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The effect of apomorphine on genital reflexes in male rats deprived of paradoxical sleep
Physiology & Behavior 80 (2003) 211 – 215
The effect of apomorphine on genital reflexes in male rats deprived of paradoxical sleep Monica L. Andersen*, Magda Bignotto, Sergio Tufik Department of Psychobiology, Universidade Federal de Sa˜o Paulo, Escola Paulista de Medicina (UNIFESP/EPM), Rua Napolea˜o de Barros, 925 Vila Clementino, Sa˜o Paulo SP-04024-002, Brazil Received 29 July 2002; received in revised form 13 June 2003; accepted 23 July 2003
Abstract Drugs that stimulate dopamine (DA) systems can stimulate sexual arousal in male rats and humans, and previous work has shown that cocaine enhances genital reflexes [penile erection (PE) and ejaculation (EJ)] in rats deprived of paradoxical sleep (PS). The present study sought to expand the latter finding by assessing the effects of DA receptor agonist apomorphine in sleep-deprived rats. Apomorphine in doses ranging from 10 to 240 Ag/kg was administered intraperitoneally to rats that had been deprived of sleep for 4 days and to normal controls, and the incidence of PEs and EJs was measured for 60 min. Sleep deprivation alone induced PE and this effect was potentiated by apomorphine, with maximal effects occurring with the 120 Ag/kg dose; results for this dose group differed from those of PSD groups treated with 0, 10, 20, 40, 80, and 240 Ag/kg of apomorphine. Sleep deprivation alone also induced spontaneous EJ, but this response was not potentiated by apomorphine in the dose range tested. We suggest that the potentiating effects of apomorphine on PE are likely due to PSD-induced DA receptor supersensitivity. D 2003 Elsevier Inc. All rights reserved. Keywords: Paradoxical sleep deprivation; Genital reflexes; Apomorphine; Dopamine; Male rat
1. Introduction A variety of dopaminergic agonists have been described as being able to elicit and/or amplify sexual behavior in male rats, whereas dopamine (DA) receptor blockers have the opposite effect (for a review, see Ref. [33]). The physiological significance of these findings has been emphasized recently by the assessment of neurochemical changes in mating animals. The use of the D1/D2 DA receptor agonist apomorphine for the treatment of erectile dysfunction provides further strong support for an involvement of dopaminergic systems in the control of sexual function [16]. Behavioral studies in rats support the hypothesis that apomorphine in relatively low doses increases the number of erections [11] and penile anteroflexions in restrained supine rats [22,37]. A number of investigators have found pronounced behavioral supersensitivity to directly and indirect-
* Corresponding author. Tel.: +55-11-5539-0155; fax: +55-11-55725092. E-mail address: [email protected] (M.L. Andersen). 0031-9384/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2003.07.006
ly acting DA agonists following prolonged paradoxical sleep deprivation (PSD). These findings have led to the hypothesis that modification in sensitivity to dopaminergic drug by PSD is due to a functional hyperactivity of the DA systems. Early studies on the effect of PSD on sexual behavior produced controversial results. Some reports described increased sexual performance in PSD male rats [34,44] and facilitation by PSD of the effects of testosterone [43] or estrogen [12] on rat sexual behavior. In another study, however, 4 days of PSD did not significantly alter male sexual performance [20]. Experiments in this laboratory indicated that PSD for 96 h in rats induced genital reflexes after cocaine injection, as reflected in the number of animals that spontaneously displayed penile erections (PEs) and ejaculations (EJs) in the absence of a female [1– 5]. Furthermore, these genital effects were observed in old [3] as well as in young PSD rats [5]. These experiments indicated that PSD and cocaine elicit genital reflexes much more markedly together than separately [2]. In order to better elucidate the effect of PSD on genital reflexes, we evaluated the effects of the DA agonist apomorphine following PSD in male rats.
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2. Methods 2.1. Animals Three-month-old Wistar male rats from our facilities were used in this experiment. The vivarium was maintained on a 12:12-h light– dark photoperiod (lights on 0700 –1900 h) at an ambient temperature of 22 jC. Laboratory chow and water were provided ad libitum. All procedures used in the present study complied with the Guide for the Care and Use of Laboratory Animals, and the experimental protocol was approved by the Ethical Committee of UNIFESP (CEP N. 205/02).
less effective than the subcutaneous route, but since prior observations indicated that sleep deprivation by itself can induce spontaneous genital reflexes, the intraperitoneal route was chosen in order to preclude the possibility of ceiling effects. Controls were handled in the same way as the PSD animals. Testing was conducted in a transparent cylindrical cage (32 cm in diameter and 30 cm in height). The number of spontaneous PEs and EJs was assessed for 60 min. PEs were counted only when the rat displayed and bent down to lick its penis in full erection. EJs were scored by the number of ejaculatory plugs. The behavioral observations were carried out between 0900 and 1100 h. Different groups of rats and different cages were used in each series of experiments; cages were cleaned thoroughly after each test.
2.2. Drugs 3.2. Statistical analysis Apomorphine HCL was purchased from Sigma (St. Louis, MO, USA). The drug was mixed immediately before testing in saline solution containing 0.2% ascorbic acid. The solution was injected intraperitoneally in a volume of 1 ml/ kg bw.
Data were expressed as mean F S.E.M. Differences between groups were statistically assessed by two-way analysis of variance (ANOVA), followed by the Duncan test for a posteriori comparison of means. The significance level was set at P < .05.
3. Paradoxical sleep deprivation 4. Results One hundred and forty sexually naive rats were randomly divided into PSD (n = 70) and home cage groups (n = 70). Seven dosage levels of apomorphine were used and each group consisted of 10 rats. For PSD, 14 narrow circular platforms (6.5 cm in diameter) were placed inside a tiled tank (123 44 44 cm) filled with water to within 1 cm of their upper surface. Ten animals were placed on the platforms in each tank, an arrangement that allowed them to move around by leaping from one platform to another. One tank was used for each group. In this procedure, when an animal reaches the paradoxical phase of sleep (PS), the accompanying muscle atonia causes it to touch the water and wake up. Under these conditions, this procedure causes a complete loss of PS in each of the 4 days (Machado RB, Hipolide DC, Benedito AA, Tufik S, submitted for publication). Throughout the study, the experimental room was maintained at a controlled temperature (23 F 1 jC) and light– dark cycle (lights on at 0700 h and off at 1900 h). Food and water were provided ad libitum by placing chow pellets and water bottles on a grid located on top of the tank. The water in the tank was changed daily throughout the PSD period. The cage control group was maintained in the same room as the experimental rats for the duration of their respective PSD group. 3.1. Genital reflexes evaluation Immediately after the end of the experimental period, the animals were tested once for genital reflexes induced by an intraperitoneal injection of apomorphine (10, 20, 40, 80, 120, and 240 Ag/kg) or saline. The intraperitoneal route is
Sleep-deprived rats injected with saline showed both PE (0.5 F 0.1) and EJ (0.2 F 0.1) during the 60-min observation period. As illustrated in Fig. 1A, the frequency of PE responses gradually increased with increasing doses of apomorphine, with maximal effects occurring at the 120Ag dose. With this dose regimen, apomorphine did not induce genital reflects in nondeprived control animals. For PEs, the ANOVA revealed significant main effects of groups [ F(1,126) = 82.8, P < .0000] and doses [ F(6,126) = 2.42, P < .02] and a significant interaction between groups and doses [ F(6,126) = 2.42, P < .02]. Responses to the lowest apomorphine dose (10 Ag/kg) did not differ from those in the control group. However, it differed from the following groups: PSD-20 ( P < .03); PSD-40 ( P < .04); PSD-80 ( P < .01), and PSD-120 Ag/kg of apomorphine ( P < .0001). Clearly, the most effective dose was 120 Ag/kg. This dose induced the highest frequency of PEs (1.1 F 0.2) and differed from the respective control group ( P < .0002); PSDsaline ( P < .002); PSD-10 ( P < .0001); PSD-20 ( P < .008); PSD-40 ( P < .006); PSD-80 ( P < .05), and PSD-240 Ag/kg of apomorphine ( P < .002). The 240 Ag/kg dose resulted in 0.5 F 0.1 episodes, and these values were similar to those observed after saline injections in PSD rats (0.5 F 0.1 and 0.2 F 0.1, for PEs and EJs, respectively). Some sleep-deprived rats also displayed ejaculatory responses. However, as shown in Fig. 1, this response was not enhanced by apomorphine in the dose range tested. Fig. 1B shows the percentage of animals displaying genital reflexes elicited by saline or apomorphine. Chisquare tests revealed that the percentage of animals in all
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Fig. 1. Effect of intraperitoneal apomorphine doses (Ag/kg) on genital reflexes (PE and EJ) in sleep-deprived or home-cage-maintained (control) rats. The control group represented all nondeprived groups since none showed genital reflexes independent of the dose administered. Panel A: Frequency of genital reflexes events expressed as mean F S.E.M. of 10 rats per group (PSD-80, n = 9). * Different from control rats; #Different from PSD-saline, PSD-20, -40, -80, and -240; §Different from PSD-10 (ANOVA followed by Duncan’s test). Panel B: Percentage of animals displaying genital reflexes. * Different from control rats (chi-square test).
PSD groups (except for the PSD-10 Ag/kg group) was significantly higher than in the respective nondeprived control groups (all Ps < .01). No significant group differences in percentage of animals showing EJ were seen among sleep-deprived groups.
5. Discussion The present results are in agreement with our previous observations that DA agonists, such as cocaine [1– 5] and amphetamine [6], increase the effects of PSD on genital reflexes in male rats. Apomorphine given intraperitoneally in doses between 10 and 120 Ag significantly potentiated genital reflexes in sleep-deprived rats. The absence of apomorphine effects in rats that were not sleep deprived is likely due to the route of administration used. Studies using subcutaneous apomorphine consistently report induction of PE within this range dose [9,17]. An early study by Barros et al. [8] demonstrated that apomorphine given subcutaneously was 7.5 times as effective as similar doses given by the intraperitoneal route. On the other hand, the use of the intraperitoneal route is useful in
providing a zero baseline in nondeprived animals against which to observe effects of sleep deprivation without concerns about possible ceiling effects. In the present experiment, sleep deprivation alone elicited spontaneous PEs, and this effect was potentiated by apomorphine, especially at the 120 Ag/kg dose. This potentiating effect of apomorphine in PSD rats was dose dependent when frequency was evaluated, although not when percentages of animals displaying erections were considered. The potentiating effect of apomorphine in PSD rats is consistent with other indications of DA receptor supersensitivity induced by sleep deprivation [40 – 42]. DA agonists have long been known to facilitate masculine sexual behavior [10,16,31]. Three major integrative systems control sexual motivation and genital and somatomotor responses in male rats [24]; sensory input from a receptive female and/ or the act of copulation are thought to elicit a release of DA in each of these systems [25,29]. The nigrostriatal DA system is involved in initiation and execution of movements and is thought to contribute to the somatomotor patterns of pursuit and mounting of the female [38]. The mesolimbic DA system is critical for appetitive behavior and reinforcement, including sexual behavior (for a review, see Ref.
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[26]). As an example, DA-depleting lesions (6-OHDA) of the nucleus accumbens decreased the numbers of both apomorphine-induced and noncontact erections [28]. The preoptic area (POA), the third integrative system for sexual behavior, is implicated in both copulatory mechanisms [30] and sleep generation, suggesting that it may be important for paradoxical sleep (PS) erectile control. Lesions of the lateral POA result in a severe disruption in PS erectile activity while leaving PS architecture otherwise intact, whereas similar lesions restricted to the medial POA had little effect on genital reflexes or sleep –wake architecture. Neither type of lesion affects the occurrence of PS, although lateral POA lesions significantly decrease slow wave sleep [39]. Finally, the disruptive effects of lateral POA lesioning on erectile activity were specific to PS, leaving waking-state erections intact. This suggests that the POA may be a potential locus for the effects observed here. Moreover, apomorphine injected into the medial POA increased erections and anteroflexions as well as seminal emissions [22] and facilitated copulation by decreasing mount latency and increased number of EJ in rats that copulate [21]. A stimulatory role for DA at this site has been suggested by the increase in DA level in the medial POA during precopulatory phase, which points to a potential role in the anticipatory phase in addition to its well-established role on performance [23]. A final potential site of action of apomorphine in the brain in causing erection is thought to be the paraventricular nucleus (PVN) of the hypothalamus. Lesions of this area prevent erections induced by apomorphine [7]. Injections of apomorphine into the PVN stimulate erections [13] without inducing stereotypy and hypermotility in the rat [31]. Systemic apomorphine has PVN-selective effects at doses relevant to erectile responses, as verified by c-fos labeling techniques [15]. Moreover, apomorphine also activates oxytocinergic neurons in the PVN [32]. Bilateral electrolytic lesions of the PVN prevent yawning and penile induction induced by oxytocin, although these lesions appear to have little effect on copulation behavior [7]. These findings suggest that the PVN may be a specific site where DA and oxytocin agonists act to induce PE in rats [19]. Several studies have suggested a complex functional interaction between D1 and D2 DA receptors in the expression of many DA-mediated behaviors. Some reports state that PSD induces substantially different effects on D1 and D2 binding sites throughout the brain, such as an increased density of D1 type receptors in the mesolimbic pathway, in particular in nucleus accumbens, olfactory tubercle, and septum, and no alteration in the corpus striatum in rats [14]. However, after examining binding in 30 brain regions, an autoradiographic study from our laboratory [35] did not detect D1 binding changes in the nucleus accumbens or in the olfactory tubercle, whereas D2 binding was significantly increased after PSD in all brain regions examined. The most pronounced effect of PSD was observed in the
nucleus accumbens, which is involved in male sexual motivation and may be yet another site mediating the present effects. DA D2 receptor supersensitivity may provide a possible mechanism to account for the PSD-induced effects on spontaneous genital reflexes and their potentiation by apomorphine. Facilitation of sexual behavior can be induced by stimulation of postsynaptic D2 receptors, which play the main role in the control of male copulatory behavior, although a modulatory role of D1 receptors cannot be ruled out [31]. Indeed, supersensitivity of D2 receptors after PSD may be sufficient to account for increases in other apomorphine-induced effects in sleep-deprived rats, including stereotypy, aggressive behaviors [40,41], and decreased yawning behavior [41,42] in response to low doses of apomorphine in these animals. Apomorphine has been extensively studied in animals (in particular rodents) and is a prototypical dopaminergic activator of brain pathways involved in erectile function. Since it has also been used extensively in human clinical trials [18,27,36], comparisons can be drawn directly between human and animal data. Moreover, studies involving apomorphine can also be used as a point of departure for examining the control of central erectile pathways and their putative role in other aspects of sexual response [19]. In summary, the present results are consistent with other evidence of dopaminergic involvement in sexual function and more specific effects of sleep deprivation on genital reflexes. The potentiating effects of apomorphine on the latter are consistent with evidence of altered DA receptor function induced by sleep deprivation. While D2 supersensitivity appears to be a reasonable mechanism for these effects, future work should address the specific identity of DA receptors involved in this effect. Acknowledgements The authors would like to express their cordial thanks to Marilde Costa, Waldemarks Leite, and Tome Pimentel for assistance during the project. The valuable critical comments of Prof. Jose´ N. Nobrega are greatly appreciated. This work was supported by grants from Associacß a˜o Fundo de Incentivo a` Psicofarmacologia (AFIP) and FAPESP (#01/ 04329-0 to M.L.A. and CEPID #98/14303-3 to S.T.).
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The effect of apomorphine on genital reflexes in male rats deprived of paradoxical sleep Monica L. Andersen*, Magda Bignotto, Sergio Tufik Department of Psychobiology, Universidade Federal de Sa˜o Paulo, Escola Paulista de Medicina (UNIFESP/EPM), Rua Napolea˜o de Barros, 925 Vila Clementino, Sa˜o Paulo SP-04024-002, Brazil Received 29 July 2002; received in revised form 13 June 2003; accepted 23 July 2003
Abstract Drugs that stimulate dopamine (DA) systems can stimulate sexual arousal in male rats and humans, and previous work has shown that cocaine enhances genital reflexes [penile erection (PE) and ejaculation (EJ)] in rats deprived of paradoxical sleep (PS). The present study sought to expand the latter finding by assessing the effects of DA receptor agonist apomorphine in sleep-deprived rats. Apomorphine in doses ranging from 10 to 240 Ag/kg was administered intraperitoneally to rats that had been deprived of sleep for 4 days and to normal controls, and the incidence of PEs and EJs was measured for 60 min. Sleep deprivation alone induced PE and this effect was potentiated by apomorphine, with maximal effects occurring with the 120 Ag/kg dose; results for this dose group differed from those of PSD groups treated with 0, 10, 20, 40, 80, and 240 Ag/kg of apomorphine. Sleep deprivation alone also induced spontaneous EJ, but this response was not potentiated by apomorphine in the dose range tested. We suggest that the potentiating effects of apomorphine on PE are likely due to PSD-induced DA receptor supersensitivity. D 2003 Elsevier Inc. All rights reserved. Keywords: Paradoxical sleep deprivation; Genital reflexes; Apomorphine; Dopamine; Male rat
1. Introduction A variety of dopaminergic agonists have been described as being able to elicit and/or amplify sexual behavior in male rats, whereas dopamine (DA) receptor blockers have the opposite effect (for a review, see Ref. [33]). The physiological significance of these findings has been emphasized recently by the assessment of neurochemical changes in mating animals. The use of the D1/D2 DA receptor agonist apomorphine for the treatment of erectile dysfunction provides further strong support for an involvement of dopaminergic systems in the control of sexual function [16]. Behavioral studies in rats support the hypothesis that apomorphine in relatively low doses increases the number of erections [11] and penile anteroflexions in restrained supine rats [22,37]. A number of investigators have found pronounced behavioral supersensitivity to directly and indirect-
* Corresponding author. Tel.: +55-11-5539-0155; fax: +55-11-55725092. E-mail address: [email protected] (M.L. Andersen). 0031-9384/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2003.07.006
ly acting DA agonists following prolonged paradoxical sleep deprivation (PSD). These findings have led to the hypothesis that modification in sensitivity to dopaminergic drug by PSD is due to a functional hyperactivity of the DA systems. Early studies on the effect of PSD on sexual behavior produced controversial results. Some reports described increased sexual performance in PSD male rats [34,44] and facilitation by PSD of the effects of testosterone [43] or estrogen [12] on rat sexual behavior. In another study, however, 4 days of PSD did not significantly alter male sexual performance [20]. Experiments in this laboratory indicated that PSD for 96 h in rats induced genital reflexes after cocaine injection, as reflected in the number of animals that spontaneously displayed penile erections (PEs) and ejaculations (EJs) in the absence of a female [1– 5]. Furthermore, these genital effects were observed in old [3] as well as in young PSD rats [5]. These experiments indicated that PSD and cocaine elicit genital reflexes much more markedly together than separately [2]. In order to better elucidate the effect of PSD on genital reflexes, we evaluated the effects of the DA agonist apomorphine following PSD in male rats.
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2. Methods 2.1. Animals Three-month-old Wistar male rats from our facilities were used in this experiment. The vivarium was maintained on a 12:12-h light– dark photoperiod (lights on 0700 –1900 h) at an ambient temperature of 22 jC. Laboratory chow and water were provided ad libitum. All procedures used in the present study complied with the Guide for the Care and Use of Laboratory Animals, and the experimental protocol was approved by the Ethical Committee of UNIFESP (CEP N. 205/02).
less effective than the subcutaneous route, but since prior observations indicated that sleep deprivation by itself can induce spontaneous genital reflexes, the intraperitoneal route was chosen in order to preclude the possibility of ceiling effects. Controls were handled in the same way as the PSD animals. Testing was conducted in a transparent cylindrical cage (32 cm in diameter and 30 cm in height). The number of spontaneous PEs and EJs was assessed for 60 min. PEs were counted only when the rat displayed and bent down to lick its penis in full erection. EJs were scored by the number of ejaculatory plugs. The behavioral observations were carried out between 0900 and 1100 h. Different groups of rats and different cages were used in each series of experiments; cages were cleaned thoroughly after each test.
2.2. Drugs 3.2. Statistical analysis Apomorphine HCL was purchased from Sigma (St. Louis, MO, USA). The drug was mixed immediately before testing in saline solution containing 0.2% ascorbic acid. The solution was injected intraperitoneally in a volume of 1 ml/ kg bw.
Data were expressed as mean F S.E.M. Differences between groups were statistically assessed by two-way analysis of variance (ANOVA), followed by the Duncan test for a posteriori comparison of means. The significance level was set at P < .05.
3. Paradoxical sleep deprivation 4. Results One hundred and forty sexually naive rats were randomly divided into PSD (n = 70) and home cage groups (n = 70). Seven dosage levels of apomorphine were used and each group consisted of 10 rats. For PSD, 14 narrow circular platforms (6.5 cm in diameter) were placed inside a tiled tank (123 44 44 cm) filled with water to within 1 cm of their upper surface. Ten animals were placed on the platforms in each tank, an arrangement that allowed them to move around by leaping from one platform to another. One tank was used for each group. In this procedure, when an animal reaches the paradoxical phase of sleep (PS), the accompanying muscle atonia causes it to touch the water and wake up. Under these conditions, this procedure causes a complete loss of PS in each of the 4 days (Machado RB, Hipolide DC, Benedito AA, Tufik S, submitted for publication). Throughout the study, the experimental room was maintained at a controlled temperature (23 F 1 jC) and light– dark cycle (lights on at 0700 h and off at 1900 h). Food and water were provided ad libitum by placing chow pellets and water bottles on a grid located on top of the tank. The water in the tank was changed daily throughout the PSD period. The cage control group was maintained in the same room as the experimental rats for the duration of their respective PSD group. 3.1. Genital reflexes evaluation Immediately after the end of the experimental period, the animals were tested once for genital reflexes induced by an intraperitoneal injection of apomorphine (10, 20, 40, 80, 120, and 240 Ag/kg) or saline. The intraperitoneal route is
Sleep-deprived rats injected with saline showed both PE (0.5 F 0.1) and EJ (0.2 F 0.1) during the 60-min observation period. As illustrated in Fig. 1A, the frequency of PE responses gradually increased with increasing doses of apomorphine, with maximal effects occurring at the 120Ag dose. With this dose regimen, apomorphine did not induce genital reflects in nondeprived control animals. For PEs, the ANOVA revealed significant main effects of groups [ F(1,126) = 82.8, P < .0000] and doses [ F(6,126) = 2.42, P < .02] and a significant interaction between groups and doses [ F(6,126) = 2.42, P < .02]. Responses to the lowest apomorphine dose (10 Ag/kg) did not differ from those in the control group. However, it differed from the following groups: PSD-20 ( P < .03); PSD-40 ( P < .04); PSD-80 ( P < .01), and PSD-120 Ag/kg of apomorphine ( P < .0001). Clearly, the most effective dose was 120 Ag/kg. This dose induced the highest frequency of PEs (1.1 F 0.2) and differed from the respective control group ( P < .0002); PSDsaline ( P < .002); PSD-10 ( P < .0001); PSD-20 ( P < .008); PSD-40 ( P < .006); PSD-80 ( P < .05), and PSD-240 Ag/kg of apomorphine ( P < .002). The 240 Ag/kg dose resulted in 0.5 F 0.1 episodes, and these values were similar to those observed after saline injections in PSD rats (0.5 F 0.1 and 0.2 F 0.1, for PEs and EJs, respectively). Some sleep-deprived rats also displayed ejaculatory responses. However, as shown in Fig. 1, this response was not enhanced by apomorphine in the dose range tested. Fig. 1B shows the percentage of animals displaying genital reflexes elicited by saline or apomorphine. Chisquare tests revealed that the percentage of animals in all
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Fig. 1. Effect of intraperitoneal apomorphine doses (Ag/kg) on genital reflexes (PE and EJ) in sleep-deprived or home-cage-maintained (control) rats. The control group represented all nondeprived groups since none showed genital reflexes independent of the dose administered. Panel A: Frequency of genital reflexes events expressed as mean F S.E.M. of 10 rats per group (PSD-80, n = 9). * Different from control rats; #Different from PSD-saline, PSD-20, -40, -80, and -240; §Different from PSD-10 (ANOVA followed by Duncan’s test). Panel B: Percentage of animals displaying genital reflexes. * Different from control rats (chi-square test).
PSD groups (except for the PSD-10 Ag/kg group) was significantly higher than in the respective nondeprived control groups (all Ps < .01). No significant group differences in percentage of animals showing EJ were seen among sleep-deprived groups.
5. Discussion The present results are in agreement with our previous observations that DA agonists, such as cocaine [1– 5] and amphetamine [6], increase the effects of PSD on genital reflexes in male rats. Apomorphine given intraperitoneally in doses between 10 and 120 Ag significantly potentiated genital reflexes in sleep-deprived rats. The absence of apomorphine effects in rats that were not sleep deprived is likely due to the route of administration used. Studies using subcutaneous apomorphine consistently report induction of PE within this range dose [9,17]. An early study by Barros et al. [8] demonstrated that apomorphine given subcutaneously was 7.5 times as effective as similar doses given by the intraperitoneal route. On the other hand, the use of the intraperitoneal route is useful in
providing a zero baseline in nondeprived animals against which to observe effects of sleep deprivation without concerns about possible ceiling effects. In the present experiment, sleep deprivation alone elicited spontaneous PEs, and this effect was potentiated by apomorphine, especially at the 120 Ag/kg dose. This potentiating effect of apomorphine in PSD rats was dose dependent when frequency was evaluated, although not when percentages of animals displaying erections were considered. The potentiating effect of apomorphine in PSD rats is consistent with other indications of DA receptor supersensitivity induced by sleep deprivation [40 – 42]. DA agonists have long been known to facilitate masculine sexual behavior [10,16,31]. Three major integrative systems control sexual motivation and genital and somatomotor responses in male rats [24]; sensory input from a receptive female and/ or the act of copulation are thought to elicit a release of DA in each of these systems [25,29]. The nigrostriatal DA system is involved in initiation and execution of movements and is thought to contribute to the somatomotor patterns of pursuit and mounting of the female [38]. The mesolimbic DA system is critical for appetitive behavior and reinforcement, including sexual behavior (for a review, see Ref.
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[26]). As an example, DA-depleting lesions (6-OHDA) of the nucleus accumbens decreased the numbers of both apomorphine-induced and noncontact erections [28]. The preoptic area (POA), the third integrative system for sexual behavior, is implicated in both copulatory mechanisms [30] and sleep generation, suggesting that it may be important for paradoxical sleep (PS) erectile control. Lesions of the lateral POA result in a severe disruption in PS erectile activity while leaving PS architecture otherwise intact, whereas similar lesions restricted to the medial POA had little effect on genital reflexes or sleep –wake architecture. Neither type of lesion affects the occurrence of PS, although lateral POA lesions significantly decrease slow wave sleep [39]. Finally, the disruptive effects of lateral POA lesioning on erectile activity were specific to PS, leaving waking-state erections intact. This suggests that the POA may be a potential locus for the effects observed here. Moreover, apomorphine injected into the medial POA increased erections and anteroflexions as well as seminal emissions [22] and facilitated copulation by decreasing mount latency and increased number of EJ in rats that copulate [21]. A stimulatory role for DA at this site has been suggested by the increase in DA level in the medial POA during precopulatory phase, which points to a potential role in the anticipatory phase in addition to its well-established role on performance [23]. A final potential site of action of apomorphine in the brain in causing erection is thought to be the paraventricular nucleus (PVN) of the hypothalamus. Lesions of this area prevent erections induced by apomorphine [7]. Injections of apomorphine into the PVN stimulate erections [13] without inducing stereotypy and hypermotility in the rat [31]. Systemic apomorphine has PVN-selective effects at doses relevant to erectile responses, as verified by c-fos labeling techniques [15]. Moreover, apomorphine also activates oxytocinergic neurons in the PVN [32]. Bilateral electrolytic lesions of the PVN prevent yawning and penile induction induced by oxytocin, although these lesions appear to have little effect on copulation behavior [7]. These findings suggest that the PVN may be a specific site where DA and oxytocin agonists act to induce PE in rats [19]. Several studies have suggested a complex functional interaction between D1 and D2 DA receptors in the expression of many DA-mediated behaviors. Some reports state that PSD induces substantially different effects on D1 and D2 binding sites throughout the brain, such as an increased density of D1 type receptors in the mesolimbic pathway, in particular in nucleus accumbens, olfactory tubercle, and septum, and no alteration in the corpus striatum in rats [14]. However, after examining binding in 30 brain regions, an autoradiographic study from our laboratory [35] did not detect D1 binding changes in the nucleus accumbens or in the olfactory tubercle, whereas D2 binding was significantly increased after PSD in all brain regions examined. The most pronounced effect of PSD was observed in the
nucleus accumbens, which is involved in male sexual motivation and may be yet another site mediating the present effects. DA D2 receptor supersensitivity may provide a possible mechanism to account for the PSD-induced effects on spontaneous genital reflexes and their potentiation by apomorphine. Facilitation of sexual behavior can be induced by stimulation of postsynaptic D2 receptors, which play the main role in the control of male copulatory behavior, although a modulatory role of D1 receptors cannot be ruled out [31]. Indeed, supersensitivity of D2 receptors after PSD may be sufficient to account for increases in other apomorphine-induced effects in sleep-deprived rats, including stereotypy, aggressive behaviors [40,41], and decreased yawning behavior [41,42] in response to low doses of apomorphine in these animals. Apomorphine has been extensively studied in animals (in particular rodents) and is a prototypical dopaminergic activator of brain pathways involved in erectile function. Since it has also been used extensively in human clinical trials [18,27,36], comparisons can be drawn directly between human and animal data. Moreover, studies involving apomorphine can also be used as a point of departure for examining the control of central erectile pathways and their putative role in other aspects of sexual response [19]. In summary, the present results are consistent with other evidence of dopaminergic involvement in sexual function and more specific effects of sleep deprivation on genital reflexes. The potentiating effects of apomorphine on the latter are consistent with evidence of altered DA receptor function induced by sleep deprivation. While D2 supersensitivity appears to be a reasonable mechanism for these effects, future work should address the specific identity of DA receptors involved in this effect. Acknowledgements The authors would like to express their cordial thanks to Marilde Costa, Waldemarks Leite, and Tome Pimentel for assistance during the project. The valuable critical comments of Prof. Jose´ N. Nobrega are greatly appreciated. This work was supported by grants from Associacß a˜o Fundo de Incentivo a` Psicofarmacologia (AFIP) and FAPESP (#01/ 04329-0 to M.L.A. and CEPID #98/14303-3 to S.T.).
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