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Chronic corticosterone regulates 5-HT₁A but not 5-HT₂A agonist-induced serotonin receptor-mediated functions Lamarre, Amanda Kathleen 2004

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CHRONIC CORTICOSTERONE REGULATES 5-HT, BUT NOT 5-HT AGONISTINDUCED SEROTONIN RECEPTOR-MEDIATED FUNCTIONS A  2A  by AMANDA KATHLEEN LAMARRE B A . Hon., University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in THE FACULTY OF GRADUATE STUDIES Department of Psychology, Clinical Psychology Programme We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July 2004 © Amanda Kathleen LaMarre, 2004  FACULTY OF GRADUATE STUDIES  Library Authorization  A  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Amanda,  Name of Author (please print)  LaKarre  Date (dd/mm/yyyy)  OJrvrartic No-V S-ViT-^y  Title of Thesis  Degree:  A-cVs  VAciST^JT p f  Year:  to1 O  Department of T%>v/f\V) jlumbia The University of British Colombia Vancouver, BC Canada  ,.  j  last updated: 20-Jul-04  11  Abstract There has been much research on serotonin (5-HT) 1A and 2A receptor activity, and speculation on how these receptors are implicated in the etiology of depression. Moreover, interest has been focused on mechanisms through which corticosteroids interact with these receptors to produce biochemical, physiological and behavioural changes. The 5 - H T i and 5 - H T 2 A receptors appear A  to be controlled by corticosteroids in a biphasic manner; low corticosteroid levels exert influence on 5 - H T I A receptor activity, while high levels activate 5 - H T 2 A receptors. It is therefore reasonable to speculate that 5 - H T I A receptor activity predominates during normal conditions, while during times of prolonged stress, receptor activity shifts to 5 - H T 2 A - The purpose of this research was to investigate the hypothesis that elevated corticosterone levels cause a shift from predominantly 5 - H T i to 5 - H T A receptor activity and that this happens A  2  over a particular time course. Corticosterone (20 mg/kg) or its vehicle was chronically administered to rats for 14 days. During this time, rats were tested at six time points on a number of 5 - H T I A and 5 - H T 2 A receptor-mediated physiological and behavioural measures. Overall, it was found that after 12 days of administration, rats receiving corticosterone exhibited an attenuation of the hypothermic response produced by the 5 - H T J A agonist 8-hydroxy-2(di-«propylamino) tetralin. However, over the entire course of administration, corticosterone did not potentiate any effects produced by the 5 - H T 2 A receptor-agonist l-(2,5-dimethoxy-4iodophenyl)-2-aminopropane on 5 - H T A receptor-mediated behaviours. These results are 2  discussed in relation to the interaction between serotonin and HPA-axis functioning in Major Depression.  Ill  Table of Contents Abstract  ii  Table of Contents  iii  List of Figures  iv  List of Nomenclature and Abbreviations  v  Acknowledgements  vi  Introduction  1  Method  7  Animals and Housing Materials Apparatus Procedure 5-HTIA Receptor Activation Testing 5-HT2A Receptor Activation Testing Statistics  7 7 8 8 9 9 10  Results 11 Analysis of Weight 11 Analysis of 5-HTi A Receptor Agonist Effects on Temperature Response 11 Analysis of 5-HT2A Receptor Agonist Effects on Temperature and Behavioural Responses 13 Discussion  15  References  22  Figure Captions  29  Appendix  ;  41  iv  List of Figures Figure 1: Mean weight change following corticosterone or vehicle treatment  31  Figure 2: Mean temperature change 10 minutes after 5-HTi agonist administration  32  Figure 3: Mean temperature change 20 minutes after 5-HTi agonist administration  33  Figure 4: Mean temperature change 30 minutes after 5-HTi agonist administration  34  Figure 5: Mean temperature change 15 minutes after 5 - H T 2 agonist administration  35  A  A  A  A  Figure 6: Mean frequency of wet dog shakes during sexual behaviour testing after  5-HT2  A  agonist administration  36  Figure 7: Mean intromission latency during sexual behaviour testing after 5 - H T 2 agonist A  administration  37  Figure 8: Mean intromission frequency during sexual behaviour testing after 5-HT agonist 2A  administration  38  Figure 9: Mean ejaculation latency during sexual behaviour testing after 5 - H T 2 agonist A  administration  39  Figure 10: Mean ejaculation frequency during sexual behaviour testing after 5 - H T 2 agonist A  administration  40  List of Nomenclature and Abbreviations hr: hour cm: centimetre g: gram jig: microgram kg: kilogram mg: milligram min: minute ml: millilitre sec: second 0  C: degrees celcius  vi  Acknowledgements This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Grant to Boris B. Gorzalka and a NSERC Postgraduate Scholarship to Amanda K. LaMarre. I would like to thank Jane Sun for all of her hard work and assistance with running the experiment, Matthew Hill for his friendship and methodological opinion, and Dr. Boris Gorzalka for his advice and support throughout my undergraduate and graduate career.  1 Introduction The central serotonergic system, and its neurotransmitter, serotonin (5-HT), have been the focus of extensive research for the past 20 years. With the advent of molecular biology techniques, 7 families of 5-HT receptors have been identified, comprising a total of 14 structurally and pharmacologically distinct receptor sub-types (Hoyer & Martin, 1996). Research on the particular receptor sub-types of the 5-HT system has elucidated many of their physiological roles, ranging from initiating changes at the neuronal level, to quantifiable behavioural stereotypies. In particular, the 5-HT and 5-HT receptors have been the two most ]A  2A  extensively researched in light of their clinical relevance (den Boer, Bosker, & Slaap, 2000). Radioligand binding studies have shed light on the 5-HTi receptor's central distribution, A  highlighting major densities in the limbic system of the brain, especially the hippocampus (Hoyer, et al., 1994). The limbic system has been implicated in a number of primary functions such as arousal, feeding, sleep, and hedonic activities such as sexual behaviour. Moreover, the limbic system is strongly associated with emotion and mood. In relation, pharmacological activation of the 5-HTi receptor in rats produces behavioural effects such as hypothermia, A  hyperphagia, and increased sexual behaviour in the male rat (Gorzalka, Mendelson, & Watson, 1990; for a review, see Lucki, 1992). Pharmacological investigations with 5-HT receptor ]A  agonists such as buspirone have also shed light on the receptor's potential anxiolytic/antidepressant role (for a review, see Lucki, 1992). These findings emphasize the involvement of 5HT] receptor activity in regulation of limbic system modalities. A  While the  5-HT2  A  receptor is also found in the hippocampus, it has been extensively  mapped in many other areas of the forebrain, particularly the cortex (Pazos, Cortes, & Palacios, 1986; Pazos, Probst, & Palacios, 1987). Interestingly, increased activity of the  5-HT2  A  receptor  is associated with functionally opposite effects of those associated with increased 5-HTi  A  receptor activity. For example, increases in 5 - H T 2 receptor activity have been demonstrated to A  2 induce hyperthermia and hypophagia, and to decrease sucrose preference and sexual behaviour in male rats (for review, see Barnes and Sharp, 1999; Gorzalka, et al., 1990). Biochemical and behavioural effects mediated via the serotonin system must also be considered in relation to its interaction with the hypothalamic-pituitary-adrenal (HPA) axis. Both systems seem to exert regulatory roles over the other, especially within the stress response (McEwen, 1987). For example, Owens, Edwards, and Nemeroff (1990) demonstrated that selective 5-HTIA agonists are able to increase corticotrophin releasing hormone (CRH) concentrations in the piriform cortex and the hippocampus. Furthermore, release of adrenocorticotropin hormone (ACTH) from the anterior pituitary is stimulated by serotonin via action mediated at the 5-HTi and 5-HT2A receptors in the paraventricular nucleus of the A  hypothalamus (Pan & Gilbert, 1992; Fuller, 1992). Reciprocally, corticosteroids control brain trytophan (the precursor to serotonin) availability, neuronal 5-HT release, electrical activity of 5-HT neurons, and responses to 5-HT receptor stimulation (Meijer & de Kloet, 1998). In fact, it appears that corticosterone is necessary to maintain serotonin levels, an effect that can be demonstrated in rats that have undergone adrenalectomy (ADX) (i.e., their source of corticosterone has been removed) (van Loon, Shum, & Sole, 1981). In the absence of circulating endogenous corticosterone, 5-HT metabolism is severely disrupted. This effect is reversed via administration of exogenous corticosterone (Singh, Corley, Phan, & Boadle-Biber 1989). Two types of corticosteroid receptors have been described in the brain based on their differing biochemistry and affinity for particular steroid ligands (Reul & de Kloet, 1985). Type I corticosteroid receptors, also called mineralocorticoid receptors (MR), are highly specific to which type of corticoid they will bind, that being Cortisol in humans and corticosterone in rats. MR receptors are found most densely in the hippocampus and septal region of the limbic system (Reul & de Kloet, 1985). Type II receptors, or glucocorticoid receptors (GR), will bind  3 corticosterone, however they tend to have higher affinity for potent exogenous corticosteroids such as dexamethasone. In addition, unlike MR receptors, they are widely distributed throughout the brain, including the hippocampus, hypothalamus, and pituitary (Reul & de Kloet, 1985). During non-stress conditions, corticosterone differentially binds to the MR receptors; during activation of the HPA-axis, these receptors become saturated by elevated levels of circulating corticosterone, whereby the GR receptors will bind the excess corticosterone. In this way, GR receptors regulate the HPA-axis by exerting negative feedback. In relation to serotonin, research suggests that GR activation is necessary for stress-induced synthesis of serotonin; however chronically high levels of circulating corticosterone have also been shown to suppress the activity of 5-HT neurons (Meijer & de Kloet, 1998). In terms of the 5-HTIA and 5-HT2A receptors, evidence suggests that they are differentially regulated by the MR and GR corticosteroid receptors, respectively. When 5-HTIA receptor mRNA expression is dysregulated, low levels of circulating corticosteroids normalize this expression, indicating MR receptor mediation (Chalmers, Kwak, Mansour, Akil, & Watson, 1993; Meijer & de Kloet., 1994). In addition, Meijer et al. (1997), found that in transgenic mice which lack functional GR receptors, their 5-HTIA receptor levels do not significantly differ from those in the wild-type control mice, suggesting that MR activation is sufficient to regulate 5HTIA  mRNA, and therefore receptor number, to normal levels. Moreover, stimulation of the 5-  HTIA  receptor with the agonist flesinoxan causes a downregulation of the GR receptor (Sibug, et  al., 2000). Reciprocally, GR activation stimulates mRNA expression, receptor density, and responsiveness of the 5-HT2A receptor. For example, chronic treatment with ACTH or dexamethasone, or chronic social stress can exert these effects on the 5-HT2A receptor (Kuroda, Mikuni, Ogawa, & Takahashi, 1992; Kuroda, Mikuni, Nomura, & Takahashi, 1993; McKittrick, Blanchard, Blanchard, McEwen, & Sakai, 1995). In addition, transcriptional control of the gene  4  for the 5-HT2A receptor is influenced by activation of the GR receptor by corticosteroids (Garlow &Ciaranello, 1995). Overall, it seems reasonable to speculate that 5-HTi receptor activity predominates A  under non-stress conditions, whereas during times of stress, 5 - H T 2 A activity overrides the biochemical and behavioural effects of  5-HTIA  activation. These changes appear to be mediated  by elevated levels of circulating corticosteroids. Indeed, Reul & de Kloet (1985) estimate that a dose of more than 10 mg/kg of corticosterone is needed in order to occupy 95% of the total GR receptors. With respect to the 5-HTi and A  5-HT2  A  receptors, studies have demonstrated  5-HTIA  downregulation with administration of chronic corticosterone at a dose of 1 mg/kg, but not elevated 5-HT2A receptor density (Crayton, Joshi, Gulati, Arora, & Wolf, 1996). However, it has been demonstrated by Kuroda et al. (1992) that doses of corticosterone at 20mg/kg and 50 mg/kg have the ability to both decrease 5-HTIA receptor density, and increase  5-HT A 2  density.  In relation, circulating corticosteroids have been shown to control numerous 5-HTIA and 5-HT2A  receptor-mediated physiological and behavioural indexes. Takao, Nagatani, Kitamura  and Yamawaki (1997) found that chronic treatment with corticosterone in rats (at levels that are similar to endogenous output during times of stress) both decreased  5-HTIA  receptor binding in  the hippocampus and increased 5-HT2A binding in the frontal cortex. In addition, behavioural indexes of 5-HTIA and attenuated the  5-HTJA  5-HT2A  receptor activity were affected. In particular, corticosterone  "serotonin syndrome" (forepaw treading, lower-lip retraction, flat body  posture), and increased wet dog shakes (WDS), a paroxysmal shudder of the neck and trunk region which is a validated behavioural assay of activation of 5-HT2A receptors (Bedard & Pycock, 1977). Furthermore, in a series of studies investigating the effects of endogenous/exogenous corticosterone on 5 - H T  2  A  receptor activation, Gorzalka and Hanson  (1998), demonstrated that 1) there is a significant reduction in the number of spontaneous WDS in ADX rats compared to controls, 2) chronic corticosterone administration (50 mg/kg) in the  5 ADX rats blocked the ADX-induced reduction of spontaneous WDS, and 3) chronic corticosterone administration (50 mg/kg) in adrenally intact rats significantly facilitated both spontaneous and l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced WDS, as well as inhibited male rat sexual behaviour. Similarly, Young, Dow, Goodwin and Fink (1993) found that ADX potentiated 8-hydroxy-2(di-«-propylamino) tetralin (DPAT) induced hypothermia while corticosterone implants attenuated this effect. In the chronic unpredictable stress (CUS) model of depression, where corticosterone levels are significantly elevated (for review, see Willner & Mitchell, 2002), the CUS procedure has been shown to increase 5-HT2A receptor density by 52% in the cerebral cortex (Ossowska, et al, 2001). In relation, CUS was also shown to decrease hippocampal binding of 5-HTi  A  receptors (Lopez, Chalmers, Little, & Watson, 1998), and these effects could be blocked by administration of 5-HTIA agonists such as buspirone and WAY 100135 (Przegalinski, Moryl, & Papp, 1995). Fernandes, McKittrick, File, and McEwen (1997) found similar results in terms of 5-HTIA and 5-HT2A receptor densities following chronic stress. Gorzalka, Hanson, and Brotto  (1998) have also demonstrated that male rats subjected to chronic psychosocial stress, exhibit increases in DOI-induced WDS and inhibition of sexual behaviour similar to that of chronic corticosterone administration. This has also been established in the CUS paradigm (Brotto, Gorzalka, & LaMarre, 2001). While previous research has focused on either 5-HTi and 5-HT2A receptor density A  changes or physiological and behavioural changes at baseline and one time point following exogenous administration of corticosterone, no one has examined what this complex pattern of change looks like with respect to physiological and behavioural stereotypies over the entire course of administration. If the hypothesis is correct that 5-HT2A receptor activity predominates under conditions of stress, whereas 5-HTIA predominates under non-stress conditions, it seems reasonable to predict that following an elevation in corticosterone levels, 5-HT2A receptor-  6 mediated behaviours would increase, while  5-HTIA  receptor-mediated behaviours would decline.  Even though the stress response involves a complex pattern of neurochemical, hormonal and physiological changes in the body besides elevating corticosteroids, it is still reasonable to assume that corticosterone is an important mediator of the stress reaction. Demonstration of the link between the 5 - H T system, HPA-axis, and behaviour has important implications with respect to the etiology and treatment of stress-induced affective disorders such as depression. Consequently, it is not only important to address the issue of how elevated corticosteroids change receptor function but when this manifests itself in behavioural changes. Therefore, the purpose of the present investigation was to demonstrate a time course model of shifting control from predominant 5 - H T J A receptor activity to  5-HT2A  receptor activity  based on examination of discrete changes in the physiological and behavioural stereotypies associated with 5-HT] and A  5-HT2A  receptors. To examine this, chronic corticosterone was  administered at a dose known to downregulate  5-HTIA  and upregulate  5-HT2A  receptor activity  (20 mg/kg) in comparison with a vehicle-administered control group for 14 days. 5-HT)  A  receptor activity was measured via temperature changes following acute administration of the 5HTIA  receptor agonist DPAT at six time points, and  5-HT2A  receptor activity was measured via  temperature changes and behavioural observation of sexual behaviour and the 5 - H T 2 A stereotypy WDS, following acute administration of the 5 - H T  2  A  receptor agonist DOI at six time points  during chronic corticosterone/vehicle administration. Examination of changes with respect to time will be used to determine the threshold for shifting control from 5 - H T I A to 5 - H T 2 A activity. Over time, it was predicted that 5 - H T I A receptor activity should diminish, as measured by an attenuation of hypothermic response to DP AT; while  5-HT2  A  receptor activity should increase, as  measured by potentiation of hyperthermic response to DOI, an increase in WDS, and a concurrent decrease in measures of sexual behaviour.  7 Method Animals and Housing 84 male Long Evans rats (Charles River Canada Inc., Quebec) were obtained at 3 weeks of age. The rats were 15 weeks old at the time of the experiment and ranged in weight between 400-600 g. Rats were weighed in the first third of the dark cycle at baseline, and then every 2 days, for a total of eight measurements. In addition, 30 sexually experienced female rats were used to elicit sexual behaviour from the male rats exposed to the sexual behaviour testing. The female rats were 12 months of age at the time of the experiment. Prior to the experiments, the females were bilaterally ovariectomized using standard surgical procedures while anesthetized under halothane (MTC Pharmaceuticals, Cambridge, Ont). A l l rats were housed in same-sex groups of three, in hanging mesh triple wire cages. Rats were allowed free access to Purina Rat chow and water ad libitum. Colony conditions were maintained at 21±1°C, on a reverse 12/12h light cycle (lights off at 0900 h). Materials Corticosterone-21-acetate (corticosterone) (Research Plus., Bayonne, New Jersey) was dissolved in propylene glycol to a dose of 20 mg/kg/ml by heating to 40°C, and stirring thoroughly. Its vehicle, propylene glycol was also heated to 40°C and stirred to facilitate injection. DP A T (Sigma Chemical Co., St. Louis, MO) was stirred in saline until dissolved, to a dose of 0.1 mg/kg/ml. DOI (Sigma Chemical Co., St. Louis, MO) was stirred in saline until dissolved, to a dose of 1 mg/kg/ml. The vehicle for both DP A T and DOI was saline. Estradiol benzoate (Sigma Chemical Co., St. Louis, MO) was dissolved to a dose of 10 ng by stirring thoroughly in 0.1 ml peanut oil. Progesterone (Sigma Chemical Co., St. Louis, MO) was dissolved to a dose of 500 \xg by stirring thoroughly in 0.1 ml peanut oil. A l l injections were performed using Vi inch, 26 gauge stainless steel needles, except corticosterone, for which 3/8 inch, 25 gauge needles were used.  8  Apparatus  All transportation and handling of animals was performed within the confines of standard plastic maternity bins filled to a height of 5 cm with wood fiber chip bedding. Testing chambers for sexual behaviour were made of Plexiglas (30 x 30 x 45 cm in height), with 10 cm of wood fiber chip bedding in the bottom. Rectal temperature was recorded using a long thin plastic probe attached to a standard digital thermometer (Fisher Scientific Ltd., Nepean, Ont). Standards of care were in accordance with the requirements of the Canadian Council on Animal Care. Procedure  Male rats were randomly assigned to either the corticosterone or vehicle treatment group with 42 rats per group. Corticosterone or its vehicle (1.0 ml/kg) was injected subcutaneously (sc) once daily for a period of 14 days during the middle third of the dark cycle. The first day of the corticosterone treatment began one day before vehicle in order to stagger testing days for each of these groups. In order to investigate 5-HTi and 5-HT2A receptor activation, the rats were further A  divided into three treatment groups within the corticosterone and vehicle group. Each of the three groups included 14 rats that received one of three treatments on testing days: (a) DPAT, (b) DPAT vehicle and DOI vehicle or (c) DOI. The rats used as controls (i.e., were administered vehicle) were utilized in both the 5-HTiAand 5-HT2 receptor activation testing in order to A  decrease the number of rats needed. The three treatment groups were further divided into an A and a B group with 7 rats each. In this way, all rats in the corticosterone or vehicle group began and received corticosterone or vehicle treatment at the same time; however groups A and B for each DPAT, vehicle, or DOI group were tested on alternate days. For example, group A was tested at baseline, group B on day 3, group A on day 6, and so on. Therefore, rather than being tested every three days, each A and B group was tested every six days. This methodology was employed in order to reduce the  9 risk of possible DOI and DP AT drug potentiation over the course of the experiment. Furthermore, it allowed testing time to remain manageable by reducing the number of rats that were to be tested on any one day to a maximum of 21 (see Appendix for flow chart of experimental design). 5-HTIA receptor activation testing. 5-HTiAreceptor activity was indirectly measured via  hypothermic temperature changes in response to agonist administration. This measure is a valid and reliable assay for 5-HTi receptor activity (Martin, Phillips, Hearson, Prow, & Heal., 1992). A  Agonist-induced hypothermia was tested at baseline, and then every third day until day 15 (for a total of six testing sessions), prior to administration of corticosterone or vehicle in the first third of the dark cycle. Baseline temperature was taken, and then each rat was injected intraperitoneally (ip) with either DP AT or its vehicle (1 ml/kg). Temperature was then recorded 10, 20, and 30 min after injection, and difference scores between baseline and each time point were calculated. 5-HT2 receptor activation testing. 5-HT2A receptor activity was indirectly assayed via A  three measures: agonist-induced hyperthermic response, agonist-induced WDS, and sexual behaviour/WDS testing after agonist administration. Baseline temperature was taken, and then DOI or its vehicle (1 ml/kg) was injected ip. Measurement occurred once, 15 min after injection. 30 min following injection, WDS testing occurred in which rats were placed in glass testing chambers (which are also used for sexual behaviour testing) and the frequency of WDS was tallied for a 5 min observation period. Immediately following the WDS testing, sexual behaviour testing occurred. Sexual behaviour testing involved recording male copulatory responses following presentation of receptive female rats. Receptivity in the 30 ovariectomized female rats was induced via sc injections of 10 ug, estradiol benzoate 2 days before testing, and sc injections of 500 ug progesterone 3 hr before  10  sexual behaviour testing. Measures o f sexual behaviour were recorded for 30 m i n and included: mount, intromission (successful penile intromission by the male), and ejaculation (emission o f semen following intromission) frequencies and latencies. Frequency o f spontaneous W D S was also tallied during the thirty m i n sexual behaviour observation period given the inverse relationship between the two.  Statistics A repeated measures Analysis of Variance ( A N O V A ) was performed in order to analyze weight changes, with time as the within-subjects factor and corticosterone/vehicle as the between-subjects factors.  The p value was set at 0.05. A repeated measures A N O V A was also  performed in order to analyze the difference scores obtained from temperature measurement 10, 20, and 30 m i n following administration o f D P A T or its vehicle. Significant interactions were analyzed using simple main effects analysis, and post-hoc analysis was performed using Tukey's multiple comparisons. A l l p values for these data were set at 0.02 (.05/3) i n order to adjust for Type I Error inflation due to three dependent variables being analyzed. A repeated measures A N O V A was also performed i n order to analyze the dependent measures associated with 5-HT2A receptor activity. Difference scores were computed for temperature change after D O I administration. Significant interactions were investigated using simple main effects analysis, and post-hoc analysis was performed using Tukey's multiple comparisons. While a repeated measures Multivariate Analysis o f Variance ( M A N O V A ) would have been a better choice in terms of the overall analysis o f the multiple dependent variables measuring 5-HT2A receptor activity, there was not sufficient degrees o f freedom to perform this analysis. Therefore, p values for these data were set at 0.01. The rationale was to preserve family wise error rate without sacrificing overall power (i.e., given that there are 9 dependent variables, use o f the Bonferroni correction would significantly reduce power). For all repeated measures A N O V A s performed, Mauchly's Test o f Sphericity was also employed. In cases where sphericity was violated, the  11 Greenhouse-Geisser corrected values were used. One rat from the DOI/ corticosterone-vehicle group died during the course of the experiment and was thus dropped from analysis. Missing latency scores for sexual behaviour testing were set to a maximum of 1800s. Results Analysis of weight Rats receiving corticosterone (C) had a significant weight decrease over the course of the experiment, compared to rats who received corticosterone vehicle (CV). Repeated measures analysis of weight change over the 14 days of C or CV treatment revealed a significant time X condition interaction [F (3.44, 278.41) = 146.38, p < 0.001]. Furthermore, the main effects of time and condition were also significant, [F (3.43, 278.41) = 9.67, p <0.001; F (1, 81 = 18.02, p < 0.001]. Simple main effects analysis revealed that beginning on day 5 of the experiment, rats administered C weighed significantly less than the CV treated rats (p < 0.001). Moreover, these differences continued over the course of the remainder of the experiment. The effects of C on weight change appear in Figure 1. Analysis of 5-HTi receptor agonist effects on temperature response A  Repeated measures analysis of difference scores between baseline temperature and temperature 10 min following DP AT (DT) or DP AT vehicle (DTV) administration over the course of the entire experiment revealed a significant main effect of condition on temperature change [F(3, 24) = 122.88, p < 0.001]. Follow up analysis indicated that over the course of the experiment, the treatment with DT/CV produced a significantly greater reduction in temperature at 10 min than treatment with DTV/C. This was the expected effect. There was no other between group differences in temperature change at 10 min post administration. Data for temperature changes 10 min following DT administration are presented in Figure 2.  12 By 12 days of C or CV administration, rats receiving C/DT had a complete attenuation of DT-induced hypothermia at 20 min following administration, compared to rats receiving CV/DT. Repeated measures analysis of difference scores between baseline temperature and temperature 20 min following DT or DTV administration over the six levels of the within-subjects factor, time, revealed a significant time X condition interaction [F (15, 120) = 3.23, p < 0.001]. Furthermore, the main effect of condition was also significant, [F (3, 24) = 41.78, p <0.001]. Simple main effects analysis for the between-subjects factor, condition, at levels of time revealed significant between group differences at each level of time (p < 0.001), except for day 6 (after six days of C or CV treatment). Tukey's multiple comparisons were conducted and the following was found: at baseline (time zero), both groups of rats receiving DT, as well as the DTV/CV group had significantly lower temperatures than rats in the DTV/C group. At day 3 and 9, rats receiving DT/CV had significantly lower temperatures than those receiving DTV. The two groups of rats which received DT did not differ significantly from one another in amount of temperature decline; however, the rats in the DT/C group also did not differ significantly from the two groups of rats which received DTV. Lastly, at days 12 and 14, rats receiving DT/CV exhibited a greater drop in temperature than rats in the other three groups. Significance levels were all p < 0.001. Figure 3 indicates the time course of DT or DTV administration on temperature difference scores 20 min following drug administration over the course of the experiment. Generally, over the course of the experiment, rats receiving C/DT had a complete attenuation of DT-induced hypothermia at 30 min following administration, compared to rats receiving CV/DT. Repeated measures analysis of difference scores between baseline temperature and temperature 30 min following DT or DTV administration over the six levels of the within-subjects factor, time, revealed a significant time X condition interaction [F (9.51, 76.06) = 2.22, p < 0.02]. There was also a significant main effect of condition [F (3, 24) = 12.37,  13. p < 0.001]. Simple main effects analysis for the between-subjects factor, condition, at levels of time revealed significant between group differences at each time (p < 0.001), except again for day 6. Tukey's multiple comparisons were conducted and the following was found: at baseline (time zero), both groups of rats receiving DT had significantly lower temperatures than rats in the DTV group. At days 6 and 9, rats receiving DT/CV had significantly lower temperatures than those in any of the other three groups. At day 12, the two groups of rats receiving DT had a significantly greater drop in temperature than rats receiving DTV. Finally, at day 14, rats receiving DT/CV had significantly lower temperatures than those in any of the other three groups. Significance levels were all p < 0.001. Figure 4 indicates the time course of DT or DTV administration on temperature difference scores 30 min following drug administration over the course of the experiment. Analysis of 5-HT2A receptor agonist temperature and behavioural  responses  Over the entire course of the experiment, rats receiving DOI (D), had a significant increase in temperature after D administration, compared to rats receiving DOI-vehicle (DV). Repeated measures analysis of agonist-induced hyperthermic response revealed a significant main effect of time [F (3.04, 69.97) = 5.53, p < 0.002], and condition [F (3, 23) = 23.12, p < 0.001]. Follow-up analysis indicated that there was a mean temperature increase for all groups combined (p < 0.001). In addition, compared to DV, D administration was able to significantly induce hyperthermia (p < 0.001). Data representing mean hyperthermic responses of rats in response to D or DV are presented in Figure 5. Generally, over the course of the entire experiment, rats receiving D had significantly more WDS during sexual behaviour testing than those who received DV. Repeated measures analysis of WDS tallied during sexual behaviour testing indicated a significant main effect of condition [F (3, 23) = 12.02, p < 0.001]. Tukey's multiple comparisons revealed that both groups of rats  14 receiving D had significantly more WDS than those receiving DV (p < 0.01). Those rats treated with D/C also had significantly more WDS than those rats treated with C alone (p < 0.01). There was no significant difference between rats which received DV/C, and rats which received D/CV. Data indicating mean WDS during sexual behaviour testing are presented in Figure 6. Repeated measures analysis of agonist-induced WDS measured prior to sexual behaviour testing did not reveal any significant effects with respect to time or condition [F (3.21, 24.60) = 0.87, p > 0.05). Repeated measures analysis of the six measures of sexual behaviour did not reveal any significant interactions between condition and time [F (9.89, 75.81) = 2.34; F (8.33, 63.90) = 1.08; F (11, 84.33) = 1.83; F (10.85, 83.18) = 1.56; F (10.26, 78.68) = 1.33; F (7.66, 58.69) = 1.00, all p > 0.01). For mount latency, a significant main effect occurred for condition [F (3, 23) = 18.55, p < 0.001]. Follow up analysis revealed that rats in the D/CV group had significantly higher mount latencies compared with rats administered DV (p < 0.001). Analysis of mount frequency revealed a significant main effect of time [F (2.78, 63.90) = 5.58, p < 0.002], and condition [F (3, 23) = 10.00, p < 0.001]. However, follow-up analyses did not indicate any significant differences between levels of time or condition (p > 0.01). Over the course of the entire experiment, rats administered D had significantly longer intromission/ejaculation latencies and significantly less intromissions/ejaculations compared to rats administered DV. For intromission latency and frequency, there was a significant main effect of condition [F (3, 23) = 28.18, p < 0.001; F (3, 23) = 61.74, p < 0.001]. There was also a main effect of condition for both ejaculation latency and frequency F (3, 23) = 15.83, p < 0.001; F (3,23) = 27.01, p < 0.001]. As mentioned above, follow up analysis revealed significantly longer intromission/ejaculation latencies in rats receiving D, and significantly less intromissions/ejaculations compared to rats administered DV (p < 0.001). Data representing the effects of D on intromission latency and frequency, and ejaculation latency and frequency are  15 presented in Figure 7, Figure 8, Figure 9, and Figure 10, respectively. Overall, it appears that there was no effect of C on any of the measures of agonist-induced  5-HT2A  receptor activity.  Discussion The purpose of this experiment was to determine the time course for shifting  5-HTIA  receptor activation to 5 - H T 2 A receptor activation following chronic administration of corticosterone at a dose known to downregulate  5-HTIA  and upregulate  5-HT2A  receptors  (Kuroda et al., 1992). Overall, it was found that after 12 days of corticosterone treatment, 5HTIA  receptor activation had shifted, as indicated by the complete attenuation of the hypothermic  response to DP AT in those rats receiving corticosterone. However, the progression of this shift is not at all clear with respect to  5-HT2A  receptor activity. It appears that 5 - H T 2 A receptor  agonism alone elicited ceiling effects on both hyperthermic and WDS responses, and floor effects on measures of sexual behaviour. Therefore, it is difficult to assess at what point, if any, corticosterone increased 5 - H T  2  A  receptor activity.  The results of this experiment indicate that the time course for shifting  5-HTIA receptor  activity in terms of hypothermic response to DP AT is relatively rapid when circulating levels of corticosterone are high (20 mg/kg). After only 3 days of chronic corticosterone treatment, the hypothermic response induced by DP AT after 20 min had been partially attenuated in rats receiving corticosterone. This effect was seen again after 9 days of corticosterone treatment. Furthermore, by day 12, the hypothermic effect of DPAT at 20 min had been attenuated by chronic corticosterone treatment, thereby suggesting that receptor activity had been shifted from 5-HTiAto 5 - H T 2 A receptor activation. A similar pattern was seen 30 min following DPAT administration over the course of the experiment. These results are consistent with data from other investigators who have examined the relationship between corticosterone administration and hypothermic responses to DP AT. For  16  example, Fone and Topham (2002) demonstrated that after 4 days of chronic corticosterone treatment in the form of a slow release implant (approximately 50 mg/kg/day), DPAT-induced hypothermia was completely attenuated in those rats receiving corticosterone. Similarly, it has been shown that rats receiving a daily dose of corticosterone at 10 mg/kg show this attenuation after 10, but not 3 days of administration. Interestingly, rats tested again at 14 days into the experiment (but with only 10 days of chronic corticosterone treatment) do not display an attenuation of the hypothermic response (Young, MacDonald, St John, Dick and Goodwin, 1991). This finding provides further evidence to suggest that when circulating corticosterone returns to baseline levels, 5-HTi receptor activity returns to normal functioning. A  In contrast to the above findings, we did not see a complete attenuation of response until after 12 days of corticosterone administration; however, there are several factors that may account for this result. For example, Fone and Topham (2002) implemented both a higher dose of corticosterone, and a different mode of administration. This would tend to facilitate a more rapid shift. Indeed, Haleem (1992) demonstrated an attenuation of the DPAT hypothermic response in rats after 5 days of chronic corticosterone treatment at a dose of 50 mg/kg twice daily. Furthermore, it can not be discounted that continuous, sc release of corticosterone may have differential effects with respect to serotonin receptor agonism, in comparison to daily injections that induce one large surge per day. Man, Young, and McAllister-Williams (2002) found that mode of administration of corticosterone (i.e., daily injection vs. osmotic microdialysis) had differential effects on corticosterone's ability to attenuate DPAT-induced hypothermia; specifically, central administration was not as effective at attenuating the hypothermic response as was sc injection. Young, et al.'s (1991) description of attenuation after 10 days but not after 3 in rats receiving lOmg/kg corticosterone fits nicely into the framework for shifting control which we have demonstrated. In this experiment, rats received a dose of 20 mg/kg daily. After 3 days, a noticeable shift occurred (versus no change with 10 mg/kg at this  17  point), which was again present following 9 days of administration. Had we tested our rats after 10 days as did Young et al., this may have been sufficient to observe a complete attenuation in our experiment as well. The results of the current experiment extend the above findings in that, rather than considering physiological changes at simply one or two time points, this design attempts to capture the dynamic changes occurring over the entire course of administration. In particular, we were able to demonstrate a smooth progression of shifting response which is not illustrated when simply measuring hypothermic response once or twice. Besides trying to delineate a comprehensive time course for shifting  5-HTIA  receptor  physiology mediated by chronic corticosterone treatment, the current study was interested in discerning the complex manner in which 5 - H T 2 A receptor activity changes in direct relation to shifting HT2A  5-HT)A  receptor activity. Unfortunately, as previously mentioned, it appears that  5-  receptor-agonist administration produced both ceiling and floor effects, thereby preventing  elucidation of the time course for corticosteorne's effects on 5 - H T 2 A receptor activity. While a main effect of DOI was established in terms of its ability to induce hyperthermia, which was maintained over the course of the experiment, we did not see a potentiation of this response in those rats treated with corticosterone. These results are consistent with Takao et al. (1997)  who found that in rats treated with chronic corticosterone for  14  days  (50  mg/kg), there  was no significant facilitation of corticosterone on hyperthermic response to DOI compared with rats treated with DOI alone. Given that Takao et al.  (1997)  administred the same dose of DOI as  was employed in the present study (1.0 mg/kg), it seems possible that the dosage may produce ceiling effects, regardless of further agonism by corticosterone. Indeed, Salmi and Ahlenius (1998)  demonstrated a statistically significant hyperthermic effect with a dose of DOI at  0.4  mg/kg. Furthermore, augmentation with the 5 - H T 2 A receptor agonist W A Y - 1 0 0 , 6 3 5 potentiated  18 the 0.4 mg/kg hyperthermic effect, providing further evidence to suggest that 1.0 mg/kg of DOI is able to completely agonize the 5 - H T 2 A receptor. Similarly, complete agonism of the 5 - H T 2 A receptor by 1.0 mg/kg of DOI may have caused the overall main effect of increased WDS during sexual behaviour. For example, Nagayama and Lu (1996) demonstrated a dose-dependent increase in WDS with the highest frequency achieved at a dose of 1.0 mg/kg. However, several studies investigating the relationship between chronic corticosterone administration and WDS have demonstrated an ability of corticosterone to significantly facilitate WDS above that of 1.0 mg/kg DOI alone (Fone & Topham, 2002; Gorzalka & Hanson, 1998; Takao, et al., 1997; Berendsen, Kester, Peeters, & Broekkamp, 1996). While each of these studies was able to demonstrate this effect at differing time points of chronic corticosterone administration, it should be noted that in each of these studies the dose of corticosterone was 50 mg/kg - a dose that is significantly higher than that received by rats in the present study. However, analysis did reveal that in those rats treated with corticosterone, the mean number of WDS was not significantly different than that obtained in rats receiving DOI, suggesting that corticosterone did have some ability to agonize  5-HT2A  receptors alone.  Nevertheless, given the present findings, it appears that a dose of 20 mg/kg corticosterone is not sufficient to further agonize  5-HT2A  receptors above and beyond the effects of 1.0 mg/kg DOI.  A surprising result of this study was the failure to observe any effect of DOI on WDS during the 5 min period prior to sexual behaviour testing. Previous findings from our laboratory indicate that at a dose starting at 1.0 mg/kg, maximal effects of DOI on WDS occur 30 min following administration. However, evidence also suggests that measurement of WDS during sexual behaviour provides a better behavioural index of 5-HT A receptor activity (Watson & 2  Gorzalka, 1990), which was evident in the current study. However, it is possible that the length  19 of the testing session was simply too short to capture any between group differences which were clearly present during the sexual behaviour testing. As an inverse relationship exists between frequency of spontaneous WDS and decreased sexual behaviour in male rats (Watson & Gorzalka, 1990), and is enhanced by 5-HT2A receptor agonism (Watson & Gorzalka, 1990; Gorzalka & Hanson, 1998) it is not surprising that the current ceiling effects on measures of hyperthermia and WDS during sexual behaviour testing also produced a floor effect on measures of sexual behaviour. While this effect occurred over the entire course of the current experiment, similar findings have been described in rats receiving 13 days of daily corticosterone (50 mg/kg) on tests of sexual behaviour in both spontaneous and DOI-induced conditions (Gorzalka & Hanson, 1998). Given that a dose of corticosterone at 20 mg/kg is able to upregulate 5-HT2A receptors as much as a dose of 50 mg/kg, and that DOI has such a robust effect at 1.0 mg/kg, these findings are to be expected. Given the limitations that occurred in this study due to methodological problems with respect to ceiling/floor effects in response to the particular dose of DOI used in this experiment, it may be helpful in future studies to implement a lower, efficacious dose of DOI when testing the 5-HT2 receptor-mediated behaviours. Alternatively, or in conjunction with, it might be A  important to establish this time course with a 50 mg/kg dose of corticosterone which has already been demonstrated to potentiate the effects of DOI on these measures. Finally, given the robust negative effects of both corticosterone and DOI on sexual behaviour, this behavioural index may not be worthwhile investigating unless one employs 5-HT2A receptor antagonists. For example, the effects of DOI can be completely reversed by 5 - H T A antagonists such as ritanserin, 2  pirenperone and ketanserin (Watson & Gorzalka, 1990, 1992). Similarly, Gorzalka, Hanson, & Hong (2001) have shown that the 5-HT2A antagonist ketanserin completely reversed the  20 inhibition of sexual behaviour and facilitation of WDS following treatment with chronic corticosterone (20 mg/kg). More generally, research suggests that 5-HTi and 5-HT receptors are heavily A  2A  implicated in the etiology of depression. Evidence from pharmacological studies suggests that either hypoactivity of the 5-HTi receptor, and/or hyperactivity of the 5-HT receptor, may play A  2A  an important role in serotonin's involvement in depression. It has been postulated that a "balance" between 5-HTi receptors and 5-HT receptors is essential to antidepressant action. A  2A  This stems from the fact that the net effect of selective serotonin reuptake inhibitors (SSRIs) appears to be a down-regulation of 5-HT receptors in the cortex, with the opposite effect 2A  observed for 5-HTi receptors in the hippocampus (Lopez et al, 1998). A  In addition, dysregulation of the HPA-axis; more specifically, hypercortisolemia, is one of the most robust findings associated with depression in biological psychiatry. Many investigators have examined Cortisol concentrations in the cerebrospinal fluid of depressed patients and found that levels of free Cortisol are significantly elevated upon comparison with a control group (e.g., Carroll, Curtis, & Mendels., 1976; Casper et al., 1987), and that the natural diurnal rhythm of secretion of Cortisol is suppressed (Wong, et al., 2000). Furthermore, evidence suggests that severity of depression is associated with increasing levels of Cortisol (Traskman et al., 1980; Gerner & Wilkins., 1983), which has also been reported in non-clinical populations (Pruessner, Hellhammer, Pruessner, & Lupien, 2003). Increasingly, the serotonergic changes seen in depression appear to be secondary to Cortisol abnormalities. For example, Meijer, Oosten, and De Kloet (1997) demonstrated that elevated basal trough levels of corticosterone, similar to those observed in depressed individuals with hypercortisolemia has the ability to suppress 5-HTi receptor mRNA expression in the A  hippocampus in rats via occupation of co-localized MR receptors. Furthermore, Leitch, Ingram, Young, McQuade, and Gartside (2003) describe functional desensitization of 5-HT) autoA  21 receptors in response to DPAT with flattened corticosterone rhythm in rats. Clinically, attenuation of 5-HT receptor mediated neuroendocrine and temperature response has been )A  demonstrated in depressed individuals as well (Shapira, Newman, Gelfin, & Lerer, 2000): Chronic stress has been shown in to increase cortical 5 - H T 2 A receptors and downregulate hippocampal 5-HTi receptors via the HPA-axis (for review, see Lopez, et al., 1997). In A  relation, stress-induced downregulation of brain-derived neurotophic factor expression in rat hippocampus is blocked by 5 - H T 2 A selective antagonists but not by 5 - H T I A agonists (Vaidya, Terwilliger, & Duman, 1999). This reinforces the idea of a necessary "balance" between these two receptors, and also suggests that 5 - H T 2 A receptor blockade could protect hippocampal neurons from stress-induced damage, especially within the context of hypercortisolemia. Given that the disturbed balance between these two receptors in relation to stress-induced HPA-axis hyperactivity has been postulated to be a significant contributor to the affective and behavioural changes seen in major depression (Lopez, Vazquez, Chalmers, & Watson, 1997; Meijer & de Kloet, 1998), the current research is important with respect to gaining insight into the rate at which these neurological, physiological and behavioural changes take place in the serotonin system. 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Mean temperature change 10 min following DP AT (DT) or DP AT-vehicle (DTV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. * denote groups which are significantly different from one another, p < 0.001. Figure 3. Mean temperature change 20 min following DPAT (DT) or DPAT-vehicle (DTV) administration over the course of the experiment in rats receiving corticosterone (C), or corticosterone-vehicle (CV). Error bars represent ± S.E.M.'s. Boxes denote groups which are significantly different from one another, p < 0.001. Figure 4. Mean temperature change 30 min following DPAT (DT) or DPAT-vehicle (DTV) administration over the course of the experiment in rats receiving corticosterone (C), or corticosterone-vehicle (CV). Error bars represent ± S.E.M.'s. Boxes denote groups which are significantly different from one another, p < 0.001. Figure 5. Mean temperature change 15 min following DOI (D) or DOI-vehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Scores of rats receiving DOI were significantly different from scores of rats receiving DOI-vehicle, p < 0.001. Figure 6. Mean frequency of wet dog shakes (WDS) occurring during sexual behaviour testing after DOI (D) or DOI-vehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. * denote groups which are significantly different from one another, p < 0.01. Figure 7. Mean intromission latency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Latencies of rats receiving DOI were significantly different from rats receiving DOI-vehicle, p < 0.001. Figure 8. Mean intromission frequency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Frequencies of rats receiving DOI were significantly different from those of rats receiving DOI-vehicle, p < 0.001. Figure 9. Mean ejaculation latency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error  30 bars represent ± S.E.M.'s. Ejaculation latencies of rats receiving DOI were significantly different from those of rats receiving DOI-vehicle, p < 0.001. Figure 10. Mean ejaculation frequency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Ejaculation frequencies of rats receiving DOI were significantly different from those of rats receiving DOI-vehicle, p < 0.001.  31  450  400  H  1  0  1  3  1  5  1  7  1  9  ~~  1  11  1  13  15  Day  Figure 1. Mean weight change for rats treated with corticosterone (C) or corticosterone-vehicle (CV) over the course of the experiment. Errors bars represent ± S.E.M.'s. * denote significant differences between groups, p < 0.05.  32  C/DT  C/DTV  CV/D"  CV/DTV  o CD  o (/}  CD CD CD CD Q  -0.5  CD  CD i— CD  Q.  E  CD  I-  Figure 2 M e a n temperature change 10 min following D P A T (DT) or D P A T - v e h i c l e ( D T V ) administration in rats receiving corticosterone (C) or corticosterone-vehicle ( C V ) . Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S E M ' s . * denote groups which are significantly different from one another, p < 0.001.  33  CO  3  co o CO  CD CD CD CD Q  1.5 1 0.5  —  1  ^  C/DT  r  0  -i=:  41  -dB—  - ± - CV/DT  C D 0.5 l_ zz  ^—'  CO  0 Q.  E  -®— C / D T V  CV/DTV  -1 1.5  CD 0  3  6  9  12  14  N u m b e r of days of C administration  Figure 3. Mean temperature change 20 min following DPAT (DT) or DPAT-vehicle (DTV) administration over the course of the experiment in rats receiving corticosterone (C), or corticosterone-vehicle (CV). Error bars represent ± S.E.M.'s. Boxes denote groups which are significantly different from one another, p < 0.001.  34  -r O  d) o (/) (D  —+~ C/DT  0.5 0  —B—-C/DTV  O) 0  Q 0  ro i—  -0.5  /  -1  CD  em  a.  -2  •  i  -1.5 0  3  6  -CV/DT  9  12  -x-  CV/DTV  14  Number of days of C administration Figure 4. Mean temperature change 30 min following DPAT (DT) or DPAT-vehicle (DTV) administration over the course of the experiment in rats receiving corticosterone (C), or corticosterone-vehicle (CV). Error bars represent ± S.E.M.'s. Boxes denote groups which are significantly different from one another, p < 0.001.  35  1.5 o CD  O  w CD CD L—  CD CD  Q  CD  1  0  Q.  E cu  r-  i  ^  r  C O i_  CD  X  0.5  -0.5 -1  C/DV  C/D  CV/DV  CV/D  Figure 5. Mean temperature change 15 min following DOI (D) or DOI-vehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Scores of rats receiving DOI were significantly different from scores of rats receiving DOI-vehicle, p < 0.001.  36  16 14 12 CO 10 8 6 4 c 2 C O cu 0 2  WD  E o  CV/DV  DV/C  D/CV  D/C  Figure 6. Mean frequency of wet dog shakes (WDS) occurring during sexual behaviour testing after DOI (D) or DOI-vehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. * denote groups which are significantly different from one another, p < 0.01.  37  1800 1600 1400 o cu 1200 CO  E  1000 800 600  ±  400 200 0 -I C/DV  C/D  CV/DV  CV/D  Figure 7. Mean intromission latency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Latencies of rats receiving DOI were significantly different from those of rats receiving DOI-vehicle, p < 0.001.  Figure 8. Mean intromission frequency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Frequencies of rats receiving DOI were significantly different from those of rats receiving DOI-vehicle, p < 0.001.  39  Figure 9. Mean ejaculation latency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Ejaculation latencies of rats receiving DOI were significantly different from those of rats receiving DOI-vehicle, p < 0.001.  40  2.5  co  ro o  ro LU  1 0.5 0  X C/DV  C/D  CV/DV  CV/D  Figure 10. Mean ejaculation frequency during sexual behaviour testing after DOI (D) or DOIvehicle (DV) administration in rats receiving corticosterone (C) or corticosterone-vehicle (CV). Data are not presented over time as there was no interaction between time and condition. Error bars represent ± S.E.M.'s. Ejaculation frequencies of rats receiving DOI were significantly different from those of rats receiving DOI-vehicle, p < 0.001.  41 Appendix Experimental Design  84 rats 1 Corticosterone vehicle (42)  Corticosterone (42)  DPAT (14)  1 DPAT/DOI vehicle  +  DOI (14)  DPAT (14)  DPAT/DOI vehicle  DOI (14)  I A&B (7 each)  i  A&B (7 each)  A&B (7 each)  A&B (7 each)  |  A&B (7 each)  A&B (7 each)  

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