3.4.3. CB1

Our findings regarding the involvement of β-catenin in the effects of shock and reminders on the amygdala-hippocampal-cortico-striatal circuit sugges<sup>t</sup> a key role for the mPFC and the NAc in mediating the effects on behavior. Hence, we also examined the expression of CB1r in the mPFC and NAc. A two-way ANOVA on CB1r protein levels revealed significant main effects of shock [mPFC: F(1,31) = 14.405, *p* < 0.01; NAc: F(1,31) = 6.839, *p* < 0.05] and virus [mPFC:F(1,31) = 10.727, *p* < 0.01; NAc: F(1,31) = 4.030, *p* = 0.05], as well as a shock × virus interaction [mPFC: F(1,31) = 20.238, *p* < 0.001; NAc: F(1,31) = 24.915, *p* < 0.001]. Post hoc analysis revealed increased CB1r levels in the mPFC (Figure 5i) and NAc (Figure 5j) in the Shock/GFP group compared with the NoShock/GFP and Shock/OE groups (mPFC: both *p* < 0.001; NAc: both *p* < 0.01). Hence, exposure to shock and reminders upregulated CB1r levels in the mPFC and NAc, and overexpressing β-catenin in the NAc normalized these effects.

#### *3.5. Experiment 4: The Effects of NAc β-Catenin Downregulation on Behavior of Rats Exposed to Shock and Reminders and Treated with URB597*

#### 3.5.1. Verifying β-Catenin Downregulation

In a preliminary experiment, we delivered an HSV P1005 vector, which expresses a mutant of β-catenin, into the NAc (Figure 3a). In one set of rats (n = 13) we measured β-catenin expression in the NAc using WB (Figure 3d). An independent sample t-test revealed that β-catenin protein levels in the NAc were downregulated (t(11) = 4.179, *p* < 0.01) compared to the HSV-GFP group. In another set of rats (n = 12), we verified the accuracy of injection in the NAc using GFP detection (Figure 3e).

Next, we examined whether the effects of URB597 on behavior in rats exposed to shock and reminders are mediated by β-catenin. To do so, we used a viral approach to downregulate (DR) β-catenin in the NAc (see Section 2.13 Experimental Design).
