiment 3, pirenperone treatments yielded a dose— response effect that was approximately biphasic. Compared to the complete reversal of DOl-induced inhibition that occurred with the lowest dose of pirenperone, higher doses of pirenperone exerted a significantly inhibitory influence. That is, although pirenperone significantly attenuated DOl induced inhibition at all doses tested, there was significantly less improvement when animals received higher doses of pirenperone than when they received the lowest dose (50 ig/kg). This pattern of results may reflect some of pirenperone’s binding to non5—HT receptors. It is possible that the data obtained with the lowest dose reflect selective binding of pirenperone to 5-HT2 receptors, with saturation of 5—HT2 receptors and the behavioural expression of increased binding to other types of receptors occurring  117  at higher doses of pirenperone. Support for this idea comes from the report that coadministration of the alpha— adrenergic antagonist prazosin with the 5-HT2/1C antagonist LY 53857 abolishes the facilitation of male rat sexual observed after treatment with LY 53857 alone (Foreman et al.,  1989). Thus, the adrenergic activity of ketanserin and  pirenperone might account for the inhibition of male rat copulation that these drugs have been reported to induce, when administered in the absence of other drugs (Foreman et al.,  1989; Mendelson & Gorzalka, 1985). In Experiment 4,  however, ketanserin produced a dose-dependent reversal of DOl-induced inhibition, and did not parallel pirenperone in producing a diminished blockade of the inhibitory effects of DCI when given in higher doses. As I have discussed, binding data suggest that ketanserin lacks the affinity for dopamine receptors that pirenperone displays. It therefore seems reasonable to conclude that the biphasic effect of pirenperone, resulting in attenuated effectiveness of high doses of pirenperone compared to the low dose, may be due to pirenperone  ‘S  dopaminergic activity.  As has been mentioned, doses of ritanserin, pirenperone, and ketanserin that were effective in reversing the DCI— induced inhibition did not affect the sexual behaviour of male rats when given in the absence of other drugs. This finding conflicts with previous reports, which are  118  themselves contradictory: namely, that the 5-HT2/1C antagonist LY 53857 facilitates sexual behaviour in male rats (Foreman, et al.,  1989), whereas the 5-HT2/1C  antagonists ketanserin and pirenperone are inhibitory when given in the absence of other drugs (Mendelson & Gorzalka, 1985). Consideration of the affinities of these compounds, and the doses administered, may allow some reconciliation of these reports. Pirenperone has been reported to be moderately inhibitory in otherwise untreated male rats, at a dose of 75 rig/kg and to be highly inhibitory at 200 tg/kg (Mendelson & Gorzalka, 1985). Others have found 100 pjg/kg to be without effect, and report a pirenperone—induced inhibition only at a dose of 1000 pg/kg (Foreman et al., 1989). This is congruent with the findings of Experiment 3, in which doses of pirenperone exceeding 50 pjg/kg were found to be inhibitory, compared to the effects observed with 50 FIg/kg. As I have argued, the inhibitory effects of pirenperone at higher doses may be due to its actions at non—5—HT2/1C receptors. The effects of ketanserin are more difficult to evaluate, as the sole report in which an inhibitory effect of ketanserin was noted provided only preliminary data collected using a single dose of ketanserin (5.0 mg/kg), ruling out any evaluation of dose-related phenomena (Mendelson & Gorzalka,  1985). Nevertheless, I note  that, in Experiment 4, a dose of just 0.2 mg/kg ketanserin  119  adequately blocked the receptors through which DOl exerts its inhibitory influence, yet when given to otherwiseuntreated males, this dose of ketanserin did not affect their sexual behaviour. Furthermore, males treated with 5.0 mg/kg of ketanserin plus DOl performed at drug-free baseline levels in Experiment 4. It may be the case that the reported facilitatory effect of LY 53857, when given alone (Foreman et al., 1989), is attributable to antagonism at 5-HT1C rather than 5-HT2 receptors; recent reports indicate that LY 53857 exhibits higher affinity for 5-HT1C than 5-HT2 receptors (foyer, 1888a, 1988b,  1992; Zifa & Fillion, 1992).  It is noteworthy  that the mixed 5-HT1B/1C agonists TFMPP and mCPP reportedly inhibit sexual behaviour in the male rat (Fernandez-Guasti, Escalante,  & Agmo,  1989; Mendelson & Gorzalka, 1988,  1990),  an effect which is consonant with the data collected in Experiments 1-4. Although the data collected with pirenperone and ketanserin in Experiments 3 and 4 suggest that 5-HT2 receptors, in particular, mediate an inhibition of male rat copulation, precise dissociation of the respective roles of 5-HT2 and 5-HT1C receptors must await the development of more selective ligands. Several reports have appeared in which drugs that are selective for subtypes of serotonin receptors have been evaluated for their effects on ex copula penile reflexes. It  120  is interesting to note that the mediation of these reflexes by subtypes of serotonin receptors does not appear to parallel the roles of these receptors in the regulation of. reproductive behaviour in the male rat. In fact, the relation of penile reflexes to activity at subtypes of 5-HT receptors appears to be the opposite of the relation between 5—HT receptor subtypes and copulatory behaviour. For instance, it has been reported that the administration of DOl facilitates penile reflexes, apparently via 5-HT1C receptor activation in the spinal cord (Berendsen, Jenck, & Broekkamp,  1990). The 5-HT1A agonist 8-OH-DPAT and 5-HT1A  partial agonist buspirone reportedly inhibit penile reflexes (Mathes et al.,  1990; Schnur et al.,  1990). This is in  marked contrast to the facilitation of male rat sexual behaviour obtained with these compounds in in copula mating tests (e.g., Ahienius et al.,  1981; Mathes, et al  1990).  The relation of penile reflexes to overt sexual behaviour is thus unclear, but it does appear that copulatory behaviour and actual seminal emission may be physiologically separable events (Berendsen et al.,  1990).  The data collected in Experiments 1-4 provide evidence that 5-HT2/1C receptor activation mediates an inhibition of overt in copula sexual behaviour in male rats. Since these experiments were conducted, confirmatory data have appeared in the literature. In a study employing essentially the same  121  procedure as in Experiments 3 and 4 of this dissertation, the efficacy of ritanserin and ketanserin in reversing DCIinduced inhibition of male rat sexual behaviour was recently confirmed, and a similar effect was demonstrated with the moderately selective 5-HT2 antagonist amperozide (Klint, Dahigren, & Larsson, 1992). Amperozide was also reported to produce a moderate attenuation of male-rat copulatory behaviour when given in the absence of other drugs, but once again, this effect appeared at a dose of amperozide (5.0. mg/kg) that far exceeded the dose of amperozide required to reverse 001-induced inhibition (0.5 mg/kg) 1992).  (Klint et al.,  It is probable that this inhibitory effect of high  doses of amperozide is attributable to nonselective binding.  2. General Discussion: Experiments 5—8. Experiments 5-8 were designed to explore the possibility that the incidence of spontaneous WDS in male rats during mating tests might provide an index of concurrent 5—HT2 activation that converges on the pharmacological data. The results of Experiment 5 and 6 indicated that, compared to Duds, Studs produce very few spontaneous shakes in mating • tests, and that this effect is limited to situations where a receptive female rat is present. The finding of Experiment 7, that DCI exerted reciprocal effects on WDS and copulatory behaviour that paralleled the data collected with untreated  122  males, provided convergent evidence that the relation between sexual behaviour and WDS in male rats is mediated by 5—HT2 receptors (and perhaps, to an unknown extent, by 5— HT1C receptors). Taken together, the results of Experiments 5—7 support the conclusion that copulatory behaviour in the male rat is incompatible with the expression of WDS. It is interesting to contrast the effects of partner-type on Duds and Studs in Experiment 6. Duds maintained high levels of WDS regardless of the types of partners they were paired with. By contrast, Studs displayed virtually no WDS when paired with receptive females, but displayed significantly increased WDS when paired with nonreceptive females and still higher levels when paired with males. Thus, in Studs, the characteristics of the partner in the testing chamber yielded an effect on the Studs’ expression of WDS that was not unlike a doseresponse function, relating the sexual stimuli of the partner rats to the expression of WDS in the Studs. The significant difference in Studs’ WDS scores between the nonreceptive-feiuale-partner condition and the male—partner condition is particularly important in this respect. The nonreceptive females lacked receptive and proceptive behaviours, and presumably also lacked olfactory cues associated with estrus. Nevertheless, it seems reasonable to suppose that the nonreceptive females still possess a  123  greater number of sexually stimulating characteristics than do the male partners. It is therefore plausible that the expression of WDS in this context specifically reflects the operation of a neural mechanism that tonically inhibits the expression of sexual behaviour in male rats. Presumably, the sexual stimuli presented by a nonreceptive female rat might partially reduce this inhibitory influence, resulting in an intermediate level of WDS expression in the male rats. In any case, given the dependency of WDS on 5—HT2 receptor activation, the data collected with Studs in Experiment 6 suggests greater 5-HT2 receptor .activity in the presence of male partners than in the presence of nonreceptive females. The finding that WDS levels differed systematically across the experimental conditions, within the Studs group, further argues that the differences in WDS between Studs and Duds measured in Experiment 5 is not readily explained as a reflection of differential levels of activity or arousal. In fact, recent evidence suggests that, if anything, Duds are more active than Studs (Kohlert & Bloch, 1993). It might be argued that a change in the expression of WDS under certain conditions, and particularly when induced by drug treatments, may be an epiphenomenon of somatosensory changes.  In particular, WDS is morphologically similar to  the pinna reflex displayed by most mammals in response to mechanical stimulation of the auditory canal, such as the  124  reaction of a cat to having a fly land on its ear. The relationship between the pinna reflex and the expression of WDS has been studied by Lucki and associates, using an experimental preparation in which a drop of a viscous liquid is placed in the ear of a rat (Lucki, Eberle, & Minugh Purvis,  1987). This treatment causes the rat to shake its  head vigorously in an attempt (usually fruitless) to expel the droplet. Comparison of this type of head shake with the WDS induced by 5-HTP loading or treatment with 5-HT2 agonists suggests that the pinna reflex and WDS are separable phenomena. Local anaesthesia of the pinnae, but not treatment with 5-HT2 receptor antagonists, was reported to be effective in attenuating the head shaking associated with the pinna reflex. Conversely, treatment with 5-HT2 antagonists, but not anaesthesia of the pinnae, was reported to be effective in reversing the WDS induced by administration of 5-HTP or 5-HT2 agonists (Lucki, et al., 1987). Building on the evidence collected in Experiment 7, which implicated 5-HT2 receptor activity in the relation between WDS and copulatory behaviour in male rats, the object of Experiment 8 was to identify a putative 5-11T2receptor—dependent mechanism important in WDS and male rat sexual behaviour. The available anatomical data suggested that this mechanism might be located in the ventromedial  125  brainstem. Microinjection of DOl in the region of the nucleus raphe obscurus and inferior olivary complex was found to produce a pattern of augmented WDS and concurrent copulatory inhibition that paralleled the results of Experiment 7, in which systemic treatments were employed. The effects of intra-R0b/IOC DOl were effectively reversed by ritanserin. The obtained data thus support the conclusion that WDS and male rat copulation are both modulated by a shared neural substrate in the Rob/Icc. Recently, neuroanatomical and lesion data have appeared which strongly support the suggestion that WDS and male rat sexual behaviour rely on a common neural mechanism in the region of the Rob/Icc (Leanza, Pellitteri, Russo, & Stanzani,  1991; Yamanouchi & Kakeyama,  1992). Using a  fluorescent retrograde double-labelling technique, it has been demonstrated that individual neurons in the Rob/Icc have bifurcating axons which project to both the medial preoptic area and to the ventral horns of the cervical (Cl C2) spinal cord (Leanza et al.,  1991). The degree to which  this anatomical finding agrees with the behavioural data collected in Experiments 5-8 cannot be over—emphasized: The MPOA is well established to be of critical importance in male rat copulation (e.g., Gorzalka & Mogenson,  1977; Rose,  1990), and the ventral horns of the cervical spine contain the motoneurons involved in the expression of WDS. It seems  126  highly probable that the relation of WDS and male rat copulatory behaviour arises from this collateralized projection from the brainstem to the MPOA and cervical spinal cord.  3. Conclusions, speculations, and implications for future research. Taken together, the investigations reported in this thesis provide strong support for the conclusion that 5-HT2/1C receptors mediate an inhibitory influence of 5-HT on the copulatory behaviour of male rats. It is probable that this influence is attributable to 5-HT2 receptor activity more than to 5-HT1C receptor activity, but precise dissociation of the roles of these two receptor subtypes in the control of male rat sexual behaviour must await the development of more selective ligands. Furthermore, the present data suggest that the incidence of WDS during sexual behaviour reflects concurrent 5-HT2 receptor activity, and implicates a ventromedial brainstem region as the site of a common 5-HT2-dependent neural mechanism that contributes to both WOS and copulatory behaviour in the male rat. Although it appears that activity at 5-HT2 receptors mediates an inhibition of sexual behaviour in male rats, it must be acknowledged that the results of contemporary experiments probing the functional significance of subtypes  127  of receptors for serotonin will require frequent review and re—evaluation in the future. During the past 5 years or so there has been an explosion in the number of new putative 5HT receptor subtypes being reported, and increasingly fine distinctions are being made within existing subtype classifications. At least 11 discrete 5-HT binding sites have now been described (e.g., Glennon & Dukat, 1991; Bradley, et al., 1992; Zifa & Fillion, 1992), and evidence for new subtypes continues to mount. It must be emphasized that, aside from the 6 established 5-HT.receptor subtypes mentioned in the General Introduction, many of the newly described sites are thus far strictly pharmacological entities that have been characterized only in isolated tissues  ——  no selective ligands have been described, and  some sites have yet to be established as functional receptors. Nevertheless, this burgeoning field has important consequences for studies of the behavioural concomitants of activity at particular 5-HT receptor subtypes, in two inter related ways. First, desàriptions of new distinctions within existing receptor subtypes complicates the attribution of behavioural effects to specific receptor subtypes. For example, pharmacological evidence has suggested that there may be two forms of 5-HT2 receptors, labelled 5-HT2A and 5-HT2B (McKenna & Peroutka, 1989). Until selective ligands are  128  developed that discriminate between these sites, it is impossible to determine whether behavioural effects currently attributed to 5-HT2 activity are due to activity at one or both of these usubsubtypes. I agree with Glennon and Dukat (1991) that until such time as pharmacological probes are available that discriminate between subtypes of 5—HT2 receptors, it is needlessly confusing to discuss any possible functional significance of 5-HT2 receptor heterogeneity; however, with the development of such ligands in the future, it will become necessary to examine the respective contributions of activity at subtypes of 5-HT2 receptors in behaviours that have been established to be 5-HT2-receptor-dependent. Second, the description of new 5-HT binding sites sometimes has the net effect of turning selective ligands into nonselective ligands. That is, re-evaluation of a serotonergic drug’s binding affinities may reveal that the drug has affinity for newly described binding sites, in addition to its established affinities. For example, quipazine was believed to be a selective 5-HT2 agonist until it was discovered that quipazine binds with equivalent affinity to the more recently described 5-HT1B receptors (Hoyer, Perry,  1988a) and 5-HT3 receptors (Peroutka & Hamik, 1988; 1990). As new affinities are described for current  129  serotonergic drugs, the functional effects of such drugs will require re-evaluation. It is also interesting to note that evidence is accumulating that certain established subtypes of serotonin receptors may exhibit functional interactions. In particular, recent behavioural evidence suggests that activity at 5-HT1A receptors may modulate 5-HT2 mediated behaviours,  and vice versa. DOl administration has been  reported to facilitate 8-OH-DPAT-induced reciprocal forepaw treading (Arnt & Hyttel,  1989). Conversely, administration  of the selective 5-HT1A agonist 8-OH-DPAT reportedly inhibits DOl-induced head twitching in mice (Darmani et al., 1990). This inhibition of DOl-induced behaviour is particularly interesting in the context of the present experiments, because 8-OH-DPAT has been found to potently facilitate the sexual behaviour of male rats et al.,  1981).  (e.g., Ahienius  It is plausible that the stimulatory effects  of 8-OH-DPAT on male rat copulatory behaviour may be at least partly due to a 5-HT1A receptor-mediated suppression of a 5-HT2-mediated inhibitory influence. As yet, no reports have appeared examining the possible effects of interactions between 5-HT1A and 5-HT2 receptor activity on the control of male rat copulatory behaviour, although DOl has been reported very recently to attenuate 8-OH-DPAT-induced inhibition of lordosis in female rats  (Uphouse, Andrade,  130  Moore,  & Caldarola-Pastuszka,  evaluate 5—HT1A  —  1993).  I have attempted to  5—HT2 interactions in male rats on several  occasions, by examining the efficacy of 8-OH-DPAT, as well as the selective 5-HT1A partial agonists buspirone and gepirone, in reversing the inhibition of male rat sexual behaviour induced by DCI. To my dismay, however,  I have  found the combination of moderate doses of DOl plus a 5-UT1A agonist or partial agonist to be uniformly lethal in male rats  (Watson & Gorzalka, unpublished data). The issue of  interactions between 5-HT1A and 5-HT2 receptors in the control of male rat sexual behaviour thus remains unresolved. It remains somewhat puzzling that doses of 5-HT2/1C antagonists that are effective in reversing DOl—induced inhibition of male rat sexual behaviour were not found to affect the sexual behaviour of male rats when given in the absence of other drugs, in Experiments 2-4 and in the study of Klint et al.  (1992). Since these drugs exerted a  facilitatory affect on male rat copulation by blocking the binding of an exogenous agonist, DCI, it might be expected that a similar facilitation would ensue from blocking the binding of the endogenous ligand, 5-UT, in otherwise untreated male rats. The absence of such effects is not easily attributed to differential affinities of DCI and 5-HT for 5-HT2/1C sites; in fact,  5—HT displays lower affinity  131  for 5-HT2 receptors than any of the drugs used in Experiments 1-4, and would thus be more completely displaced from 5-HT2 receptors by the 5-HT2 antagonists than would be DOl. The effects of 5-HT2 receptor antagonists on reproductive behaviour in otherwise untreated male rats could be evaluated in more detail in future experiments, by administering these drugs to castrated males maintained on doses of testosterone that have been titrated to yield intermediate levels of copulatory proficiency. These males would thus provide more range in which to demonstrate any facilitation of sexual behaviour that may obtain following treatment with 5-HT2 receptor antagonists. In this dissertation, a model of serotonergic participation in the control of reproductive behaviour in the male rat has been described, in which a tonic inhibition of male rat sexual behaviour has been attributed to a 5-HT2receptor-dependent mechanism located in the ROb/bC, which also participates in the expression of WDS via collateralized neuronal projections. A neural adaptation that inhibits copulatory behaviour under inappropriate conditions  (Experiment 6) would make ecological sense, since  inappropriate sexual advances would be at best somewhat costly, in terms of energy expended in attempting to inseminate an unreceptive female, or in provoking aggression if directed at a male. This model also provides numerous  132  opportunities for further investigation. In particular, nothing is yet known about the manner in which the Rob/bc mechanism is modulated by more rostral structures, or the manner in which this system is integrated with other neural sites of established importance in the control of male rat sexual behaviour, such as the MPOA (Rose, 1990).  It is  presumed that the function of the ROb/bC mechanism is governed by descending serotonergic neurons, as the data collected in Experiment 8 suggest that the operation of this mechanism is 5-HT2-receptor-dependent. It is noteworthy that the region of the raphe obscurus may receive serotonergic projections from the midbrain dorsal raphe (Steinbusch & Nieuwenhuys, 1983). Lastly, although it is not clear to what extent the results of the present experiments offer generalizations about the control of sexual behaviour in the human male, it is self-evident that, across mammalian species, copulatory behaviour is more similar than it is different. Relatively little is known about the role of activity at particular subtypes of serotonin receptors in the control of sexual behaviour in the human male, primarily because many of the receptor—selective ligands that have been developed thus far are still being used exclusively for experimental purposes in nonhuman animals. In general, however, treatments that elevate levels of synaptic 5-HT, such as MAO inhibitors,  133  tricyclic antidepressants, and serotonin reuptake inhibitors have been reported to impair sexual behaviour in human males, while 5-HT1A agonists such as buspirone and lisuride and 5-HT2 antagonists such as cyproheptadine reportedly facilitate sexual behaviour (for review: Meston & Gorzalka, 1992), in agreement with data collected using rats. As drugs that are selective for subtypes of serotonin receptors come into clinical use in the next few years, it will become possible to assess the extent to which behavioural changes associated with modulation of 5-HT2 receptor activity in human males mirror the behavioural concomitants of 5—HT2 activity in male rats.  134  REFERENCES  Abraham, E.M., Viesca, P.M., Plaza, A.V.,  & Mar, B.  (1988). Modifications of the sexual activity in male rats following administration of antiserotonergic drugs. Behavioral Brain Research,  ,  251-258.  Ahienius, S., Eriksson, H., Larsson, K., Modigh, K., and Soderston, P.  (1971). Mating behavior in the male rat  treated with p-chlorophenylalanine methyl ester alone and in combination with pargyline. PsycholDharmacologia,  20,  383—388. Ahlenius, S., Heimann, M., & Larsson, K.  (1979).  Prolongation of the ejaculation latency in the male rat by thoridazine and chlorimipramine. Psychopharmacology, ,  137—140.  Ahlenius, S.,  & Larsson, K.  (1984a). Failure to antagonize  the 8-hydroxy-2-(di-n-propylamino)tetralin-induced facilitation of male rat sexual behavior by the administration of 5—HT receptor antagonists. European Journal of Pharmacology, Ahlenius, S.,  & Larsson, K.  ,  279-286.  (1984b). Lisuride, LY-141865,  and 8-OH-DPAT facilitate male rat sexual behavior via a non—dopaminergic mechanism. Psychopharmacology, 334.  ,  330—  135  Ahienius, S.,  & Larsson, K.  (1985). Antagonism by lisuride  and 8-OH-DPAT of 5-HTP induced prolongation of the performance of male rat sexual behavior. European Journal of Pharmacology,  110, 379-381.  Ahlenius, S., Larsson, K.,  & Svenson, L.  (1980). Further  evidence for an inhibitory role of central 5-HT in the male rat sexual behavior. Psychopharmacology,  68, 217..-  220. Ahienius, S., Larsson, K., Svensson, L., Hjorth, S., Carisson, A., Lindberg, P., Wikstrom, H., Arvidsson, L.E., Hacksell, U.,  Sanchez, D.,  & Nilsson, J.L.G.  (1981).  Effects of a new type of 5-HT receptor agonist on male rat sexual behavior. Pharmacology Biochemistry & Behavior, j5., Araki, H.,  785—792.  & Aihara, H.  (1986). Effects of neuroleptics on  hippocampal stimulation induced rats. Physiology & Behavior, Arnt, J.,  & Hyttel, J.  ‘wet-dog shaking’  ,  in  69-74.  (1989). Facilitation of 8-OH-DPAT-  induced forepaw treading of rats by the 5-HT2 agonist DCI. European Journal of Pharmacology, Baum, M.J.,  & Starr, M.S.  (1980).  161,  45-51.  Inhibition of sexual  behaviour by dopamine antagonist or serotonin agonist drugs in castrated male rats given estradiol or dihydrotestosterone. Pharmacology Biochemistry & Behaviour,  U,  57-67.  136  Beach, F.A.  (1956). Characteristics of masculine “sex  drive”. Nebraska Symposium on Motivation (pp.  1-41).  Lincoln, NB: Nebraska University Press. Bedard, P.,  & Pycock, C.J.  (1977).  “Wet dog” shake behaviour  in the rat: A possible quantitative model of central 5-hydroxytryptamine activity. Neuropharmacologv,  16,  663—670. Berendsen,  H.H.G., Jenck, F.,  & Broekkamp, C.L.E.  (1990).  Involvement of 5—HT1C-receptors in drug-induced penile erections in rats. Psychopharmacology, jQj, 57—61. Bevan, P.,  Cools, A.R.,  & Archer, T.  (Eds.).  (1989).  Behavioral pharmacology of 5-HT Hilisdale, NJ: Lawrence Erlbaum Associates Bowker, R.M., Westlund, K.N., Sullivan, M.C., J.D.  & Coulter,  (1982). Organization of descending serotonergic  projections to the spinal cord. Progress in Brain Research,  i,  239-265.  Bradley, P.B, Handley, S.L., Cooper, S.J., Key, B.J., Barnes, N.M.,  & Coote, J.H.  (Eds.).  (1992). Serotonin,  CNS receptors and brain function. Advances in the biosciences, vol. Clark,  85 Oxford: Pergamon.  J.T., Smith, E.R., Stefanick, M.L., Arneric, S.P.,  Long, J.P.,  & Davidson, J.M.  (1982). Effects of a novel  dopamine receptor agonist RDS-127  (2-N,N-di-n-  propylamino-4,7—dimethoxyindane), on hormone levels and  137  sexual behavior in the male rat. Physiology & Behavior,  aa,  1—6.  Colpaert, F.C.,  & Janssen, P.A.J.  (1983). The head-twitch  response to intraperitoneal injection of 5— hydroxytryptophan in the rat: Antagonist effects of purported 5-hydroxytryptamine antagonists and of pirenperone, an LSD antagonist. Neuropharmacoloctv, 22, 993—1000. Cooley, R.K., & Vanderwoif, C.H.  (1978). Stereotaxic surgery  in the rat: A Ihotographic series  (2nd ed.. London,  Ont.: A.J. Kirby, Co. Come, S.J., Pickering, R.W.,  & Warner, B.T.  (1963). A  method for assessing the effects of drugs on the central actions of 5-hydroxytryptantine. British Journal of Pharmacology,  ,  106-120.  Crowley, W.R., Popolow, H.R., & Ward, O.B., Jr.  (1973). From  dud to stud: Copulatory behavior elicited through conditioned arousal in sexually inactive male rats. Physiology & Behavior,  10, 391-394.  Dabiré, H., Chaouche-Teyara, K., Cherqui, C., Fournier, B., Laubie, M., & Schmitt, H.  (1989). Characterization of  DCI, a putative 5-HT2 receptor agonist in the rat. European Journal of Pharmacology, j, 369-374.  138  Dahiof, L.-G.  (1980). PCPA and the sexual behavior of the  pudendectomized male rat. Biology of Behaviour,  5,  211-  218. Dahiof, L.—G., Ahienius, S.,  & Larsson, K.  (1988).  Copulatory performance of penile desensitized male rats •  following the administration of 8-OH-DPAT. Physiology & Behavior,  ,  841-843.  Da Prada, M., Carruba, M., O’Brien, R.A., Saner, A., Pletscher, A.  &  (1972). The effect of 5,6-  dihydroxytryptamine on sexual behaviour of male rats. European Journal of Pharmacology,  19,  Darmani, N.A., Martin, B.R., Pandey, U.,  288-290. & Glennon, R.A.  (1990). Do functional relationships exist between 5-HT1A and 5-HT2 receptors? Pharmacology Biochemistry & Behavior, 36,  901-906.  Dewsbury, D.A., Davis, H.N., Jr., & Jansen, P.E.  (1972).  Effects of monoamine oxidase inhibitors on the copulatory behavior of male rats. Psychopharmacologia, j, 209-217. Eison, A.S., Yocca, F.D., & Gianutsos, G.  (1988).  Noradrenergic denervat ion alters serotonin2 —mediated behaviour but not serotonin2 receptor number in rats: Modulatory role of beta adrenergic receptors. Journal of Pharmacoloy and Experimental Therapeutics, Ellis, H. H.  (1898).  2..4.,  571-577.  Studies in the psycholocrv of sex (Vols.  1—2). New York: Random House.  139  Fernandez—Guasti, A., Escalante, A.,  &  Agmo,  A.  (1989).  Inhibitory action of various 5-HT1B receptor agonists on rat masculine sexual behaviour. Pharmacology Biochemistry & Behavior, j,  811-816.  Fernandez—Guasti, A., Escalante, A.,  Hong, E., & Agmo, A.  (1990). Behavioural actions of the anxiolytic indorenate, Pharmacology Biochemistry & Behavior,  fl,  83-88.  Fernandez—Guasti, A., Hansen, S., Archer, T., & Jonsson, G. (1986). Noradrenalin—serotonin interactions in the control of sexual behavior in the male rat: DSP4-induced noradrenalin depletion antagonizes the facilitatory effect of serotonin receptor agonists 5—MeODMT and lisuride. Brain Research, Fernandez-Guasti, A.,  fl,  112-118.  & Rodriguez-Manzo, G.  (1992). Further  evidence that the inhibitory action of serotonin on rat masculine sexual behavior is mediated by the stimulation of 5-HT1B receptors. Pharmacology, Biochemistry & Behavior,  ia,  529-533.  Fone, KC.F., Johnson, J.V., Bennett, G.W., & Marsden, C.A. (1989).  Involvement of 5-HT2 receptors in the behaviours  produced by intrathecal administration of selected 5-HT agonists and the TRH analogue (CG 3509) to rats. British Journal of Pharmacology,  ,  599-608.  140  Foreman, N.M., Hall, J.L., Love, R.L.  (1989). The role of  the 5-HT2 receptor in the regulation of sexual performance of male rats. Life Sciences, Frazer, A., Maayani, S., & Wolfe, B.B.  ,  1263  1270.  —  (1990). Subtypes of  receptors for serotonin. Annual Review of Pharmacology and Toxicology, Gaddum, J.H..,  Q,  307-348.  & Picarelli, Z.P.  (1957). Two kinds of  tryptamine receptors. British Journal of Pharmacology & Chemotherapeutics, j, 323-328. Genedani, S., Bernardi, M., Tagliavini, S., Botticelli, A., & Bertolini, A.  (1987). Shaking behaviour induced by  putrescine in naive rats: A pharmacological and histological study. Pharmacology & Toxicology,  61, 224-  227. Gessa, G.L.  (1970). Brain serotonin and sexual behavior in  male animals. Annals of Internal Medicine, Glennon, R.A.  (1990).  ll  622—629  Serotonin receptors: Clinical  implications. Neuroscience & Biobehavioral Reviews,  li  35—47. Glennon, R.A.,  & Dukat, M.  (1991). Serotonin receptors and  their ligands: A lack of selective agents. Pharmacology Biochemistry & Behavior,  jQ,  1009-1017.  Glennon, R.A., McKenney, J.D., Lyon, R.A., & Titeler, M. (1986).  5-HT1 and 5-HT2 binding characteristics of  141  1-(2 , 5-dimethoxy- 4-bromophenyl)-2-aminopropane analogues. Journal of Medicinal Chemistry, Goodwin, G.M., Green, A.R.,  & Johnson, P.  ,  194-199.  (1984). 5-HT2  receptor characteristics in frontal cortex and 5—HT2 receptor-mediated head-twitch behaviour following antidepressant treatment to mice. British Journal of Pharmacology,  235-242.  ,  Gorzalka, B.B., Mendelson, S.D., & Watson, N.y.  (1990).  Serotonin receptor subtypes and sexual behavior. Annals of the New York Academy of Sciences, Gorzalka, B.B., & Mogenson, G.J.  (1977).  QQ,  435-446.  Sexual behavior. In  G.J. Mogenson (Ed.), The Neurobiology of Behavior (pp. 151-186). Hilisdale NJ: Lawrence Eribaum Associates. Grabowska, N.  (1975).  Influence of quipazine on sexual  behavior in male rats. In M. Sandler & G.L. Gessa (Eds.), Sexual behavior: Pharmacology and biochemistry (pp.  59-  62). New York: Raven Press. Gray, D.S.,  & Gorzalka, B.B.  (1979). An easily constructed  durable chronic intracerebral cannula system. Pharmacology Biochemistry & Behavior, Grahame-Smith, D.G.  (1971).  jJ,  463-466.  Studies in vivo on the  relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L—tryptophan. Journal of Neurochemistry,  ,  1053-1066.  142  Green, A.R.  (1989). Behavioural pharmacology of 5-UT: An  introduction. In P. Bevan, A.R. Cools,  & T. Archer  (Eds.), Behavioural Iharmacolocry of 5-.HT (pp. 3-20). Hilisdale, NJ: Lawrence Erlbauiu Associates. Green, A.R.,  & Graham-Smith, D.G.  (1976).  (-)-Propranolol  inhibits the behavioural responses of rats to increased 5—hydoxytryptamine in the central nervous system. Nature,  2..i,  594—596.  Green, A.R.,  & Heal, D.J.  (1985). The effects of drugs on  serotonin-mediated behavioural models.  In A.R. Green  (Ed.), Neuropharmacolociv of serotonin (pp. 326-365), Oxford: Oxford University Press. Green, A.R., O’Shaughnessy, K., Hammond, M., Schachter, M., & Grahame-Smith, D.G.  (1983).  Inhibition of 5-  hydroxytryptamine mediated behavior by the putative 5-HT2 antagonist pirenperone. Neuropharmacoloqv, 22, Hartig, P.R.  (1989). Molecular biology of 5-HT receptors.  Trends in Pharmacological Science, Heym, J.,  573—578.  & Jacobs, B.L.  (1988).  64—69.  5-HT2 agonist activity as a  common action of hallucinogens. Gudelsky (Eds.),  ,  In R.H. Rech,  & G.A.  5-HT agonists as psychoactive drugs  (pp.  95-106). Ann Arbor, MI: NEP Books. Hillegaart, V., Ahienius,  S.,  & Larsson, K.  (1989). Effects  of local application of 5-HT into the median and dorsal  143  raphe nuclei on male rat sexual and motor behavior. Behavioural Brain Research, Holmes, G.M., Holmes, D.G.,  33, 279-286.  & Sachs, B.D.  (1988). An IBM-PC  based data collection system for recording rodent sexual behavior and for general event recording. Physiology & Behavior, Floyer, D.  ,  825—828.  (1988a). Functional correlates of serotonin 5-HT 1  recognition sites. Journal of Receltor Research,  59-  ,  81. foyer, D.  (1988b). Molecular pharmacology and biology of 5—  HT1c receptors. Trends in Pharmacological Science,  9, 89-  94, foyer, D.  (1992). Agonists and antagonists at 5-fT receptor  subtypes.  In Bradley, P.B, Handley, S.L., Cooper, S.J.,  Key, B.J., Barnes, N.M.,  & Coote, J.H.  (Eds.), Serotonin,  CNS receptors and brain function. Advances in the biosciences, vol. Janssen, P.A.J.  85.  (1983).  (pp. 29-47). Oxford: Pergamon. 5-HT2 receptor blockade to study  serotonin-induced pathology. Trends in Pharmacological Science,  ,  198—206.  Johansson, C.E., Meyerson, B.J.,  & Hacksell, U.  (1991). The  novel 5-HT1A receptor antagonist (S)-UH-301 antagonizes 8-OH-DPAT-induced effects on male as well as female copulatory behaviour. European Journal of Pharmacology,  .aQ.a,  81—87.  144  Kalant, H., Roschlau, W.H.E.,  & Sellers, E.M.  (Eds.).  (1985). Principles of medical pharmacology. Toronto: The University of Toronto Press. Kandel, E.R., & Schwartz, J.H.  (1992). Principles of neural  science (3rd ed.). New York: Elsevier. Kleinrok,  Z., & Juszkiewicz, M.  (1986). The influence of  insulin on 5-methoxytryptam.ine-induced wet dog shake behavior in the rat. Polish Journal of Pharmacoloav & Pharmacy,  ,  443—448.  Klint, T., Dahigren,  I.L, & Larsson, K.  (1992). The  selective 5-HT2 receptor antagonist amperozide attenuates 1- (2 ,5-dimethoxy-4-iodophenyl ) -2-aminopropane-induced inhibition of male rat sexual behavior. European Journal of Pharmacology, Koe, B.K.,  212,  241-246.  & Weissman, A.  (1966). p-Chlorophenylalanine: a  specific depletor of brain serotonin. Journal of Pharmacology & Experimental Therapeutics, Kohiert, J.G.,  & Bloch, G.J.  hyperactive male rats. Abstracts,  1A.,  499-516.  (1983). Hyposexuality in  Society for Neurosciences  19, 241.2.  Larsson, K., Fuxe, K., Everitt, B.J., Holmgren, M., & Sodersten, P.  (1978).  Sexual behavior in male rats after  intracerebral injection of 5,7-dihydroxytryptamine. Brain Research,  14J.,  293-303.  145  Leanza, C., Pellitteri, R., Russo, A.,  & Stanzani, S.  (1991). Neurons in raphe nuclei pontis and magnus have branching axons that project to medial preoptic area and cervical spinal cord. A fluorescent retrograde double labeling study in the rat. Neuroscience Letters, 195—199. Lucki,  I., Eberle, K.M.,  & Minugh-Purvis, N.  (1987). The  role of the aural head shake reflex in serotonin—mediated head shaking behavior. Psychopharmacology, Lucki,  I., &  Minugh-Purvis, N,  92,  150-156.  (1987). Serotonin-induced  head shaking behavior in rats does not involve receptors located in the frontal cortex. Brain Research, j, 403406. Lyon, R.A.,  & Titeler, M.  (1988). Pharmacology and  biochemistry of the 5-HT2 receptor. In E. Sanders-Bush (Ed.), The serotonin receptors  (pp. 59-88). Clifton, NJ:  The Humana Press. Malmnas, C.O., & Meyerson, B.J.  (1970). Monoamines and  testosterone-activated copulatory behaviour in the castrated male rat. Acta Pharmacoloqica Toxicologica, 28 (suppl.  1),  Maimnas, C.O.,  1—67. & Meyerson, B.J.  (1971). p  Chlorophenylalanine and copulatory behaviour in the male rat. Nature.  232,  398-399.  146  Mathes, C.W., Smith, ER., Popa, B.R.,  & Davidson, J.M,  (1990). Effects of intrathecal and systemic administration of buspirone on genital reflexes and mating behavior in male rats. Pharmaco1oy Biochemistry & Behavior,  ,  McIntosh, T.K.,  63—68. & Barfield, R.J.  (1984). Brain monoaminergic  control of male reproductive behavior. I. Serotonin and the post—ejaculatory interval. Behavioural Brain Research,  12,  McKenna, D.J.,  255—265.  & Peroutka, S.J.  (1989). Differentiation of  5—hydroxytryptamine2 receptor subtypes using [‘ 1]-R-(-) 25 2, 5-dimethoxy-4-iodo-phenylisopropylamine and [ H 3 ]ketanserin. Journal of Neuroscience,  ,  3482—3490.  Meert, T.F., Niemegeers, C.J.E., Gelders, Y.G., P.A.J.  & Janssen,  (1989). Ritanserin (R 55 667), an original  thymosthenic. In P. Bevan, A.R. Cools,  & T. Archer  (Eds.), Behavioural pharmacology of 5-HT (pp.  235-238).  Hilisdale, NJ: Lawrence Eribaum Associates. Mendelson, S.D.,  & Gorzalka, B.B.  (1985). Serotonin  antagonist pirenperone inhibits sexual behavior in the male rat: Attenuation by guipazine. Pharmacoloqy Biochemistry & Behavior, Mendelson, S.D.,  ,  565-571.  & Gorzalka, B.B.  (1986).  5-HT1A receptors:  Differential involvement in female and male sexual behavior in the rat. Physiology &  Behavior,  fl,  345-351.  147  Mendelson, S.D.,  & Gorzalka, B.B.  (1988). Differential  effects of 5—HT1B agonists on female and male sexual behavior in the rat. Society for Neuroscience Abstracts,  ii,  372.  Mendelson,  S.D.,  & Gorzalka, B.B.  (1990). Sex differences in  the effects of 1-(m-trifluoromethylphenyl)piperazine and 1-(m-chlorophenyl)piperazine on copulatory behavior in the rat. Neuropharmacology, Meston, C.,  & Gorzalka, B.B.  ,  783-786.  (1992). Psychoactive drugs and  human sexual behavior: The role of serotonergic activity. Journal of Psychoactive Drugs, Middlemiss, D.N.,  ,  & Tricklebank, M.D.  1-40. (1992). Centrally  active 5—HT receptor agonists and antagonists. Neuroscience & Biobehavioral Reviews, j, Morali, C.,  & Larsson, K.  75—82.  (1984). Differential effects of a  new serotoninomimetic drug,  8—OH-DPAT, on copulatory  behavior and pelvic thrusting pattern in the male rat. Pharmacology Biochemistry & Behavior, Nabeshima, T., Kameyaina, T.  ,  185-187.  Ishikawa, K., Yamaguchi, K., Furukawa, H., (1987). Phencyclidine-induced head-twitch  responses as 5-HT2 receptor-mediated behavior in rats. Neuroscience Letters,  2&,  Osborne, N.N., & Hamon, M.  335—338.  (Eds.).  (1988). Neuronal  serotonin. New York: John Wiley & Sons.  &  148  Palacios, J.M, Mengod, G., Hoyer, D., Waeber, C., M., Niclou, S.,  & Bruinvels, A.  (1992).  Pompeiano,  In Bradley, P.B,  Handley, S.L., Cooper, S.J., Key, B.J., Barnes, N.M., & Coote, J.H.  (Eds.), Serotonin, CNS receptors and brain  function. Advances in the biosciences, vol.  85.  (pp.  61—  72). Oxford: Pergamon. Palacios, J.M., Waeber, C., Hoyer, D.,  & Mengod, G.  (1990).  Distribution of serotonin receptors. Annals of the New York Academy of Sciences, Paxinos, G.,  & Watson, C.  36-52.  (1986). The rat brain in  stereotaxic coordinates. Sydney: Academic Press. Peroutka, S.J.,  & Hamik, A.  (1988).  HjQuipazine labels 53 [  3 recognition sites in rat cortical membranes. European UT Journal of Pharmacology. Peroutka, S.J.,  & Snyder,  148,  S.H.  of [ H]5-hydroxytryptamine, 3  297-299.  (1979). Differential binding Hjlysergic acid 3 [  diethylamide and [ H]spiroperidol. Molecular 3 Pharmacology,  li  687-699.  Peroutka, S.J., Lebovitz, R.M., & Snyder,  S.H.  (1981). Two  distinct central serotonin receptors with different physiological functions. Science, Perry, D.C.  827—829.  (1990). Autoradiography of [ HJquipazine in 3  rodent brain. European Journal of Pharmacology, ji, 85.  75-  149  Pranzatelli, M.R.  (1989). Benzodiazepine—induced shaking  behavior in the rat: Structure-activity and relation to serotonin and benzodiazepine receptors. Experimental Neurology, 1QAI 241-50. Pranzatelli, M.R.  (1990). Evidence for involvement of 5-HT2  and 5-HT1C receptors in the behavioral effects of the 5HT agonist 1- (2 ,5-dimethoxy-4-iodophenyl aminopropane)(DOI). Neuroscience Letters, Pottier, J.J.G., & Baran, D.  115,  74—80.  (1973). A general behavioral  syndrome associated with persistent failure to mate in the male laboratory rat. Journal of Comparative and Physiological Psychology,  ,  499-509.  Rodriguez, M., Castro, R., Hernandez, C.,  & Mas, M.  (1984).  Different roles of catecholaminergic and serotonergic neurons of the median forebrain bundle on male rat sexual behavior. Physiology & Behavior, Rose, J.D.  ,  5-11.  (1990). Brainstem influences on sexual behaviour.  In In W.R. Klemm & R.P. Vertes  (Eds.), Brainstem  mechanisms of behavior (pp. 407-463). New York: Wiley. Sachs, B.D.,  & Barfield, R.J.  (1976). Functional analysis of  masculine copulatory behavior in the rat. Advances in the Study of Behavior, Salis, P.J.,  Z,  91-154.  & Dewsbury, D.A.  (1971). p-Chlorophenylalanine  facilitates copulatory behavior in male rats. Nature, 232, 400—401.  150  Schmidt, C.J,  & Black, C.K.  (1989). The putative 5-HT3  agonist phenylbiguanide induces carrier—mediated release of [ H]dopamine. European Journal of Pharmacology, 3  167,  309—310. Schnur, S.L., Smith, E.R., Lee, R.L., Mas, M., & Davidson, J.M.  (1989). A component analysis of the effects of DPAT  on male rat sexual behavior. Physiology & Behavior, 45, 897—901, Sheard, M.H.  (1969) The effect of p-chlorophenylalanine on  behavior in rats: Relation to brain serotonin and 5— hydroxyindoleacteic acid. Brain Research, ,j5.., Sheard, M.H.  524-528.  (1973). Brain serotonin depletion by p  chiorophenylalanine or lesions of raphe neurons in rats. Physiology & Behavior, Shillito, E.E.  10,  809-811.  (1969). The effect of p-chlorophenylalanine  on social interactions of male rats. British Journal of Pharmacology, Siegel, S.,  ,  193-194.  & Castellan, N.J., Jr.  (1988). Nonparametric  statistics for the behavioral sciences. New York: McGraw— Hill. Sodersten, P., Larsson, K., Ahlenius, S.,  & Engel, J.  (1976). Stimulation of mounting behavior but not lordosis behavior in ovariectomized female rats by p. chiorophenylalanine. Pharmacology Biochemistry & Behavior,  .,  329-333.  151  Sjoerdsma, A.,  & Paifreyman, M.G.  (1990). History of  sérotonin and serotonin disorders. Annals of the New York Academy of Sciences,  &.Q.,  1-8.  Sodersten, P., Berge, O.G., & Hole, K.  (1978). Effects of p  chioroamphetamine and 5,7-dihydroxytryptan’tine on the sexual behavior of gonadectomized male and female rats. Pharmacology, Biochemistry & Behavior, Steinbusch, H.W.M.,  & Nieuwenhuys, R.  ,  499-508.  (1983). The raphe  nuclei of the rat brainstem: A cytoarchitectonic and immunohistochemical study. neuroanatomy (Pp.  In P.C. Emson (Ed.), Chemical  131—206). New York: Raven Press.  Svensson, K., Larsson, K., Ahienius, Carisson, A.  S., Arvidsson, L.—E., &  (1987). Evidence for a facilitatory role of  central 5—HT in male mouse sexual behavior. Dourish, S. Ahienius,  In C.T.  & P.H. Hutson (Eds.), Brain 5-HT1A  receptors. Chichester, U.K.: Ellis Horwood Ltd. Tagliamonte, A., Fratta, W., Mercuro, G., Biggio, G., Cainba, R.C.,  & Gessa, G.L.  tryptophan,  (1972).  5-Hydroxytryptophan, but not  inhibits copulatory behaviour in male rats.  Rivisita di Farmacologia e Terapia, 3, 405-409. Tagliamonte, A., Tagliamonte, P., Gessa, G.L., & Brodie, B.B.  (1969). Compulsive sexual activity induced by p  chlorophenylalanine in normal and pinealectomized male rats. Science, j,  1433—1435.  152  Tanco, S.A., Watson, N.y.,  & Gorzalka, B.B.  (1993a). Lack of  effects of 5—HT3 antagonists on normal and morphine— attenuated sexual behaviours in female and male rats. Experientia, j.,  238-241.  Tanco, S.A., Watson, N.y., & Gorzalka, B.B.  (1993b). Effects  of 5—HT3 aqonists on rat sexual behavior. Manuscript submitted for publication. Tork I.  (1985). Raphe nuclei and serotonin containing  systems. Vol.2 Tork,  I.  In G. Paxinos (Ed.), The rat nervous sytem,  (pp. 43-78). Sydney: Academic Press. (1990). Anatomy of the serotonergic system. Annals  of the New York Academy of Sciences, Uphouse, L., Andrade, M., Moore, N., M.  QQ.,  9-35.  & Caldarola—Pastuszka,  (1993). The 5-HT2/1C agonist, DCI, attenuates the  inhibitory effects of VMN infusions with 8-OH-DPAT on female lordosis behavior. Society for Neuroscience Abstracts, Watson, N.y.,  19,  242.4.  & Gorzalka, B.B.  (1991). DOl-induced  inhibition of copulatory behavior in male rats: Reversal by 5-11T2 antagonists. Pharmacology Biochemistry & Behavior, 37,  825-829.  Watson, N.V., Tanco, S.A.,  & Gorzalka, B.B.  (1991). Effects  of 5—HT3 receptor selective drugs on rat sexual behaviour. Society for Neuroscience Abstracts, j, 59.8.  153  Whalen, R.E., Beach, F.A.,  & Kuehn, R.E.  (1961). Effects of  exogenous androgen on sexually responsive and unresponsive male rats. Endocrinology, Whalen, R.E.,  & Luttge, W.G.  ,  373—380.  (1970). p-Chlorophenylalanine  methyl ester: An aphrodisiac? Science,  1000-1001.  Wieland, S., Kreider, M.S., Mcconigle, P., & Lucki,  I.  (1990). Destruction of the nucleus raphe obscurus and potentiation of serotonin-mediated behaviors following administration of the neurotoxin 3-acetylpyridine. Brain Research,  291—302.  Wing, L.L., Tapson, G.S.,  & Geyer, M.A.  (1990).  5-HT2  mediation of acute behavioral effects of hallucinogens in rats. Psychopharmacology,  100, 417-425.  Yamaguchi, K., Nabeshima, T., Ishikawa, K., Yoshida, S., & Kameyama, T.  (1987). Phencyclidine-induced head-weaving  and head-twitch through interaction with 5-HT1 and 5-HT2 receptors in reserpinized rats. Neuropharmacology,  26,  1489—1497. Yamanouchi, K.,  & Kakeyama, M.  (1992). Effect of medullary  raphe lesions on sexual behavior in male rats with or without treatments of p-chlorophenylalanine. Physiology & Behavior, Yap, C.Y.,  51,  575—579.  & Taylor, D.A.  (1983).  Involvement of 5-HT2  receptors in the wet-dog shake behaviour induced by  154  5-hydroxytryptophan in the rat. Neuropharmacology,  ,  801—804. Zamble, E., Hadad, G.M., Mitchell, J.B., & Cutmore, T.R.H. (1985). Pavlovian conditioning of sexual arousal: Firstand second—order effects. Journal of Experimental Psycho1ocy: Animal Behavior Processes, Zifa, E.,  & Fillion, G.  (1992).  598-610.  5-Hydroxytryptamine  receptors. Pharmacological Reviews,  44,  402—458.  

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