**4. Discussion**

Our findings sugges<sup>t</sup> a potentially novel mechanism for the stress-protective effects of URB597 through β-catenin activation in the NAc in a rat model for PTSD and depression.

We found that exposing rats to shock and reminders induced anxiety-like behavior (i.e., increased freezing and startle response) and depressive-like behavior (i.e., decreased social behavior, induced anhedonia, and despair-like behavior), and also impaired memory function in the social recognition and water maze tasks. Administering the FAAH inhibitor URB597 1 h after shock exposure prevented these effects, as had been previously demonstrated [3,17]. Most of the effects of URB597 on behavior were found to be CB1rdependent, as co-administration of the CB1r antagonist AM251 with URB597 prevented the ameliorating effects of URB597.

Importantly, exposure to shock and reminders decreased β-catenin levels in the NAc and mPFC. Decreased β-catenin levels in the NAc were associated with the behavioral phenotype, including enhanced freezing and startle response, anhedonia, and impaired performance in the WTM. This association led us to focus on upregulating and downregulating β-catenin levels in the NAc. Overexpression of NAc β-catenin in rats exposed to shock and reminders resulted in intact behavior (restores freezing and startle response, no indication of depressive-like behavior, intact memory function) and restored expression of β-catenin, mGluR5, and CB1r in the NAc. It is interesting that overexpressing β-catenin in the NAc also restored these same normal levels in the mPFC. Future studies might test the overexpression of β-catenin in the mPFC. Another future study is to examine the potential therapeutic effects of URB597 and the involvement of β-catenin in female rats.

Increased β-catenin levels were reported to promote resilient responses to stress in the NAc [26]. Mice with stabilized β-catenin in the hippocampus showed resilience to some anxious/depressive manifestations when subjected to the corticosterone model of depression [49]. NAc β-catenin upregulation in mice exposed to social defeat stress resulted in a pro-resilient phenotype, demonstrating less social avoidance and better performance in the FST and elevated plus maze (Dias et al., 2014). Similarly, intra-NAc LiCl (2 μg/side), which upregulates β-catenin activity via inhibition of GSK-3β, facilitated inhibitory extinction [26].

In rats exposed to shock and reminders, we found that β-catenin overexpression restored the decrease in mGluR5 and the increase in CB1r expression in the NAc and mPFC. Decreased mGluR5 levels were observed in animal models of depression and the mPFC of depressed individuals [34,35], and blocking mGluR5 has therapeutic effects in PTSD patients and animal models [34,50]. Increased CB1r in the BLA and CA1 were observed in animal models for PTSD [4,15,17], and increased CB1r availability in the amygdalahippocampal-cortico-striatal circuit in human subjects with PTSD [10].

Other studies have shown a stress-induced decrease in β-catenin levels in the mPFC and NAc that was accompanied by highly susceptible behavioral responses [22,51–53]. Mice with dysfunctional NAc Wnt signaling demonstrated increased depression-like behavior and susceptibility to social defeat stress [23], and β-catenin inactivation in the astrocytespecific glutamate transporter (GLAST)-expressing cells enhanced anxious/depressive-like responses [49]. In depressed patients, lowered β-catenin protein levels, but not mRNA levels were found in the NAc, suggesting that depression may be associated with reduced activity of β-catenin, and perhaps not a defect at the transcriptional level [49,54].

Importantly, URB597 did not prevent the effects of exposure to shock and reminders on behavior in rats with viral-mediated NAc downregulation of β-catenin. This indicates that β-catenin is crucial for URB597 to exert its ameliorating effects on behavior. In a previous study, we showed that downregulating β-catenin using sulindac prevented the facilitating effect of the CB1/2 agonist WIN55,212-2 on extinction [26]. Taken together, these findings sugges<sup>t</sup> a strong functional interaction between CB1r and β-catenin. Indeed, cannabinoids regulate neuronal precursor proliferation via β-catenin; the activation of CB1r enhances the activity of PI3K/AKT; this results in AKT-mediated phosphorylation of GSK-3β, followed by the stabilization of β-catenin that translocates into the nucleus; in the

nucleus, β-catenin regulates transcription and gene expression such as cyclin D1 that is involved in cell proliferation regulation [55]. β-catenin may activate TCF/Lef transcription factors [56] and microRNAs [57], which promote anti-stress responses. This could be a possible explanation for the therapeutic-like effects of URB597, acting through CB1r to modulate β-catenin and produce pro-resilient responses.

There are other relevant pathways that might play a critical role in explaining our results. For example, AEA affects CB1r but also has other targets that might be involved in the effects of URB597 [i.e., AEA is a full agonist of TRPV1, which probably participates in ECB signaling [58]]. Garro-Martinez et al. (2020) suggested a link between β-catenin levels and 5-HT1A receptor functionality underlying the vulnerability or resilience to stress-related disorders [59].
