Suppression of copulatory behavior in male rats following central administration of clonidine

NeuropharmacologyVol. 30, No. 4, pp. 373-382, 1991 Printed in Great Britain. All rights reserved

0028-3908/91 $3.00+ 0.00 Copyright © 1991 PergamonPress plc

SUPPRESSION OF COPULATORY BEHAVIOR IN MALE RATS FOLLOWING CENTRAL ADMINISTRATION OF CLONIDINE J. T. CLARK Department of Physiology, Meharry Medical College, Nashville, TN 37208, U.S.A. (Accepted 31 August 1990)

Summary--Adrenergic transmitters have been implicated in the regulation of male sexual behavior. In the present study the contribution of ct2-adrenoceptors, located within the central nervous system, was evaluated. Sexually experienced male Long-Evans rats were implanted with a permanent cannula in either the third cerebral ventricle or, unilaterally, in the medial preoptic area. Baseline mating tests were administered 4, 7 and I0 days after surgery and only males ejaculating (at least) in the 7 and 10 day tests were used. Clonidine-evoked dose-dependent decrements in the number of males mounting, intromitting and ejaculating, with administration into the medial preoptic area more effective than into the 3rd ventricle. In those animals mating, administration of 20 nmol into the 3rd ventricle was associated with decreases in the latency to ejaculation and intercopulatory interval, whereas administration of 2 nmol into the medial preoptic area was associated with increases in these parameters and decreases in the ejaculatory threshold. Administration of yohimbine into the medial preoptic area attenuated the effects of systemically-administered clonidine and the systemic administration of yohimbine completely prevented the copulatory suppression induced by administration of clonidine into the medial preoptic area. It is suggested that central ~t2-adrenoceptors are important in the control of male sexual behavior and that alterations in adrenergic mechanisms in the medial preoptic area may underlie sexual dysfunction of various etiologies. Key words--sexual behavior, ejaculation, copulation, medial preoptic area, clonidine, yohimbine, ~2-adrenoceptors, catecholamines.

Central monoaminergic neurotransmitters have long been implicated in the regulation of male sexual behavior (Bitran and Hull, 1987; Kalra, Clark and Kalra, 1988 for reviews). Recent work has led to the suggestion that specific components of male sexual behavior (e.g. arousal, erection, ejaculation) are differentially regulated by monoamines interacting with specific subtypes of binding sites (Clark, Kalra and Kalra, 1987; Clark, Peroutka, Ciaranello, Smith and Davidson, 1985; Clark and Smith, 1986, 1987, 1990; Clark, Smith and Davidson, 1984, 1985a, 1985b; Kwong, Smith, Davidson and Peroutka, 1986; Smith, Lee, Schnur and Davidson, 1987a, 1987b). Work with systemically-administered ~-adrenoceptor agonists clearly supports the suggestion that stimulation of ct2-adrenoceptors is associated with a decrement in sexual behavior. This inhibitory effect appears to be specific and tonic, since it is competitively inhibited by prior treatment with selective ~t:-adrenoceptor antagonists which, on their own, enhance sexual motivation. Although sexual function is dependent on the actions of testosterone, within the central nervous system, administration of an ct2adrenoceptor antagonist, yohimbine, is capable of inducing sexual activity in long-term castrated male rats, indicating that the ct-adrenergic regulation of sexual behavior is downstream from the effects of testosterone (Clark et al., 1985b).

Centrally active antihypertensive agents have become well established as hypotensive agents. Clonidine is a centrally acting hypotensive agent. Although the actions of clonidine are complex, its major actions are believed to be mediated by stimulation of ct2-adrenoceptors (e.g. Hoffman and Lefkowitz, 1980; van Zwieten, Thoolen and Timmermans, 1983). Previous work has demonstrated that systemically-administered clonidine, in a dose-related manner, inhibits sexual behavior in male rats (Clark et al., 1985a). It was subsequently suggested that postsynaptic cq-adrenoceptors, located within the brain, were the primary substrate for this suppression of male sexual behavior (Clark et al,, 1987; Kalra et al., 1988). A well recognized problem in the elucidation of mechanisms in the central nervous system, of systemically-administered drugs, are the effects on peripheral tissues. A better resolution of the neuropharmacological substrates for the adrenergic regulation of male sexual behavior, may be achieved by site-specific injection of drugs interacting with ~2-adrenoceptors. Various hypothalamic and adjacent preoptic areas have been implicated in the regulation of male sexual behavior (see Sachs and Meisel, 1988) and thus, in the present study, the effects of injections of clonidine into the third cerebral ventricle (IIIV; to assess global hypothalamic influences) or the medial preoptic area (mPOA; to assess

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J.T. CLARK

a role for this region) were examined on copulatory behavior in male rats. Subsequently, the effects of pretreatment with yohimbine (an ~2-adrenoceptor antagonist) on cionidine-induced changes in copulatory behavior in the male rat were examined. METHODS

Animals Adult male Long-Evans rats (N = 148; Charles River Laboratories, COBS, Wilmington, Massachusetts), aged 3-6 months on the first day of treatment, were used for these studies. Rats were maintained 2-3 per cage, under controlled light (lights off 1130-2130) and temperature (21-23°C), with food and water always available. Rats were either naive to drugs (N = 66) or were used in an earlier study where they received one intraperitoneal treatment with drug (N = 82; either clonidine or naloxone; Clark and Smith, 1990). At least 3 weeks elapsed between this treatment and stereotaxic surgery. Experimental groups were balanced for prior treatment history. Behavioral screening tests All animals were tested for masculine copulatory behavior prior to the initiation of experiments. Testing was performed in glass aquaria (10 × 20 × 12 inches), in the dark phase of the light:dark cycle (1300-1600), under dim illumination. Stimulus females of the same strain were rendered sexually receptive by subcutaneous injection of 100/zg estradiol benzoate and 500 #g progesterone, 52 and 4-6 hr before testing, respectively. All mounts, intromissions and ejaculations were recorded. Each male was given a maximum of 3 tests per week with a minimum of 3 days between positive (those with ejaculation) tests. Implantation of cannula and injection After three successive tests with ejaculation, pentobarbital-anesthetized rats (42 mg/kg, with supplementation of 7.5mg/kg atropine sulfate) were stereotaxically implanted with a stainless steel cannula (22Ga) in either the 3rd ventricle (coordinates according to DeGroot, 1959: A6.4, L0.0, and 8.0 mm below the dural surface) or the right medial preoptic area (A7.4, L0.65, and 6.5mm below the dural surface). Placement of the cannula within the ventricle was achieved using a previously described procedure (Gallo and Drouva, 1979; Clark, Kalra, Crowley and Kalra, 1984; Clark, Kalra and Kalra, 1985; Clark, 1989). Following placement of the cannula an obturator (26Ga) was inserted. Placement of the cannula within the 3rd ventricle was verified by the efflux of clear cerebrospinal fluid following removal of the obturator on the experimental days. Placement of the cannula within the medial preoptic area was histologically verified (see below). Injections were accomplished in unanesthetized rats by removing the obturator and using an injection cannula (26Ga), which extended 0.75 mm beyond the perma-

nent guide cannula and was connected to a Hamilton microsyringe (25 pl), fitted to a Sage infusion pump by PE20 tubing. The injection cannula was left in place for 1 rain preceding and after the injection. Injection volumes were 3.0/~1 (IIIV) or 0.5gl (mPOA). In an attempt to minimize neuronal damage, only one intracranial injection was administered in each experimental test. Definitive behavior tests Behavioral observation was conducted as above and the occurrence of each mount, intromission and ejaculation was recorded on a computer. Values for the following parameters were obtained from the record: mount (ML) and intromission (IL) latencies, time from introduction of female to the initial mount or intromission; ejaculation latency (EL), time from the initial intromission to ejaculation; postejaculatory interval (PEI), time from ejaculation to the next intromission; mount (MF) and intromission (IF) frequencies, the number of mounts or intromissions preceding ejaculation. Additionally, intercopulatory interval (ICI), was calculated as EL divided by IF and a measure of copulatory efficacy (CE) was calculated as IF divided by (MF + IF). Positive tests were terminated after the postejaculatory intromission. Negative tests were terminated when the intromission latency, postejaculatory interval or the time between successive intromissions exceeded 15 min or when the ejaculation latency exceeded 30 min. Baseline mating tests were administered 4, 7 and 10 days after implantation of the cannula. Only males which ejaculated on (at least) the 7 and 10 day tests were used. Experimental tests were conducted 11 and 14 days after surgery, with each animal serving as its own control. Each animal received only one drug/ combination treatment. Treatment order (drug/ combination or vehicle) was balanced within each treatment. Drugs Clonidine hydrochloride was generously provided by Boehringer-Ingelheim Ltd (Ridgefield, Connecticut) and yohimbine hydrochloride was purchased from Sigma (St Louis, Missouri). All drugs were dissolved in deionized water and diluted to the appropriate concentrations immediately prior to use. For injections into the medial preoptic area, solutions of yohimbine were sonicated. Intraperitoneal (i.p.) injections were in a volume of 0.1ml/100g body weight using doses which have previously been demonstrated to effectively suppress (for clonidine) or enhance (for yohimbine) intromissive and ejaculatory behavior in the male rat (Clark et al., 1984, 1985a, 1985b; Clark and Smith, 1990). Motor behavior Gross behavioral observations of the animals were performed immediately prior to the introduction of the receptive female for mating tests. Motor activity

ct2-Adrenoceptors and sexual behavior was scored as increased ( + 1, +2), normal (0), or decreased ( - 1 , - 2 ) , as recently described by Hillegaart, Ahlenius and Larsson (1989). Further, after the introduction of the female, activity was scored in a similar manner.

Histology After the second experimental test, males with cannulae in the medial preoptic area were given a large dose of pentobarbital (65 mg/kg) and the brains perfused transcardially with normal saline, followed by buffered 10% formalin. Brains were stored in buffered 10% formalin for at least 4 days, frozen on dry ice and mounted in a microtome cryostat. Sections (50 #m thick) were cut, mounted on glass slides and examined using a projection microscope. Only data obtained from animals with histologically verifi-

375

able cannula in the medial preoptic area were included in the analyses.

Experimental protocol (Table 1) To evaluate the effect of stimulation of ~t2-adrenoceptors in the hypothalamus and medial preoptic area, sexually experienced animals (N = 43 with IIIV cannula; N = 42 with cannula aimed at the mPOA) were divided into 6 groups (3 with IIIV, and 3 with mPOA cannula) such that no differences in parameters of baseline copulatory behavior were evident. All animals were given two experimental behavior tests, with 4 days between successive tests. One-half of the rats in each group received an injection of one dose of clonidine (group A: 2 nmol (0.533/~g); group B: 20 nmol; group C: 200 nmol) 5 min prior to mating tests. The remaining males received an injection of

Table 1. Experimental groups and treatments [N]'

Experimental treatments 1

2

Effects of administration of clonidine or vehicle into the third cerebral ventricle: Group A, Group B, Group C,

i ii i ii i ii

[6] [7] [7] [7] [7] [7]

2 nmol clonidine 3/~1 vehicle 20 nmol clonidine 3/~1 vehicle 200 nmol clonidine 3/zl vehicle

3/zl vehicle 2 nmol clonidine 3 #1 vehicle 20 nmol clonidine 3/~1 vehicle 200 nmol clonidine

Effects of unilateral administration of clonidine or vehicle into the medial preoptic area: Group A, i [7] 2 nmol clonidine 0.5/zl vehicle ii [7] 0.5/~1 vehicle 2 nmol clonidine Group B, i [7] 20 nmol clonidine 0.5 #1 vehicle ii [7] 0.5/~1 vehicle 20 nmol clonidine Group C, i [6] 200 nmol clonidine 0.5/~1 vehicle ii [6] 0.5/~1 vehicle 200 nmol clonidine Effects of unilateral injection into the medial preoptic area of yohimbine (12.5nmoO or vehicle (0.5#1), followed by intraperitoneal administration of clonidine (0.25mg/kg) or vehicle (0.1 ml/ lOOg body weight): Group A, i [5] yohimbine vehicle + vehicle + vehicle ii [5] vehicle yohimbine + vehicle + vehicle Group B, i [5] vehicle vehicle + clonidine + vehicle ii [5] vehicle vehicle + vehicle + clonidine Group C, i [5] yohimbine vehicle + clonidine + vehicle ii [5] vehicle yohimbine + vehicle + clonidine Effects of intraperitoneal administration of yohimbine (2mg/kg) or vehicle, followed by unilateral administration of clonidine (20nine0 or vehicle (0.5#0 into the medial preoptic a r e a : Group A, i [6] yohimbine vehicle + vehicle + vehicle ii [5] vehicle yohimbine + vehicle + vehicle Group B, i [6] vehicle vehicle + clonidine + vehicle ii [5] vehicle vehicle + vehicle + clonidine Group C, i [6] yohimbine vehicle + clonidine + vehicle ii [5] vehicle yobimbine + vehicle + clonidine "N = the number of rats with properly placed cannula which were histologically verified. For each group, the subgroups i and ii were combined.

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J.T. CLARK

vehicle (0.5#1). Treatment was reversed for experimental test 2. Next, the effects of blockade of =~2-adrenoceptors in the medial preoptic area was evaluated, using sexually experienced animals with cannula in the medial preoptic area (N = 30). These rats were divided into 3 groups, such that no differences in parameters of baseline copulatory behavior were evident. All animals were given two experimental tests, with 4 days between them. One half of the rats in each group received an experimental treatment; group A: 12.5nmol yohimbine into the mPOA, followed 15min later by vehicle given intraperitoneally (0.1 ml/100g body weight); group B: 0.5#1 vehicle into the medial preoptic area, followed 15 min later by clonidine intraperitoneally (0.25 mg/kg); group C: 12.5 nmol yohimbine into the medial preoptic area, followed 15 rain later by clonidine intraperitoneally (0.25 mg/kg), with the remaining rats all receiving injections of vehicle (0.5#1 into the mPOA and 0.1 ml/100 g body weight). Treatments were reversed for the second experimental test. Mating tests were initiated 5min after the intraperitoneal injection. This dose of yohimbine was chosen, based on solubility. Without sonication, solutions of 2 mg/ml could be typically achieved; with sonication this could be increased to approximately 10 mg/ml. In the present study, a 9.8 mg/ml solution was achieved, such that each 0.5 #1 contained 12.5 nmol. Due to the limited solubility of yohimbine, it was decided to further evaluate the effects of blockade of ~2-adrenoceptors on clonidine-induced suppression of copulatory behavior. To this end, sexually-experienced animals with cannula in the medial preoptic area (N = 33) were divided into 3 groups, such that no differences in parameters of baseline copulatory behavior were evident. All animals were given two experimental tests, with 4 days between them. One half of the rats in each group received an experimental treatment; group A: 2 mg/kg yohimbine intraperitoneally, followed 15 min later by vehicle into the medial preoptic area (0.5#I); group B: 0.1 ml/kg vehicle intraperitoneally, followed 15min later by clonidine into the medial preoptic area (20 nmol); group C: 2mg/kg yohimbine intraperitoneally, followed 15 min later by clonidine into the medial preoptic area (20nmol), with the remaining rats receiving injections of vehicle (0.1ml/100g body weight plus 0.5 #1 into the mPOA). Treatments were reversed for the second experimental test. Mating tests were initiated 5 min after the injection into the medial preoptic area.

Statistical analyses Each rat received two experimental treatments. Therefore, two types of statistical analyses were conducted, within groups to determine the effects of treatments and between groups to compare the treatments. For within-group comparisons of the number

of animals displaying mounts, intromissions and ejaculations, the McNemar Test for Significance of Changes was used. Between-group comparisons for the number of rats exhibiting a specific behavior were done using a Chi-square analysis for 3 independent samples (group A vs B vs C), followed by Fisher exact probability (group A vs B, B vs C and C vs A) tests. Data for the treatment tests are presented as the percentage of rats displaying the behavior. Maximum values of 15min were used for the latency of mount and intromission for those rats failing to initiate copulatory behavior and are presented as mean _+ SEM. For all other measures, data are included only for those rats displaying the behavior. Data for these measures of copulatory behavior are presented as mean _+ SEM, except in those circumstances where fewer than four rats in a group failed to display ejaculatory behavior. In these cases, the arithmetic mean is presented. Values for measures of copulatory behavior within each treatment group were analyzed using Wilcoxon Matched-Pairs Signed-Ranks tests. Comparisons between groups were made using Newman-Keuls (groups A vs B vs C) and Mann-Whitney U-tests (group A vs B, B vs C and A vs C). Motor activity data are presented as mean _+ SEM and were analyzed in a similar way to measures of copulatory behavior.

RESULTS

Effects o f administration o f clonidine into the 3rd ventricle or medial preoptic area on male copulatory behavior Data from 5 rats were excluded for the following reasons: (a) one male with a cannula in the 3rd ventricle and one with a cannula directed at the medial preoptic area, dislodged the cannula assemblies during the course of the study; (b) 2 rats with cannula aimed at the medial preoptic area were outside the area; and (c) one rat with a cannula in the 3rd ventricle was unable to receive the second experimental injection, due to blockage. The remaining rats all mated to ejaculation on the experimental test after injection(s) of vehicle. Motor activity was normal after treatment with vehicle, but was reduced after the largest dose of clonidine (IIIV score of -0.81 _+ 0.07, P < 0.01; mPOA score of - 0 . 4 + 0.05, P > 0.05). In mating tests, initiated 5min after treatment, clonidine evoked dose-dependent decreases in the number of males mounting, intromitting and ejaculating (Fig. 1). Those males mounting and intromitting after administration of clonidine did so with normal latencies. Other parameters of copulatory behavior were affected. However, the effects of injection into the 3rd ventricle vs the medial preoptic area were not always identical. For those males mating to ejaculation after administration of clonidine into the

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Yohimbine (12.5nmol), administered into the medial preoptic area, 20 rain prior to mating tests, was without significant effect on any of the parameters of copulatory behavior. Conversely, in agreement with previous reports (Clark et al., 1985a; 1987; Clark and Smith, 1990), systemic administration of 0.25 mg/kg clonidine was associated with a suppression of male copulatory behavior, demonstrated by a reduced number of males mounting, intromitting and ejaculating (Fig. 3). Unilateral pretreatment with yohimbine into the medial preoptic area tended to attenuate the suppressive effects of systemically administered clonidine, shown by decreases in the

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Fig. 1. Effects of various doses of clonidine, administered into either the third cerebral ventricle (IIIV; 3/zl volume) or unilaterally into the medial preoptic area (mPOA; 0.5/zl volume), on male rat mounting, intromissive and ejaculatory behavior. Treatments were administered to unanesthetized rats 5 min prior to mating tests. All rats mated to ejaculation in tests conducted after administration of saline. Administration of 200 nmol clonidine into the 3rd ventricle was followed by a dramatic reduction in the number of males mounting, intromitting or ejaculating (McNemar test, P < 0.01). In contrast, administration of clonidine into the medial preoptic area evoked a dose-dependent decrease in the number of males exhibiting copulatory behavior (Chisquare, P < 0.01), with 20nmol and 200nmol clonidine significantly decreased the number of males mounting, intromitting and ejaculating (McNemar test, P <0.01). Administration of 200 nmol clonidine into the medial preoptic area had a greater effect than did 20 nmol (Fisher exact, P < 0.01). 3rd ventricle, decreased latencies to ejaculation were observed after 20 and 200 nmol (Fig. 2A) and a decreased intercopulatory interval was evident after the 20 nmol dose (Fig. 2B). In contrast, those males mating to ejaculation after administration into the medial preoptic area exhibited increased ejaculation latencies and increased intercopulatory intervals after 2 or 20 nmol clonidine. Further, 2 nmol clonidine was associated with a decreased postejaculatory interval (Fig. 2C).

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Fig. 2. Effects of various doses of clonidine, administered into the 3rd ventricle (IIIV) or medial preoptic area (mPOA) on the interval from intromission to ejaculation (ejaculation latency), the average interval between successive intromissions (intercopulatory interval) and the ejaculatory threshold (the number of intromissions preceding ejaculation). Data are included only for those rats ejaculating in mating tests. A counterbalanced design was utilized with each rat serving as its own control (*P < 0.05; **P < 0.01, relative to values obtained after treatment with vehicle, using the Wilcoxon matched-pairs, signed-ranks test).

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exerted suppressive effects on male copulatory behavior, shown by a decreased number of males mounting, intromitting and ejaculating (Fig. 6). Pretreatment with yohimbine (2 mg/kg, i.p.) blocked the cionidine-induced reduction in the number of males mounting, intromitting and ejaculating (Fig. 6). Further, pretreatment with yohimbine reversed the effects of clonidine on the latency of intromission (Fig. 7A). Conversely, a reciprocal interaction was suggested, due to clonidine-induced attenuation of the reductions in the latency to ejaculation and intercopulatory interval, seen after yohimbine given alone (Fig. 8).

0 Cl F . , . ~ O N S

Several lines of evidence strongly support a suggested role for adrenergic (utilizing norepinephrine or epinephrine) systems in the brain in the regulation of male sexual behavior. First, it is well documented that the medial preoptic area is richly innervated by adrenergic fibers from perikarya in the

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Fig. 3. Effects of yohimbine (12.5 nmol, 15 min prior to clonidine) administered unilaterally into the medial preoptic area (mPOA) on alterations in copulatory behavior, induced by systemic administration of clonidine (0.25 mg/kg, 5 min prior to mating tests). Treatment with clonidine was associated with significantlyfewer rats mounting, intromitting and ejaculating and yohimbine, injected into the medial preoptic area, attenuated the effects on mounting and intromissive, but not ejaculatory, behavior (*P < 0.05; **P < 0.01 using McNemar tests comparing treatment to control; N = 10 per group). latency to initiate copulatory behavior (Fig. 4). Too few animals ejaculated after systemic administration of clonidine to allow comparison of other parameters (Fig. 5).

Effects o f systemic pretreatment with yohimbine on alterations in copulatory behavior induced by clonidine administered into the medial preoptic area In agreement with previous reports (Clark et al., 1984, 1985a), yohimbine facilitated aspects of copulatory behavior; reduced the latency to ejaculation without a reduction in the number of intromissions preceding ejaculation, resulting in a reduced interval between successive intromissions (Fig. 8). In agreement with the findings in Study 1, unilateral injection of clonidine into the medial preoptic area (20 nmol)

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Fig. 4. Effects of yohimbine (12.5 nmol), administered unilaterally into the medial preoptic area (mPOA) on alterations in intromission latency and postejaculatory interval, induced by systemic administration of clonidine (0.25 mg/ kg). For those rats not intromitting, a maximum of 15 min was used for introanissionlatency. Only data from those rats ejaculating and reinitiating sexual behavior within 15 min of ejaculation are included in (B) (N = 3 for intraperitoneal clonidine + mPOA vehicle group and N = 4 for intraperitoneal clonidine+mPOA yohimbine group; for these groups, the number of animals exhibiting differences in behavior on control and treatment tests precluded statistical analysis and the data are presented as the arithmetic mean; **P < 0.01 by Wilcoxon matched-pairs, signed-ranks test).

a2-Adrenoceptors and sexual behavior A] Ed/CULATIONLATENCY 5.000

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competitively inhibited by pretreatment with the 62adrenoceptor antagonist, yohimbine (Clark et al., 1985a). Treatment with yohimbine alone increased the pacing of copulatory events. Subsequently, it was found that administration of yohimbine [a] increased the percentage of rats ejaculating in their first heterosexual encounter, [b] induced sexual behavior in rats exhibiting idiopathic sexual inactivity, [c] increased mounting behavior in rats after genital desensitization (Clark et al., 1984) and [d] induced sexual behavior in rats that had been castrated 91 days previously (Clark et al., 1985b). Recent reports indicate that other ~2-adrenoceptor antagonists, imoloxan and idazoxan, exert similar effects (Smith et al., 1987a, 1987b). Thus, ct2-adrenoceptor blockade, achieved by systemic administration of antagonists, is effective in stimulating motivational comA] MOUNTS 100

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Fig. 5. Effects of yohimbine (12.5 nmol), administered unilaterally into the medial preoptic area (mPOA) on alterations in ejaculation latency, intercopulatory interval and the ejaculatory threshold (intromissions preceding ejaculation), induced by systemic administration of clonidine (250 #g/kg). Only data from those rats ejaculating in mating tests are included. Too few animals, receiving systemically administered clonidine, exhibited differences in the behavior to allow statistical comparison and the data for these groups are presented as the arithmetic mean.

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brainstem and hypothalamus (Hokfelt, Johansson and Goldstein, 1984; Day, Blessing and Willoughby, 1980) and contains a high density of ~t-adrenoceptors (Young and Kuhar, 1980). Also, humans treated with drugs affecting adrenergic transmission, often exhibit iatrogenic sexual dysfunction (e.g. Bulpitt and Dollery, 1973; Hogan, Wallin and Baer, 1980; Buffum, 1982). Earlier work demonstrated that acute systemic administration of clonidine produced a long-lasting, dose-dependent suppression of copulatory behavior and erectile function (Clark et al., 1985a; Clark and Smith, 1990). This effect was not prevented by the prior administration of phentolamine (a nonspecific or-receptor antagonist (unpublished), prazosin [a selective ~q-adrenoceptor antagonist (Clark et al., 1985a)], nor naloxone [an opiate antagonist (Clark and Smith, 1990)] but was

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Fig. 6. Effects of systemic administration of yohimbine (2 mg/kg, 15 min prior to clonidine) on alterations in copulatory behavior induced by unilateral administration of clonidine into the medial preoptic area (mPOA) (20 nmol, 5 min prior to mating tests). (*P < 0.01 vs control [McNemar tests], systemically administered yohimbine, or systemic yohimbine plus clonidine into the medial preoptic area [Chi-Square test for 3 groups followed by Fisher exact tests comparisons between two treatments; N = 11 per treatment group.)

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lowed by a suppression of sexual behavior in male rats. One locus for the suppression of sexual behavior by clonidine appears to be the medial preoptic area. The observation of subtle differences in the parameters of sexual behavior, when the effects of injections into the 3rd ventricle and medial preoptic area are compared is indicative that clonidine, presumably by interacting with ~2-adrenoceptors, acting at other neural sites, plays a role in the modulation of sexual function. It is suggested that the effects of systemic administration of clonidine are mediated, at least in part, by ct2-adrenoceptors within the medial preoptic

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Contro I, VehicLe (mPOA)+ Yohimbine (i.p) BC~lCLonidine (mPOAl+Vehic[e (i,pl m CLonidine (mPOA) + Yohimbine (i.p)

Fig. 7. Effects of systemic pretreatment with yohimbine on alterations in intromission latency and postejaculatory interval, induced by unilateral administration of clonidine into the medial preoptic area (mPOA). For those rats not intromitting, a maximum of 15 min was used for intromission latency. Administration of clonidine into the medial preoptic area evoked a dramatic increment in intromission latency (P < 0.01 vs control, using Wilcoxon matched-pairs, signed-ranks tests and vs vehicle into the m P O A + systemically administered clonidine or vs clonidine into the m P O A + systemic yohimbine, using Mann-Whitney Utests). Only data from those rats ejaculating and reinitiating sexual behavior within 15 min of ejaculation are included in (B). Data for postejaculatory interval after clonidine (into the mPOA)+ systemic vehicle are presented as the arithmetic means since the number of rats exhibiting differences precluded statistical evaluation. Systemic administration of yohimbine was followed by an abbreviated postejaculatory interval (*P < 0.01 as compared to control tests, using Wilcoxon matched-pairs, signed-ranks tests or compared to values obtained after clonidine (into the mPOA) + systemic yohimbine, using Mann-Whitney U-tests).

ponents of male sexual behavior. The importance of a2-adrenoceptors within the central nervous system was indicated by the observations that systemic administration of a polar analog of clonidine, was ineffective in suppressing sexual behavior (Clark et al., 1987) and a recent report on the stimulatory effects of administration of yohimbine into the lateral ventricular (Sala, Braida, Leone, Calcaterra, Monti and Gori, 1990). Further, a suppression of male copulatory behavior was observed after administration of 50 nmol epinephrine into the medial preoptic area (unpublished). The present study shows that administration of clonidine into the central nervous system was fol-

0.000 B] INTERCOPULATORYINTERVAL 1.000.

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0.500.

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Control VehicLe (mPOA]+ Yohimbine (i.p.) Cl.onidine (mPOA)-I-VehicLe (Lp,) CLonidine (mPOA)+Yohimbine (i.p.)

Fig. 8. Effects of systemic pretreatment with yohimbine on alterations in ejaculation latency, intercopulatory interval and the ejaculatory threshold, induced by unilateral administration of clonidine into the medial preoptic area (mPOA). Only data from those rats ejaculating are included. Data for ejaculation latency, intercopulatory interval and the number of intromissions preceding the ejaculation after clonidine (into the mPOA)+ systemically administered vehicle are presented as the arithmetic means since the number of rats exhibiting differences between treatment and control tests precluded statistical evaluation. (*P < 0.01 compared to control tests using Wilcoxon matched-pairs, signed-ranks tests.) The reduction in ejaculation latency and intercopulatory interval after systemic yohimbine was attenuated by clonidine (into the mPOA) (P < 0.05, treatment values compared, using Mann-Whitney U-tests).

~t2-Adrenoceptors and sexual behavior area, since unilateral administration of a small dose of yohimbine into this region attenuated the copulatory effects. There is, however, a caveat: although it is generally accepted that clonidine interacts with ct2-adrenoceptors, clonidine also binds with high affinity to non-adrenergic, so-called imidazoline binding sites (e.g. Ernsberger, Meeley, M a n n and Reis, 1987; Michel, Brodde, Schnepel, Behrendt, Tschada, Motulsky and Insel, 1989). However, recent reports (e.g. Kamisaki, Ishikawa, Takao, Omodani, K u n o and Itoh, 1990) indicate that these imidazoline binding sites account for less than 30% of the binding of [3H]p-aminoclonidine in hypothalamus of the rat. Further support for the involvement of ct2-adrenoce ptors comes from the observation that systemic administration of yohimbine completely prevented the suppression of copulation induced by clonidine injected into the medial preoptic area. A reciprocal interaction was observed and a competitive interaction is suggested, between an agonist and an antagonist. Injection of clonidine into the medial preoptic area attenuated the facilitatory effects of systemically administered yohimbine. While the medial preoptic area and adrenergic transmitters are certainly involved in other neuroendocrine (including behavioral) and autonomic functions, the observation of alterations in copulatory behavior, at doses which did not induce decrements in locomotor behavior, would argue for a physiological role in the regulation of copulatory function. Thus, the present data provide strong support for the suggestion that ~2-adrenoceptors, located within the central nervous system, especially the medial preoptic area, are tonically active in the inhibition of sexual behavior. This inhibition appears to affect both motivational (or the co-ordination of motivation with erection) and performance aspects of the behavior. It is suggested that changes in adrenergic mechanisms in the medial preoptic area may be involved in sexual dysfunction, associated with various disorders. Acknowledgements--This research was supported by funds from NIH grants RCMI-RR03032 and BRSG-SO7RR05422, NSF grant RIMI-RII04121 and a Minority School Faculty Development Award K14-HL02482. I am grateful to Boehringer Ingelheim for the generous gift of clonidine hydrochloride and to Drs Satya P. Kalra, Julian M. Davidson, Pushpa S. Kalra and Erla R. Smith for their continuous intellectual support. Preliminary data from these studies were presented at the Annual Meeting of the Endocrine Society in New Orleans, June 1988.

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