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Sexual behaviour and serotonergic type 2A stereotypic behaviour in male and female rats : the effects… Hanson, Laura A. 1999

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S E X U A L B E H A V I O U R A N D SEROTONERGIC T Y P E 2A STEREOTYPIC B E H A V I O U R IN M A L E A N D F E M A L E RATS : THE EFFECTS OF STRESS A N D CORTICOSTEROIDS by L A U R A A . H A N S O N B.A. , T H E UNIVERSITY OF BRITISH C O L U M B I A , 1992 M . A . , T H E UNIVERSITY OF BRITISH C O L U M B I A , 1996 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Psychology) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A August 1999 © Laura A . Hanson, 1999 U B C Special Collections - Thesis Authorisation Form http://www.library.ubc.ca/spcoll/thesauth.html In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada 1 of 1 9/3/1999 1:32 PM 11 A B S T R A C T Both chronic psychosocial stress and chronic administration of corticosterone have been shown to alter serotonergic type 2A (5-HT2A) receptor activity. A non-invasive behavioural index of 5-HT2A receptor activity is the frequency of "wet dog shakes" (WDS) or serotonergic stereotypy. In addition to WDS, 5-HT2A receptors mediate effects on sexual behaviour in the rat, in particular, inhibition in the male and stimulation in the female. In the present series of experiments, the potential involvement of stress and corticosterone in the regulation of WDS and sexual behaviour in the male and female rat was investigated. In Experiments 1-4, chronic exposure to several different forms of psychosocial stress was found to facilitate female and inhibit male rat sexual behaviour while concurrently increasing the display of WDS in both sexes. In Experiment 5, nefazodone, an antidepressant with 5-HT2A antagonistic properties, blocked the effects of stress on WDS but not sexual behaviour in female rats. In Experiments 6-7, the corticosterone synthesis inhibitor, metyrapone, blocked the effects of stress on sexual proceptivity and WDS in female rats. Metyrapone blocked the effects of stress on WDS but not sexual behaviour in male rats. In Experiments 8-9, high doses of corticosterone administered chronically facilitated female and inhibited male rat sexual behaviour while concurrently increasing WDS in both sexes. In Experiments 10-11, the 5-HT2A antagonist ketanserin was found to completely attenuate the effects of corticosterone on sexual behaviour and WDS in both male and female rats. In Experiments 12-13, the acute administration of corticosterone was found to exert no effect on either sexual behaviour or WDS in male or female rats. The present results indicate that both chronic corticosterone Ill treatment and exposure to chronic stress inhibit male and facilitate female sexual behaviour while concurrently increasing WDS behaviour. The stress-induced facilitation of WDS appears to be related to elevated corticosterone levels and is suggestive of increased 5-HT2A activity. Both corticosterone and stress exerted effects on sexual behaviour in the direction that would be predicted by increased 5-HT2A activity. While the effects of corticosterone on sexual behaviour appear to be mediated by 5-HT2A activity, the effects of stress on sexual behaviour do not appear to be related to either elevations in corticosterone levels or alterations in 5-HT2A activity. T A B L E OF CONTENTS iv Abstract i i Table of contents iv List of Figures vi List of Tables x Acknowledgements xii Introduction.. 1 5-HT 2 A: Pharmacological Considerations 4 5-HT 2 A and Sexual Behaviour 6 5-HT 2 A and Stereotypy 8 Stereotypy and Sexual Behaviour 10 Corticosteroids and 5-HT 2 A Receptor Regulation 11 Corticosteroids and Sexual Behaviour 14 Objectives of the Present Dissertation 16 Experiments General Methods 20 Objective 1 - Effects of Chronic Stress Experiment 1 26 Experiment 2 41 Experiment 3 52 Experiment 4 65 V Objective 2 - Effects of Chronic Stress and 5-HT 2 A Receptor Antagonism Experiment 5 78 Objective 3 - Effects of Chronic Stress and Corticosterone Synthesis Inhibition Experiment 6 91 Experiment 7 99 Objective 4 - Effects of Varying Doses of Chronically Administered Corticosterone Experiment 8 108 Experiment 9 122 Objective 5 - Effects of Chronically Administered Corticosterone and 5-HT 2 A Receptor Antagonism Experiment 10 132 Experiment 11 140 Objective 6 - Effects of Acutely Administered Corticosterone Experiment 12 150 Experiment 13 154 General Discussion Objective 1 159 Objective 2 165 Objective 3 167 Objective 4 169 Objective5 172 Objective 6 173 Conclusions, speculations, and implications for future research 175 References 183 LIST OF FIGURES Figure 1. Wet dog shakes in estrogen and progesterone-primed female rats as a function of stress and adrenalectomy 37-38 Figure 2. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of stress and adrenalectomy 37-38 Figure 3. Wet dog shakes following chronic exposure to stress in the DOI-treated male rat 48-49 Figure 4. Ejaculatory behaviour following chronic exposure to stress in the DOI-treated male rat 48-49 Figure 5. Copulatory efficiency following differential housing conditions in the male rat 55-56 Figure 6. Wet dog shakes following differential housing conditions in the DOI-treated male rat 61-62 Figure 7. Wet dog shakes following 3 days ofdifferential housing conditions in the estrogen-primed DOI-treated female rat 67-68 Figure 8. Receptive behaviour following 3 days ofdifferential housing conditions in the estrogen-primed DOI-treated female rat 67-68 Figure 9. Proceptive behaviour following 3 days ofdifferential housing conditions in the estrogen-primed DOI-treated female rat 69-70 vii Figure 10. Rejection behaviour following 3 days ofdifferential housing conditions in the estrogen-primed DOI-treated female rat 69-70 Figure 11. Proceptive behaviour following 10 days ofdifferential housing conditions in the estrogen-primed DOI-treated female rat 73-74 Figure 12. Wet dog shakes in estrogen-primed female rats as a function of stress and nefazodone treatment 81-82 Figure 13. Proceptive behaviour in estrogen-primed female rats as a function of stress and nefazodone treatment 83-84 Figure 14. Receptivity in estrogen-primed female rats as a function of stress and nefazodone treatment 83-84 Figure 15. Wet dog shakes in estrogen and progesterone-primed female rats as a function of stress and nefazodone treatment 87-88 Figure 16. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of stress and nefazodone treatment 89-90 Figure 17. Receptivity in estrogen and progesterone-primed female rats as a function of stress and nefazodone treatment 89-90 Figure 18. Wet dog shakes in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment ..95-96 Figure 19. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment 95-96 viii Figure 20. Receptivity in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment 97-98 Figure 21. Rejection behaviour in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment 97-98 Figure 22. Wet dog shakes in DOI-treated male rats as a function of stress and metyrapone treatment 101-102 Figure 23. Ejaculation latency in DOI-treated male rats as a function of stress and metyrapone treatment 103-104 Figure 24. Copulatory efficiency in DOI-treated male rats as a function of stress and metyrapone treatment 103-104 Figure 25. Receptivity in estrogen-primed female rats as a function of corticosterone dose 111-112 Figure 26. Proceptive behaviour in estrogen-primed female rats as a function of corticosterone dose 111-112 Figure 27. Wet dog shakes in estrogen and progesterone-primed female rats as a function of corticosterone dose 116-117 Figure 28. Receptivity in estrogen and progesterone-primed female rats as a function of corticosterone dose 118-119 Figure 29. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of corticosterone dose 118-119 Figure 30. Wet dog shakes in male rats as a function of corticosterone dose 124-125 ix Figure 31. Ejaculations in male rats as a function of corticosterone dose 126-127 Figure 32. Ejaculation latency in male rats as a function of corticosterone dose 126-127 Figure 33. Wet dog shakes in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment 134-135 Figure 34. Receptivity in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment 136-137 Figure 35. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment 136-137 Figure 36. Wet dog shakes in DOI-treated male rats as a function of corticosterone and ketanserin treatment 142-143 Figure 37. Ejaculations in DOI-treated male rats as a function of corticosterone and ketanserin treatment 144-145 Figure 38. Ejaculation latency in DOI-treated male rats as a function of corticosterone and ketanserin treatment 144-145 LIST OF TABLES x Table 1. Sexual behaviour measures and wet dog shakes in estrogen-primed female rats as a function of stress and adrenalectomy 31-32 Table 2. Receptivity and rejection behaviour in estrogen and progesterone-primed female rats as a function of stress and adrenalectomy 39-40 Table 3. Measures of spontaneous sexual behaviour and wet dog shakes following chronic exposure to stress in the male rat 44-45 Table 4. Ejaculation latency, post-ejaculatory interval, and copulatory efficiency following chronic exposure to stress in the DOI-treated male rat 50-51 Table 5. Ejaculatory behaviour and wet dog shakes following differential housing conditions in the male rat 57-58 Table 6. Ejaculatory behaviour and copulatory efficiency following differential housing conditions in the DOI-treated male rat 63-64 Table 7. Receptivity, rejection behaviour, and wet dog shakes following 10 days of differential housing conditions in the estrogen-primed DOI-treated female rat...75-76 Table 8. Post-ejaculatory interval and ejaculations in DOI-treated male rats as a function of stress and metyrapone treatment 105-106 Table 9. Wet dog shakes and rejection behaviour in estrogen-primed female rats as a function of corticosterone dose 113-114 xi Table 10. Rejection behaviour in estrogen and progesterone-primed female rats as a function of corticosterone dose 120-121 Table 11. Copulatory efficiency and post-ejaculatory interval in male rats as a function of corticosterone dose 128-129 Table 12. Rejection behaviour in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment 138-139 Table 13. Copulatory efficiency and post-ejaculatory interval in DOI-treated male rats as a function of corticosterone and ketanserin treatment 146-147 Table 14. Sexual behaviour measures and wet dog shakes in estrogen and progesterone-primed female rats after the acute administration of corticosterone 152-153 Table 15. Sexual behaviour measures and wet dog shakes in DOI-treated male rats after the acute administration of corticosterone 156-157 A C K N O W L E D G E M E N T S xn There are so many people whom I wish to acknowledge that this list can in no way be complete. First and foremost, I wish to acknowledge my mentor - Dr. Boris Gorzalka - for all his support and guidance throughout my graduate career. What might have been a long and arduous journey was transformed into one of the most enjoyable times of my life thanks to his patience, his humour, and his faith in my abilities. I would also like to acknowledge the many undergraduate students that assisted me with this research and express my gratitude towards my committee - Dr. Don Wilkie, Dr. Del Paulhus, Dr. Anita Delongis, and Dr. Bi l l Maurice - for their useful advice on ways to improve this dissertation. To all my friends and family - thank you - for always being there, for understanding my absence, and for valuing my presence. I dedicate this dissertation to the memory of my mother whose unconditional love and support allowed me to pursue my dreams. INTRODUCTION 1 The substance known as serotonin (5-hydroxytryptamine; 5-HT) was first isolated in mammalian serum in the laboratory of Irvine H. Page in 1948 and was so named because of it's "serum tonic" vasoconstrictive effects (for review see Folk & Long, 1988). The presence of serotonin in the brain was first detected in 1953 by Twarog and Page (reviewed in Twarog, 1988). The development of novel serotonergic agonists and antagonists in recent years has resulted in an explosion of research on serotonin's role in diverse functions including sleep, aggression, learning and memory, feeding, thermoregulation, hallucinogenesis, depression, and sexual behaviour. The majority of studies prior to the early 1980's described serotonin's role as that of an exclusively inhibitory neurotransmitter (for review see Bevan, Cools & Archer, 1989). The past 2 decades have seen major advances in the understanding of serotonin's putative role in various aspects of behaviour. These recent developments in the behavioural pharmacology of 5-HT can be attributed to the identification of multiple 5-HT receptors and the increasing availability of selective drugs that act on the range of 5-HT receptors (for review see Elliott, Flanigan, Newberry, Zettersom & Leslie, 1994; Murphy, Andrews, Wichems, L i , Tohda & Greenberg, 1998). Approximately 20 years ago, radioligand binding studies revealed that the neurotransmitter serotonin had two distinct receptors which were named the 5-HT, and 5-HT2 receptors (Peroutka & Snyder, 1979; Peroutka, Lebovitz & Snyder, 1981). Subsequently, at least 14 5-HT binding sites have been described in the literature, and the present system of classification 2 involves the differentiation of 5-HT receptors into four major families: 5-HT,, 5-HT2, 5-HT3 and 5-HT 4 (Hoyer & Martin, 1996). Within both the 5HT, and 5HT 2 families exist a number of functional receptor subtypes. For example, the 5-HT2 family consists of three distinct subtypes: 5-HT 2 A 5-HT 2 B and 5-HT 2 C (Hoyer & Martin, 1996). The Serotonin Receptor Nomenclature Committee has reclassified the 5-HT 2 A (formerly 5-HT2), 5-HT 2 B (formerly 5-HT 2 F), and 5-HT 2 C (formerly 5-HT l c ) receptors (Hoyer & Martin, 1996). Once viewed as an exclusively inhibitory neurotransmitter, serotonin now appears as likely to be excitatory as inhibitory, depending on which receptor subtype is activated. It was initially proposed that the 5-HT, receptor inhibited and the 5-HT2 receptor facilitated sexual behaviour. This model has since rapidly evolved to encompass other 5-HT receptor subtypes, including subtypes of the 5-HT2 receptor family. Additionally, research has revealed dramatic sex differences in the consequences of activating specific serotonin receptor subtypes (for review see Gorzalka, Mendelson & Watson, 1990). It has now been demonstrated that 5-HT 2 A receptor activity mediates a facilitation of female sexual behaviour (James, Lane, Hole & Wilson, 1989; Mendelson & Gorzalka, 1985; Mendelson & Gorzalka, 1986; Wilson & Hunter, 1985) and an inhibition of male sexual behaviour (Foreman, Hall & Love, 1989; Watson & Gorzalka, 1991) while increasing "wet dog shakes" (WDS; also called head shakes or head twitches), a stereotyped motor behaviour induced by 5-HT 2 A receptor activation in both the male and female rat (Goodwin, Green & Johnson, 1984; Yap & Taylor, 1983). 3 Strong interactions exist between hormones of the hypothalamic-pituitary-adrenal (HP A) axis and the central 5-HT system: glucocorticoid receptors are present on almost all 5-HT neurons, blood levels of HP A axis hormones affect 5-HT neurotransmission and, conversely, 5-HT transmission modulates blood levels of HP A hormones (for review see Chaouloff, 1993). Since the discovery of multiple serotonin receptors, investigators have begun to narrow their focus to HP A interactions with specific 5-HT receptors. Specifically, recent research has demonstrated that the adrenal steroid corticosterone upregulates 5-HT 2 A receptors (Kuroda, Mikuni, Ogawa & Takahashi, 1992). The effects of chronic corticosterone treatment, at doses which create plasma levels that are equivalent to those seen during times of chronic stress, on sexual behaviour and WDS have been found to be consistent with an upregulation in 5-HT 2 A receptor activity (Gorzalka & Hanson, 1998; Hanson & Gorzalka, 1999). This suggests a functional neuroendocrine link between 5-HT 2 A receptor activity and stress-induced increases in corticosterone which may have implications for a range of different behaviours. The current dissertation proposes the hypothesis that 5-HT 2 A receptor activity plays a role in an organism's response to chronic stress. The demonstration of a potential link between the HPA axis, central serotonergic systems, and behaviour, may have important implications for the pathophysiology and treatment of stress-induced behavioural disorders including sexual dysfunction and depression (for review see Lopez, Vazquez, Chalmers & Watson, 1997; van Pragg, 1996). The following sections comprise a review of the putative role of the 5-HT 2 A receptor and it's relationship to sexual behaviour, stereotypic behaviour, and the hypothalamic-pituitary-adrenal axis. l ^ H T ^ : Pharmacological Considerations A l l members of the 5-HT, receptor family are directly coupled to phosphatidyl inositol hydrolysis, by which the intracellular second messengers inositol triphosphate and diacylglycerol are formed (Julius, 1991). 5-HT 2 A receptors in the central nervous system are located postsynaptically and are most concentrated in the neocortex but are also distributed in the caudate nucleus, hypothalamus, and hippocampus (Pazos, Probst & Palacios, 1987). The 5-HT 2 A receptor is unique in that it has relatively low ligand sensitivity (Leysen, 1990). This suggests that in vivo, this receptor is activated transiently when the local 5-HT concentration is high and probably receives little stimulation under normal physiological conditions. It has been suggested that the 5-HT 2 A receptor may only function under emergency or in pathological conditions (Leysen, 1990). Recent data have demonstrated that the 5-HT 2 A receptor is also unlike all the other serotonergic receptors in that it does not follow the classical rules of receptor regulation (for review see Eison & Mullins, 1996). Serotonergic receptors normally downregulate in response to increased stimulation and upregulate in response to decreased stimulation. However, evidence has now revealed that 5-HT 2 A receptors are resistant to upregulation; it has been shown that most pre- and post-synaptic manipulations fail to promote an upregulation (for review see Sanders-Bush, 1990). 5-HT 2 A receptors downregulate in response to the chronic administration of 5-HT 2 A receptor agonists. Uniquely among monoamine receptors, chronic administration of 5-HT 2 A receptor antagonists also downregulate 5-HT 2 A receptors (Eison, Eison, Yocca & Gianutsos, 1989; Leysen, VanGempel, 5 Gommeren, Woestenborghs & Janssen, 1986). Additionally, the chronic administration of all effective antidepressant drugs results in a downregulation of 5-HT 2 A receptors (Blackshear & Sanders-Bush, 1982; Eison, et al, 1989). The downregulation of 5-HT 2 A receptors following the administration of 5-HT 2 A antagonists and antidepressants occurs even following presynaptic 5-HT denervation, suggesting that this effect is not dependent on overall alterations of serotonin levels (Blackshear, Steranka & Sanders-Bush, 1981; Eison, et al, 1989). These findings call into question the theory that elevated levels of 5-HT 2 A receptors in the brains of patients with major depressive disorder (Mann, Stanley, McBride & McEwen, 1986; Yates, Leake, Candy, Fairbairn, McKeith & Ferrier, 1990) are due to lowered levels of serotonergic activity. Therefore, it has been suggested that non-serotonergic systems may play a role in the regulation of 5-HT 2 A receptor density - in particular, there is evidence suggesting that hormones of the HPA axis may influence this regulation (for review see Lopez, et al, 1997). The exact mechanisms responsible for 5-HT 2 A receptor regulation by either endogenous or exogenous agents remain to be elucidated. Current hypotheses suggest that transcriptional processes are responsible for the developmental alterations seen in 5-HT 2 A levels, and that post-translational modifications of receptor proteins (e.g., phosphorylation, proteolysis) are responsible for the changes seen with pharmacological agents (Roth, Hamblin & Ciaranello, 1990; Toth, 1996). 6 5-HT M and Sexual Behaviour Studies employing 5-HT agonists and antagonists relatively selective for specific receptors have demonstrated the behavioural complexity of the serotonergic system. It is now known that specific 5-HT receptors can mediate diverse effects on the same behaviour. For example, the expression of sexual behaviour can be either inhibited or facilitated depending on which 5-HT receptor subtype is activated (Gorzalka, et al, 1990). It has also been demonstrated that specific 5-HT receptor subtypes may exert differential effects on female and male sexual behaviour (Gorzalka, et al, 1990). Specifically, the 5-HT 2 A receptor has been shown to have different effects on the expression of sexual behaviour depending on the sex of the animal. Increased 5-HT 2 A receptor activity appears to facilitate female rat sexual behaviour and inhibit male rat sexual behaviour. In the female rat, systemic administration of relatively selective 5-HT 2 A receptor agonists, such as (±) 1 -(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), has been shown to consistently stimulate sexual behaviour (Hanson & Gorzalka, 1999; James, et al, 1989; Maswood, Andrade, Caldarola-Pastuszka & Uphouse, 1996). Additionally, the administration of less selective 5-HT 2 A receptor agonists generally produce a facilitation of female sexual behaviour (for review see Gorzalka, et al., 1990; Mendelson, 1992). Similarly, recent research has demonstrated that ventromedial hypothalamic (Wolf, Caldorola-Pastuszka & Uphouse, 1998) or preoptic area (Gonzalez, Greengrass, Russell & Wilson, 1997) infusions of 5-HT 2 A agonists increase sexual receptivity, while infusions of 5-HT 2 A antagonists inhibit lordosis behaviour (Gonzalez, et al, 1997; 7 Uphouse, Colon, Cox, Caldarola-Pastuszka & Wolf, 1996). A review of research employing a wide variety of 5-HT 2 A receptor agonists and antagonists supports the suggestion that 5-HT 2 A receptor activation in the female rat facilitates sexual behaviour (Gorzalka, et al., 1990). Additional research has shown that ovariectomized rats primed with estradiol benzoate and progesterone to induce sexual receptivity, had a significantly higher density of 5-HT 2 A receptors in the preoptic area and median eminence of the hypothalamus than ovariectomized rats not made sexually receptive (Gonzalez, et al, 1997). By contrast, in the male rat, systemic administration of the selective 5-HT 2 A antagonists, LY237733, cyproheptadine, and LY53857 facilitate male rat sexual behaviour suggesting that 5-H T 2 A activation is inhibitory (Abraham, Viesca, Plaza & Marin, 1988; Foreman, et al, 1989; Foreman, Fuller, Nelson, Calligaro, Kurz, Misner, Garbrecht & Parli, 1992). Additionally, the administration of the 5-HT 2 A agonist, DOI, inhibits male rat sexual behaviour (Foreman, et al, 1989; Klint, Dahlgren & Larsson, 1992; Klint & Larsson, 1995; Watson & Gorzalka, 1991), and this effect can be blocked by the administration of 5-HT 2 A antagonists (Foreman, et al, 1989; Watson & Gorzalka, 1991). Consistent with these findings, the central administration of varying doses of DOI in the ventromedial brainstem of male rats produced a dose-dependent inhibition of sexual behaviour which was attenuated by the administration of the 5-HT 2 A receptor antagonist, ritanserin (Watson & Gorzalka, 1992). The attenuation of DOI's inhibitory effect on male rat sexual behaviour seen following the administration of the antipsychotic drug clozapine has been used as evidence that clozapine acts as a 5-HT 2 A antagonist (Klint & Larsson, 1995). Overall, the available data suggest inhibitory influence of 5-HT 2 A receptor activity on male rat sexual behaviour. 8 Taken together, the available data suggest that 5-HT 2 A receptor activation mediates a facilitation of female sexual behaviour and an inhibition of male sexual behaviour. 5-HT 2 A and Stereotypy Prior to the discovery of multiple 5-HT receptors, the pattern of behaviour known as wet dog shakes (WDS) or head twitches was identified and described in the rat. The frequency of WDS, a quivering shudder of the head, neck, and trunk, is correlated with increases in 5-HT activity (Bedard & Pycock, 1977). Subsequent to the identification of multiple 5-HT receptors, it was revealed that WDS are primarily mediated by 5-HT 2 A receptor activity (Yap & Taylor, 1983). WDS can be induced pharmacologically with 5-HT 2 A receptor agonists in rats and mice (Darmani, Martin, Pandey & Glennon, 1990; Goodwin, et al, 1984; Gorzalka & Hanson, 1998; Pranzatelli, 1990; Schreiber, Brocco, Audinot, Gobert, Veiga & Millan, 1995; Yap & Taylor, 1983), and 5-HT 2 A antagonists inhibit the production of WDS (Colpaert & Janssen, 1983; Darmani, et al., 1990; Lucki, Eberle & Minugh-Purvis, 1987; Meert, Niemegeers, Gelders, & Janssen, 1989; Pranzentelli, 1990; Schreiber, et al, 1995; Yamaguchi, Nabeshima, Ishikawa, Yoshida & Kameyama, 1987). Central administration of DOI in the ventromedial brainstem (Watson & Gorzalka, 1992) or the prefrontal cortex (Willins & Meltzer, 1997) of male rats produces a dose-dependent increase in WDS which can be attenuated by the administration of 5-HT 2 A receptor antagonists. 9 The 5-HT 2 A and 5-HT 2 C receptors have been shown to be 51% homologous in terms of overall amino acids and 80% identical in the transmembrane regions (Hartig, 1989). In addition, these receptors share similar pharmacology and coupling to the same second messenger system. Therefore, it is not surprising that many 5-HT 2 A "selective" drugs also have moderate to high affinity for the 5-HT 2 C receptor. Although the 5-HT 2 A receptor agonist DOI also demonstrates high affinity for 5-HT 2 C receptors (Hoyer, 1988), studies with antagonists varying in 5-HT 2 C and 5-HT 2 A receptor activity indicate that the action of DOI on 5-HT 2 C receptors is most likely not relevant to the display of DOI-induced WDS (Skingle, Malcolm, Cole & Nicola, 1991; Willins & Meltzer, 1997). WDS can occur following treatments that do not directly influence the 5-HT 2 A receptor including the administration of phencyclidine (Yamaguchi, et al, 1987), the benzodiazepine clonazepam (Pranzatelli, 1989), the noradrenergic neurotoxin DSP4 (Eison, Yocca & Gianutsos, 1988), insulin (Kleinrok & Juskiewicz, 1986), melatonin (Eison, Freeman, Guss, Mullins & Wright, 1995), putrescine (Genedani, Bernardi, Tagliavini, Botticelli & Bertolini, 1987), cocaine (Essman, Singh & Lucki, 1994), adrenocorticotrophic hormone (ACTH; Kuroda, et al, 1992), and corticosterone (Berendsen, Kester, Peeters & Broekkamp, 1996; Gorzalka & Hanson, 1998; Hanson & Gorzalka, 1999; Hanson, Gorzalka & Brotto, 1998). Additionally, WDS can occur as a consequence of hippocampal stimulation (Araki & Aihara, 1986) and chronic administration of electroconvulsive shock (Moorman, Grahame-Smith, Smith & Leslie, 1996). DOI-induced WDS can be blocked by the administration of the 5-HT I A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (Willins & Meltzer, 1997) or by the administration of a variety of dopaminergic type 1 and 2 receptor 10 antagonists (Schreiber, et al, 1995). Nevertheless, where it has been tested, modulation of central 5-HT 2 A receptor activity has been implicated in WDS produced by non-5-HT manipulations (e.g., Eison, et al, 1988; Eison, et al, 1995; Fone, Johnson, Bennett, & Marsden, 1989; Hanson, et al, 1998; Kleinrok & Juskiewicz, 1986; Kuroda, et al, 1992; Pranzatelli, 1989; Yameguchi, et al, 1987). While 5-HT 2 A receptor activity is normally measured through post-mortem neurochemical methods, the measure of WDS has gained acceptance as a non-invasive behavioural index of central 5-HT 2 A receptor activity in the scientific literature (e.g., Eison, et al, 1995; Essman, et al, 1994; Gorzalka & Hanson, 1998; Hanson & Gorzalka, 1999; Kuroda, et al, 1992; Nagayama & Lu, 1996; Pranzatelli, 1990; Robson, Gower, Kendall & Marsden, 1993; Schreiber, et al, 1995; Watson & Gorzalka, 1990; Yap & Taylor, 1983). Stereotypy and Sexual Behaviour Spontaneously occurring WDS have been used as a noninvasive measure of 5-HT 2 A receptor activity in the context of male sexual behaviour (Watson & Gorzalka, 1990). An inverse relationship exists between frequency of male copulatory activity and spontaneous WDS. Moreover, this inverse relationship is magnified by the administration of DOI. Additionally, the central administration of varying doses of DOI in the ventromedial brainstem of male rats results in a dose dependent decrease in sexual behaviour and a concurrent increase in WDS, which can be effectively blocked by pretreatment with the 5-HT 2 A antagonist ritanserin (Watson & Gorzalka, 1992). These results support 11 the hypothesis that increased 5-HT 2 A receptor activity mediates a facilitation of WDS behaviour and an inhibition of sexual behaviour in the male rat. Similar research remains to be performed in the female. Given the facilitatory role of 5-HT 2 A receptor activity in female sexual behaviour, it is reasonable to expect that spontaneous WDS would be positively correlated with sexual behaviour, and that a 5-HT 2 A receptor agonist would enhance both sexual behaviour and WDS in the female. Corticosteroids and 5-HT 2 A Receptor Regulation Since the discovery of multiple serotonin receptors, research has revealed that the HPA axis interacts with specific 5-HT receptors (for review see Chaouloff, 1995). The interactions between the HPA axis and 5-HT 2 A receptor activity are of considerable interest due to the unusual regulation of the 5-HT 2 A receptor. Chronic stress has been shown to activate the HPA axis. HPA axis activation is part of the body's physiological response to stress, and plasma corticosterone levels are frequently used as an indication of the degree to which an animal is stressed (Nelson, 1980). It has been suggested that a state of chronic stress has been achieved when prolonged exposure to stress has induced both sustained corticosterone elevation and behavioural disruption (Ottenweller, Servatius, Tapp, Drastal, Bergen & Natelson, 1992). During chronic stress, the HPA axis fails to return corticosterone to baseline levels, and these sustained corticosterone levels have morphological and behavioural effects. 12 Recently, the effects of stress on 5-HT 2 A receptor activity have been examined. Subordinate rats subjected to chronic social stress (McKittrick, Blanchard, Blanchard, McEwen & Sakai, 1995), rats chronically subjected to inescapable shock (Nankai, Yamada, Muneoka & Toru, 1995), rats exposed to chronic forced swim stress (Takao, Nagatani, Kitamura, Kawasaki, Hayakawa & Yamawaki, 1995), rats exposed to a chronic variate stress (Ferretti, Blengio, Gamalero & Ghi, 1995) and rats exposed to repeated defeat (Berton, Aguerre, Sarrieau, Mormede & Chaouloff, 1998) have significantly increased serum corticosterone levels and increased 5-HT 2 A receptors in cerebral cortex, and these changes in 5-HT 2 A receptor number are proportional to the extent of HP A axis activation and corticosterone secretion (McKittrick, et al, 1995). Consistent with these findings, chronic stress has also been found to significantly increase the number of WDS (Takao, et al, 1995). The overall concentrations of 5-HT and 5-HT metabolites appear to be unaltered by long-term exposure to stress (Takao, et al, 1995) and therefore it is likely that a non-serotonergic mechanism is involved in the stress-induced upregulation of the 5-HT 2 A receptors. The effects of acute stress are remarkably inconsistent in that published reports reveal increases (Stanford, 1996), decreases (Ferretti, et al., 1995), and no effects on 5-HT 2 A receptor levels (Chaouloff, Baudrie & Coupry, 1994; Takao, et al, 1995; Yamada, Watanabe, Nankai & Toru, 1995). Additionally, acute immobilization stress induced by taping four limbs, applying tail pinch stress and electric foot shock has been found to immediately reduce the frequency of DOI-induced WDS (Yamada, et al, 1995), while in another study 3 hours of restraint stress was found to increase the frequency of DOI-induced WDS (Chaouloff, et al, 1994). Consistent with the effects of chronic stress, chronic administration of high doses of 13 corticosterone (i.e., 20 and 50 mg/kg for 10 days) significantly increases 5-HT 2 A receptor density in the rat brain as measured by radioligand binding using 3H-ketanserin (Fernandes, McKittrick, File & McEwen, 1997; Kuroda, et al, 1992; Takao, Nagatani, Kitamura & Yamawaki, 1997), and behaviourally by increased WDS (Berendsen, et al, 1996; Gorzalka & Hanson, 1998; Hanson & Gorzalka, 1999; Hanson, et al, 1998; Kuroda, et al, 1992). To date, radioligand binding studies performed after chronic corticosterone treatment have examined 5-HT 2 A receptor density in the cerebral cortex and not in other brain regions. The acute administration of corticosterone (50 mg/kg) is apparently without effect on either 5-HT 2 A receptor levels or WDS (Takao, et al, 1997). These studies suggest that chronic adrenal secretion of corticosterone may trigger 5-HT 2 A receptor upregulation. However, inconsistent with this are reports of either no change in 5-HT 2 A receptor-mediated head twitches after chronic corticosterone treatment (30 mg/kg for 10 days) in mice (Young, MacDonald, St. John, Dick & Goodwin, 1992), or no change in 5-HT 2 A receptor binding in the cortex after corticosterone removal by adrenalectomy (Chaouloff, Baudrie & Coupry, 1993). Although there is strong evidence that chronically elevated corticosterone is implicated in the regulation of 5-HT 2 A receptor activity, the conflicting findings render this suggestion equivocal. Corticosteroids and Sexual Behaviour Stress has been postulated to affect sexual behaviour in both the female and the male rat. Chronic sequential exposure to a variety of mild stresses produces a significant decrease in sexual behaviour in the male rat (DAquila, Brain, & Willner, 1994; Retana-Marquez, Salazar & Velazquez-14 Moctezuma, 1996; Sato, Kumamoto & Suzuki, 1992; Sato, Suzuki, Horita, Wada, Shibuya, Adachi, Tsukamoto, Kumamoto & Yamamoto, 1996). One study demonstrated that the chronic stress-induced inhibition of male rat sexual behaviour can be restored by the administration of indeloxazine hydrochloride, a drug that increases monoamine neurotransmission (Sato, et al, 1996). However, unlike the consistent inhibitory effects on male rat sexual behaviour seen following chronic stress, the effects of acute stress appear to be biphasic. The form of acute stress appears to be particularly relevant to the effect seen on sexual behaviour. For example, acute stress by electrical shock or tail pinching facilitates masculine sexual behaviour (Caggiula, 1972; Wang & Hull, 1980), while acute stress by other means has been shown to inhibit male sexual behaviour (Beach, Conovitz, Steinberg & Goldstein, 1956; Menendez-Patterson, Flores-Lozano, Fernandez & Marin, 1978) In contrast to results obtained in the male, chronic psychosocial stress has been demonstrated to increase sexual receptivity and proceptivity in ovariectomized female rats primed with estrogen (Williams, McGinnis & Lumia, 1992). One type of chronic psychosocial stress in the rat, individual housing, has been found to facilitate sexual receptivity in ovariectomized, but not ovariectomized-adrenalectomized, females, suggesting that adrenocortical secretions may be a mediating factor (Gorzalka & Raible, 1981). To date, no studies looking at the effects of acute stress on female rat sexual behaviour have been reported in the literature. The observation that chronic stress can inhibit male or facilitate female sexual behaviour in the rat suggests that corticosterone might influence sexual behaviour. To date, two studies looking 15 at the effects of chronic corticosterone administration on male rat sexual behaviour have been reported in the literature and have provided contradictory results (Gorzalka & Hanson, 1998; Retana-Marquez, Bonilla-Jaime & Velazquez-Moctezuma, 1998). These two studies used very different doses of corticosterone (20 and 50 mg/kg vs. 0.5, 1, 2 and 4 mg per rat) and were administered over different periods of time (13 days vs. 4 days). Since the study using higher doses over a longer period of time demonstrated an inhibition of male rat sexual behaviour (Gorzalka & Hanson, 1998) and the study using lower doses over a shorter period of time found no change in sexual behaviour (Retana-Marquez, et al, 1998) it is possible that different paradigms accounted for the results. The higher doses of corticosterone produce plasma levels similar to those seen following chronic stress and are consistent with the stress-induced inhibition of male rat sexual behaviour (D'Aquila, et al, 1994; Retana-Marquez, et al, 1996). The effects of adrenalectomy on male rat sexual behaviour have been reported to depend on the period of time that has elapsed since the adrenalectomy was performed. Short-term adrenalectomy (i.e., 35 days after surgery) did not modify any of the parameters of male rat sexual behaviour; however, long-term adrenalectomy (i.e., 420 days after surgery) appeared to prevent the age-related decline in sexual performance (Poggioli, Vergoni, Santi, Carani, Baraghini, Zini, Marrama & Bertolini, 1984). In the female rat, the chronic systemic administration of corticosterone has been shown both to facilitate (Hanson & Gorzalka, 1999; Hanson, et al, 1998) and to have no effect (deCatanzaro & Gorzalka, 1980) on sexual behaviour. Similar to the discrepant reports of chronic corticosterone administration on male rat sexual behaviour, these two studies used very different doses of 16 corticosterone (20 and 50 mg/kg vs. 5 pg/kg to 4 mg/kg). Therefore, it remains possible that the effects of chronic corticosterone administration on female rat sexual behaviour are bimodal and that low doses have no effect while high doses mediate a facilitation. A facilitation of female rat sexual behaviour following chronic administration of high doses of corticosterone would be consistent with the results seen following chronic stress (Williams, et al, 1992). However, the one study to examine the effects of centrally administered corticosterone reported an inhibition of sexual receptivity (deCatanzaro, 1987) although the doses used likely produce brain levels of corticosterone that are lower than those seen during times of chronic stress. The acute administration of corticosterone has been reported to both increase (Kubli-Garfias, 1990; Plas-Roser & Aron, 1981) and not affect (Gorzalka & Whalen, 1977) sexual receptivity. Similarly, the effects of adrenalectomy on female sexual behaviour have provided mixed results. Adrenalectomy has been reported to facilitate sexual receptivity in some studies (Eriksson & Sodersten, 1973; Gorzalka & Raible, 1981; Gray & Gorzalka, 1980), and to have no effect in other studies (Erskine, 1985; Kow & Pfaff, 1975; Larsson, Feder & Komisaruk, 1974; Tennent, Smith & Davidson, 1980). Conversely, adrenalectomy appears to inhibit sexual proceptivity (Kow & Pfaff, 1975; Gorzalka & Moe, 1994; Tennent, et al, 1980). Objectives of the Present Dissertation Evidence to date suggests that both chronic stress and chronically administered corticosterone exert opposite effects on sexual behaviour, that is, a facilitation of female and an inhibition of male rat sexual activity. Increased 5-HT 2 A receptor activity also exerts opposite effects on male and 17 female sexual behaviour. Therefore, the overall objective of this dissertation is to determine i f upregulation of 5-HT 2 A receptors serves as a common mechanism for the effects of stress and elevated corticosterone levels on sexual behaviour. This goal was formulated as six general objectives. 1) The first objective was to determine the effects of chronic stress on male and female rat sexual behaviour and WDS. Numerous studies have indicated that chronic stress inhibits male rat sexual behaviour. To date, only one study has directly examined the effects of chronic stress on sexual behaviour in the female rat. The results of this study suggest a significant sex difference in the response to stress. Experiments 1-4 were designed to test whether this sex difference is consistent across a variety of different forms of chronic stress. 2) The second objective was to determine if the effects of stress on sexual behaviour are mediated by a serotonergic mechanism. Given that many different forms of stress alter 5-HT 2 A receptor activity, and that both stress and 5-HT 2 A receptor activity exert differential effects on male and female rat sexual behaviour, it remains possible that the effects of stress on sexual behaviour are mediated by changes in 5-HT 2 A receptor levels. Experiment 5 was designed to test this hypothesis by attempting to prevent the stress-induced alterations in female sexual behaviour using the 5-HT 2 A antagonist, nefazodone. 3) The third objective was to determine whether both the stress induced changes in 5-HT-18 stereotypy and sexual behaviour are mediated, at least in part, by the secretion of corticosterone. Experiments 6-7 were designed to test this hypothesis by attempting to prevent the stress-induced alterations in sexual behaviour and 5-HT 2 A stereotypy with the corticosterone synthesis inhibitor, metyrapone. 4) The fourth objective was to determine i f the effects of corticosterone on sexual behaviour are dose dependent. It has previously been demonstrated in our laboratory that high doses of chronically administered corticosterone facilitate female sexual behaviour and inhibit male sexual behaviour while concurrently increasing WDS in both sexes. However, high doses mimic stress-induced levels of corticosterone and it remains to be determined whether non-stress levels would produce similar results. Reports in the literature are mixed and appear to indicate that the effects of corticosterone on sexual behaviour are dependent on dose. Experiments 8-9 were designed to examine the effects of varying doses of chronically administered corticosterone on both sexual behaviour and WDS. 5) The fifth objective was to determine i f the effects of corticosterone on sexual behaviour are mediated by an upregulation of 5-HT 2 A receptors. It has previously been suggested that the effects of high doses of chronically administered corticosterone on sexual behaviour may be mediated by alterations in 5-HT 2 A receptor activity. This suggestion followed from evidence that concurrent WDS was increased by corticosterone administration. Experiments 10-11 were designed to test this hypothesis by attempting to block the effects of chronic corticosterone administration on sexual behaviour by using the 5-HT 2 A antagonist, ketanserin. 19 6) The sixth objective was to determine if the acute administration of high doses of corticosterone exerts any effect on sexual behaviour. The acute administration of corticosterone has no detectable effect on 5-HT 2 A receptor density. If the effects of chronic corticosterone on sexual behaviour and WDS are mediated via changes in 5-HT 2 A receptor density, the acute administration of corticosterone should not exert an effect on either of these behaviours. Experiments 12-13 were designed to test this hypothesis by testing both sexual behaviour and WDS following the acute administration of a high dose of corticosterone. 21 Hormone and Drug Treatments Estradiol benzoate (EB) and progesterone (P) (Sigma Chemical Company, St. Louis, MO) were dissolved in 0.1 ml peanut oil at the concentrations mentioned in the individual experimental methods sections. (±)l-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (Research Biochemicals International, Natick, M A ) was freshly dissolved in 0.9% saline (1 mg/ml) prior to the commencement of each experiment. Animals were injected 30 minutes prior to behavioural testing at the concentrations mentioned in the individual experimental methods sections. Nefazodone (Pfizer, Groton, PA) was freshly dissolved in 0.9% saline (50 mg/ml) and was injected subcutaneously (2 ml/kg) for a final dose of 100 mg/kg. Metyrapone (2-methyl-l,2-di-3-pyridil-l-propanone; Sigma Chemical Co., St. Louis, MO) was dissolved in propylene glycol at a concentration of 37.5 mg/ml and was injected subcutaneously (2 ml/kg) for a final dose of 75 mg/kg. Corticosterone-21-acetate (Sigma Chemical Co., St. Louis, MO) was dissolved in propylene glycol at the concentrations described in the individual experimental methods sections. 22 Ketanserin tartrate (Research Biochemicals Inc., Natick, M A ) was stored at 5 °C in dark conditions and was freshly dissolved in 0.9% saline at a concentration of 1 mg/ml and was injected intraperitoneal ly (1 ml/kg) for a final dose of 1 mg/kg. A l l injection syringes had 26 gauge and one-half inch stainless steel needles attached. Procedure A l l behavioural testing occurred during the middle third of the dark cycle. Behavioural testing was not videotaped and was performed by a trained observer who remained blind to the experimental condition of the subjects. Behavioural testing consisted of recording male and female copulatory behaviours with the assistance of a microcomputer program designed for this purpose (Holmes, Holmes & Sachs, 1988). When testing males, females were used to elicit sexual behaviour (stimulus females). Receptivity was induced in stimulus females by subcutaneous injection of 10 pg EB 48 hours prior to testing, and 500 pg P 4 hours prior to testing. Receptivity was induced in female subjects by the hormone regimen described in the individual experimental methods and sexually proficient male rats (stud males) were used to elicit sexual behaviour. Rats were tested in Plexiglas chambers (30 X 30 X 45 cm) for the display of sexual behaviours and WDS. Male rats were allowed to habituate to the testing chambers for 10 minutes prior to the commencement of testing. 23 Behavioural Testing of Male Rats The following male sexual behaviour parameters were scored: frequency of mounts with pelvic thrusting prior to ejaculation, frequency of penile intromissions prior to ejaculation, frequency of ejaculations, ejaculation latency (i.e., the period between the first intromission and the first ejaculation) and the post-ejaculatory interval (i.e., the period between ejaculation and the first intromission of the next copulatory bout). Copulatory efficiency was calculated by dividing the number of intromissions by the sum of the number of intromissions and mounts. A high score for copulatory efficiency indicates that more copulatory attempts resulted in intromissions rather than mounts; this measure is relatively independent of motivational changes (Parrott, 1975). WDS behaviour was scored by measuring the number of WDS observed during the testing interval and dividing this number by the test duration to achieve a frequency score of WDS/minute. The test interval was 30 minutes in length. Stimulus females were rotated between males every 10 minutes to maintain the sexual interest of the males. Male rats that failed to achieve ejaculation during the test session were dropped from the data analyses of copulatory efficiency and post-ejaculatory interval, and ejaculation latency scores for these animals were set to the maximum (1800 seconds). Behavioural Testing of Female Rats Test sessions began with the presentation of the female to a sexually experienced male in individual test chambers. Proceptive, receptive, rejection and WDS behaviours were recorded 24 simultaneously. Sexual receptivity was assessed by scoring lordosis behaviour and calculating the lordosis quotient (LQ), defined as the proportion of full lordotic responses exhibited by a female in response to 10 mounts with pelvic thrusting by a male. Full lordosis consisted of a significant downward arching of the back, neck and head stretched upwards, and tail deviated to the side. If a male did not mount, the female was placed with a different male in another test chamber. Proceptive behaviours included ear wiggles (vibrations of the external ears symmetrically around an erected position for 1-2 seconds), and darts and hops (rapid movements followed by an abrupt halt). Rejection behaviours included kicking, boxing, vocalizations, and rolling onto the back. The frequency of rejection behaviours was tallied throughout the session. The test session ended after the female had been mounted 10 times. WDS behaviour was scored by recording the frequency of concurrent WDS during the testing interval. The length of each test session was timed in order that both rejection behaviours and WDS per minute could be calculated. Data for each of the proceptive behaviours, darts and ear wiggles, were combined to form a composite proceptivity score of solicitations per minute (Gorzalka & Moe, 1994). Statistical analysis Data from each of the experiments were analyzed using either analysis of variance (ANOVA) or independent samples t-tests with a significance criterion set at .05. Prior to data analysis for t-tests and unbalanced A N O V A designs, a Levene's test for equality of variance was performed on each variable. A Welch's correction to the degrees of freedom was performed if the variance was 25 found to be heterogenous. Significant A N O V A results were subsequently analyzed using Newman-Keuls pairwise comparisons. Effect sizes were calculated, using Cohen's d, for all statistically significant effects as well as for all results in Experiment 1 where small sample sizes were likely to result in insufficient statistical power. The means and standard errors for all significant results are presented in figures and all other data are presented in tables. OBJECTIVE 1: EFFECTS OF CHRONIC STRESS 26 Chronic stress results in a significant activation of the HPA axis and elevated plasma corticosterone levels. Exposure to a variety of different forms of chronic stress (e.g., individual housing, strobe and noise, cage rotation) has consistently been shown to lead to an inhibition of male sexual behaviour (D'Aquila, et al, 1994; Retana-Marquez, et al, 1996; Sato, et al, 1992). The only study to date designed to directly examine the effects of chronic stress on female sexual behaviour revealed a significant facilitation of sexual behaviour (Williams, et al, 1992). This is consistent with the effects of chronic corticosterone administration at doses that create plasma levels similar to the levels seen during chronic stress (Hanson, et al, 1998). At the neurochemical level, studies have revealed that chronic stress can lead to an upregulation in 5-HT 2 A receptor activity (Takao, et al, 1995). Given that 5-HT 2 A activity exerts opposite effects on male and female rat sexual behaviour, it is plausible that the effects of stress on sexual behaviour are in part mediated by 5-HT 2 A receptor activity. We propose to examine the effects of a variety of different forms of chronically administered stress on both male and female rat sexual behaviour and concurrent WDS behaviour. EXPERIMENT 1A The first experiment was designed to determine whether chronic psychosocial stress administration had an effect on the display of sexual behaviour in estrogen-treated, ovariectomized female rats. Potential adrenal involvement was determined by comparing adrenalectomized and sham-adrenalectomized animals. Finally, the possible role of 5-HT2A-mediation was examined 27 through concurrent measurement of spontaneous WDS. Subjects Wistar female rats served as subjects (n = 22) and at the time of testing, females were 18 months of age (400-450 g). Surgeries Half of the female subjects (n = 11) were randomly chosen to be adrenalectomized at 12 months of age. The remaining female subjects underwent sham surgery at the same time. A l l females were previously ovariectomized at 3 months of age as outlined in the General Methods section. Adrenalectomies were performed via a bilateral lumbar incision under a combination of the 75 mg/kg ketamine hydrochloride and 7 mg/kg xylazine anaesthesia. For the next 3 postoperative days, adrenalectomized rats were maintained on 10% sucrose solution (ad libitum) and 0.9% saline solution, and no solid food was made available. The combination of sucrose and saline solutions immediately following adrenalectomy has been shown to improve the electrolyte status of the animals (Will & Barnett, 1983). Four days after surgery, rats were put on a diet consisting of Purina Rat Chow and 0.9% saline solution ad libitum. Sham adrenalectomies were performed with the surgical procedure identical to that employed for the adrenalectomized rats, except that the adrenals 28 were left intact. Given that large quantities of saline would be harmful to rats with intact adrenals, sham adrenalectomized rats were returned to a normal diet of Purina Rat Chow and tap water (ad libitum) immediately after surgery. Stress Apparatus Animals were placed in a black wooden arena ( 1 1 8 X 1 1 8 X 3 0 cm) covered with Sanicel. A strobe light (7 Hz) and white noise (100 dB ± 5 dB) emitted from a microcassette recorder mounted approximately 1 meter above the wooden arena, were used to administer the chronic stress regimen. Hormone and Drug Administration The experimental females were administered both 0.8 pg EB and 20 ug P. The hormonal doses were determined from a pilot study conducted on adrenally-intact females to arrive at doses which created a moderate level of receptivity and proceptivity. Stress Procedure Five adrenalectomized females and 6 sham-adrenalectomized females were randomly chosen 29 and assigned to the chronic stress group. The stress regimen followed a procedure previously described (Williams, et al, 1992), which was shown to significantly increase sexual behaviour and elevate serum corticosterone levels in the female rat. It consisted of 30 minutes/day of crowding (1 rat per square foot) in the black arena and simultaneous exposure to strobe lighting and white noise in a dark testing room. Immediately following stress exposure, all females were transferred back to their home cages until the next day's stress period. Stress was delivered during the middle third of the dark cycle, and was continued for 59 consecutive days. The remaining 6 adrenalectomized and 5 sham-adrenalectomized females formed the control group, and were removed from their home cages daily but not exposed to stress. Behavioural testing procedure On Day 58, 48 hours prior to behavioural testing, all female rats were injected with 0.8 pg EB. On Day 60, females received 0.9% saline (1 ml/kg) 30 minutes prior to testing. Sexual behaviour parameters and WDS were scored as described in the General Methods section. Results were subsequently analyzed using two-way ANOVAs on each variable with a significance level set at .05. Results and Discussion The means and standard errors for the effects of stress and surgery on sexual behaviour and WDS are presented in Table 1. 30 An examination of WDS results suggests that stress increased WDS and that this effect was blocked by adrenalectomy. However, the WDS interaction failed to reach statistical significance (p = .34). Similarly, there were no statistically significant main effects on WDS of either stress (p = .17) or surgery (p = .74). However, the effect of stress in non-adrenalectomized animals was medium in size (d = .67) and may have reached statistical significance with adequate statistical power. Although inspection of Table 1 suggests that the stressed females displayed more rejection behaviour (p = .32; d = .51), a higher frequency of lordosis (p = .63; d = .30), and more solicitations (p = .44; d = .27) than the control females, these effects did not reach statistical significance and were small to medium in magnitude. Furthermore, there was no effect of adrenalectomy on any measure of sexual behaviour (p > .10), nor was there an interaction between stress and adrenalectomy (p > .10). 31 Table 1. Sexual behaviour measures and wet dog shakes (WDS) in estrogen-primed female rats as a function of stress and adrenalectomy (ADX) or sham-adrenalectomy (SHAM). Data are presented as mean scores (+/- standard error). Receptivity was measured using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. A l l other values are the frequency of the behaviour per minute. The numbers of animals in each group are presented in the left column in parentheses (total n = 22). 32 WDS Solicitation LQ (%) Rejection NO STRESS SHAM(5) 0.00 ±0.00 2.12 ± 2.12 26.7 ± 15.0 0.50 ±0.34 ADX(6) 0.15 ± 0.11 0.05 ± 0.03 14.0 ± 6.8 0.40 ± 0.24 STRESS SHAM(6) 0.24 ±0.18 3.82 ±2.61 38.3 ± 14.5 0.83 ±0.17 ADX(5) 0.09 ± 0.05 1.02 ±0.46 52.0 ±21.8 1.40 ±0.51 33 These results fail to replicate the findings of Williams, et al (1992) that chronic psychosocial stress increased both sexual receptivity and proceptivity and decreased sexual rejection as well as increased WDS (Takao, et al, 1995). While this lack of replication may be accounted for by the small sample sizes and low statistical power in the present experiment, it may also reflect differences in methodology. The study that found an effect of stress on sexual behaviour used subcutaneously implanted estrogen capsules and a high dose of progesterone (500 pg) to induce sexual behaviour, whereas the present study used a very low, acute dose of estrogen (0.8 pg) and no progesterone. This suggests that if stress does affect the display of female sexual behaviour, it is not apparent after the administration of acute estrogen alone. It may be that progesterone is required, that estrogen needs to be administered chronically, or both. Although females show partial receptivity without progesterone, complete sexual responsiveness, including significant displays of proceptivity, may require the presence of both estrogen and progesterone (Fadem, Barfield & Whalen, 1979; Tennent, et al, 1980). Similarly, the administration of both estrogen and progesterone may be necessary to reveal the effects of stress on sexual behaviour. It is surprising that adrenalectomy had no significant effect on the display of either receptive, proceptive or rejection behaviours in the estrogen-treated rat. However, the literature regarding the effects of adrenalectomy on female sexual behaviour remains controversial with reports of both facilitatory effects (e.g., deCatanzaro & Gorzalka, 1980; deCatanzaro, Knipping & Gorzalka, 1981; Gorzalka & Raible, 1981) or no effects (Davidson, Rodgers, Smith & Bloch, 1968; Gorzalka & Moe, 1994; Tennent, etal, 1980). EXPERIMENT IB 34 The results of Experiment 1A indicate that there is no effect of either stress or adrenalectomy on the display of sexual behaviour and WDS in ovariectomized females treated acutely with a low dose of estrogen. It is possible that low statistical power may account, at least in part, for the lack of any effect of either stress or adrenalectomy. However, it may be that the effects of stress or adrenalectomy on WDS or sexual behaviour are progesterone-dependent. Therefore, in the present experiment, WDS and sexual behaviour were measured in ovariectomized rats treated with both estrogen and progesterone. Procedure Female subjects were each injected with 20 pg P immediately following testing in Experiment 1 A. After 2 hours, each female was injected with 0.9% saline (1 ml/kg) and was tested 30 minutes later for sexual behaviour and WDS as described in the General Methods section. Immediately following testing, females were returned to their home cages and the stress administration was terminated. Results were statistically analyzed as in Experiment 1 A. Results and Discussion A significant interaction was found between stress and surgery on WDS behaviour, F(l,18) 35 = 7.74, p = .012. Stress significantly increased WDS (d = 1.34), and this effect was completely attenuated by adrenalectomy as shown in Figure 1. There also was a significant interaction between stress and surgery for proceptive behaviour, F(l,18) = 6.02, p = .019. Chronic stress elevated proceptivity (d = 1.57), and this effect was completely attenuated by adrenalectomy as shown in Figure 2. Examination of Table 2 suggests that chronic stress increased receptive behaviour (p = .08; d = 1.45) and decreased rejection behaviour (p = .21; d = 0.90). However, both of these effects of stress were relatively large in magnitude and may have reached statistical significance with adequate statistical power. Furthermore, there was no effect of adrenalectomy on either receptive or rejection behaviour (p > .10), nor was there an interaction between stress and adrenalectomy (p > .10). These results suggest that stress significantly increases proceptivity and WDS in females treated with estrogen and progesterone, and that this effect is prevented by adrenalectomy. This replicates previous findings (Williams, et al, 1992) of a facilitation in sexual behaviour following chronic stress in females treated with estrogen and progesterone. The blocking of this effect by adrenalectomy indicates that the adrenals or adrenal secretions are necessary for the stress-induced facilitation of proceptivity. The significant interaction between stress and surgery on WDS, and the higher WDS scores of the stressed, sham-adrenalectomized group, support the hypothesis of 5-HT 2 A involvement in female sexual behaviour. 36 The effect of stress on sexual behaviour and WDS became apparent only after the administration of progesterone. Evidence suggests that the effects of stress on serotonin levels are partially dependent on progesterone. Ovariectomized rats pretreated with progesterone and exposed to electric footshock had higher serotonin concentrations than control animals exposed to footshock (Ladisich, 1975); therefore, it may that progesterone acts to enhance serotonergic activity at the 5-H T 2 A receptor. 37 Figure 1. Wet dog shakes (WDS) in estrogen and progesterone-primed female rats as a function of stress and adrenalectomy (ADX). Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 11 animals in both the No Stress (sham = 5; adx = 6) and Stress (sham = 6; adx = 5) conditions. Figure 2. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of stress and adrenalectomy (ADX). Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 11 animals in both the No Stress (sham = 5; adx = 6) and Stress (sham = 6; adx = 5) conditions. No Stress Stress 39 Table 2. Receptivity and rejection behaviour in estrogen and progesterone-primed female rats as a function of stress and adrenalectomy (ADX). Data are presented as mean scores (+/- standard error). Receptivity was measured by the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. Rejection behaviour is the frequency of the behaviour per minute. The numbers of animals in each group are presented in the left column in parentheses (total n = 22). LQ(%) Rejection NO STRESS SHAM(5) 41.7 ±20.1 0.50 ±0.34 ADX(6) 62.0 ± 8.0 0.40 ± 0.24 STRESS SHAM(6) 91.7 ± 5.4 0.00 ±0.00 ADX(5) 68.0 ± 18.6 1.00 ±0.45 41 EXPERIMENT 2A It has been observed that stress has differential effects on male and female sexual behaviours. The results from Experiment 1 suggest that the stress-induced facilitation of female rat sexual behaviour is likely the result of an interaction between progesterone, the HPA axis and the 5-HT 2 A system. This is supported by the concurrent, significant increase in WDS, thus reflecting an increase in 5-HT 2 A activity. It has been noted that males show decreased sexual behaviour and increased WDS in response to 5-HT 2 A activation (Watson and Gorzalka, 1990). If it were shown that chronic stress inhibited sexual behaviour while concurrently increasing WDS in the male, this would strengthen the hypothesis of 5-HT 2 A involvement in the stress-induced inhibition of sexual behaviour in the male. The present study was designed to test this hypothesis. Subjects Male Wistar rats (n = 34) served as subjects and were approximately 7 months of age (600 - 700 g) when tested. Stress procedure A cage rotation scheme was used to administer stress to the male rats according to a procedure that has been described elsewhere, and has been shown to significantly elevate plasma corticosterone levels and disrupt reproduction (Taylor, Weiss & Rupich, 1987). This stress 42 procedure for males differed from that employed for females in Experiment 1. The psychosocial stress procedure which has been found to significantly increase glucocorticoids and sexual behaviour in female rats (Williams, et al, 1992), has yet to be tested for it's effects on glucocorticoids and sexual behaviour in male rats. Animals in the stressed group received random cage rotation into a new cage with unfamiliar cage mates each day. The number of rats per cage and all other housing conditions remained constant. In the control group, males were also moved into new cages; however, familiarity of cage mates was maintained by transferring all members from one cage together into the new cage. The stress procedure was carried out for 27 consecutive days during the middle third of the dark cycle. Aggression screening Male subjects were tested for their relative aggression according to a procedure previously described by Taylor and colleagues (1987). This was performed prior to stress administration so that the possible confounding effect of aggressive behaviour on corticosterone secretion could be eliminated. Only males displaying equivalent levels of aggression and dominance were selected for the study. Males were paired in a partial round-robin manner for three 5 minute sessions over a 1 week period. Each male was allowed to habituate for 10 minutes to a testing chamber before being placed into a new chamber with one other male. Each was scored for typical offensive behaviours (e.g., lateral attack, chase, biting, piloerection, standing on top), and defensive behaviours (e.g., lying on back, flight, freezing, upright posture). Each rat was tested three times with a new male each 43 time, and at least 1 of the 3 opponents was a cage mate. One rat was removed from the study due to illness, thus leaving 33 male subjects. Rats who displayed moderate, but equal, aggression were assigned either to the stressed group (n=17), or the control group (n=16). Behavioural testing procedure On Day 28, each male was injected with a control injection of 0.9% saline solution (1 ml/kg) 30 minutes prior to testing. Behavioural testing for sexual behaviour and WDS occurred as described in the General Methods section. Results were subsequently analyzed using an independent samples t-test for each variable with a significance level set at .05. Results and Discussion Independent samples t-tests performed between the stressed and control groups showed no statistically significant differences on frequency of ejaculations (p = .47), ejaculation latency (p = .62), post-ejaculatory interval (p = .56), copulatory efficiency (p = .73), or WDS (p = .81). A l l results for sexual behaviour measures and WDS are expressed as means and standard errors in Table 3. However, on most measures the trend was in the hypothesized direction of stressed males 44 Table 3. Measures of spontaneous sexual behaviour and wet dog shakes (WDS) following chronic exposure to stress in the male rat. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation. Ejaculations is the total number of ejaculations during the test interval. WDS is the frequency of the behaviour per minute. Ejaculation latency is the period between the first intromission and ejaculation and postejaculatory interval is the time interval in seconds between ejaculation and the first intromission of the next copulatory bout. 45 NO STRESS (n =16) STRESS (n= 17) Ejaculations 1.71 ±0.31 1.47 ±0.31 Ejaculation latency 918.9 ± 158.2 1117.2 ± 140.9 Postejaculatory interval 778.9 ± 165.1 969.3 ± 174.3 Copulatory efficiency 0.35 ±0.07 0.29 ±0.13 WDS 0.04 ±0.02 0.05 ± 0.02 46 showing impaired sexual performance and increased WDS. The present results fail to replicate previous findings that chronic stress significantly decreases male sexual behaviour (D'Aquila, et al, 1994; Sato, et al, 1992). The reasons for this are not obvious. Furthermore, the WDS results do not support the hypothesis that stress increases 5-HT 2 A activity in the male rat. EXPERIMENT 2B In the present experiment, pharmacological manipulation of 5-HT 2 A receptor activity was employed in order to enhance any potential effect of chronic stress on sexual behaviour and WDS. Previous research has shown that the relationship between sexual behaviour and WDS is maximal following the administration of a 5-HT 2 A agonist (Watson & Gorzalka, 1990). Therefore, in the present experiment, the effects of chronic stress on sexual behaviour and WDS were examined following DOI administration. Procedure Immediately following the behavioural test on Day 28 of Experiment 2A, the daily stress regimen was administered and continued until Day 34. Thirty minutes prior to testing, males were injected with DOI (1 mg/kg). Subjects were scored for sexual behaviour and WDS as described in the General Methods section. After testing was completed, the stress regimen was terminated and 47 rats were reassigned to their original housing conditions with no further cagemate rotations. Results were analyzed as in Experiment 2A. Results and Discussion It is apparent from Figure 3 that chronic stress increased the frequency of concurrent WDS measured during sexual behaviour testing. This effect was statistically significant, t(32) = 2.56, p = .015, and relatively large in magnitude (d = 0.88). Statistical analysis revealed that following chronic stress exposure, males displayed significantly fewer ejaculations, t(32) = 2.40, p = .02, d = 0.85, as shown in Figure 4. Males did not differ on measures of ejaculation latency (p = .19), copulatory efficiency (p = .83), or post-ejaculatory interval (p = .46). The inhibitory effects of chronic stress on sexual behaviour are consistent with previous reports in the male rat (D'Aquila, et al, 1994; Sato, et al, 1992). The finding that some, but not all measures are affected, is also consistent with previous reports (Watson & Gorzalka, 1991). Comparison of the results of Experiments 2A and 2B suggests that DOI inhibited sexual behaviour and increased WDS, and that chronic stress enhanced both effects. This supports previous findings that chronically stressed male rats have increased 5-HT 2 A receptor activity (McKittrick, et al, 1995). 48 Figure 3. Wet dog shakes (WDS) following chronic exposure to stress in the DOI-treated male rat. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 16 animals in the No Stress condition and 17 animals in the Stress condition. Figure 4. Ejaculatory behaviour following chronic exposure to stress in the DOI-treated male rat. Data are presented as mean scores (+/- standard error) for the total number of ejaculations during the test interval. There were 16 animals in the No Stress condition and 17 animals in the Stress condition. 49 0.50 I — — , 0.40 h No Stress Stress 50 Table 4. Ejaculation latency, post-ejaculatory interval, and copulatory efficiency following chronic exposure to stress in the DOI-treated male rat. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation. Ejaculation latency is the period between the first intromission and ejaculation in seconds, and post-ejaculatory interval is the time interval in seconds between ejaculation and the first intromission of the next copulatory bout in seconds. 51 NO STRESS (n = 16) STRESS (n = 17) Ejaculation latency Postejaculatory interval Copulatory efficiency 1178.1 ± 148.8 1498.8 ±117.6 1188.1 ±162.5 1411.2 ±150.9 0.27 ±0.09 0.25 ±0.06 EXPERIMENT 3A 52 Individual housing or social isolation acts a stress and has been shown to produce marked physiological disturbances (Hatch, Wiberg, Zawidzka, Carm, Airth & Grice, 1965), significant increases in corticosterone levels (Bennett & Gardiner, 1978; Lovely, Pagano & Paolino, 1972), and marked behavioural changes (Singh, D'Souza & Singh, 1991). Although there have been numerous studies addressing the effects of prepubertal housing conditions on adult sexual behaviour in the male rat, relatively few have been concerned with postpubertal housing conditions. In general, the former have concluded that the observed decrements in male sexual behaviour after individual housing are caused by a lack of social experience during development (e.g., Duffy & Hendricks, 1973; Spevak, Quadagno & Knoeppel, 1973). However, this theory has been weakened by evidence that postpubertal individual housing of male rats, even those reared in a social environment, also exhibited deficits in adult sexual behaviour (deCatanzaro & Gorzalka, 1979). It seems reasonable to speculate that individual housing per se, rather than individual housing during a critical period, constitutes stress which produced the observed deficits in adult sexual behaviour. The available evidence suggests that individual housing of the male rat results in elevated corticosterone levels and impaired sexual performance. Considerable evidence that corticosterone may be involved in the regulation of 5-HT 2 A receptors provide the impetus for the present study. By measuring behavioural changes in WDS and male sexual behaviour concurrently, this study was 53 designed to assess, in a relatively non-invasive manner, the effects of individual housing on 5-HT 2 A receptor activity. It is possible that the effects of individual housing on sexual behaviour are mediated, in part, by the upregulation of 5-HT 2 A receptor density in response to increased corticosterone secretion. Subjects Male Wistar rats (n = 34) were employed as subjects and were tested for behaviour at 7 months of age (500-600 g). Apparatus Standard triple wire mesh cages were used for group housing of rats, and single wire mesh cages for individual housing of rats. Procedure When males were approximately 27 weeks of age, 18 of them were randomly assigned to the experimental group and were housed individually in single wire mesh cages. The remaining 17 males formed the control group and were housed in groups of three or four in triple wire mesh cages. A l l subjects were housed in the same colony room. After 34 days of differential housing, males were tested for sexual behaviour and spontaneous WDS as outlined in the General Methods section with 54 several exceptions. Testing was terminated at 30 minutes or after ejaculation, and the percentage of animals ejaculating within the 30 minute period was calculated (rather than the number of ejaculations). Given that animals were not allowed to proceed to a second copulatory bout, the postejaculatory interval was not calculated. Therefore, four variables were selected to be analyzed a priori. Data were analyzed using independent samples t-tests and the significance criterion was set at .05. Results and Discussion The frequency of WDS did not differ between groups (p = .35) as shown in Table 5. The frequency of WDS in both groups was extremely low with most animals not displaying the behaviour during the test interval. Copulatory efficiency was significantly lower in individually-housed rats, t(22) = 3.92, p < .01, d = 1.6, as shown in Figure 5. No significant difference between groups was found for ejaculation latency (p = .41). Of the group-housed animals, 4 of 17 did not ejaculate within the 30 minute test period. Of the individually-housed animals, 7 of 18 did not attain ejaculation. Data for ejaclulation latency and percent of animals ejaculating are shown in Table 5. 55 Figure 5. Copulatory efficiency following differential housing conditions in the male rat. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation. There were 18 animals in the Group Housed condition and 17 animals in the Individually Housed condition. 0.60 0.50 0.40 0.30 h 0.20 0.10 0.00 Group Housed Individually Housed 57 Table 5. Ejaculatory behaviour and wet dog shakes (WDS) following differential housing conditions in the male rat. Data are presented as mean scores (+/- standard error). Ejaculations is the percentage of animals that ejaculated during the test interval and ejaculation latency is the period between the first intromission and ejaculation in seconds. WDS is the frequency of the behaviour per minute. 58 Group Housed (n = 18) Individually Housed (n = 17) % Ejaculating Ejaculation latency WDS 76 61 875.5 ± 150.1 1122.7 ±155.6 0.002 ±0.000 0.005 ±0.003 59 The present results replicate previous findings (deCatanzaro & Gorzalka, 1979) that specific components of the sexual response pattern, such as copulatory efficiency, rather than all sexual responses, are affected by individual housing. Casual observation of individually-housed rats revealed that relative to group-housed rats, they exhibited the following symptoms: (i) aggressiveness and a tendency to bite when handled, (ii) caudal dermatitis near the base of the tail, and (iii) some loss of hair from the back of the head. This is consistent with the observations of Hatch and colleagues (1965) that individual housing of rats is stressful. The lack of significant differences for WDS is not surprising since only four rats displayed any WDS. Spontaneous WDS behaviour occurs relatively infrequently and the period of behavioural testing may have been too brief to provide sufficient data for statistical analysis. Because of the low frequency of WDS, observed differences in sexual behaviour cannot be attributed to altered 5-HT 2 A receptor activity. EXPERIMENT 3B It was shown in Experiment 3 A that individually-housed male rats were impaired in their sexual performance. The lack of significant differences in spontaneous WDS behaviour may be attributed to the overall infrequent display of this behaviour. It is possible to increase the frequency of WDS either by testing for a significantly longer period of time or by administering a 5-HT 2 A 60 agonist (Watson & Gorzalka, 1990). In Experiment 3B, both sexual behaviour and WDS were observed following the administration of the 5-HT 2 A agonist, DOI. Procedure Animals from Experiment 3A were maintained under their previously assigned housing condition for an additional 4 days. Thirty minutes before testing, each male was injected with 1 mg/kg DOI. Behavioural testing and statistical analysis were performed as in Experiment 3A. Results and Discussion The frequency of WDS was significantly higher in the individually-housed rats, t (33) = 2.68, p < .01, d = .95, as shown in Figure 6. Nine of 17 group-housed animals and 12 of 18 individually-housed animals did not ejaculate. Table 6 suggests a general trend in the direction of reduced copulatory efficiency (p = . 17) and longer ejaculation latency (p = .53) in the individually-housed group but these effects were not statistically significant. 61 Figure 6. Wet dog shakes (WDS) following differential housing conditions in the DOI-treated male rat. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 17 animals in the Group Housed condition and 18 animals in the Individually Housed condition. Group Housed Individually Housed 63 Table 6. Ejaculatory behaviour and copulatory efficiency following differential housing conditions in the DOI-treated male rat. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation, and ejaculation latency is the period between the first intromission and ejaculation in seconds. Ejaculations is the percentage of animals that ejaculated during the test interval. 64 Group Housed (n = 18) Individually Housed (n = 17) % Ejaculating Ejaculation latency Copulatory efficiency 47 33 1245.5 ± 156.9 1399.3 ±159.1 0.32 ±0.04 0.23 ±0.03 65 Results from this experiment indicate that while a trend was present, there was no significant decrease in the sexual behaviour of individually-housed rats relative to group-housed rats. Nevertheless, differences in WDS frequency between individually- and group-housed animals were significant. This may reflect increased 5-HT 2 A activity in the individually-housed rat. EXPERIMENT 4A While numerous studies have examined the effects of individual housing as a means of inducing stress in the male rat, comparatively few studies have utilized individual housing as stress in the female rat. It has been demonstrated that chronic individual housing in postpubertal female rats is an effective stress that significantly increases corticosterone levels (Brown & Grunberg, 1995). It has also been demonstrated that individual housing facilitates sexual receptivity in the female rat (Gorzalka & Raible, 1981). The current study will extend that finding by examining the impact of individual housing on receptivity, proceptivity, rejection and concurrent WDS in the female rat. Subjects. Long-Evans female rats (n = 40) served as subjects and were approximately 8 months of age (300-350 g) at the time of testing. A l l rats were housed in groups of four until the start of the experimental procedure. 66 Procedure. At 8 months of age, 20 female rats were randomly chosen to be individually housed in single wire mesh cages. The other 20 female rats remained housed in groups of four in triple wire mesh cages. A l l 40 rats were kept in the same colony room. Injections of 0.8 pg EB were given daily for three days to all rats. According to Gorzalka and Raible (1981), Day 3 of EB administration is when facilitation of sexual behaviour is most pronounced. On Day 3, rats were tested 3 hours after the last EB injection. DOI (1 mg/kg) was administered 30 minutes prior to behavioural testing. Behavioural testing was performed as described in the General Methods section. Results were analyzed using independent samples t-tests with a significance level set at .05 as outlined in the General Methods section. Results and Discussion Analyses revealed that individual housing significantly increased the display of WDS behaviour, t(38) = 3.73, p = .001, d = 1.14, as shown in Figure 7. As displayed in Figures 8 and 9, individual housing also significantly facilitated both receptive behaviour, t(31.8) = 3.17, p = .001, d = 1.22, and proceptive behaviour, t(38) = 2.86, p = .007, d = 0.88. Paradoxically, individually housed females displayed significantly more rejection 67 Figure 7. Wet dog shakes (WDS) following 3 days of differential housing conditions in the estrogen-primed DOI-treated female rat. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 20 animals in each group. Figure 8. Receptive behaviour following 3 days of differential housing conditions in the estrogen-primed DOI-treated female rat. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. There were 20 animals in each group. 2.00 68 1.60 Group Housed Individually Housed 69 Figure 9. Proceptive behaviour following 3 days of differential housing conditions in the estrogen-primed DOI-treated female rat. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 20 animals in each group. Figure 10. Rejection behaviour following 3 days of differential housing conditions in the estrogen-primed DOI-treated female rat. Data are presented as mean scores (+/- standard error) for the frequency of rejections per minute. There were 20 animals in each group. Group Housed Individually Housed behaviour than females that were grouped housed, t(38) = 2.50, p = .002, d = Figure 10. 71 1.08, as shown in These results suggest that short-term individual housing increases both sexual behaviour and WDS. The finding that sexual rejection was increased even though the animals were more receptive and proceptive is surprising. Typically, a treatment which increases receptivity or proceptivity tends to decrease sexual rejection measures. However, since individual components of female sexual behaviour may depend on different hormonal or neural mechanisms, it is possible that these measures may be dissociated at times. It is also possible that rejection behaviours more accurately reflect a measure of aggression than a measure of sexual behaviour. It is unlikely that stress per se is responsible for this increase in rejection behaviour given that this was not a finding for females exposed to stress in Experiment 1. Perhaps a period of social isolation leads to increased rejection and aggressive behaviours in rats when they are placed with a conspecific of either sex. EXPERIMENT 4B Experiment 4A revealed that a short period of individual housing (3 days) led to a significant increase in sexual behaviour and WDS. However, an overwhelming majority of studies that have examined the behavioural and physiological impact of individual housing have employed a longer period of time (between 7 and 30 days). Experiment 4B was designed to examine whether the behavioural effects of individual housing would be maintained over a longer duration. 72 Animals Female subjects from Experiment 4A were maintained in their housing conditions after behavioural testing on Day 3. Procedure Injections of 0.8 ug EB were continued daily for another seven days to all rats. On Day 10, rats were tested on 3 hours after the last EB injection. DOI (1 mg/kg) was administered 30 minutes prior to behavioural testing. Behavioural testing was performed as described in the General Methods section. Results were analyzed as in Experiment 4A. Results and Discussion Results for WDS behaviour are presented in Table 7. No significant effect of housing conditions was found for WDS (p = .17). Analyses revealed that individual housing significantly increased proceptive behaviour, t(28.3) = 2.97, p = .006, d = 0.92, as displayed in Figure 11. As shown in Table 7, there was no significant group difference on either the measure for receptive behaviour (p = .38) or rejection behaviour (p = .24). 73 Figure 11. Proceptive behaviour following 10 days of differential housing conditions in the estrogen-primed DOI-treated female rat. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 20 animals in each group. 74 Group Housed Individually Housed 75 Table 7. Receptivity, rejection behaviour, and wet dog shakes (WDS) following 10 days of differential housing conditions in the estrogen-primed DOI-treated female rat. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. A l l other values are the frequency of the behaviour per minute. 76 Group Housed (n = 20) Individually Housed (n = 20) LQ (%) Rejection WDS 93.5 ± 1.8 97.5 ±4.12 0.80 ±0.54 0.29 ±0.13 0.67 ±0.32 1.20 ±0.17 77 On Day 3 of isolation, both the display of proceptive and receptive sexual behaviour was facilitated. On Day 10 of isolation, only the display of proceptive behaviour was significantly facilitated. The lack of significant effects for LQ on Day 10 is likely due to a ceiling effect as rats in the control group had an average LQ of 93.5 out of a possible 100. This high level of receptivity is likely the result of the cumulative effects of the chronic estrogen treatment. Although individually housed females displayed nearly double the number of WDS than grouped housed females on Day 10, this measure failed to reach statistical significance. It is unlikely that the use of two consecutive DOI injections over a period of 7 days would have affected WDS testing at Day 10. While it has been demonstrated that the acute administration of DOI results in a downregulation of 5-HT 2 A receptors and that a single injection of DOI significantly reduces WDS following a second injection of DOI, these effects are only apparent over a 24 hour period (Darmani, Martin & Glennon, 1992). Because of the high level of receptivity demonstrated by both groups on Day 10, the test duration was relatively brief compared to that of Day 3, and frequently no WDS occurred within that brief interval. It is conceivable that similar WDS scores would have been obtained had the test duration been identical to that of Day 3. 78 OBJECTIVE 2: EFFECTS OF CHRONIC STRESS A N D 5-HT 2 A RECEPTOR ANTAGONISM Nefazodone, a potent antidepressant that affects serotonergic neurotransmission by both inhibiting the reuptake of serotonin and by antagonizing 5-HT 2 A receptors (Taylor, Carter, Eison, Mullins, Smith, Torrente, Wright & Yocca, 1995), decreases the frequency of WDS in rats treated with a 5-HT 2 A agonist (Eison, Eison, Torrente, Wright & Yocca, 1990). Since chronic stress and nefazodone apparently exert opposite effects on 5-HT 2 A receptor activity as seen by their effects on WDS in the rat, it appears reasonable that nefazodone would antagonize any increase in WDS induced by chronic stress. In addition, i f nefazodone and chronic stress both alter 5-HT 2 A receptor activity, nefazodone may attenuate any effects of stress on sexual behaviour that are mediated by alterations in 5-HT 2 A receptors. Therefore, the purpose of the present experiment was to investigate the chronic effects of nefazodone and stress, singly and in combination, on sexual behaviour and WDS. EXPERIMENT 5A The purpose of the current study is both to replicate the findings that chronic stress facilitates WDS and female sexual behaviour and to attempt to attenuate the effects of stress on these two behaviours through the chronic administration of nefazodone. Although the effects of stress on WDS and female sexual activity are consistent with a common 5-HT 2 A mechanism, attenuation of both by nefazodone would provide stronger evidence. On the other hand, i f nefazodone had no 79 effect on sexual activity, this would suggest that the effects of stress were not acting via a common 5-HT 2 A mechanism. Subjects Long-Evans female rats (n = 40) were employed as subjects and were approximately 6 months of age (325-375 g) at the time of behavioural testing. Stress and injection procedure Female subjects were randomly assigned to one of four treatment groups: no stress and saline; no stress and 100 mg/kg nefazodone; stress and saline; or stress and 100 mg/kg nefazodone. Females assigned to the stress group (n=20), were subjected to 30 minutes stress/day for 30 days as outlined in Experiment 1 A . Nefazodone was injected subcutaneously for 30 days in half of the females randomly selected from each of the stress and no-stress conditions. The remaining females received an injection of 0.9% saline. A l l injections were performed either immediately following stress administration, or after removal of the animal from the home cage. Procedure 80 On Days 23, 28, and 29 all female subjects were injected with 0.8 |ig EB. On Day 31, 24 hours after the last stress and nefazodone administration, females were tested for both sexual behaviour and WDS as described in the General Methods section with the exception that sexual rejection was not measured. Therefore, three variables were chosen a priori to be analyzed. Data were analyzed using a two-way A N O V A with a significance criterion set at .05. Results and Discussion There was a significant interaction between stress and nefazodone on WDS, F(l,36) = 35.3, p < .001, as shown in Figure 12. Chronic stress greatly increased the display of WDS (d = 3.90) and this was completely attenuated by treatment with nefazodone. Females treated with nefazodone alone did not differ on the display of WDS from control females. As displayed in Figures 13 and 14, chronic stress significantly increased proceptivity, F( 1,3 6) = 27.1, p < .001, d = 1.73, and receptivity, F(l,36) = 65.8, p < .001, d = 3.41. Nefazodone treatment alone had no significant effect on either proceptivity or receptivity, nor did it attenuate the effects of stress on these behaviours. 81 Figure 12. Wet dog shakes (WDS) in estrogen-primed female rats as a function of stress and nefazodone treatment. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group. 82 No Stress Stress 83 Figure 13. Proceptive behaviour in estrogen-primed female rats as a function of stress and nefazodone treatment. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 10 animals in each group. Figure 14. Receptivity in estrogen-primed female rats as a function of stress and nefazodone treatment. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. There were 10 animals in each group. No Stress Stress 85 The present results replicate the finding that chronic stress facilitates both female sexual behaviour and WDS. That chronic stress managed to facilitate these behaviours in the absence of progesterone is surprising given that progesterone was necessary for the effects to become apparent in Experiment 1. However, differences in methodology between the two experiments may account for this discrepant finding. In the present experiment, stress was administered for a shorter period of time, a subchronic regimen of estrogen was used, and a greater number of subjects were employed. EXPERIMENT 5B It has previously been demonstrated that some of the effects of antidepressant medications are dependent on moderate circulating levels of the gonadal hormones, testosterone, estrogen, and progesterone (Kendall, Stancel & Enna, 1980; 1981). Additionally, it has been demonstrated that progesterone enhances the effects of nefazodone on WDS and female sexual behaviour (Hanson, et al, 1998). Therefore, it is possible that in the presence of progesterone, nefazodone might attenuate the effects of stress on sexual behaviour. Procedure Immediately following testing in Experiment 5A, females were injected with 50 pg P and tested 3 to 4 hours later. Receptivity, proceptivity, rejection, and WDS were scored as in Experiment 5A. There were 39 females in Experiment 5B because one was excluded due to illness. 86 Results and Discussion There was a significant interaction between stress and nefazodone on WDS, F(l,35) = 13.6, p < .001, as shown in Figure 15. Chronic stress greatly increased the display of WDS (d = 1.78) and this was attenuated by treatment with nefazodone. Females treated with nefazodone alone did not differ on the display of WDS from control females. As displayed in Figures 16 and 17, chronic stress significantly increased proceptivity, F(l,35) = 27.9, p < .001, d = 1.89, and receptivity, F(l,35) = 75.4, p < .001, d = 3.20. Nefazodone treatment alone had no significant effect on either proceptivity or receptivity, nor did it attenuate the effects of stress on these behaviours. The results from Experiment 5 suggest that the effects of stress on WDS behaviour are likely mediated by 5-HT 2 A receptor activity, while the effects of stress on sexual behaviour are not. Chronic stress results in numerous neurochemical and neuroendrocrine changes including activation of the HP A axis, activation of the sympathetic nervous system, increases in beta-endorphin levels, and alterations in serotonergic, dopaminergic and GABAergic transmitter and receptor levels (for review see Lopez, Young, Herman, Aki l & Watson, 1991). Therefore, other mechanisms, singly or in combination, may be responsible for the effects of stress on female sexual behaviour. 87 Figure 15. Wet dog shakes (WDS) in estrogen and progesterone-primed female rats as a function of stress and nefazodone treatment. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group except for the No Stress/vehicle group in which there were 9 animals. 88 1.50 No Stress Stress 89 Figure 16. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of stress and nefazodone treatment. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 10 animals in each group except for the No Stress/vehicle group in which there were 9 animals. Figure 17. Receptivity in estrogen and progesterone-primed female rats as a function of stress and nefazodone treatment. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. There were 10 animals in each group except for the No Stress/vehicle group in which there were 9 animals. No Stress Stress 91 OBJECTIVE 3: EFFECTS OF CHRONIC STRESS A N D CORTICOSTERONE SYNTHESIS INHIBITION One of the primary physiological effects of chronic stress is the activation of the HPA axis. The activation of the HPA axis leads to a significant elevation in plasma corticosteroid levels. Given that the chronic administration of corticosterone has been shown to facilitate female sexual behaviour, inhibit male sexual behaviour, and facilitate WDS in both sexes, it is possible that chronic stress exerts similar effects via elevated corticosterone levels. The objective of the next two experiments is to attempt to block the effects of stress by the corticosterone synthesis inhibitor metyrapone. Metyrapone acts to block corticosterone synthesis through the inhibition of 11-beta-hydroxylase, and pretreatment with metyrapone has been demonstrated to prevent some of the behavioural and neurochemical effects of stress (Calvo, Martijena, Molina & Volosin, 1998). EXPERIMENT 6 Chronic psychosocial stress has been shown to increase female sexual activity (Williams, et al, 1992) and this has been replicated in Experiments 1 and 5 via the same methodology. Experiment 5 demonstrated that the effects of stress on sexual behaviour were not prevented by nefazodone administration but the effects of stress on WDS were. This indicates that stress probably does not alter sexual behaviour via alterations in 5-HT 2 A receptor activity. However, it remains possible that the effects of stress on sexual behaviour are mediated by increases in corticosterone levels. Therefore, the present experiment was designed to test the hypothesis that stress-induced increases 92 in female rat sexual behaviour are mediated by increases in corticosterone levels. This hypothesis was tested by administering the corticosterone synthesis inhibitor metyrapone during the period of chronic stress. Subjects Long-Evans female rats (n = 40) were employed as subjects and were approximately 7 months of age (375-400 g) at the time of behavioural testing. Stress and injection procedure Female subjects were randomly assigned to one of four treatment groups: no stress and propylene glycol; no stress and 75 mg/kg metyrapone; stress and propylene glycol; or stress and 75 mg/kg metyrapone. Females assigned to the stress group (n=20), were subjected to 30 minutes of the stress regimen daily for 30 days as described in Experiment 1 A . Metyrapone was subcutaneously injected daily for 30 days. Injections were performed 3 hours prior to the administration of the stress. Half of the females from each of the stress and no-stress conditions were randomly selected to receive metyrapone. The remaining females received an injection of 40% propylene glycol. 93 Procedure On Days 23, 28, and 29 all female subjects were injected with 0.8 ug EB. Three hours prior to behavioural testing on Day 31, females were administered 20 pg P and were tested for both sexual behaviour and WDS as described in the General Methods section. Data were analyzed using two-way analysis A N O V A s with a significance criterion set at .05 as outlined in the General Methods section. Results and Discussion There was a significant interaction between stress and metyrapone treatment on WDS, F(l,36) = 15.00, p < .001, as shown in Figure 18. Stress was found to increase WDS (d = 2.1) and this was completely attenuated by treatment with metyrapone. Metyrapone treatment had no effect on animals that were not stressed. Similarly, a significant interaction between stress and metyrapone was found for proceptive behaviour, F(l,36) = 7.71, p = .009, as shown in Figure 19. Stress increased the number of solicitations made by the female subjects (d = 1.2) and this effect was attenuated by treatment with metyrapone. Metyrapone treatment on it's own had no effect on proceptive behaviour. Significant main effects of stress were found for both receptivity, F(l,36) = 40.21, p < .001, and rejection behaviour, F(l,36) = 15.81, p < .001. Stressed females were more sexually receptive 94 (d = 1.7) and displayed more rejection behaviour (d = 1.1) than non-stressed females as shown in Figures 20 and 21. Metyrapone treatment had no effect on either of these behaviours, p > .10. These results replicate the finding that stress significantly increases both WDS and the display of female rat sexual behaviour. Interestingly, stress also had the effect of increasing sexual rejection. Metyrapone treatment attenuated the effects of stress on WDS and proceptive behaviour, but had no effect on receptive behaviour or rejection behaviour. This suggests that corticosterone may mediate the effects of stress on WDS and proceptive behaviour, but that receptive behaviour and rejection behaviour are mediated via another mechanism. 95 Figure 18. Wet dog shakes (WDS) in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group. Figure 19. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 10 animals in each group. 96 97 Figure 20. Receptivity in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. There were 10 animals in each group. Figure 21. Rejection behaviour in estrogen and progesterone-primed female rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error) for the frequency of rejections per minute. There were 10 animals in each group. No Stress Stress EXPERIMENT 7 99 Chronic stress has been shown to consistently decrease male rat sexual behaviour (D'Aquila, et al, 1994; Retana-Marquez, et al, 1996; Sato, et al, 1992; Sato, et al, 1996). Chronic corticosterone administration at doses that create plasma levels comparable to those seen during chronic stress has also been shown to decrease male rat sexual behaviour (Gorzalka & Hanson, 1998). Therefore, it is possible that chronic stress acts to inhibit male rat sexual behaviour by increasing corticosterone levels. In the present experiment, the corticosterone synthesis inhibitor, metyrapone, was administered concurrently with the stress regimen. It was hypothesized that metyrapone would attenuate the effects of stress on male rat sexual behaviour as well as WDS. Animals Long-Evans male rats (n = 40) were employed as subjects and were approximately 10 months of age (475-525 g) at the time of behavioural testing. Stress and injection procedure Male subjects were randomly assigned to one of four treatment groups (n = 10 per group): no stress and propylene glycol; no stress and 75 mg/kg metyrapone; stress and propylene glycol; or stress and 75 mg/kg metyrapone. Stress was administered as described in Experiment 2 for 30 days. 100 Metyrapone was injected subcutaneously for 30 days in a randomly selected half of the males from each of the stress and no-stress conditions. The remaining males received an injection of 0.9% propylene glycol. A l l injections were performed 3 hours prior to the day's cage changing. DOI at a concentration of 0.25 mg/kg was administered to all subjects 30 minutes prior to behavioural testing. Procedure Male subjects were tested for both sexual behaviour and WDS as described in the General Methods on Day 31, 24 hours after the last cage rotation occurred. Data were analyzed using two-way A N O V A s with a significance criterion set at .05. Results and Discussion There was a significant interaction between stress and metyrapone treatment on WDS, F(l,36) = 28.60, p < .001, as shown in Figure 22. Stress was found to increase WDS (d = 1.9) and this was completely attenuated by treatment with metyrapone. Metyrapone treatment had no effect on animals that were not stressed. 101 Figure 22. Wet dog shakes (WDS) in DOI-treated male rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group. 1 0 2 No Stress Stress 103 Figure 23. Ejaculation latency (EL) in DOI-treated male rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error). E L is the period between the first intromission and ejaculation measured in seconds. There were 10 animals in each group. Figure 24. Copulatory efficiency in DOI-treated male rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation. There were 10 animals in each group. 104 105 Table 8. Post-ejaculatory interval (PEI) and ejaculations in DOI-treated male rats as a function of stress and metyrapone treatment. Data are presented as mean scores (+/- standard error). Ejaculations is the total number of ejaculations during the test interval. PEI is the time interval in seconds between ejaculation and the first intromission of the next copulatory bout. There were 10 animals in each group (total n = 40). 106 Ejaculations PEI NO STRESS metyrapone 0.40 ±0.16 433.5 ±38.4 vehicle 0.40 ±0.16 464.3 ± 46.2 STRESS metyrapone 0.30 ± 0.15 vehicle 0.20 ±0.13 412.0 ±00.0 107 Eight males in the no-stress condition achieved ejaculation (metyrapone treatment n = 4; no metyrapone treatment n = 4) and 5 males in the stress condition achieved ejaculation (metyrapone treatment n = 3; no metyrapone treatment n = 2). Only 1 male in the stress condition achieved a post-ejaculatory intromission, while 8 of the males in the no-stress condition achieved a post-ejaculatory intromission. Male rats that underwent stress had a longer ejaculation latency, F(l ,36) = 8.69, p = .006, d = 1.1, and were less efficient copulators, F (1,9) = 13.23, p = .005, d = 1.5, as shown in Figures 23 and 24. Metyrapone treatment had no effect on either of these behaviours, p > .10. Stress did not have any effect on the number of ejaculations (p = .34) or the post-ejaculatory interval (p = .60), as shown in Table 8. Additionally, metyrapone treatment had no effect on either of these behaviours, p > . 10. The present results replicate the finding that stress significantly increases WDS and inhibits male rat sexual behaviour. Metyrapone treatment attenuated the effects of stress on WDS, but had no effect on sexual behaviour. This suggests that corticosterone is likely involved in the mediation of WDS by stress, but the effects of stress on male rat sexual behaviour are not mediated by stress-induced elevations in corticosterone. 108 OBJECTIVE 4: EFFECTS OF V A R Y I N G DOSES OF C H R O N I C A L L Y ADMINISTERED CORTICOSTERONE It has previously been shown in our laboratory that the chronic administration of corticosterone facilitates female sexual behaviour, inhibits male sexual behaviour, and facilitates WDS in both sexes (Gorzalka & Hanson, 1998; Hanson & Gorzalka, 1999). Prior reports in the literature of no change in female sexual behaviour (deCatanzaro & Gorzalka, 1980) and no change in male rat sexual behaviour after chronic corticosterone administration (Retana-Marquez, et al, 1998) render these findings equivocal. The studies in which these contradictory findings were reported employed doses that were significantly lower than the doses used in our laboratory (0.5-4 mg/kg vs. 20-50 mg/kg). Additionally, the available evidence suggests that a dose of at least 20 mg/kg is necessary to see an upregulation in 5-HT 2 A receptors (Kuroda, et al, 1992). Therefore, it remains possible that the effects of corticosterone on sexual behaviour are dose-dependent. The current objective is to examine the effects of corticosterone administration on sexual behaviour and WDS for a variety of doses. EXPERIMENT 8A The low doses of chronically administered corticosterone used in the chronic systemic studies (deCatanzaro & Gorzalka, 1980; deCatanzaro, et al, 1981) do not result in the high plasma levels of corticosterone that are seen during times of stress nor do they result in changes in 5-HT 2 A receptor 109 activity (Kuroda, et al, 1992). It has been demonstrated that high doses (i.e., 20 mg/kg) lead to a facilitation of female rat sexual behaviour and concurrently increase WDS (Hanson & Gorzalka, 1999). The present study was designed to see i f the effects of corticosterone on female sexual behaviour and WDS are dose-dependent. Animals Long-Evans female rats (n = 50) served as subjects and were approximately 6 months of age (275-325 g) at the time of testing. Procedure Female rats were randomly assigned to one of five treatment groups: 20 mg/kg corticosterone, 10 mg/kg corticosterone, 4 mg/kg corticosterone, 2 mg/kg corticosterone, or vehicle. Corticosterone injections were given once daily for a period of 10 days and behavioural testing occurred on Day 11, 24 hours after the last corticosterone injection. Forty-eight hours prior to behavioural testing, all subjects were given 2 pg EB. Females were tested for both sexual behaviour and WDS as described in the General Methods section. Results were analyzed using a one-way A N O V A for each variable with a significance level set at .05. Results and Discussion 110 Results for WDS are shown in Table 9. Statistical analysis found no significant effect of corticosterone on WDS behaviour (p = .26). A significant effect of corticosterone dose was found for both receptive behaviour, F(4,45) = 5.71, p = .0008, and proceptive behaviour, F(4,45) = 3.72, p = .011, as shown in Figures 25 and 26. The 20 mg/kg dose of corticosterone increased both receptivity (d = .89) and proceptivity (d = .86) relative to all other doses. No other dose had a significant effect on these behaviours. Results for rejection behaviour are shown in Table 9. Statistical analysis found no significant effect of corticosterone on rejection behaviour (p = .08). The high dose of corticosterone facilitated female sexual behaviour, replicating previous findings (Hanson & Gorzalka, 1999; Hanson, et al, 1998). The present experiment failed to reveal any effect of low doses on sexual behaviour consistent with prior reports (e.g., deCatanzaro & Gorzalka, 1980). The failure to find a significant effect of corticosterone on WDS in females primed with estrogen and not progesterone has been reported previously (Hanson & Gorzalka, 1999). I l l Figure 25. Receptivity in estrogen-primed female rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. There were 10 animals in each group. Figure 26. Proceptive behaviour in estrogen-primed female rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 10 animals in each group. 70 60 50 h 40 30 20 10 h MB 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 1.00 0.80 0.60 0.40 0.20 0.00 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 113 Table 9. Wet dog shakes (WDS) and rejection behaviour in estrogen-primed female rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error) for the frequency of the behaviour per minute. There were 10 animals in each group (total n = 50). WDS Rejection Corticosterone Dose 0 mg/kg 0.17 ±0.05 1.61 ±0.26 2 mg/kg 0.38 ±0.11 2.13 ±0.97 4 mg/kg 0.17 ±0.05 1.11 ±0.75 10 mg/kg 0.19 ±0.06 1.52 ±0.47 20 mg/kg 0.41 ±0.18 1.48 ±0.51 EXPERIMENT 8B 115 Previous research has demonstrated that the effect of corticosterone on WDS is progesterone dependent (Hanson & Gorzalka, 1999). However, that study employed only one dose of corticosterone. The purpose of Experiment 8B was to see i f the addition of progesterone would reveal a dose-dependent effect of corticosterone on either WDS or sexual behaviour. Procedure Immediately following testing in Experiment 8A, females were injected with 50 pg P and tested 3 to 4 hours later. Behavioural testing and statistical analyses were performed as in Experiment 8A. Results and Discussion A significant effect of corticosterone dose was found for WDS behaviour, F(4,45) = 11.09, p < .0001. The 20 mg/kg dose of corticosterone increased WDS behaviour (d = 1.6) relative to all other doses as shown in Figure 27. No other dose had a significant effect on WDS behaviour. 116 Figure 27. Wet dog shakes (WDS) in estrogen and progesterone-primed female rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group. 117 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 118 Figure 28. Receptivity in estrogen and progesterone-primed female rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. There were 10 animals in each group. Figure 29. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 10 animals in each group. 100 119 80 60 40 20 I r I 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 2.00 1.60 1.20 § 0.80 0.40 0.00 •Hfe» 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 120 Table 10. Rejection behaviour in estrogen and progesterone-primed female rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error) for the frequency of rejection behaviours per minute. There were 10 animals in each group (total n = 50). Rejection Corticosterone Dose 0 mg/kg 0.34 ±0.16 2 mg/kg 0.27 ± 0.09 4 mg/kg 0.41 ± 0.06 10 mg/kg 0.25 ± 0.07 20 mg/kg 0.32 ±0.12 122 Significant effects of corticosterone were also found for both receptive, F(4,45) = 5.51, p = .0011, and proceptive behaviour, F(4,45) = 5.28, p = .0014. The 20 mg/kg dose of corticosterone increased both receptivity (d = 1.3) and proceptivity (d = 1.2) relative to all other doses as can be seen in Figures 28 and 29. No other dose of corticosterone had any effect on either receptive or proceptive behaviour. No significant effect of corticosterone was found for rejection behaviour (p = .22). The current results suggest that a high dose of corticosterone facilitates female sexual behaviour and, in the presence of progesterone, increases WDS behaviour. EXPERIMENT 9 Chronic corticosterone treatment has been found to both inhibit male rat sexual behaviour (Gorzalka & Hanson, 1998) and to have no effect on male rat sexual behaviour (Retana-Marquez, et al, 1998). These discrepant findings may reflect the different doses used in each experiment. Relatively high doses (20 and 50 mg/kg) were used in the study that found an inhibitory effect, while lower doses (0.5, 1, 2, and 4 mg per rat) were used in the study that found no effect. The current study was designed to investigate if the effects of corticosterone on male rat sexual behaviour and concurrent WDS are dose dependent. 123 Animals Long-Evans male rats (n = 50) served as subjects. At the time of testing, rats were approximately 6 months of age (400-450 g). Procedure Male rats were randomly assigned to one of five treatment groups: 20 mg/kg corticosterone, 10 mg/kg corticosterone, 4 mg/kg corticosterone, 2 mg/kg corticosterone, or vehicle. Corticosterone injections were given once daily for a period of 10 days and behavioural testing occurred on Day 11, 24 hours after the last corticosterone injection. On Day 11, males were tested for both sexual behaviour and WDS as described in the General Methods section. Results were analyzed using a one-way A N O V A for each variable with a significance level set at .05. Results and Discussion A l l 10 animals achieved ejaculation at least once in the 0,4, and 10 mg/kg dose groups. Nine of 10 animals achieved ejaculation in the 2 mg/kg dose group, but only 2 animals reached ejaculation in the 20 mg/kg dose group. 124 Figure 30. Wet dog shakes (WDS) in male rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group. 0.80 0.70 0.60 0.50 S 0.40 Q. 0.30 0.20 0.10 0.00 i i i i i i 4 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 126 Figure 31. Ejaculations in male rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error). Ejaculations is the number of ejaculations attained during the behavioural test interval. There were 10 animals in each group. Figure 32. Ejaculation latency (EL) in male rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error). EL is the period between the first intromission and ejaculation measured in seconds. There were 10 animals in each group. 2.00 1 2 7 1.60 h v> c o 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 1800 0 mg/kg 2 mg/kg 4 mg/kg 10 mg/kg 20 mg/kg 128 Table 11. Copulatory efficiency and post-ejaculatory interval (PEI) in male rats as a function of corticosterone dose. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation. PEI is the time interval in seconds between ejaculation and the first intromission of the next copulatory bout. There were 10 animals in each group (total n = 50). PEI Copulatory Efficiency Corticosterone Dose 0 mg/kg 435.6 ±67.6 0.31 ± 0.09 2 mg/kg 523.3 ± 134.0 0.24 ± 0.06 4 mg/kg 461.1 ±95.6 0.35 ± 0.08 10 mg/kg 508.7 ± 112.7 0.29 ± 0.05 20 mg/kg 513.8 ±73.4 0.30± 0.11 130 Analysis revealed a significant effect of corticosterone on WDS behaviour, F(4,45) = 12.56, p < .0001. The 20 mg/kg dose increased WDS (d = 1.7) relative to all other doses as shown in Figure 30. No other dose had any effect on WDS behaviour. A significant effect of corticosterone dose was found for the number of ejaculations, F(4,45) = 3.72, p = .011, and ejaculation latency, F(4,45) = 5.82, p = .0007. The 20 mg/kg dose of corticosterone decreased the number of ejaculations (d = 1.5) and increased latency to ejaculation (d = 2.0) relative to all other doses as shown in Figures 31 and 32. No other dose had any effect on these measures. No significant effect of corticosterone was found for copulatory efficiency (p = .63) or post-ejaculatory interval (p = .42). Data for copulatory efficiency and post-ejaculatory interval are presented in Table 11. The current findings indicate that a relatively high dose (20 mg/kg) of corticosterone is required to exert inhibitory effects on male rat sexual behaviour and facilitatory effects on WDS. This dose is significantly higher than the highest dose (equivalent to 11.4-13.3 mg/kg) used in the study that failed to find any effect of corticosterone (Retana-Marquez, et al, 1998). The effects of corticosterone are apparent without the administration of DOI. This strongly suggests that a high dose of corticosterone exerts an even more powerful effect than the stress regimen previously employed. In Experiments 2 and 3, where the stress regimen was used, DOI administration was required for the effects of stress to become apparent. 131 OBJECTIVE 5: EFFECTS OF C H R O N I C A L L Y ADMINISTERED CORTICOSTERONE A N D 5-HT 2 A RECEPTOR A N T A G O N I S M Chronic administration of corticosterone at high doses (20 and 50 mg/kg) has been shown to increase 5-HT 2 A receptor density (Kuroda, et al, 1992), increase WDS behaviour (Berendsen, et al, 1996), inhibit male rat sexual behaviour (Gorzalka & Hanson, 1998), and facilitate female rat sexual behaviour (Hanson, et al, 1998; Hanson & Gorzalka, 1999). Additionally, increased 5-HT 2 A receptor activity has been demonstrated to inhibit male rat sexual behaviour and facilitate female rat sexual behaviour (for review see Gorzalka, et al, 1990) while concurrently increasing WDS in both sexes (Hanson & Gorzalka, 1999; Watson & Gorzalka, 1992). Therefore, it is possible that corticosterone mediates opposite effects on male and female rat sexual behaviour through it's action on the 5-HT 2 A receptor. Additionally, the drug nefazodone, which acts to both inhibit serotonergic reuptake and antagonize 5-HT 2 A receptors (Taylor, et al, 1995), has been shown to completely attenuate the effects of corticosterone on female sexual behaviour and WDS (Hanson, et al, 1998). The drug ketanserin is a potent and'more selective 5-HT 2 A receptor antagonist (Glennon & Dukat, 1991). Moreover, ketanserin is more specific than nefazodone in it's actions and does not alter overall 5-HT levels. The objective of the following experiments was to test the hypothesis that the effects of corticosterone on sexual behaviour and WDS are mediated by an upregulation in 5-HT 2 A receptors. In order to test this hypothesis, ketanserin and corticosterone were administered concurrently. The chronic administration of a 5-HT 2 A antagonist, such as ketanserin, results in a down-regulation of 5-HT 2 A receptors (Eison & Mullins, 1996) which theoretically should counteract any upregulating effect of corticosterone. 132 EXPERIMENT 10 Chronic corticosterone administration at a dose of 20 mg/kg has been shown to increase female rat sexual activity and WDS. If these effects are mediated by an upregulation in 5-HT 2 A receptors, administration of the 5-HT 2 A receptor antagonist ketanserin at the time of corticosterone administration should attenuate these effects by counteracting 5-HT 2 A receptor upregulation. Subjects Long-Evans female rats (n = 36) were employed as subjects and were approximately 6 months of age (300-350 g) at the time of behavioural testing. Procedure Female subjects were randomly assigned to four treatment groups: saline and propylene glycol; 1 mg/kg ketanserin and propylene glycol; saline and 20 mg/kg corticosterone; and 1 mg/kg ketanserin and 20 mg/kg corticosterone. Injections were given once daily for a period of 10 days. On Day 9, female subjects were injected with 0.8 ug EB. On Day 11, females received 50 pg P four hours prior to behavioural testing. Behavioural testing occurred 24 hours after the last ketanserin or corticosterone injection. Females were scored for sexual behaviour and WDS as described in the 133 General Methods section. Results were subsequently analyzed using two-way A N O V A s with a significance criterion set at .05. Results and Discussion A significant interaction between corticosterone and ketanserin was found for WDS, F(l,36) = 11.69, p = .002. Corticosterone facilitated WDS behaviour (d = 1.5) and this was attenuated by ketanserin treatment as shown in Figure 33. Ketanserin treatment alone had no effect on WDS. Similarly, significant interactions between corticosterone and ketanserin were found for both receptivity, F(l,36) = 8.26, p = .007, and proceptivity, F(l,36) = 11.96, p = .001. Corticosterone treatment increased both receptivity (d = 1.9) and proceptivity (d = 1.3) and this effect was attenuated completely by ketanserin treatment as displayed in Figures 34 and 35. Ketanserin on it's own did not have any effect on receptivity and proceptivity. Data for rejection behaviour are presented in Table 12. Neither corticosterone (p = .29) nor ketanserin (p = .85) treatment had any significant effect on rejection behaviour. 134 Figure 33. Wet dog shakes (WDS) in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group. 135 No Corticosterone Corticosterone 136 Figure 34. Receptivity in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error). Receptivity was assessed using the lordosis quotient (LQ) which is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat. There were 10 animals in each group. Figure 35. Proceptive behaviour in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error) for the frequency of solicitations per minute. There were 10 animals in each group. No Corticosterone Corticosterone 138 Table 12. Rejection behaviour in estrogen and progesterone-primed female rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error) for the frequency of rejections per minute. There were 10 animals in each group (total n = 40). Rejection No corticosterone ketanserin 0.17 ±0.06 saline 0.23 ± 0.07 Corticosterone ketanserin 0.30 ±0.09 saline 0.27 ±0.08 140 The current findings indicate that ketanserin attenuates the corticosterone-induced facilitation of female sexual activity and WDS behaviour. These results suggest that the effects of corticosterone on these behaviours are mediated by changes in 5-HT 2 A receptors. The finding that ketanserin on it's own exerted no effect on female sexual behaviour is surprising given that previous studies have indicated that ketanserin inhibits female sexual behaviour (Mendelson & Gorzalka, 1985; Uphouse, et al, 1996). These contradictory findings may be the result of different dosing schedules. Studies that have found an inhibitory effect of ketanserin on female rat sexual behaviour have generally examined the effects within an hour after it's administration (Uphouse, et al, 1996). The current experiment measured sexual behaviour 24 hours after the last administration of ketanserin and therefore it is possible that the level of 5-HT 2 A antagonism was low at the time of behavioural testing. EXPERIMENT 11 Chronic corticosterone administration at a dose of 20 mg/kg has been shown to decrease male rat sexual behaviour and increase WDS. If these effects are mediated by an upregulation in 5-HT 2 A receptors, administration of the 5-HT 2 A antagonist ketanserin at the time of corticosterone administration should attenuate these effects by preventing 5-HT 2 A receptor activation. 141 Subjects Long-Evans male rats (n =36) were tested at 6 months weeks of age (400-450 g). Procedure Male subjects were randomly assigned to four treatment groups: saline and propylene glycol; 1 mg/kg ketanserin and propylene glycol; saline and 20 mg/kg corticosterone; and 1 mg/kg ketanserin and 20 mg/kg corticosterone. Injections were given once daily for 10 days. Behavioural testing occurred on Day 11, 24 hours after the last ketanserin or corticosterone injection. DOI (0.25 mg/kg) was administered 30 minutes prior to behavioural testing. DOI was administered both to prevent a ceiling effect and to ensure that either an inhibition or a facilitation could be seen. Males were scored for sexual behaviour and WDS as described in the General Methods section. Results were subsequently analyzed using two-way A N O V A s with a significance level set at .05. Results and Discussion A significant interaction between corticosterone and ketanserin was found for WDS, F(l,36) = 27.76, p < .0001. Corticosterone facilitated WDS behaviour (d = 1.9) and this was attenuated by ketanserin treatment as shown in Figure 36. Ketanserin treatment alone had no effect on WDS. 142 Figure 36. Wet dog shakes (WDS) in DOI-treated male rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error) for the frequency of WDS per minute. There were 10 animals in each group. 143 144 Figure 37. Ejaculations in DOI-treated male rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error). Ejaculations is the total number of ejaculations attained during the test interval. There were 10 animals in each group. Figure 38. Ejaculation latency (EL) in DOI-treated male rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error). EL is the period between the first intromission and ejaculation measured in seconds. There were 10 animals in each group. No Corticosterone Corticosterone 146 Table 13. Copulatory efficiency and post-ejaculatory interval (PEI) in DOI-treated male rats as a function of corticosterone and ketanserin treatment. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation. PEI is the time interval in seconds between ejaculation and the first intromission of the next copulatory bout. There were 10 animals in each group (total n = 40). 147 PEI Copulatory Efficiency No corticosterone ketanserin 554.5 ±40.9 0.30 ±0.06 saline 573.7 ±77.3 0.29 ±0.05 Corticosterone ketanserin 538.8 ±69.0 0.34 ±0.06 saline 0.15 ±0.00 148 Only one of 10 animals in the group treated with corticosterone alone achieved ejaculation. In each of the other three groups, seven of 10 animals achieved ejaculation at least once. A significant interaction between corticosterone and ketanserin was found for the number of ejaculations, F(l,36) = 6.68, p = .014. Corticosterone treatment decreased ejaculatory behaviour (d = 1.3) and this effect was attenuated completely by ketanserin treatment as shown in Figure 37. Ketanserin on it's own did not have any effect on the number of ejaculations but did slightly but significantly decrease the latency to ejaculation, F(l,36) = 6.55, p = .015, d = .31, as can be seen in Figure 38. Corticosterone did not significantly effect either the post-ejaculatory interval (p = .86) or copulatory efficiency (p = .58). Additionally, ketanserin treatment had no effect on either of these behaviours, p > .10. Data for post-ejaculatory interval and copulatory efficiency are shown in Table 13. The current findings indicate that ketanserin attenuates the corticosterone-induced inhibition of male ejaculatory behaviour and WDS behaviour. These results suggest that the effects of corticosterone on these behaviours are mediated by changes in 5-HT 2 A receptors. Ketanserin treatment alone did exert a facilitatory effect on rats in that it decreased the latency for the DOI-treated males to reach ejaculation. Previous research results have been contradictory with respect to the effects of ketanserin on male rat sexual behaviour (for review see Gorzalka, et al, 1990). Subsequent research revealed that ketanserin can block the DOI-induced inhibition of male rat sexual behaviour but is without effect when animals are not pretreated with DOI (Watson & Gorzalka, 149 1991). Given that the 5-HT 2 A receptor is believed to receive extremely low levels of stimulation under physiological conditions, behavioural effects of antagonists are less likely to be observed without first agonizing the receptor (Leysen, 1990). 150 OBJECTIVE 6: EFFECTS OF A C U T E L Y ADMINISTERED CORTICOSTERONE It has been demonstrated that the acute administration of corticosterone exerts no effect on 5-HT 2 A receptor density or WDS (Takao, et al, 1997). If the effects of chronic corticosterone on sexual behaviour are mediated via changes in 5-HT 2 A receptor density, the acute administration of corticosterone would be less likely to exert an effect on this behaviour. Experiments 12 and 13 were designed to examine this hypothesis by testing both sexual behaviour and WDS following the acute administration of a high dose of corticosterone. EXPERIMENT 12 The acute administration of 300 ug of corticosterone has been found to exert no effect on female rat sexual behaviour when administered subcutaneously (Gorzalka & Whalen, 1977) but a similar dose exerts a significant facilitatory effect when administered intravenously (Kubli-Garfias, 1990). Both studies employed relatively low doses of corticosterone. The current experiment was designed to test the effect of the acute administration of a high dose (20 mg/kg) of corticosterone on both female sexual behaviour and WDS. Subjects Long-Evans female rats (n = 40) were employed as subjects and were approximately 4 months of age (200-300 g) at the time of behavioural testing. 151 Procedure Half of the female subjects (n = 20) were randomly assigned to receive 20 mg/kg of corticosterone which was injected 4 hours prior to behavioural testing. The remaining 20 females were assigned to the control group and received an injection of propylene glycol (1 ml/kg) 4 hours prior to behavioural testing. Forty-eight hours prior to behavioural testing, female subjects were injected with 2 pg EB, and 4 hours prior to behavioural testing, females received 50 pg P. Four hours after corticosterone and progesterone administration, female subjects were tested for sexual behaviour and for the frequency of WDS as described in the General Methods section. Results were subsequently analyzed using independent samples t-tests with a significance level set at .05. Results and Discussion Statistical analyses failed to reveal significant differences between female rats receiving corticosterone and females not receiving corticosterone on WDS (p = .80), receptivity (p = .12), proceptivity (p = .37), or sexual rejection (p = .61). 152 Table 14. Sexual behaviour measures and wet dog shakes (WDS) in estrogen and progesterone-primed female rats after the acute administration of corticosterone. Data are presented as mean scores (+/- standard error). Lordosis quotient (LQ) is the proportion of full lordoses to 10 mounts (x 100%) made by a male rat, all other measures are the frequency of the behaviour per minute. There were 20 animals in each group (total n = 40). 153 WDS Solicitation LQ (%) Rejection Corticosterone 0.04 ± 0.02 0.29 ±0.11 72.5 ± 5.4 0.14 ±0.04 Propylene glycol 0.04 ±0.01 0.42 ±0.10 61.0 ±4 .6 0.11 ±0.04 154 The means and standard errors for measures of sexual behaviour and WDS are presented in Table 14. These results indicate that high doses of corticosterone administered acutely do not have an effect on female rat sexual behaviour or WDS. The finding that acute administration of corticosterone has no effect on WDS replicates previous findings (Takao, et al, 1997). Both the mode of administration and the time interval between administration and behavioural testing may have obscured any effect of corticosterone on female sexual behaviour. This is particularly relevant given that previous research has demonstrated that an effect of corticosterone is only apparent when the hormone is administered intravenously (Kubli-Garfias, 1990) and not subcutaneously (Gorzalka & Whalen, 1977). Additionally, when administered intravenously corticosterone's effect on sexual receptivity is apparent 5 minutes after injection, peaks between 30-60 minutes post-injection, and is on the decline at 2 hours post-injection (Kubli-Garfias, 1990). EXPERIMENT 13 The only study to examine the effects of the acute administration of corticosterone on male rat sexual behaviour failed to find any effect (Retana-Marquez, et al, 1998). The study used low doses of corticosterone ranging between 0.5-4 mg per rat. The current study was designed to examine the effects of the acute administration of a high dose (20 mg/kg) of corticosterone on male rat sexual behaviour and concurrent WDS behaviour. 155 Subjects Long-Evans male rats (n = 40) were employed as subjects and were tested at 6 months of age (400-450 g). Procedure Twenty male subjects were randomly assigned to receive 20 mg/kg of corticosterone which was injected 4 hours prior to behavioural testing. The remaining 20 males were assigned to the control group and received an injection of propylene glycol (1 ml/kg) 4 hours prior to behavioural testing. Thirty minutes prior to behavioural testing, all males were injected with 0.25 mg/kg DOI. Four hours after corticosterone administration, male subjects were tested for sexual behaviour and for the frequency of WDS as described in the General Methods section. Results were subsequently analyzed using independent samples t-tests with a significance level set at .05. Results and Discussion Statistical analyses failed to reveal significant differences between male rats receiving corticosterone and males not receiving corticosterone on WDS (p = .83), ejaculations (p = .98), ejaculation latency (p = .28), copulatory efficiency (p = .44), or post-ejaculatory interval (p = .26). 156 Table 15. Sexual behaviour measures and wet dog shakes (WDS) in DOI-treated male rats after the acute administration of corticosterone. Data are presented as mean scores (+/- standard error). Copulatory efficiency is the number of intromissions prior to ejaculation divided by the sum of the number of mounts and intromissions prior to ejaculation. Ejaculations is the total number of ejaculations during the test interval. WDS is the frequency of the behaviour per minute. Ejaculation latency (EL) is the period between the first intromission and ejaculation and postejaculatory interval (PEI) is the time interval in seconds between ejaculation and the first intromission of the next copulatory bout. There were 20 animals, in each group (total n = 40). 157 WDS Ejaculations EL PEI Copulatory Efficiency Corticosterone 0.24 ± 0.05 0.70 ±0.57 1313.5 ± 121.9 403.9 ±28.2 0.46 ±0.04 Propylene glycol 0.26 ± 0.05 0.70 ±0.66 1120.1 ± 129.0 450.3 ±27.1 0.42 ±0.04 158 The means and standard errors for sexual behaviours and WDS are presented in Table 15. These results indicate that high doses of corticosterone (20 mg/kg) administered acutely do not have an effect on male rat sexual behaviour or WDS. This replicates the finding of no effect of acute corticosterone administration on WDS (Takao, et al, 1997), and extends the findings of Retana-Marquez and colleagues (1998) who found lower doses of corticosterone (where the highest dose used by the authors was 4 mg per rat and was equivalent to between 11.4-13.3 mg/kg) had no effect on male rat sexual behaviour. 159 G E N E R A L DISCUSSION OBJECTIVE 1: EFFECTS OF CHRONIC STRESS The first objective was to determine the effects of chronic stress on male and female rat sexual behaviour and concurrent WDS behaviour. Taken together, the results from Experiments 1-4 suggest that the stress-induced facilitation and inhibition of female and male rat sexual behaviour, respectively, and the increased WDS in both females and males, reflect increased 5-HT 2 A activity. Evidence that chronic corticosterone treatment significantly increases 5-HT 2 A receptor activity (Berendsen, et al, 1996; Kuroda, et al, 1992) support the present observation that increased WDS reflect increased 5-HT 2 A activity in response to stress-induced corticosterone secretion. The concurrent effect on sexual behaviour may be induced by the same mechanism, with corticosterone secretion increasing 5-HT 2 A activity, which would exert effects on sexual behaviour. The effects on sexual behaviour can in turn be predicted by whether 5-HT 2 A activity is stimulatory, as in the female, or inhibitory, as in the male (for review see Gorzalka, et al, 1990). Because adrenalectomy blocked the enhancing effect of stress on proceptivity in Experiment 1, the adrenal response to psychosocial stress may be responsible for the enhancement. It has been suggested that corticosterone of adrenal origin is responsible for the increase in stress-induced receptivity and proceptivity via competitive binding to progestin receptors (Williams, et al, 1992). Notwithstanding this possibility, the significant increase in WDS evident in the present study suggests an alternative mechanism. Adrenal corticosterone may instead be interacting with the 5-HT 2 A system. Support for this has been demonstrated by an increase in radioactively labelled 160 5-HT 2 A receptors and WDS after both stress and corticosterone administration ( Berendsen, et al, 1996; Kuroda, et al, 1992; Takao, et al, 1995; Takao, et al, 1997). These physiological and behavioural changes occur in the absence of any changes in 5-HT and 5-HT metabolite levels (e.g., 5-hydroxyindole acetic acid), thus indicating that if corticosterone regulates or is otherwise involved in 5-HT 2 A receptor activity, it does so independently of changes in circulating 5-HT levels. The results of Experiment IB indicate that exposure to chronic stress significantly increases proceptivity and WDS in non-adrenalectomized females pretreated with both estrogen and progesterone. The significant increase in proceptivity, but not receptivity, in the stressed females after progesterone administration, may reflect the differential effects of estrogen and progesterone on these behaviours and the likely possibility that these hormones act on different neural circuits to influence aspects of sexual behaviour (Tennent, et al, 1980). Results from Experiment 2 suggest that chronic stress has the potential to decrease sexual behaviour and increase WDS in the male rat. The stress created by cage rotation did not affect the spontaneous expression of sexual behaviour and WDS (Experiment 2A), but did result in physiological changes that become behaviourally apparent upon pharmacological manipulation (Experiment 2B). Presumably, one of these physiological changes was an increase in 5-HT 2 A density. It has also been suggested that the best indicator of effective chronic stress is a combination of increased corticosterone levels and alterations in behaviour, and that some stress paradigms are not sufficiently intense to elicit behavioural changes despite elevations in corticosterone levels (Natelson, Ottenweller, Cook, Pitman, McCarty & Tapp, 1988). It is possible that the current 161 paradigm was not sufficiently intense to evoke behavioural changes in WDS and sexual behaviour even though HPA axis arousal triggered an elevation in corticosterone levels and concurrent physiological changes. That these behavioural changes became significant after stimulation of the 5-HT 2 A receptor with DOI supports this suggestion. Although physiological changes had occurred, the corresponding behavioural changes became apparent only after pharmacological manipulation. The results of Experiment 3 indicate that short-term individual housing of male rats has inhibitory effects on the spontaneous expression of sexual behaviour and facilitatory effects on the DOI-induced expression of WDS. Previous work has shown that central activation of 5-HT 2 A receptors concurrently induces WDS and inhibits male sexual behaviour (Watson & Gorzalka, 1990). Therefore it is possible that individual housing acts, at least in part, via a 5-HT 2 A receptor mechanism and this may involve an increase in the density of postsynaptic 5-HT 2 A receptors. In addition to impairing male sexual performance, individual housing in the rat activates the H P A axis and causes an elevation in corticosterone levels (Bennett & Gardiner, 1978; Handa, Burgess, Kerr & O'Keefe, 1994). Therefore, the increase in corticosterone levels following chronic individual housing may contribute to the observed reduction in sexual behaviour perhaps by elevating 5-HT 2 A receptor activity. The increase in DOI-induced WDS evident in Experiment 3 suggests activation of 5-HT 2 A receptors. This is consistent with findings that rearing rats in isolation produces a significantly greater number of 5-HT 2 A receptor mediated back muscle contractions (Wright, Ismail, Upton & Marsden, 1991). 162 In Experiment 4 it was shown that short-term individual housing facilitated sexual behaviour and WDS at Day 3, but failed to significantly facilitate all measures of sexual behaviour and WDS at Day 10. As mentioned previously, the lack of significant effects on Day 10 may be related to the high level of receptivity seen in both groups and the associated brief test duration. The high level of receptivity may have been due to a combination of the subchronic administration of estrogen and the stress-induced release of progestins and corticosteroids. The finding that WDS were significantly increased at Day 3 is surprising. Prior studies have demonstrated that the chronic administration of stress or corticosterone is necessary to increase 5-HT 2 A receptor density (Kuroda, et al, 1992; Takao, et al, 1997). These studies have generally looked at 5-HT 2 A receptor density after a minimum of 10 days of stress or corticosterone treatment. While a single administration of stress or corticosterone has been shown to be without effect on 5-HT 2 A receptors (Takao, et al, 1997), it is unknown precisely when changes in 5-HT 2 A receptor activity occur. The current findings suggest that perhaps stress can induce changes in 5-HT 2 A receptors in as little as 3 days. Evidence indicates that short-term individual housing alters central serotonergic function. Three months of isolation has been shown to result in diminished neuronal sensitivity to serotonin in the striatum and nucleus raphis in the rat (Oehler, Jahkel & Schmidt, 1987). Additionally, isolation increases synaptosomal serotonin uptake in the striatum of alcohol preferring rats (Daoust, Chretien, Moore, Saligaut, Lhuintre & Boismare, 1985). It is likely that increases in WDS in the present study reflect increases in 5-HT 2 A receptor density. Moreover, it is unlikely that decreased serotonergic function is responsible for an increase in 5-HT 2 A receptor density. As noted earlier, the regulation of 5-HT 2 A receptors does not follow the classical rules of receptor regulation and 5-HT 2 A 163 receptors do not upregulate in response to most pre- and post-synaptic manipulations (Sanders-Bush, 1990). Therefore, it is possible that the upregulation of 5-HT 2 A receptors occurs independently of presynaptic events. To date, no studies have directly investigated the effects of social isolation on 5-HT 2 A receptor density. However, studies have demonstrated that other serotonergic receptors are altered in response to isolation. Individual housing of rats for three months significantly decreased the affinity of 5-HT, receptors (St. Popova & Petkov, 1990). In addition, evidence connotes that another receptor of the 5-HT2 family, the 5-HT 2 C receptor, is more responsive in rats that were reared in isolation (Fone, Shalders, Fox, Arthur & Marsden, 1996). Further studies need to be done to clarify directly what effect individual housing has on 5-HT 2 A receptor density or sensitivity. The adrenal glands are hyperactive during the stress response and usually release numerous steroid hormones from the adrenal cortex (Nelson, 1980), some of which may have effects on sexual activity, particularly in the female rat. For example, adrenal deoxycorticosterone, testosterone, progesterone, or estrogen secretion during stress may combine additively with exogenous estrogen to facilitate proceptivity. Testosterone has been shown to increase female sexual behaviour, presumably after being aromatized (Whalen, Battie & Luttge, 1972), while deoxycorticosterone has been shown to facilitate sexual receptivity (Gorzalka & Whalen, 1977). In addition to the adrenal cortex, the adrenal medulla is also hyperactive during the stress response. The adrenal medulla produces the catecholamines epinephrine and norepinephrine which may also influence female rat sexual behaviour. The adrenal medulla is innervated by the sympathetic nervous system and decreased sympathetic activity has been shown to inhibit sexual receptivity and proceptivity (Meston, Moe & Gorzalka, 1996). Furthermore, stress results in P-endorphin release which has also 164 been demonstrated to facilitate receptivity in high doses (Pfaus & Gorzalka, 1987). There is no evidence to suggest that an increase in adrenal hormones other than corticosterone would significantly have an impact on male rat sexual behaviour. However, another effect of chronic stress is to decrease the release of gonadal hormones. This would not occur in the experiments with females given that they were ovariectomized, but this could theoretically influence male rat sexual behaviour providing that chronic stress sufficiently reduced testosterone. Testosterone levels have been found to be decreased (Johnson, Kamilaris, Chrousos & Gold, 1992) or increased (Taylor, et al, 1987) in male rats following exposure to chronic stress. The method of stress used in Experiment 2 has been shown actually to increase testosterone levels (Taylor, et al, 1987) which would not be associated with an inhibition of male rat sexual behaviour. Chronic stress increases adrenal steroid output by increasing the release of A C T H from the pituitary. A C T H administration has been shown to facilitate female (deCatanzaro, Gray & Gorzalka, 1981), inhibit male (Spruijt, Hoglund, Gispen & Meyerson, 1985) rat sexual behaviour, and increase WDS behaviour (Kuroda, et al, 1992). However, A C T H does not have any of the aforementioned effects in adrenalectomized rats. This suggests that A C T H is exerting behavioural effects by increasing the output of adrenal steroid hormones rather than acting directly. It is possible that the GABA-benzodiazepine system is involved in some effects of stress on sexual behaviour. Research has demonstrated that the GABA-benzodiazepine and serotonergic systems are both involved in the regulation of anxiety and stress (Lopez-Rubalcava, Saldivar & Fernandez-Guasti, 1992). Additionally, it has been shown that G A B A agonists inhibit while 165 antagonists facilitate male sexual behaviour (Fernandez-Guasti, Larsson & Beyer, 1986a). Data suggest that G A B A antagonists also facilitate female sexual behaviour and that this effect is dependent on adrenal secretions (Fernandez-Guasti, Larsson & Beyer, 1986b). The role of G A B A in the effects of stress on sexual behaviour in the present study needs to be further investigated. The stresses in Experiments 1-4 are not comparable across sex due to the method of inducing stress, the duration of stress, the administration of DOI, and the hormonal treatment of the females. The stresses that were used in Experiments 1 -4 were chosen because they have been demonstrated to both increase corticosterone levels and have an impact on sexual behaviour. Further research is needed in order to demonstrate whether any single method of inducing stress would exert opposite behavioural effects in male and female rats. OBJECTIVE 2: EFFECTS OF CHRONIC STRESS A N D 5-HT 2 A RECEPTOR A N T A G O N I S M The second objective was to determine i f the effects of stress on sexual behaviour are mediated by a serotonergic mechanism. Overall, the findings from Experiment 5 replicate previous studies which demonstrate that chronic psychosocial stress increases both sexual behaviour (Williams, et al, 1992) and WDS (Takao, et al, 1995) in the female rat. Additionally, these findings are consistent with the findings from Experiments 1 and 4. In contrast to the results of other studies, however, these effects were apparent in both females treated with estrogen alone and in females treated with both estrogen and progesterone. 166 The results of Experiment 5 demonstrated that the stress-induced facilitation of WDS behaviour can be completely attenuated by nefazodone treatment but the stress-induced facilitation of sexual behaviour cannot. How nefazodone was able to attenuate the stress-induced facilitation of WDS, but not the facilitation of sexual behaviour is unclear. The WDS results are consistent with previous findings that nefazodone attenuated quipazine-induced increases in WDS (Eison, et al, 1990). It appears that the effects of stress on female sexual behaviour and WDS may be mediated by different mechanisms. Since chronic stress is known to produce an elevation in all adrenal hormones (Nelson, 1980), including corticosterone, it may be that these other hormones are influencing sexual behaviour independent of changes in 5-HT 2 A receptor activity. Therefore, i f other adrenal or non-adrenal hormones are contributing to the increase in sexual behaviour, and their effect does not involve 5-HT 2 A receptor activity, then it follows that nefazodone, which specifically antagonizes the 5-HT 2 A receptor and inhibits serotonergic reuptake, would not attenuate those effects. Further research is necessary to elucidate the mechanism by which chronic stress facilitates female sexual behaviour. A recent study has shown that chronic psychosocial stress increased both sexual receptivity and proceptivity in estrogen-primed females (Williams, et al, 1992). The psychosocial stress used in that study significantly elevated serum corticosterone levels. The authors suggested that adrenal corticosterone was responsible for the increase in stress-induced receptivity and proceptivity by competitively binding to progestin receptors. Both glucocorticoids and progestins have receptors that are similar in structure (Carlstedt-Duke, Stromstedt, Persson, Cederlund, Gustafsson & Jornvall, 167 1988) and the hormones have been shown to have comparable affinities for some protein receptors (Trueba, Guantes, Vallejo, Sancho, Marino & Macarulla, 1987). However, acute administration of glucocorticoids fails to exert any effect on female sexual receptivity when given at doses comparable to those at which progesterone is effective (Gorzalka & Whalen, 1977). These latter findings suggest that it is unlikely that the stress-induced facilitation of female sexual behaviour is the result of glucocorticoids binding to progesterone receptors. Another possibility is that stress-induced increases in adrenal progestin secretion act to facilitate female sexual behaviour. Ovariectomized, estrogen-primed female rats that were chronically stressed by intense exercise showed increased receptive behaviour and evidence of weight loss (Schwartz, Nunez & Axelson, 1983). The authors of that study argued that stress-induced elevations of adrenal progestin were not responsible for the effect on sexual behaviour since estrogen possesses weight reducing properties (Bernstein, Courtney & Braget, 1986) which are reversed by progesterone (Tchekmedyian, Hickman & Heber, 1991). OBJECTIVE 3: EFFECTS OF CHRONIC STRESS A N D CORTICOSTERONE SYNTHESIS INHIBITION The third objective was to determine whether both the stress induced changes in 5-HT 2 A receptor levels and in sexual behaviour are mediated, at least in part, by the secretion of corticosterone. Results from Experiments 6 and 7 indicate that the stress-induced facilitation of WDS can be effectively blocked by the concurrent administration of metyrapone suggesting that this effect is dependent on increased plasma levels of corticosterone. In Experiment 6, metyrapone blocked the effects of stress on proceptive behaviour but not receptive or rejection behaviour in 168 female rats. These results suggest that unlike proceptivity, receptivity and rejection are not affected by stress-induced increases in corticosterone levels. While the facilitation of receptivity by corticosterone has been previously demonstrated (Hanson & Gorzalka, 1999), it appears that the effects of stress on receptivity are not mediated solely by increased levels of corticosterone. Stress also appears to increase rejection behaviour, yet this does not occur following the chronic administration of corticosterone (Hanson & Gorzalka, 1999). This suggests that stress acts completely independently of corticosterone to increase rejection behaviour. It was suggested in Experiment 4 that perhaps isolation per se rather than stress was necessary to increase rejection behaviour. However, the results from Experiment 6 suggest that it may be stress per se that increases the display of rejection behaviour. The finding of increased rejection behaviour when receptivity and proceptivity are facilitated is not unprecedented (Gorzalka & Gray, 1981). Further research is necessary to determine the relevance of rejection behaviour to other sexual behaviours. In Experiment 7, metyrapone did not block the effect of stress on male rat sexual behaviour. Although corticosterone administered chronically has been shown to inhibit male rat sexual behaviour (Gorzalka & Hanson, 1998), stress appears to inhibit sexual behaviour independent of it's effect on corticosterone levels. As mentioned previously, there are multiple hormonal and neural effects of stress and some of these other effects may lead to an inhibition of male rat sexual behaviour. Prior research has shown that the chronic administration of metyrapone significantly facilitates lordosis in estrogen-primed, ovariectomized female rats (deCatanzaro, Knipping & 169 Wigmore, 1983). This was not found in the current study and these contradictory findings are likely the result of different doses and dosing schedules. The current study used a dose of 75 mg/kg administered once daily for 30 days. A facilitation of lordosis has been reported using a dose of 12 mg/kg administered 5 times daily for 5 days (deCatanzaro, et al, 1983). Those authors also reported a trend towards an inhibition of lordosis when higher doses were administered chronically (36 and 108 mg/kg). OBJECTIVE 4: EFFECTS OF V A R Y I N G DOSES OF C H R O N I C A L L Y ADMINISTERED CORTICOSTERONE The fourth objective was to determine if the effects of corticosterone on sexual behaviour are dose dependent. In Experiments 8-9, the chronic administration of corticosterone at high doses was found to facilitate female and inhibit male rat sexual behaviour while concurrently increasing WDS in both sexes, replicating previous findings (Gorzalka & Hanson, 1998; Hanson & Gorzalka, 1999). Low doses of corticosterone were not found to have any effect on sexual behaviour or WDS. One might speculate that the increased WDS observed in Experiments 8-9 reflects an increase in 5-HT 2 A receptor affinity as opposed to an actual increase in receptor number or density. However, radioligand binding studies have demonstrated that manipulations of the HPA axis alter the number of 5-HT 2 A receptors as opposed to the affinity of the receptor (Kuroda, et al, 1992; Martire, et al, 1989). This suggests that the results of the present study reflect an upregulation in 5-H T 2 A density. 170 The mechanism of 5-HT 2 A upregulation by chronic corticosterone treatment has yet to be delineated. Lowered presynaptic serotonergic activity could be the cause of the increase in 5-HT 2 A density, however, this is not likely. The regulation of 5-HT 2 A receptors does not follow the classical rules of receptor regulation. Instead of upregulating after antagonist administration, 5-HT 2 A receptors downregulate (Sanders-Bush, 1990). Also, the lowered levels of 5-HT by denervation, neurotoxins, and synthesis inhibitors do not modify 5-HT 2 A receptor density (Eison & Mullins, 1996). Additionally, increased 5-HT 2 A receptors are seen in chronically ACTH-treated rats without any modifications in the levels of 5-HT or its major metabolite, 5-hydroxyindole acetic acid (Kuroda, et al, 1992). Therefore, it is possible that the upregulation of 5-HT 2 A receptors occurs independently of presynaptic events. The existence of two distinct glucocorticoid receptor (GR) systems in the rat brain has been well established. The type I GR displays a high affinity for corticosterone and is primarily located in the lateral septum and hippocampus. The type II GR displays a low affinity for corticosterone and a much higher affinity for synthetic glucocorticoids such as dexamethasone, and is widely distributed throughout the brain (Reul & deKloet, 1985). The type II GR only becomes fully occupied with corticosterone when endogenous levels of corticosterone attain stress levels (Dallman, Akana, Cascio, Darlington, Jacobsen & Levin, 1987). The corticosterone doses used in the present study are sufficiently high to occupy both GR systems (Reul & deKloet, 1985). Chronic treatment with dexamethasone has been found to significantly increase 5-HT 2 A receptor density in the cerebral cortex but not in the hippocampus (Kuroda, Mikuni, Nomura & Takahashi, 1993). These results suggest that the type II GR is involved in the regulation of 5-HT 2 A receptor density. However, as 171 mentioned previously, glucocorticoids and progestins may compete for the same receptor site (Trueba, et al, 1987). Additionally, progesterone has been shown to alter 5-HT 2 A receptor density in female rats (Biegon, Reches, Snyder & McEwen, 1983);and in the present study, alter the 5-HT 2 A receptor mediated behaviour WDS in estrogen-primed females. Therefore, it is possible that dexamethasone affects 5-HT 2 A receptor density via interactions on the progesterone receptor. If the effects of dexamethasone on 5-HT 2 A receptor density could be blocked by the administration of a selective type II GR antagonist, this would provide further evidence that the type II GR modulates 5-HT 2 A receptor density. Because no selective type II GR antagonist is available, this study cannot be performed at present. A l l currently available type II GR antagonists, such as RU-28362 and R U -38486, also possess anti-progestin activities (Mao, Regelson & Kalimi, 1992). Further investigation is necessary to reveal the interaction between glucocorticoids and progestins in modulating 5-HT 2 A receptor activity. There is evidence to suggest that chronic administration of corticosteroids can downregulate 5-HT 1 A receptors in rodents (Young, et al, 1992), Since data indicate that decreased 5-HT 1 A receptor activity results in a facilitation of female rat sexual behaviour and an inhibition of male rat sexual behaviour (Gorzalka, et al, 1990), it is possible that the current effects on sexual behaviour may, in part, be mediated by alterations in 5-HT 1 A receptor activity. However, it is unlikely that this would account for the present WDS results. 172 OBJECTIVE 5: EFFECTS OF C H R O N I C A L L Y ADMINISTERED CORTICOSTERONE A N D 5-HT 2 A RECEPTOR A N T A G O N I S M The fifth objective was to determine i f the effects of corticosterone on sexual behaviour are mediated by an upregulation of 5-HT 2 A receptors. Results from Experiments 10 and 11 replicate previous findings that the chronic administration of corticosterone, at a dose of 20 mg/kg, facilitates female and inhibits male rat sexual behaviour while concurrently increasing WDS in both sexes. The administration of the 5-HT 2 A antagonist ketanserin attenuated the effect of corticosterone on both sexual behaviour and WDS in both male and female rats. Ketanserin when administered alone facilitated male rat sexual behaviour but had no effect on either female rat sexual behaviour or WDS. As mentioned previously, the lack of effects of ketanserin may be the result of either the time interval between administration and behavioural testing or due to the difficulties inherent in antagonizing a receptor that normally receives relatively low levels of stimulation. Presumably, ketanserin acted to prevent an upregulation of 5-HT 2 A receptors by chronically antagonizing the receptors. The chronic administration of 5-HT 2 A antagonists has been shown to result in a downregulation of 5-HT 2 A receptors (Eison & Mullins, 1996). These results provide strong support for the hypothesis that corticosterone mediates effects on sexual behaviour and WDS via an upregulation in 5-HT 2 A receptor activity. 173 OBJECTIVE 6: EFFECTS OF A C U T E L Y ADMINISTERED CORTICOSTERONE The sixth objective was to determine i f the acute administration of high doses of corticosterone exerts any effect on sexual behaviour. Results from Experiments 12 and 13 indicate that the acute administration of corticosterone exerts no effect on either male or female rat sexual behaviour or WDS behaviour. The lack of effect of a high acute dose of corticosterone on WDS has been reported previously (Takao, et al, 1997). This suggests that corticosterone levels must be elevated for some period of time before 5-HT 2 A receptors upregulate. The minimum length of this time period remains to be determined. To date, published studies have only examined the effect of corticosterone on 5-HT 2 A receptor activity after 1 day (acute) or 10 days (chronic). The lack of effect of acute corticosterone treatment on female sexual behaviour is not surprising given that it was administered subcutaneously and there was a four hour interval between administration and behavioural testing. The only study to demonstrate an acute effect used intravenous administration and a maximum interval of 2 hours between administration and behavioural testing (Kubli-Garfias, 1990). However, this study did use a significantly lower dose (200 ug per rat) than was used in Experiment 12 (20 mg/kg). Therefore, it is possible that the higher dose of corticosterone could have exerted an effect on sexual behaviour as blood levels of corticosterone presumably would have been higher and remained that way for a longer period of time than they would have with a lower dose. Regardless, the lack of effect of a high dose of corticosterone 4 hours after administration suggests that the effects of chronic corticosterone on female rat sexual behaviour 24 hours after the last administration are not due to any acute effect. 174 How corticosterone would act to exert an effect after a single administration remains unknown. Corticosteroids and progestins possess a similar molecular ring configuration, and both corticosteroids and progestins have been shown to compete for the same receptors (Trueba, et al, 1987). Therefore, it is possible that any apparent acute effect of corticosterone is due to it's action at the progesterone receptor. This raises another important methodological discrepancy between the study that found an effect (Kubli-Garfias, 1990) and the design of Experiment 12. Whereas Kubli-Garfias (1990) reported an effect in females primed with estrogen alone, Experiment 12 used females that were primed with both estrogen and progesterone. The presence of progesterone could potentially have obscured any facilitatory effect of corticosterone if the effect of corticosterone was dependent on it's ability to mimic progesterone. The lack of effect of acute corticosterone treatment in Experiment 13 on male rat sexual behaviour extends previous findings (Retana-Marquez, et al, 1998). The time interval between administration of corticosterone and behavioural testing was four hours in both studies. The only difference in methodologies was that Experiment 13 employed a higher dose than has been used previously. The lack of effect of corticosterone administered acutely suggests that the inhibition of male rat sexual behaviour seen after chronic corticosterone administration is not due to any acute effects of corticosterone. It has been demonstrated that acute stress can exert mixed effects on male rat sexual behaviour depending on the form of stress (Retana-Marquez, et al, 1996). Given that effects of acute stress are usually seen almost immediately after the administration of stress, it is unlikely that corticosterone mediates the effects of acute stress on male rat sexual behaviour. For example, acute stresses such as tail pinch, electric shock, and immobilization all have been shown 175 to exert effects on male sexual behaviour. These stresses are extremely brief in duration and behavioural testing usually occurs immediately after application of the stress. The HPA axis tends to be relatively slow to respond to stress (taking hours not minutes) and therefore, corticosterone levels are likely not elevated at the time of behavioural testing. The nervous system as opposed to the endocrine system, responds almost immediately to acute stress. Therefore, it is more probable that the effect of acute stress on male rat sexual behaviour is mediated by the immediate response of the sympathetic nervous system or some other mechanism that responds immediately to stress. It has been suggested that the facilitation of male rat sexual behaviour seen following acute stress is related to increased activity of the mesencephalic reticular formation which would increase the activity of other brain areas including the amygdala, hippocampus, and septum that are potentially involved in the male sexual response (Menendez-Patterson, et al, 1978). CONCLUSIONS, SPECULATIONS, A N D IMPLICATIONS FOR FUTURE R E S E A R C H Taken together, the current series of experiments suggest that chronic elevation of corticosterone, at plasma levels that are equivalent to levels after exposure to chronic stress, increases 5-HT 2 A receptor activity as indicated by an increase in WDS behaviour. The current results suggest that the increase in WDS seen after exposure to chronic stress is mediated by an increase in corticosterone levels as these effects are attenuated by concurrent administration of the corticosterone synthesis inhibitor metyrapone. While both chronic corticosterone and chronic stress were found to exert similar effects on sexual behaviour in both male and female rats, it is likely that they do so via different mechanisms. The effects of corticosterone on sexual behaviour are most 176 likely mediated by an upregulation in 5-HT 2 A receptor activity. Corticosterone acts to alter female and male rat sexual behaviour in the directions that would be predicted by an upregulation in 5-HT 2 A receptor density. Additionally, the effects of chronic corticosterone treatment on sexual behaviour are only apparent at the same doses that increase WDS, and are completely attenuated by concurrent treatment with the 5-HT 2 A receptor antagonist ketanserin. Although the effects of chronic stress on sexual behaviour are also in the direction that would be predicted by an upregulation in 5-HT 2 A receptor density, it is unlikely that chronic stress alters sexual behaviour either by increasing corticosterone levels or altering 5-HT 2 A receptor activity. While concurrent administration of the 5-H T 2 A antagonist nefazodone or the corticosterone synthesis inhibitor metyrapone blocked the effects of stress on WDS, neither substance was able to significantly attenuate the effects on sexual behaviour. Given that stress exerts multiple physiological effects, stress most likely acts on other neuroendocrine systems to alter male and female rat sexual behaviour. Evidence to date indicates that both chronic corticosterone treatment and chronic stress cause an upregulation of 5-HT 2 A receptors in the cerebral cortex. However, no radioligand binding studies have examined the effects of corticosterone or stress in other brain regions. Given that research has demonstrated that adrenalectomy alters 5-HT 2 A receptor density only in select brain regions (Martire, et al, 1996) it remains possible that corticosterone and stress differentially alter 5-HT 2 A receptor density in distinct brain regions. It has been suggested that WDS and copulatory behaviour rely on overlapping neural mechanisms. Brainstem administration of DOI, in the region of the raphe obscurus, resulted in a 177 dose-dependent decrease in sexual behaviour and a concurrent increase in WDS (Watson & Gorzalka, 1992). The fact that neurons in the raphe nuclei have bifurcating axons that project to the hypothalamus/medial preoptic area (MPOA) and the ventral horns of the cervical spinal cord (Leanza, Pellitteri, Russo & Stanzani, 1991) is of particular interest since the hypothalamus/MPOA is a brain region important for the display of both male and female copulatory behaviour (Rose, 1990) and the cervical spinal cord contains motor neurons which control the display of WDS. These results suggest the possibility that corticosterone or stress may affect WDS and sexual behaviour by altering 5-HT 2 A receptor density in the raphe nuclei. No radioligand binding studies have yet looked at the effect of stress or corticosterone treatment on 5-HT 2 A receptor density in this region of the brain. The available evidence indicates that chronic elevations of corticosterone act to upregulate 5-HT 2 A receptors. This may have implications for other behaviours that are mediated by 5-HT 2 A receptor activity. Feeding is a behaviour that is influenced both by altered 5-HT 2 A receptor activity and corticosterone. For example, it has been demonstrated that 5-HT 2 A agonists induce hypophagia (Price, Gorzalka, White & Arkinstall, 1998) and that adrenalectomy completely blocked the effect of DOI on feeding (Yamada, Sugimoto, Yoshikawa & Horisaka, 1996). These data suggest that the effects of 5-HT 2 A receptors on feeding may be dependent on the presence of corticosterone. 5-HT 2 A receptors have also been implicated in learning (Meneses & Hong, 1997) and 5-HT 2 A agonists enhance conditioned avoidance responding (Harvey, 1996). Other evidence indicates that adrenalectomy produces major learning deficits in learning paradigms which are stressful and elevate corticosterone levels (Peeters & Broekkamp, 1994). Therefore, it is possible that the learning of 178 stressful events is mediated via corticosterone altering 5-HT 2 A receptor activity. The relevance of the present findings to human sexual behaviour remain to be determined. While it has been demonstrated that an overall increase in 5-HT levels leads to sexual dysfunction in both men and women (Meston & Gorzalka, 1992), the effects that specific serotonergic receptors mediate on human sexual behaviour are not well understood. However, to some extent, selective serotonin receptor activation has similar effects on the sexual behaviour of humans and rats which may account for at least some instances of sexual dysfunction induced by psychotherapeutic drugs (Meston & Gorzalka, 1992). Several studies have demonstrated that the antidepressant nefazodone does not result in sexual dysfunction (Taylor, et al, 1995), which is a side effects seen with other serotonergic antidepressants. The lack of sexual dysfunction seen following treatment with nefazodone may be due to it's direct antagonism of the 5-HT 2 A receptor. Additionally, the 5-HT 2 A and alpha-2 adrenergic receptor antagonist mianserin, and the 5-HT 2 A antagonist cyproheptadine are both effective in reversing the sexual dysfunction induced by serotonin reuptake inhibitor antidepressants (Aizenberg, Gur, Zemishlany, Granek, Jeczmien & Weizman, 1997; Aizenberg, Zemishlany & Weizman, 1995). To date, studies examining the sexual effects of selective serotonergic receptor agonists and antagonists have been performed primarily in men, and little is know about the sexual effect of these agents in women. The finding that a particular treatment exerts opposite effects on female and male sexual behaviour is not unprecendented. Previous research has demonstrated that in both humans and rats, increased sympathetic nervous system activity inhibits male and facilitates female sexual arousal 179 (Heiman & Rowland, 1983; Meston & Gorzalka, 1995; Meston, Moe & Gorzalka, 1996). However, the usefulness of predicting human sexual response using a rat model remains problematic particularly with regard to the female. Sexual behaviour in the female rat is completely dependent on the ovarian hormones estrogen and progesterone, while sexual behaviour in women has no apparent relationship to estrogen and progesterone (Wallen, 1990). The relevance of the sexual behaviour findings remain limited with respect to human sexual function. The changes in sexual behaviour seen in the present series of experiments are more pertinent when viewed as a behavioural assay of 5-HT 2 A receptor activity. Alterations in rat sexual behaviour have previously been used as a means for evaluating the affinity of pharmaceutical agents for 5-HT 2 A receptors in vivo (Foreman, Fuller, Nelson, Calligaro, Kurz, Misner, Garbrecht & Parli, 1992). It was concluded that the antipsychotic medication clozapine has 5-HT 2 A receptor antagonist properties using the male rat sexual behaviour model (Klint & Larsson, 1994). Additionally, WDS has gained acceptance as a behavioural measure of 5-HT 2 A receptor activity (e.g., Eison, et al, 1995; Essman, et al, 1994; Kuroda, et al, 1992; Schreiber, et al, 1995). Therefore, it is likely that the combination of rat sexual behaviour and concurrent WDS would provide a more robust and reliable behavioural assay of 5-HT 2 A receptor activity. Ultimately, the demonstration of a functional link between the HPA axis, central serotonergic systems, and behaviour, may have important implications for the neurobiology and treatment of certain behavioural disorders, particularly depression (for review see Lopez, et al, 1997). Accumulating animal and human evidence suggests that the 5-HT 2 A receptor changes observed in 180 the brains of depressed patients may be the result of HPA overactivity. It is well-established that stress increases activity of the HPA axis and elevates plasma levels of the corticosteroids (corticosterone and Cortisol) in both rats and humans (Lopez, et al, 1991). Chronic stress can lead to an impairment in the feedback mechanisms of corticosteroids to the hypothalamus and pituitary, resulting in a failure to return corticosteroid levels to the normal physiological range (Lopez, et al, 1991). Similar feedback impairments have been demonstrated in depressed patients (Gold, Goodwin & Chrousos, 1988). Multiple studies have reported that patients with depressive illness tend to show HPA hormonal changes consistent with the changes seen during times of stress. Patients tend to have elevated basal Cortisol levels (Murphy, 1991a), higher levels of corticotropin-releasing hormone in the cerebrospinal fluid (Nemeroff, Widerlov & Bissette, 1984), and impaired Cortisol suppression in response to dexamethasone (Arana & Mossman, 1988). Chronic corticosterone elevations and chronic stress in rats upregulate 5-HT 2 A (Kuroda, et al, 1992; Martire, et al, 1989; Takao, et al, 1995; Takao, et al, 1997) and downregulate 5-HT 1 A receptors (Kuroda, Watanabe, Albeck, Hastings & McEwen, 1994; Lopez, et al, 1997). Increased 5-HT 2 A receptor density and decreased 5-HT,A receptor density have also been reported in postmortem studies of depressed individuals (Arora & Meltzer, 1989; Lopez, et al, 1997; Mann, Arango, Marzuk, Theccanat & Reis, 1989). The interaction between the H P A axis and the serotonergic system is bi-directional, and these changes in serotonergic receptors act to further increase corticosteroid plasma levels in both rats and humans (for review see Fuller, 1996). This 181 interaction may resemble a supraphysiological feedback loop such that corticosteroids maintain abnormal serotonergic function, and vice versa. The chronic administration of all serotonergic antidepressant medications revert or prevent the 5-HT receptor changes seen after stress (Heninger & Charney, 1987) and depression (Deakin, 1988). It has been suggested that depression in humans is the result of an imbalance between 5-HT 1 A and 5-HT 2 A receptors, and that antidepressants work by restoring this imbalance (Berendsen, 1995). Other investigators have suggested that the correct balance between 5-HT 1 A and 5-HT 2 A receptors are essential for an animal's ability to respond to stress (McKittrick, et al, 1995). Currently, only the drug flibanserin (BIMT 17) has been reported to be a full 5-HT ] A agonist and 5-HT 2 A antagonist (Borsini, Giraldo, Monferini, Antonini, Parenti, Bietti & Donetti, 1995). When compared to the antidepressants imipramine and fluoxetine, flibanserin has demonstrated rapid antidepressant effects in several animal paradigms (Borsini, Cesana, Kelly, Leonard, McNamara, Richards & Seiden, 1997; D'Aquila, Monleon, Borsini, Brain & Willner, 1997). In these studies, flibanserin exhibited antidepressant-like activity after a single administration, while both imipramine and fluoxetine had to be administered chronically before demonstrating antidepressant-like activity. To date, no clinical trials using flibanserin for depression have been reported. Chronic antidepressant treatment also reverses the overactivity and disrupted feedback mechanism of the HPA axis in rats after stress and in depressed patients (for review see Holsboer & Barden, 1996). Antidepressants appear to normalize feedback of the HPA axis by upregulating type II GR in the limbic system (Mitchell, Betito, Rowe, Boksa & Meaney, 1992). It has been 182 suggested that the normalization of feedback inhibition to corticosteroids is a primary action of antidepressants (Barden, Reul & Holsboer, 1995). A number of small scale clinical trials have demonstrated the effectiveness of corticosterone synthesis inhibitors as antidepressants (Thakore & Dinan, 1995; Murphy, 1991b). Additionally, preliminary data suggests that both corticotrophin releasing hormone and glucocorticoid receptor antagonists may be useful in the pharmacological treatment of depression (Lopez, et al, 1997). The interactions between the HPA axis and the 5-HT system form a neuroendocrine loop through which both stress and behavioural responses are mediated. The available evidence suggests that malfunctions in this loop can lead to maladaptive behavioural patterns and psychopathological states including depression. 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