Selegiline Enhances Erectile Activity Induced by Dopamine Injection in the Paraventricular Nucleus of the Hypothalamus in Anesthetized Rats

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Selegiline enhances erectile activity induced by dopamine injection in the paraventricular nucleus of the hypothalamus in anesthetized rats

J Allard1, J Bernabe1, F Derdinger1, L Alexandre1, K McKenna1,2 and F Giuliano1,3*

1PELVIPHARM Laboratories, Domaine INRA, Bures-sur-Yvette, France; 2Northwestern University Medical School, Department of Physiology and Urology, Chicago, IL, USA; and 3Groupe de Recherche en Urologie, UPRES, EA 1602, Medical University of Paris South, Le Kremlin Biceˆtre Cedex, France

Apomorphine delivered in the paraventricular nucleus of the hypothalamus (PVN) induces penile erection in rats, suggesting a role of dopaminergic projection to the PVN in the control of penile erection. We assessed whether the selective inhibitor of monoamine oxidase B, selegiline, could enhance the erectile activity induced by dopamine delivery in the PVN. Intracavernous and blood pressure (ICP and BP) were monitored in anesthetized rats to quantify ICP rises (number and percentage of ICP maximum=mean BP (ICPmax=BP Â 100)) elicited by 10 mg dopamine injection in the PVN after saline or 3 mg=kg i.v. selegiline (8 rats per group). The number of ICP rises (mean Æ s.d.: 4.5 Æ 2.9 vs 1.4 Æ 1.9; P ¼ 0.017) and their ICPmax=BP Â 100 (49 Æ 8% vs 34 Æ 9%; P ¼ 0.015) were signiﬁcantly greater upon dopamine injection in the PVN than upon vehicle. Compared to saline i.v., 3 mg=kg selegiline pretreatment signiﬁcantly increased the number of ICP rises induced by dopamine injection in the PVN (9.4 Æ 2.6 vs 4.5 Æ 2.9; P < 0.001), without affecting their amplitude. This suggests that drugs potentiating dopaminergic responses in the central nervous system might enhance proerectile commands of supraspinal origin. International Journal of Impotence Research (2002) 14, 518–522. doi:10.1038=sj.ijir.3900934

Keywords: penile erection; monoamine oxidase; l-deprenyl; dopamine

Introduction A corollary of the aforementioned statement is that indirect dopaminergic agonist might have some proerectile activities. Selegiline hydrochloride Peripheral delivery of apomorphine, a non-specific (registered as L-deprenyl) is a selective monoamine D1-like as well as D2-like dopamine receptor ago- oxidase inhibitor B (MAO B), which has been suc- nist, induces penile erections in rats by acting in the cessfully used alone or as an adjunctive treatment to paraventricular nucleus of the hypothalamus 3,4-dihydroxy-L-phenylalanine (L-DOPA, the pre- 1 (PVN). Injection of doses of apomorphine as low cursor of dopamine) in Parkinson’s disease.4 Experi- as 5 ng in the PVN can induce penile erections in mental data suggest that selegiline enhances 2 conscious rats. Conversely, penile erections dopaminergic responses in rats. For example, pre- induced by peripheral delivery of apomorphine are treatment with selegiline potentiated individual antagonized by a preceding injection of SCH23390, a neuronal responses of single striatal neuron to D1-like receptor antagonist or sulpiride, a D2-like iontophoretically applied dopamine in vivo.5 It receptor antagonist, in the PVN of anesthetized also increased contralateral rotations induced by 3 rats. This suggests that dopaminergic release intraperitoneal L-DOPA injections in rats with within the PVN play a key role in the control of unilateral lesion of the substantia nigra.6 The penile erection. mechanisms responsible for the effect of selegiline on dopaminergic responses differ in rats and humans. In rats, catabolism of dopamine is not affected by inhibition of MAO B by selegiline, pro- *Correspondence: F Giuliano, Department of Urology, CHU bably because of a high ratio of MAO A=MAO B de Biceˆtre, 78 rue du Geńe´ral Leclerc, 94270 Le Kremlin concentration.5,7 – 10 The enhanced dopaminergic Biceˆtre Cedex, France. E-mail: [email protected] responses are therefore not due to an increased Received 12 May 2002; revised 10 July 2002; availability of dopamine caused by a decreased accepted 23 July 2002 degradation by MAO B. The ability of selegiline to Enhancement of dopamine-induced erection by selegiline J Allard et al 519 increase dopaminergic responses in rats has been injection site at the end of the experiment. After explained by the amphetamine-like properties of sacrifice, the brain was removed and serially cut in L-methamphetamine, a major metabolite of selegi- 20 mm thick sections on a microtome. One in 5 slices line.11,12 It was also proposed that in the rat brain was mounted on gelatinized slides and dyed with selegiline could increase the level of b-phenylethyl- cresyl violet. Rats for which the tip of the injection amine, an endogenous trace amine which potenti- was outside the PVN were eliminated from the ates neuronal response to dopamine.5,13 – 15 study. Taking into account the potentiating activity of BP and ICP tracings were analyzed retrospec- selegiline on dopaminergic responses in rats, we tively using software designed in our laboratory. hypothesized that erectile responses induced by Only ICP rises with a maximal value superior to central dopamine could be enhanced by systemic the sum of the average þ 3 standard deviations of the selegiline pretreatment. We therefore assessed the ICP recorded during the 5 min before the injection of ability of selegiline delivered intravenously (i.v.) to drug or vehicle were quantified. Such ICP rises were enhance erectile responses induced by dopamine considered as erectile events. Latency was the time injections in the PVN in anesthetized rats. separating the beginning of the PVN injection from the first ICP rise. ICPmax=BP Â 100 corresponded to the percentage of the ratio: maximal value reached Materials and methods by ICP during the rise (mm=Hg)=mean BP(mm=Hg) for the duration of the ICP rise. The area under the curve of ICP rise divided by the mean BP, termed Thirty two adult male Sprague-Dawley rats (Charles- AUC=BP (s) and the sum of the AUC=BP (s) of all the River, St-Aubin-le`s-Elbeuf, France) weighing 200 – ICP rises, which represents an index of overall 250 g, were anesthetized with an intraperitoneal erectile activity, were also calculated for each rat. injection of urethane (1.2 g=kg, Sigma-Aldrich, St- All parameters were averaged per rat, except the Quentin-Fallavier, France). Their temperature was sum of AUC=BP, and then per treatment. Rats maintained at 37C using a homeothermic blanket. which did not display any ICP rise (non-responder Rats were tracheotomized to prevent aspiration of rats) during the recording were attributed the value saliva, and, when required, to perform artificial 0 for the sum of AUC=BP and number of ICP rises. ventilation. The carotid artery and jugular vein For non-responder rats, ICPmax=BP Â 100 and were catheterized with polyethylene tubings filled AUC=BP were considered as missing data for the with heparinized saline (25 IU=ml) to record the statistical analysis. mean systemic arterial blood pressure (BP) via a Data were compared using two way analysis of pressure transducer (Elcomatic 750, Glasgow, UK) variance (ANOVA) followed by post-hoc analysis and inject drugs into the bloodstream, respectively. with the Student – Newman – Keuls method when The prepuce was circumcised and the penis required. All tests were conducted using SigmaStat1 denuded of skin. The animal was placed on a software, version 2.03 (SPSS, Erkrath, Germany). stereotaxical apparatus. A heparinized saline-filled Differences were considered significant when catheter connected to a pressure transducer (Elco- P < 0.05. All results are expressed as mean Æ standard matic EM 750, UK) was inserted in the left corpus standard deviation (s.d.). cavernosum to record intracavernous pressure (ICP). Selegiline (Sigma-Aldrich) was dissolved in saline and dopamine (Sigma-Aldrich) in saline sup- Results plemented with 0.05% ascorbic acid to retard oxidation. Intravenous injection of selegiline (3 mg=kg) or the corresponding vehicle injection was performed In the groups injected with vehicle in the PVN, selegi- 5 min after the start of recording and 10 min prior to line i.v. injection resulted in an increase in the the PVN injection. Dopamine (10 mgin1ml) or vehi- number of rats presenting at least one ICP rise com- cle were injected into the PVN (stereotaxic coordi- pared to saline i.v. injection (7=8 vs 3=8 respectively). nates: 0.3 mm posterior to bregma; 0.4 mm lateral to All rats displayed at least one ICP rise in the groups the midline; 8.4 mm below the skull surface, with injected with 10 mg dopamine in the PVN. Original the upside of the incisor bar at the level of the recordings showings the effect of dopamine injection interaural line) over 1 – 2 min using a 1 ml Hamilton in the PVN after i.v. pretreatment with either saline or microsyringe. Eight rats were included in each of the 3mg=kg selegiline are displayed in Figure 1. 4 groups (2 groups injected with selegiline i.v. and Two way ANOVA revealed a significant effect of with either dopamine or the corresponding vehicle dopamine injection in the PVN on the mean number in the PVN, 2 groups injected with saline i.v. and and sum of AUC=BP of the ICP rises (F[1,28] ¼ 30.13 with either dopamine or the corresponding vehicle and 33.91 respectively, P < 0.001 for both), as well as in the PVN). BP and ICP were recorded for 30 min on ICPmax=BP Â 100 and latency (F[1,22] ¼ 22.94, after the injection of dopamine. Evans blue was P < 0.001 and 4.39, P ¼ 0.048, respectively). There was microinjected for histological identification of the also a significant effect of selegiline pretreatment on

International Journal of Impotence Research Enhancement of dopamine-induced erection by selegiline J Allard et al 520

Figure 1 Representative recordings of BP and ICP after intra PVN dopamine injection. Anesthetized rats were placed on a stereotaxic frame. BP (upper trace) and ICP (lower trace) were continu- ously monitored. Rats were injected i.v. (arrow) with either saline (A) or 3 mg=kg selegiline (B). Both rats were injected 10 min later with 10 mg dopamine in the PVN (arrow head). Note the appearance of phasic ICP rises after dopamine injection, and Figure 2 Quantification of the erectile activity induced by dopa- the greater number of ICP rises after i.v. pretreatment with selegi- mine injection in the PVN after i.v. selegiline. Anesthetized rats line (B) compared to saline (A). Note also the transient decrease in were injected with either selegiline (3 mg=kg) or saline i.v., blood pressure induced by selegiline injection in B. followed 10 min later with vehicle or dopamine (10 mg) injection in the PVN. Eight rats were used in each condition. ICP rises were quantified for 30 min after the onset of the PVN injection. *P < 0.05 and **P < 0.01 refers to post-hoc analysis with Stu- dent – Newman – Keul’s test after 2 way ANOVA. Stars above the number and sum of AUC=BP of the ICP rises horizontal bars indicate a significant effect of selegiline i.v. (F ¼ [1,28] ¼ 14.10 and 15.89 respectively, P < 0.001 treatment for a given PVN injection (eg in A, selegiline injection for both) and latency (F[1,22] ¼ 5.82, P ¼ 0.025). significantly increased the number of ICP rises induced by dopamine injection in the PVN). Stars above mean standard deviation Post-hoc analysis within factor with Student – refer to a significant effect of dopamine injection in the PVN for a Newman – Keuls’ test allowed analysis of: 1) the given i.v. injection (eg in C, the ICPmax=BP Â 100 of the ICP rises effects of dopamine injection in the PVN after was significantly increased by dopamine injection in the PVN in saline i.v. pretreatment; 2) the effects of selegiline either vehicle or selegiline treated rats compared to vehicle pretreatment on saline PVN delivery; and 3) the injection in the PVN). effect of selegiline pretreatment on dopamine PVN delivery. of the ICP rises was also increased in the group injected with dopamine compared to its control group Dopamine injection in the PVN (14.7 Æ 9.3 s vs 9.1 Æ 4.6 s respectively, Figure 2B), but this was not significant. The increase of the number and amplitude of the ICP rises upon 10 mg The proerectile activity of dopamine delivered in the dopamine injection in the PVN was reflected in a PVN was analyzed by comparing the 2 groups pre- four fold increase of the sum of AUC=BP (52 Æ 27 s in treated with i.v. saline (Figure 2), and injected with the dopamine group vs 13 Æ 21 s in the control either 10 mg dopamine or the corresponding vehicle group, P ¼ 0.031, Figure 2D). The latency of the in the PVN. The mean number (Figure 2A) and first ICP rise was significantly decreased by ICPmax=BP Â 100 (Figure 2C) of the ICP rises were dopamine injection in the PVN (138 Æ 55 s in the significantly greater upon 10 mg dopamine delivery dopamine group vs 362 Æ 90 s in the control compared to the corresponding vehicle injection group, P ¼ 0.046, Figure 2E). Dopamine injection (4.5 Æ 2.9 vs 1.4 Æ 1.9, P ¼ 0.017, and 49 Æ 8% vs in the PVN had no effect on blood pressure at a 34 Æ 9%, P ¼ 0.015, respectively). The mean AUC=BP dose of 10 mg.

International Journal of Impotence Research Enhancement of dopamine-induced erection by selegiline J Allard et al 521 Selegiline i.v. dopamine receptors than dopamine itself; and 2) the considerable catabolic inactivation and reuptake of extracellular dopamine after its release in the In rats injected with vehicle in the PVN, selegiline extracellular space, which considerably reduces its 3mg=kg i.v. resulted in a significant reduction of the bioavailability compared to apomorphine.16,17 latency of the first ICP rise compared to i.v. saline We hypothesized that drug enhancing dopaminer- (117 Æ 114 s vs 362 Æ 438 s respectively, P ¼ 0.033, gic responses, such as selegiline, should increase the Figure 2E). AUC=BP and ICPmax=BP Â 100 were not erectile activity induced by dopamine injected in the affected (Figure 2B and C). The mean number of ICP PVN. Such dopamine injection is supposed to mimic rises and sum of AUC=BP tended to be increased by i.v. the release of dopamine occurring in the PVN upon selegiline compared to i.v. saline (Figure 2A and D). It sexual arousal in physiological conditions. Accord- is noteworthy that when only rats displaying at least ingly, a significant and consistent enhancement of one erectile event are considered, the number of ICP the number of erectile events induced by dopamine rises in the saline and selegiline i.v. groups injected injected in the PVN was observed after pretreatment with vehicle in the PVN were similar (3.6 Æ 0.6 with 3 mg=kg i.v. selegiline, a dose which selectively and 3.4 Æ 2.0 respectively). Selegiline 3 mg=kg i.v. inhibits MAO B in rats.5 In rats, the potentiation of induced a transient decrease in blood pressure, dopaminergic response by selegiline is likely to be which returned to its baseline value after 3 to 4 min. caused by an increase in the availability of b-phenylethylamine, a trace amine which has been shown to be a specific substrate for monoamine oxidase B and Selegiline i.v. and dopamine injection to potentiate dopaminergic responses.5,13 – 15 Our in the PVN results are in agreement with several studies report- ing an enhancement of mating performance in old and sexually sluggish rats treated with deprenyl, The ability to selegiline to enhance central dopamine- although Ferrari and Giuliani could not provide induced erectile activity was analyzed by comparing evidence for an enhancer effect of selegiline on results obtained in rats injected with 10 mgdopamine copulatory performance in sexually-active and inac- in the PVN and pretreated with i.v. saline or selegiline tive male rats.18 – 21 The reason for this discrepancy is (grouped empty and filled bars on the right of each unclear, but it may be related to the difference in graph in Figure 2). Selegiline i.v. 3 mg=kg 10 min prior strains and age of rats used. to 10 mg dopamine injection in the PVN significantly The efficacy of apomorphine in the treatment of enhanced the number (9.4 Æ 2.6 vs 4.5 Æ 2.9, erectile dysfunction further supports an involve- Figure 2A) and the sum of AUC=BP (132 Æ 54 vs ment of endogenous central dopamine in the control 52 Æ 27, Figure 2D) of ICP rises compared to i.v. of penile erection.22 To our knowledge, the impact saline injection (P < 0.001 for both). AUC=BP of selegiline treatment on sexual activity has never and ICPmax=BP Â 100 of the ICP rises induced by been assessed in humans. Nevertheless, the present dopamine injection in the PVN were barely affected data suggest that indirect dopaminergic agonists by i.v. selegiline pretreatment (Figure 2B and C). could facilitate sexual response, by specifically enhancing dopaminergic responses at the sites invol- ved in the control of penile erection. Discussion

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