3.1.7. β-Catenin

Punches were extracted from the rat mPFC, NAc, CA1, and BLA (Figure 1g–j, respectively; also shown are brain sites from where the tissue samples were extracted).

For β-catenin levels, a two-way ANOVA [shock × drug; 2 × 2] revealed significant effects of shock [NAc: F(1,36) = 8.52, *p* < 0.01], drug [mPFC: F(1,36) = 15.324, *p* < 0.001; NAc: F(1,36) = 24.099, *p* < 0.001; CA1: F(1,36) = 8.749, *p* < 0.01], and shock × drug interaction [mPFC: F(1,36) = 4.320, *p* < 0.05; NAc: F(1,36) = 16.888, *p* < 0.001; CA1: F(1,36) = 10.863, *p* < 0.01]. Post hoc comparisons revealed a significant decrease in β-catenin levels in the mPFC and NAc in the Shock/Veh group compared with the NoShock/Veh group (mPFC: *p* < 0.05; NAc: *p* < 0.01) and the Shock/URB597 group (mPFC: *p* < 0.01; NAc *p* < 0.001). In CA1, the Shock/URB597 group demonstrated increased β-catenin levels compared with the NoShock/URB597 group and the Shock/Veh group (both *p* < 0.01). No significant effects were observed in the BLA. Hence, exposure to shock and reminders downregulated β-catenin levels in the mPFC and NAc, and URB597 normalized these effects.

The same blots were rehybridized with antibodies specific for β-actin in order to confirm equal protein loading. As there were no differences between the groups in the levels of β-actin in the brain regions we examined, we concluded that the treatment had no effect on the levels of β-actin.

#### 3.1.8. Correlation between β-Catenin Levels and Behavior

We conducted Pearson bivariate correlation tests (Supplementary File; Table S1) between the expression of β-catenin and behavior to explore the association between the β-catenin levels and the anxiety- and depressive-like phenotype of the rats. The most robust correlations were found between β-catenin levels in the NAc and the following behaviors: freezing (SR1: r = −0.560; SR2: r = −0.550; SR3: r = −0.610; SR4: r = −0.572; SR1: r = −0.541; all *p* < 0.01); ASR (r = −0.461, *p* < 0.01); WTM acquisition (r = −0.437, *p* < 0.01) and reversal (r = −0.694, *p* < 0.01), and saccharin preference (Day 2: r = 0.618, *p* < 0.01; Day 7: r = 0.597, *p* < 0.01). This suggests that decreased β-catenin levels in the NAc are associated with enhanced freezing and startle response, anhedonia, and impaired

performance in the WTM. A robust correlation was also found between mPFC β-catenin levels and climbing in the FST (r = 0.419, *p* < 0.01).

#### *3.2. Experiment 2: The Preventing Effects of URB597 on Behavior of Rats Exposed to Shock and Reminders Are Mediated by CB1 Receptors*

As URB597 is a FAAH inhibitor, we aimed to examine whether its effects on stress behavior are mediated through CB1r-dependent mechanisms. We used a low dose of the CB1r antagonist AM251 (am; 0.3 mg/kg) to block CB1r, as previous results have demonstrated that a low dose of this antagonist had no effect on behavior by itself but prevented the therapeutic effects of the cannabinoid agonists (Segev et al., 2018). Therefore, AM251 and URB597 were administered concurrently in order to examine the involvement of CB1rs in the effects of the FAAH inhibitor on behavior (for a detailed study design, see Section 2.13 Experimental Design).
