Pharmacology, Biochemistry and Behavior 103 (2012) 345–352 Contents lists available at SciVerse ScienceDirect Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh Effect of dexmedetomidine on ejaculatory behavior and sexual motivation in intact male rats Eelke M.S. Snoeren a,⁎, Jyrki Lehtimäki b, Anders Ågmo a a b Department of Psychology, University of Tromsø, Tromsø, Norway Research and Development, Orion Corporation, Orion Pharma, Finland a r t i c l e i n f o Article history: Received 8 May 2012 Received in revised form 29 August 2012 Accepted 8 September 2012 Available online 12 September 2012 Keywords: Sexual incentive motivation Ejaculatory behavior Male rats Dexmedetomidine Noradrenaline a b s t r a c t Premature ejaculation is the most common sexual disorder in young men. Consequently, there is an intense search for efficient and safe pharmacological treatments. Insofar, almost no effective treatment with acute effects is available. In this study, we evaluated the effects of the noradrenergic α2 receptor agonist dexmedetomidine on sexual incentive motivation and copulatory behavior in male rats. Sexual incentive motivation was tested in a large rectangular arena connected to two small incentive stimulus cages containing either a male or sexually receptive female rat. There was no sexual interaction possible between the experimental subjects and the incentives during this test. Approach to the incentives constituted the measure of sexual incentive motivation. After the sexual incentive motivation test, the subjects were tested for copulatory behavior in a regular copulation test for 30 min. Doses of 0.1 and 1 μg/kg of dexmedetomidine (i.p.) had no effect on any of the indices of locomotor activity or on parameters of sexual incentive motivation. With regard to copulatory behavior, it was found that the dose of 1 μg/kg prolonged the latency to the first ejaculation, while the latency to second ejaculation showed a tendency to increase. The absence of an effect on indices of sexual incentive motivation or general activity showed that the actions of dexmedetomidine in this study were limited to ejaculatory mechanisms. Insofar as the ejaculation latency in the male rat is predictive of prolonged ejaculation latency in men, it can be proposed that dexmedetomidine is of potential utility for the treatment of premature ejaculation. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Premature ejaculation is the most common sexual disorder in young men (Dunn et al., 1998; Laumann et al., 1999; Althof, 2006). As a consequence, there is an intense search for efficient and safe pharmacological treatments. So far, the only compounds appearing to be clinically effective are some of the specific serotonin reuptake inhibitors (SSRI), with paroxetine as the most efficient (Waldinger et al., 1998, 2003). One of the disadvantages of the traditional SSRIs is that they require multiple dosing before becoming effective (Waldinger, 2007). This has led to the search for other compounds with acute effects on sexual functioning. At the moment, a few compounds have been used for the treatment of premature ejaculation (Giuliano and Hellstrom, 2008; Powell and Wyllie, 2009). A short acting SSRI, dapoxetine, has been reported to be efficient in men after acute treatment (McMahon, 2010). It is presently registered as a treatment for premature ejaculation in several European countries. An on-demand treatment with the tricyclic antidepressant clomipramine has also been shown to increase the intravaginal ejaculatory latency time compared to placebo ⁎ Corresponding author. E-mail address: eelke.snoeren@uit.no (E.M.S. Snoeren). 0091-3057/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2012.09.007 in patients with premature ejaculation (Segraves et al., 1993; Strassberg et al., 1999). However, the use of clomipramine is limited by its associated side-effects (Kim and Seo, 1998). Tramadol, a centrally acting agonist of μ-opioid receptors and in generic form available in most countries, might also be effective for acute treatment (Safarinejad and Hosseini, 2006; Salem et al., 2008). Although its potential mode is not completely understood, tramadol might inhibit noradrenaline and serotonin reuptake in addition to its antinociceptive actions on μ-opioid receptors. However, like with all opioids, there might be concerns about the risk of abuse and dependence. Another compound, modafinil, has been evaluated in a study in male rats (Marson et al., 2010). Modafinil increases the release of monoamines, specifically noradrenaline and dopamine. It prolonged the ejaculation latency and increased the number of preejaculatory intromissions. However, there are no human data concerning possible effects of modafinil on premature ejaculation. Short ejaculation latencies in male rats are considered to be comparable to premature ejaculation in men (Pattij et al., 2005; Chan et al., 2008). Many drugs acting on copulatory behavior in rats have similar effects in humans; e.g. the inhibiting effects of SSRIs on copulatory behavior (Waldinger et al., 1998; Chan et al., 2008) and the stimulating effects of sildenafil (Ottani et al., 2002; Steidle et al., 2007). It is, therefore, reasonable to suppose that compounds delaying ejaculation in rats will also do so in men. Thus, rats can be used as a tool to 346 E.M.S. Snoeren et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 345–352 evaluate drugs potentially useful for the treatment of premature ejaculation. Serotonin is not the only neurotransmitter involved in sexual behavior. There is much evidence showing that dopaminergic, as well as noradrenergic compounds modify many aspects of this behavior (Clark et al., 1984; Bitran and Hull, 1987; Agmo and Picker, 1990; Benelli et al., 1993; Hull et al., 2004), including the ejaculation latency. Dopaminergic drugs are probably not appropriate for treatment of premature ejaculation because of the abuse potential (agonists) or tranquilizing (antagonists) effects. To the contrary, drugs acting on noradrenergic systems may prove to be helpful, provided sympathetic effects are kept to a minimum. The noradrenaline system consists of different receptor types, including α1, α2, and β adrenoceptors. Adrenergic α2 receptors are located in the brain, spinal cord and periphery. The receptors are localized both post- and presynaptically, as inhibitory receptors on non-adrenergic neurons (heteroceptors) and on the terminals and dendrites of the noradrenergic neurons themselves (autoreceptors) (Frankhuyzen and Mulder, 1982; Nasseri and Minneman, 1987). The α2 receptors manifest a high level of tonic activity and their blockade markedly accelerates the synthesis and release of noradrenaline in the cortex and elsewhere (Dennis et al., 1987; Millan et al., 1994; Kiss et al., 1995). To the contrary, agonists such as dexmedetomidine resulted in a decrease in NA release and synthesis (Gobert et al., 1998; Millan et al., 2000b). A substantial amount of data suggests that blockade of adrenergic α2 receptors stimulate rat sexual behavior, while stimulation of this receptor inhibits copulation (Clark et al., 1984, 1985; Clark and Smith, 1990; Clark, 1991; Benelli et al., 1993). Stimulation of α1 receptors may also have an inhibitory effect on sexual behavior (Clark, 1995). The role of β adrenergic receptors is not entirely clear, but data exists suggesting that nonselective antagonists inhibit sexual behavior, while agonists stimulate copulation (Smith et al., 1995; Mallick et al., 1996; Gulia et al., 2002). It is probably the β2 receptor causing this effect, because the selective β1 receptor antagonists atenolol, labetalol and metoprolol seemed to be ineffective (Smith et al., 1990, 1996). It has previously been reported that a dose of 8 μg/kg of dexmedetomidine slightly reduced sexual incentive motivation and locomotor activity in male rats (Viitamaa et al., 2006). The effects on copulation were not investigated in that study. However, other α2 receptor agonists seem to reduce copulatory behavior in male rats. Guanabenz, for example, increased mount and intromission latencies in both sexually experienced and inexperienced male rats, while mount and intromission frequencies were decreased (Benelli et al., 1993). Also, clonidine had similar effects on the amount of copulatory behavior when administered into the third ventricle or preoptic area (Clark, 1991). Administration of clonidine directly to the preoptic area increased the ejaculation latency, while large doses inhibited all aspects of copulatory behavior in a way similar to guanabenz. Systemic administration of clonidine appeared to cause a general suppression of copulatory behavior (Clark and Smith, 1990). In conclusion, these studies showed that agonists at the adrenergic α2 receptor generally inhibit male rat sexual behavior. To the contrary, studies with α2 receptor antagonists showed opposite effects. Yohimbine, for instance, increased sexual motivation in male rats as evidenced by increased mounting performance in mating tests conducted after genital anesthetization (Clark et al., 1984) and facilitated copulatory behavior by drastic decreases in ejaculation latency and intercopulatory and postejaculatory intervals (Clark et al., 1985). In the present study we determined the effects of low doses of the adrenergic α2 receptor agonist dexmedetomidine on copulatory behavior. This compound is far more selective for the α2 receptor than clonidine, with faster onset and shorter duration of action (Bol et al., 1997). It binds only marginally to other receptors, including the dopamine, serotonin and histamine receptor families (Virtanen, 1989; Millan et al., 2000b). This makes this compound a good candidate for clinical use. Ideally, a compound suitable for treating premature ejaculation should have a specific effect on ejaculation latency; meaning that all other aspects of sexual behavior should remain unaffected. Therefore, we investigated whether low doses of dexmedetomidine could specifically prolong the ejaculation latency without modifying other behaviors. 2. Materials and methods 2.1. Animals Twelve experiment-, and drug-naive male Wistar rats (Charles River, Sulzfeld, Germany, 280–300 g) were used. Some other males of the same strain and from the same provider were used as social incentives in the experiments. Twelve females (250–300 g) were used either as sexual incentives in the motivation tests or copulation partners in the copulatory behavior tests. The rats were housed in pairs in Macrolon IV cages on a reversed 12 h light/dark cycle (lights on 23:00–11:00), in a room with controlled temperature (21 ± 1 °C) and relative humidity (55 ± 10%). Standard rodent food and tap water were available ad libitum. The females were ovariectomized under isoflurane anesthesia at least 2 weeks before use. At the same time, they were subcutaneously implanted with a 5 mm long Silastic capsule (medical grade Silastic tubing, 0.0625 in. inner diameter, 0.125 in. outer diameter, Degania Silicone, Degania Bet, Israel) containing 10% 17β-estradiol (Sigma, St. Louis, MO, USA) in cholesterol (Sigma, St. Louis, MO, USA). The ends of the capsules were sealed with medical grade adhesive silicone (Nusil Silicone Technology, Carpinteria, CA USA). The females were given progesterone (Sigma, St Louis, MO, USA) in a dose of 1 mg/rat at least 3.5 h prior to testing. The steroid was dissolved in peanut oil (Apoteksproduskjon, Oslo, Norway) and injected subcutaneously in a volume of 0.2 ml/rat. This hormonal treatment assures maximum receptivity and proceptivity (Whalen, 1974). All experimentation was approved by the National Animal Research Authority of Norway. 2.2. Apparatus 2.2.1. Sexual incentive motivation test Sexual motivation was tested in a rectangular arena (100 × 50 × 45 cm) with rounded corners. The walls consisted of metal sheet covered with a black plastic surface and the floor was made of dark-gray polyvinyl chloride. At the long sides, 15 cm from opposite corners, there were openings (25 × 25 cm) linked to two incentive stimulus cages connected from the outside of the observation arena. The incentive stimulus was separated from the experimental subject by a wire mesh. Thus, the animals could see, smell and hear the stimulus. Outside each incentive stimulus cage, a virtual zone of 30× 21 cm was defined. The subject was considered to be within the zone whenever its point of gravity was inside. The tests were performed in a room that was illuminated with dim white light, about 5 lx at the bottom of the arena. A video camera located in the ceiling above the observation arena was connected to a computer and a video tracking system (Ethovision XT, Noldus, Wageningen, The Netherlands) determined the experimental subject's position with a frequency of 5 Hz. The program determined the time the experimental subjects spent in each incentive zone, the distance moved during the test, the mean velocity of movement, and the time moving (Agmo, 2003; Agmo et al., 2004). 2.2.2. Copulation test Tests for copulatory behavior were performed in rectangular cages (40 × 60 × 40 cm high) of black sheet steel with a Plexiglas front. These tests were performed in a room different from that used for E.M.S. Snoeren et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 345–352 347 the test of sexual incentive motivation. The light intensity within the cage was about 60 lx. mounts and intromissions as well as the intromission ratio and interintromission interval were determined. 2.3. Drugs 2.5. Statistics Dexmedetomidine (Orion, Finland) was provided as a ready-made solution (Dexdomitor®). Shortly before use it was diluted with physiological saline to the appropriate concentrations. The drug was administered in the doses of 0.1 and 1 μg/kg. These doses were based on data from earlier studies (Bol et al., 1997; Millan et al., 2000a, 2000b; Viitamaa et al., 2006). The large dose is known to be subeffective with regard to locomotor activity (Viitamaa et al., 2006), while being effective in some models of analgesia (Bol et al., 1997; Millan et al., 2000a) and in tests of aggression (Millan et al., 2000b) The low dose is not known to have effects but it was considered worthwhile to determine potential actions on sexual behaviors. Physiological saline was used as vehicle. All injections were given intraperitoneally in a volume of 1 ml/kg. At the experimental tests, treatments were administered according to a Latin square within subject design. There was an interval of 1 week between drug treatments. The rats were injected 15 min before the start of sexual incentive motivation test. Sexual incentive motivation was quantified in two ways. First, a preference score (time spent in the female incentive zone / (time spent in the female incentive zone + time spent in the male incentive zone)) was calculated. Second, the time spent in the female incentive zone and the time spent in the male incentive zone were used. Furthermore, the number of entries into each incentive zone as well as the mean duration of each visit was determined. As indicators of ambulatory activity we employed the total distance moved during the test, the mean velocity of movement while moving, and the time spent moving. The preference score and indices of ambulatory activity were evaluated with one-factor repeated measures ANOVAs. In case of significance, a posteriori comparisons were made with Tukey's HSD test. The time spent with the incentives as well as the number of visits to them were evaluated with two-factor ANOVAs for repeated measures on both factors (incentive and treatment). Sex behavior data were analyzed with one-factor ANOVAs for repeated measures. Some of the variables were not normally distributed according to the Shapiro–Wilk test. These variables were analyzed with Friedman's one-way ANOVA. In case of significance, a posteriori tests were made with the Wilcoxon test, appropriately modified for multiple comparisons. All probabilities mentioned are two-tailed. 2.4. Design and procedure 2.4.1. Sexual incentive motivation test Prior to the experiment, the animals were familiarized to the observation arena during 3 sessions of 10 min each, separated by 48 h. During these sessions, no incentive animals were present. During the drug tests, a sexually receptive female and an intact male were employed as incentives. All incentive animals were sexually experienced. Before each session the arena and the incentive animal cages were carefully cleaned with a 0.1% glacial acetic acid solution in water. The incentive animals were then placed in their respective cages. About 5 min later the first experimental subject was introduced into the middle of the arena and the 10 min of observation started. The subject was then removed from the arena, and the following rat was immediately introduced. The position of the incentive animals was semi-randomly changed throughout the experimental session. 2.4.2. Copulation test Prior to the experiment, the twelve subject male rats were allowed to copulate with a receptive female on 3 occasions to become sexually experienced. At drug tests, the male subject was transferred to the copulation room where it was put into the copulation cage immediately after the sexual incentive motivation test. Five minutes later, a sexually receptive female was introduced. Observation in the copulation test lasted for 30 min after the introduction of the female. The following behavioral parameters were recorded or calculated with the Observer XT software (Noldus, Wageningen, the Netherlands) for behaviors associated with the first ejaculation: Mount latency (time from introduction of the female until the first mount with pelvic thrusting), intromission latency (time from introduction of the female until the first mount with vaginal penetration), ejaculation latency (time from the first intromission until ejaculation), the postejaculatory interval (time between the ejaculation and the next intromission), number of mounts, and number of intromissions for the first ejaculation. In addition, we calculated the intromission ratio (number of intromissions / (number of intromissions + number of mounts)) and the interintromission interval (ejaculation latency / number of intromissions). The intromission ratio is considered to be an indicator of the quality of erection and activity in the penile striated muscles while the interintromission interval expresses the intensity of copulatory behavior (Agmo, 1997). For subsequent ejaculations, only the ejaculation latency, postejaculatory interval, the number of 3. Results 3.1. Sexual incentive motivation No drug effects were found on the preference score (F(2,22) = 0.29, NS) or on the time spent in the incentive zones (F(2,22) = 0.48, NS) (Fig. 1). There was a difference between incentives (F(1,11) = 80.54, P b 0.001), showing that the subjects spent far more time in the vicinity of the receptive female than in vicinity of the male. The ineffectiveness of dexmedetomidine was further confirmed by the lack of an interaction between treatment and incentive (F(2,22) = 0.43, NS). Dexmedetomidine had no effect on the number of visits to the incentive zones and the duration of visits (F(2,22) = 1.30, NS and F(2,22)=0.84, NS, respectively ), while there was an effect of incentive (number of visits: F(1,11)=45.99; Pb 0.001 and duration of visits: F(1,11)=54.85, Pb 0.001) (Fig. 2). The subjects made much more and longer visits to the female incentive zone than to the male zone. The interaction incentive×treatment was nonsignificant for the number of visits (F(2,22)=0.35, NS) and the duration of visits (F(2,22)=0.21, NS). With regard to the distance moved during the test (Fig. 3), there was no treatment effect (F(2,22) = 1.15, NS). The same results were found with other indicators of motor function, such as the mean velocity of movement while moving and the time spent moving (F(2,22) = 1.30, NS and (F(2,22)) = 1.53, NS, respectively). It is quite clear that dexmedetomidine does not modify any aspect of ambulatory activity at the doses used in the present experiment. 3.2. Copulatory behavior The results of the tests for copulatory behavior are summarized in Table 1. All animals ejaculated at least once at all tests, 10 ejaculated twice at all tests, and only 5 rats made 3 or more ejaculations at all tests. This number is too low for statistical evaluation, and therefore only data from the first and second ejaculatory series are displayed. In the first ejaculatory series, the latencies to mount and intromission were not distributed normally, and the Friedman test was used for analysis. There was no treatment effect on these latencies 348 E.M.S. Snoeren et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 345–352 Fig. 1. Mean ± SEM preference score (A) and time in incentive zone (B) after treatment with vehicle or two doses of dexmedetomidine (n = 12). One-factor (A) and two-factor (B) repeated measures ANOVA was used for the statistical analysis, with Tukey's HSD test for post hoc analysis. ⁎Significantly different between incentives, p b 0.001. (mount, χ 2(2) = 1.17), NS; intromission, (χ 2(2) = 0, NS). The number of mounts preceding the first ejaculation had also a non-normal distribution. Nevertheless, it was unaffected by dexmedetomidine (χ 2(2) = 1.17, NS), as was the number of intromissions preceding Fig. 3. Mean ± SEM ambulatory activity expressed either as distance moved (A), mean velocity of movement (B), or time spent moving (C) during the sexual incentive motivation test after treatment with vehicle or two doses of dexmedetomidine (n = 12). One-factor repeated measures ANOVA was used for the statistical analysis. Fig. 2. Mean ± SEM number of visits to the incentives (A) and duration of visits to the incentives (B) after treatment with vehicle or two doses of dexmedetomidine (n = 12). Two-factor repeated measures ANOVA was used for the statistical analysis, with Tukey's HSD test for post hoc analysis. ⁎Significantly different between incentives, p b 0.001. the first ejaculation. (F(2,22) = 1.09, NS). To the contrary, there was a treatment effect on the latency to the first ejaculation (F(2,22) = 6.01, P = 0.027). Tukey's HSD test revealed that the 1 μg/kg dose differed from vehicle while the 0.1 μg/kg dose lacked effect compared to vehicle. The postejaculatory interval was unaffected by the treatment (F(2,22) = 0.64, NS). The data for the interintromission interval were not normally distributed. Friedman's test showed that there was no treatment effect (χ 2(2) = 4.17, NS), despite the trend to an increase after 1 μg/kg of dexmedetomidine. This is probably a consequence of the increase in ejaculation latency, since the intromission number was also unaffected by treatment (F(2,22) = 0.90, NS). The preceding analyses show that the only aspect of copulatory behavior preceding the first ejaculation that was modified by dexmedetomidine was the ejaculation latency. It is noteworthy that neither the number of preejaculatory intromissions nor the interval between intromissions was significantly modified. In the second ejaculatory series (based on 10 rats), there was no treatment effect on the number of mounts (χ 2(2) = 3.77, NS) or the number of intromissions (χ 2(2) = 2.59, NS). Both these variables 349 E.M.S. Snoeren et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 345–352 Table 1 Parameters for copulatory behavior after treatment with vehicle or two doses of dexmedetomidine. ⁎Different from vehicle, pb0.05; ⁎⁎pb0.01. Parameter Vehicle 0.1 μg/kg 1 μg/kg Latency to 1st mount Latency to 1st intromission Number of mounts 1st series Number of intromissions 1st series Ejaculation latency 1st series Postejaculatory interval 1st series Interintromission interval 1st series Intromission ratio 1st series Number of mounts 2nd series Number of intromissions 2nd series Ejaculation latency 2nd series Postejaculatory interval 2nd series Interintromission interval 2nd series Intromission ratio 2nd series Number of mounts in test Number of intromissions in test Number of ejaculations in test Intromission ratio in the test 8.5 ± 3.4 12.6 ± 5.6 9.5 ± 2.6 13.3 ± 1.4 371.9 ± 65.7 326.5 ± 17.8 26.8 ± 3.5 0.67 ± 0.06 6.3 ± 1.7 5.6 ± 0.6 150.1 ± 16.3 393.9 ± 19.9 27.6 ± 2.5 0.58 ± 0.08 23.6 ± 5.8 26.9 ± 1.9 3.1 ± 0.2 0.60 ± 0.05 4.5 ± 0.72 11.8 ± 4.5 7.8 ± 1.6 12.0 ± 1.4 344.5 ± 43.9 306.6 ± 10.5 31.6 ± 4.5 0.64 ± 0.06 4.4 ± 1.7 4.8 ± 0.5 151.7 ± 18.3 392.1 ± 17.2 34.2 ± 4.3 0.61 ± 0.07 20.3 ± 3.5 24.5 ± 1.7 3.3 ± 0.2 0.57 ± 0.04 7.2 ± 1.27 10.8 ± 3.0 10.5 ± 2.0 13.9 ± 1.9 502.7 ± 87.0⁎⁎ 327.1 ± 22.3 41.7 ± 9.8 0.59 ± 0.03 6.4 ± 1.5 6.4 ± 0.8 220.1 ± 57.8 401.8 ± 15.6 29.8 ± 3.9 0.56 ± 0.05 21.3 ± 2.9 26.1 ± 2.5 2.6 ± 0.3* 0.56 ± 0.03 did not have a normal distribution. Likewise, the ejaculation latency was unaffected by the treatment (χ 2(2) = 2.60, NS). At difference to the first ejaculation, the latency to the second ejaculation was not normally distributed. The postejaculatory interval had a normal distribution for the second ejaculation while it did not for the first. In any case, there was no treatment effect (F(2,16) = 0.44, NS). Please note that two rats did not reach a second ejaculation and that one male did not resume copulation after the second ejaculation, meaning that the postejaculatory interval was obtained from nine animals only. The interintromission interval was unaffected by treatment (F(2,18) = 0.88, NS) as was the intromission ratio (F(2,22) = 0.36, NS). Thus, there was no drug effect at all on any parameter in the second ejaculatory series. The number of mounts displayed during the entire 30 min period of testing was not altered by dexmedetomidine (F(2,22) = 0.48, NS). This also applies to the number of intromissions (F(2,22) = 0.62, NS). The number of ejaculations achieved during the test had a non-normal distribution, and was consequently analyzed by the Friedman test. It turned out that dexmedetomidine had a significant effect (χ 2(2) = 6.25, P = 0.044). Comparisons between vehicle and the two doses of the compound revealed that the 0.1 μg/kg dose was ineffective while the 1 μg/kg dose reduced the number of ejaculations. The intromission ratio during the entire 30 min test was unaffected by treatment (F(2,22) = 0.72, NS). To summarize, when the entire 30 min test period is considered, only the number of ejaculations was affected by dexmedetomidine. 4. Discussion The present study shows that dexmedetomidine has no effect on any of the indices of locomotor activity or on parameters of sexual incentive motivation. With regard to copulatory behavior, it was found that the dose of 1 μg/kg enhanced the latency to the first ejaculation. The latency to the second ejaculation showed a tendency to be enhanced by this dose. The dose of 0.1 μg /kg, on the other hand, was ineffective. The number of ejaculations achieved during the 30 min test was slightly but significantly reduced by the dose of 1 μg/kg dexmedetomidine. This was a direct consequence of the prolonged ejaculation latency, and does not indicate a general inhibition of copulatory behavior. The fact that there was no significant modification of other parameters of copulatory behavior supports this assertion. Dexmedetomidine had a specific effect on ejaculation latency, without modifying the intromission ratio. This ratio is often considered to be an indicator of the quality of erection. Although the method is not sufficient for conclusions, it can be suggested that dexmedetomidine did not interfere with the quality of erection. In addition, the fact that its effect on copulatory behavior was independent of any effect on general activity suggests that the prolonged ejaculation latency is quite specific and not a result of sedation. Likewise, potential analgesia effects of the 1 μg/kg dose cannot explain the prolonged ejaculation latency. Potent analgesics like morphine fail to enhance this latency (Pfaus and Gorzalka, 1987; Agmo and Paredes, 1988). Overall, present data are consistent with the results of an earlier study (Viitamaa et al., 2006) in which a dose of at least 8 μg/kg of dexmedetomidine was needed for a modest reduction of sexual incentive motivation and 4 μg/kg for a small reduction of locomotor activity. Our results also coincide with data reported after treatment with another noradrenalin α2 receptor agonist, clonidine. This compound increased ejaculation latencies after systemic (Clark et al., 1985) as well as local injections in the preoptic area (Clark, 1991). In addition, the effects of clonidine were blocked by pretreatment with the α2 receptor antagonist yohimbine (Clark et al., 1985). Yohimbine by itself decreased ejaculation latency (Clark et al., 1985) and thereby showed the opposite effect of dexmedetomidine on copulatory behavior. An important issue that needs to be considered is whether the observed effect of dexmedetomidine can be entirely attributed to the noradrenergic α2 receptor. The lack of significant binding to other receptors potentially important for sexual behavior (Virtanen, 1989; Millan et al., 2000b) as well as the fact that dexmedetomidine has no effect in mice lacking α2 receptors (Hunter et al., 1997; Altman et al., 1999) suggest that this indeed is the case. Furthermore, whenever dexmedetomidine has been administered together with a selective α2 receptor antagonist, its actions have been blocked (see, e.g. (Boyce-Rustay et al., 2008; Bell et al., in press)). For these reasons, no effect was made to block the effect of dexmedetomidine with an antagonist in the present study. Dexmedetomidine's specific action on ejaculation latency can be understood only if we consider potential noradrenergic modulations of the ease by which this viscerosomatic response is activated. Ejaculation is mediated by a spinal control center, also called the spinal ejaculation generator (Marberger, 1974; McKenna et al., 1991; Carro-Juarez and Rodriguez-Manzo, 2008). Besides the coordination of sympathetic, parasympathetic and motor outflow to induce ejaculation, the spinal ejaculation generator integrates this information with inputs from the genitals that are required to trigger ejaculation (Coolen et al., 2004; Carro-Juarez and Rodriguez-Manzo, 2005). The lumbar spinothalamic cells (LSt cells) have been shown to play a fundamental role in generation of ejaculations and may be part of the spinal ejaculation generator (Truitt and Coolen, 2002). This was 350 E.M.S. Snoeren et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 345–352 Fig. 4. Simplified schematic display of the connections in the brain and spinal cord involved in ejaculation. Pathways between the lumbosacral spinal cord and the genitals are not illustrated. The dotted lines are confirmed noradrenergic projections which could involve the effect of α2 receptors. (LC = locus coeruleus; nPGI = nucleus paragigantocellularis; SPF = subparafascicular nucleus; LH = lateral hypothalamus; PVN = paraventricular nucleus of the hypothalamus; POA = preoptic area; BNST = bed nucleus of stria terminalis; MeA = medial amygdala). suggested by the fact that LSt cells are specifically activated during ejaculation and not after any other component of male sexual behavior (Heeb and Yahr, 1996; Kollack-Walker and Newman, 1997; Truitt and Coolen, 2002). Besides, lesions of these LSt-neurons caused dramatic disruptions in ejaculatory behavior (Truitt and Coolen, 2002). In general, the ejaculatory reflex, activating the LSt-neurons, is complex and involves multiple afferent and efferent systems. An important center for the regulation of sexual behavior is the subparafascicular nucleus (SPFp). The medial part of the SPFp receives afferents directly from the LSt cells in the lumbosacral spinal cord, but it is also connected to brain areas like the medial preoptic area (Pehek et al., 1989; Markowski et al., 1994), paraventricular nucleus of the hypothalamus (PVN) (Marson and McKenna, 1994) and the nucleus paragigantocellularis (nPGi) in the caudal brainstem (Marson et al., 1992; Marson and McKenna, 1992; Truitt and Coolen, 2002; Coolen et al., 2003a). Overall, the medial SPFp conveys copulation-related information to a number of areas, which in turn provide feedback to the medial SPFp (Heeb and Yahr, 2001; Coolen et al., 2003b). Furthermore, the SPFp receives inputs from the motor cortex that is involved in the control of locomotor patterns associated with ejaculation (Coolen et al., 2003b). The nPGI, on the other hand, is also heavily involved in the control of ejaculation, by composing the brake of the spinal ejaculation center (Yells et al., 1992). Lesions of the nPGI result in the facilitation of sexual behavior (Marson et al., 1992; Yells et al., 1992). It is thought that the nPGI inhibits the spinal cord via serotonergic neurons and that this tonic inhibition has to be removed first before ejaculation becomes possible. The activation of the medial preoptic region seems to be necessary for the release of the ejaculatory response, apparently by ‘inhibiting the inhibitor’ (Yells et al., 1992). This excitatory effect might act via dopamine and oxytocin systems (Hull et al., 1992; Wagner and Clemens, 1993). The PVN has been previously implicated as the source of descending excitatory input to genital musculature (Chen et al., 1997). Interestingly, it seems that adrenergic innervation is also affecting the spinal ejaculation generator. A study performed in spinal cord-transected and urethane-anesthetized male rats showed that stimulation of α1 adrenoceptors by methoxamine, as well as blockade of α2 adrenoceptors by yohimbine, initiated and modulated the rhythmic expression of the genital motor pattern of ejaculation (Smith et al., 1987; Carro-Juarez and Rodriguez-Manzo, 2006). In addition, yohimbine provoked the immediate expression of single ejaculatory genital motor pattern in the exhausted coital reflex model (CarroJuareza and Rodriguez-Manzo, 2003). Activation of α2 adrenoceptors with clonidine or blockade of α1 adrenoceptors by prazosin, on the other side, prevented the activity of the spinal generator for ejaculation, without inducing any other genital motor activity (Carro-Juarez and Rodriguez-Manzo, 2003, 2006). This suggests that noradrenergic agents could target the spinal generator involved in the control of ejaculation (Carro-Juarez and Rodriguez-Manzo, 2006). The spinal and supraspinal circuitry modulating sexual function, including the nPGI, receives a dense noradrenergic innervation from either the lateral tegmental or the locus cerulean noradrenergic cell groups (Kojima et al., 1985; Lyons et al., 1989; Rajaofetra et al., 1992). The innervation is particularly dense in the pudendal nucleus of motoneurons which supply the striated genital muscles involved in ejaculation (Kojima et al., 1985; Lyons et al., 1989). Some of the noradrenergic innervation of the spinal cord may also originate from spinal cells and play a role in motor coordination (Kjaerulff and Kiehn, 1997). The connections in the brain and spinal cord involved in ejaculation are schematically displayed in Fig. 4. It is likely that the coordinated, rhythmic contractions of the muscles involved in ejaculation are modulated by noradrenergic pathways acting on the spinal generator to release ejaculation. Potential candidate areas for the noradrenergic effect on ejaculation besides a direct effect in the spinal cord might be the nPGI, LC and the PVN. α2 receptors are widely distributed in the central nervous system (Wamsley et al., 1992; Alburges et al., 1993), and the localization of this receptor subtype in these specific brain areas have been confirmed, in addition to noradrenergic connections with other brain areas (Kojima et al., 1985; Lyons et al., 1989; Rajaofetra et al., 1992). Thus, noradrenergic agents acting on α1 and α2 adrenoceptor subtypes could affect the spinal generator, with an inhibitory role for α2 adrenoceptors agonists in ejaculation. It would, therefore, be reasonable to link the effects of dexmedetomidine in our study to this mechanism. It must be noted, though, that results from the present single-drug study are far from sufficient for any firm conclusion concerning the role of noradrenergic systems in ejaculation. Nevertheless, by outlining the potential circuitry involved in this process we provide the bases for future systematic studies. Insofar as the ejaculation latency in the male rat is predictive of enhanced ejaculation latency in men, it can be proposed that dexmedetomidine is of potential utility for the treatment of premature ejaculation. The fact that its effect on copulatory behavior is independent of any effect on general activity suggests that the prolonged ejaculation latency is quite specific and not a result of sedation. 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