The endocannabinoid system exerts multiple and complex modulatory physiological functions (Kendall and Yudowski, 2016; Ligresti et al., 2016; Argueta and DiPatrizio, 2017; Bennett et al., 2017; Dos Anjos-Garcia et al., 2017; Sun et al., 2017). For instance, cumulative evidence has suggested that the elements of the endocannabinoid system, including FAAH, control the sleep-wake cycle (Santucci et al., 1996; Murillo-Rodríguez et al., 1998, 2001, 2003, 2008a, 2011a, 2013, 2016; Herrera-Solis et al., 2010; Rueda-Orozco et al., 2010; Pava et al., 2014, 2016). In this regard, an experimental approach to explore the role of FAAH in sleep modulation has consisted in the characterization of the properties of FAAH inhibitors, such as URB597 (Murillo-Rodríguez et al., 2007b, 2011a, 2016). Among FAAH inhibitors, several compounds such as 1-heteroarylpropan-2-ones, piperidinyl thiazole isoxazoline, PF-04457845, JNJ-42165279, BIA 10-2474, AM3506, URB694, ARN146333-carboxamido-5-aryl-isoxazole, N-aryl 2-aryloxyacetamides have been reported with significant activity. It is worthy to mention that URB597 has been described as one of the most potent FAAH inhibitors so far (Carnevali et al., 2015; Keith et al., 2015; Lodola et al., 2015; Doenni et al., 2016; Panlilio et al., 2016; Pember et al., 2016; Tong et al., 2016; Tuo et al., 2016; Zahov et al., 2017; Sunduru et al., 2017; Wilkerson et al., 2017). However, full description of these FAAH inhibitors in sleep control requires attention.

AA-5-HT is a dual blocker of FAAH/TRPV1 that shows multiple pharmacological effects. For instance, this compound produces anxiolytic-like effects in mice tested in the open arms (Micale et al., 2009; John and Currie, 2012). Moreover, AA-5-HT also reduces depression-like behaviors without changing locomotor activity (Navarria et al., 2014; Sartim et al., 2017). It is worthy to note that to our knowledge, the current report provides for the very first time direct evidence about the effects of AA-5-HT in sleep modulation. In this regard, the systemic injection of this drug increased sleep during the lights-off, but not in the lights-on period. Moreover, we found that AA-5-HT enhanced power spectra linked to sleep such as delta and theta whereas neurotransmitters associated to waking were found decreased during the lights-off period. Furthermore, in our experiments, AA-5-HT prevented the blockade of the effects of CBD or MOD on sleep rebound as well as neurotransmitters changes after TSD. Taking together, these data suggest that either FAAH or TRPV1 or both might be modulating sleep, sleep homeostasis and neurotransmitter contents in the presence of wake-promoting compounds such as CBD or MOD. Although a mechanistic approach was not addressed in the current report, we would like to draw the following hypothetical scenario (Figure 13): AA-5-HT inhibits FAAH leading to the enhancement in contents of oleoylethanolamide (OEA), palmitoylethanolamide (PEA) as well as AEA (de Lago et al., 2005; Palazzo et al., 2010). Whereas OEA binds to PPARα in the nucleus and promotes waking (Mijangos-Moreno et al., 2016), AEA is able to diffuse across the cellular membrane by AMT to bind either CB₁ cannabinoid receptors or TRPV1 (Leung et al., 2013; Nicolussi and Gertsch, 2015). While activation of CB₁ cannabinoid receptor has been linked with sleep generation (Murillo-Rodríguez et al., 2001, 2003, 2016), further evidence is required to fully determine whether activation of TRPV1 modulates sleep. As mentioned previously, FAAH inhibition may also lead to the increase in the levels of other FAAH substrates. Indeed, Wollank et al. (2015) demonstrated higher levels of AEA, 2-AG, OEA, and PEA in cells incubated with AA-5-HT. These data were confirmed by LC-MS analyses in in vitro studies showing an enhancement of the levels of AEA-like lipids, including OEA and PEA with URB597 (Winkler et al., 2016). If FAAH inhibition promotes waking (Murillo-Rodríguez et al., 2007b, 2011a), how can we reconcile the current results with available evidence in the literature? Since AEA modulates multiple neurobiological functions also acting via TRPV1 (Di Marzo and De Petrocellis, 2012; Starowicz et al., 2012), then a hypothetical explanation would be that AA-5-HT increases sleep via the involvement of TRPV1 activated by AEA. However, the TRPV1 agonist capsaicin increases the firing rate of rat DA neurons (Marinelli et al., 2005; Chakraborty et al., 2016). Moreover, AA-5-HT promotes the electrophysiological activity of locus coeruleus neurons (de Novellis et al., 2008). It is widely accepted that both brain areas are related with waking since their firing rates are higher across alertness rather than during sleep (Berridge et al., 2012). Thus, discrepancies between current results and available evidence might involve –besides methodological differences between comparable studies- a more complex mechanism of action. It can be proposed that AA-5-HT induces sleep not only by facilitating endogenous AEA action at CB1 cannabinoid receptors but also by antagonizing its activation of TRPV1. Thus our data lead us to speculate that AA-5-HT acts as a dual FAAH inhibitor and TRPV1 antagonist, but this should not necessarily be viewed as predictable, because: (1) the activity of AA-5-HT is exerted at higher concentrations comparted to other more potent FAAH inhibitors, such as URB597, and (2) the role of AEA as a full or partial agonist of TRPV1 to promote sleep lacks full investigation. Nevertheless, AA-5-HT could show higher preference for binding to TRPV1 resulting in an increase in AEA levels which in turn will lead to sleep promotion via activation of CB₁ cannabinoid receptors and not to sleep inhibition via TRPV1. Studies addressing the displacement of AA-5-HT from binding sites of TRPV1 simultaneously with AEA will provide new insights regarding endocannabinoid system function.

Lastly, although there are no comparative studies between the different experimental approaches on what could be the most reliable strategy for the manipulation of the endocannabinoid system (endogenous ligands, receptors, transporters, synthesizing/degrading enzymes, etc.) in sleep modulation, the current report provides further evidence for the role of AA-5-HT in sleep control. At this date, the neuromolecular role of the endocannabinoid system in sleep-wake cycle regulation has provided important insights regarding the involvement of AEA, CB₁ cannabinoid receptor, AMT, as well as FAAH (Murillo-Rodríguez et al., 1998, 2001, 2003, 2007b, 2008a, 2011a, 2016; Herrera-Solis et al., 2010; Pava et al., 2014, 2016; Rueda-Orozco et al., 2010). However, the identification of new compounds targeting the endocananbinoid system will help validating the role of this system in sleep control highlighting the most pertinent drug with highest efficiency and lowest side effects in animal models (Lauria et al., 2015; Nimczick and Decker, 2015; Bertini et al., 2016; Aizpurua-Olaizola et al., 2017; Chicca et al., 2017).

Indeed, studies are needed to fully describe the mechanism of action by which AA-5-HT promotes sleep and modulates power spectra activity. Moreover, the regulation of enzymes responsible for neurotransmitter synthesis and/or degradation under the influence of AA-5-HT is also unknown. In addition, the role of AA-5-HT as a blocker of alertness caused by CBD or MOD during the sleep rebound period after TSD still requires further studies.