The main findings of the present series of experiments can be summarized as follows: (a) sexually exhausted male rats exhibit a reduced sexual motivation 24 h after copulation to satiety, when the sexual inhibitory period is well established; (b) low doses of the eCB 2-AG reverse sexual satiety through a CB1 receptor-dependent mechanism, but do not modify copulatory behavior of sexually experienced male rats; (c) the eCBs AEA and 2-AG interact with the dopaminergic system to induce sexual behavior expression in sexually exhausted male rats; (d) D2-like, but not D1-like DA receptors, participate in the 2-AG-induced sexual satiety reversal; and (e) AEA-induced satiety reversal is mediated by D1-like DA receptors. Notwithstanding, D2-like receptor agonists also synergize with AEA to induce sexual activity in sexually exhausted males.

The sexual incentive motivation test revealed that sexually exhausted male rats do not show the preference for a sexually receptive female exhibited by sexually experienced animals, thereby confirming that during the sexual inhibitory period that characterizes sexual satiety, male rats have a reduced sexual motivation. As mentioned in the introduction section, previous data implied that sexual motivation might play a role in the sexual satiety phenomenon (Rodríguez-Manzo, 1999a; Guadarrama-Bazante and Rodríguez-Manzo, 2019), including a report on a diminished sexual motivation measured in male rats immediately after reaching sexual satiety (Ågmo et al., 2004). However, the sexual motivational state of the satiated animals during the long-lasting sexual inhibitory period (i.e., 24–72 h later) had not been directly assessed. This assessment is important, because 24 h after copulation to satiety, not only the characteristic sexual inhibitory state is well established, but also other possible confounding factors are absent. For instance, the fatigue due to intense copulation is no longer present, since the animals rested overnight. Males had free access to water and food when returning to their home-cages after copulation to satiety, eliminating hunger and thirst as factors playing a role in their lack of interaction with the receptive female rat. Finally, during the sexual incentive motivation test, the sexually satiated rats are exposed to a new sexually receptive female, eliminating the possible habituation to the sexual partner as another factor involved in the absence of preference. Under these conditions, we believe that the lack of interest for the sexually receptive female exhibited by sexually satiated males 24 h after copulation to satiety reflects an actual decrease in sexual motivation, thus validating the notion that this factor is involved in the long-lasting sexual inhibitory state that characterizes the sexual satiety phenomenon.

eCB signaling in the brain has been found to regulate the motivation for natural rewards (Parsons and Hurd, 2015). Reinforcing this notion, previous data from our group showed that the eCB AEA reversed sexual satiety after its direct infusion into the VTA (Canseco-Alba and Rodríguez-Manzo, 2016). This result supports the idea that eCBs’ actions at the MSL system might modify the sexual motivational tone of sexually satiated rats. AEA and 2-AG are the best-characterized eCBs and both activate CB1 receptors; however, AEA binds with moderate affinity and is a partial agonist at CB1 receptors, whereas 2-AG binds with low affinity but exhibits full efficacy at these receptors (Hillard, 2000). Besides, 2-AG is more abundant than AEA in the brain (Stella et al., 1997; Nomura et al., 2008) and is considered as the key eCB released on demand by VTA DA neurons to modulate its own activity (Melis et al., 2004; Tanimura et al., 2010). Therefore, it was crucial to determine if 2-AG was also capable of reversing the sexual inhibitory state of sexually exhausted rats. Results showed that similar to AEA’s effects (Canseco-Alba and Rodríguez-Manzo, 2014), low doses of 2-AG reversed sexual satiety through the activation of CB1 receptors. However, 2-AG was capable of inducing copulation in satiated rats within a broader dose range than AEA (0.1–3.0 mg/kg vs. 0.1–0.3 mg/kg, respectively), which is compatible with the higher efficacy of the former at CB1 receptors. Interestingly, 2-AG induced the display of sexual behavior in sexually satiated rats but did not modify the sexual performance of sexually experienced animals. A possible explanation for this differential action could be based on the proposal that the net effect of CB1 receptor-mediated actions at the MSL system depend on the level of baseline activity of midbrain DA neurons, such that enhancing DA neuronal firing may have a larger effect when baseline frequency is low compared to when neurons are burst firing (Covey et al., 2017). Following this idea, when sexually experienced rats are exposed to sexually receptive females as well as during copulation, DA neurons fire in the bursting mode rendering a phasic DA release in the NAcc (Robinson et al., 2002). Although, eCBs are capable of removing the tonic inhibition exerted by GABAergic inputs onto midbrain dopaminergic neurons to promote the phasic mode of DA release (Oleson et al., 2012), in the sexually experienced males this effect is also elicited by the rewarding stimulus (Grace, 1991; Grace et al., 2007), i.e., the presence and interaction with the sexually receptive female, which could account for the lack of 2-AG sexual facilitative effects in these animals. By contrast, in sexually satiated rats the inhibition of their sexual responsiveness suggests that DA neuron baseline activity is low, explaining the ability of 2-AG to produce sexual behavior facilitative effects in animals with this sexual condition.

A central finding of the present work is that both AEA and 2-AG interact with the dopaminergic system to reverse sexual satiety. Sub-optimal doses of each of these eCBs synergized with a dose of apomorphine that was subthreshold for reversing sexual satiety, whereas the reversal of the sexual inhibitory state, induced by effective doses of AEA or 2-AG, was canceled by the DA receptor antagonist haloperidol. These results contribute to strengthening the notion of a relationship between eCB and DA signaling in the facilitation of reward-motivated behaviors (Oleson and Cheer, 2012; Wenzel and Cheer, 2018). They also suggest that the deficient motivational tone of sexually exhausted rats might be increased by eCBs’ actions, through the modulation of DA activity, enabling sexually satiated males to respond with sexual activity to the rewarding stimulus, represented by the receptive female.

It has been proposed that the dopaminergic system might be the common final pathway for the pharmacological reversal of sexual satiety. This idea emerged from the fact that several drugs, acting at different neurotransmitter systems, are capable of reversing sexual satiety by interacting, directly or indirectly, with the dopaminergic system (Rodríguez-Manzo and Fernández-Guasti, 1995; Rodríguez-Manzo, 1999b; Hull and Rodríguez-Manzo, 2017). Present results are in line with this proposal ascribing to the MSL system a central position in the dopaminergic-mediated regulation of male rat sexual behavior expression.

Midbrain DA neurons are involved in the signaling of reward-related stimuli by changing their firing pattern (Grace, 1991; Grace et al., 2007). The basal activity of these neurons involves low-frequency firing resulting in a dopaminergic tone capable of activating high-affinity D2-like DA receptors in the NAcc. Upon the presentation of a rewarding stimulus, this firing pattern changes to a high-frequency burst firing that is accompanied by an increase in NAcc DA release, which activates low-affinity D1-like DA receptors (Grace et al., 2007; Dreyer et al., 2010). On these bases, establishing the DA receptor family/families involved in the 2-AG/DA and AEA/DA interactions to induce sexual behavior display in sexually satiated rats appeared relevant. Interestingly, our data showed that there was a differential interaction between each of these eCBs and the two DA receptor families. 2-AG synergized with D2-like, but not with D1-like DA receptor agonists, to induce sexual satiety reversal; an effect that was completely prevented by the D2-like receptor antagonist raclopride. In contrast, AEA synergized with both D1- and D2-like DA receptor agonists to reverse the sexual inhibition, but only the D1-like DA receptor antagonist SCH23390 was able to block the AEA-induced sexual satiety reversal.

It has been documented that 2-AG and AEA, in spite of activating the same cannabinoid receptors and signal transduction pathways (Janero et al., 2009), do not always play the same physiological role, acting sometimes in concert and sometimes not (Di Marzo and Cristino, 2008; Luchicchi and Pistis, 2012). It is important to recall that in the present work eCBs were exogenously administered and, therefore, could activate CB1 receptors in different brain regions. Within the MSL system, CB1 receptors are expressed both in the NAcc (Pickel et al., 2006) and in the VTA (Herkenham et al., 1991); therefore, systemically administered eCBs might have reversed satiety by acting at each of these brain regions, where they behave as retrograde messengers suppressing presynaptic glutamate and GABA release (Lupica and Riegel, 2005). However, substantial evidence indicates that exogenously administered cannabinoids increase DA release in rat NAcc (Chen et al., 1990; Gardner and Vorel, 1998; Gessa et al., 1998) and excite midbrain DA neurons in the VTA (French et al., 1997). These data suggest that the actions of exogenously administered eCBs in the present work would be exerted at the VTA; a proposal supported by the finding that intra-VTA infusion of AEA reverses sexual satiety (Canseco-Alba and Rodríguez-Manzo, 2016).

In relation to DA receptors, D1-like and D2-like receptors within the MSL system are mainly expressed in the NAcc, segregated in different populations of medium spiny neurons, which constitute 95% of the cells in this brain region (Yang et al., 2018). According to present data, the 2-AG/DA interaction for the reversal of sexual satiety clearly involves only the activation of D2-like receptors, probably from the NAcc, since in the VTA these receptors are somatodendritic autoreceptors that regulate the firing rate of DA neurons and DA release in terminal fields. D2 autoreceptor activation reduces DA release at the NAcc and also limit somatodendritic DA release in the VTA (Rice and Patel, 2015). In support of the notion that the 2-AG/DA interaction takes place in the NAcc is the finding that direct infusion of the selective D2-like DA receptor agonist, quinpirole, into this brain region reverses the sexual inhibition of satiated rats (Guadarrama-Bazante and Rodríguez-Manzo, 2019).

Results of this work also suggest that AEA’s interaction with DA transmission in sexually satiated rats is essentially, although not exclusively, mediated by D1-like DA receptor activation. This conclusion derives from the fact that D1-like DA receptor blockade with the antagonist SCH23390 canceled AEA-induced reversal of sexual satiety and a sub-effective dose of the D1-like receptor agonist SKF33939 synergized with a dose of AEA, that was subthreshold for reversing sexual satiety, to promote sexual behavior display. In line with this finding, our group observed that systemically administered DA receptor agonists reverse sexual satiety through the activation of D1-like DA receptors (Guadarrama-Bazante et al., 2014).

AEA also synergized with D2-like DA receptor activation to reverse sexual satiety, but reversal of sexual satiety induced by an effective dose of AEA, was not canceled by the D2-like receptor antagonist raclopride. These results evidence that AEA effects are mediated by the modulation of D1-like DA receptors, though the independent activation of D2-like DA receptors with quinpirole could synergize with the AEA-mediated activation of D1-like DA receptors to reverse satiety. Interestingly, activation of D2-like DA receptors in the NAcc increases the extracellular levels of AEA in this brain region (Giuffrida et al., 1999). In fact, DA exerts modulatory effects on both AEA and 2-AG content in the NAcc (Patel et al., 2003), where medium spiny neurons synthesize and release these eCBs in response to DA stimulation. Remarkably, the NAcc’s content of AEA and 2-AG is differentially modulated by the activation of D1-like and D2-like DA receptors, respectively (Patel et al., 2003). These data further support the relationships described in the present work, between AEA and D1-like DA receptors and between 2-AG and D2-like DA receptors. Thus, a possible contribution to the reversal of sexual satiety of changes in NAcc’s content of these two eCBs, resulting from the activation of the two DA receptor families, cannot be discarded.

In spite of the differential interaction found for each eCB with the DA receptor families, it has been considered that the simultaneous activation of D1-like and D2-like DA receptors in the NAcc is required for the processing of reward relevant information (Ikemoto et al., 1997). Moreover, it has been reported that only the combination of D1-like and D2-like receptor agonists is able to enhance NAcc core cell firing in vitro—an effect in which the participation of eCBs is required—, while the independent activation of each of these receptor families does not reproduce this result (Seif et al., 2011). Remarkably, we found that the direct infusion of the non-selective DA receptor agonist, apomorphine, into the NAcc induced a full sexual satiety reversal, not obtained with any other pharmacological treatment so far tested in sexually satiated rats. In this case, the most effective apomorphine dose induced sexual behavior to ejaculation and copulation resumption after ejaculation in every sexually satiated animal. Besides, the copulatory performance of these males was as efficient as that of sexually experienced males, an outcome not commonly seen with other treatments reversing satiety (Guadarrama-Bazante and Rodríguez-Manzo, 2019). This result reinforces the notion that the regulation of the rewarding properties and motivational tone at the NAcc is mediated by the cooperative actions of the two DA receptor families.

Based on the data here presented, it could be proposed that repeated activation of the MSL system by intense copulation during sexual satiety development, induces a diminished sexual motivational tone, responsible for their characteristic long-lasting sexual behavior inhibition. AEA and 2-AG seem to reverse this sexual inhibitory state by modulating dopaminergic transmission, presumably at the MSL system. Specific experiments further analyzing the role played by eCBs in the regulation of DA-mediated motivation for natural rewards at the MSL system are warranted.

The motivational component of male rat sexual behavior and the initiation of the copulatory behavioral pattern (Blackburn et al., 1992) have not only been linked to mesolimbic DA, but also to DA transmission at the hypothalamic medial preoptic area (mPOA; Pfaus and Phillips, 1991; Hull et al., 1999). Indeed, DA levels increase simultaneously in the NAcc and mPOA in response to the presence of an inaccessible sexually receptive female (Blackburn et al., 1992; Hull et al., 1999). However, present and previous data from our lab do not suggest a participation of this brain region in the eCB/DA-mediated increase in sexual motivation that leads to the reversal of sexual satiety. To the extent of our knowledge, eCBs have not been associated to the control of mPOA DA neuron activity and, infusion of DA receptor agonists into this brain region does not reverse sexual satiety, except for a specific low dose of the D2-like receptor agonist quinpirole (Guadarrama-Bazante and Rodríguez-Manzo, 2019). However, the systemic administration of several quinpirole doses failed to reverse the sexual inhibition of satiated rats (Guadarrama-Bazante et al., 2014). Notwithstanding, the indirect participation of the mPOA, a brain region that is crucial for male sexual behavior expression in all vertebrate species (Hull et al., 1999), through its efferent projections, which have been considered essential for the initiation of the copulatory pattern (Everitt, 1990), cannot be discarded. Actually, some of these projections target specifically the VTA (Simerly and Swanson, 1988; Stolzenberg and Numan, 2011; Zahm et al., 2011) and might influence the activity of mesolimbic DA neurons favoring an increase in sexual motivation of sexually satiated rats.

The present work uses sexual satiety as an animal model to provide new evidence showing that eCBs modulate the MSL system with an impact on sexual motivation. This model offers the opportunity to explore the nature of adaptive inhibitory mechanisms (Bancroft, 1999) controlling the expression of an innate behavior normally triggered by specific incentive stimuli, i.e., copulation in the presence of an accessible sexually receptive female, which appears to involve changes in the male’s motivational tone. This research also suggests that CB1 receptor activation might have therapeutic potential to positively influence sexual arousal and desire, which deficiency underlies several human sexual disorders, the hypoactive sexual desire disorder (American Psychiatric Association, 2013) among them; this possibility should be explored.

Like all studies, the present work has its limitations. Since all drug treatments in this study were systemically administered, the involvement of brain structures outside the mesolimbic system as well as peripheral effects of eCBs cannot be discarded. The participation of the MSL system in the eCB-mediated actions here described has to be confirmed, however, this study is a good starting point motivating more specific experiments directed to identify the brain loci within the mesolimbic system where the described eCB/DA interactions occur.