3.5.3. Saccharin Preference

For saccharin preference (Figure 6b), a repeated measures ANOVA (shock × virus × drug × time; 2 × 2 × 2 × 4) revealed significant main effects of shock [F(1,56) = 36.663, *p* < 0.001] and time [F(3,168) = 15.689, *p* < 0.001]. We also identified the following interactions: shock × virus [F(1,56) = 4.108, *p* < 0.05], shock × time [F(3,168) = 17.943, *p* < 0.001], and drug × time [F(3,168) = 3.263, *p* < 0.05]. Post hoc analysis revealed that on postshock days 2 and 7, a significant decrease in saccharin preference was observed in the Shock/GFP + Veh group compared with the Shock/GFP + URB group (day 2: *p* < 0.05; day 7: *p* < 0.01) and the NoShock/GFP + Veh group (days 2 and 7: both *p* < 0.01). Additionally, a significant decrease was observed in the Shock groups (GFP + URB, DR + Veh, and DR + URB) compared their corresponding NoShock groups: Shock/GFP + URB (day 2: *p* < 0.01), Shock/DR + Veh (day 2: *p* < 0.01; day 7: *p* < 0.001), and Shock/DR + URB (day 2: *p* < 0.05; day 7: *p* < 0.01). Hence, downregulating β-catenin in the NAc had no effect on saccharin preference-shocked rats by itself; however, it blocked the restoring effects of URB597.

#### 3.5.4. Acoustic Startle Response

For ASR (Figure 6c), a repeated measures ANOVA revealed significant main effects of shock [F(1,56) = 108.120, *p* < 0.001], virus [F(1,56) = 19.008, *p* < 0.001], and drug [F(1,56) = 24.204, *p* < 0.001], as well as the following interactions: shock × virus [F(1,56) = 5.597, *p* < 0.05], shock × drug [F(1,56) = 34.653, *p* < 0.001], and shock × virus × drug [F(1,56) = 11.692, *p* < 0.01]. Post hoc analysis on ASR2 revealed decreased amplitude in shocked rats treated with URB597 (Shock/GFP + URB597) compared to the Shock/GFP + Veh and Shock/DR + URB597 groups (both *p* < 0.001). In addition, the shocked groups (Shock/GFP + Veh, Shock/DR + URB597, and Shock/DR + Veh) demonstrated increased startle compared to the non-shocked groups (NoShock/GFP + Veh, NoShock/DR + URB597, and NoShock/DR + Veh; all *p* < 0.001). The Shock/DR + URB597 group showed decreased amplitude compared to the Shock/DR + Veh group (*p* < 0.05). Hence, downregulating β-catenin in the NAc prevented the URB597 normalization of the startle response in shocked rats.

**Figure 6.** Nucleus accumbens (NAc) β-catenin downregulation (DR) blocked the preventive effects of URB597 on behavior in rats exposed to shock and reminders. Compared to shocked rats injected with vehicle (Shock/GFP + Veh) or shocked rats with β-catenin downregulation (DR) and URB597 injection (Shock/DR + URB597), shocked rats treated with URB597 (Shock/GFP + URB597) demonstrated the following: decreased freezing on SR1 to SR5 (SR: situational reminder; GFP: green fluorescent protein) (**a**); higher saccharin preference (only compared with the Shock/DR + URB group) (**b**); decreased acoustic startle response (ASR) on ASR2 (**c**); increased social preference and social recognition (**d**); intact performance in the acquisition and reversal phases in the water T-maze (WTM) (**e**); and decreased immobility in the forced swim test (FST) (**f**) (data is shown as mean ± sem; \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001).

## 3.5.5. Social Tests

For the social tests (Figure 6d), a three-way ANOVA revealed significant main effects of shock [preference: F(1,56) = 19.525; recognition: F(1,56) = 46.854; both *p* < 0.001], virus [preference: F(1,56) = 8.982, *p* < 0.01; recognition: F(1,56) = 4.958, *p* < 0.05], and drug [preference:

F(1,56) = 4.008; recognition: F(1,56) = 4.447; both *p* < 0.05]. We also identified the following interactions: shock × drug [recognition: F(1,56) = 8.885, *p* < 0.01]; virus × drug [recognition: F(1,56) = 8.744, *p* < 0.01]; and shock × virus × drug [preference: F(1,56) = 15.375, *p* < 0.001]. Post hoc analysis revealed a significant increase in the exploration ratio in both tasks in the Shock/GFP + URB597 group compared to the Shock/GFP + Veh group (preference: *p* < 0.001; recognition: *p* < 0.01) and the Shock/DR + URB597 group (*p* < 0.01). This suggests that downregulation blocked the effects of URB597 on social behavior.

In addition, we observed a decrease in the shocked groups (Shock/GFP + Veh, Shock/ DR + URB597, Shock/DR + Veh) compared to the non-shocked groups (NoShock/GFP + Veh: preference, *p* < 0.05, recognition: *p* < 0.001; NoShock/DR + URB597: preference and recognition, *p* < 0.01; NoShock/DR + Veh: recognition, *p* < 0.01). In the preference task, an increase was observed in the NoShock/GFP + Veh group compared with NoShock/DR + Veh group (*p* < 0.05). Hence, NAc β-catenin downregulation blocked the preventive effects of URB597 on social behavior in rats exposed to shock and reminders. For total exploration time, see the Supplementary File, Figure S6.
