logical Psychiatry 31 (2007) 652–657www.elsevier.com/locate/pnpbp

Progress in Neuro-Psychopharmacology & Bio

Involvement of nitric oxide in cocaine-induced erections and ejaculationsafter paradoxical sleep deprivation

Monica L. Andersen ⁎, Juliana C. Perry, Isabela B. Antunes, Sergio Tufik

Department of Psychobiology — Universidade Federal de São Paulo, Escola Paulista de Medicina (UNIFESP/EPM), R. Napoleão de Barros,925, V. Clementino 04024-002, São Paulo, SP, Brazil

Received 20 September 2006; received in revised form 14 December 2006; accepted 16 December 2006Available online 28 December 2006

Abstract

Objectives: As nitric oxide (NO) is involved in penile erectile (PE) function and also influences the sleep–wake cycle, we speculated that NOcould play a role in PE and ejaculation of paradonical sleep deprivation (PSD) rats.Methods: Animals were pretreated with NG-nitro-L-arginine methyl ester (L-NAME, ip) and L-arginine (ip and icv) prior to saline or cocaineinjection.Results: Cocaine-induced PE in 90% of PSD rats, 60% of which ejaculated. L-NAME reduced the frequency of erection, but had no effect in theproportion of PSD-cocaine-injected rats displaying this response. L-NAME had no effect in saline groups. L-Arginine in PSD-saline rats reducedthe proportion of animals displaying PE at the highest dose and reduced the frequency of PE at all doses in both saline and cocaine groups. The icvadministration of L-arginine reduced PE only in PSD-cocaine rats. Results indicate that common to both drugs, whether it was NO synthase (NOS)inhibitor or NO precursor, was their capacity to strongly reduce PE frequency in cocaine-treated rats. Moreover, L-arginine (ip) played a relevantinhibitory role in the erection displayed by PSD rats.Conclusions: Our findings suggest that the stimulating effects of PSD associated or not with cocaine on erection can be modified by alterations inthe NO system.© 2006 Elsevier Inc. All rights reserved.

Keywords: Cocaine; Erection; L-Arginine; L-NAME; Nitric oxide; Rat; Sleep deprivation

1. Introduction

It has already been demonstrated that nitric oxide (NO) playsa crucial role in the control of erectile function (Burnett, 2005).Sexual stimulation leads to NO production that in turnstimulates guanylate cyclase that converts guanosine triphos-phate to cGMP resulting in relaxation of the smooth muscles ofpenile arteries and corpus cavernosum (Burnett, 1997, 2005).

The implication of NO in sexual behavior is derived from thefact that systemic administration of NO synthase (NOS)inhibitor NG-nitro-L-arginine methyl ester (L-NAME) impairedcopulation (Hull et al., 1994, 1997). This probably was due to

⁎ Corresponding author. Department of Psychobiology— UniversidadeFederal de São Paulo, Rua Napoleão de Barros, 925, Vila Clementino— SP-04024-002, São Paulo, Brazil. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092.

E-mail address: mandersen@psicobio.epm.br (M.L. Andersen).

0278-5846/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.pnpbp.2006.12.012

an inhibition of erectile ability. Drug-treated males were unableto achieve vaginal intromissions during most mounts, and hadfewer penile erections (Moses and Hull, 1999) suggesting thatNO promotes erection in intact males probably by mediating thefilling of the corpora cavernosa of the penis (Rajfer et al., 1992).

Besides peripheral mechanisms, central effects of NO af-fecting erection in male rats have not been completely ruled out(Bialy et al., 1996). NO may influence these behaviors throughrelease of a variety of neurotransmitters, among them, dopamine(DA) (Lorrain and Hull, 1993). In fact, NO promotes DA releasein the medial preoptic area (MPOA) (Lorrain et al., 1996; Hullet al., 1997). In rats, injection of NOS inhibitors intracerebroven-tricularly or in the paraventricular nucleus (PVN) preventedpenile erection (PE) responses induced by dopaminergic agonists(Melis and Argiolas, 1993). Finally, the L-NAME inhibitoryeffects on sexual behavior have been attributed to a decrease ofblood flow within the penis, an increase in the adrenergic drive to

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the corpora cavernosa and a decrease in DA released inhypothalamic MPOA (McCann et al., 1999).

Behaviorally, we have demonstrated that paradoxical sleepdeprivation (PSD—Tufik et al., 1978) induces PE andejaculation (EJ) in male rats (Andersen et al., 2003). It hasbeen shown that such PSD effect is dramatically potentiated bydopaminergic drugs like cocaine (Andersen et al., 2003, 2004,2005a,b). In an attempt to further elucidate the mechanismsbehind these cocaine-induced genital events in PSD male ratsthe neurochemical basis of such behaviors has been the focus ofour investigations. The systemic administration of dopaminer-gic (Andersen et al., 2005b), cholinergic (Andersen et al.,2004), noradrenergic (Andersen et al., 2005a) and GABAergic(Andersen and Tufik, 2004) drugs alters the expression ofgenital reflexes and suggests that cocaine-induced PE and EJ inPSD rats is related to a complex chain of events ruled by varioussystems.

Based upon the description of the role of DA and NOtransmission in the modulation of sexual behavior in theliterature, this study attempted to address the question as towhether the NO systems also participate in the cocaine-inducedPE and EJ in PSD male rats. To this purpose, male rats weresubmitted to the PSD method for 96 h (Andersen et al., 2003)and were treated with NO synthase (NOS) inhibitor or NOprecursor drugs prior to cocaine or saline injection.

2. Methods

2.1. Subjects

Male rats (Department of Psychobiology, UNIFESP) usedin this study were maintained and treated in accordance withthe guidelines established by the Ethical and Practical Prin-ciples of the Use of Laboratory Animals. The experimentalprotocol was approved by the Ethical Committee of UNIFESP(CEP N. 482/02).

2.2. Paradoxical Sleep Deprivation (PSD)

The animals were submitted to PSD (96 h) using themodified multiple platform (systemic injection) and singleplatform (icv injection) methods. For the multiple platformmethod, rats were placed inside a water tank containing 14circular platforms, 6.5 cm in diameter. The rats could thus movearound inside the tank by jumping from one platform to another.When they reached the paradoxical phase of sleep, muscleatonia set in and they fell into the water and woke. The singleplatform method consisted in maintaining the animal in a cagewith a single platform in water up to within 1 cm of the uppersurface of the platform.

2.3. Drugs

Three doses of L-NAME (50, 100 and 200 mg/kg) and L-arginine (50, 100 and 200 mg/kg) were administered ip allowingfor the assessment of the dose–response curve in terms ofpercentage and frequency of PE and EJ (N=10/dose). Frequency

was defined as the number of PE or EJ per rat. L-NAME wasadministered 60 min and L-arginine 30 min prior to cocaine orsaline. The route and latency of administrations were selectedbased on Benelli et al. (1995). The data indicated that the chosendoses did not result in motor impairment.

2.4. Intracerebroventricular (icv) administration

Rats were anesthetized with ketamine and diazepam and theguide cannula was kept in place with stainless screws and acrylicdental cement. The guide cannula was implanted in the rightlateral brain ventricule: (AP): −1.0 mm; (ML): −1.6 mm; (DV):−3.9 mm, adapted from Paxinos and Watson (1986). L-Argininewas administered at the doses of 10 nmol and 100 nmol, 2 μl ofsterile saline, 0.5 μl/min (Plech et al., 2003) through a needle thatwas kept in place for 90 s after the injection. The animalsreturned to the PSD environment until the injection of saline(N=9/group) or cocaine (7 mg/kg, ip, N=10/group) that tookplace 15 min after the icv administration.

The brains were removed quickly, frozen over dry ice, andstored at −80 °C. Sixty-micron coronal sections were cut on aLeica cryostat at −20 °C, thaw-mounted onto slides andstained with Cresyl Violet. The position of the cannula wasverified with reference to the atlas of Paxinos and Watson(1986).

2.5. Penile erection/ejaculation evaluation and experimentaldesign

PE was counted only when the rat stood on its hind limbs,bent its body forward, bent its head down to reach the genitalarea, held and licked its penis in full erection and displayed hipmovements. The erect penis was always visible. EJ was scoredby the number of ejaculatory plugs. The frequency ofspontaneous PE and EJ (total number of genital reflex dividedby the number of rats) was assessed for 45 min. Each animalwas tested only once. Observations of the genital reflexes ofeach animal took place immediately after acute i.p. saline orcocaine injection (7 mg/kg) which was applied immediatelyafter each animal was removed from the tank and after theadministration of the NO drugs according to its respectivelatency. Since we had found, in previous studies, that there wereno genital reflexes in home-cage non-sleep deprived (saline andcocaine) control animals, the PSD group designed as controlwas pretreated with saline and dubbed vehicle group to avoidmisinterpretation with the group challenged with saline, thusavoiding the use of a large number of animals. This protocol isin accordance with our experimental design established inprevious studies (Andersen and Tufik, 2004; Andersen et al.,2004, 2005a,b).

In a first experiment, three doses of L-NAME were admin-istered allowing for the assessment of the dose–response curvein terms of percentage and frequency of genital reflexes in PSD-rats. In the second experiment, the same doses of L-NAME weretested in PSD-cocaine-treated animals. In the subsequent exper-iments, L-arginine was injected prior to saline (Experiment 3)and cocaine (Experiment 4) in PSD rats.

Fig. 1. Effects of L-NAME on the proportion of animals (Panel A) displayinggenital behaviors (expressed in percentage) and on the frequency (number ofgenital events per rat (Panel B) of penile erection (PE) and ejaculation (EJ) inparadoxical sleep deprived rats challenged with saline. Data are expressed asmean±SEM (N=10/group).

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2.6. Statistical analysis

For the statistical analysis of the proportion of animalsdisplaying PE and EJ (expressed in percentage), the FisherExact Probability test (two-tailed) was used to assess differ-ences between groups. Frequency of PE and EJ and latency datawere analysed by one-way ANOVA test followed by Duncanmultiple range test for comparison between the treatment and

Fig. 2. Effects of L-NAME on the proportion of animals (Panel A) displayinggenital behaviors (expressed in percentage) and on the frequency (number ofgenital events per rat (Panel B) of penile erection (PE) and ejaculation (EJ) inparadoxical sleep deprived rats challenged with cocaine. *pb0.001 and**pb0.0001, relative to vehicle (ANOVA followed by Duncan's test). Dataare expressed as mean±SEM (N=10/group).

vehicle groups. Values are expressed as mean±SEM. The levelof significance was set at pb0.05.

3. Results

3.1. Effects of L-NAME pretreatment in PSD male rats

Figs. 1 and 2 show the effect of L-NAME on the proportionof animals expressed in percentage and frequency of PE and EJafter saline (Fig. 1A and B) or cocaine (Fig. 2A and B)administration. Four days of PSD induced PE in 50% of themale rats, and 10% ejaculated after saline injection. In those ratsin which L-NAME+saline was administered, there was nosignificant alteration neither in the proportion nor frequency ofanimals exhibiting these behaviors (Fig. 1). L-NAME did notaffect latency of PE or EJ. The latency to the first PE or EJ didnot reach a level of significance because of a large variance andthe low proportion of animals exhibiting these behaviors (datanot shown).

3.2. Effects of L-NAME pretreatment in PSD+cocaine malerats

As expected, cocaine administration induced PE and EJ inmore animals than saline. Nine of the ten that were administeredcocaine (7 mg/kg) responded with PE and six of thoseejaculated (Fig. 2A). There was no statistical alteration in theproportion of animals exhibiting genital events after thepretreatment with L-NAME. When the frequency was evaluat-ed, the administration of L-NAME at all doses significantlydecreased the frequency of PE events [F(3,36) =11.17;

Fig. 3. Effects of L-arginine (ip) on the proportion of animals (Panel A)displaying genital behaviors (expressed in percentage) and on the frequency(number of genital events per rat (Panel B) of penile erection (PE) andejaculation (EJ) in paradoxical sleep deprived rats challenged with saline(N=10/group). *pb0.03, relative to vehicle (Panel A, Fisher exact test) and*pb0.01, relative to vehicle (Panel B, ANOVA followed by Duncan's test).Data are expressed as mean±SEM (N=10/group).

Fig. 5. Effects of L-arginine (icv) on the proportion of animals (Panel A)displaying genital behaviors (expressed in percentage) and on the frequency(number of genital events per rat (Panel B) of penile erection (PE) andejaculation (EJ) in paradoxical sleep deprived rats challenged with saline andcocaine (ip). *pb0.05 and **pb0.001, relative to saline; #pb0.001, relative tococaine; §pb0.03, relative to their respective dose saline group (ANOVAfollowed by Duncan's test). Data are expressed as mean±SEM (N=10/group).

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pb0.001] as shown in Fig. 2B. No dose-dependent effects wereobserved between the doses tested. No statistically significantalterations were found in the ejaculatory parameters after theinjection of L-NAME plus cocaine at any dosage level.Similarly, there were no significant differences in PE or EJlatency.

3.3. Effects of intraperitoneal L-arginine pretreatment in PSDmale rats

Fisher exact test revealed a significant decrease of theproportion of rats treated with the highest dose of L-arginine(200 mg/kg) displaying PE when compared to vehicle (pb0.03)as depicted in Fig. 3A. A one-way ANOVA [F(3,36)=4.25;pb0.01] revealed significant reductions in the frequency ofPE after saline injections in all the three doses of L-argininetested in comparison to vehicle group (pb0.01) as shown inFig. 3B. No statistically significant alterations were found in thelatencies for PE and EJ, percentage of animals ejaculating or inits frequency after the injection of L-arginine at any dose tested(Fig. 3A and B).

3.4. Effects of intraperitoneal L-arginine pretreatment in PSD+cocaine male rats

Animals pretreated with L-arginine had no significantdifferences in the proportion of animals displaying PE andEJ when compared with vehicle group at any dose tested(Fig. 4A). ANOVA followed by Duncan test for PE [F(3,36)=4.95; pb0.005] revealed that the PE frequencies observed inthe L-arginine pretreated groups were significantly lower at allthree dose groups than in vehicle-pretreated rats (pb0.01 for50 and 100 mg/kg; pb0.001 for 200 mg/kg).

Fig. 4. Effects of L-arginine (ip) on the proportion of animals (Panel A)displaying genital behaviors (expressed in percentage) and on the frequency(number of genital events per rat (Panel B) of penile erection (PE) andejaculation (EJ) in paradoxical sleep deprived rats challenged with cocaine.*pb0.01 and **pb0.001, relative to vehicle (ANOVA followed by Duncan'stest). Data are expressed as mean±SEM (N=10/group).

3.5. Effects of intracerebroventricular L-arginine pretreatmentin PSD male rats

As previously illustrated, PSD induced erection in 50% of therats and 10% ejaculated, and the administration of cocaine-induced PE in 90% of PSD rats, 60% of which ejaculated. An icvinjection of L-arginine prior to saline or cocaine did not produce adifferent statistical significance in the proportion of PSD ratsdisplaying PE (Fig. 5A). Further analysis revealed that the icvadministration pretreatment 15 min prior to cocaine significantlyreduced PE frequency in all doses used compared to ip cocaineinjection as revealed by one-way ANOVA [F(5,52)=8.98;pb0.001] as depicted in Fig. 5B. In addition, the 100 nmol ofL-arginine+cocaine group differed from the respective saline-injected rats (pb0.03). By examining PE latency, one-wayANOVA revealed that icv L-arginine administered rats did notexhibit significant difference among the groups (data not shown).

4. Discussion

This experiment demonstrated that the systemic adminis-tration of L-NAME did not influence PE and EJ induced bysleep deprivation. However, when PSD was associated tococaine, the frequency of PE was reduced after all dosestested. In contrast, L-arginine (ip) did affect PE in PSD-salinerats by reducing the proportion of animals (200 mg/kg) as wellas the frequency of this behavior. In addition, in the PSD-cocaine rats, all doses of L-arginine decreased PE. However,the icv administration of L-arginine only reduced the erectionfrequency in PSD-cocaine rats. Thus, based on our findings,

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the NO system plays a relevant influence on the erectileresponse induced by PSD and cocaine.

NO has been proposed to serve multiple functions in thecentral nervous system, which may in part be related to itsfacilitatory influence on neurotransmitter release (Aoki et al.,1995). It is currently believed that NO is involved in themediation of nociception, sexual behavior, neuroendocrinefunction and sleep–wake cycle (Burlet and Cespuglio, 1997;Datta et al., 1997; Schutz et al., 2004).

Studies have shown that erection induced by the electricalstimulation of the cavernous nerve was prevented by inhibitorsof NOS, which synthesize NO in several tissues (Marletta et al.,1988), including brain tissue (Bredt and Snyder, 1990). It isgenerally accepted that NO originates in nonadrenergicnoncholinergic nerve fibers of the penis, and is the principalvasodilator during the erectile response (Reilly et al., 1997). Theinhibition of NO synthesis by L-NAME impaired copulation(Hull et al., 1994; Ferraz et al., 2003), caused marked reductionof precoital sexual behavior (Ratnasooriya et al., 2000) andreduced the percentage of mounting and ejaculating rats (Plechet al., 2003). In accordance to our data, in which we alsoobserved reduction in PE frequency in the PSD-cocaine group,this overall impairment appears to reflect primarily inhibition oferectile function, since L-NAME also decreased the number oferection in rats (Hull et al., 1994). NOS is highly concentrated instructures directly or indirectly involved in sexual behavior(Bredt et al., 1991). Such suggests a more comprehensive rolefor NO in copulation, not limited to the induction of PE by anaction in the periphery but that also extends to the behavioralcomponent of sexual activity (Benelli et al., 1995).

Cocaine exerts its pharmacological effects via both centraland peripheral mechanisms. The central stimulatory actionderives from blocking the uptake of neurotransmitters at thesynaptic cleft thus producing a sustained action on the receptorsystem. One of the few neuromodulatory systems that have notyet been extensively examined in relation to cocaine is that ofNO. L-NAME blocked the development of cocaine oramphetamine induced behavioral sensitization (Kim and Park,1995) suggesting that NO may play a major role in the processof cocaine-induced behavioral response (Park et al., 2001).Chang et al. (1996) reported that the increased and prolongedintracavernous pressure induced by cocaine is not related todopaminergic or noradrenergic neurotransmission, but is likelymediated by NO at the corpus cavernosum. Although thisinterpretation should be considered, the complete comprehen-sion of the interactions between NO and cocaine on sexualbehavior warrant further examination.

Based on our observations, L-NAME blocked PE induced bycocaine+PSD, while L-NAME had no effect on PE induced byPSD+saline. If that is the case then NO might be important forerections stimulated by cocaine and not by PSD. This alternativeinterpretation would be in line with studies showing that central(Melis and Argiolas, 1993) and systemic (Andersson et al., 1999)administration of L-NAME prevented apomorphine-induced PE.Indeed, it is well documented that blocking the synthesis of NOwith L-NAME inhibits erectile functions (Burnett et al., 1992). Inour experiments, L-NAME did not inhibit erections after PSD

(without cocaine), suggesting that different mechanisms maymodulate such genital events in distinct manners. Intraperitonealadministration of L-arginine, in turn, inhibited erections in all testscarried out in saline and cocaine PSD groups, suggesting thatincreased NO inhibits erections. Although a previous studyreported amarginal increase in the number of penile reflexes (Hullet al., 1994), this apparent discrepancy with the presentobservations may be due to methodological differences and tothe PSD effects in the animals present in the current study.However, this contradiction requires a clearer explanation.

Although the proportion of PSD-saline animals displayingPE was practically unchanged (except by the highest dose ofL-arginine), the frequency was overall affected. The percentageof animals displaying genital behaviors seems to be a param-eter that is less subject to alteration whereas the frequency isquite altered by different drugs (Andersen and Tufik, 2004;Andersen et al., 2004) suggesting that it is more difficult tomodify the triggering mechanism for sexual behavior than thefrequency of sexual activity itself.

The systemic administration of L-arginine resulted in amarked alteration in PSD-saline as well as in PSD ratschallenged with cocaine. In turn, the icv injection of thiscompound led to significant reduction in PE frequency only inthe animals submitted to PSD associated to cocaine, suggestingthe central participation of NO in the male sexual behavior. Lowdoses of L-arginine (10 nmol) decreased locomotion activity(Plech et al., 2003) suggesting a dopaminergic involvement inthis behavior. In addition to the dopaminergic system, it hasbeen suggested that NO modulates motor behavior, probably byinterfering with serotonergic, cholinergic and glutamate neuro-transmission in the striatum (Del Bel et al., 2005).

The unexpected inhibitory effect of the NO precursor,L-arginine on PE events in PSD groups is similar to previousdata showing that sildenafil did not potentiate the genitalreflexes in PSD animals (Andersen et al., submitted forpublication). Although knowledge of the mechanisms involv-ing L-arginine behind such behavior is still patchy, it can beinterpreted that the effects of L-arginine are possibly associatedto alterations produced by PSD on the organism.

In respect to EJ, it has been reported that injections of L-NAMEincreased the number of ex copula seminal emissions (Hull et al.,1994) as a probable result from stimulation of sympathetic nerves(Benson, 1988) indicating that NO maintains a tonic inhibitoryinfluence on sympathetic function (Hull et al., 1994). In ourexperiments, EJ was not affected by L-NAME or L-argininepretreatments. The reasons may be attributed to the ceiling effectof cocaine on the drug-induced PE and EJ and to the possibilitythat it would only modify sexual behavior when such is inhibitedor disrupted. In the case of saline groups, more studies arerequired for the complete understanding of the consequence ofsystemically administered NO agents on genital/sexual behavior.NOS is highly concentrated in structures directly or indirectlyinvolved in sexual behavior (Bredt et al., 1991) lending additionalsupport for the further investigations on this subject.

The search for the elements that are involved in PE has led tothe discovery of NO (Burnett, 1997). A direct relationshipseems to exist between this chemical and genital behavior as

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shown by PSD-cocaine rats. The results of our previous studiesdemonstrated a decreased trend in PE frequency in PSD ratschallenged with cocaine in different pharmacologicalapproaches (Andersen and Tufik, 2004; Andersen et al., 2004)supporting the interpretation that genital reflexes may be aneffect of a range of intricate and intertwined events rather than aresult from a single mechanism. Like sleep deprivation, cocainealso alters many systems leading to the concept that a complexneurochemical and behavioral alteration is involved in suchbehaviors. As noted, several systems have been found to beinvolved so far, but the participation of other neurotransmittersshould also be considered.

In summary, based on our data that both NO precursor andNO inhibitor agents reduce the frequency of PE, it isconceivable that any alteration in the NO system may impairgenital reflexes in PSD-cocaine male rats. Moreover, thestimulating action of cocaine in potentiating the sexual effectsin PSD rats does not override the inhibitory effects of NOcompounds on genital reflexes.

Acknowledgments

The excellent technical assistance of Waldemarks Leite,Tomé Pimentel, Marilde Costa and Tathiana Alvarenga isgratefully acknowledged. This work was supported by grantsfrom Associação Fundo de Incentivo à Psicofarmacologia(AFIP) and FAPESP (#01/04329-0 to M.L.A.; #04/04008-7 toJ.C.P.; #04/03979-9 I.B.A., and CEPID #98/14303-3 to S.T.).

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