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Association of paradoxical sleep deprivation and ecstasy (MDMA) enhances genital reflexes in male rats
Behavioural Brain Research 170 (2006) 287–292
Research report
Association of paradoxical sleep deprivation and ecstasy (MDMA) enhances genital reflexes in male rats M.L. Andersen a,∗ , J.C. Perry a , M.C. Battisti a , M.B. Calzavara b , J.L. Costa c , O.N. Neto c , R. Frussa-Filho b , S. Tufik a a
Department of Psychobiology - Universidade Federal de S˜ao Paulo, Rua Napole˜ao de Barros, 925, Vila Clementino - SP - 04024-002, S˜ao Paulo, Brazil b Department of Pharmacology - Universidade Federal de S˜ ao Paulo (UNIFESP), S˜ao Paulo, SP, Brazil c Instrumental Analysis Laboratory, Criminalistic Institute, S˜ ao Paulo, SP, Brazil Received 8 December 2005; received in revised form 24 February 2006; accepted 7 March 2006 Available online 18 April 2006
Abstract Ecstasy ((±)3,4-methylenedioxymethamphetamine, MDMA) is a psychostimulant and a synthetic derivative of amphetamine that, according to its consumers, promotes the enhancement of sexual pleasure. This study sought to investigate the effects of ecstasy in the genital reflexes of paradoxical sleep deprived (PSD) male rats. Distinct groups of PSD rats were administered with saline or different doses of ecstasy. The incidence of genital reflexes was verified for 100 min. The four doses that were used induced genital reflexes in PSD animals and these significantly differed from their respective treated control groups. Under the influence of two intermediary doses (2.5 and 5 mg/kg), all animals displayed erection and ejaculation. The frequency of genital reflexes was also significantly greater than in relation to the PSD-saline group. The comparison between cocaine and ecstasy in PSD rats revealed that ecstasy induced more erections and ejaculations than cocaine. Thus, the present results showed a great enhancement of the genital reflexes of PSD rats that might have occurred due to serotoninergic alterations induced by this illicit substance when associated to sleep deprivation. © 2006 Elsevier B.V. All rights reserved. Keywords: Sleep deprivation; Ecstasy; Cocaine; Erection; Ejaculation; Serotonin
1. Introduction Recreational use of (±)3,4-methylenedioxymethamphetamine (MDMA, known as “Ecstasy”), a ring-substituted amphetamine derivative that acts indirectly by stimulating the release of brain monoamines [40] was elicited substantial interest over the last few years since its consumption is widespread and growing in western Europe and north and south America [2,16]. The predominant mechanism of action of MDMA has been associated with the serotonergic system. Indeed, the binding of MDMA to its paramount target in the brain, the presynaptic 5-HT transporter (SERT), inhibits 5-HT uptake, and enhances SERTmediated exchange and release of 5-HT [26,42]. Following the early release of 5-HT, there is a long-term depletion of the neurotransmitter in the brain. Mutant mice deficient in SERT were insensitive to MDMA [12], confirming a major role of that trans-
∗
Corresponding author. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092. E-mail address: [email protected] (M.L. Andersen).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.03.005
porter in the mode of action of MDMA [42]. Ample evidence indicates that the effect of MDMA on the serotonergic system is closely associated with activation of dopaminergic pathways and release of DA [38,46]. The enhanced release of DA by MDMA may partially result from reversal of DA transport [30]. Considering the particular concern of the elevated rate of ecstasy use among the young [14,33], the effect of this drug on sexual behaviour becomes evident. This association is pertinent since MDMA may increase sexual receptivity [15], which has led some to call it the “love drug” [35] or the “hug drug” [43]. Our group has documented the effects of psychostimulant drugs like cocaine and methamphetamine on genital reflexes (erection-PE and ejaculation-EJ) in paradoxical sleep deprived rats (PSD) [3,9] as well as the neurochemical and hormonal basis of these behaviours potentiated by drugs in PSD male rats [10]. In fact, sleep deprivation is a consequent event related to psychostimulant use [29]. Since MDMA has been related to affect male sexuality (PE and EJ) [15], and ecstasy users are more likely to have multiple sex partners that in turn can be associated with increased rates of sexually transmitted diseases
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[14], and finally, to better comprehend the interaction of the 5HT and DA systems that are altered by sleep deprivation [25,44] the association of this substance and sleep deprivation needs to be further investigated.
studies. Penile erection (PE) was counted only when the rat stood on its hindlimbs, bent its body forward, bent its head down to reach the genital area, held and licked its penis in full erection and displayed hip movements. The erect penis was always visible. Ejaculation (EJ) was scored as the number of ejaculatory plugs. The number of spontaneous PE and EJ, as well as latencies (time elapsed between injection and the first genital reflex) were assessed for 100 min.
2. Materials and methods 2.1. Animals One hundred twelve adult male Wistar rats bred in our facilities weighing 300–360 g were used. They were maintained in a 12-h light/12-h dark cycle photoperiod (lights on 07:00–19:00 h) at room temperature of 22 ◦ C. Laboratory chow and water were provided ad libitum. Rats used in this study were maintained and treated in accordance with the guidelines established by the Ethical and Practical Principles of the Use of Laboratory Animals [5] and all procedures used in the present study complied with the Guide for the Care and Use of Laboratory Animals. Each animal was tested only once.
2.2. Drugs Four doses of Ecstasy (1.25, 2.5, 5 and 10 mg/kg) were administered allowing for the derivation of dose–response for the percentage and frequency of genital reflexes, with independent groups of 10 rats being used for each dose. The drug was dissolved in sterile saline (and two drops of tween) and administered i.p. immediately before the behavioural observation in volumes of 1 mL/kg. For comparison, the dose of 7 mg/kg (i.p.) of cocaine was elected based on our previous studies. The PSD group designed as control group was challenged with sterile saline injection. All the rats were administered intraperitoneally with ecstasy and placed immediately in the observation cages for the evaluation of genital reflexes. Since our proposal was to investigate the effects of ecstasy on erectile function, and further, to draw parallels to human studies, we elected to use ecstasy pills that were donated to us by the Criminalistic Institute (S˜ao Paulo). The material was quantified by a validated high performance liquid chromatography/fluorescence detection (HPLC/FD) method. To confirm the absence of other active substance, the material was analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) screening method. Since our previous studies have documented that cocaine potentiated genital reflexes in PSD rats, we made use of homecage and PSD animals challenged with cocaine in an attempt to compare the effects induced by ecstasy. Cocaine was used at the dose of 7 mg/kg since our previous studies showed that this dose was the most effective in potentiating genital reflexes in PSD rats.
2.5. Statistical analyses For statistical analyses of the numbers of animals displaying PE and EJ, the Fisher Exact Probability test was used to assess differences between groups. One-way analysis of variance (ANOVA) was used to analyze the frequency and latency data to determine possible group effects. Comparison between cocaine and ecstasy on genital reflexes frequency were analyzed by ANOVA with treatment as one factor and the group as the second factor. In order to make specific group comparisons, post hoc Duncan test was performed. Kolmogorov–Smirnov test showed of normal distribution between the groups. Values are expressed as mean ± S.D. The level of significance was set at p < 0.05.
3. Results 3.1. Effects of ecstasy on penile erections Fig. 1A and B shows the effects of four doses of ecstasy on PE. In the control groups a single ecstasy injection regardless the dose did not modify the number of rats that displayed PE compared to CTRL-saline group. Forty percent of PSD-saline rats present PE. The injection of ecstasy at 1.25 mg/kg resulted
2.3. Paradoxical sleep deprivation The animals (n = 10 group) were subjected to PSD for a period of 96 h using the modified multiple platform method. Rats were placed in a tilled water tank (123 cm × 44 cm × 44 cm) containing 14 circular platforms, 6.5 cm in diameter, in water up to within 1 cm of their upper surface. The rats could thus move inside the tank by jumping from one platform to another. When they reached the paradoxical phase of sleep, muscle atonia caused them to fall into the water and wake up. Throughout the study, the experimental room was maintained under controlled temperature (23 ± 1 ◦ C) and a 12-h light/12-h dark cycle (lights on 07:00–19:00 h). Food and water were provided ad libitum by placing chow pellets and water bottles on a grid located on top of the tank. Tank water was changed daily throughout the PSD period. Duration of deprivation was chosen on the basis of our previous work indicating that genital reflexes are maximal after 96 h of PSD [4].
2.4. Assessment of genital reflexes The animals were observed in experimental wire mesh cages (15 cm × 31 cm × 26 cm) containing neither water nor food. Behavioural observations were carried out between 08:00 and 10:00 h in a temperature controlled room, where animals were monitored by trained observers unaware of group membership; inter-rater reliability had been established in previous
Fig. 1. Effects of ecstasy (1.25, 2.5, 5 and 10 mg/kg) administration on the proportion of animals displaying penile erection (PE) (panel A) and frequency of PE (panel B) in rats. * p < 0.05 compared to respective control group, # p < 0.05 compared to PSD-saline (ANOVA followed by Duncan test). Data are expressed as mean ± S.E.M. (n = 10 group).
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in erection in 70% of the rats but did not statistically differ from PSD-saline group. Notably, the intermediary doses (2.5 and 5 mg/kg) induced PE in all animals (p’s < 0.01 compared to PSD-saline group). The dose of 10 mg/kg resulted in erection in 70% of the rats. An analysis of the proportion of PE in CTRL versus PSD rats after ecstasy treatment showed that the PSD potentiated the genital reflex in all doses, as depicted in Fig. 1A. One-way ANOVA revealed a significant group effect on PE frequency [F(9,90) = 17.014; p < 0.001]. Ecstasy injection induced an increase of PE frequency in PSD rats compared to PSD-saline in all doses 1.25 (p < 0.03), 2.5 (p < 0.001), 5 (p < 0.001) and 10 (p < 0.02), as shown in Fig. 1B. The data indicate that there were no differences in PE frequency of CTRL animals treated with saline or ecstasy. When compared to CTRL group, PE was significantly increased in all PSD-ecstasy groups. As for latency of PE in the PSD rats only the higher dose (10 mg/kg) induced an increase in this parameter compared to other groups [F(4,35) = 17.04; p < 0.001] (data not shown).
the rats showed an increase of EJ compared to PSD-saline (p < 0.001). An ANOVA test showed a highly significant effect of drug treatment in the frequency of animals exhibiting genital reflexes [F(9,90) = 20.40; p < 0.001]. There was no significant difference between CTRL groups after ecstasy administration and neither did PSD alter the frequency of EJ in saline treated rats. When PSD rats received 2.5, 5 or 10 mg/kg injection of ecstasy, the results showed an increase of EJ frequency compared to PSDsaline (p < 0.001). Moreover, EJ frequency in PSD animals was significantly increased after ecstasy administration (all doses) compared to CTRL groups that received the same treatment. No significant differences were observed in EJ latency among rats after ecstasy administration [F(4,32) = 1.76; p = 0.16] (data not shown).
3.2. Effects of ecstasy on ejaculation
As shown in Fig. 3A and B, 4 days of PSD induced PE in 40% of the male rats, and 20% ejaculated. A single cocaine injection (7 mg/kg) potentialized the genital reflexes in PSD rats. Of the 10 animals that were administered cocaine, 9 responded with PE
Fig. 2A and B shows the effects of ecstasy on EJ. Although no statistically significant alterations were found in the percentage of CTRL animals treated with ecstasy displaying EJ, the ejaculatory response was completely modified after ecstasy administration in PSD rats. At the three higher doses of ecstasy
Fig. 2. Effects of ecstasy (1.25, 2.5, 5 and 10 mg/kg) administration on the proportion of animals displaying ejaculation (EJ) (panel A) and frequency of EJ in rats (panel B). * p < 0.05 compared to respective control group, # p < 0.05 compared to PSD-saline (ANOVA followed by Duncan test). Data are expressed as mean ± S.E.M. (n = 10 group).
3.3. Comparison between cocaine and ecstasy in genital reflexes
Fig. 3. Effects of saline, cocaine (7 mg/kg) or ecstasy (5 mg/kg) administration on the proportion of animals and frequency of PE (panel A) and EJ (panel B) in paradoxical sleep deprived rats. Data are expressed as mean (n = 10 group).
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(p < 0.001) and 7 of them ejaculated (p < 0.05). Similar results were observed after ecstasy administration (5 mg/kg). In fact, the three intermediary doses of ecstasy induced PE and EJ in 100% of the rats (p < 0.001). ANOVA for PE and EJ frequencies revealed significant main effects for group [F(1,54) = 81.98; p < 0.001 and F(1,54) = 37.55; p < 0.001], drug [F(2,54) = 21.43; p < 0.001 and F(2,54) = 12.05; p < 0.001] and a significant interaction between group × drug [F(2,54) = 19.58; p < 0.001 and F(2,54) = 7.05; p < 0.01], respectively. Post hoc test showed that there were no differences of PE and EJ frequencies among CTRL groups regardless the drug. Acute cocaine administration in PSD rats increases frequency of PE and EJ compared to CTRL-cocaine and PSD-saline rats (p < 0.001). PSD rats challenged with ecstasy injection displayed more erection and ejaculation events than the CTRL-ecstasy, PSD-saline and PSD-cocaine groups (p < 0.001). One-way ANOVA showed a treatment effect in latency time to first PE in PSD rats [F(2,21) = 5.36; p < 0.02]. Post hoc test indicated that PSD-ecstasy (10 mg/kg) groups had significantly higher PE latency compared to PSD-saline and PSD-cocaine rats (p < 0.03). No statistically significant differences were observed in the latency of PE after cocaine administration. For EJ, no statistically significant alterations were found in this parameter after cocaine or ecstasy administration [F(2,16) = 0.11; p = 0.89]. 4. Discussion Our results showed that ecstasy is clearly implicated with sexual enhancement in PSD male rats by markedly increasing not only the number of animals displaying PE/EJ but also the frequencies of their occurrence. In fact, at doses of 2.5 and 5 mg/kg all rats had erections and ejaculated. In comparison to cocaine, another drug that also potentiated genital reflexes in male PSD rats, ecstasy produced a greater frequency of erections (2.3 ± 0.8 versus 4.7 ± 0.8). Curiously, the period in which the rats displayed erection was more prolonged in ecstasy (100 min) challenged rats than in cocaine (30 min). The results of this study have enabled us to extend previous findings by assessing the effects of ecstasy on the genital reflexes in PSD male rats. In the 1980’s MDMA became a popular recreational drug especially among the younger population, and was dubbed the “love drug” because it elicited empathy and euphoria [41]. Recently, however, MDMA has received special attention due to its potential for abuse and neurotoxicity [34,40]. Its reputed aphrodisiacal properties have been questioned but it is accepted that MDMA evokes great psychomotor excitation, a rise of selfesteem, enhanced alertness and promotes greater receptivity in sensual aspects, however, without the desire to engage in sexual activity [15]. In view of the widespread degeneration of presynaptic serotoninergic axon terminals and 5-HT depletion in brain tissue after ecstasy (for review [27]), and the expressive participation serotoninergic receptors have in the genital reflexes of PSD rats (Andersen et al., submitted for publication), we proposed to investigate the effects of ecstasy within the confines of this
context. The current data demonstrated the marked effect on erectile function, since the two intermediary doses (2.5 and 5 mg/kg) resulted in all animals displaying PE and the frequency of these erections was significantly higher than that observed in rats under the influence of cocaine. Interestingly, latency of the first erection in PSD rats was longer in ecstasy-injected rats (46.4 min after the 10 mg/kg dose) than in PSD-saline (5.6 min) and PSD-cocaine (6.6 min) administered rats. The grounds for such effect, although still unknown, allow for speculation of the differences in neurotransmissions pathways, since the action of ecstasy is attributed to the serotonergic system, whereas cocaine is known to act mainly on DA pathways. Yet another factor that deserves attention is the expressive increase in the number of animals that ejaculated during the execution of the experiment. All animals injected with the three highest doses of ecstasy had a significant higher frequency of ejaculatory reflexes (1.7 ± 0.2; for 2.5 mg/kg) that those administered with saline (0.2 ± 0.1; mean values and standard error are expressed). Besides the reports of MDMA eliciting spontaneous EJ in rats [13], there is no report of this drug inducing EJ in men. In fact, MDMA might inhibit the ability to produce an erection and delay EJ in men [15] as well as produced a transient disruption of male copulatory behaviour in rats [18]. MDMA’s unique effect in the elicitation of seminal plugs in PSD rats may be due to differences in the absorption, disposition and metabolism of the drug between rats and humans. The investigation of the interaction of this drug with the sleep deprivation paradigm in the light of genital reflexes, as well as with other behaviours, opens an entirely new line of research. In the last decade there has been considerable research on the pharmacology of MDMA in experimental animals and humans (e.g. Psychopharmacology, vol. 173, 2004), and there are clear indications that much of the information is relevant to the clinical pharmacology and toxicology of the drug. It is, of course, always difficult to extrapolate data in animals to predict problems that may be encountered in man [21]. It is well-documented that ecstasy produces dose-related reductions in brain concentrations of 5-HT [34,40]. Similarly, in humans, positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging studies have demonstrated sustained decrements in brain 5-HT transporter density [37,39]. In turn, cocaine, considered by most to be the world’s most addictive drug, is a powerful euphoriant that acts on monoamine neurons to produce acute psychomotor activation and long-term alterations including psychosis and addiction. These effects are the result of cocaine-induced activity in DA pathways that project to the neostriatum and nucleus accumbens [1]. Although cocaine has a variety of pharmacological actions, one of its major effects is binding to the DA transporter to prevent DA uptake into presynaptic neurons, thus, increasing synaptic concentrations of DA [24] leading to behavioural effects. In this context, it can be stated that both drugs albeit present facilitatory effects on genital reflexes in PSD males, they present distinct mechanisms of action on the central nervous system. Although progress has been made in understanding the neurobiological basis of the effects of drugs of abuse in rats, further studies are needed at the behavioural level of interaction between
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psychostimulants and sleep deprivation, a consequent event related to cocaine use [29], and further, an inherent condition associated to ecstasy use in all-night dancing parties [22,35,36]. In fact, alertness and sleep disturbances are one of the problems most commonly reported by drug users [28]. Sleep deprivation results in a constellation of generalized symptoms leading to several catecholamine [25,31,44,45], hormonal [7,9,10] and behavioural [6,11,17,20] alterations. In particular, PSD is known to alter the functioning of several neurotransmitter systems, including DA [31,44] and 5-HT [32]. More recently, Hip´olide et al. [25] demonstrated significant decreases in 5-HT transporters providing further evidence that sleep deprivation results in rapid and widespread alterations in the brain. Sexual behaviour is complex and results from an intricate, well-balanced string of neurochemical events that involve a network of neurotransmitters. Additional factors may be involved since both PSD and drugs of abuse like cocaine and ecstasy stimulation also alters other systems, besides dopaminergic and serotoninergic ones. Insofar, our studies have suggested that DA D2 receptor supersensitivity [31] may provide a possible mechanism to account for the PSD induced effects on spontaneous genital reflexes and their potentiation by cocaine [8]. Indeed, facilitation of sexual behaviour can be induced by stimulation of postsynaptic D2 receptors, which play the main role in the control of male copulatory behaviour [19] although influence of 5-HT should be considered [23]. Notwithstanding, the alterations in the serotoninergic system promoted by PSD should be taken into account as an important factor in the expression of genital reflexes in ecstasy-treated males. Taken together, it can be assumed that PSD potentiates the action of the substance of abuse over genital reflexes. This effect should be interpreted with the consideration that enhancement of sexual components (e.g. erection) is but one of the effects subject to alteration by psychostimulant drugs. It must be taken into account, that several other alterations occur as consequence of these drugs such hyperthermia, tachicardia and convulsion. Before youngsters resort to drugs for enhanced sexual performance, they should re-evaluate the deleterious consequences of substances of abuse. Finally, sleep deprivation is a determinant in the action the drugs of abuse. Acknowledgements The authors would like to express their cordial thanks to Waldemarks Leite, Tome Pimentel, Alice Lima and Marilde Costa, for assistance during the project. This work was supported by grants from Associac¸a˜ o Fundo de Incentivo a` Psicofarmacologia (AFIP) and FAPESP (#01/04329-0 to M.L.A.; #04/04008-7 to J.C.P. and CEPID #98/14303-3 to S.T.). References [1] Adams DH, Hanson GR, Keefe KA. Differential effects of cocaine and methamphetamine on neurotensin/neuromedin N and preprotachykinin messenger RNA expression in unique regions of the striatum. Neuroscience 2001;102:843–51. [2] Almeida SP, Silva MTA. Ecstasy (MDMA): effects and patterns of use reported by users in S˜ao Paulo. Rev Brasil Psiquiatria 2003;25:11–7.
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Research report
Association of paradoxical sleep deprivation and ecstasy (MDMA) enhances genital reflexes in male rats M.L. Andersen a,∗ , J.C. Perry a , M.C. Battisti a , M.B. Calzavara b , J.L. Costa c , O.N. Neto c , R. Frussa-Filho b , S. Tufik a a
Department of Psychobiology - Universidade Federal de S˜ao Paulo, Rua Napole˜ao de Barros, 925, Vila Clementino - SP - 04024-002, S˜ao Paulo, Brazil b Department of Pharmacology - Universidade Federal de S˜ ao Paulo (UNIFESP), S˜ao Paulo, SP, Brazil c Instrumental Analysis Laboratory, Criminalistic Institute, S˜ ao Paulo, SP, Brazil Received 8 December 2005; received in revised form 24 February 2006; accepted 7 March 2006 Available online 18 April 2006
Abstract Ecstasy ((±)3,4-methylenedioxymethamphetamine, MDMA) is a psychostimulant and a synthetic derivative of amphetamine that, according to its consumers, promotes the enhancement of sexual pleasure. This study sought to investigate the effects of ecstasy in the genital reflexes of paradoxical sleep deprived (PSD) male rats. Distinct groups of PSD rats were administered with saline or different doses of ecstasy. The incidence of genital reflexes was verified for 100 min. The four doses that were used induced genital reflexes in PSD animals and these significantly differed from their respective treated control groups. Under the influence of two intermediary doses (2.5 and 5 mg/kg), all animals displayed erection and ejaculation. The frequency of genital reflexes was also significantly greater than in relation to the PSD-saline group. The comparison between cocaine and ecstasy in PSD rats revealed that ecstasy induced more erections and ejaculations than cocaine. Thus, the present results showed a great enhancement of the genital reflexes of PSD rats that might have occurred due to serotoninergic alterations induced by this illicit substance when associated to sleep deprivation. © 2006 Elsevier B.V. All rights reserved. Keywords: Sleep deprivation; Ecstasy; Cocaine; Erection; Ejaculation; Serotonin
1. Introduction Recreational use of (±)3,4-methylenedioxymethamphetamine (MDMA, known as “Ecstasy”), a ring-substituted amphetamine derivative that acts indirectly by stimulating the release of brain monoamines [40] was elicited substantial interest over the last few years since its consumption is widespread and growing in western Europe and north and south America [2,16]. The predominant mechanism of action of MDMA has been associated with the serotonergic system. Indeed, the binding of MDMA to its paramount target in the brain, the presynaptic 5-HT transporter (SERT), inhibits 5-HT uptake, and enhances SERTmediated exchange and release of 5-HT [26,42]. Following the early release of 5-HT, there is a long-term depletion of the neurotransmitter in the brain. Mutant mice deficient in SERT were insensitive to MDMA [12], confirming a major role of that trans-
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Corresponding author. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092. E-mail address: [email protected] (M.L. Andersen).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.03.005
porter in the mode of action of MDMA [42]. Ample evidence indicates that the effect of MDMA on the serotonergic system is closely associated with activation of dopaminergic pathways and release of DA [38,46]. The enhanced release of DA by MDMA may partially result from reversal of DA transport [30]. Considering the particular concern of the elevated rate of ecstasy use among the young [14,33], the effect of this drug on sexual behaviour becomes evident. This association is pertinent since MDMA may increase sexual receptivity [15], which has led some to call it the “love drug” [35] or the “hug drug” [43]. Our group has documented the effects of psychostimulant drugs like cocaine and methamphetamine on genital reflexes (erection-PE and ejaculation-EJ) in paradoxical sleep deprived rats (PSD) [3,9] as well as the neurochemical and hormonal basis of these behaviours potentiated by drugs in PSD male rats [10]. In fact, sleep deprivation is a consequent event related to psychostimulant use [29]. Since MDMA has been related to affect male sexuality (PE and EJ) [15], and ecstasy users are more likely to have multiple sex partners that in turn can be associated with increased rates of sexually transmitted diseases
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[14], and finally, to better comprehend the interaction of the 5HT and DA systems that are altered by sleep deprivation [25,44] the association of this substance and sleep deprivation needs to be further investigated.
studies. Penile erection (PE) was counted only when the rat stood on its hindlimbs, bent its body forward, bent its head down to reach the genital area, held and licked its penis in full erection and displayed hip movements. The erect penis was always visible. Ejaculation (EJ) was scored as the number of ejaculatory plugs. The number of spontaneous PE and EJ, as well as latencies (time elapsed between injection and the first genital reflex) were assessed for 100 min.
2. Materials and methods 2.1. Animals One hundred twelve adult male Wistar rats bred in our facilities weighing 300–360 g were used. They were maintained in a 12-h light/12-h dark cycle photoperiod (lights on 07:00–19:00 h) at room temperature of 22 ◦ C. Laboratory chow and water were provided ad libitum. Rats used in this study were maintained and treated in accordance with the guidelines established by the Ethical and Practical Principles of the Use of Laboratory Animals [5] and all procedures used in the present study complied with the Guide for the Care and Use of Laboratory Animals. Each animal was tested only once.
2.2. Drugs Four doses of Ecstasy (1.25, 2.5, 5 and 10 mg/kg) were administered allowing for the derivation of dose–response for the percentage and frequency of genital reflexes, with independent groups of 10 rats being used for each dose. The drug was dissolved in sterile saline (and two drops of tween) and administered i.p. immediately before the behavioural observation in volumes of 1 mL/kg. For comparison, the dose of 7 mg/kg (i.p.) of cocaine was elected based on our previous studies. The PSD group designed as control group was challenged with sterile saline injection. All the rats were administered intraperitoneally with ecstasy and placed immediately in the observation cages for the evaluation of genital reflexes. Since our proposal was to investigate the effects of ecstasy on erectile function, and further, to draw parallels to human studies, we elected to use ecstasy pills that were donated to us by the Criminalistic Institute (S˜ao Paulo). The material was quantified by a validated high performance liquid chromatography/fluorescence detection (HPLC/FD) method. To confirm the absence of other active substance, the material was analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) screening method. Since our previous studies have documented that cocaine potentiated genital reflexes in PSD rats, we made use of homecage and PSD animals challenged with cocaine in an attempt to compare the effects induced by ecstasy. Cocaine was used at the dose of 7 mg/kg since our previous studies showed that this dose was the most effective in potentiating genital reflexes in PSD rats.
2.5. Statistical analyses For statistical analyses of the numbers of animals displaying PE and EJ, the Fisher Exact Probability test was used to assess differences between groups. One-way analysis of variance (ANOVA) was used to analyze the frequency and latency data to determine possible group effects. Comparison between cocaine and ecstasy on genital reflexes frequency were analyzed by ANOVA with treatment as one factor and the group as the second factor. In order to make specific group comparisons, post hoc Duncan test was performed. Kolmogorov–Smirnov test showed of normal distribution between the groups. Values are expressed as mean ± S.D. The level of significance was set at p < 0.05.
3. Results 3.1. Effects of ecstasy on penile erections Fig. 1A and B shows the effects of four doses of ecstasy on PE. In the control groups a single ecstasy injection regardless the dose did not modify the number of rats that displayed PE compared to CTRL-saline group. Forty percent of PSD-saline rats present PE. The injection of ecstasy at 1.25 mg/kg resulted
2.3. Paradoxical sleep deprivation The animals (n = 10 group) were subjected to PSD for a period of 96 h using the modified multiple platform method. Rats were placed in a tilled water tank (123 cm × 44 cm × 44 cm) containing 14 circular platforms, 6.5 cm in diameter, in water up to within 1 cm of their upper surface. The rats could thus move inside the tank by jumping from one platform to another. When they reached the paradoxical phase of sleep, muscle atonia caused them to fall into the water and wake up. Throughout the study, the experimental room was maintained under controlled temperature (23 ± 1 ◦ C) and a 12-h light/12-h dark cycle (lights on 07:00–19:00 h). Food and water were provided ad libitum by placing chow pellets and water bottles on a grid located on top of the tank. Tank water was changed daily throughout the PSD period. Duration of deprivation was chosen on the basis of our previous work indicating that genital reflexes are maximal after 96 h of PSD [4].
2.4. Assessment of genital reflexes The animals were observed in experimental wire mesh cages (15 cm × 31 cm × 26 cm) containing neither water nor food. Behavioural observations were carried out between 08:00 and 10:00 h in a temperature controlled room, where animals were monitored by trained observers unaware of group membership; inter-rater reliability had been established in previous
Fig. 1. Effects of ecstasy (1.25, 2.5, 5 and 10 mg/kg) administration on the proportion of animals displaying penile erection (PE) (panel A) and frequency of PE (panel B) in rats. * p < 0.05 compared to respective control group, # p < 0.05 compared to PSD-saline (ANOVA followed by Duncan test). Data are expressed as mean ± S.E.M. (n = 10 group).
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in erection in 70% of the rats but did not statistically differ from PSD-saline group. Notably, the intermediary doses (2.5 and 5 mg/kg) induced PE in all animals (p’s < 0.01 compared to PSD-saline group). The dose of 10 mg/kg resulted in erection in 70% of the rats. An analysis of the proportion of PE in CTRL versus PSD rats after ecstasy treatment showed that the PSD potentiated the genital reflex in all doses, as depicted in Fig. 1A. One-way ANOVA revealed a significant group effect on PE frequency [F(9,90) = 17.014; p < 0.001]. Ecstasy injection induced an increase of PE frequency in PSD rats compared to PSD-saline in all doses 1.25 (p < 0.03), 2.5 (p < 0.001), 5 (p < 0.001) and 10 (p < 0.02), as shown in Fig. 1B. The data indicate that there were no differences in PE frequency of CTRL animals treated with saline or ecstasy. When compared to CTRL group, PE was significantly increased in all PSD-ecstasy groups. As for latency of PE in the PSD rats only the higher dose (10 mg/kg) induced an increase in this parameter compared to other groups [F(4,35) = 17.04; p < 0.001] (data not shown).
the rats showed an increase of EJ compared to PSD-saline (p < 0.001). An ANOVA test showed a highly significant effect of drug treatment in the frequency of animals exhibiting genital reflexes [F(9,90) = 20.40; p < 0.001]. There was no significant difference between CTRL groups after ecstasy administration and neither did PSD alter the frequency of EJ in saline treated rats. When PSD rats received 2.5, 5 or 10 mg/kg injection of ecstasy, the results showed an increase of EJ frequency compared to PSDsaline (p < 0.001). Moreover, EJ frequency in PSD animals was significantly increased after ecstasy administration (all doses) compared to CTRL groups that received the same treatment. No significant differences were observed in EJ latency among rats after ecstasy administration [F(4,32) = 1.76; p = 0.16] (data not shown).
3.2. Effects of ecstasy on ejaculation
As shown in Fig. 3A and B, 4 days of PSD induced PE in 40% of the male rats, and 20% ejaculated. A single cocaine injection (7 mg/kg) potentialized the genital reflexes in PSD rats. Of the 10 animals that were administered cocaine, 9 responded with PE
Fig. 2A and B shows the effects of ecstasy on EJ. Although no statistically significant alterations were found in the percentage of CTRL animals treated with ecstasy displaying EJ, the ejaculatory response was completely modified after ecstasy administration in PSD rats. At the three higher doses of ecstasy
Fig. 2. Effects of ecstasy (1.25, 2.5, 5 and 10 mg/kg) administration on the proportion of animals displaying ejaculation (EJ) (panel A) and frequency of EJ in rats (panel B). * p < 0.05 compared to respective control group, # p < 0.05 compared to PSD-saline (ANOVA followed by Duncan test). Data are expressed as mean ± S.E.M. (n = 10 group).
3.3. Comparison between cocaine and ecstasy in genital reflexes
Fig. 3. Effects of saline, cocaine (7 mg/kg) or ecstasy (5 mg/kg) administration on the proportion of animals and frequency of PE (panel A) and EJ (panel B) in paradoxical sleep deprived rats. Data are expressed as mean (n = 10 group).
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(p < 0.001) and 7 of them ejaculated (p < 0.05). Similar results were observed after ecstasy administration (5 mg/kg). In fact, the three intermediary doses of ecstasy induced PE and EJ in 100% of the rats (p < 0.001). ANOVA for PE and EJ frequencies revealed significant main effects for group [F(1,54) = 81.98; p < 0.001 and F(1,54) = 37.55; p < 0.001], drug [F(2,54) = 21.43; p < 0.001 and F(2,54) = 12.05; p < 0.001] and a significant interaction between group × drug [F(2,54) = 19.58; p < 0.001 and F(2,54) = 7.05; p < 0.01], respectively. Post hoc test showed that there were no differences of PE and EJ frequencies among CTRL groups regardless the drug. Acute cocaine administration in PSD rats increases frequency of PE and EJ compared to CTRL-cocaine and PSD-saline rats (p < 0.001). PSD rats challenged with ecstasy injection displayed more erection and ejaculation events than the CTRL-ecstasy, PSD-saline and PSD-cocaine groups (p < 0.001). One-way ANOVA showed a treatment effect in latency time to first PE in PSD rats [F(2,21) = 5.36; p < 0.02]. Post hoc test indicated that PSD-ecstasy (10 mg/kg) groups had significantly higher PE latency compared to PSD-saline and PSD-cocaine rats (p < 0.03). No statistically significant differences were observed in the latency of PE after cocaine administration. For EJ, no statistically significant alterations were found in this parameter after cocaine or ecstasy administration [F(2,16) = 0.11; p = 0.89]. 4. Discussion Our results showed that ecstasy is clearly implicated with sexual enhancement in PSD male rats by markedly increasing not only the number of animals displaying PE/EJ but also the frequencies of their occurrence. In fact, at doses of 2.5 and 5 mg/kg all rats had erections and ejaculated. In comparison to cocaine, another drug that also potentiated genital reflexes in male PSD rats, ecstasy produced a greater frequency of erections (2.3 ± 0.8 versus 4.7 ± 0.8). Curiously, the period in which the rats displayed erection was more prolonged in ecstasy (100 min) challenged rats than in cocaine (30 min). The results of this study have enabled us to extend previous findings by assessing the effects of ecstasy on the genital reflexes in PSD male rats. In the 1980’s MDMA became a popular recreational drug especially among the younger population, and was dubbed the “love drug” because it elicited empathy and euphoria [41]. Recently, however, MDMA has received special attention due to its potential for abuse and neurotoxicity [34,40]. Its reputed aphrodisiacal properties have been questioned but it is accepted that MDMA evokes great psychomotor excitation, a rise of selfesteem, enhanced alertness and promotes greater receptivity in sensual aspects, however, without the desire to engage in sexual activity [15]. In view of the widespread degeneration of presynaptic serotoninergic axon terminals and 5-HT depletion in brain tissue after ecstasy (for review [27]), and the expressive participation serotoninergic receptors have in the genital reflexes of PSD rats (Andersen et al., submitted for publication), we proposed to investigate the effects of ecstasy within the confines of this
context. The current data demonstrated the marked effect on erectile function, since the two intermediary doses (2.5 and 5 mg/kg) resulted in all animals displaying PE and the frequency of these erections was significantly higher than that observed in rats under the influence of cocaine. Interestingly, latency of the first erection in PSD rats was longer in ecstasy-injected rats (46.4 min after the 10 mg/kg dose) than in PSD-saline (5.6 min) and PSD-cocaine (6.6 min) administered rats. The grounds for such effect, although still unknown, allow for speculation of the differences in neurotransmissions pathways, since the action of ecstasy is attributed to the serotonergic system, whereas cocaine is known to act mainly on DA pathways. Yet another factor that deserves attention is the expressive increase in the number of animals that ejaculated during the execution of the experiment. All animals injected with the three highest doses of ecstasy had a significant higher frequency of ejaculatory reflexes (1.7 ± 0.2; for 2.5 mg/kg) that those administered with saline (0.2 ± 0.1; mean values and standard error are expressed). Besides the reports of MDMA eliciting spontaneous EJ in rats [13], there is no report of this drug inducing EJ in men. In fact, MDMA might inhibit the ability to produce an erection and delay EJ in men [15] as well as produced a transient disruption of male copulatory behaviour in rats [18]. MDMA’s unique effect in the elicitation of seminal plugs in PSD rats may be due to differences in the absorption, disposition and metabolism of the drug between rats and humans. The investigation of the interaction of this drug with the sleep deprivation paradigm in the light of genital reflexes, as well as with other behaviours, opens an entirely new line of research. In the last decade there has been considerable research on the pharmacology of MDMA in experimental animals and humans (e.g. Psychopharmacology, vol. 173, 2004), and there are clear indications that much of the information is relevant to the clinical pharmacology and toxicology of the drug. It is, of course, always difficult to extrapolate data in animals to predict problems that may be encountered in man [21]. It is well-documented that ecstasy produces dose-related reductions in brain concentrations of 5-HT [34,40]. Similarly, in humans, positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging studies have demonstrated sustained decrements in brain 5-HT transporter density [37,39]. In turn, cocaine, considered by most to be the world’s most addictive drug, is a powerful euphoriant that acts on monoamine neurons to produce acute psychomotor activation and long-term alterations including psychosis and addiction. These effects are the result of cocaine-induced activity in DA pathways that project to the neostriatum and nucleus accumbens [1]. Although cocaine has a variety of pharmacological actions, one of its major effects is binding to the DA transporter to prevent DA uptake into presynaptic neurons, thus, increasing synaptic concentrations of DA [24] leading to behavioural effects. In this context, it can be stated that both drugs albeit present facilitatory effects on genital reflexes in PSD males, they present distinct mechanisms of action on the central nervous system. Although progress has been made in understanding the neurobiological basis of the effects of drugs of abuse in rats, further studies are needed at the behavioural level of interaction between
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psychostimulants and sleep deprivation, a consequent event related to cocaine use [29], and further, an inherent condition associated to ecstasy use in all-night dancing parties [22,35,36]. In fact, alertness and sleep disturbances are one of the problems most commonly reported by drug users [28]. Sleep deprivation results in a constellation of generalized symptoms leading to several catecholamine [25,31,44,45], hormonal [7,9,10] and behavioural [6,11,17,20] alterations. In particular, PSD is known to alter the functioning of several neurotransmitter systems, including DA [31,44] and 5-HT [32]. More recently, Hip´olide et al. [25] demonstrated significant decreases in 5-HT transporters providing further evidence that sleep deprivation results in rapid and widespread alterations in the brain. Sexual behaviour is complex and results from an intricate, well-balanced string of neurochemical events that involve a network of neurotransmitters. Additional factors may be involved since both PSD and drugs of abuse like cocaine and ecstasy stimulation also alters other systems, besides dopaminergic and serotoninergic ones. Insofar, our studies have suggested that DA D2 receptor supersensitivity [31] may provide a possible mechanism to account for the PSD induced effects on spontaneous genital reflexes and their potentiation by cocaine [8]. Indeed, facilitation of sexual behaviour can be induced by stimulation of postsynaptic D2 receptors, which play the main role in the control of male copulatory behaviour [19] although influence of 5-HT should be considered [23]. Notwithstanding, the alterations in the serotoninergic system promoted by PSD should be taken into account as an important factor in the expression of genital reflexes in ecstasy-treated males. Taken together, it can be assumed that PSD potentiates the action of the substance of abuse over genital reflexes. This effect should be interpreted with the consideration that enhancement of sexual components (e.g. erection) is but one of the effects subject to alteration by psychostimulant drugs. It must be taken into account, that several other alterations occur as consequence of these drugs such hyperthermia, tachicardia and convulsion. Before youngsters resort to drugs for enhanced sexual performance, they should re-evaluate the deleterious consequences of substances of abuse. Finally, sleep deprivation is a determinant in the action the drugs of abuse. Acknowledgements The authors would like to express their cordial thanks to Waldemarks Leite, Tome Pimentel, Alice Lima and Marilde Costa, for assistance during the project. This work was supported by grants from Associac¸a˜ o Fundo de Incentivo a` Psicofarmacologia (AFIP) and FAPESP (#01/04329-0 to M.L.A.; #04/04008-7 to J.C.P. and CEPID #98/14303-3 to S.T.). References [1] Adams DH, Hanson GR, Keefe KA. Differential effects of cocaine and methamphetamine on neurotensin/neuromedin N and preprotachykinin messenger RNA expression in unique regions of the striatum. Neuroscience 2001;102:843–51. [2] Almeida SP, Silva MTA. Ecstasy (MDMA): effects and patterns of use reported by users in S˜ao Paulo. Rev Brasil Psiquiatria 2003;25:11–7.
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