3.2.4. Social Tests

For the social tests performed on day 27, a two-way ANOVA revealed significant main effects of shock [preference: F(1,42) = 419.047, *p* < 0.001; recognition: F(1,42) = 315.307, *p* < 0.001] and drug [preference: F(2,42) = 9.021, *p* < 0.01], with a shock × drug interaction [preference: F(2,42) = 5.161, *p* < 0.05; recognition: F(2,42) = 6.969, *p* < 0.01] (Figure 2d). Post hoc analysis revealed a significant decrease in the exploration ratio in both tasks in the shocked groups (Shock/Veh, Shock/am, Shock/am + URB597) compared to the non-shocked groups (NoShock/Veh, NoShock/am, NoShock/am + URB597; all *p* < 0.001). Hence, AM251 had no effect on social behavior by itself; shocked rats co-administrated with URB597 and AM251 behaved similarly to shocked rats treated with vehicle, suggesting that the effects of URB597 on social behavior were blocked by AM251 treatment. For total exploration time, see Supplementary File, Figure S4.

## 3.2.5. Water T-Maze

Rats were tested in the WTM on days 28–29. Repeated measures ANOVA (2 × 2 × 2) revealed significant main effects of shock [F(1,54) = 85.307, *p* < 0.001], drug [F(2,54) = 5.788, *p* < 0.01], and time [F(1,54) = 9.179, *p* < 0.01]. We also detected two interactions: shock × drug [F(2,54) = 5.840, *p* < 0.01] and time × shock [F(1,54) = 6.950, *p* < 0.01] (Figure 2e). Post hoc analysis revealed that fewer trials were needed to reach the criterion in the NoShock groups (Veh, am, am + URB) compared with their corresponding shock groups: Shock/Veh (acquisition and reversal, both *p* < 0.001), Shock/am (reversal, *p* < 0.01), and Shock/am + URB (acquisition and reversal, both *p* < 0.01). In addition, the NoShock/Veh group needed fewer trials to reach criterion compared with the NoShock/am group (acquisition, *p* < 0.001; reversal, *p* < 0.01) and the NoShock/am + URB group (acquisition, *p* < 0.001; reversal, *p* = 0.05) groups. Hence, the co-administration of URB597 + AM251 shows that the restoring effect of URB597 on performance in this task was blocked by AM251 treatment.

#### 3.2.6. Forced Swim Test

For FST performed on days 30–31, a two-way ANOVA on immobility revealed a significant main effect of shock [F(1,42) = 70.807, *p* < 0.001] as well as a shock × drug interaction [F(1,42) = 3.660, *p* < 0.05] (Figure 2f). Post hoc analysis revealed that, compared to the non-shocked groups, the shocked groups demonstrated a significant increase in immobility (Shock/Veh, *p* < 0.01; Shock/am, Shock/am + URB597, *p* < 0.001). Hence, the effects of URB597 on immobility were blocked by AM251 treatment.

No significant differences were observed between the Shock/Veh and Shock/am groups in any of the tests, suggesting that this low dose of AM251 had no effect on behavior by itself. We did detect differences between the Noshock/Veh and Noshock/am groups in the saccharin and WTM tests, suggesting an effect of AM251 in these tests in control rats. The fact that AM251 had an effect by itself in these two tests in non-stressed rats could sugges<sup>t</sup> that under these conditions, the co-administration of URB with AM251 has additive effects (i.e., the combining effects of the two drugs equal the sum of the effects of the two drugs acting independently); hence, the effects of URB597 in WTM performance and social preference are not necessarily mediated by CB1r.

#### *3.3. Experiment 3: The Effects of NAc β-Catenin Overexpression on Behavior in Rats Exposed to Shock and Reminders*

We found that exposure to shock and reminders induced a behavioral phenotype that includes anxiety- and depressive-like behaviors, impaired memory performance, and decreased expression of β-catenin in the NAc and mPFC compared to non-shocked rats. Pearson correlations indicated that the behavioral phenotype was highly associated with decreased levels of β-catenin in the NAc. Hence, we next examined whether the overexpression (OE) of β-catenin in the NAc would prevent the effects of exposure to shock and reminders on behavior.

3.3.1. Verifying β-Catenin Overexpression and Accuracy of Injection

In a preliminary experiment, we delivered overexpression (OE) vectors into the NAc (Figure 3a). In one set of rats (n = 12) we measured β-catenin expression in the NAc using WB (Figure 3b). An independent sample t-test revealed that overexpressing β-catenin in the NAc resulted in significant upregulation of β-catenin levels in the NAc 5 days after viral delivery [t(10) = 3.230, *p* < 0.05] compared to the GFP group. In the second set of rats (n = 12) we verified the accuracy of injection in the NAc using GFP detection (Figure 3c).

**Figure 3.** The delivery of the viral overexpression (OE) or downregulation (DR) vectors affects β-catenin levels in the nucleus accumbens (NAc). On day 0, overexpression (OE), downregulation (DR),

or green fluorescent protein (GFP) vectors were injected bilaterally into the rat nucleus accumbens (NAc). After five days of recovery, brains were removed and taken for GFP detection and β-catenin expression evaluation (**a**); the overexpression (OE) group demonstrated increased β-catenin levels compared to the green fluorescent protein (GFP) group (**b**); GFP detection revealed successful delivery of the OE vector to NAc (**b**); the OE group demonstrated increased β-catenin levels compared to the GFP group (**c**); GFP detection revealed successful delivery of the DR vector to the NAc (**d**); the DR group demonstrated decreased β-catenin levels compared to the GFP group (**e**); the expression of β-catenin in the NAc, after the delivery of viral vectors (OE/DR) were quantified by western blotting (**f**,**g**); bilateral expression of GFP in the NAc area (**h**); (data is shown as mean ± sem (\*, *p* < 0.05; \*\*, *p* < 0.01). (NA coordinates are relative to Bregma; AP: anterior–posterior; ML: medial–lateral; NAc: nucleus accumbens; OE: overexpression; DR: downregulation; V: ventral; WB: western blotting).

We examined whether viral-mediated OE of β-catenin in the NAc can restore the effects of shock and reminders on behavior, compared to rats injected with GFP (see Section 2.13 Experimental Design).
