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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: IMPORTANCEOF 5—HT2 RECEPTOR ACTIVITY AND RELATION TO5-HT2-DEPENDENT SEROTONERGIC STEREOTYPYbyNEIL VERNE WATSONB.A., The University of Western Ontario, 1985M.A., The University of Western Ontario, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Psychology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1993© Neil Verne Watson, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of_______________The University of British ColumbiaVancouver, CanadaDate_______DE-6 (2/88)11ABSTRACTIt is well established that the neurotransmitterserotonin participates in the control of sexual behaviour inthe male rat. Recently, it has been found that serotonergicactivity may either inhibit or facilitate sexual behaviour,depending on the subtypes of serotonin receptors involved.However, the participation of 5-HT2 receptors in the controlof male rat copulation has received little experimentalattention, and the published data are equivocal.In Experiments 1-4, it was established that the 5-HT2/1C agonist DCI inhibits sexual behaviour in male rats;this inhibition is effectively reversed by the antagonistsritanserin, pirenperone, and ketanserin. Comparison of theseeffects , with reference to the binding profiles of eachdrug, provided strong evidence that 5-HT2/1C receptorsmediate an inhibitory influence on sexual behaviour in malerats. In addition, a tentative claim may be made that theeffects of these drugs may be more attributable to 5-HT2activity than 5-UT1C activity.‘Wet dog shake’ behaviour in rats is known to be 5-HT2-dependent. Experiments 5—7 evaluated the novel propositionthat the incidence of spontaneous wet dog shaking (WDS) bymale rats in mating tests may provide a behavioural assay ofconcurrent 5—HT2 activity. WDS was found to be associatediiiwith copulatory inhibition in noncopulating males, comparedto normal copulators, and this relationship was specific tomating situations. Activating 5-HT2/1C receptors with DOlsimultaneously induced WDS and inhibited copulation. Thus,the incidence of spontaneous WDS in untreated males mayreflect the function of a 5—HT2—mediated neural mechanismthat tonically inhibits copulation in male rats. InExperiment 8, DOl microinjection in the nucleus rapheobscurus/inferior olivary complex also induced WDS andinhibited copulation. This suggests that the hypothesized 5-flT2-dependent inhibitory mechanism is vested in theventromedial brainstem. Recent anatomical findings supportthis suggestion: cells in this region have bifurcatingaxons, projecting collaterally to both the medial preopticarea (implicated in sexual behaviour) and to the ventralcervical spinal cord (implicated in WDS). Overall, theresults of the eight experiments provide strong evidencethat 5-HT2 receptors mediate some of the inhibitory effectsof serotonin on male rat sexual behaviour.ivTABLE OF CONTENTS.I.bstract. iiTable of Contents . . .. . . . . . . . ivList of Tables viList of Figures viiAckno1edgeent . .. . . . . . . . ixIntroduction 11. Serotonin: Multiple subtypes ofreceptors in the CNS .. . . 62. Serotonin receptor subtypes andmaleratsexualbehaviour 123. Serotonin receptor subtypes andserotonin stereotypy 164. Objectives 19ExperimentsGeneral Method: Experiments 1—4 . . . . . . . . 22Experiment 1 . . . .. . . . . . 26Experiment 2 33Experiment 3. 45Experiment 4. . . . . 63GeneralMethod:Experiments5-7 77Experiment 5 . . . . . . . . . . . . . 80Experinient 6 . .. . . 87Experiment 7... . . . . . 95Experiment 8• 100Discussion 1131. General Discussion: Experiments 1-4 . 1142. General Discussion: Experiments 5—8 .. 1213. Conclusions, speculations, andimplications for future research 126References. . . . . . . . 134VviLIST OF TABLESTable 1, The effects of increasing doses ofritanserin on the copulatory behaviourof male Sprague—Dawley rats 40—41Table 2. The effectiveness of pirenperone inreversing DOl-induced inhibition ofsexual behaviour in male Sprague—Dawley rats 57—58Table 3. The effectiveness of pirenperone inreversing DOl-induced inhibition ofsexual behaviour in male Long-Evansrats. . . . . . . 59—60Table 4. The effectiveness of ketanserin inreversing DOI-induced inhibition ofsexual behaviour in male Sprague—D awley rats . . . . . . . 72—73Table 5. The effectiveness of ketanserin inreversing DOl-induced inhibition ofsexual behaviour in male Long—Evansrats. . 74—75LIST OF FIGURESFigure 1. Proportion of males displaying mounts,intromissions, or ejaculations followingtreatment with DOI 31—32Figure 2. Effectiveness of ritanserin in reversingDOl-induced inhibition of sexual behaviourinmale rats 42—43Figure 3. Effectiveness of pirenperone in reversingDOl-induced inhibition of sexual behaviourin male Sprague—Dawley rats 50—51Figure 4. Effectiveness of pirenperone in reversingDOl-induced inhibition of sexual behaviourin male Long—Evans rats 52—53Figure 5. Effectiveness of ketanserin in reversingDOl-induced inhibition of sexual behaviourin male Sprague—Dawley rats . . . . . . 66—67Figure 6. Effectiveness of ketanserin in reversingDOl-induced inhibition of sexual behaviourinmaleLong—Evansrats . 68—69Figure 7, Incidence of WDS in male rats of varyingcopulatory proficiency . .. 84—85Figure 8. Comparison of spontaneous WDS in malerats paired with differing types ofpartners. . . . . . 91—92viiFigure 9. Effects of DOl treatment on concurrentWDS and ejaculations 97—98Figure 10. Intracerebral microinjection ofDOl into the region of the rapheobscurus and inferior olivary complex:Effects on WDS and ejaculatorybehaviourinmalerats ...110—111viiiixACKNOWLEDGMENTI am indebted to my supervisor, Dr. Boris Gorzalka, forhis advice, encouragement and friendship. Thanks also to thefaculty of the Biopsychology area, particularly Drs. DaveAlbert, Ariane Coury, and John Pinel. Dr. Doreen Kimura ofU.w.O. introduced me to biopsychology, and has been myfriend and mentor throughout my academic career.Fellow graduate students have been a source of supportand stimulation in far too many ways to catalogue. This isespecially true of (in no particular order!): Ingrid Moe,Bill Mah, Sheryl Tanco, Georg Schulze, Scott Mendelson, andTheresa Newlove. I particularly wish to single out David(Jimmeh) Carey, most recently of St. Andrews University, formy thanks—- his e-mail nagging, while we were writing ourtheses in parallel, kept me going at times when I thoughtthe well was dryLastly, I am fortunate to have family who have beentolerant of my somewhat mercurial behaviour—— my brotherPete, Ingrid & Nic Cuk and family, and most especially myparents, Kathleen and Dennis, to whom I owe so much.This thesis is dedicated to my wife, Maria, thelove of my life, and the answer to an ancient question.S’tu ma cholil.1INTRODUCT IONHavelock Ellis (1898) maintained that “Sex lies at theroot of life, and we can never learn to reverence life untilwe know how to understand sex” (preface, p. xxx). Althoughone may dispute Ellis’ lofty ambitions for the pursuit, itis axiomatic that the study of sexual behaviour is the studyof a fundamental life process of major adaptive andorganizational significance across species. The sexualinteraction of animals, and the exchange of genetic materialthat ensues, is the currency of natural selection and thebasis of biological diversity. Comprehensive understandingof the control of sexual behaviour has remained elusive.Attempts to specify the physiological determinants ofsexual behaviour have frequently focussed on the copulatorybehaviour of the laboratory rat. Males and females of thisspecies display highly stereotyped patterns of matingbehaviour that are easily and reliably quantified andtherefore are potentially sensitive to experimentalmanipulations. When a male rat encounters a sexuallyreceptive female rat, copulation proceeds in a highlyregular manner (Beach, 1956; Sachs & Barfield, 1976). Themale grasps the female’s flanks, mounts her, and commencespelvic thrusting. In response to this tactile stimulation bythe male, the female adopts the lordosis posture, arching2her back, elevating her rump and diverting her taillaterally. A typical copulatory bout consists of a series ofsuch mounts, during some of which penile intromission of thevagina occurs. After a number of intromissions (usuallybetween 6 and 10), the male ejaculates intravaginally. Thecopulatory bout is followed by a brief period ofrefractoriness in the male, following which a new copulatorybout is commenced. Normal male rats will usually completebetween three and five copulatory bouts in 1 hour. The threemale copulatory behaviours—— mounts, intromissions, andejaculations -- are readily and reliably discriminated fromone another, and they form the basis of a variety ofmeasures of copulatory behaviour in the male rat (describedin General Method: Experiment 1-4).Early work on the neurochemical bases of male ratsexual behaviour particularly implicated the monoamineneurotransmitters: 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 toproduce an overall inhibition of the sexual behaviour ofmale rats (e.g. Dewsbury, Davis, & Jansen, 1972; Maimnas &Meyerson, 1970). MAO inhibitors exert their effects byinterfering with the enzyme that renders monoamine moleculesinert, resulting in supranormal concentrations of these3transmitters in brain tissue. Consequently, the findingsthat MAO inhibitors reduce male rat sexual behaviour weretaken as evidence that increased monoaluinergic activity hasa 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 apparentwith the development of methods for selectively altering 5-HT concentrations in the brain. When rats are treated withthe metabolic precursor of serotonin, 5-aT?(5-hydroxytryptophan), plus an enzyme inhibitor thatprevents it from being metabolized peripherally, serotoninsynthesis in the central nervous system (CNS) is acceleratedand levels of 5-HT in the brain become elevated. Thistreatment has been repeatedly reported to inhibit copulatorybehaviour in male rats (e.g., Ahienius & Larsson, 1984;Ahlenius, Larsson, & Svensson, 1980; Gessa, 1970; Maimnas &Meyerson, 1971; Tagliamonte, Fratta, Mercuro, Biggio, &Cainba, 1972), suggesting a general inhibitory influence ofserotonin on male rat sexual behaviour. Similarly, it wasreported that the sexual behaviour of male rats wasinhibited by treatment with PCA (p-chloroamphetamine), whichinduces serotonergic neurons to release larger than normalquantities of 5-HT (Sodersten, Berge, & Hole, 1978).4Serotonin reuptake inhibitors are drugs that preventserotonergic neurons from reabsorbing serotonin after it hasbeen released, resulting in supranormal serotoninconcentrations in the brain. Treatment of male rats withdrugs 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 beenreported to inhibit mating behaviour in male rats.The drug PCPA (para-chiorophenylalanine) blocks theactions of the rate-limiting enzyme in the synthesis ofserotonin; administration of a few large doses of PCPAproduces rats whose brains are nearly devoid of serotonin(Koe & Weissman, 1966). Dozens of reports in the late 1960’sand 1970’s, using a variety of experimental preparations,noted a facilitation of copulatory behaviour in male rats asa 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, 1976; Dahiof,1980). In some cases, however, PCPA was found to have littleor no effect, especially in sexually—experienced male rats(McIntosh & Barfield, 1984; Whalen & Luttge, 1970).5The availability of serotonin in the CNS may also bereduced by the administration of neurotoxins thatselectively destroy serotonergic neurons. When injected intothe lateral ventricles of the brain, which are filled withcerebrospinal fluid that circulates throughout the CNS, theserotonergic neurotoxin 5, 6-DHT (5 ,6-dihydroxytryptamine)was found to induce a general facilitation of male ratsexual behaviour (Da Prada, Carruba, O’Brien, Saner &Pletscher, 1972). A similar facilitation was reported withthe closely related neurotoxin 5,7-DHT, when injected intoeither the lateral ventricles (Sodersten et al., 1978;McIntosh & Barfield, 1984) or directly into the region ofdorsal and median raphe nuclei, which contain large numbersof serotonergic cells (Larsson, Fuxe, Everitt, Holmgren, &Sodersten, 1978; Rodriguez, Castro, Hernandez & Mas, 1984).The direct application of 5-lIT into the raphe nuclei causesa decrease in serotonin release through a feedbackmechanism, and it also facilitates copulatory behaviour inmale rats (Hillegaart, Ahienius, & Larsson, 1989).Taken together, investigations in which brain-wideserotonin levels have been manipulated have led to thegeneral conclusion that serotonergic activity in the CNSinhibits copulation in male rats. Male rats that receivedtreatments that induced serotonin accumulation in the braindisplayed deficient sexual behaviour, and males whose brains6were depleted of serotonin displayed facilitated copulation.Pharmacological and physiological discoveries since 1980,however, have revealed a remarkable degree of diversitywithin the serotonergic systems of the CNS, and it nowappears that the general conclusion that serotonin inhibitssexual behaviour is too simple.1. Serotonin: Multiple subtypes of receptors in the CNSSerotonin derives its name from its “serum tonic”vasoconstrictive effects; it was for these effects that 5-HTwas first studied (for review: Sjoerdsma & Palfreyman, 1990;Zifa & Fillion, 1992). By the 1950s, 5-UT had beendemonstrated to be heterogeneously distributed in the CNSand to bind to at least two different kinds of receptors inperipheral tissues (Gaddum & Picarelli, 1957). During theperiod from the 195Os to the 1970s, studies usingpharmacological tools that altered brain-wide serotoninconcentrations revealed that serotonin plays a role in suchdiverse 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 serotonergicparticipation in the control of behaviour has begun toemerge, as multiple subtypes of receptors for 5-HT have been7described in the CNS and their functional significanceinvestigated. Molecules of serotonin, which serve aschemical messengers in the CNS, may be released from theterminals or axonal varicosities of serotonergic neurons.Like other neurotransmitters, serotonins actions on thesurface of an individual neuron alters its excitability. Inorder to do this, serotonin binds to membrane—boundproteins, or receptors, on the surface of the cell. Thesereceptors have an electrochemical configuration that iscomplementary to that of the serotonin molecule; as aresult, the receptor is particularly selective forserotonin, and is less likely to bind other types ofmolecules. When serotonin binds to the receptor, itselectrochemical interaction with the receptor causes changeswithin the neuron that alter its excitability, that is,alter the probability that it will release its ownneurotransmitter) (for review: Kandel & Schwartz, 1992).The first intimations that more than one type ofserotonin receptor exists on CNS neurons came with theadvent of radioligand binding analyses (see, e.g., Lyon &Titeler, 1988). A ligand’ is a substance that has affinityfor the type of receptor being investigated. In radioligandbinding analysis, homogenized brain tissue is incubated witha radioactive form of a ligand for the type of receptors inquestion. In the case of serotonin receptors, this might be8radioactive serotonin ([3H]5-HT) or a radiolabelled form ofa drug that mimics serotonin. The labelled compoundselectively binds to the serotonin receptors in the braintissue, and any leftover radioactive material is rinsedaway. In a binding competition analysis, which is the mostcommon type, a nonradioactive compound that selectivelybinds to the same type of receptors as the labelled compoundis added to the brain homogenate in increasingconcentrations. If the unlabelled competitor has a highaffinity for the same type of receptors as the labelledcompound, it will effectively displace the labelled compoundeven at low concentrations. If the test compound’s affinityfor the labelled receptors is low, high concentrations willbe required to displace the labelled compound from thereceptor sites.Mathematical analyses of the manner in which drugscompete for labelled receptors reveal two things: theaffinity of the test compound for the type of receptor thatis labelled, and the selectivity of the test compound acrossdifferent types of receptors. Importantly, the pattern ofthese two characteristics may suggest the presence of morethan one subtype of receptor that is labelled by theradioactive substance. An additional consideration with anynew ligand is its intrinsic activity. When a drug binds to areceptor, its ability to produce the same effect as the9endogenous ligand (e.g., serotonin) reflects its intrinsicactivity. If the drug ‘turns on’ the receptor, therebyaltering the activity of the cell, it has high intrinsicactivity and is called a receptor agonist. If, instead, thedrug binds with high affinity but simply blocks thereceptor, preventing any other ligand from activating it,the drug has low intrinsic activity and is called a receptorantagonist. Drugs that have moderate intrinsic activityproduce partial activation of the receptors and are referredto as partial agonists (or partial antagonists) (see, e.g.,Kalant, Roschlau, & Sellers, 1985).In the early 1980 us, this type of radioligand bindinganalysis was applied to binding at serotonin receptors. Bycomparing the efficacy of unlabelled serotonin in displacinglabelled serotonin and labelled spiperone, which binds toserotonin receptors, with the efficacy of unlabelledspiperone in the same preparation, Peroutka and hiscollaborators (Peroutka & Snyder, 1979; Peroutka, Lebovitz,& Snyder, 1981) found that there existed two populations ofreceptors which, while differing in their pharmacologicalproperties and anatomical distribution, were neverthelesssubtypes of serotonin receptors. These they named the. 5-HT1receptor (displaying high affinity for labelled 5-HT) andthe 5-11T2 receptor (displaying high affinity for labelledspiperone).10In the last decade or so, further binding studies haverevealed a remarkable profusion of different subtypes ofserotonin binding sites in the CNS. Recent accounts suggestthat there may be as many as 11 distinct subtypes of 5-HTbinding sites, and evidence of new subtypes continues toaccumulate (Bradley, Handley, Cooper, Key, Barnes, & Coote,1992; Frazer, Maayani, & Wolfe, 1990; Glennon & Dukat, 1991;Zifa & Fillion, 1992). In many cases, however, these bindingsites have been characterized only pharmacologically, inisolated tissue preparations, and their functionalsignificance remains unknown. Drugs that are truly selectivefor particular subtypes of serotonin receptors in the intactorganism have yet to be developed, making it difficult toattribute particular functions to modulation of activity atparticular 5-HT receptor subtypes (Glennon & Dukat, 1991;Zifa & Fillion, 1992). Nevertheless, ligands that aremoderately selective do exist, and they have been used forin vivo studies of the functions of the six best-established5-HT receptor subtypes: the 5-HT1A, 1B, 1C, and 10receptors; 5-HT2 receptors; and 5-HT3 receptors (Bradley etal., 1992; Glennon & Dukat, 1991; Zifa & Fillion, 1992).Quantitative autoradiography has demonstrated that thesesubtypes of 5-HT receptors are differentially distributedthroughout the brain (e.g., Palacios, Waeber, Hoyer, &Mengod, 1990); however, it is important to keep in mind that11the distribution of the various subtypes of serotoninreceptors has provided little indication of their respectivefunctions.It should be mentioned that pharmacological data suggestthe existence of multiple subtypes of 5-HT2 binding sites,having in common a phosphoinositol hydrolysis secondmessenger system. In particular, both the classical 5—HT2receptor and the 5-HT1C receptor belong to this class (e.g.,Glennon & Dukat, 1991; Hoyer, 1988b, 1992). Indeed, cloningstudies have revealed an 80% homology between the 5-HT2 and5-HT1C receptor subtypes (Hartig, 1989; Glennon & Dukat,1991). Given the great physical similarity of these tworeceptor subtypes, it is not surprising that ligands haveyet to be developed that can potently differentiate betweenthem (Hoyer, 1988b, 1992; Glennon & Dukat, 1991) -- drugsthat bind to 5-HT2 receptors also tend to bind to 5-HT1Creceptors with high affinity. However, because eachserotonergic drug possesses its own unique profile ofaffinities for the various 5-HT receptor subtypes, comparingthe behavioural effects of a spectrum of serotonergic drugs,that share affinity for a target subtype of receptor butdiffer in affinity for other types of receptors, does allowlimited inferences about the behavioural roles of the targetsubtype. In particular, pharmacological probes have becomeavailable in recent years that provide a means of specifying12some behavioural effects of 5-HT2/1C receptor activity, andto a much more limited extent allow distinctions to be madebetween 5-HT2 and 5-HT1C-mediated functions (reviewed inBradley et al., 1992; Zifa & Fillion, 1992).2. Serotonin receptor subtypes and male rat sexualbehaviour.Although early work suggested a general inhibitoryeffect of serotonergic activity on the sexual behaviour ofmale rats, in the last 12 years or so research usingreceptor-selective ligands has suggested that 5-HT mayinhibit or facilitate male rat copulation, depending on thesubtypes of receptors involved (reviewed in Gorzalka,Mendelson, & Watson, 1990; Zifa & Fillion, 1992).The majority of this work has focussed on the roles ofsubtypes of 5—HT1 receptors in the control of male ratcopulation. In particular, treatment of male rats with drugsthat are agonists at the 5-HT1A site has been found toproduce a net facilitation of sexual behaviour. Doses of theselective 5-HT1A agonist 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] ranging from 0.025 to 4.0 mg/kg areuniformly facilitatory, in some cases producing male ratsthat ejaculate on the first intromission of a copulatorybout (Ahlenius & Larson, 1984a, 1984b, 1985; Ahienius,Larsson, Svensson, Hjorth, Carlsson, Lindberg, et al., 1981;13Dahiof, Ahlenius, & Larsson, 1988; Mendelson & Gorzalka,1986; Morali & Larsson, 1984; Schnur, Smith, Lee, Mas, &Davidson, 1989). Administration of the related compound8-OMe-DPAT is reported to produce a comparable facilitation(Ahlenius et al., 1981). Treatment with 8-OH-DPAT has beenreported to restore copulatory function in castrates and inmales that have undergone penile deaf ferentation (Dahlof etal., 1988). Recent evidence suggests that the stimulatoryeffects of 8-OH-DPAT are effectively reversed by theselective 5-HT1A receptor antagonist UH-301 (Johansson,Meyerson, & Hacksell, 1991), supporting the conclusion that5-HT1A receptors mediate a facilitation of male ratcopulatory behaviour. Interestingly, there also exists amarked sex difference in the effects of 8-OH-DPAT on sexualbehaviour; in female rats, 8-OH-DPAT is reportedlyinhibitory (e.g., Mendelson & Gorzalka, 1986). In agreementwith the data collected using 8-OH-DPAT, facilitation ofsexual behaviour has also been observed in male rats treatedwith the less selective 5-HT1A agonists lisuride (Ahienius,Larsson, & Svensson, 1980; Fernandez—Guasti, Hansen, Archer,& Jonsson, 1986) and RDS-127 (Clark, Smith, Stefanick,Arneric, Long, & Davidson, 1982). The 5-HT1A partialagonists buspirone (Mathes, Smith, Popa, & Davidson, 1990)and ipsapirone (Fernandez-Guasti, Escalante, & Agmo, 1989)likewise appear to facilitate copulation in male rats, as14does the novel 5-HT1A agonist indorenate (Fernandez-Guasti,Escalante, Hong, & Agmo, 1990).The 5-HT agonists TFMPP and mCPP display mixedaffinities, but particularly bind with high affinity to5-HT1C sites. Administration of these drugs to male rats hasbeen reported to inhibit their sexual behaviour (Fernandez—Guasti et al., 1989; Fernandez-Guasti & Rodriguez-Manzo,1992; Mendelson & Gorzalka, 1990). The mixed 5-HT1A/1Breceptor agonist RU 24969 reportedly also produces aninhibition of copulation in male rats, presumably due to itsactivity at 5-HT1B receptors given that 5-HT1A receptoragonists are facilitatory (Fernandez-Guasti et al., 1989;Mendelson & Gorzalka, 1988).Recent evidence suggests that brain 5—HT3 receptorsplay no role in regulating reproductive behaviour in rats.We have found that the highly selective 5-HT3 receptorantagonists ondansetron and granisetron are without effectin 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 malerats, and although the 5-HT3 agonist 1-PBG(1-phenylbiguanide) induces a slight facilitation in matingtests (Watson, Tanco & Corzalka, 1991; Tanco, Watson, &Gorzalka, 1993b), this effect may be attributable to 1-PBG-induced dopamine release (Schmidt & Black, 1989).15The present thesis is concerned with some possiblefunctional effects of 5-HT2 receptor activity. In contrastto the attention that has been directed at the roles of5-HT1 receptors on the regulation of copulatory behaviour inthe male rat, relatively few studies have directlyinvestigated the effects of 5-HT2/1C-selective compounds.The data that have been collected thus far have beenequivocal, and in some cases contradictory, and arediscussed in greater detail in the introductory sections ofExperiments 1-4. Briefly, the 5-HT2/1C receptor antagonistsketanserin and pirenperone have been reported to inhibitmale rat sexual behaviour (Mendelson & Gorzalka, 1985),suggesting that 5-HT2/1C receptors mediate a facilitation ofsexual behaviour in the male rat. Conversely, the 5-HT2/1Cantagonists cyproheptadine and LY 53857 reportedlyfacilitate male rat copulation, suggesting that 5-HT2/1Cactivation is inhibitory (Abraham, Viesca, Plaza, & Mann,1988; Foreman, Hall, & Love, 1989). This latter conclusionis supported by the report that the 5-HT2/1C receptoragonist DOl [1-(2 ,5-dimethoxy-4-iodophenyl)-2-aminopropane]inhibits copulation in male rats (Foreman et al., 1989).Given the contradictory conclusions of the publishedstudies, therefore, one of the major objectives of thepresent dissertation is to attempt to clarify the role of5-HT2 receptor activity in male rat sexual behaviour.163. Serotonin receptor subtypes and serotonin stereotypy.It has been known for some time that a highlystereotyped “Serotonin Behavioural Syndrome” ensues whenrats receive treatments that augment serotonergic activityin the brain (Come, Pickering, & Warner, 1963; GrahameSmith, 1971). This is most frequently accomplished by givingrats large doses of the metabolic precursor of serotonin,5-HTP, plus an inhibitor of peripheral decarboxylation suchas benserazide or carbidopa, or by giving the animals largeamounts of the amino acid tryptophan plus an MAO inhibitor.The syndrome consists of a variety of bizarre motorpatterns, including reciprocal forepaw treading, Straub tail(a stiff or “poker” tail), abnormal hindlimb abduction,flattened body posture, and ‘wet dog shaking’ (WDS), It ispresumed 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 refiningthe pharmacology of the multiple subtypes of CNS receptorsfor serotonin have been made in the last decade, but theeffects of activity at these receptor subtypes on overtbehaviour remains largely uncharacterized. Nevertheless,evidence has accumulated that the different elements of theserotonin behavioural syndrome are mediated by different175-HT receptor subtypes (for review: Green & Heal, 1985). Inparticular, it appears that WDS is primarily mediated by theactivation of 5-HT2 receptors (Green, 1989; Green & Heal,1985; Pranzatelli, 1990; Yap & Taylor, 1983). WDS may bedefined as a paroxysmal, rapid rotational shudder of thehead and shoulders; as its name suggests, the behaviour isreminiscent 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 ofthe serotonin behavioural syndrome by administration of 5-fiTagonists which act at 5-HT2 receptors, such as thenonselective compounds LSD and quipazine (Green & Heal,1985; Lucki & Minugh-Purvis, 1987) and the more highlyselective agonist DOl (Darmani, Martin, Pandey, & Glennon,1990; Pranzatelli, 1990). Conversely, 5—HT antagonists suchas ritanserin, ketanserin, mianserin, methysergide,pirenperone, cinanserin, cyproheptadine, and pizotifen, allof which display some selectivity for 5-11T2 receptors, havegenerally been found to inhibit the production of WDS(Colpaert & Janssen, 1983; Darmani, et al., 1990; Lucki,Eberle, & Minugh-Purvis, 1987; Meert, Niemegeers, Gelders, &Janssen, 1989; Pranzatelli, 1990; Yamaguchi, Nabeshima,Ishikawa, Yoshida, & Kameyama, 1987).WDS has also been reported as a consequence of a varietyof experimental treatments that are not direct manipulations18of 5-HT2 activity. For example, increased WDS has beenreported 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 hormoneanalogue CC 3509 (Fone, Johnson, Bennett, & Marsden, 1989).Interestingly, large systemic injections of putrescine (asubstance produced by decaying bodies!) have also beenreported to induce WDS (Genendani, Bernardi, Tagliavini,Botticelli, & Bertolini, 1987). WDS has also been reportedas a consequence of hippocampal stimulation (Araki & Aihara,1986). Nevertheless, where it has been tested, modulation ofcentral 5-HT activity has frequently been implicated in WOSproduced by non5-HT manipulations (e.g., Pranzatelli, 1989;Fone et al., 1989; Kleinrok & Juskiewicz, 1986; Nabeshima,et al., 1987; Yameguchi, et al. 1987). Overall, the evidencegenerally supports the conclusion that WDS is mediated by5—HT2 receptors, with other transmitters or mechanismsserving ancillary or permissive functions (Green & Heal,1985). It is for this reason that pharmacologically inducedWDS, in conjunction with the serotonin behavioural syndrome,has gained some acceptance as a model of neuronalserotonergic activity. Examination of the effectiveness of19novel psychoactive compounds in modulating the expression ofWDS has gained acceptance as an index of central 5-HT2receptor activity (e.g., Meert et al., 1989; Pranzatelli,1990; Yap & Taylor, 1983).4. ObiectivesAs was discussed earlier, the existing data stronglysuggest serotonergic participation in the control of malerat sexual behaviour, but the way in which subtypes ofserotonin receptors contribute to this regulation is poorlyunderstood. In particular, the effects of changes inactivity at 5-HT2 receptors on the reproductive behaviour ofmale rats have received little attention. Therefore, theoverall objective of the present dissertation was to attemptto elucidate the role of 5-HT2 activity in male rat sexualbehaviour. This goal was formulated as three generalobjectives.a) The two published studies that had focussed on thecontributions of 5-HT2 receptor activity in the control ofthe reproductive behaviour of male rats had led tocontradictory conclusions. Accordingly, the first fourexperimental sections of this dissertation report theresults of studies examining the effects of a spectrum of5—HT2 receptor ligands on sexual behaviour in male rats. Theagents employed in these studies were selected on the basis20of common affinity for 5-HT2 (and in some cases, 5-HT1C)receptors, but different affinities for other types ofreceptors, in an effort to reconcile the conflicting datareported by others.b) In Experiments 5-7, the issue of 5-HT2 receptormediation of male rat sexual behaviour was addressed in anentirely behavioural fashion by incorporating behaviouralmeasures of central serotonergic activity with measures ofmale rat copulatory behaviour. Informal observationssuggested to me that untreated normal rats spontaneouslydisplay WDS while engaging in other types of behaviour. Inthis set of experiments, I evaluated a novel approach inwhich WDS was employed as a behavioural index of 5-HT2receptor activation during copulation. Given the establisheddependency of WDS on 5-HT2 receptor activity, it seemedreasonable to suppose that the occurrence of spontaneous WDScould constitutea “behavioural assay” of concurrent 5-HT2activation that would converge on the pharmacological data.In Experiment 5-7, measures of .WDS by male rats were takenduring various types of mating tests.c) Very little is known about the participation ofbrainstem mechanisms in the control of male rat sexualbehaviour; most work has focussed on the critical importanceof more rostral structures, such as the medial preoptic areaand rostral midbrain (e.g., Rose, 1990). Likewise, the21neural substrate of WDS has yet to be established. In aninitial attempt to identify neural sites involved in thecontrol of WDS and sexual behaviour, measures of thesebehaviours were taken in male rats after direct manipulationof 5-HT2 receptor activity in the ventromedial brainstem, inExperiment 8. The degree to which WDS and male rat sexualbehaviour rely on a common neural substrate was alsoevaluated.22GENERAL METHOD: EXPERIMENTS 1-4AnimalsMale and female Sprague—Dawley and Long—Evans rats werebred from stock originally obtained from Charles RiverCanada Inc., Montreal. Rats were housed in our colonies, ingroups of 6, in standard wire mesh cages, segregated by sexand strain. Free access to food and water was provided.Colony rooms were maintained on a reversed 12/12 hrlight/dark cycle.At approximately 70 days of age, females underwentbilateral ovariectomy while under sodium pentobarbitalanaesthesia (65 mg/kg). Males were between 90 and 180 daysof age when tested. All males used in these experiments weresexually experienced, having been exposed to receptivefemales on at least three occasions prior to testing.Steroid treatmentsReceptivity was induced in the ovariectomized females bysubcutaneous injection of 10 [tg estradiol benzoate(Steraloids Inc.) 48 hr prior to testing, and 500 gprogesterone (Steraloids Inc.) 4 hr prior to testing.Steroids were dissolved in 0.1 ml peanut oil.23Behavioural TestingWith the aid of a microcomputer program (Holmes, Holmes,& Sachs, 1988), the copulatory behaviours of male rats wererecorded after they had been presented with a receptivefemale rat. Behavioural recording was performed by anobserver blind to experimental hypotheses and treatmentconditions. All testing was conducting during the middle 1/3of the rats’ dark cycle, at which time rats are most active.Males were placed individually into clear Plexiglastesting arenas measuring 30 cm in width and length and 45 cmin height, and containing a layer of fresh San-i-cel beddingmaterial. A period of 5 mm was then allowed for the malesto habituate to their new surroundings. During each session,4 or 5 males were scored simultaneously, in separate testingchambers.Following the habituation period, receptive females wereintroduced into the males’ testing chambers and recording ofthe males’ copulatory behaviours commenced. For eachsession, the proportion of males displaying mounts (%M),intromissions (%I), or ejaculations (%E) was quantified. InExperiments 3 and 4 the following additional measures ofmale rat copulatory behaviour were recorded: the number ofmounts (M) and intromissions (I) preceding ejaculation; thelatency from presentation of the stimulus female to theoccurrence of the first mount (ML) or intromission (IL) by24the male; the latency from the first intromission toejaculation (EL); and the postejaculatory interval (PEI)between ejaculation and the first intromission of the nextcopulatory bout. Scoring of the males sexual behaviour wasdiscontinued either after the first intromission of thesecond copulatory bout, or after a maximum of 30 mm hadelapsed. Stimulus females were rotated among the males every10 mm during the test session.Under some experimental conditions, male rats fail toachieve an ejaculation, or exhibit no sexual behaviourwhatsoever. In such cases, behaviour counts such as M, I,and E were set to zero. Missing latency scores (ML, IL, ELand PEI) were assigned the maximum value possible: 1800 sec.Additionally, because group variances of 0.0 grossly violatethe assumption of normalcy in parametric tests, such asanalysis of variance, the data were analysed usingnonparametric 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 inproportions across multiple groups. In the event of astatistically significant Cochran Q test, the significanceof the differences between individual pairs of group25proportions was assessed with paired McNemar tests. Allother behavioural measures were submitted to a Friedmannonparametric analysis of variance for k related samples.Significant Friedman tests were followed by Wilcoxon pairedcomparisons.26EXPERIMENTSExperiment 1Although many reports have appeared regarding theeffects of potentiated overall serotonergic transmission onthe sexual behaviour of male rats, few data have beencollected using compounds that selectively bind to, andactivate, 5-HT2 receptors. Agonists and antagonists for5-HT2 receptors have been either characterized orsynthesized only relatively recently, and even thesecompounds exhibit the problem, discussed earlier, ofcomparable binding to 5-HT2 and 5-HT1C receptors (Glennon &Dukat, 1991; Hoyer, 1988a,b).The 5—fiT agonist quipazine has been reported to induce anet facilitation of male rat sexual behaviour (Mendelson &Gorzalka, 1985). This finding was attributed to 5-HT2receptor activation, on the basis of functional analysesavailable at that time which suggested that quipazineexhibits 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-HT2receptor activity, as more recent binding analyses haverevealed that quipazine shows little selectivity between the27various 5-HT receptor subtypes (Glennon & Dukat, 1991;Hoyer, 1988a; Peroutka & Hamik, 1988; Perry, 1990). In fact,[3H]quipazine is now used in radioligand binding studies of5-HT3 receptors, suggesting that, if anything, it issomewhat more selective for 5-HT3 receptors than for 5—HT2receptors (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 aciddiethylamide], are now believed to exhibit a degree ofselectivity for 5-HT2/1C binding sites (Glennon, 1990; Heym& Jacobs, 1988; Wing, Tapson & Geyer, 1990; Zifa & Fillion,1992). Administration of these compounds to male rats hasbeen reported to have no effect on their copulatorybehaviour (Ahienius et al., 1981). However, it should benoted that while LSD appears to bind with some selectivityto 5-HT2/1C receptors, its activity at those receptors isdisputed. Although LSD has most frequently been reported toact as an agonist , evidence also exists suggesting anantagonistic action of LSD at 5-HT2 receptors (eg. Heym &Jacobs, 1988).More recently, it has been reported that treatment withthe serotonin agonist DOl [1-(2 , 5-dimethoxy-4-iodophenyl)-2-aiuinopropane] induces a net inhibition of copulatorybehaviour in male rats (Foreman et al., 1989). DOl is aprototypic 5-HT2/1C agonist (Dabiré, Chaouche-Teyara,28Cherqui, Fournier, Laubie, & Schmitt, 1989; Glennon & Dukat,1991; Glennon, McKenney, Lyon, & Titeler, 1986), and thusexhibits high and selective affinity for 5-HT2 and 5-HT1Csites (Hoyer, 1988a). When administered to male rats indoses ranging from 0.1 mg/kg to 1.0 mg/kg, DOl has beenreported to systematically reduce or eliminate copulatorybehaviour (Foreman et al., 1989). In view of theinconsistent findings regarding the effects of 5-HT2receptor stimulation on male rat sexual behaviour, theobjectives of Experiment 1 were to confirm the inhibitoryaction of DOl and to establish an effective dose of DOl foruse in the subsequent experiments.MethodDrugsDOl HC1 was obtained from Research Biochemicals, Inc.,Natick, MA, and stored at 5° C, protected from light. Freshsolutions of DOl were prepared before each test session,dissolved in sterile physiological saline and administeredsubcutaneously (SC) in a total volume of 1.0 ml per kg ofbody weight (mi/kg). DOl was administered 30 mm prior totesting (Foreman et al., 1989).29ProceduresIn Experiment 1, 20 sexually-experienced Sprague Dawleymale rats were tested following administration of 0.0, 0.1,or 1.0 mg/kg DOl SC. Doses were administered in a randomcounterbalanced fashion over three consecutive sessions,such that by the end of the experiment the sexual behaviourof each male (% M, %I and %E) had been evaluated once ateach dose. An interval of 1 week separated successive testsessions.ResultsThe results of Experiment 1 are presented in Figure 1,in the form of dose—response curves for each behaviouralmeasure. One rat failed to initiate normal copulatorybehaviour under any treatment condition, and was excludedfrom analyses.Treatment with DOl inhibited the sexual behaviour of themale rats in a dose—dependent manner (Fig 1). Cochran Qanalyses revealed a significant decrement in the proportionsof males exhibiting mounts (Q(2) = 21.0, p < 0.0001),intromissions (Q(2) = 16.9, p = 0.0002), and ejaculations(Q(2) = 13.0, p = 0.0015). Subsequent McNemar tests revealedthat this inhibition was most pronounced in the 1.0 mg/kgDOl condition (see Fig 1 for group comparisons). Indeed,30after 1.0 mg/kg DCI, only one rat displayed mounting andintromitting behaviour, and no animal achieved ejaculation.DiscussionThe results of Experiment 1 confirm the previous finding(Foreman et al., 1989) that DCI induces a profoundinhibition of copulatory behaviour in male rats, andinformal observation suggested that this inhibition did notreflect a generalized motor deficit. To the extent that theeffects of DCI on copulatory behaviour are attributable to5-HT2 specific activation, the data provide preliminaryevidence that 5-HT2 receptors mediate an inhibitoryinfluence on copulation in male rats. Additionally, theresults provide an empirical basis for selecting 1.0mg/kgDCI as an effective dose for use in the subsequentexperiments of this series.31Fiaure 1. Proportion of male rats displaying mounts,intromissions or ejaculations following treatment withvarying 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 < .05(McNemar test).0 > ci 0 -c C.) Q a) C >‘ 0 ci)-o (I) 0* *100• 80-60-40 20 0•*** D.*0.00—0Mounts—IntromissionsQEjaculations0.1DoseofDOl,mg/kgs.c.1.0t’j33Experiment 2In Experiment 1, the administration of DOl to male ratswas found to profoundly inhibit their copulatory behaviour.The binding profile of DOl suggests that its inhibitoryeffect on male rat sexual behaviour is attributable to thehigh affinity of DOl for 5-HT2 and 5-HT1C receptors (Hoyer,1988a). One method of substantiating this interpretationwould be to examine the reversibility of the effect of DOl,using other pharmacological probes that share high affinityand selectivity for 5-HT2/1C receptors. This may beaccomplished by administering DOI and coadministering a5-HT2/1C antagonist, which blocks receptors rather thanactivating them. Like all receptor-selective drugs, theseantagonists display unique affinity profiles across avariety of types of receptors, but nevertheless display highaffinity for 5-11T2/1C receptors, in common with DOl (forreview: Hoyer, 1988a, b; Jansson, 1983; Glennon & Dukat,1991; Zifa & Fillion, 1992).Previous work with 5-HT2 antagonists has yieldedequivocal results. Drugs of this class bind to 5-HT2/1Creceptors without activating them, and thereby reduceactivity at these receptor populations to subnormal levels.Inhibition of sexual behaviour after treatment with a5-HT2/1C antagonist would therefore imply that, in the34untreated male rat, 5-HT2/1C receptors mediate afacilitation of copulatory behaviour. Conversely,facilitation of sexual behaviour after such treatment wouldimply that 5-HT2/1C receptors mediate an inhibition ofsexual behaviour in male rats.Although the 5-HT antagonist pirenperone was initiallyreported to be without effect on male rat sexual behaviour(Ahienius & Larsson, 1984), later studies have reported aninhibition of sexual behaviour in male rats followingpirenperone treatment (Mendelson & Gorzalka, 1985; Foremanet al., 1989). A similar inhibition has been reported withthe structurally related compound ketanserin (Mendelson &Gorzalka, 1985). Pirenperone and ketanserin are serotoninreceptor antagonists displaying high affinity for 5-HT2/1Creceptors and moderate selectivity (Janssen, 1983); thefinding that they inhibit the sexual behaviour of male ratstherefore suggests that 5-HT2/1C receptor activityfacilitates male rat copulation.However, data collected using other antagonists havesuggested the reverse. Cyproheptadine is a 5-HT antagonistthat, while it is not very selective overall, displays amoderate 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 highly35selective 5-11T2/1C antagonist LY 53857, and its congeners LY237733 and LY 281067, has been reported to induce a similarfacilitation of sexual behaviour in male rats (Foreman etal., 1989). The results of these studies therefore suggestthat 5-HT2/1C receptor activity inhibits, rather thanfacilitates, the sexual behaviour of male rats.Studies employing a combination of agonists andantagonists have added to the confusion. As mentionedbefore, the agonist quipazine inhibits sexual behaviour inmale rats when given alone (Ahlenius et al., 1981;Grabowska, 1975; Mendelson & Gorzalka, 1985). Paradoxically,when coadministered with pirenperone, quipazine isreportedly effective in reversing the inhibitory effects ofpirenperone (Mendelson & Gorzalka, 1985). In this case,then, quipazine acted in a facilitatory fashion.Furthermore, pirenperone has been reported to effectivelyreverse the inhibition of male rat copulatory behaviourinduced by treatment with the precursor of serotonin, 5-HTP(Ahlenius & Larsson, 1984). Pirenperone thus exerted afacilitatory action in this study, which would be congruentwith an inhibitory effect of 5-HT2/1C receptor activity.Taken together, the data from these studies are completelycontradictory; both pirenperone and quipazine have displayedboth inhibitory and facilitatory profiles in the variousstudies.36The conflicting results of previous studies are atleast partly attributable to the relative lack ofselectivity of the agonists employed. In the one previousstudy in which the selective 5-HT2/1C agonist DOl wasemployed in a competitive manner, it was found that DOlinduced inhibition of male rat sexual behaviour waseffectively blocked in rats that had been pretreated withthe 5-HT2I1C antagonist LY 53857 (Foreman, et al., 1989).The 5-UT antagonist ritanserin displays very highaffinity and relatively high selectivity for 5-HT2/1Creceptors (Hoyer, 1988a; Meert, Niemegeers, Gelders, &Janssen, 1989). The effects of ritanserin on male rat sexualbehaviour have not been determined, but the unique bindingprofile of ritanserin suggests that it might be of value inassessing the particular contributions of 5-HT2/1C receptorsto the control of male rat copulation. In Experiment 2, theeffects of varying doses of ritanserin on male ratcopulation were examined. In addition, the efficacy ofritanserin in reversing the inhibition of sexual behaviourinduced by DOl was examined.37MethodsDrugsRitanserin was obtained from Research Biochemicals, andstored at 5°C in dark conditions. Fresh solutions ofrita.nserin and DCI were prepared before each test session.DCI was prepared as in Experiment 1. Ritanserin wasinitially dissolved in a minimal amount of lactic acid anddiluted with sterile distilled water. Both drugs wereadministered SC in a total volume of 1.0 mi/kg; ritanserinwas administered 90 mm prior to testing (Meert et al.,1989) and DCI was administered 30 mm prior.ProceduresExperiment 2 was conducted in two parts. In the firstpart, Experiment 2a, 18 sexually experienced male SpragueDawley rats were observed following the administration ofritanserin alone, in the following doses: 0.0, 0.3, 0.6, or1.2 mg/kg. The doses were administered in a randomcounterbalanced fashion over successive test sessions, suchthat by the end of the experiment each male rat had beenevaluated (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 DOlinduced inhibition were evaluated. The same male rats as had38been used in Experiment 2a received a pretreatment ofritanserin (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 inproducing 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 malerats received each of the five treatments in counterbalancedfashion over the course of 5 weeks, and their copulatorybehaviour was quantified (%M, %I, %E) under each treatmentcondition.ResultsThe results of Experiment 2a are presented in Table 1.Friedman tests revealed that, when given alone, ritanserindid not significantly affect the sexual behaviour of themale rats, at any dose tested (x2 values ranging from 2.09 to4.61, NS). To assess the possibility that male rat sexualbehaviour may be affected only by a high dose of ritanserin,an additional follow—up study was performed in which the 18male rats received 0.0 or 3.0 mg/kg of ritanserin, followingthe procedure of Experiment 2a. The 3.0 mg/kg dose did notsignificantly affect the male rats’ sexual behaviour, forany measure (Wilcoxon test; z values ranging from 0.2068 to1.5148, NS).39The results of Experiment 2b are presented in Figure 2,in the form of a dose—response graph. In the absence ofritanserin, 1.0 mg/kg 001 completely abolished sexualbehaviour in the male rats. Cochran Q tests revealed thatpretreatment of the male rats with ritanserin potentlyblocked this inhibitory influence of DCI, in a dosedependent manner. This was reflected in systematic increasesin %M (Q(3) = 31.2, p < .0001), %I (Q(3) = 31.4 p < .0001),and %E (Q(3) 16.1, p = .003). Indeed, with ritanserinpretreatments of 1.0 or 5.0 mg/kg, the copulatory behaviourof the male rats was maintained at baseline levels (Figure2).40Table 1. The effects of various doses of ritanserin on thecopulatory behaviour of male Sprague-Dawley rats. Mountsand intromissions are total counts, all other values aretime in seconds. Data are presented as mean scores (S.E.M.in parentheses) after substitution of missing values. Thenumbers of rats that displayed each behaviour are presentedin square brackets.41Effects of Ritanserin on Sexual Behaviour in Male RatsRitanserin treatment, mg/kgBehaviouralMeasure 0.0 0.3 0.6 1.2Mounts 10.3 9.9 9.8 7.15(1.8) (1.6) (1.4) (0.9)[20] [20] [20] [20]Intromissions 10.0 8.9 8.7 9.9(0.7) (0.6) (0.9) (0.9)[20] [20] [19] [20]Mount Latency 56.3 38.3 117.3 47.4(19.0) (10.6) (89.2) (28.0)[20] [20] [19] [20]Intromission 126.6 62.9 161.2 67.4Latency (42.5) (14.3) (88.0) (41.1)[20] [20] [19] [20]Ejaculation 567.8 441.9 629.9 428.7Latency (99.1) (48.4) (117.2) (31.4)[18] [20] [18] [20]Postejaculatory 499.0 373.9 572.4 422.0Interval (100.1) (16.0) (118.7) (74.1)[18] [20] [17] [19]42Ficiure 2. Effectiveness of increasing doses of ritanserin inblocking the inhibition of male rat copulatory behaviourinduced by DOl. In all but the No-Drugs condition, ratsreceived a test dose of ritanserin 90 mm prior to testingand 1.0 mg/kg DOl 30 mm prior. In the No-Drugs condition,animals received the drug vehicles 90 and 30 mm prior totesting.*Differs from 0.0 mg/kg condition, p<.05 (McNemar test).**Differs from 0.0 and 0.2 iug/kg conditions, p<.O5 (McNemartest).L 0 > 0 U) 0 C-) 0 U) C >‘ 0 U) 0 U) U) 0100 80 60 40 20 0** 0*/*****o-o* * El/**//El./*0.00—0Mounts—LtntromissjonsEl-[1Ejaculations0.2Ritanserinpretreatment,mg/kgS.C.1.05.0(I) 01 I 0 zw44DiscussionRitanserin effectively competes with 5-HT agonists for5-HT2 and 5-HT1C binding sites (eg. Hoyer, 1988a, 1992;Meert, Niemegeers, Gelders, & Janssen, 1989). In the presentexperiment, ritanserin potently blocked the inhibitingeffects of DOl on the copulatory behaviour of male rats.Because both DOl and ritanserin exhibit high affinity for5-HT2 and 5-HT1C receptors, but differ in their affinitiesfor other types of receptors, the present data suggest thatthe efficacy of ritanserin in blocking D01-inducedinhibition specifically reflects the displacement byritanserin of DOl from 5-HT2/1C receptors. The present datatherefore support the tentative conclusion, from Experiment1, that activation of 5-HT2/1C receptors mediates aninhibition of sexual behaviour in the male rat.It is somewhat curious that ritanserin was withouteffect when given alone. The 5-HT2/1C antagonists LY 53857,pirenperone, and ketanserin have all been reported tosignificantly influence the copulatory behaviour of malerats, albeit in opposing directions (Foreman et al., 1989;Mendelson & Gorzalka, 1985). One possibility is that thesedrugs exert their effects through their interactions withother types of receptors, rather than through their effectsat 5-HT2/1C binding sites.45Experiment 3Although the results of Experiments 1 and 2 support thecontention that 5-HT2/1C receptor activity mediates aninhibition of male rat copulatory behaviour, the findingthat ritanserin did not affect sexual behaviour when givenalone is not congruent with the previous reports in which5-HT2/1C antagonists have been found to influence male ratcopulation (Foreman et al., 1989; Mendelson & Gorzalka,1985). Two additional considerations may reconcile thisapparent discrepancy.One possible explanation is related to the uniquebinding profiles of the different drugs that have beenemployed. Ritanserin and DOl both display high affinity andselectivity for 5-HT2 and 5-HT1C receptors relative to othertypes of receptors. However, neither drug displaysappreciable selectivity between 5-HT2 and 5-HT1C sites(Moyer, 1988a; Glennon & Dukat, 1991). Pirenperone, bycontrast, is less selective overall than is ritanserin, butit does display somewhat more selectivity for 5-HT2 sitesthan for 5-HT1C sites. That is, although pirenperone bindsto a wider variety of non5-HT2/1C receptors than doesritanserin (Janssen, 1983), it does display slightly moreaffinity for 5-HT2 than 5-UT1C receptors (Moyer, 1988a,b;Glennon & Dukat, 1991). Although the reported effects of46pirenperone on male rat copulation (Mendelson .& Gorzalka,1985) may reflect the occupation of non5-HT2/1C sites athigher concentrations, low doses of pirenperone mightselectively occupy 5-HT2 receptors, and to a lesser extent5-HT1C receptors. This proposition is amenable toexperimental evaluation, by examining the efficacy ofpirenperone in reversing the inhibitory effects of DOl.Given the results of Experiments 1 and 2, it is probablethat the smallest dose of pirenperone found to be effectivein blocking DOl-induced inhibition would also be a dose atwhich pirenperone preferentially occupied 5-HT2 sites. Theeffects of this dose of pirenperone, in the absence of DOl,could then be evaluated for effects on male rat sexualbehaviour.A second consideration is that the conflicting resultsof the previously published reports may be at least partlydue to genetic differences between their respectiveexperimental samples. Specifically, whereas Mendelson andGorzalka (1985) used rats of the Long—Evans strain, Foremanet al. (1989) employed Sprague-Dawley rats. Although straindifferences in the serotonergic control of sexual behaviourhave not been established, such differences do exist betweenrelated species. For example, muroid species are known todiffer in their response to serotonergic drugs; 8-OH-DPAT, a5-HT1A agonist, reportedly facilitates the sexual behaviour47of male rats (Ahlenius & Larsson, 1984; Mendelson &Gorzalka, 1986) but inhibits the sexual behaviour of malemice (Svensson, Larsson, Ahlenius, Arvidsson, & Carisson,1987).In Experiment 3, the effects of pirenperone pretreatmenton DOl—induced copulatory inhibition were assessed. Parallelexperiments were run in the two genetic strains of rats,Long—Evans and Sprague—Dawley, in order to evaluate thehypothesis that the conflicting data reported by others maybe a consequence of strain differences.MethodDrugsPirenperone was obtained from Janssen Pharmaceutica,Beerse, Belgium, and stored at 5° C in dark conditions.Fresh solutions of pirenperone and DCI were prepared beforeeach test session; DCI was prepared as in Experiment 1.Pirenperone was initially dissolved in a. minimal quantity ofwarm 0.0007 M citrate solution, and diluted to finalconcentration with sterile physiological saline. Pirenperonewas administered intraperitoneally (IP) 60 mm prior totesting, and DCI was administered SC 30 mm prior totesting.48ProceduresA total of 13 male Sprague-Dawley rats and 14 male Long-Evans rats were tested as in Experiment 2b. All males weresexually experienced. Prior to each test session, the malesreceived a pretreatment dose of pirenperone: 0.0, 20.0,100.0, or 200.0 pg/kg, plus 1.0 mg/kg DCI. A controlcondition was also included in which animals were testedafter administration of the drug vehicles alone. Thetreatments were administered in a random counterbalancedfashion, such that by the end of the experiment, all themale rats had been evaluated (%M, %I, M, I, ML, IL, EL, PEI)once under each treatment condition, At the conclusion ofthe experiment, a follow—up session was conducted in whichthe males were tested after receiving a dose of pirenperonethat corresponded to the smallest dose that significantlyreversed DOl-induced inhibition. In this session,pirenperone was administered without DCI, in order to assessthe effects of pirenperone on sexual behaviour in otherwise—untreated males.ResultsThe effects of the pirenperone pretreatment on theproportion of Sprague—Dawley males showing each of the threecopulatory behaviours (%M, %I, %E) are presented in49Figure 3. Figure 4 presents the data for the Long-Evansmales. In both strains, pirenperone potently blocked theinhibition of sexual behaviour induced by DCI. In neitherstrain was the effect of pirenperone dose dependent. Maximalreversal of the inhibitory effect of DCI occurred with thelowest dose of pirenperone (50.0 ig/kg).50Figure 3. Effectiveness of pirenperone in reversing DOlinduced inhibition of copulatory behaviour in male SpragueDawley rats.*Differs from 0.0 rig/kg condition, p < 0.05 (McNemar test)**Differs from all other doses, p < 0.05 (McNemar test)100.080.060.040.020.0 0.00.0LIntromissionsQEjaculations* 0—0 > 0-c a) 0 o a) >1 ci 0 Ci)-o Cl) ci) ci/ / /* U.*U•DU0—0MountsPirenperonepretreatment,pg/kg,60mmpriorU)Q)50100200:1(J152Figure 4. Effectiveness of pirenperone in reversing DOl—induced inhibition of copulatory behaviour in male LongEvans rats.*Differs from 0.0 pg/kg condition, p < 0.05 (McNemar test)100.0-80.0-60.0-40.0-20.00.0-*** 0U)‘)ñfl0)C 01...a) 00 zL.a-**L. 0 > 0-c a) 0 C.) 0 a) >‘ 0 ci (I) -o (I) a) ci** *0.0500—0Mounts—LIntromissions0Ejaculations100Pirenperonepretreatment,/Lg/kgU’54The means and standard errors for the various parametersof male rat sexual behaviour are given in Table 2 forSprague-Dawley males, and in Table 3 for Long—Evans males.For the Sprague—Dawley males, overall Friedman analysesrevealed that pirenperone pretreatment had a significanteffect for M (x23) 15.3, p = 0.0016), I (x23) = 10.5, p =0.0148), ML (x23) = 15.5, p = 0.0014), and IL (x23) = 8.6,p = 0.0354). subsequent Wilcoxon pairwise comparisonsrevealed that the 50.0, 100.0, and 200.0 pig/kg pirenperoneconditions significantly differed from the 0.0 pig/kg (i.e.,DOl plus pirenperone vehicle) condition (Z ranging from 2.2to 2.9, p < .02) for all four behavioural measures.Pirenperone’s overall effect on EL and PEI was notstatistically significant (x23) = 1.03, NS, and x2(3) = 0.6,NS, respectively).In the Long-Evans rats, pirenperone had a pronouncedeffect on all measures of sexual behaviour in the DCI—treated male rats (Table 3). This was reflected insignificant overall Friedman analyses for M (x23) = 19.5,p < 0.0002), I (x23) = 19.8, p < 0.0002), ML (x23) = 20.25,p < 0.0002), IL (x23) = 20.0, P < 0.0002), EL (x23) = 16.4,p = 0.001), and PEI (x23) = 17.8, p = 0.0005). Subsequentpairwise Wilcoxon tests revealed that the males displayedsignificantly facilitated sexual behaviour on all 6 of thesemeasures, relative to the 0.0 fig/kg pirenperone condition,55when 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 betweenthe 50.0 and 200.0 i.g/kg pirenperone pretreatmentconditions, for ML, IL, EL, and PEI (Z ranging from 2.0 to2.4, p < 0.05). Similarly, The 50.0 and 100.0 tg/kg dosessignificantly differed on EL (Z = 2.6, p = 0.011). Thesedifferences thereby statistically confirmed the trend,apparent in Figures 3 and 4, that pirenperone pretreatmentwas maximally efficacious in reversing the inhibitingeffects of DCI at the low 50.0 p.g/kg dose. Behaviouralmeasures of copulatory proficiency were significantlydiminished with higher concentrations of pirenperone (100.0and 200.0 pig/kg).In order to assess the effects of pirenperone on malerat sexual behaviour, when given in the absence of DCI, afollow—up session was conducted in which the male rats weretested following injections of 50.0 g/kg pirenperone 60 mmprior to testing, and physiological saline (DCI vehicle) 30mm prior. This dose of pirenperone did not significantlyaffect the male rats’ sexual behaviour when given alone(Figures 1 and 2), despite being effective in reversing DCIinduced inhibition.The two genetic strains of rats displayed comparableresponses to the drug treatments, although they differed56somewhat in scope and variability. Mann-Whitney U tests ofthe baseline data revealed Long—Evans males to be much morevigorous copulators than the Sprague—Dawley males across thevarious behavioural measures (p values ranging from 0.0427to 0.0013). Nevertheless, it does not appear that straindifferences account for the contradictory findings ofMendelson & Gorzalka (1985) and Foreman et al. (1989), sincethe pattern of results in the present study was in the samedirection for both strains.57Table 2. The effectiveness of pirenperone in reversing DCIinduced inhibition of sexual behaviour in male rats, of theSprague-Dawley strain. In all but the ‘No-Drugs’ condition,1.0 mg/kg DCI was administered 30 mm prior to testing.Data are presented as mean scores (S.E.M. in parentheses);the number of animals displaying each number are given insquare brackets.58Effects of Pirenperone Pretreatment on DOl—InducedInhibition of Male Rat Sexual Behaviour: Spracrue-DawlevStrain.Pirenperone pretreatment, tg/kgBehaviouralMeasure 0.0 50.0 100.0 200.0 No DrugsMounts 0.0 10.6 7.3 9.4 20.4(0.0) (2.3) (2.4) (2.7) (3.3)[0] [10] [10] [11] [13]Intromissions 0.0 3.9 3.2 3.6 10.4(0.0) (1.3) (1.4) (1.2) (0.8)[0] [9] [7] [6] [13]Mount Latency 1800.0 490.9 783.3 534.5 62.9(0.0) (207.4) (199.5) (170.7) (16.2)[0] [10] [10] [11] [13]Intromission 1800.0 922.8 1198.3 1194.5 179.7Latency (0.0) (209.7) (205.2) (208.0) (54.8)[0] [9] [7] [6] [13]Ejaculation 1800.0 1668.0 1551.4 1583.4 927.6Latency (0.0) (98.0) (138.3) (122.7) (144.0)[0] [2] [3] [3] [11]Postejaculatory 1800.0 1700.3 1578.7 1574.9 894.9Interval (0.0) (99.7) (149.8) (152.5) (206.7)[0] [1] [2] [2] [8]59Table 3. The effectiveness of pirenperone in reversing DOlinduced inhibition of sexual behaviour in male rats, of theLong-Evans strain. In all but the ‘No-Drugs’ condition, 1.0mg/kg DCI was administered 30 mm prior to testing. Data arepresented as mean scores (S.E.M. in parentheses); the numberof animals displaying each number are given in squarebrackets.60Effects of Pirenperone Pretreatment on DOl—InducedInhibition of Male Rat Sexual Behaviour: Long-Evans Strain.Pirenperone pretreatment, ig/kgBehaviouralMeasure 0.0 50.0 100.0 200.0 No DrugsMounts 0.1 5.1 8.9 4.1 9.7(0.1) (1.1) (2.3) (1.2) (3.2)[1] [13) [12] [10] [13]Intromissions 0.2 8.1 6.5 5.0 8.1(0.2) (0.8) (1.2) (1.1) (1.2)[1] [13] [12] [9] [13]Mount Latency 1680.7 260.6 475.4 560.4 149.9(120.3) (147.4) (177.6) (217.9) (127.0)[1] [13] [12] [10] [13]Introinission 1720.8 264.6 517.4 709.6 164.1Latency (81.2) (147.0) (171.6) (226.2) (126.1)[1] [13] [12] [9] [13]Ejaculation 1677.5 363.6 634.6 820.1 378.7Latency (123.5) (113.4) (171.4) (203.5) (122.1)[1] [13] [11] [9] [13]Postejaculatory 1800.0 404.2 753.4 860.3 391.9Interval (0.0) (108.2) (184.6) (194.8) (110.1)[0] [13] [10] [9] [13]61DiscussionLike ritanserin, pirenperone was found to effectivelyreverse the inhibition of male rat sexual behaviour inducedby systemic administration of DCI. Because the effects ofDCI are believed to be exerted via 5-HT2/1C receptoractivation, and pirenperone’s main pharmacological effect isantagonism of these sites (Dabiré et al., 1989; Glennon &Dukat, 1991; Glennon et al., 1986) this finding supports thecontention that 5-HT2/1C receptor activation inhibits malerat copulation.At a concentration of 50.0 tg/kg, pirenperone reversedthe effects of DOl on male rat copulation. Becausepirenperone and DCI share high affinity for 5-HT2/1Creceptors, but differ in their affinity for other types ofreceptors (Dabiré et al., 1989; Janssen, 1983; Middlemiss &Tricklebank, 1992; McKenna & Peroutka, 1989), it may beassumed that pirenperone’s behavioural effects were exertedthrough effective competion with DOl for binding at thereceptor sites through which the inhibiting effects of DCIare realized (for review, e.g., Kalant et al., 1985). Thus,it may be assumed that when given in the absence of DCI, a50.0 ig/kg dose of pirenperone saturates this samepopulation of receptors. When given alone, 50.0 rig/kg ofpirenperone was found to be without effect on the sexualbehaviour of male rats in the present experiment. This is62congruent with the results of Experiment 2, in which dosesof ritanserin also were without effect when given tootherwise untreated male rats. This pattern of resultssuggests that the previously reported facilitatory effectsof pirenperone in otherwise untreated male rats (Mendelson &Gorzalka, 1985) may be attributable to binding ofpirenperone to other non—5-HT2/1C receptor types when givenin higher doses.Lastly, evidence exists that pirenperone exhibits amoderate degree of selectivity for 5-HT2 versus 5-HT1Creceptors (Janssen, 1983; Hoyer, 1992), unlike ritanserin(foyer, 1992). This selectivity of pirenperone for 5-HT2receptors, coupled with its efficacy in reversing theeffects of DOl, provides preliminary evidence that theinhibitory effects of DOl on male rat sexual behaviour maybe attributable to 5-HT2, rather than 5-HT1C, activation.63Experiment 4In Experiments 2 and 3, the 5-HT2/1C antagonistsritanserin and pirenperone were found to effectively blockDOl-induced inhibition of male rat sexual behaviour. Theefficacy of pirenperone was evident at a dose that did notin itself affect male rat copulatory behaviour when given inthe absence of DOl. Because this finding conflicts with thepublished data (Mendelson & Gorzalka, 1985), furtherinvestigation with an additional 5-HT2/1C antagonist waswarranted. Comparing the data collected with a variety ofantagonists, given their differing binding profiles, mightclarify the unique contributions of 5-HT2/1C receptors tothe control of male rat copulation, and may also permit somedegree of discrimination between the effects of 5-HT2 and5-HT1C activity.Ketanserin is considered to be a prototypic 5-11T2receptor antagonist (Glennon & Dukat, 1991). Likepirenperone, it possesses moderately higher selectivity for5-HT2 receptors, compared to 5-HT1C receptors, than doesritanserin (Hoyer, 1988a, 1988b, 1992; Middlemiss &Tricklebank, 1992). Pirenperone and ketanserin differ intheir affinity for other types of monoaminergic receptors,however. In particular, ketanserin displays less affinityfor dopamine receptors than does pirenperone (Janssen,641983). The efficacy of ketanserin in reversing theinhibition of male rat sexual behaviour induced by DOl wasexamined in Experiment 4. As in Experiment 3, thepossibility of different responses between the SpragueDawley and Long-Evans genetic strains was also evaluated,MethodDrugsKetanserin tartrate was obtained from JanssenPharmaceutica, Beerse, Belgium, and stored at 5°C in darkconditions. Fresh solutions of ketanserin and DOl wereprepared before each session; DOl was prepared as in thepreceding experiments. Ketanserin was dissolved in warmphysiological saline and injected IP in a total volume of1.0 mi/kg, 60 mm prior to testing. DOl was administered 30mm prior to testing.ProceduresExperiment 4 was conducted in a fashion similar toExperiment 3. As before, 13 Sprague-Dawley and 14 Long-Evansmales were used; all were sexually experienced and ofcomparable age to the animals tested in Experiment 3. Thecopulatory behaviour of the male rats (%M, %I, %E, M, I, ML,65IL, EL, PEI) was evaluated after administration of 1.0 mg/kgDOl plus a test dose of ketanserin: 0.0, 0.2, 1.0, or 5.0mg/kg -- or after the administration of the drug vehiclesonly. The treatment conditions were randomly counterbalancedover the course of the experiment, such that each animal wastested once under each treatment condition. Consecutivetesting sessions were separated by a period of 1 week.ResultsKetanserin potently blocked DOl-induced inhibition ofmale rat sexual behaviour, as was found with pirenperone inExperiment 3. In contrast to the data collected withpirenperone, however, the facilitatory effects of ketanserinwere dose—dependent. The effects of ketanserin on theproportions 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 arepresented in Table 4 for Sprague-Dawley males, and in Table5 for Long—Evans males. Baseline (‘No—Drugs’) data areincluded for comparison.In the Sprague—Dawley group, overall Friedman analysesrevealed a significant treatment effect of ketanserin for M(x23) = 20.1, p < 0.0002), I (x23) = 10.1, p = 0.0181),66Figure 5. Effectiveness of ketanserin in reversing DOl—induced inhibition of copulatory behaviour in male SpragueDawley rats.*Differs from 0.0 pjg/kg condition, p < 0.05 (McNemar test)**100.0*0o/•5/-80.0//*E]*/**0060./-**I/,/*D40.0//.0—0MountsII /..—ISIntromissions-20.0/Q••QEjaculaUonsKetanserinpretreatment,mg/kggz68Figure 6. Effectiveness of ketanserin in reversing DOl—induced inhibition of copulatory behaviour in male LongEvans rats.*Differs from 0.0 tg/kg condition, p < 0.05 (McNemar test)*.**100.0*-oL)—f80.0*a)*C.) *40.0/0—0Mounts60.0I./U)/—LIntromissionsU)-20.0/“LiQEjaculations00.0•U)C0.00.21.05.00wOKetanserinpretreatment,mg/kgo—U)0•70ML (x23) =14.8, p = 0.002), and IL (x23) = 11.2, p =0.0108). Subsequent Wilcoxon tests revealed that for thesebehavioural measures, scores for the 0.2, 1.0 and 5.0 mg/kgketanserin pretreatments all significantly differed from the0.0 mg/kg (i.e. vehicle plus DCI) condition (Z ranging from2.4 to 3.1, p < 0.02).In the Long—Evans males, overall Friedman tests revealedthat ketanserin pretreatment significantly reversed DOlinduced inhibition of male rat copulatory behaviour on all 6behavioural measures: M (x23) = 13.6, p = .0035), I (2(3) =19.1, p = 0.0003), ML (x23) = 25.8, p < 0.0002), IL (x23) =24.7, p < 0.0002), EL (x23).= 12.8, p = 0.0052), and PEI(x23) = 29.4, p < 0.0002). Subsequent Wilcoxon testsindicated that, for each of these measures, the scoresobtained for the 0.2, 1.0, and 5.0 mg/kg ketanserinpretreatment groups significantly differed from the scoresobtained with 0.0 mg/kg (i.e. vehicle plus DCI) ketanserinpretreatment (Z ranging from 2.4 to 3.3, p < 0.02).Moreover, dose-dependency was clearly evident in the patternof results. Scores for ML and PEI were significantlyimproved (i.e. decreased) when the males received 1.0 mg/kgketanserin compared to when they received 0.2 mg/kg (Z =2.43 and 1.96, p = 0.015 and 0.049, respectively). Asignificant facilitation of PEI was also evident for the 5.0mg/kg ketanserin pretreatment condition, relative to the 1.071mg/kg condition (Z = 1.98, p = 0.048). Lastly, ML, IL and ELwere all improved in the 5.0 mg/kg ketanserin pretreatmentcondition, when compared with the scores for 0.2 mg/kg ofketanserin ( Z = 2.98, 3.11, and 3.3; p = 0.0029, 0.0019,and 0.001, respectively). Taken together, these resultsindicate that the reversal by ketanserin of DOl—inducedinhibition of sexual behaviour in the male rats wassignificantly greater with successively higher doses ofketanserin,As in Experiment 3, a follow-up session was conductedin which a minimal effective dose of ketanserin wasadministered to the male rats, in the absence of DOl, andtheir sexual behaviour was quantified. The low dose (0.2mg/kg) of ketanserin was selected for this follow-up,because it had a significant effect on DOl-inducedinhibition of male rat sexual behaviour in the presentexperiment. As is evident in Figures 5 and 6, treatment withthis dose of ketanserin had no effect on the expression ofsexual behaviour in otherwise untreated male rats. As wasfound in Experiment 3, Long—Evans males were more vigorouscopulators than the Sprague—Dawley males (p values rangingfrom 0.022 to < 0.0001), but the males of the two strainsresponded in comparable fashion to the drug treatments.72Table 4. Effectiveness of ketanserin in reversing DOl—induced inhibition of sexual behaviour in male rats of theSprague-Dawley strain. In all but the No-Drugs condition,1.0 mg/kg DCI was administered 30 mm prior to testing. Dataare presented as mean scores (S.E.M. in parentheses); thenumber of rats displaying each behaviour is given in squarebrackets.73Effects of Ketanserin Pretreatment on DOl-Induced Inhibitionof Male Rat Sexual Behaviour: Sprague—Dawley Strain.Ketanserin pretreatment, mg/kgBehaviouralMeasure 0.0 0.2 1.0 5.0 No DrugsMounts 0.5 9.9 15.5 10.8 22.3(0.5) (3.2) (4.4) (2.3) (4.5)[1] [8] [12] [13] [13]Intromissions 0.0 3.7 4.9 3.9 6.6(0.0) (1.2) (1.3) (1.0) (1.3)[0] [7] [9] [8] [11]Mount Latency 1662.9 829.3 430.1 547.2 214.9(137.1) (230.9) (174.8) (158.6) (113.2)[1) [8] [12] [13] [13]Intromission 1800.0 916.5 821.8 902.9 502.4Latency (0.0) (240.2) (228.1) (222.4) (178.6)[0] [7] [9] [8] [11]Ejaculation 1800.0 1592.7 1272.2 1337.0 1245.0Latency (0.0) (141.1) (195.9) (163.1) (168.7)[0] [2] [5] [6] [7]Postejaculatory 1800.0 1578.5 1471.4 1454.3 1245.2Interval (0.0) (150.0) (173.4) (182.3) (202.8)[0] [2] [3] [3] [5]74Table 5. Effectiveness of ketanserin in reversing DOl—induced inhibition of sexual behaviour in male rats of theLong-Evans strain. In all but the No-Drugs condition, 1.0mg/kg DOl was administered 30 mm prior to testing. Data arepresented as mean scores (S.E.M. in parentheses); the numberof animals displaying each behaviour is given in squarebrackets.75Effects of Ketanserin Pretreatment on DOl-Induced Inhibitionof Male Rat Sexual Behaviour: Long-Evans Strain.Ketanserin pretreatment, mg/kgBehaviouralMeasure 0.0 0.2 1.0 5.0 No DrugsMounts 0.8 2.1 6.2 5.4 6.1(0.6) (0.6) (2.3) (1.6) (1.8)[2] [9] [12] [14] [14]Intromissions 0.4 4.9 5.6 7.5 9.6(0.4) (1.2) (1.0) (0.5) (1.0)[1] [9] [11] [14] [14]Mount Latency 1594.0 728.0 311.4 66.1 41.1(144.4) (225.9) (171.3) (37.6) (21.3)[2] [9] [12] [14] [14]Intromission 1676.4 731.9 471.9 74.5 50.9Latency (123.6) (224.9) (196.9) (37.4) (21.5)[1] [9] [11] [14] [14]Ejaculation 1679.2 746.2 564.0 194.1 229.8Latency (120.8) (218.4) (184.0) (21.6) (25.9)[1] [9] [11] [14] [14]Postejaculatory 1706.9 904.6 648.4 291.6 308.2Interval (93.1) (186.8) (168.2) (14.0) (19.6)[1] [9] [11] [14] [14]76DiscussionIn a similar manner to ritanserin and pirenperone,ketanserin effectively reversed the inhibitory influence ofDOl on male rat sexual behaviour. That it did so at aconcentration that did not in itself affect the males’sexual behaviour suggests that previously reported effectsof 5-HT2/1C antagonists, when given alone (Abraham et al.,1988; Mendelson & Gorzalka, 1985), may be attributable tononselective activity at other types of receptors. Moreover,the data collected in Experiment 4 reinforce the notion that5-HT2/1C receptors mediate an inhibition of male rat sexualbehaviour. To the extent that ketanserin particularly sharesaffinity for 5-HT2 receptors with ritanserin andpirenperone, while differing from them in affinity for otherreceptor types, including 5-HT1C (Hoyer, 1992; Janssen,1983), the present data allow a further limited inference:Specifically, the alterations in male rat sexual activityseen in Experiments 1 through 4 may be primarilyattributable to modulation of a mechanism dependent on 5-HT2receptor activity, rather than 5-HT1C receptor activity. Theresults of Experiments 1—4, taken together, are consideredin greater detail in the General Discussion.77GENERAL METHOD: EXPERIMENTS 5-7AnimalsMale and female Sprague-Dawley rats were bred from stockoriginally 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 meshlaboratory cages. Free access to food and water wasprovided, and a reversed 12/12 hr light/dark cycle wasmaintained.At approximately 70 days of age, the female ratsunderwent bilateral ovariectomy while under sodiumpentobarbital anaesthesia (65 mg/kg IP). Males wereapproximately 100 days old at the beginning of behaviouraltesting.Steroid TreatmentsAs in the preceding experiments, receptivity was inducedin the ovariectomized female rats by subcutaneous injectionof 10.0 pg estradiol benzoate (Steraloids) and 500.0progesterone (Steraloids), 48 hr and 4 hr prior to testing,respectively. Steroids were dissolved in 0.1 ml peanut oilvehicle. The nonreceptive females employed in Experiment 6were ovariectomized females that had not received steroidreplacement for a minimum of 2 weeks prior to testing.78Behavioural TestingIn order to evaluate the possibility that WDS andcopulatory behaviour are systematically related in malerats, the coincidence of these behaviours was measured by anobserver who was blind to the experimental hypotheses andunaware of individual treatment conditions. As before, alltesting occurred during the middle 1/3 of the dark phase ofthe rats light-dark cycle.The experimental males were placed individually intoclear 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 freshSan—i—Cel bedding material before each testing session, andthe arenas were washed weekly. During each testing session,between 2 and 4 males were scored simultaneously in separatetesting chambers.Following the 5-mm habituation period, a stimulus ratwas introduced into each male’s testing chamber andbehavioural recording commenced. During each 60-mm testingsession, the occurrence of WDS was scored for each male, bymeans of a verbal recording on a voice—activatedmicrocassette recorder (Sony model M-660V). In addition, the79incidence of mounts, intromissions, and ejaculations wasrecorded in Experiment 5 and 6, and the incidence ofejaculations was recorded in Experiment 7. These recordingswere transcribed immediately following each testing session.80Experiment 5The evidence discussed in the General Introductionsuggests that the generation of WDS in rats is criticallydependent on a 5-HT2-mediated mechanism. It is possible,therefore, that spontaneously occurring WDS could be used asa type of behavioural assay of concurrent 5—HT2 activationduring the performance of other types of behaviour. Informalobservation during the course of other experiments hassuggested that males generate WDS during sexual behaviour.Consequently, it is reasonable to suppose that combiningmeasures of WDS with measures of male rat copulatorybehaviour might yield a behavioural index of 5—HT2 activityduring copulation that converges on the pharmacological datacollected in Experiments 1-4.In any group of male rats, a certain proportion reliablyfails to initiate normal copulation when confronted withreceptive females, even after numerous trials (Whalen,Beach, & Kuehn, 1961; Crowley, Popolow, & Ward, 1973). Thispresented the opportunity to perform a natural experiment indrug-free animals, in which the sexual and WDS responses ofnoncopulators (‘Duds’: Crowley et al., 1973) were comparedwith those of normal copulators (‘Studs’: Crowley et al.,1973). The extent to which WDS and male rat copulatorybehaviours rely on a shared 5-HT2 mediated mechanism is81unknown. However, to the extent that such a mechanismunderlies the WDS and copulatory behaviours, WDS frequencymay be systematically related to copulatory proficiency andreflect concurrent 5-HT2 activation; specifically, based onthe results of Experiments 1-4, it was hypothesized thatincreased WDS, reflecting augmented 5-HT2 activity, would beobserved in males that failed to copulate.MethodProceduresA total of 58 sexually inexperienced male rats wereevaluated in Experiment 5. Previous observations suggestedthat this number would yield approximately 10 noncopulators.Following habituation to the observation chambers, receptivefemale rats were introduced into the chambers andbehavioural recording commenced. During each 60-mm session,4 males were evaluated simultaneously for the incidence ofWDS, mounts, intromissions, and ejaculations. The receptivefemale rats were rotated among the males every 15 mm.ResultsUpon completion of the experiment, the experimentalmales were divided into groups on the basis of their82measured 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 oftheir sexual proficiency. The levels of WDS recorded inthese groups are presented in Figure 7. The incidence ofspontaneously occurring WDS differed markedly between theDuds (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 thanthe Studs. The third group, Slugs (males that mounted orintromitted at least once, but failed to achieveejaculation; n=7) displayed an intermediate level of WDS.Slugs displayed very little copulatory behaviour, comparedto Studs, and thus appeared to be a subset of the Dudscategory.Overall tests of the WDS scores across the three groupsrevealed significant differences, both by the nonparametricKruskal—Wallis test (x2) 20.1, p < O.OOO1), andparametric analysis of variance (F(255) = 14.46,p < 0.00005). Subsequent comparisons of pairs of groups,using the the Mann-whitney U test, revealed that Studsproduced significantly fewer WDS than either Duds (U = 33.5,P < 0.0004) or Slugs (U = 44.5, p < 0.0004), and when testedusing the Newman—Keuls procedure, all paired comparisonswere significant (p < 0.05). Alternatively, the relationbetween WDS and sexual behaviour was also evident in asimple correlation of E with WDS, collapsed across thegroups (r (56) = —0.40., p < 0.001).8384Figure 7. Incidence of WDS in male rats of varyingcopulatory proficiency, during 60 mm interactions withreceptive female rats (Mean ± S.E.M.).mU(1)LAcoSPfl4Ss6nispn0z.990I.86DiscussionTotal WDS scores for the three groups of males were allsignificantly different from one another. Both sexualbehaviour and the incidence of WDS appeared to discriminateamong the Duds, Slugs, and Studs groups. The results ofExperiment 5 thus suggest that normal copulatory behaviouris incompatible with the expression of WDS in the male rat.Given the established dependency of WDS on 5-HT2 receptoractivity (e.g., Green & Heal, 1985), this pattern of resultsprovides preliminary, entirely behavioural, evidence thatWDS and male rat sexual behaviour are directly related insome fashion, and therefore supports the proposition that a5-HT2 receptor mediated mechanism exerts an inhibitoryinfluence on sexual behaviour in male rats.87Experiment 6The results of Experiment 5 suggested that theexpression of WDS is incompatible with normal copulatorybehaviour in male rats. However, the nature of this relationbetween the two types of behaviour remains unknown.In order to infer, with confidence, that the increasedincidence of WDS in Duds during mating tests is attributableto activity of a 5-HT2-dependent mechanism that alsocontributes, at least in part, to the control of copulatorybehaviour, it is necessary to examine the specificity of theeffect. It is possible that the effect observed inExperiment 5 may be an artifact of some other mediatingvariable. For instance, it has been suggested thatnoncopulating males may exhibit a generalized syndromecharacterized by diminished arousability (Pottier & Baran,1973). In the context of the present experiments, asystematic difference between Duds and Studs in autonomicactivity or general arousal levels might predispose certainmales to fail to copulate and also to produce excessive WDS,without the two behaviours being directly related to oneanother. If this were true, one would expect the differencein WDS observed in Experiment 5 to obtain regardless of thetype of stimulus rat presented. In Experiment 6, theexpression of WDS was measured while the males were paired88with other types of stimulus rats, and the degree to whichgroup differences in WDS emerge specifically during matingtests was assessed.MethodProcedureThe 58 male Sprague-Dawley rats used in Experiment 5also served as subjects in Experiment 6. As in Experiment 5,an observer, blind to experimental condition, made verbalmicrocassette recordings of WDS and sexual behavioursexhibited by the male rats during 60 mm test sessions. Therecorded data were transcribed at the end of each testingsession.As in Experiment 5, the experimental males wereevaluated following presentation of receptive females. Datawere also collected while the experimental males were pairedwith another male, or with a nonreceptive female.Experimental males were paired with each of the three typesof stimulus animals in a counterbalanced fashion, such that,over three consecutive test sessions, each experimental malehad been tested once with each type of partner in theobservation chaniber. Stimulus rats were rotated among theexperimental males every 15 mm during behavioural89observation. The experimental males were allowed at least 1week between consecutive test sessions.ResultsThe results of Experiment 6 are presented in Figure 8.The data collected in Experiment 5 suggested that Slugs area subset of the Duds category, as evidenced by themarginally significant differences between Duds and Slugs onWDS incidence. Given their minimal and very deficientcopulatory behavIour, Slugs were combined with Duds for thepurposes of the present experiment, with Duds defined asthose males (n=15) which failed to achieve ejaculationduring 60 mm mating tests with a receptive female (eg.Zainble, Hadad, Mitchell, & Cutmore, 1985).The mean incidence of WDS was significantly greater forDuds than for Studs, when the males were paired withreceptive females (Mann-whitney U test: U = 78.0, P <0.0001). In contrast, levels of WDS were comparable betweenDuds 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 thatwhile the mean WDS scores for the Duds group did notsignificantly differ across the three treatment conditions(x2) = 0.433, NS), the types of partners which Studs were90paired with significantly affected their WDS scores (x2) =43.41, p < 0.0001). Subsequent pairwise comparisons usingthe Wilcoxon test revealed that the Studs’ mean WDS scoressignificantly differed for each of the treatment conditions(p < 0.0005 for all comparisons). Thus, not only did Studsproduce significantly more WDS when paired with anonreceptive female than they did when paired with areceptive female, but they also displayed significantly moreWDS when paired with a male than when paired with anonreceptive female. The overall pattern of WDS productionin Studs may thereby be characterized as minimal in thepresence of receptive females, moderate in the presence ofnonreceptive females, and maximal in the presence of males.Although not directly quantified, subjective observationsuggested that the obtained results were not reducible tooverall activity levels. Duds explored their cagemates quiteactively throughout the test sessions, as did Studs.Similarly, although it might be argued that WDS appears tobe related to grooming behaviour, group differences in thefrequency of grooming do not appear to explain the presentresults, either. In fact, copulating males engage inconsiderably more grooming behaviour than do noncopulatingmales.91Figure 8. Comparison of spontaneous WOS production in malerats paired with differing types of partners in the testingchambers. Results are displayed as mean WDS scores ± S.E.M.“WetDogShake&’undervariousconditions10 8+_ riDuds+ci2__j__0•—________________ReceptiveNonreceptiveMaleFemaleFemaleeD 1%)93DiscussionThe results of Experiment 6 support and extend thesuggestion, from Experiment 5, that male rat copulation andthe expression of WDS are almost mutually exclusive.Moreover, the present data indicate that the inverserelationship between copulation and WDS is highly specificto the copulatory milieu; Duds and Studs generate comparablelevels of WDS when paired with either nonreceptive femalesor with males. This is the first demonstration of abehavioural correlate, that manifests specifically duringmating tests, of the copulatory failure phenomenon in malerats.It is also important to note that Studs displayedsignificantly fewer WDS in the presence of nonreceptivefemales than in the presence of males. The Stud males werenot engaging in copulation in either of these conditions.This suggests that the marked suppression of WDS productionby Studs, measured when they were paired with receptivefemales, is not readily explicable on the basis ofdifferential activity levels, or as an epiphenomenon of thecopulatory behaviour itself. The present data thereforestrengthen the argument that the in copula expression ofspontaneous WDS, which is believed to be 5-HT2-mediated(Green & Heal, 1985; Zifa & Fillion, 1992), reflects levelsof activation of a 5-HT2--dependent neural mechanism thatmodulates both the WDS and copulatory behaviours.9495Experiment 7In light of the evidence implicating 5-HT2 receptoractivation in the expression of WDS (e.g., Green, 1989;Green & Heal, 1985) the results of Experiments 5 and 6support the idea that spontaneous WDS may reflect concurrentserotonergic activity, at least in mating tests. These dataare also consonant with the pharmacological data collectedin Experiments 1-4, which suggest that 5-HT2 receptoractivity mediates an inhibition of copulatory behaviour inthe male rat.Although Experiments 5 and 6 demonstrated a relation ofWDS to copulatory proficiency that was specific to matingsituations, and it was inferred that this stemmed fromactivity in a 5-HT2-mediated mechanism, it has yet to bedirectly established that the relation between these twobehaviours is a reflection of 5-HT2 receptor activity. Thisissue could be addressed by assessing WDS and copulatorybehaviours while using a pharmacological probe to modulate5-HT2 receptor activity. Therefore, in Experiment 7, WDS andcopulatory behavjours were evaluated in male rats thatreceived varying doses of DCI.96MethodDrugsDOl was prepared in physiological saline, as inExperiments 1-4. All injections were given Sc, 30 mm priorto behavioural testing.ProceduresSixty sexually experienced male rats served asexperimental subjects. The males were randomly assigned toone of three treatment conditions; they received either 0.1mg/kg DCI, 1.0 mg/kg DOl, or the saline vehicle in anequivalent volume (approximately 0.4 ml). Each male rat wasthen paired with a receptive female rat, and the incidenceof WDS and ejaculatory behaviour was scored for a period of60 mm,ResultsThe effects of DCI treatment on the sexual behaviour andsimultaneous WDS of the male rats is depicted in Figure 9.Overall analyses of these measures were performed using theKruskal-Wallis test for k independent samples. DOl had asignificant effect on both the number of ejaculationsachieved by the experimental males (x2) = 21.02,97Figure 9. Effects of DOl treatment on concurrent WDS andejaculations in male rats paired with receptive female ratsfor 60 mm. Data are presented as mean ± S.E.M.MeanWDS/hro0)000III0oII\1213ICo____I-.--’IICDC.,.11I0mCC)0CMTotalEjaculations8699p < 0.0001), and on the amount of WDS they produced (x2) =32.24, p < 0.0001). Subsequent paired comparisons (MannWhitney U test) revealed that WDS scores in each conditionwere significantly different from the scores in the otherconditions (p < 0.0005): Minimal WDS was produced in the 0.0mg/kg DCI condition, moderate levels were evident with 0.1mg/kg DCI, and high levels of WDS occurred with the 1.0mg/kg dose of DCI. Concurrently, ejaculatory behaviour wassignificantly suppressed in male rats treated with 1.0 mg/kgDCI relative to the other two treatments (p < 0.0001).DiscussionThe inverse relation of WDS and copulatory proficiencyin male rats that was demonstrated in Experiments 5 and 6was also evident in Experiment 7, this time induced using apharmacological probe. The fact that DCI is a 5-HT2/1Cagonist (Glennon et al., 1986) extends the previous findingsby providing direct evidence that the relation between WDSand copulatory behaviour in the male rat is 5-HT2/1C-dependent. It is noteworthy that the behaviour of the DOltreated animals in the present experiment strongly resembledthat of the untreated male rats in Experiments 5 and 6; inessence, the present data indicate that 5-HT2/1C activationcauses Studs to become Duds.100Experiment 8In Experiments 1-4, pharmacological manipulations of 5-HT2/1C activity led to the conclusion that 5-HT2 receptoractivation mediates an inhibition of male rat copulation. InExperiments 5 and 6, convergent behavioural evidencedemonstrated that the incidence of spontaneous WDSdiscriminates between males of differing copulatoryproficiency, and that the behavioural assay provided byconcurrent WDS counts is quite specific to sexual behaviourtesting, rather than being a component of a more generalizeddifference between subpopulations of male rats. Thepharmacological data collected in Experiment 7 stronglysuggest that differential WDS expression during mating testsdoes indeed reflect 5—HT2 receptor activation.It remains to be established, however, that the effectsof 5-HT2 activity on WDS and copulatory behaviours aremediated by a unitary population of 5-HT2 receptors. Nodirect evidence has been collected demonstrating that thetwo behaviours rely on a shared neural substrate. It ispossible, for example, that the relation between WDS andmale rat sexual behaviour is somewhat coincidental;activation of one population of 5-HT2 receptors may inhibitsexual behaviour, while simultaneous activation of adifferent population of 5-HT2 receptors may induce WDS. If101this were so, the use of spontaneous WDS as a behaviouralassay would not necessarily be invalidated, but theinference that WDS and male rat sexual behaviour areuniquely related would be weakened.The existence of overlap between neural structures thatcontrol the WDS and male rat copulatory behaviours may beassessed by employing a focal intracerebral manipulation, inwhich 5-HT2 activity is manipulated in only a circumscribedregion of the brain. If WDS and male rat sexual behaviourrely on overlapping neural mechanisms, then a focalintracerebral manipulation that affects one behaviour shouldalso affect the other. If the two behaviours are neurallyindependent, however, and artifactually correlated in testsusing systemic drug injections, a dissociation of thebehaviours should appear with discrete intracerebraltreatments.The precise brain regions involved in the generation ofWDS are unknown. However, research by others has narrowedthe range of candidate regions through the process ofelimination. The neurotoxin 5,7-dihydroxytryptantine (5,7-DHT) destroys serotonergic neurons with reasonableselectivity. When injected intrathecally (i.e., into thefluid-containing space in the spine), 5,7-DHT treatmentreportedly has no effect on the production of WDS (Fone,Johnson, Bennett, & Marsden, 1989). This suggests that WDS102is mediated by structures rostral to the spinal cord. Thegeneration of WDS is also apparently unaffected by completeablation 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 transectionsof the brain rostral to the posterior coimuissure, none ofwhich affected WDS production. Thus, it would appear fromthese studies that WDS must be mediated by a mechanismlocated in the brainstem.Very little is known about the possible involvement ofthe brainsteni in the control of copulatory behaviour in themale rat (Rose, 1990); most of the work on central nervoussystem mechanisms contributing to mating behaviour in malerats has focussed on the critical role of the medialpreoptic area (MPOA), which is an area that lies anterior tothe hypothalamus, and thus also anterior to the posteriorcommissure. However, given the evidence suggesting that theneural substrate for WDS behaviour must be located in thebrainstem, it follows that if WDS and male rat sexualbehaviour are influenced by a common neural mechanism, thatmechanism must be located in the brainstem.Experiment 8 was therefore intended to address severalissues. It is of interest to attempt to identify neural103mechanisms mediating the effects of drug treatments on malerat copulation and simultaneously occurring WDS for at leasttwo reasons. First, this would provide insights into thecentral mechanisms participating in the inhibition of malerat sexual behaviour observed after treatment with 5-HT2/1Cagonists (Foreman et al. 1991; Watson & Gorzalka, 1990,1991). Second, it would elucidate the neural basis of WDS --pharmacologically-induced WDS is increasingly employed inpharmacological research as an index of 5-HT2 activity, andthe ability of new drugs to modulate 5-HT2 receptoractivation is assessed on the basis of the ability of suchdrugs to elicit/inhibit WDS (e.g., Bedard & Pycock, 1977;Meert et al., 1989). The utility of WDS as a pharmacologicalscreening tool would be improved by a more completeunderstanding of the neural mechanisms that contribute tothe production of WDS. In previous pharmacological studies,for example, attempts have been made to correlate changes inWDS after drug treatment with changes in 5-HT2 receptordensity in cortical tissues (Goodwin, Green, & Johnson,1984). Inconsistent results have been obtained, presumablybecause the brain tissues being assayed are not from thestructures that actually mediate the WDS behaviour (Lucki &Minugh-Purvis, 1987).In Experiment 8, copulatory behaviour and WDS weremeasured in male rats that received focal microinjections of104DOl in the brainstem, in the region of the nucleus rapheobscurus (ROb) and inferior olivary complex (ICC). Thisregion was selected for two reasons. First, anatomicalresearch has demonstrated that the neurons of this regionproject to the ventral horns of the spinal cord (Bowker,Westlund, Sullivan, & Coulter, 1982; Tork, 1985, 1990),which contain the motor neurons that would ultimately beinvolved in the production of WDS. Second, it has beendemonstrated that moderate densities of 5-HT2 receptorsexist in the region of the Rob/Icc (Wieland, Krieder,Mcconigle, & Lucki, 1990), supporting the possibility of arole of cells in the Rob/Icc in the expression of WDS.MethodAnimalsTwelve female and 24 male Long—Evans rats, derived fromstock originally obtained from charles River Inc.(Montreal), were employed. Females were prepared as in thepreceding experiments. Animals were housed in wire meshcages, with free access to food and water, and weremaintained on a reversed 12/12 hr light/dark cycle.At approximately 70 days of age, each male rat wasanaesthetized (1.0 mg/kg atropine sulfate 12, as apreanaesthetic treatment to minimize respiratory secretions,105followed by sodium pentobarbital, 45.0 mg/kg IP, plusketamine hydrochloride, 60.0 mg/kg IP) and placed in a Kopfstereotaxic instrument. Using standard stereotaxic surgicalprocedures (Cooley & Vanderwoif, 1978; Gray & Gorzalka,1979), a single guide cannula constructed from 23 gaugestainless steel tubing was implanted in the Rob/bc. The tipof the guide cannula was aimed at a point on the midline,12.6 mm posterior to bregma and 9.8 mm ventrally from thedural surface. Stereotaxic coordinates were derived from theatlas of Paxinos and Watson (1986). Following implantation,the cannula was affixed to the skull using dental acrylicand jeweller’s screws, and the rat was returned to its homecage for 1 week of postoperative recovery. All experimentalmale rats were housed individually following surgery.DrugsFresh drug solutions were prepared immediately prior toeach testing session. Ritanserin (Research Biochemicals) wasprepared as in Experiment 2. Doses of DOl nd (ResearchBiochemicals) were dissolved in physiological saline andinjected in a total volume of 1.0 tl, via the brainstemcannula, using a 30 gauge injection needle. Each dose wasdelivered using a Sage Instruments infusion pump (Model341A), at a flow rate of 4 p1/mm for 15 s. The injectionneedle was left in place for a further 45 s following106injection, to allow the drug solution to fully diffuse awayfrom the needle tip. Ritanserin was administered 90 mmprior to testing; intracerebral injections of DOl were made15 mm prior to testing.ProcedureFollowing surgery, and prior to behavioural scoring, theexperimental male rats were paired with receptive femalerats for 45 mm, on at least three separate occasions, toallow screening of the male rats on the basis of copulatoryproficiency. Males that failed to display normal copulatorybehaviour (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 conductedin five testing sessions, over the course of 5 consecutiveweeks. All behavioural testing occurred during the middle1/3 of the dark phase of the rats’ light-dark cycle. In arepeated measures design, the incidence of WDS and sexualbehaviour was recorded after the experimental males receivedintracerebral microinjection of 0.0 (vehicle-only control),0.1, 1.0, or 10.0 tg of DOl, or after they had received 10.0ig DOl intracerebrally plus a SC injection of 5.0 mg/kgritanserin. The five drug treatments were administered tothe male rats in random counterbalanced fashion, such that107by the conclusion of the experiment each animal had beentested once at each dose.Behavioural observations were conducted in much the samefashion as in Experiments 5-7. After the administration ofdrugs, the male rats were placed in Plexiglas observationchambers (30 X 30 X 45 cm) and allowed 5 mm to habituate totheir new surroundings. Receptive female rats were thenintroduced into the chambers for an observation period of 30iuin. As in Experiments 5-7, an observer blind toexperimental condition recorded the incidence of WDS andsexual behaviour on microcassette tape; the data weretranscribed from tape at the end of each session.At the conclusion of the experiment, each male rat wasdeeply anaesthetized (sodium pentobarbital, 65 mg/kg IP),and a 1 il volume of 1% Evans blue solution was injected viathe brainstem cannula, to aid in histological verificationof the injection site. Each male rat was then transcardiallyperfused with 10% formalin, and the brain was removed,sectioned (35 tm slices), and the slices were mounted ongelatin-coated microscope slides. Following staining of theslices with Cresyl violet, cannula placement wasmicroscopically verified. Examination of the diffusion ofEvans blue at the injection site revealed stain depositionthroughout a spherical area 1-2 mm in diameter.108ResultsA total of 7 experimental rats were excluded from theexperiment, either on the basis of a persistent failure toinitiate copulation in the pre-experimental screening trials(n=5), or due to inaccurate placement of the Rob/bccannula. The effects of the various drug treatments on theWDS and copulatory behaviours of the male rats are depictedin Figure 10. The statistical significance of the treatmenteffects were assessed using overall Friedman tests, followedby Wilcoxon paired comparisons where appropriate.Microinjection of DOl into the region of the ROb/bC hada pronounced effect on both the sexual behaviour and WDS ofthe male rats. Thus, the overall test of the effect ofintracerebral DOl on WDS was statistically significant(x24) = 32.04, p < 0.001). With microinjection of 10.0DOl, almost 400% more WDS was expressed than under the othertreament conditions (p < 0.001). Simultaneously, theintracerebral microinjections of DOl induced a dose—dependent decrease in the mean number of ejaculationsachieved during the 30-mm test sessions (x24) = 36.25, p <0.001). Subsequent paired comparisons revealed that when themales received intra-ROb/IOC injections of DOl in doses ofeither 1.0 or 10.0 ig they displayed significantly lessejaculatory behaviour than they did when they received the1090.0 ig DOl control injection (p < 0.03 and p < 0.01,respectively).Pretreatment of the male rats with a systemic injectionof ritanserin effectively reversed the effects of intraROb/bC microinjection of 10.0 tg DOl on WDS (p < 0.001). Infact, the ritanserin treatment almost completely abolishedthe WDS response, lowering WDS to a level significantlylower than in the 0.0 tg DOl control condition (p < 0.004).Concurrently, ritanserin treatment significantly reversedthe inhibitory influence of intra-ROb/IOC injection of 10.0tg DCI on the copulatory behaviour of the male rats (p <0.02), although baseline levels of ejaculatory behaviourwere not re-established (p < 0,001).110Figure 10. Effects on WDS and ejaculatory behaviour of malerats that received intracerebral microinjection of DOl intothe region of the nucleus raphe obscurus and inferiorolivary complex. In the Ritanserin + DOl condition, the malerats received a subcutaneous injection of ritanserin (5.0mg/kg) in addition to an intra-ROb/IOC microinjection of 10ig DOl. Results are presented as mean ± S.E.M.4C.z0 U) C2.2-I a C.) a1•C 0 00TT•0IC 0 U) C 0 020 15 10 5 00—-OEJACI—IWDS\0.0-p\0.11.0-p.T-• .1.DOlj..tg/rat,brainsteminjection10.0Rltanserin+DOltOjg/ratI-I112DiscussionIn Experiment 7, it was found that systemic injection ofDCI both inhibited sexual behaviour and stimulated WDS,presumably through D0Is agonistic actions at 5-HT2/1Creceptors. These findings were mirrored in the presentexperiment. DCI microinjection into a discrete region of theventromedial brainstem of male rats not only stimulated WDSbut also inhibited their copulatory behaviour. That nodissociation of the effects of DOl emerged with the focalbrainstem manipulation suggests that the two behavioursrely, to some degree, on a common neural substrate vested inthe region of the nucleus raphe obscurus and inferiorolivary complex. Furthermore, the efficacy of ritanserin inreversing the effects of intra Rob/ICC DCI supports theargument that the hypothesized brainstem mechanism ismediated by 5-HT2/1C receptors, given the known 5-HT2/1Cselectivity of both DOl (Glennon, 1986; Hoyer, 1992) andritanserin (Meert et al., 1989).113DISCUSSIONBack to Havelock Ellis (1898) for a moment: He furthernoted that, “I regard sex as the central problem of life”(preface p.xxx). Fortunately, it would seem that thisproblem, at least in the academic sense, is tractable; someof the mysteries of the biochemical control of sexualbehaviour are yielding to ever more detailed levels ofanalysis.In the present experiments, I have investigated therelationship between alterations of activity at 5-HT2receptors (and, to some unknown extent, 5—HT1C receptors)and the control of reproductive behaviour in male rats. Inorder to do this, two different experimental approaches wereemployed. In Experiments 1—4, a systemic pharmacologicalapproach was used to evaluate the sexual behaviour of malerats after the administration of various 5-HT2/1C receptorligands. The drugs varied in their affinities, selectivity,and intrinsic activity. A novel behavioural approach waselaborated in Experiments 5-8: the incidence of WDS in malerats during mating tests was evaluated as a behaviouralassay of concurrent 5—HT2 activity, and, furthermore, theneural basis of this relationship was explored. The resultsof the two sets of experiments that make up these approachesare reviewed and discussed separately in two following114sections. In the third section of the Discussion, theresults of the two approaches are integrated, and it isargued that the data generally support the suggestion that5—HT2 receptors mediate an inhibition of copulatorybehaviour in the male rat. Implications for future researchare also considered.1. General Discussion: Experiments 1—4.In Experiment 1, the 5-HT2/1C receptor agonist DOl wasfound to inhibit the copulatory behaviour of male rats in adose-dependent manner. This finding is consistent with thatof Foreman and associates (1989), and it provided initialevidence that 5-HT2/1C receptor activation inhibited malerat sexual behaviour. In Experiments 2—4, the efficacy ofthe 5—HT2/1C receptor antagonists ritanserin, ketanserin,and pirenperone in reversing DOl-induced inhibition ofcopulation was assessed. All were highly effective inreversing this inhibition, and, interestingly, none had aneffect on male rat sexual behaviour when administered aloneat a dose that was effective in reversing DOl—inducedinhibition. Inconsistencies in the literature regarding theeffects of 5-HT2/1C antagonists on the sexual behaviour ofmale rats may be attributable to the use of excessively highdoses, resulting in behavioural expression of nonselectivebinding to non5-HT2/1C receptors.115Like DOl, ritanserin has very high affinity for 5-HT2and 5-HT1C receptors, but does not discriminate between thetwo subtypes (Hoyer, 1988b, 1992; Meert et al., 1989;Middlemiss & Tricklebank, 1992; Zifa & Fillion, 1992;Glennon & Dukat, 1991). Pirenperone and ketanserin bothdisplay moderate selectivity for 5-HT2 receptors over 5-HT1Creceptors (Hoyer, 1988a, 1988b, 1992; Glennon & Dukat, 1991;Janssen, 1983; Zifa & Fillion, 1992). Basic pharmacologicalprinciples (e.g., Kalant, et al., 1985) suggest that thesethree antagonists are effective in reversing 001-inducedinhibition by competing with 001 for binding at thereceptors through which 001’s effects are exerted. Thebinding data cited here thus suggest that it is the 5-HT2receptor subtype that mediates the effects of these drugs onmale rat sexual behaviour, because it is the subtype forwhich all four drugs have the highest affinity. However, asI have repeatedly noted, it is also important to keep inmind that all these drugs have high affinity for 5-HT1Creceptors as well.An additional complication should be noted. Althoughpirenperone and ketanserin are moderately selective for5-HT2 receptors, both drugs have appreciable affinity forother types of receptors, particularly histaminergic andalpha—adrenergic receptors (Janssen, 1983; Glennon & Dukat,1991). Pirenperone also displays affinity for dopamine116receptors, but ketanserin lacks this activity, suggestingthat the reversal of DOl-induced inhibition by the threeantagonists is not attributable to dopamine receptorblockade. Furthermore, the 5-HT/1C antagonist LY 53857,which has also been reported to reverse DOl—inducedinhibition of copulatory behaviour in male rats, reportedlylacks appreciable activity at alpha-adrenergic receptors(Foreman et al., 1989). This suggests that the effectsobserved 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 tothe complete reversal of DOl-induced inhibition thatoccurred with the lowest dose of pirenperone, higher dosesof pirenperone exerted a significantly inhibitory influence.That is, although pirenperone significantly attenuated DOlinduced inhibition at all doses tested, there wassignificantly less improvement when animals received higherdoses of pirenperone than when they received the lowest dose(50 ig/kg). This pattern of results may reflect some ofpirenperone’s binding to non5—HT receptors. It is possiblethat the data obtained with the lowest dose reflectselective binding of pirenperone to 5-HT2 receptors, withsaturation of 5—HT2 receptors and the behavioural expressionof increased binding to other types of receptors occurring117at higher doses of pirenperone. Support for this idea comesfrom the report that coadministration of the alpha—adrenergic antagonist prazosin with the 5-HT2/1C antagonistLY 53857 abolishes the facilitation of male rat sexualobserved after treatment with LY 53857 alone (Foreman etal., 1989). Thus, the adrenergic activity of ketanserin andpirenperone might account for the inhibition of male ratcopulation that these drugs have been reported to induce,when administered in the absence of other drugs (Foreman etal., 1989; Mendelson & Gorzalka, 1985). In Experiment 4,however, ketanserin produced a dose-dependent reversal ofDOl-induced inhibition, and did not parallel pirenperone inproducing a diminished blockade of the inhibitory effects ofDCI when given in higher doses. As I have discussed, bindingdata suggest that ketanserin lacks the affinity for dopaminereceptors that pirenperone displays. It therefore seemsreasonable to conclude that the biphasic effect ofpirenperone, resulting in attenuated effectiveness of highdoses of pirenperone compared to the low dose, may be due topirenperone ‘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 ofmale rats when given in the absence of other drugs. Thisfinding conflicts with previous reports, which are118themselves contradictory: namely, that the 5-HT2/1Cantagonist LY 53857 facilitates sexual behaviour in malerats (Foreman, et al., 1989), whereas the 5-HT2/1Cantagonists ketanserin and pirenperone are inhibitory whengiven in the absence of other drugs (Mendelson & Gorzalka,1985). Consideration of the affinities of these compounds,and the doses administered, may allow some reconciliation ofthese reports. Pirenperone has been reported to bemoderately inhibitory in otherwise untreated male rats, at adose of 75 rig/kg and to be highly inhibitory at 200 tg/kg(Mendelson & Gorzalka, 1985). Others have found 100 pjg/kg tobe without effect, and report a pirenperone—inducedinhibition 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 foundto be inhibitory, compared to the effects observed with 50FIg/kg. As I have argued, the inhibitory effects ofpirenperone at higher doses may be due to its actions atnon—5—HT2/1C receptors. The effects of ketanserin are moredifficult to evaluate, as the sole report in which aninhibitory effect of ketanserin was noted provided onlypreliminary data collected using a single dose of ketanserin(5.0 mg/kg), ruling out any evaluation of dose-relatedphenomena (Mendelson & Gorzalka, 1985). Nevertheless, I notethat, in Experiment 4, a dose of just 0.2 mg/kg ketanserin119adequately blocked the receptors through which DOl exertsits inhibitory influence, yet when given to otherwise-untreated males, this dose of ketanserin did not affecttheir sexual behaviour. Furthermore, males treated with 5.0mg/kg of ketanserin plus DOl performed at drug-free baselinelevels in Experiment 4.It may be the case that the reported facilitatory effectof LY 53857, when given alone (Foreman et al., 1989), isattributable to antagonism at 5-HT1C rather than 5-HT2receptors; recent reports indicate that LY 53857 exhibitshigher affinity for 5-HT1C than 5-HT2 receptors (foyer,1888a, 1988b, 1992; Zifa & Fillion, 1992). It is noteworthythat the mixed 5-HT1B/1C agonists TFMPP and mCPP reportedlyinhibit sexual behaviour in the male rat (Fernandez-Guasti,Escalante, & Agmo, 1989; Mendelson & Gorzalka, 1988, 1990),an effect which is consonant with the data collected inExperiments 1-4. Although the data collected withpirenperone and ketanserin in Experiments 3 and 4 suggestthat 5-HT2 receptors, in particular, mediate an inhibitionof male rat copulation, precise dissociation of therespective roles of 5-HT2 and 5-HT1C receptors must awaitthe development of more selective ligands.Several reports have appeared in which drugs that areselective for subtypes of serotonin receptors have beenevaluated for their effects on ex copula penile reflexes. It120is interesting to note that the mediation of these reflexesby subtypes of serotonin receptors does not appear toparallel the roles of these receptors in the regulation of.reproductive behaviour in the male rat. In fact, therelation of penile reflexes to activity at subtypes of 5-HTreceptors appears to be the opposite of the relation between5—HT receptor subtypes and copulatory behaviour. Forinstance, it has been reported that the administration ofDOl facilitates penile reflexes, apparently via 5-HT1Creceptor activation in the spinal cord (Berendsen, Jenck, &Broekkamp, 1990). The 5-HT1A agonist 8-OH-DPAT and 5-HT1Apartial agonist buspirone reportedly inhibit penile reflexes(Mathes et al., 1990; Schnur et al., 1990). This is inmarked contrast to the facilitation of male rat sexualbehaviour obtained with these compounds in in copula matingtests (e.g., Ahienius et al., 1981; Mathes, et al 1990).The relation of penile reflexes to overt sexual behaviour isthus unclear, but it does appear that copulatory behaviourand actual seminal emission may be physiologically separableevents (Berendsen et al., 1990).The data collected in Experiments 1-4 provide evidencethat 5-HT2/1C receptor activation mediates an inhibition ofovert in copula sexual behaviour in male rats. Since theseexperiments were conducted, confirmatory data have appearedin the literature. In a study employing essentially the same121procedure as in Experiments 3 and 4 of this dissertation,the efficacy of ritanserin and ketanserin in reversing DCI-induced inhibition of male rat sexual behaviour was recentlyconfirmed, and a similar effect was demonstrated with themoderately selective 5-HT2 antagonist amperozide (Klint,Dahigren, & Larsson, 1992). Amperozide was also reported toproduce a moderate attenuation of male-rat copulatorybehaviour when given in the absence of other drugs, but onceagain, this effect appeared at a dose of amperozide (5.0.mg/kg) that far exceeded the dose of amperozide required toreverse 001-induced inhibition (0.5 mg/kg) (Klint et al.,1992). It is probable that this inhibitory effect of highdoses of amperozide is attributable to nonselective binding.2. General Discussion: Experiments 5—8.Experiments 5-8 were designed to explore the possibilitythat the incidence of spontaneous WDS in male rats duringmating tests might provide an index of concurrent 5—HT2activation that converges on the pharmacological data. Theresults of Experiment 5 and 6 indicated that, compared toDuds, Studs produce very few spontaneous shakes in mating• tests, and that this effect is limited to situations where areceptive female rat is present. The finding of Experiment7, that DCI exerted reciprocal effects on WDS and copulatorybehaviour that paralleled the data collected with untreated122males, provided convergent evidence that the relationbetween sexual behaviour and WDS in male rats is mediated by5—HT2 receptors (and perhaps, to an unknown extent, by 5—HT1C receptors).Taken together, the results of Experiments 5—7 supportthe conclusion that copulatory behaviour in the male rat isincompatible with the expression of WDS. It is interestingto contrast the effects of partner-type on Duds and Studs inExperiment 6. Duds maintained high levels of WDS regardlessof the types of partners they were paired with. By contrast,Studs displayed virtually no WDS when paired with receptivefemales, but displayed significantly increased WDS whenpaired with nonreceptive females and still higher levelswhen paired with males. Thus, in Studs, the characteristicsof the partner in the testing chamber yielded an effect onthe Studs’ expression of WDS that was not unlike a dose-response function, relating the sexual stimuli of thepartner rats to the expression of WDS in the Studs. Thesignificant difference in Studs’ WDS scores between thenonreceptive-feiuale-partner condition and the male—partnercondition is particularly important in this respect. Thenonreceptive females lacked receptive and proceptivebehaviours, and presumably also lacked olfactory cuesassociated with estrus. Nevertheless, it seems reasonable tosuppose that the nonreceptive females still possess a123greater number of sexually stimulating characteristics thando the male partners. It is therefore plausible that theexpression of WDS in this context specifically reflects theoperation of a neural mechanism that tonically inhibits theexpression of sexual behaviour in male rats. Presumably, thesexual stimuli presented by a nonreceptive female rat mightpartially reduce this inhibitory influence, resulting in anintermediate level of WDS expression in the male rats. Inany case, given the dependency of WDS on 5—HT2 receptoractivation, the data collected with Studs in Experiment 6suggests greater 5-HT2 receptor .activity in the presence ofmale partners than in the presence of nonreceptive females.The finding that WDS levels differed systematically acrossthe experimental conditions, within the Studs group, furtherargues that the differences in WDS between Studs and Dudsmeasured in Experiment 5 is not readily explained as areflection of differential levels of activity or arousal. Infact, recent evidence suggests that, if anything, Duds aremore active than Studs (Kohlert & Bloch, 1993).It might be argued that a change in the expression ofWDS under certain conditions, and particularly when inducedby drug treatments, may be an epiphenomenon of somatosensorychanges. In particular, WDS is morphologically similar tothe pinna reflex displayed by most mammals in response tomechanical stimulation of the auditory canal, such as the124reaction of a cat to having a fly land on its ear. Therelationship between the pinna reflex and the expression ofWDS has been studied by Lucki and associates, using anexperimental preparation in which a drop of a viscous liquidis placed in the ear of a rat (Lucki, Eberle, & MinughPurvis, 1987). This treatment causes the rat to shake itshead vigorously in an attempt (usually fruitless) to expelthe droplet. Comparison of this type of head shake with theWDS induced by 5-HTP loading or treatment with 5-HT2agonists suggests that the pinna reflex and WDS areseparable phenomena. Local anaesthesia of the pinnae, butnot treatment with 5-HT2 receptor antagonists, was reportedto be effective in attenuating the head shaking associatedwith the pinna reflex. Conversely, treatment with 5-HT2antagonists, but not anaesthesia of the pinnae, was reportedto be effective in reversing the WDS induced byadministration 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 relationbetween WDS and copulatory behaviour in male rats, theobject of Experiment 8 was to identify a putative 5-11T2-receptor—dependent mechanism important in WDS and male ratsexual behaviour. The available anatomical data suggestedthat this mechanism might be located in the ventromedial125brainstem. Microinjection of DOl in the region of thenucleus raphe obscurus and inferior olivary complex wasfound to produce a pattern of augmented WDS and concurrentcopulatory inhibition that paralleled the results ofExperiment 7, in which systemic treatments were employed.The effects of intra-R0b/IOC DOl were effectively reversedby ritanserin. The obtained data thus support the conclusionthat WDS and male rat copulation are both modulated by ashared neural substrate in the Rob/Icc.Recently, neuroanatomical and lesion data have appearedwhich strongly support the suggestion that WDS and male ratsexual behaviour rely on a common neural mechanism in theregion of the Rob/Icc (Leanza, Pellitteri, Russo, &Stanzani, 1991; Yamanouchi & Kakeyama, 1992). Using afluorescent retrograde double-labelling technique, it hasbeen demonstrated that individual neurons in the Rob/Icchave bifurcating axons which project to both the medialpreoptic area and to the ventral horns of the cervical (ClC2) spinal cord (Leanza et al., 1991). The degree to whichthis anatomical finding agrees with the behavioural datacollected in Experiments 5-8 cannot be over—emphasized: TheMPOA is well established to be of critical importance inmale rat copulation (e.g., Gorzalka & Mogenson, 1977; Rose,1990), and the ventral horns of the cervical spine containthe motoneurons involved in the expression of WDS. It seems126highly probable that the relation of WDS and male ratcopulatory behaviour arises from this collateralizedprojection from the brainstem to the MPOA and cervicalspinal cord.3. Conclusions, speculations, and implications for futureresearch.Taken together, the investigations reported in thisthesis provide strong support for the conclusion that5-HT2/1C receptors mediate an inhibitory influence of 5-HTon the copulatory behaviour of male rats. It is probablethat this influence is attributable to 5-HT2 receptoractivity more than to 5-HT1C receptor activity, but precisedissociation of the roles of these two receptor subtypes inthe control of male rat sexual behaviour must await thedevelopment of more selective ligands. Furthermore, thepresent data suggest that the incidence of WDS during sexualbehaviour reflects concurrent 5-HT2 receptor activity, andimplicates a ventromedial brainstem region as the site of acommon 5-HT2-dependent neural mechanism that contributes toboth WOS and copulatory behaviour in the male rat.Although it appears that activity at 5-HT2 receptorsmediates an inhibition of sexual behaviour in male rats, itmust be acknowledged that the results of contemporaryexperiments probing the functional significance of subtypes127of receptors for serotonin will require frequent review andre—evaluation in the future. During the past 5 years or sothere has been an explosion in the number of new putative 5-HT receptor subtypes being reported, and increasingly finedistinctions are being made within existing subtypeclassifications. At least 11 discrete 5-HT binding siteshave now been described (e.g., Glennon & Dukat, 1991;Bradley, et al., 1992; Zifa & Fillion, 1992), and evidencefor new subtypes continues to mount. It must be emphasizedthat, aside from the 6 established 5-HT.receptor subtypesmentioned in the General Introduction, many of the newlydescribed sites are thus far strictly pharmacologicalentities that have been characterized only in isolatedtissues—— no selective ligands have been described, andsome sites have yet to be established as functionalreceptors. Nevertheless, this burgeoning field has importantconsequences for studies of the behavioural concomitants ofactivity at particular 5-HT receptor subtypes, in two interrelated ways.First, desàriptions of new distinctions within existingreceptor subtypes complicates the attribution of behaviouraleffects to specific receptor subtypes. For example,pharmacological evidence has suggested that there may be twoforms of 5-HT2 receptors, labelled 5-HT2A and 5-HT2B(McKenna & Peroutka, 1989). Until selective ligands are128developed that discriminate between these sites, it isimpossible to determine whether behavioural effectscurrently attributed to 5-HT2 activity are due to activityat one or both of these usubsubtypes. I agree with Glennonand Dukat (1991) that until such time as pharmacologicalprobes are available that discriminate between subtypes of5—HT2 receptors, it is needlessly confusing to discuss anypossible functional significance of 5-HT2 receptorheterogeneity; however, with the development of such ligandsin the future, it will become necessary to examine therespective contributions of activity at subtypes of 5-HT2receptors in behaviours that have been established to be5-HT2-receptor-dependent.Second, the description of new 5-HT binding sitessometimes has the net effect of turning selective ligandsinto nonselective ligands. That is, re-evaluation of aserotonergic drug’s binding affinities may reveal that thedrug has affinity for newly described binding sites, inaddition to its established affinities. For example,quipazine was believed to be a selective 5-HT2 agonist untilit was discovered that quipazine binds with equivalentaffinity to the more recently described 5-HT1B receptors(Hoyer, 1988a) and 5-HT3 receptors (Peroutka & Hamik, 1988;Perry, 1990). As new affinities are described for current129serotonergic drugs, the functional effects of such drugswill require re-evaluation.It is also interesting to note that evidence isaccumulating that certain established subtypes of serotoninreceptors may exhibit functional interactions. Inparticular, recent behavioural evidence suggests thatactivity at 5-HT1A receptors may modulate 5-HT2 mediatedbehaviours, and vice versa. DOl administration has beenreported to facilitate 8-OH-DPAT-induced reciprocal forepawtreading (Arnt & Hyttel, 1989). Conversely, administrationof the selective 5-HT1A agonist 8-OH-DPAT reportedlyinhibits DOl-induced head twitching in mice (Darmani et al.,1990). This inhibition of DOl-induced behaviour isparticularly interesting in the context of the presentexperiments, because 8-OH-DPAT has been found to potentlyfacilitate the sexual behaviour of male rats (e.g., Ahieniuset al., 1981). It is plausible that the stimulatory effectsof 8-OH-DPAT on male rat copulatory behaviour may be atleast partly due to a 5-HT1A receptor-mediated suppressionof a 5-HT2-mediated inhibitory influence. As yet, no reportshave appeared examining the possible effects of interactionsbetween 5-HT1A and 5-HT2 receptor activity on the control ofmale rat copulatory behaviour, although DOl has beenreported very recently to attenuate 8-OH-DPAT-inducedinhibition of lordosis in female rats (Uphouse, Andrade,130Moore, & Caldarola-Pastuszka, 1993). I have attempted toevaluate 5—HT1A— 5—HT2 interactions in male rats on severaloccasions, by examining the efficacy of 8-OH-DPAT, as wellas the selective 5-HT1A partial agonists buspirone andgepirone, in reversing the inhibition of male rat sexualbehaviour induced by DCI. To my dismay, however, I havefound the combination of moderate doses of DOl plus a 5-UT1Aagonist or partial agonist to be uniformly lethal in malerats (Watson & Gorzalka, unpublished data). The issue ofinteractions between 5-HT1A and 5-HT2 receptors in thecontrol of male rat sexual behaviour thus remainsunresolved.It remains somewhat puzzling that doses of 5-HT2/1Cantagonists that are effective in reversing DOl—inducedinhibition of male rat sexual behaviour were not found toaffect the sexual behaviour of male rats when given in theabsence of other drugs, in Experiments 2-4 and in the studyof Klint et al. (1992). Since these drugs exerted afacilitatory affect on male rat copulation by blocking thebinding of an exogenous agonist, DCI, it might be expectedthat a similar facilitation would ensue from blocking thebinding of the endogenous ligand, 5-UT, in otherwiseuntreated male rats. The absence of such effects is noteasily attributed to differential affinities of DCI and 5-HTfor 5-HT2/1C sites; in fact, 5—HT displays lower affinity131for 5-HT2 receptors than any of the drugs used inExperiments 1-4, and would thus be more completely displacedfrom 5-HT2 receptors by the 5-HT2 antagonists than would beDOl. The effects of 5-HT2 receptor antagonists onreproductive behaviour in otherwise untreated male ratscould be evaluated in more detail in future experiments, byadministering these drugs to castrated males maintained ondoses of testosterone that have been titrated to yieldintermediate levels of copulatory proficiency. These maleswould thus provide more range in which to demonstrate anyfacilitation of sexual behaviour that may obtain followingtreatment with 5-HT2 receptor antagonists.In this dissertation, a model of serotonergicparticipation in the control of reproductive behaviour inthe male rat has been described, in which a tonic inhibitionof male rat sexual behaviour has been attributed to a 5-HT2-receptor-dependent mechanism located in the ROb/bC, whichalso participates in the expression of WDS viacollateralized neuronal projections. A neural adaptationthat inhibits copulatory behaviour under inappropriateconditions (Experiment 6) would make ecological sense, sinceinappropriate sexual advances would be at best somewhatcostly, in terms of energy expended in attempting toinseminate an unreceptive female, or in provoking aggressionif directed at a male. This model also provides numerous132opportunities for further investigation. In particular,nothing is yet known about the manner in which the Rob/bcmechanism is modulated by more rostral structures, or themanner in which this system is integrated with other neuralsites of established importance in the control of male ratsexual behaviour, such as the MPOA (Rose, 1990). It ispresumed that the function of the ROb/bC mechanism isgoverned by descending serotonergic neurons, as the datacollected in Experiment 8 suggest that the operation of thismechanism is 5-HT2-receptor-dependent. It is noteworthy thatthe region of the raphe obscurus may receive serotonergicprojections from the midbrain dorsal raphe (Steinbusch &Nieuwenhuys, 1983).Lastly, although it is not clear to what extent theresults of the present experiments offer generalizationsabout the control of sexual behaviour in the human male, itis self-evident that, across mammalian species, copulatorybehaviour is more similar than it is different. Relativelylittle is known about the role of activity at particularsubtypes of serotonin receptors in the control of sexualbehaviour in the human male, primarily because many of thereceptor—selective ligands that have been developed thus farare still being used exclusively for experimental purposesin nonhuman animals. In general, however, treatments thatelevate levels of synaptic 5-HT, such as MAO inhibitors,133tricyclic antidepressants, and serotonin reuptake inhibitorshave been reported to impair sexual behaviour in humanmales, while 5-HT1A agonists such as buspirone and lisurideand 5-HT2 antagonists such as cyproheptadine reportedlyfacilitate sexual behaviour (for review: Meston & Gorzalka,1992), in agreement with data collected using rats. 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