In the present study, we suggest that the antidepressant-like behavior and the pro-neurogenic effect promoted by Esc in stressed mice are, at least in part, dependent of CB2 receptors. The pharmacological reduction of CB₂ receptors activity have attenuated the behavioral deficits induced in chronically stressed mice, since the lower doses of AM630 (Figure 3) were able to promote anxiolytic-like and antidepressant-like effects, suggesting the CB2 spontaneous activity as an important regulator of behaviors. Interestingly, several lines of evidence demonstrate controversial effects of inverse agonists/antagonists of CB2. The overexpression of CB2 in mice increases resistance to anxiogenic-like stimuli in the hippocampus and amygdala (García-Gutiérrez and Manzanares, 2011). On the other hand, acute administration of CB2 antagonist/inverse agonist was shown to induce anxiogenic-like behavioral, whereas chronic pharmacological blockade of this receptor produced anxiolytic-like effects in parallel with increased expression of the CB2 in the amygdala and prefrontal cortex (García-Gutiérrez et al., 2012). In a recent study, the acute administration of the association of CB2 inverse agonist/antagonist, AM630, and atypical antidepressants (agomelatine and tianeptine) in ineffective doses, promoted antidepressant-like effects in the forced swimming test (Poleszak et al., 2020).

In our model, the pharmacological modulation of the activity of CB₂ receptors with AM630 prior to ESC was not able to prevent the stress-induced depressive-like behavior but promoted anxiolytic-like effect, suggesting the anxiolytic-like effect seems to be dependent of the spontaneous activity of CB2 receptors but not the antidepressant-like behavior. Our findings concerning the behavioral effects of CB₂ modulation are consistent with the molecular results induced by chronic AM630 treatment (Figure 3). The mTOR signaling have been shown to be an integrative protein hub that couples environmental cues, such as stress, to the activation of intracellular pathways to assemble and optimize the inflammatory responses (Laplante and Sabatini 2012). mTOR reconfigures the cellular metabolism and regulates translation, cytokine release, macrophage and mitochondrial polarization and cell migration (Laplante and Sabatini 2012). In the CNS, mTORC1 is considered an important inductor of neurogenesis in neurogenic niches and in vitro models (Palazuelos et al., 2012). Accordingly, chronic AM630 treatment enhanced the expression of phospho-mTOR in stressed mice, suggesting the recruitment of pro-neuroplastic input aiming to counteract the stress effects.

Recent efforts shed light into the contribution of CB₂ receptor activation during the stress-induced neuroendocrine adaptations (García-Gutiérrez et al., 2010; Zoppi et al., 2014). The increase of the full agonist of CB₂ ligand, 2-AG, is reported as a classical chronic stress-related response in several brain regions: amygdala (Patel et al., 2005a; Hill et al., 2010), pre-frontal cortex (Dubreucq et al., 2012; Patel et al., 2005b), hypothalamus (Dubreucq et al., 2012; Patel et al., 2004), and hippocampus (Dubreucq et al., 2012). Enhanced HPA-axis activation appears to be the primary mechanism by which stress increases 2-AG levels (Morena et al., 2016). In stressed mice, we observed increased hippocampal DAGL protein expression, the main enzyme responsible for the synthesis of 2-AG, which exerts pro-inflammatory actions. This stress-induced effect was prevented by chronic CB₂ blockade, suggesting that coping stress effects might include reduction of 2-AG synthesis and signaling by regulation of DAGL expression via buffering HPA-axis activation. HPA-axis disruption promoted by stress is a key factor related to mood disorders that include depletion of monoamines and growth factors, neuroinflammation and alteration in adult hippocampal neurogenesis (Fujioka 2010; Kohl 2011; Busse 2015; Bai 2019). Hence, the classical cannabinoid role in regulation of anti-inflammatory responses in the CNS is especially important since neuroimmunomodulatory processes have been proposed to underlie the pathophysiology of a variety of stress-related neuropsychiatric disorders (Madrigal et al., 2006; Wager-Smith and Markou, 2011). In this sense, several studies have reported that mice lacking the CB₂ receptor have an exacerbated pro-inflammatory phenotype (Turcote 2016).

Regarding neuroplastic effects of Esc, our results showed increased DCX + cells in the hippocampus of mice chronically treated with the SSRI (Figure 5). Preclinical models of chronically stressed and treated with Fluoxetine (FLX), a classical SSRI, have shown a more complex dendritic arborization of DCX +, indicating that the amount of DCX + cells is not necessarily related to antidepressant chronic effects, whereas the microenvironment modulation promoted by this cell subtype might be more relevant to the antidepressant effects than its absolute number (Wang et al., 2008). Additionally, a refined work of Hill and colleagues (2015) with inducible transgenic mice in which the pro-apoptotic gene Bax was deleted from NSC´s, therefore enhancing adult neurogenesis through decreasing in progenitor cell death, has shown reduced anxiety- and depression-like behaviors in stressed mice (Hill et al., 2015). These data indicates that the increasing in adult neurogenesis is sufficient to promote stress resilience. Moreover, both cannabinoid receptors modulate adult neurogenesis by acting at distinct neurogenic phases (Palazuelos et al., 2006; Palazuelos et al., 2012; Prenderville et al., 2015). CB₂ is expressed on NSCs in vitro and in vivo models and it plays a role in the regulation of cell proliferation, neuronal differentiation and maturation (Palazuelos et al., 2006; Palazuelos et al., 2012). In our work, we found DCX + cells number to be increased in mice treated with ESC parallel to the antidepressant-like behavior, an effect dampened by the pre-treatment with AM630, suggesting a complex neuroplastic modulation of chronic antidepressant and CB₂ receptor activity during stress. The CB₂ role in neurogenesis was assessed by Mensching and colleagues by using an CB₂-KO mouse model. They reported that CB2-KO did not present alterations in SGZ proliferation nor DCX + cells compared to WT (Mensching et al., 2019). On the other hand, previous studies have shown reduced basal levels of cell proliferation in the SGZ of CB2-KO mice at 2 months of age after exposure to neurotoxic drug Kainic acid, a potent agonist of glutamate receptors (Palazuellos et al., 2012). These results indicate that CB₂ might not regulate basal levels of adult hippocampal neurogenesis, but rather this refined modulation appears to be more significant in the modulation of neurogenesis during dynamically regulated states, such as in neuroinflammation, drug treatment or even stress. Taking this into account, our data indicate that Esc may be up-regulating some important steps of neurogenesis in a CB2-dependent fashion which is reflected by the altered profile of DCX+ in stressed and treated mice.

We found CB2 chronic blockade to dampen BDNF signaling in the hippocampus of stressed mice. The interactions between BDNF signaling and cannabinoid receptors have been shown to independently modulate neurogenesis (Aguado et al., 2005, 2007; Aso 2008), but how they may interact remains poorly understood. In vitro approaches performed by Ferreira and colleagues have interestingly demonstrated an interplay between BDNF and cannabinoid receptors, especially regarding the CB₂ receptor as a pivotal modulator of BNDF expression and effects in the DG (Ferreira et al., 2018). Thus, our results may reflect direct AM630 actions on the reduction of BDNF signaling which is required to the neurogenic effects of antidepressants, thereafter, leading to the neurogenic disturbance observed in the group whose received chronic Esc.

Esc is classified as an SSRI because of its mechanism of action, but the monoaminergic actions are not enough to completely explain its behavioral and neuroplastic effects (Delgado, 2000; Dale et al., 2015). There are few evidences investigating the possible supplementary Esc mechanisms of action, but comparisons with other SSRIs are conceivable. For instance, FLX has pro-neurogenic effects assigned to the expression of 5HT1A in mature granule cells of the adult mice DG (Samuels et al., 2015). FLX has been shown to accelerate the maturation of young neurons by promoting a faster transition between the DCX + stage to the type 3 cells (NeuN + stage), possibly because of the 5-HT1A expression in these specific cells (Malberg & Duman, 2003; Wang et al., 2008). Furthermore, the FLX proneurogenic effect was shown to be dependent of mTORC1 signaling (Liu et al., 2015). In our model, phosphorylated mTOR have increased in mice exposed to the AM630 chronic treatment, therefore, it is possible that the sharply increase in the mTORC1 signaling promoted by AM630 might be compensated by the drug combination, resulting in the observed neurogenic imbalance. In this sense, further investigations are need to evince whether Esc and FLX share one or more mechanisms.

Alternatively, the impaired neuroplastic effect of ESC in mice prior treated with AM630 might be attributed to the anti-neurogenic effect of pro-inflammatory state induced by lacking CB₂ signaling. Previous studies reported two specific conditions where DCX expression is regulated non linearly compared to levels of adult hipocampal neurogenesis: in chronic stress model where DCX is upregulated and in inflammation models where DCX is down regulated. Considering these data, in our model, it is possible that the reduction in DCX + cells was promoted by a pro-inflammatory state evoked by the pharmacological blockade of CB₂ receptors. Other approaches such as the determination of the inflammatory profile in the DG will allow to indicate whether the effects of CB₂ receptor manipulations in the behavior and in the number of DCX cells are due to inflammation-related actions on the neurogenic niche or to a direct effect of neuronal CB₂ receptors in the Esc response.

Noteworthy, not only neurons exert important functions, but also glial cells exert pivotal roles in the CNS, as it has been highlighted by several studies in the past few years (Jäkel & Dimou, 2017). CB₂ receptor are expressed mainly in microglia, but also in astrocytes and oligodendrocytes (Scheller & Kirchhoff, 2016; Ilyasov et al., 2018). Therefore, the role of glial cells in the ESC antidepressants and neuroplastic effects should be considered in future neuropharmacological studies.

Some methodological differences in relation to other published studies should be considered, since previous investigations of targeting CB2 to promote behavioral modulations were assessed in non-stressed rodents and the neurobiological basis of the stress are determinant to the responses obtained in psychopharmacology studies with cannabinoids (to detailed information, see Morena et al., 2016).

Despite the relevance of our results, we recognize some limitations of our study. Our control versus CUS-groups that received vehicle as treatments presented different stress-induced changes in behavioral despair in the TST (Figures 2 vs Figure 3). However, it is relevant to mention that there are differences between protocols performed to the dose-response curve experiments and the experiments with the antidepressant/antagonism assay which could change the stress levels of control mice: In the first protocol (dose-response curve of AM630) mice received a single injection per day whereas in the second protocol (escitalopram) mice receive two separate injections daily (1st AM630 or vehicle; 2nd ESC or vehicle). This difference in handling and number of injections can interfere in the response observed, since it constitutes one more ‘layer’ of stress to the animals and induces more anxious-like behaviors, specially to groups of control animals (Lapin, 1995; Clarkson et al., 2018). This apparent discrepancy was observed in a recent study published by our group (Fernandes et al., 2021). In addition, chronic unpredictable stress can be used to determine sub-populations of mice that respond different to stress (resilient versus susceptible) and specific molecular markers that could be used as future tools to understand how stress influence behaviors and, translationally, psychiatric disorders (Torrisi et al., 2021; Dziedzicka-Wasylewska et al., 2021). The participation of CB2 receptors in resilience events remain to be elucidated.

Another important limitation of our results relies on the single measure of DCX as a marker of immature neurons survival without the analysis of other phases of the process of adult hippocampal neurogenesis during stress responses possibly under the influence of CB2 receptor (the initial proliferative phase and the expression of survival and mature cells. The migratory ability of DCX + cells is well established in the literature and this feature may be responsible for the complex modulation of the microenvironment during neurogenic events, mediating the connectivity profile of cells in different regions into the DG (Kempermann et al., 2015). However, the precise role of DCX + cells in the neurogenic processes remains under evaluation, which is the reason we choose to investigate this specific cell population in the context of antidepressant chronic treatment. Although the existence of other pharmacological approaches to antagonize more specifically the CB2 receptors in the periphery, such as the SR 144528 (Rinaldi-Carmona et al., 1998), some data has shown pharmacological activity (Rhee and Kim, 2002) and behavioral effects (Hassanzadeh et al., 2016) to be similar to the AM630, highlighting the need of development of new pharmacological compounds to manipulate and study the CB2 receptor role in the SNC.

In summary, our data reveals the relevance of CB₂ receptor activation on the Esc neuroplastic effects and antidepressant-like, but not anxiolytic-like. effects Our results bring new pieces of evidence for an important role of the CB₂ receptor in the mechanism of action of SSRI, supporting the hypothesis that SSRI drugs display CB₂ receptor-dependent neuroplastic effects and behavioral adaptations to promote stress coping. We fully endorse the need of further investigation of parallel mechanisms of action of antidepressants.