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The inhibitory influence of HPA axis hormones on the 5-HT2A receptor-mediated behavioural effects of… Hong, Janie J. 2001

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The inhibitory influence of HP A axis hormones on the 5-HT2A receptor-mediated behavioural effects of risperidone: Potential therapeutic implications JANIE J. HONG B.Sc, The University of British Columbia, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Psychology; Clinical Psychology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 2001 © Janie J. Hong, 2001 UBC Special Collections - Thesis Authorisation Form Page 1 of 1 I n 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 o f th e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e 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 r e f e r e n c e and s t u d y . I f u r t h e r a gree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by th e head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h C o lumbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html 24/08/01 A B S T R A C T The present series of experiments was designed to investigate the potential inhibitory influence of stress and stress-related hormones on the 5-HT2A receptor-mediated behavioural effects of risperidone, an atypical antipsychotic. Experiment 1 examined the effects of chronic risperidone treatment; Long-Evans male rats were assigned to a daily regimen of either corticosterone (20 mg/kg) or vehicle for 14 days. Risperidone was administered at a dose of 0.1 mg/kg for 14 days prior to testing. Rats were tested on measures of sexual behaviour and wet dog shakes (WDS), both 5-HT2A receptor-mediated behaviours. Consistent with previous findings, chronic corticosterone treatment induced behavioural changes indicative of an increase in 5-HT2A receptor activity. Results from Experiment 1 demonstrated the efficacy of risperidone in facilitating rat sexual behaviour and inhibiting WDS frequency. Of interest, the behavioural effects of risperidone were significantly attenuated by a chronic corticosterone regimen. In Experiment 2, the behavioural influence of chronic mild stress (CMS) on a chronic risperidone regimen was examined. Rats received daily injections of either risperidone (0.1 mg/kg) or saline and were exposed daily to either CMS or no stress for 21 days. Similar to findings using a chronic corticosterone regimen, CMS significantly attenuated the effects of risperidone on both male rat sexual behaviour and WDS expression. Experiment 3 investigated the possibility that chronic CMS treatment act via elevated corticosterone levels when influencing the behavioural efficacy of risperidone. Rats received risperidone and metyrapone (50 mg/kg), a corticosterone synthesis inhibitor, on a daily basis for a period of 14 days. Results from Experiment 3 indicated the effects of stress on WDS frequency, but not on sexual behaviour, in risperidone-treated rats are likely mediated by corticosterone. Taken together, the data implicate both corticosterone and stress as playing an inhibitory role on the behavioural efficacy of risperidone. The current i i i findings may help to explain the high incidence of psychotic patients who are non-responsive to their medications and demonstrate elevated levels of stress-related hormones. T A B L E OF C O N T E N T S Page Abstract ii Table of Contents iv List of Tables v List of Figures vi Acknowledgements vii INTRODUCTION 1 METHOD 7 Experiment 1 Effects of Chronic Risperidone and Corticosterone 1.1 Method 9 1.2 Results and Discussion 10 Experiment 2 Effects of Chronic Risperidone and Stress 2.1 Method 12 2.2 Results and Discussion 14 Experiment 3 Effects of Chronic Metyrapone, Risperidone and Stress 3.1 Method 16 3.2 Results and Discussion 18 G E N E R A L DISCUSSION 20 References 31 V LIST OF T A B L E S Page Table 1 39 Effects of Chronic Corticosterone and Chronic Risperidone on WDS and Sexual Behaviour (Mean ± SEM) in the Male Rat. Table 2 40 Weekly Effects of Chronic Stress and Chronic Risperidone on WDS and Sexual Behaviour (Mean ± SEM) in the Male Rat. Table 3 42 Weekly Effects of Chronic Metyrapone, Chronic Stress and Chronic Risperidone on WDS and Sexual Behaviour (Mean ± SEM) in the Male Rat vi LIST OF FIGURES Figure 1 44 Weekly Effects of Chronic Mild Stress and Chronic Risperidone on WDS Frequency (Means ± SEM) in the Male Rat. Treatment groups are represented as follows: control, STRESS (saline + stress), RIS (risperidone + no stress) and RIS-STR (risperidone + stress). Figure 2 45 Weekly Effects of Chronic Mild Stress and Chronic Risperidone on Ejaculation Latency (Means ± SEM) in the Male Rat. Treatment groups are represented as follows: control, STRESS (saline + stress), RIS (risperidone + no stress) and RIS-STR (risperidone + stress). Figure 3 46 Weekly Effects of Chronic Metyrapone, Stress and Risperidone treatments on WDS Frequency (Means ± SEM) in the Male Rat. Treatment groups are represented as follows: control, STRESS (saline + stress), RIS (risperidone + no stress) and RIS-STR (risperidone + stress). V l l A C K N O W L E D G E M E N T S The current document reflects the valuable contributions of my research supervisor, Dr. Boris Gorzalka. Throughout my years in the Master's program, his demonstrated patience and guidance helped facilitate my growth as both a researcher and graduate student. I am grateful for his wisdom and only hope to achieve a similar passion for scientific knowledge. As I progress in my research career, I will continue to uphold the principles of empirical and ethical excellence he taught and strive to contribute to the growing scientific community. In addition, the efforts of my undergraduate research team are duly noted. The successful completion of the current series of experiments required the assistance of the following people: Matt Hil l , Janna Kost, Amelia Wan, Damian Chan, Mark McConkey, Brandon Hiang, Jon Braunstein and Zameel Dewji. Research is a demanding process; the number of tireless hours contributed and the demonstrated efficiency of the team proved to be invaluable. The inhibitory influence of 1 I N T R O D U C T I O N The emergence of atypical antipsychotic medications marked a shift in therapeutic focus from the dopaminergic to the serotonergic (5-HT) neurotransmitter system; agents acting on the 5-HT system have demonstrated improved efficacy in treating patients with psychotic disorders (Breier, 1995; Meltzer, 1999). Within the 5-HT system, numerous receptor subtypes exist (for review see Murphy et al., 1998). Among the various 5-HT receptor subtypes, antipsychotic medications that influence 5-HT2A receptors have been the most promising (e.g., Kuoppamaki, Palvimaki, Hietala, & Syvalahti, 1995; Schreiber, Brocco, Millan, 1994). More specifically, 5-HT2A receptor antagonism has been associated with a greater reduction in positive (e.g., hallucinations and delusional thinking) and negative (e.g., anergy, apathy, and social withdrawal) psychotic symptoms, a lower incidence of extrapyramidal side effects (EPS) and improved cognitive functioning (Breier, 1995; Chouinard et al., 1993; Schmidt, Sorenson, Kehne, Carr & Palfreyman, 1995). On the forefront of therapeutic efficacy is the atypical antipsychotic risperidone (Megen et al., 1994; Song, 1997). Randomized, placebo-controlled, double-blind studies have indicated the superiority of risperidone to conventional antipsychotic medications (e.g., Chouinard et al., 1993; Claus et al., 1992). When compared to other atypical antipsychotics (e.g., clozapine), risperidone has demonstrated a greater tolerability and lower side effect profile (Heinrich, Klieser, Lehmann & Kinzler, 1991). Risperidone primarily acts as a 5-HT2A receptor antagonist and has higher affinity for 5-HT2A receptors than any other clinically approved antipsychotic medication (Breier, 1995; Canton, Verriele & Millan, 1994; Megens et al., 1994). Although, at higher doses, risperidone displays D2 receptor antagonism, its clinical efficacy has been attributed to 5-HT2A receptor blockade (Megens et al., 1994; Meltzer, 1999). For example, doses The inhibitory influence of 2 higher than 10 mg/day are associated with an increase in EPS that are characteristic of D2 receptor antagonism (Breier, 1995). At the recommended dosages, risperidone (4 mg/day) and other 5-HT2A receptor selective antipsychotics produce fewer EPS than haloperidol, a conventional D 2 receptor antipsychotic (Megens et al., 1994; Meltzer, 1999). Moreover, PET studies of dopamine receptor occupancy have suggested that high 5-HT2A receptor occupancy (relative to D2 receptor occupancy) is necessary to avoid EPS with these agents (Meltzer, 1999). Research with animals has also indicated the predominance of 5-HT2A receptor antagonism by risperidone, at low doses. In the rat, ex vivo autoradiography studies have demonstrated that doses between 0.037 and 0.12 mg/kg induce 50% receptor occupancy in the frontal cortex and maximal change in 5-HT2A receptor-mediated behaviours (Schotte, Bonaventure, Janssen, & Leysen, 1995; Schotte, Bruyckere, Janssen, & Leysen, 1989). In contrast, behavioural changes associated with D2 receptor antagonism and 50% D2 receptor occupancy do not occur until doses of 0.75 mg/kg to 2.5 mg/kg (Schotte et al., 1995; Schotte et al., 1989). Unlike other atypical antipsychotics, the relatively slow shift from 5-HT2A receptor occupancy to D2 receptors allows for the study of risperidone's influence on 5-HT2A receptors (and its associated behaviours) in isolation. Recent advances in behavioural pharmacology have helped delineate the complexities of the 5-HT neurotransmitter system and the different behaviours influenced by its specific receptor subtypes. Unique to the 5-HT2A receptor is its affect on a 5-HT behavioural stereotypy known as wet dog shakes (WDS). WDS can be described as a reflexive shudder of the head, neck and trunk and can be induced pharmacologically with 5 - H T 2 A receptor agonists in both rats and mice (Goodwin, Green, Johnson, 1984; Pranzatelli, 1990). For example, central administration of the 5-HT2A receptor agonist (+) 1- (2,5 dimethyl-4-iodophenyl)-2-aminopropane (DOI) into the The inhibitory influence of 3 ventromedial brainstem (Watson & Gorzalka, 1992) or the prefrontal cortex (Willins & Meltzer, 1997) of male rats produces a dose-dependent increase in WDS. Moreover, risperidone and other 5-HT2A receptor antagonists inhibit 5 - H T 2 A receptor agonist-induced WDS in a dose dependent manner (Barwick, Jones, Richter, Hicks & Young, 2000; Janssen et al., 1988; Pranzatelli, 1990; Wettstein, Host & Hitchcock, 1999). Administration of agonists and antagonists specific to other 5-HT receptor subtypes do not produce such an effect (Pranzatelli, 1990). Measurement of WDS frequency has gained acceptance as a non-invasive behavioural assay of central 5 - H T 2 A receptor activity (Eison, Freeman, Guss & Mullins, 1995; Gorzalka & Hanson, 1998; Kuroda, Mikuni, Ogawa & Takahashi, 1992, Watson & Gorzalka, 1990; 1992; Yap & Taylor, 1983). Also influenced by the 5-HT2A receptor is male rat sexual behaviour (e.g., Foreman, Hall & Love, 1989). Systemic administration of selective 5-HT2A receptor antagonists can facilitate male rat sexual behavior (Abraham, Viesca, Plaza & Marin, 1988). Similarly, both chronic and acute administration of risperidone, at doses relatively selective to the 5-HT2A receptor, induce an increase in male rat sexual activity (Drago et al., 1997; Genazzani, Mauceri, Valerio, Nardo & Drago, 1990). Consistent with an increase in 5-HT2A receptor activity, central administration of DOI to male rats produces a dose-dependent inhibition of sexual behaviour; 5-HT2A receptor antagonists can reverse this DOI-induced sexual inhibition (Watson & Gorzalka, 1991). Taken together, the data suggest that 5-HT2A receptor activation mediates an inhibitory influence on male rat sexual behavior. Thus, studies have implicated both the inhibition of male copulatory responses and the facilitation of WDS with increased 5 - H T 2 A receptor activity. Further research has revealed a direct, negative relationship between these two behaviors in male rats: decreases in spontaneous The inhibitory influence of 4 WDS are significantly correlated with increases in sexual behavior (Watson & Gorzalka, 1990). Additionally, the selective 5-HT2A receptor agonist, DOI, has been shown to magnify this inverse relationship (Watson & Gorzalka, 1990). The observed inverse correlation between WDS and copulatory proficiency can be attributed to overlapping neural substrates in the ventromedial brainstem (Watson & Gorzalka, 1992), suggesting that the two behaviors are neurally dependent. An intimate, interactive relationship between hormones of the hypothalamic-pituitary adrenocortical (HPA) axis and the 5-HT neurotransmitter system has been demonstrated (for review see Chaouloff, 1995). The HPA axis becomes activated during times of stress and releases the adrenocortical steroid corticosterone. Recent studies have implicated corticosterone as playing a fundamental role in the regulation of 5-HT2A receptor density (e.g., Kuroda, Mikuni, Ogawa & Takahashi, 1992; McKittrick, Blanchard, Blanchard, McEwen & Sakai, 1995). More specifically, it has been demonstrated, using radioligand binding procedures, that chronic administration of corticosterone (at levels that mimic those seen during times of stress) can induce an increase in 5-HT2A receptor density in the rat brain, which is a direct indication of an increase in 5-HT2A receptor activity (e.g, Fernandes, McKittrick, File & McEwen, 1997; Kuroda, et. al„ 1992). At a behavioural level, chronic corticosterone treatment (20 mg/kg or 50 mg/kg) induces a decrease in male rat sexual behaviour and concurrent increase in WDS (Gorzalka & Hanson, 1998). Administration of the antidepressant nefazadone, which possesses 5-HT2A receptor antagonistic properties, can attenuate the effects of corticosterone on rat sexual behaviour and WDS (Hanson, Gorzalka & Brotto, 1998). Thus, the behavioural data offer further evidence for a corticosterone-induced increase in 5-HT2A receptor activity. The effects of corticosterone on sexual behaviour and WDS may be attributed to its influence on 5-HT2A receptor density. The inhibitory influence of 5 The general effects of stress on 5-HT2A receptor activity have also been examined. Studies employing various chronic stress paradigms have demonstrated significant increases in serum corticosterone levels and increases in 5-HT2A receptors in the cerebral cortex; changes in 5-HT2A receptor density were proportional to the degree of HPA-axis activation and corticosterone secretion (e.g., McKittrick et al., 1995). Consistent with these findings, chronic stress has also been found to significantly increase WDS frequency (e.g., Gorzalka, Hanson & Brotto, 1998; Takao et al., 1995) and decrease male rat sexual behaviour (e.g., Brotto, Gorzalka & Hanson, 1998; Retana-Marquez, Salazar & Velazquez-Moctezuma, 1996). In sum, the demonstrated influence of corticosterone on 5-HT2A receptor activity can be extended to general HPA axis activation (via chronic stress procedures). It is unclear, however, i f the behavioural influences observed are actually being mediated by the same pathway. Although changes in WDS behaviour appear to be influenced by a similar pathway, there is recent indication that the effects of stress on sexual behaviour may not be mediated by the same mechanism as corticosterone (Gorzalka, Hanson & Hong, manuscript in preparation). The apparent contradiction in the findings may be explained by the relative vulnerability of rat sexual behaviour to other (unspecified) factors, but further investigation is required. The apparent influence of corticosterone on 5-HT2A receptor activity holds important implications for the effectiveness of risperidone as both a 5-HT2A receptor antagonist and an antipsychotic agent. Although it has been shown that 5-HT 2 A receptor antagonists can attenuate the 5-HT2A receptor-mediated behavioural effects of corticosterone (e.g., Hanson et al., 1998), the possibility that corticosterone may impede the efficacy of a 5-HT2A receptor antagonist alone has not been explored. There is recent indication that DOI pre-treatment can interfere with the behavioural effects of risperidone in the paw test, an established animal model of antipsychotic The inhibitory influence of 6 efficacy (Ellenbroek, Prinssen & Cools, 1994). In the same way, corticosterone-induced changes in 5 - H T 2 A receptor density may also have an inhibitory influence. Within the human literature, links between psychotic disorders and HPA axis dysfunction have been made (e.g., Gispen-de Wied, 2000; Jansen, Gispen-de Wied, Gademan, Jonge, Linden, & Kahn, 1998; Pivac, Muck-Seler & Jakovljevic, 1997). It has been suggested that subsets of the psychotic patient population suffer from elevated levels of Cortisol levels and that a high proportion of stressful life events predicts psychotic relapse (Gispen-de Wied, 2000; Jansen, Gispen-de Wied, Kahn, 2000). Given that both risperidone and HPA axis hormones influence the same receptor in opposing directions, investigation of the potential involvement of the HPA axis in affecting the pharmacological efficacy of risperidone is both scientifically and clinically relevant. The present series of experiments was designed to satisfy the following three research objectives: 1) Research has indicated that chronic risperidone treatment, at a dose of 0.1 mg/kg or less, can effectively increase sexual behaviour (Drago et al., 1997) and block WDS frequency (Wettstein et al., 1999). By contrast, chronic corticosterone treatment (at doses that mimic levels seen during times of stress) induces an increase in 5-HT2A receptor activity, as demonstrated by a concurrent inhibition of male rat sexual behaviour and facilitation of WDS (e.g, Gorzalka & Hanson, 1998; Gorzalka, Brotto & Hong, 1999). Experiment 1 was designed to examine the possible inhibitory influence of corticosterone on the 5-H T 2 A receptor-mediated behavioural effects of chronic risperidone treatment. 2) Evidence has also implicated chronic stress (and subsequent HPA axis activation) as having similar influence as corticosterone on 5-HT2A receptor-mediated behaviours and The inhibitory influence of 7 5-HT 2 A receptor density (e.g., McKittrick et al., 1995; Gorzalka & Hanson, 1998). To the extent that stress mimics the 5-HT 2 A receptor-mediated behavioural influence of corticosterone, Experiment 2 investigated the potential interference of stress on chronic risperidone treatment. 3) Although the 5-HT 2 A receptor-mediated behavioural effects of chronic corticosterone and chronic stress treatments are similar, there is recent indication that these treatments may represent different mechanisms of influence on male rat sexual behaviour (Gorzalka et al., manuscript in preparation). To the extent that the behavioural results from objective 1 and 2 are similar, Experiment 3 incorporated the corticosterone synthesis inhibitor metyrapone into the chronic stress-risperidone paradigm. Findings from this study will help elucidate the potential role of corticosterone in the behavioural influence of stress on the pharmacological efficacy of risperidone. M E T H O D Animals Animals were housed in groups of three or four in the standard triple wire mesh cages and were allowed free access to both Purina Rat Chow and water. The colony was maintained at a temperature of 21 ± 1°C and on a reverse 12/12 hour light/dark cycle (lights off at 0900h). Subjects were screened for copulatory proficiency; rats were required to ejaculate (at least once) within a 25 minute screening session. A maximum of 3 screening sessions was allowed. Sexually-experienced female Wistar rats were employed as sexual behaviour stimuli. Females were bilaterally ovariectomized, at 3 months of age, while under a combination of xylazine (8 mg/kg) and ketamine (95 mg/kg) anesthesia. Females weighed approximately 500-600 g and were 8-14 months of age when used. The inhibitory influence of 8 Drugs Doses of estradiol benzoate (EB) and progesterone (P) (Sigma Chemical Company, St. Louis, MO) were dissolved in 0.1 ml peanut oil. Corticosterone (Sigma Chemical Co., St. Louis, MO), dissolved in propylene glycol at dose of 20 mg/kg. Risperidone (Sigma Chemical Co., St. Louis, MO), dissolved in 0.9% saline at a dose of 0.1 mg/kg. (+) 1- (2,5 dimethyl-4-iodophenyl)-2-aminopropane (DOI; Sigma Chemical Co., St. Louis, MO), dissolved in 0.9% saline at a dose of 0.5 mg/kg. Metyrapone (Sigma Chemical Company, St. Louis, MO) was dissolved in propylene glycol at a dose of 50mg/kg. Apparatus A l l animals were injected in plastic maternity bins with 26 gauge and one-half inch stainless steel needles. Rats were tested in cubical (30 x 30 x 45 cm) or cylindrical (30cm in diameter x 45cm in height) Plex-i-glas chambers; all chamber floors were lined with contact bedding. Latencies were timed with a stopwatch and behavioural measures were recorded on standard score sheets by trained observers who were blind to all treatment conditions. Procedure Prior to testing, females were injected with EB (10 ug) and P (500 ug) to induce sexual receptivity. EB was injected (sc) 44-48 hours prior to testing, while P was injected (sc) 3-4 hours prior to testing; 0.1 ml of each solution was administered. Injections and behavioural testing were performed during the middle third of the dark The inhibitory influence of 9 cycle. Subjects were allowed to habituate individually to a testing chamber for 5 minutes before testing began. Test sessions were 30 minutes in length and commenced upon the introduction of a stimulus female. Females were interchanged among the subjects every 10 minutes to maintain sexual interest. The following sexual behaviour parameters were scored: 1) frequency of mounts with pelvic thrusting prior to ejaculation; 2) frequency of intromissions prior to ejaculation; 3) frequency of ejaculations; 4) mount latency, the time between the start of the test session and the first mount; 5) intromission latency, the time between the start of the test session and the first intromission; 6) ejaculation latency, the time between the first intromission and first ejaculation; 7) post-ejaculatory interval, the time between the first ejaculation and the first intromission of the next copulatory bout. The frequency of WDS was concomitantly recorded throughout the test session. Male rats that failed to ejaculate during the test session were dropped from the analyses of mounts and intromission frequencies and latency scores were set to the maximum of 1800 seconds. Rats that failed to intromit after the first ejaculation were dropped from the analyses of the post-ejaculatory interval. EXPERIMENT 1 Effects of Chronic Risperidone and Corticosterone The first experiment was designed to examine the behavioural influence of chronic corticosterone treatment on chronic risperidone administration. Both corticosterone and risperidone treatments, alone, have been shown to influence 5-HT2A receptor activity and 5-HT2A receptor-mediated behaviours (Drago et al., 1997; Gorzalka & Hanson, 1998; Kuroda et al., 1992). Given that chronic corticosterone treatment induces an increase in 5-HT2A receptor The inhibitory influence of 10 density/activity (Kuroda et al., 1992), the potential inhibitory role of corticosterone on risperidone's ability to antagonize the 5-HT2A receptor was investigated. WDS frequency and male rat sexual behaviour, both 5-HT2A receptor-mediated behaviours, were concomitantly scored to measure changes in 5-HT2A receptor activity. Animals Long-Evans male rats (n=20; Charles River Canada Inc., Montreal) were approximately 8 months old and between 600-800 g at the time of testing. Procedure Subjects were randomly divided into two equal groups (n = 10) and were administered (sc) either risperidone (RIS; 1 ml/kg) or saline once daily for a period of 10 days. On the 11 t h day, 30 minutes prior to testing, rats were administered DOI (1 ml/kg). Rats were tested on measures of sexual behaviour and WDS (TEST 1). Two weeks following the test session, rats (n= 20) were re-assigned to the same treatment groups. In addition to the original treatment regimen, rats were administered corticosterone (CORT; 1 ml/kg). Rats, again, received daily injections (sc) for a duration of 10 days and were administered DOI (1 ml/kg) 30 minutes prior to testing. Rats were re-tested on measures of sexual behaviour and WDS (TEST 2). Data were analyzed for main and interaction effects using a repeated-measures Analysis of Variance (ANOVA); significance level was set at p. < 0.05. Results and Discussion Data for WDS and sexual behaviour are presented in Table 1 (TEST 1) and Table 2 (TEST 2). The inhibitory influence of 11 A significant main effect of CORT was indicated for measures of mount latency (F(l,18) = 7.05, E < 0.016), intromission latency (F(l,18) = 8.03, p_ < 0.011), ejaculation latency (F(l,18) = 10.63, 2 < 0.004), post-ejaculatory interval (F(l,14) = 9.48, p_ < 0.008), ejaculation frequency (F(l,18) = 15.78, rj < 0.001), and WDS frequency (F(l,18) = 19.59, p < 0.0001). Changes were indicative of an increase in 5-HT2A receptor activity. The observed main effect of RJS suggested that chronic RIS effectively increased sexual activity and blocked DOI-induced WDS. RIS shortened mount latency (F(l,18) = 7.30, p < 0.015), intromission latency (F(l,18) = 7.83, p < 0.0012), ejaculation latency (F(l,18) = 11.78, p < 0.003), post-ejaculatory interval (F(l,18) = 35.64, p < 0.0001), increased the frequency of ejaculations (F(l,18) = 11.24, p < 0.004) and lowered the frequency of DOI-induced WDS (F(l,18) = 37.38, p < 0.0001). Taken together, the results indicate the effectiveness of chronic RIS treatment in decreasing 5-HT2A receptor activity, as demonstrated by a facilitation of sexual behaviour and inhibition of DOI-induced WDS frequency. An inhibitory influence of CORT on the behavioural effectiveness of RIS was suggested by significant interactions found on measures of sexual behaviour. When compared to RIS rats, rats treated with CORT-RIS showed significantly longer intromission latency (F(l,18) = 4.21, p_ < 0.05) and post-ejaculatory interval (F(l,14) = 9.48, p < 0.008) and lower frequency of ejaculations (F(l,18) = 10.57, p < 0.004). That is, CORT significantly blocked the RIS-induced facilitation of sexual behaviour. In the same way, trends toward significance (p < 0.10) were found on mount latency, ejaculation latency and WDS frequency measures; CORT inhibited the effects of RIS. The current findings support previous findings that indicate chronic corticosterone treatment as increasing 5-HT2A receptor activity, as measured by an inhibition of sexual The inhibitory influence of 12 behaviour and facilitation of WDS expression. The 5-HT2A receptor antagonistic properties of risperidone were also supported. Risperidone induced behavioural changes that suggested a decrease in 5-HT2A receptor activity- an increase in sexual behaviour and a decrease in WDS frequency. Of interest, chronic corticosterone treatment either attenuated or showed a trend to attenuate effects of risperidone on both WDS frequency and male rat sexual behaviour. The results indicate that corticosterone inhibits the 5-HT2A receptor antagonist activity of risperidone. E X P E R I M E N T 2 Effects of Chronic Risperidone and Stress Experiment 2 was designed to investigate the influence of chronic stress on the behavioural effects of a chronic risperidone regimen. Similar to a chronic corticosterone regimen, chronic stress has been shown to increase the frequency of WDS and decrease male rat sexual activity (e.g., Gorzalka, Hanson & Brotto, 1998). Also consistent with an increase 5-HT2A receptor activity, chronic stress can induce an upregulation of 5-HT2A receptors (e.g., McKittrick et al., 1995; Takao et al., 1995). Adapting the chronic mild stress (CMS) procedure outlined in Kopp et al. (1999), the present study examined the possible generalization of the inhibitory role of chronic corticosterone treatment (Experiment 1) to stress. Animals Long-Evans male rats (n=34; Charles River Canada Inc., Montreal) were 6 months old at the time of testing and weighed between 450 and 700g. Apparatus The strobe light/white noise stressor was conducted in a black wooden box (100cm x 100cm x 80cm ht.) and used a portable strobe light and radio; the floor of the apparatus was The inhibitory influence of 13 generously lined with contact bedding. For the tube confinement stressor, 20cm long x 6cm diameter plastic tubes were used. Tubes were open at one end and covered at the other with a plex-I-glas square (5cm x 5cm) with a circular air hole (1 cm dia.) in the center. Plastic maternity bins with single wire mesh lids, lined with damp contact bedding, were used in the wet bedding stressor. Procedure Males were randomly assigned to one of the following four groups: 1) saline and stress (n=7; STRESS); 2) saline and no stress (n=8; NO STRESS); 3) risperidone and stress (n=9; RIS-STRESS); 4) risperidone and no stress (n=10; RIS). Risperidone or saline was injected (sc) at a volume of 1 ml/kg, once daily for a period of 21 days. Rats assigned to one of the two stress conditions were exposed to a 21-day chronic stress schedule. The schedule was adapted from that outlined by Kopp and colleagues (1999) and included the following stressors: 1) tube confinement (lh) in which movement was restricted; 2) food and water deprivation (15h); 3) food restriction (50g/rat; 2h) after food deprivation; 4)empty water bottles after water deprivation (2h); 5)stroboscopic illumination/white noise (30 min.); 6)wet bedding/isolation (200ml water/1 OOg bedding; 18h); 7) cage (mate) rotation (4h); 8) reversal of colony light/dark cycle (48h). Stressors varied daily to minimize habituation. Prior to stress and injection exposure, rats underwent sexual and WDS behaviour testing to provide a baseline measure. Rats were tested on measures of sexual behaviour and WDS frequency every seven days; the last test session occurred on Day 22 of the experiment. On each test day, rats were not exposed to a morning stressor and injections were administered following behavioural testing. The inhibitory influence of 14 Data, collected across the 3 weeks, were analyzed using a repeated-measures A N O V A . Weekly differences were analyzed using a one-way A N O V A ; all parameters that reached significance were further analyzed using the Least Significant Difference (LSD) multiple comparisons test. The significance criterion was maintained at 0.05. Results and Discussion Significant behavioural differences were not found at baseline. Weekly data for all measures are presented in Table 2 and weekly data for WDS frequency and ejaculation latency are presented in Figures 1 and 2, respectively. Repeated measures A N O V A indicated treatment conditions varied significantly by week on mount latency (F(l,30) = 4.95, p < 0.034), intromission latency (F(l,30) = 5.77, p < 0.023) and ejaculation frequency (F(l ,30) = 3.90, p < 0.05) measures. A main effect of treatment condition was found on mount latency (F(3,30) = 6.34, p < 0.002), intromission latency (F(3,30) = 6.34, p < 0.002), ejaculation latency (F(3,30) = 11.57, p < 0.0001), post-ejaculatory interval (F(3,14)= 10.86, p< 0.001), ejaculation frequency (F(3,30) = 11.49, p < 0.0001) and WDS frequency (F(3,30) = 11.51, p < 0.0001) measures. Significant interactions among test weeks and treatment conditions were not found. Using one-way ANOVAs , differences among treatment conditions at each week were analyzed. At week 1, significant differences were found on the following measures: mount latency (F(3,30) = 5.61, p < 0.004), intromission latency (F(3,30) = 5.06,jo < 0.006), ejaculation latency (F(3,30) = 10.03, p < 0.0001), post-ejaculatory interval (F(3,23) = 8.50, p < 0.001), ejaculation frequency (F(3,30) = 5.46, p < 0.004) and WDS frequency (F(3,30) = 3.54, p < 0.026). At week 2, significant differences were found on the following measures: mount latency (F(3,30) = 3.37, p < 0.031), intromission latency (F(3,30) = 3.57, p < 0.026), ejaculation The inhibitory influence of 15 latency (F(3,30) = 9.21, p < 0.0001), post-ejaculatory interval (F(3,25) = 7.98, p_ < 0.001), ejaculation frequency (F(3,30) = 9.07, _p < 0.0001) and WDS frequency (F(3,30) = 5.75, p_ < 0.003). At week 3, significant differences were found on the following measures: mount latency (F(3,30) = 3.19, p. < 0.038), intromission latency (F(3,30) = 3.33, p. < 0.033), ejaculation latency (F(3,30) = 4.83, p < 0.007), post-ejaculatory interval (F(3,22) = 5.30, p_ < 0.007), ejaculation frequency (F(3,30) = 5.98, p_ < 0.003) and WDS frequency (F(3,30) = 11.54, p < 0.0001). At each week, significant differences on measures of mount and intromission frequencies were not found at (p_ > 0.05). Using the LSD multiple comparisons test, significant differences between treatment conditions were explored, at each week. CMS significantly decreased sexual activity from weeks 1-3; these effects strengthened with increased CMS exposure. At week 1, STRESS rats, when compared with NO STRESS rats, demonstrated increased ejaculation latency (p. < 0.01) and post ejaculatory interval (p_<0.03). At week 2, the increase in ejaculation latency (p_ < 0.003) and post-ejaculatory interval (p_ < 0.006) was maintained. At week 3, in addition to the increase the ejaculation latency (p_ < 0.008) and post-ejaculatory interval (p < 0.02), the frequency of ejaculations decreased (p < 0.03). CMS also effectively increased WDS frequency at week 1 (p_ < 0.03), week 2 (rj < 0.01) and week 3 (p_< 0.001). Chronic administration of risperidone, when compared to control, facilitated copulatory behaviour, but only reached significance at weeks 2 and 3. More specifically, risperidone significantly shortened the latency to ejaculate at both week 2 (p < 0.02) and week 3 (p < 0.002). Although significant differences in WDS behaviour between RIS rats and NO STRESS rats were The inhibitory influence of 16 not revealed, RIS rats demonstrated significantly lower WDS frequency when compared with STRESS rats at week 1 (p < 0.005), week 2 (p < 0.010) and week 3 (p < 0.0001). Significant differences were not found among rats exposed to CMS; risperidone did not attenuate the effects of CMS on sexual behaviour and WDS frequency (p> 0.05). Comparison of RIS rats to RIS-STRESS rats revealed significant differences on parameters of sexual behaviour and WDS frequency. At week 1, RIS-STRESS animals showed significantly higher mount latencies (p < 0.001), intromission latencies (p < 0.002), ejaculation latencies ( p < 0.0001), post-ejaculatory intervals (p < 0.0001) and lower ejaculation frequencies (p < 0.001) . At week 2, RIS-STRESS significantly increased intromission latency (p < 0.004), ejaculation latency (p < 0.0001), post-ejaculatory interval (p < 0.0001) and decreased ejaculation frequency (p < 0.0001). In addition, RIS-STRESS significantly increased WDS frequency (p< 0.004). At week 3, significantly longer post-ejaculatory intervals (p < 0.01) and ejaculation latencies (p < 0.03), lower ejaculation frequencies (g < 0.005) and higher WDS frequencies (p < 0.0001) were found in RIS-STRESS rats. Taken together, stress significantly attenuated the facilitatory effects of risperidone on copulatory behaviour at weeks 1,2 and 3 and blocked risperidone's inhibitory effect on WDS frequency at weeks 2 and 3. The present findings are consistent with results found in Experiment 1. Similar to chronic corticosterone administration, CMS exposure significantly inhibited sexual behaviour and facilitated WDS expression. Risperidone effectively increased sexual activity and decreased WDS expression, which is consistent with its properties as a 5-HT2A receptor antagonist. CMS exposure also mimicked the inhibitory effects of corticosterone on risperidone; stress significantly attenuated the risperidone-induced changes in 5-HT2A receptor-mediated behaviours. The inhibitory influence of 17 EXPERIMENT 3 Effects of Chronic Metyrapone, Risperidone and Stress Results from Experiment 1 and 2 suggest that both chronic corticosterone and stress treatments act to inhibit the efficacy of risperidone as a 5 - H T 2 A receptor antagonist. Experiment 3 was designed to investigate the possibility that the behavioural effects observed in both Experiments 1 and 2 can be attributed to corticosterone. The current study incorporated the corticosterone synthesis inhibitor metyrapone into a 14-day CMS-risperidone paradigm. Animals Long-Evans male rats (n = 39; Charles River Canada Inc., Montreal) were approximately 8-14 months in age and weighed between 515-800 g at the time of testing. Apparatus Stressors used are outlined in Experiment 2. Procedure Male rats were randomly assigned to one of four treatment groups: 1) saline (1 ml/kg), metyrapone (2 ml/kg) and stress (n = 10; STRESS); 2) saline (lml/kg), metyrapone (2ml/kg) and no stress (n=10; NO STRESS); 3) risperidone (1 ml/kg), metyrapone (2 ml/kg) and stress (n=9; RIS-STRESS); 4) risperidone (1 ml/kg), metyrapone (2 ml/kg) and no stress (n=10; RIS). Injections (sc) were given once daily for a period of 14 days and behavioural testing occurred 24 hours after the last injection. A baseline test was performed one week prior to the first test day. The last test session occurred on day 15 of the experiment. The stress schedule from Experiment 2 was implemented, with the exception of using only the first 14 days of the 21-day schedule. On each test day, rats were not exposed to a morning stressor and injections were administered following behavioural testing. The inhibitory influence of 18 Data, collected across the 2 weeks, were analyzed using a repeated-measures A N O V A . Weekly differences were analyzed using a one-way A N O V A ; all parameters that reached significance were further analyzed using the Least Significant Difference (LSD) multiple comparisons test. The significance criterion was maintained at 0.05. Results and Discussion At baseline, significant differences among treatment groups were not observed. Weekly data for all measures are presented in Table 3 and weekly WDS frequency data are presented in Figure 3. A repeated-measures A N O V A indicated a significant main effect of time. Changes among treatment conditions varied significantly by week on the following measures: mount latency (F(l, 35) = 42.04, p < 0.0001), intromission latency (F(l, 35) = 46.22, p < 0.0001), ejaculation latency (F(l, 35) = 36.40, p < 0.0001), post-ejaculatory interval (F(l, 26) = 35.63, p < 0.0001), ejaculation frequency (F(l, 35) = 47.47, p < 0.0001) and WDS frequency (F(l, 35) = 8.56, p < 0.006). The following measures indicated a significant main effect of treatment condition: mount latency (F(3, 35) = 7.45, p < 0.001), intromission latency (F(3, 35) = 6.92, p < 0.001), ejaculation latency (F(3, 35) = 3.01, p < 0.05), post-ejaculatory interval (F(3, 26) = 4.70, p < 0.009), ejaculation frequency (F(3, 35) = 3.93, p < 0.016) and WDS frequency (F(3, 35) = 2.93, p < 0.05). Significant interaction effects were not found. At week 1, as revealed by the one-way A N O V A , the effects of metyrapone were not apparent and results replicated those found in Experiment 2. Significant differences were found on the following measures: mount latency (F(3, 35) = 4.36, p < 0.01), intromission latency (F(3, 35) = 5.01, p < 0.005), post-ejaculatory interval (F(3, 31) = 3.21, p < 0.04), ejaculation frequency (F(3, 35) = 3.50, p < 0.03) and WDS frequency (F(3,35)=5.31, p < 0.004). The inhibitory influence of 19 The LSD multiple comparisons test indicated that STRESS significantly inhibited sexual behaviour; STRESS increased mount latency (p_ < 0.005), intromission latency (p. < 0.002), ejaculation latency (p < 0.04), and the post-ejaculatory interval (p_ < 0.01) and decreased in ejaculation frequency (p < 0.01). Significant differences between STRESS and STRESS-RIS were not found. STRESS-RIS significantly increased mount latency (p < 0.006), intromission latency (p_ < 0.002), ejaculation latency (p < 0.04), the post-ejaculatory interval (p < 0.01) and WDS expression (p_ < 0.003) and decreased ejaculation frequency (p < 0.01). RIS decreased WDS frequency (p < 0.05) and post-ejaculatory interval length (p < 0.05), suggesting an inhibition of WDS and facilitation of sexual behaviour. When compared to RIS, STRESS-RIS significantly increased the number of WDS (p < 0.001) and decreased ejaculation latency (p < 0.05). Thus, metyrapone failed to attenuate the effects of stress on risperidone, as indicated by the increase in WDS frequency and ejaculation latency. At week 2, metyrapone significantly blocked the effects of stress on WDS frequency; significant differences among treatment conditions were not observed (F(3, 35) = 0.854, p > 0.05). But, differences were maintained on mount latency (F(3, 35) = 3.814, p < 0.02), intromission latency (F(3,35) = 3.719, rj < 0.04) and the post-ejaculatory interval (F(3, 29) = 3.567, p < 0.03). More specifically, STRESS continued to increase the length of mount (p < 0.006) and intromission (p < 0.01) latencies and the post-ejaculatory interval (p < 0.008). The present study replicated the STRESS results of Experiment 2, when the effects of metyrapone, a corticosterone synthesis inhibitor, were not apparent (i.e., week 1). The observed decrease in sexual activity and increase in WDS from CMS exposure is consistent with behavioural findings linked to corticosterone-induced increases in 5 - H T 2 A receptor density (e.g., Gorzalka & Hanson, 1998; Kuroda, 1992). Following the second week of metyrapone treatment, The inhibitory influence of 20 metyrapone effectively blocked the stress-induced increase in WDS; significant differences between STRESS-RIS and RIS rats in WDS expression disappeared. Thus, the data suggest that the facilitatory influence of stress on the expression of WDS can be attributed to elevated levels of corticosterone. But, metyrapone treatment only partially blocked the inhibitory effect of CMS on sexual behaviour. The effects of stress on corticosterone levels and sexual behavior do not appear to be directly related. Several other stress-induced hormonal and neural changes may account for the inhibition in male rat sexual behaviour. G E N E R A L DISCUSSION The current series of experiments was designed to investigate and satisfy the following three experimental objectives: First, the potential inhibitory influence of chronic corticosterone treatment on the effectiveness of risperidone as a 5-HT2A receptor antagonist was examined. Recent research has suggested that chronic corticosterone administration induces an upregulation of 5-HT2A receptors and behavioural changes that are indicative of an increase in 5-HT2A receptor activity (Gorzalka & Hanson, 1998; Kuroda et al., 1992). Moreover, pretreatment with DOI can inhibit the efficacy of risperidone in the paw test (Ellenbroek et al., 1994). Given that risperidone acts to decrease 5-HT2A receptor activity, corticosterone-induced changes in 5-HT2A receptor density may also attenuate the pharmacological efficacy of risperidone. In Experiment 1, chronic corticosterone administration inhibited risperidone's ability to influence both WDS and male rat sexual behaviour. More specifically, the addition of corticosterone to chronic risperidone treatment inhibited male rat sexual behaviour and facilitated WDS expression. Taken together, systemic alteration of basal corticosterone levels can affect the behavioural efficacy of The inhibitory influence of 21 risperidone; corticosterone's ability to inhibit the risperidone-induced facilitation of sexual behaviour and inhibition of WDS is likely mediated by an upregulation of 5-HT2A receptors. Second, the effects of a chronic stress paradigm in conjunction with a chronic risperidone regimen were explored. During times of stress the HPA axis becomes activated and releases corticosterone and other adrenocortical hormones. Ottenweller and colleagues (1992) argue that a state of 'chronic stress' is achieved when prolonged stress exposure induces sustained corticosterone elevation. It is not surprising that chronic corticosterone administration (at doses that mimic levels seen during times of chronic stress) produces the same 5-HT2A receptor-mediated behavioural changes as chronic stress exposure (e.g., Gorzalka & Hanson, 1998; Gorzalka et al., 1998). It has been shown that chronic stress induces a concomitant inhibition of sexual behaviour and facilitation of WDS expression (Brotto et al., 1998; Gorzalka et al., 1998). Moreover, 5 - H T 2 A receptor density changes that are characteristic of chronic corticosterone administration have also been found following chronic stress exposure (McKittrick et al., 1995; Takao et al., 1995). To the extent that chronic stress exposure and chronic corticosterone treatment have similar 5-HT2A receptor-mediated behavioural influences, chronic stress exposure may, also, act to inhibit risperidone's pharmacological effects. Consistent with this hypothesis, Experiment 2 demonstrated the effectiveness of CMS in blocking risperidone's ability to facilitate sexual behaviour and inhibit WDS frequency. Third, the possibility that the similar behavioural findings of Experiment 1 and 2 can be attributed to elevated levels of corticosterone was investigated. Following the chronic administration of metyrapone (a corticosterone synthesis inhibitor) the inhibitory effects of chronic stress exposure on risperidone for WDS frequency disappeared. With respect to WDS behaviour, it appears that the effects of stress on risperidone are likely mediated by The inhibitory influence of 22 corticosterone elevation. In contrast, the inhibitory effects of stress on male rat sexual behaviour were only partially blocked by chronic metyrapone treatment. These findings are consistent with recent evidence that suggests the effects of stress on male rat sexual behaviour cannot be completely explained by a corticosterone-mediated mechanism (Gorzalka et al., manuscript in preparation). Evidence for a link between the HP A axis and the 5 - H T 2 A receptor is convincing. Both chronic corticosterone treatment and various chronic stress procedures induce an increase in 5-H T 2 A receptor density in the rat (e.g., Fernandes et al., 1997; Kuroda et al., 1992; McKittrick et al., 1995). Similarly, the serotonergic stereotypy WDS, identified as being unique to the 5 - H T 2 A receptor (e.g., Eison et al., 1995; Watson & Gorzalka, 1990; Yap & Taylor, 1983 ) is facilitated upon either chronic corticosterone treatment or chronic stress exposure (Gorzalka & Hanson; Gorzalka et al., 1998; Takao et al., 1995). Although sexual behaviour is affected by several different 5-HT receptor subtypes, concomitant measurement of male rat sexual behaviour and WDS expression offers a reliable behavioural assay of 5 - H T 2 A receptor activity (Brotto et al., 1998; Gorzalka & Hanson, 1998; Gorzalka et al., 1999; Watson & Gorzalka, 1990). Both chronic corticosterone treatment and chronic stress exposure induce a concurrent inhibition of male rat sexual behaviour and facilitation of WDS (Gorzalka & Hanson, 1998; Gorzalka et al., 1998; Gorzalka et al., 1999). Consistent with previous findings, results from both Experiments 1 and 2 suggested that chronic corticosterone and stress regimens act to increase 5 - H T 2 A receptor activity, as indicated by a significant increases in WDS frequency and decreases in male rat sexual activity. Taken together, the data suggest the HPA axis as playing an important role in expression of 5 - H T 2 A receptor-mediated behaviours. The inhibitory influence of 23 Presumably, the similarity in behavioural findings of both chronic stress and corticosterone regimens suggests corticosterone as mediating these changes through the upregulation of 5-HT2A receptors. Similar to an adrenalectomy, chronic metyrapone treatment effectively blocks the ability of various stressors to induce higher plasma levels of corticosterone and related behavioural changes (e.g., Deroche, Piazza, Casolini, LeMoal & Simon, 1993; Reid, Ho, Tolliver, Wolkowitz & Berger, 1998). Findings from Experiment 3 indicated that the 5-H T 2 A receptor-mediated behavioural effects of stress cannot be entirely attributed to corticosterone elevation; inhibition of corticosterone elevation failed to (completely) block the effects of stress on the risperidone-induced changes in copulatory behaviour. Other animal behaviour models support the complexity of stress and its behavioural and physiological effects. For example, the stress-induced response to amphetamine treatment is only partially blocked by a chronic metyrapone regimen (Reid et al., 1998). More specifically, in stressed animals, metyrapone decreases locomotor sensitization to amphetamine treatment but increases the dopamine-releasing effect of amphetamine (Reid et al., 1998). Despite the reduction in stress-induced behaviours, the augmentation of amphetamine-induced dopamine release indicates the potential for an exacerbation (vs. alleviation) of psychotic symptoms and EPS. In sum, corticosterone appears to play a fundamental role in the expression of particular, but not all, stress-induced responses. To the extent that stress has an inhibitory influence on antipsychotic activity, the contribution of plasma corticosterone elevation remains equivocal. Stress and corticosterone-induced alterations in 5-HT2A receptor density have been shown to be (brain) region specific (McKittrick et al., 1995; Kuroda et al., 1995; Takao et al., 1995). Using the radiolabelled ligand [3-H]ketanserin, chronic corticosterone administration has been shown to induce an increase in 5 - H T 2 A receptor density in the frontal neocortex of the rat The inhibitory influence of 24 (Kuroda et al., 1992). Chronic stress exposure also alters cortical 5-HT2A receptor density (McKittrick et al., 1995; Takao et al., 1995). For example, a forced swim stressor induces upregulation of 5-HT2A receptors in the rat frontal cortex (Takao et al., 1995), while psychosocial stressors are associated with 5 -HT2 A receptor upregulation in the parietal cortex (McKittrick et al., 1995). In addition, there is indication that an adrenalectomy only alters 5-HT2A receptor density in select brain regions. To date, no radioligand binding studies have examined the effects of corticosterone or stress in other brain regions. The standing possibility that corticosterone and stress differentially alter 5-HT2A receptor density in distinct brain regions may help to explain why chronic metyrapone treatment failed to block stress-induced changes in sexual behaviour. It has been suggested that the sexual behaviour effects of stress and corticosteone are mediated by two different mechanisms (Gorzalka et al., manuscript in preparation) and that the corticosterone-induced changes in 5-HT2A receptor density observed following a chronic stress regimen may not be sufficient in altering sexual activity. Across all three studies risperidone, when administered alone, demonstrated an ability to facilitate sexual behaviour and inhibit WDS expression; an effect that was strengthened following a chronic injection regimen. The consistency of these behavioural results offers confidence in risperidone's ability to act as a potent 5-HT 2 A receptor antagonist, at low doses. Studies employing selective 5-HT2A receptor antagonists L Y 281067 and L Y 53857 also demonstrate a facilitation of male rat sexual behaviour (Foreman et al., 1989). The current findings are also in agreement with the existing literature (e.g., Drago et al., 1997; Genazzani et al., 1990; Wettstein et a l , 1999). For example, Drago and colleagues (1997) found that both chronic and acute administration of risperidone facilitated male rat sexual behaviour, but only at doses equivalent to 0.1 mg/kg; higher doses had no effect. Ex-vivo autoradiography studies have The inhibitory influence of 25 shown the selectivity of risperidone to 5-HT2A receptors at low doses and the gradual shift in receptor occupancy to D2 receptors at higher doses (Schotte et al., 1995; Schotte et al., 1989). The fact that significant changes in male rat sexual behaviour are not found at higher doses is consistent with the receptor occupancy profile of risperidone (Drago et al., 1997). Conventional antipsychotics (e.g., haloperidol) primarily act as D2 receptor antagonists and act to inhibit male rat sexual behaviour (Drago et al., 1997). Therefore, at higher doses, the shift to D 2 receptor occupancy by risperidone likely cancels its facilitatory effect on male rat copulatory behaviour (Drago et al., 1997). Taken together, the results from the present series of experiments suggest the facilitation of sexual behaviour (and inhibition of WDS) by risperidone may be explained by its influence on 5-HT2A receptors. Recent evidence supporting the relevance of 5-HT2A receptors in antipsychotic drug action has also been demonstrated through various animal models of psychosis. Administration of phencyclidine (l(l-phenylcylcohexyl)piperidine hydrochloride; PCP), which can induce both positive and negative psychotic symptoms in humans, produces stereotyped behaviours and hyperlocomotion in rats (e.g., Castelli & Adams, 1981; Kitaichi, Yamada, Hasegawa, Furukawa & Nabeshima, 1994). Prevention of these behaviours has been linked to the therapeutic effects of different antipsychotic agents on both psychotic symptoms and EPS (e.g., Kitaichi et al., 1994). Risperidone-induced blockade of PCP-induced stereotyped behaviours has been primarily linked to its activity at the 5-HT2A receptor and presented as supportive evidence for its clinical efficacy in reducing EPS (Kitaichi et al., 1994). Similarly, selective 5-HT2A receptor antagonists have been found to be effective in blocking both amphetamine-induced (Schmidt et al., 1995) and PCP-induced locomotor activity (Gleason & Shannon, 1997) and demonstrating antipsychotic-like activity in the paw test (Ellenbroek et al., 1994). Noda and colleagues (1995) The inhibitory influence of 26 recently demonstrated the ability of clozapine, ritanserin and risperidone (all selective to the 5-H T 2 A receptor) to attenuate the immobility effects of PCP in a forced swim test and the failure of haloperidol to produce such effects. It has been suggested that the PCP- forced swim test can be used as an effective animal model of negative psychotic symptoms (Noda, Yamada, Furukawa & Nabeshima, 1995). In sum, animal behaviour models of both 5-HT2A receptor activity and psychosis implicate the importance of 5-HT2A receptor antagonism in antipsychotic activity. The 5-HT2A receptor antagonistic action of atypical antipsychotics have also been implicated in improving positive and negative symptoms of psychosis and EPS. Similar to conventional neuroleptics, double-blind, placebo-controlled studies have demonstrated the efficacy of risperidone (and other atypical antipsychotics) in reducing positive symptoms of chronic schizophrenia (e.g., Chouinard et al., 1993) One of the difficulties with conventional antipsychotic medications has been its relative ineffectiveness in treating negative psychotic symptoms (e.g., Breier, 1995). For example, chlorpromazine and high doses of loxapine, which produce high levels of D2 receptor blockade in vivo, do not improve ameliorate negative symptoms (Meltzer, 1999). The recent advent of new, atypical antipsychotic has been promising; sertindole, ziprasidone, Ml00907, ritanserin and risperidone have all been found to be effective in treating negative symptoms (Breier, 1995; Megens et al., 1994; Meltzer, 1999). Various types of analyses to partial out the effect on negative symptoms from the effect on positive and depressive symptoms and EPS suggest that the effects of 5-HT2A receptor blockade on negative symptoms are direct (Tollefson & Sanger, 1996). Also unique to 5-HT2A receptor blockade has been the observed reduction in EPS when compared with conventional antipsychotic medications (Meltzer, 1999). For example, clinical trials of risperidone have shown that, at doses less than 6 mg/day, EPS were similar to placebo (Ellis, Cudkowicz, Sexton & Growdon, 2000). Moreover, The inhibitory influence of 27 agents specifically developed for high 5-HT2A (and low D 2 ) receptor antagonist activity produce fewer EPS than haloperidol at comparable doses (for review see Meltzer, 1999). Taken together, the improved efficacy of atypical antipsychotics can, at least partially, be attributed to 5-HT 2 A receptor blockade. Although risperidone, at low doses, acts as a selective antagonist to all 5-HT 2 A receptors, its mechanism of action appears to be highly localized to the frontal cortex (Megens et al., 1994). Using the radiolabeled ligand [3-H]ketanserin, risperidone demonstrates a high affinity for rat frontal cortex 5-HT 2 A receptors and is twice as potent as ritanserin, another highly selective 5 - H T 2 A receptor antagonist, in this area (Megens et al., 1994). Also unique to risperidone is the lack of 5-HT 2 A receptor downregulation associated with a chronic risperidone regimen; chronic treatment of both conventional (i.e., chlorpromazine) and atypical (i.e., amperozide, clozapine and ORG 522) antipsychotics induce a significant decrease in 5-HT 2 A receptor binding sites (Kuppamaki et al., 1995). The results suggest that the 5-HT 2 A receptor antagonist action of risperidone cannot be explained by a downregulation of 5 - H T 2 A receptors (Kuppamaki et al., 1995), and that risperidone may be more vulnerable to the 5 - H T 2 A receptor upregulating effects of corticosterone and stress. Radioligand binding studies have demonstrated that corticosterone and stress-induced changes in 5 - H T 2 A receptor density are specific to the cortical brain regions, particularly the rat frontal cortex (McKittrick et al., 1995; Kuroda et al., 1995; Takao et al., 1995). The implicated overlap of regional specificity of both risperidone and HPA axis hormones may also explain the observed inhibitory influence of stress and corticosterone on risperidone's pharmacological efficacy. Given that both HPA axis activation and risperidone primarily act to modify 5 - H T 2 A receptor activity in the frontal cortex, it is The inhibitory influence o f 28 possible that this regional overlap is acting to enhance risperidone's susceptibility to the influence of HPA axis activation or dysfunction. The current findings may be extended to the human literature. Three major lines of evidence are emerging that implicate the HPA axis in the development of and maintenance of psychotic disorders (Gispen-de Wied, 2000; Jansen et al., 1998; Pivac et al., 1997). First, research has shown that schizophrenic patients are more sensitive to stressful life events and more likely to perceive events as stressful; this has been attributed to the lack of appropriate coping skills (e.g., Jansen et al., 2000; Yank, Bentley, Hargrove, 1993). Conversely, it has been shown that psychotic relapse can be predicted by stressful life events and the number of daily stressors experienced (Gispen-de Wied, 2000). Thus, a vicious circle develops. According to this line of research, psychotic patients are more vulnerable to chronic stress exposure and are more likely to prolong exposure with inadequate coping skills. Second, examination of HPA axis function in psychotic patients has demonstrated elevated levels of Cortisol and a blunted HPA axis response (Pivac et al., 1997; Muck-Seler, Pivac, Jakovljevic & Brzovic, 1999). For example, Muck-Seler and colleagues (1999) found that schizophrenic patients had significantly higher plasma Cortisol levels and higher rates of non-suppression on the dexamethasone suppression test (DST) when compared to healthy subjects. Reports of diminished HPA axis response to various stressors in psychotic patients has also been attributed to hypercortisolemia (e.g., Jansen et al., 2000). In addition, chronic schizophrenic patients who demonstrate non-suppression on the DST show higher levels of plasma Cortisol and score higher for negative symptoms (Kaneko et al., 1992). Elevated Cortisol levels in psychotic patients are still found after controlling for the potential confound of depressive symptoms (Muck-Selar et al., 1999). Deliberate manipulation of Cortisol levels are also associated with The inhibitory influence of 29 schizophrenia-like symptoms. The degree of Cortisol elevation in chronic schizophrenic patients, following a metachlorophenylpiperazine (m-CPP) challenge, has been associated with psychopathology severity; an increase in Cortisol response was related to an increase in psychotic symptoms (Lindenmayer et al., 1997). Administration of exogenous corticosteroids, especially in high doses, can induce symptoms of mania, depression, paranoia, catatonia and delusional thinking (Abramovicz, 1993). Similarly, an excess of circulating gluccorticoids or HPA axis hormones has been associated with loss of receptors in the hippocampus, which is important in learning, emotion and memory (Young, Kwak & Kottak, 1995). Taken together, the data suggest subsets of the psychotic population suffer from HPA axis dysregulation, which has been associated with the degree of symptomology and severity. Third, post-mortem studies of psychiatric and suicidal patients have shown increased 5-H T 2 A receptor density in the brain, particularly in the frontal cortex (Arora & Meltzer, 1989). The observed increase in 5-HT2A receptor density in the frontal cortex is consistent with evidence of corticosterone-induced density changes in the rat frontal cortex and may be explained by the reportedly elevated Cortisol levels in psychotic patients. The current findings would suggest that the observed increase in 5-HT2A receptor density, particularly in the frontal cortex, would somehow impede the therapeutic efficacy of risperidone. Although risperidone offers an improved therapeutic profile, relapse rates of up to 75% within 12 to 18 months after cessation of antipsychotic medications have been reported (Kane, 1999). Moreover, Kane (1999) reported that only 67% of risperidone-treated patients improved significantly and Remington and Kapur (2000) have documented the non-robustness of risperidone on refractory schizophrenic patients. The large number of patients who do not benefit The inhibitory influence of 30 from risperidone may be indicative of a (unidentified) factor that is operating to hinder the effects of the drug. To the extent that the clinical efficacy of risperidone is contingent upon its influence on the 5-HT2A receptor, an increase in 5 - H T 2 A receptor density from H P A axis dysfunction would attenuate the therapeutic benefits of risperidone. Results from the current series of experiments suggest that H P A axis activation can inhibit the pharmacological efficacy of risperidone, via the 5 - H T 2 A receptor. Given the growing evidence for both H P A axis dysfunction and potential lack of therapeutic efficacy of risperidone in subsets of the psychiatric population, the present findings may lend itself as a stepping stone toward elucidating the missing link between these two bodies of research. The inhibitory influence of 31 R E F E R E N C E S Abraham, E., Viesca, P., Plaza, A. & Marin, B. (1988). Modification of sexual activity in male rats following administration of antiserotonergic drugs. Behavioural Brain Research, 30, 251-258. Abramovicz, M . (1993). Drugs that cause psychiatric symptoms. The Medical Letter, 35, 65-70. Arora, R. & Meltzer, H. (1989). Serotonergic measures in the brains of suicide victims: 5-HT2 binding sites in the frontal cortex of suicide victims and control subjects. American Journal of Psychiatry, 730-736 Barwick, V. , Jones, D., Richter, J., Hicks, P. & Young, K. (2000). Subthalamic nucleus microinjections of 5-HT2 receptor antagonists suppress stereotypy in rats. Neuroreport, 11, 267-270. Breier, A . (1995). Serotonin, schizophrenia and antipsychotic drug action. Schizophrenia Research, 14, 187-202. Brotto, L., Gorzalka, B. & Hanson, L. (1998). Effects of housing conditions and 5-HT2A receptor activation on male rat sexual behavior. Physiology & Behavior, 63, 475-479. Canton, H., Verriele, L. & Millan, M . (1994) Competitive antagonism of sertotonin (5-HThc and 5-HT2A receptor-mediated phosphoinositide (PI) turnover by clozapine in the rat: A comparison to other antipsychotics. Neuroscience Letters, 181, 65-68. Castelli, S & Adams, P. (1981). Acute and chronic phencyclidine effects on locomotor activity, stereotypy and ataxia in rats. European Journal of Pharmacology, 73, 263-287. Chaouloff, F. (1995). Regulation of 5-HT receptors by corticosteroids: Where do we stand? The inhibitory influence of 32 Fundamental Clinical Pharmacology, 9, 219-23 3. Deroche, V. , Piazza, P., Casolini, P., LeMoal, M . & Simon, H. (1993). Sensitization to the psychomotor effects of amphetamine and morphine induced by food restriction depend on corticosterone secretion. Brain Research, 611, 352-356. Drago, F., Contarino, A. , Marino, R., Anzallo, C , Valerio, C , Rampello, L., Raffaele, R., & Scapagnini, U . (1997). Effects of acute or chronic administration of risperidone on motor and sexual behavior of male rats. Pharmacological Research, 35, 17-25. Eison, A.S., Freeman, R.P., Guss, V.B. , Mullins, U.L. & Wright, R.N. (1995). Melatonin agonists modulate 5-HT2A receptor-mediated neurotransmission: behavioral and biochemical studies in the rat. Journal of Pharmacology and Experimental Therapeutics, 273, 304-308. Ellenbroek, B., Prinssen, E. & Cools, A. (1994). The role of serotonin receptor subtypes in the behavioural effects of neuroleptic drugs: A paw test study in rats. European Journal of Neuroscience, 6, 1-8. Fernandes, C , McKittrick, C.R., File, S.E., & McEwen, B.S. (1997). Decreased 5-HT 1 A and increased 5-HT2A receptor binding after chronic corticosterone associated with a behavioural indication of depression but not anxiety. Psychoneuroendocrinology, 22, 477-491. Foreman, M . , Hall, J. & Love, R. (1989). The role of the 5-HT2 receptor in the regulation of sexual performance in male rats. Life Sciences, 45, 1263-1270. Genazzani, A. , Mauceri, F., Valerio, C , Nardo, L. & Drago, F. (1990). Effects of risperidone on motor and sexual activity in the rat. The Italian Pharmacological Society, 22, 61-62. Gispen-de Wied, C.C. (2000). Stress in schizophrenia: An integrative view. European Journal of The inhibitory influence of 33 Pharmacology, 405, 375-384. Gleason, S. & Shannon, H. (1997). Blockade of phencyclidine-induced hyperlocomotion by olanzapine, clozapine and serotonin receptor subtype selective antagonists in mice. Psychopharmacology, 129, 79-84. Goodwin, G., Green, A. & Johnson, P. (1984). 5-HT2 receptor characteristics in frontal cortex and 5-HT2 receptor-mediated head twitch behavior following antidepressant treatment to mice. British Journal of Pharmacology, 83, 235-242. Gorzalka, B.B., Brotto, L .A. , & Hong, J.J. (1999). Corticosterone regulation of 5-HT2A receptor-mediated behaviors: Attenuation by melatonin. Physiology & Behavior, 67, 439-442. Gorzalka, B.B. & Hanson, L .A. (1998). Sexual behavior and wet dog shakes in the male rat: regulation by corticosterone. Behavioural Brain Research, 97, 143-151. Gorzalka, B.B., Hanson, L.A. , & Brotto, L .A. (1998). Chronic stress effects on sexual behavior in male and female rats: Mediation by 5-HT2A receptors. Pharmacology, Biochemistry and Behavior, 61, 405-412. Gorzalka, B.B., Hanson, L .A. & Hong, J.J. (in preparation). Sexual and other 5-HT2A receptor-mediated behaviors: Differential effects of stress and corticosterone treatment. Hanson, L.A. , Gorzalka, B.B., & Brotto, L .A. (1998). The antidepressant, nefazodone, attenuates corticosterone-induced increases in 5 - H T 2 A receptor-mediated behaviors in the female rat. European Journal of Pharmacology, 342, 163-165. Jansen, L .M.C. , Gispen-de Wied, C.C., Gademan, P.J., De Jonge, R.C.J. (1998). Blunted Cortisol response to a psychosocial stressor in schizophrenia. Schizophrenia Research, 33, 87-94. Janssen, P., Niemegeers, C , Awouters, F., Schellekens, K., Megens, A. & Meert, T. (1988). The inhibitory influence of 34 Pharmacology of risperidone (R 64 766), a new antipsychotic with serotonin-S2 and dopamine-D2 antagonistic properties. Journal of Pharmacology and Experimental Therapeutics, 244, 685-693. Kane, J .M. (1999). Pharmacologic treatment of schizophrenia. Biological Psychiatry, 46, 1396-1408. Kaneko, M . , Yokoyama, F., Takahagi, K , Murata, S., Watanabe, M . & Kumashiro, H. (1992). Hypothalamic-pituitary-adrenal axis function in chronic schizophrenia: Association with clinical features. Biological Psychiatry, 25, 1-7. Kitaichi, K. , Yamada, K., Hasegawa, T., Furukawa, H. & Nabeshima, T. (1994). Effects of risperidone on phencyclidine-induced behaviors: Comparison with haloperidol and ritanserin. Japanese Journal of Pharmacology, 66, 181-189. Kiraly,S., Ancill, R. & Dimitrova, G. (1997). The relationship of endogenous Cortisol to psychiatric disorder: A review. Canadian Journal of Psychiatry, 42, 415-420. Kopp, C , Vogel, E., Rettori, M-C. & Misslin., R. (1999). The effects of melatonin on the behavioural disturbances induced by chronic mild stress in CjH/He mice. Behavioural Pharmacology, 10, 73-83. Kuoppamaki, M . , Palvimaki, E.P., Hietala, J., & Syvalahti, E. (1995). Differential regulation of rat 5-HT2A and 5-HT2C receptors after chronic treatment with clozapine, chlorpromazine and three putative atypical antipsychotic drugs. Neuropsychopharmacology, 13, 139-150. Kuroda, Y. , Mikuni, M . , Ogawa, T. & Takahashi, K. (1992) Effect of A C T H , adrenalectomy and the combination treatment on the density of 5-HT 2 receptor binding sites in the neocortex of rat forebrain and 5-HT2 receptor-mediated wet-dog shake behaviors. Psychopharmacology, 10, 27-32. The inhibitory influence of 35 Lindenmayer, J., Adityanjee, Vital-Herne, M . , Bark, S. & Moynihan, N . (1997). Heterogeneity of serotonergic response in treatment-refractory schizophrenic patients. Biological Psychiatry, 42, 6-12. McKittrick, C.R., Blanchard, D.C., Blanchard, R.J., McEwen, B.S., & Sakai, R.R. (1995). Serotonin receptor binding in a colony model of chronic social stress. Biological Psychiatry, 37, 383-393. Megens, A.A.H.P, Awouters, F.H.L., Schotte, A. , Meert, T.F., Dugovic, C , Niemegeers, C.J.E., & Leysen, J.E. (1994). Survey on the pharmacodynamics of the new antipsychotic risperidone. Psychopharmacology, 114, 9-23. Meltzer, H (1999). The role of serotonin in antipsychotic drug action. Neuropsychopharmacology, 21, 106-115. Muck-Seler, D., Pivac, N . , Jakovljevic, M . & Brzovic, Z. (1999). Platelet serotonin, plasma Cortisol and dexamethasone suppression test in schizophrenic patients. Biological Psychiatry, 45. 1433-1439. Murphy, D., Andrews, A. , Wichems, H., L i , Q., Tohada, M . & Greenberg, B. (1998). Brain serotonin neurotransmission: an overview and update with an emphasis on serotonin system heterogeneity, multiple receptors, interactions with other neurotransmitter systems, and consequent implications for understanding the actions of serotonergic drugs. Journal of Clinical Psychiatry,59, 4-12. Noda, Y. , Yamada, K., Furukawa, H. & Nabeshima, T. (1995). Enhancement of immobility in a forced swimming test by subacute and repeated treatment with phencyclidine: A new model of schizophrenia. British Journal of Pharmacology, 116, 2531-2537. Ottenweller, J., Servatius, R., Tapp, W., Drastal, S, Bergen, M . & Natelson, B. (1992). A The inhibitory influence of 36 chronic stress state in rats: Effects of repeated stress on basal corticosterone and behavior. Physiology & Behavior, 51,689-698. Pivac, N . , Muck-Seler, D. & Jakovljevic, M . (1997). Platelet 5-HT levels and hypothalamic -pituitary-adrenal axis activity in schizophrenic patients with positive and negative symptoms. Biological Psychiatry, 36, 19-21. Pranzatelli, M . (1990). Evidence for involvement of 5-HT2 and 5-HT ) C receptors in the behavioural effects of the 5-HT agonist l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Neuroscience Letters, 115, 74-80. Reid, M . , Ho, L., Tolliver, B., Wolkowitz, O. & Berger, S. (1998). Partial reversal of stress-induced behavioral sensitization to amphetamine following metyrapone treatment. Brain Research, 783, 133-142. Retana-Marquez, S., Salazar, E. & Velazquez-Moctezema, J. (1996). Effect of acute and chronic stress on masculine sexual behavior in the rat. Psychoneuroendocrinology, 21, 39-50. Remington, G., & Kapur, S. (2000). Atypical antipsychotics: Are some more atypical than others? Psychopharmacology, 148, 3-15. Schmidt, C , Sorenson, S.M., Kehne, J, Carr, A. & Palfreymann, M . (1995). Minireview: The role of 5-HT2A receptors in antipsychotic activity. Life Sciences, 56, 2209-2222. Schotte, A. , Bonaventure, P., Janssen, P.F.M., & Leysen, J.E. (1995). In vitro receptor binding and in vivo receptor occupancy in rat and guinea pig brain: Risperidone compared with antipsychotics hitherto used. Japanese Journal of Pharmacology, 69, 399-412. Schotte, A. , Bruyckere, K. , Janssen, P. & Leyson, J. (1989). Receptor occupancy by ritanserin and risperidone measured using ex vivo autoradiography. Brain Research, 500, 295-301. Schreiber, R., Brocco, M . , Millan, M . (1994). Blockade of the discriminative stimulus effects of The inhibitory influence of 37 DOI by M D L 100,907 and the 'atypical' antipsychotics, clozapine and risperidone. European Journal of Pharmacology, 264, 99-102. Song, F. (1997). Risperidone in the treatment of schizophrenia: A meta-analysis of randomized controlled trials. Journal of Psychopharmacology, 11, 65-71. Takao, K., Nagatani, T., Kitamura, Y. , Kawasaki, K , Hayakawa, H. & Yamawaki, S. (1995). Chronic forced swim stress of rats increases frontal cortical 5-HT2 receptors and the wet dog shakes they mediate, but not frontal cortical /^-adrenoceptors. European Journal of Pharmacology, 294, 721-726. Tollefson, G. & Sanger, T. (1996). Negative symptoms: A path analytic approach to a double-blind, placebo-and-haloperidol-controlled clinical trial with olanzapine. American Journal of Psychiatry, 154, 466-474. Watson, N.V. , & Gorzalka, B.B. (1990). Relation of spontaneous wet dog shakes and copulatory behavior in male rats. Pharmacology Biochemistry & Behavior, 37, 825-829. Watson, N . & Gorzalka, B. (1992) Concurrent wet dog shaking and inhibition of male rat copulation after ventromedial brainstem injection of the 5-HT2 agonist DOI. Neuroscience Letters, 141, 25-29. Wettstein, J., Host, M . & Hitchcock, J. (1999). Selectivity of action of typical and atypical anti-psychotic drugs as antagonists of the behavioral effects of 1- (2,5 dimethyl-4-iodophenyl)-2-aminopropane (DOI). Progress in Neuro-Psychopharmacology & Biological Psychiatry, 23, 533-544. Willins, L. & Meltzer, H. (1997). Direct injection of 5-HT2A receptor agonists into the medial prefrontal cortex produces a head-twitch response in rats. Journal of Pharmacology and Experimental Therapeutics, 282, 699-706. The inhibitory influence of 38 Yank, G., Bentley, K., Hargrove, D. (1993). The vulnerability-stress model of schizophrenia: Advances in psychosocial treatment. American Journal of Orthopsychiatry, 63, 55-69. Yap, C.Y. & Taylor, D.A. (1983) Involvement of 5-HT2 receptors in the wet-dog shake behaviour induced by 5-hydroxytryptophan in the rat. Neuropharmacology, 22, 801-804. Young, E., Kwak, S. & Kottak, J. (1995). Negative feedback regulation following administration of chronic exogenous corticosterone. Journal of Neuroendocrinology, 7, 37-45. o o CD o c o <L> H --W Z O Q a w PH GO HH u z o OS K u Q Z <3 GO GO H GO U z o u fa o GO H u w fa fa fa W fa fa GO -H z H os P o h H a w -P X fa GO Q Z GO Q Z O G O 2 i GO GO H GO GO GO H G O GO 2 w hJ u h-H W > ON O ON i n NO i n CN 0 0 NO CM CN CN +1 -H +1 .—i O ON NO NO ON •f o r-1 — 1 '—1 NO r n •—' O ON 33 ±6 ON -H ON -H CN 33 ±6 +! +! CN CN +i r n CN ,_; o CN ON o NO CN ,—i r-: m m 1 — 1 1 — 1 1 — 1 r n NO NO CN -H o CN o U z w H -H Z P IT) ON ON IT) NO r n +! +! -H i n i n i n r n O ON 1 — 1 CN 1^-m -H O 0 0 CN o o •3-m m -H r n ON O N O o CN N O o o N O 0 0 i n IT) CN CN CN CN -H +1 -H CN rt i n O O N 4i -H -H ON CN i n i n r n N O m »—< CN O N i n ' — 1 m o CN -H o o o CN rn -H 0 0 i n o o ON -H m ON i n CN CN +! 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