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ORIGINAL RESEARCH—BASIC SCIENCE Anandamide Reduces the Ejaculatory Threshold of Sexually Sluggish Male Rats: Possible Relevance for Human Lifelong Delayed Ejaculation Disorder Gabriela Rodríguez-Manzo, PhD, and Ana Canseco-Alba, MSc Departamento de Farmacobiología, Cinvestav-Sede Sur, México, México DOI: 10.1111/jsm.12866

ABSTRACT

Introduction. The sexually sluggish (SLG) male rat has been proposed as an animal model for the study of lifelong delayed ejaculation, a sexual dysfunction for which no treatment is available. Low endocannabinoid anandamide (AEA) doses facilitate sexual behavior display in normal sexually active and in noncopulating male rats through the activation of CB1 receptors. Aim. To establish whether low AEA doses reduced the ejaculatory threshold of SLG male rats by acting at CB1 receptors. Methods. SLG male rats were intraperitoneally injected with different doses of AEA (0.1–3.0 mg/kg), the CB1 receptor antagonist AM251 (0.1–3.0 mg/kg), or their vehicles and tested for copulatory behavior during 60 minutes. Animals receiving AEA effective doses were subjected to a second sexual behavior test, 7 days later under drug-free conditions. To determine the participation of CB1 receptors in AEA-induced actions, SLG rats were pretreated with AM251 prior to AEA. Main Outcome Measures. The sexual parameters, intromission latency, number of mounts and intromissions, ejaculation latency, and interintromission interval. Results. All sexual behavior parameters of SLG rats were significantly increased when compared with normal sexually experienced animals. Low AEA doses (0.3 and 1 mg/kg) significantly lowered the ejaculatory threshold of SLG rats, reducing the number of pre-ejaculatory intromissions and ejaculation latency. IL, M number, and locomotor activity were unaffected by AEA. Facilitation of the ejaculatory response of SLG rats disappeared 7 days after AEA injection. AM251 lacked an effect on copulation of SLG rats but blocked the AEA-induced lowering of the ejaculatory threshold. Conclusions. AEA appears to specifically target the ejaculatory threshold of SLG rats through the activation of CB1 receptors. This specificity along with the fact that AEA’s effects are exerted acutely and at low doses makes this drug emerge as a promising treatment for the improvement of the ejaculatory response in men with primary delayed ejaculation. Rodríguez-Manzo G and Canseco-Alba A. Anandamide reduces the ejaculatory threshold of sexually sluggish male rats: Possible relevance for human lifelong delayed ejaculation disorder. J Sex Med 2015;12:1128–1135. Key Words. Ejaculatory Dysfunctions; Lifelong Delayed Ejaculation; Sluggish Male Rat; Endocannabinoids; Anandamide; CB1 Receptors

Introduction

A

mong human ejaculatory dysfunctions, retarded or delayed ejaculation (DE) appears to have a low prevalence, less than 3% [1] and is

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probably one of the least understood male sexual disorders. The fifth edition of the diagnostic and statistical manual of mental disorders of the American Psychiatric Association (DSM-V) defines DE as “a marked delay in or inability to © 2015 International Society for Sexual Medicine

AEA Reduces the Ejaculatory Threshold of Sluggish Rats achieve ejaculation. The man reports difficulty or inability to ejaculate despite the presence of adequate sexual stimulation and the desire to ejaculate.” This condition can be primary (lifelong) or secondary (acquired) [2]. There are few clinical studies on primary lifelong DE, a condition that has a profound negative impact on the affected men and their partners (it may contribute to difficulties in conception and is associated with considerable psychological distress) and for which no approved drug treatment is currently available [3,4]. The use of animal models for the study of sexual responses has increased our understanding of the neurobiology of normal and pathological sexual function. In particular, animal pharmacological research has made important contributions to the understanding and management of human sexual disorders [5]. The recent description of the biological variability in ejaculatory behavior of male rats led to suggest the existence of different ejaculatory endophenotypes in this species, with rapid and sluggish ejaculating rats represented at both ends of a Gaussian distribution [6]. These extremes were proposed to represent natural animal models for the study of human premature and retarded ejaculation, respectively [7]. Sexually sluggish (SLG) male rats have been defined on the one side as those animals consistently requiring a long time to achieve ejaculation (more than 20 minutes) or not attaining ejaculation in 30-minute tests [8] and, on the other, as those males showing between 0 and 1 ejaculations in 30-minute training sessions in contrast to normal ejaculators achieving one to three ejaculations in the same period [6]. We reported recently that low doses of the endocannabinoid anandamide (AEA) promote sexual behavior display in previously noncopulating male rats, that is, apparently normal and healthy animals that will not mate despite repeated exposure to sexually receptive females [9]. Low AEA doses were also found to facilitate the sexual behavior display of normal sexually experienced rats. In this case, the endocannabinoid reduces both the readiness to initiate copulation (intromission latency [IL]) and the ejaculatory threshold, that is, the amount of stimulation (number of preejaculatory intromissions) and time needed to achieve ejaculation (ejaculation latency)—effects mediated by the activation of CB1 receptors [10]. Based on these findings, we hypothesized that low AEA doses might improve the copulatory behavior of SLG males.

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Aims

The present study was designed to establish whether low doses of AEA reduced the ejaculatory threshold of SLG male rats, an animal model for DE, by acting at CB1 cannabinoid receptors. Materials and Methods

Animals One hundred and ten male Wistar rats classified as SLG were used for the experiments. The criterion used to consider males as SLG was that animals required more than 20 minutes after a first intromission to achieve ejaculation or did not ejaculate within the 30 minutes that followed that intromission, in five independent training sessions with a sexually receptive female. A group of sexually experienced animals (EXP, n = 8), that is, males with an optimal sexual behavior display, attaining ejaculation in less than 15 minutes after a first intromission, in at least three independent training sessions run every other day, was used as a positive control for comparison purposes. Sexually receptive intact female rats served as stimuli; receptivity was induced by the sequential s.c. injection of estradiol benzoate (4 μg/rat) followed 44 hours later by progesterone (2 mg/rat). Animals were maintained under inverted light/ dark cycle conditions (12 hours light: 12 hours dark, lights on at 22:00 hour) and with free access to rat chow and tap water. The Local Committee of Ethics on Animal Experimentation approved all experimental procedures which followed the Mexican official norm for the use and care of laboratory animals NOM-062-ZOO-1999. Sexual Behavior Tests Sexual behavior recording was conducted in a room under dim red light, 2 hours after the onset of darkness and 4 hours after injection of progesterone to the females. The animals were introduced into polycarbonate cylindrical arenas (40 cm diameter, 60 cm height), and a 5-minute period of adaptation was allowed before introducing a receptive female. The sexual behavior parameters recorded were intromission latency (IL) (time elapsing from the introduction of the female to the arena until the appearance of the first intromission), number of mounts (M) and intromissions (I) preceding ejaculation, ejaculation latency (EL) (time between the first intromission and ejaculation) and interintromission interval (III) (mean time separating I, calculated by dividing the J Sex Med 2015;12:1128–1135

1130 total number of pre-ejaculatory intromissions by the ejaculation latency).

Locomotor Activity In order to discard possible motor effects of the pharmacological treatments that could interfere with copulation, a motor activity test was conducted. Spontaneous ambulation was recorded in a box measuring 33 × 44 × 20 cm, with the bottom divided into squares (11 × 11 cm). The animals were placed in the cage, and the number of times they crossed from one square to another was recorded during a 5-minute period. Between tests, the cage was carefully cleaned. Drugs All drugs were purchased from Sigma-Aldrich (St. Louis, MO, USA). Arachidonylethanolamide (anandamide, AEA) was dissolved in a vehicle composed of a mixture of ethanol (2%), Tween80 (2%), and saline solution (96%). The CB1 receptor antagonist, AM251, was dissolved in a mixture of DMSO (one drop), Tween80 (2%), and saline solution (98%). Both drugs were i.p. injected in a volume of 1 mL/kg. Estradiol benzoate and progesterone were dissolved in sesame oil and s.c. administered to the female rats. Experimental Design SLG rats were randomly divided into 13 independent groups (n = 8–11, each); five of these groups were employed to construct a dose–response curve of the effects of AEA (0.1–3.0 mg/kg, i.p.) or its vehicle on sexual behavior display. AEA doses were chosen based on published data showing facilitative actions of this endocannabinoid on behavioral responses [10–12]. Five additional groups were used to test the effects of different doses the CB1 receptor antagonist AM251 (0.1–3.0 mg/kg) or its vehicle on copulatory behavior of SLG males. Finally, to establish the possible participation of CB1 receptors in the AEA-induced actions, three additional groups of SLG males were pretreated with a dose of AM251 that lacked an effect per se (0.1 mg/kg), followed by one of two AEA effective doses (0.3 or 1.0 mg/kg) or with the combination of vehicles. Each recording session included animals from different treatments, and the observer was blind to them. Sexual behavior recording started 5 minutes after the administration of the pharmacological treatments or the respective vehicles and behavioral observations lasted 60 minutes. The SLG J Sex Med 2015;12:1128–1135

Rodríguez-Manzo and Canseco-Alba animals in which AEA treatment reduced both I number and EL (ejaculatory threshold) were tested again for sexual behavior 7 days later, under drug-free conditions. The first ejaculatory series of a group of EXP animals injected with vehicle was used as a positive control to compare the sexual behavior parameters of vehicle-treated SLG rats.

Statistical Analyses Nonparametric statistics were used to analyze the sexual behavior data. Comparisons of drug treatment effects on the specific sexual parameters were conducted by means of a Kruskal–Wallis anova followed by Dunn’s test. The sexual parameters of the SLG rats injected with vehicle were compared with those of EXP rats using the Mann–Whitney U test. This same test was used to compare the effects of combined treatments of AM251+ AEA with those of AEA alone. Within-group analysis of the sexual behavior parameters was conducted in SLG rats comparing their performance on the day of AEA injection with that exhibited 7 days after AEA, by means of the Wilcoxon signed-rank test. Motor activity data were analyzed by means of a one-way anova. Results

Figure 1 illustrates the copulatory performance of SLG males. The vehicle-treated SLG rats showed statistically significant increases in all specific sexual behavior parameters when compared with those exhibited by normal EXP animals (solid grey and white box comparisons). As it can be observed, the duration of both the IL and EL appears to increase almost threefold in SLG rats as compared with EXP animals. The median number of preejaculatory M and I, as well as the III, is also importantly augmented. This figure also describes the effects of different doses of AEA on the copulatory behavior of SLG rats (dashed boxes). It is shown that the intermediate AEA doses tested (0.3 and 1 mg/kg) significantly reduced I number and EL in these animals when compared with vehicle treated SLG males. The 3 mg/kg AEA dose also reduced EL, but it did not affect I number. Finally, a statistically significant reduction in the III of SLG males was detected after the 1 mg/kg AEA dose. Neither IL nor M number was modified by AEA at any dose. Figure 2 compares the ejaculatory performance of SLG rats on their last training session (fifth) with that displayed in response to the injection of AEA effective doses (0.3 and 1 mg/kg) and the one

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Figure 1 Dose–response curve of the effects of anandamide (AEA) on copulatory behavior of SLG male rats. Each graph depicts the effects of different doses of AEA (0.1–3.0 mg/kg, i.p.; dashed boxes) and its vehicle (Veh; empty box) on the specific sexual parameters of SLG rats and includes data of a group of sexually experienced (EXP) animals (grey boxes) as a reference. IL = intromission latency; M = mount number; I = intromission number; EL = ejaculation latency; III = interintromission interval. Data are expressed as medians and interquartile ranges; the mean value is depicted with a discontinuous line. Paired comparisons for all parameters between vehicle-treated EXP and SLG rats are indicated by crosses, Mann–Whitney U test, ++P < 0.01; +++P < 0.001. Statistical differences between vehicle and AEA-treated SLG rats are indicated by asterisks, Kruskal–Wallis ANOVA followed by Dunn’s test, *P < 0.05.

exhibited 7 days after AEA administration. The parameters depicted are I number and EL. As it can be seen, the AEA-induced reductions in these parameters disappeared 7 days after drug treatment and their values returned to pretreatment levels. Figure 3 shows the effects of different doses of the CB1 receptor antagonist AM251 on the sexual performance of SLG rats. All AM251 doses tested (0.1–3 mg/kg) lacked an effect on the sexual parameters of this population. Figure 4 illustrates the effect of the pretreatment with AM251 (0.1 mg/kg) on the AEA-induced reductions in I number and EL. The AM251 dose used lacked an effect per se on these parameters but blocked the

facilitative actions of 0.3 and 1.0 mg/kg AEA on both measures, evidencing the participation of CB1 receptors in these effects. The analysis of locomotor activity is presented in Table 1. None of the pharmacological treatments employed affected spontaneous ambulation of SLG rats. Discussion

The main findings of the present work were that low doses of the endocannabinoid AEA are able to reduce the ejaculatory threshold of SLG rats, decreasing both the number of pre-ejaculatory intromissions and the ejaculation latency. This is J Sex Med 2015;12:1128–1135

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Figure 2 Comparison of the ejaculatory threshold parameters (intromission number [I] and ejaculation latency [EL]) of SLG rats during their last training session (fifth) with those displayed in response to AEA effective doses (0.3 and 1 mg/kg, i.p.) and those exhibited 7 days after AEA, under drug-free conditions (dose labels in italics serve only to identify the different groups). Median values and interquartile range of the data are presented. Wilcoxon signed-rank test, **P < 0.01

Rodríguez-Manzo and Canseco-Alba as compared with normal EXP rats. Notwithstanding, AEA’s sexual facilitative actions in the SLG rats consisted specifically in the lowering of the ejaculatory threshold, which relied on a reduction in both I number and EL. By contrast, these same AEA doses were reported to facilitate all sexual behavior parameters of EXP male rats, in spite of the fact that those animals already had an optimal copulatory behavior [10]. Thus, AEA’s sexual effects in SLG rats do not consist in a general improvement of sexual activity but appear to specifically target the ejaculatory threshold in an acute fashion, since animals return to their original ejaculatory phenotype. According to Waldinger and Olivier, lifelong DE in men may be characterized by a triad of symptoms: ejaculation after a large number of penile thrusts, a long duration of the intravaginal ejaculation latency and occurrence at every, or nearly every, coitus [7]. Remarkably, these symptoms were specifically “repaired” in SLG rats by AEA effective doses; I number and EL values were reduced to levels of normal EXP rats under AEA’s pharmacological influence. This specificity, along with the fact that AEA’s effects are exerted acutely and at low doses, makes this drug emerge as a promising treatment for the improvement of the ejaculatory response in men with primary DE. AEA has been recently reported to exert dosebased biphasic effects on sexual behavior display of animals with a different sexual condition (i.e., sexually experienced and sexually exhausted) [10]. In line with those results, clear facilitative actions of AEA on copulatory behavior of SLG rats were exerted only at low dose levels, whereas a tendency toward the disappearance of these effects appeared with the highest AEA dose tested. Table 1 Locomotor activity

an acute pharmacological effect, since 7 days after AEA administration, the animals return to be sluggish ejaculators, evidencing that the reduction of the ejaculatory threshold in SLG rats occurs only under AEA effects. The AEA-induced facilitation of ejaculatory behavior of SLG rats is mediated by CB1 receptor activation. Specific low AEA doses (0.3 and 1 mg/kg) facilitated the achievement of ejaculation in SLG rats without modifying other aspects of copulatory behavior. This is an interesting result since the analysis of sexual behavior display of SLG rats in the present work revealed that this population exhibits deficits in all sexual behavior parameters J Sex Med 2015;12:1128–1135

Treatment (mg/kg)

Number of counts/5 minutes mean ± SEM

AEA Veh AEA 0.1 AEA 0.3 AEA 1.0 AEA 3.0 AM251 Veh AM251 AM251 AM251 AM251 AEA Veh + AM251 Veh AEA 0.3 + AM251 0.1 AEA 1.0 + AM251 0.1

35.67 ± 2.2 35.37 ± 3.4 32.87 ± 3.1 31.25 ± 2.2 36.50 ± 3.2 34.89 ± 2.5 31.71 ± 0.9 35.00 ± 2.3 35.43 ± 3.0 37.00 ± 3.2 34.50 ± 2.7 32.17 ± 2.4 32.50 ± 1.9

One-way ANOVA, nonsignificant AEA = endocannabinoid anandamide; Veh = vehicle

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Figure 3 Dose–response curve of the effects of different doses of AM251 (0.1–3.0 mg/kg, i.p.; dashed boxes) or its vehicle (Veh; empty box) on sexual parameters of SLG rats. Data are expressed as medians and interquartile range; the mean value is depicted as a discontinuous line. Abbreviations as in Figure 1. Kruskal–Wallis ANOVA, nonsignificant

A review of the literature shows a variety of drugs that have been reported to facilitate sexual behavior of SLG rats. These included the dopaminergic drugs L-DOPA and apomorphine [13], SND-919 and sildenafil [14], the β-adrenoceptor agonist clenbuterol [15] as well as the 5-HT1A receptor agonists 8-OH-DPAT and buspirone [5,6]. However, the lack of specificity of drug actions and/or the emergence of drug-associated side effects in these studies have limited their clinical relevance. The neural commands for ejaculation are organized at the level of the spinal cord, controlled by the spinal generator for ejaculation [16]. However, descending pathways from several brain regions influence its functioning modulating the ejaculatory response. Studies in rats, using the neuronal expression of Fos, revealed the activation of several brain regions in response to ejaculation. These

included the medial preoptic area, bed nucleus of the stria terminalis, medial amygdala, the ventromedial nucleus of the hypothalamus, and the midbrain central tegmental field [17]. Later, the nucleus paragigantocellularis (nPGi), located at the brainstem, was found to exert a tonic inhibitory control on the ejaculatory response [18–20]. The nPGi receives neural inputs from several of the ejaculation-related brain activated regions [21], and most serotonergic nPGi neurons project directly to the lumbosacral spinal cord [22] where the spinal ejaculation generator is located. Although present data do not allow establishing the site at which AEA acts to reduce the ejaculatory threshold of SLG rats, this endocannabinoid can be transported through the blood–brain barrier [23], and therefore, it could be speculated that it occurs within the central nervous system, directly at the spinal cord, modulating the activity J Sex Med 2015;12:1128–1135

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Rodríguez-Manzo and Canseco-Alba AEA is a molecule synthesized and released from postsynaptic neuronal membranes that suppresses neurotransmitter release from those presynaptic terminals bearing CB1 receptors, a mechanism known as retrograde signaling [26]. Thus, AEA-induced reduction of the ejaculatory threshold of SLG rats might involve the direct or indirect suppression of the release of neurotransmitters augmenting it, such as serotonin [27]. In line with this idea, activation of CB1 receptors has been reported to inhibit brain serotonin release [28] and AEA to inhibit serotonin-mediated behavioral responses [29]. The possible interaction of AEA and serotonin in the reduction of the ejaculatory threshold of SLG rats should be explored. With the aid of brain imaging techniques the subparafascicular nucleus, zona incerta, lateral central tegmental field, thalamic nuclei, and ventral tegmental area were reported as the most active human brain regions during ejaculation [30], and CB1 receptors exert a modulatory role in some of them [31–33]. Recently, a clinical trial tested the possible aid of the dopaminergic reuptake inhibitor bupropion for the treatment of lifelong DE, finding only modest beneficial effects after its chronic administration [34]. This result leaves this ejaculatory dysfunction without viable drug treatments; the AEA data presented here offer a possibility worth being clinically explored.

Figure 4 Effect of pretreatment with AM251 on the AEAinduced sexual effects in SLG males. The graph shows the effects of AM251 (0.1 mg/kg) and of two AEA doses (0.3 and 1.0 mg/kg) per se and their combined injection on I number and EL of SLG males expressed as medians and interquartile range. Note that AM251 pretreatment prevented the reduction in both parameters induced by the AEA doses tested. Asterisks over columns indicate comparisons vs. vehicle-treated SLG rats, Mann–Whitney U test, **P < 0.01. Comparison of the combined treatments and the respective AEA dose alone are indicated with brackets, Mann–Whitney U test, **P < 0.01.

Corresponding Author: Gabriela Rodríguez-Manzo, PhD, Departamento de Farmacobiología, Cinvestav Calz. de los Tenorios 235, Col. Granjas Coapa, México, 14330 D.F., México. Tel: (5255) 5483-2871; Fax: (5255) 5483-2863; E-mail: [email protected] Conflict of Interest: The author(s) report no conflicts of interest.

Statement of Authorship

Category 1 of the central pattern generator for ejaculation and/or modulating the activity of brain regions involved in the control of ejaculation. The reduction of the ejaculatory threshold of SLG males by low AEA doses here reported was mediated by CB1 receptors. It is important to mention that AEA binds not only to CB1 receptors in the brain but also to vanilloid TRPV1 receptors [24]; however, only high AEA doses activate the vanilloid receptor [25]. J Sex Med 2015;12:1128–1135

(a) Concept and Design Ana Canseco-Alba; Gabriela Rodríguez-Manzo (b) Acquisition of Data Ana Canseco-Alba (c) Analysis and Interpretation of Data Gabriela Rodríguez-Manzo; Ana Canseco-Alba

Category 2 (a) Drafting the Article Gabriela Rodríguez-Manzo; Ana Canseco-Alba

AEA Reduces the Ejaculatory Threshold of Sluggish Rats (b) Revising It of Interllectual Content Gabriela Rodríguez-Manzo

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J Sex Med 2015;12:1128–1135

Anandamide reduces the ejaculatory threshold of sexually sluggish male rats: possible relevance for human lifelong delayed ejaculation disorder.

The sexually sluggish (SLG) male rat has been proposed as an animal model for the study of lifelong delayed ejaculation, a sexual dysfunction for whic...
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