3.1.1. Freezing

Freezing behavior was monitored during the 1 min exposure to the SR conditions (baseline and SR1-SR5) (Figure 1a). Repeated measures ANOVA (shock × drug × SR; 2 × 2 × 6) revealed significant main effects of drug [F(1,36) = 37.737, *p* < 0.001], shock [F(1,36) = 78.650, *p* < 0.001], SR day [F(5,180) = 11.773, *p* < 0.001]. We also detected the following interactions: shock × drug [F(1,36) = 36.301, *p* < 0.001], shock × SR [F(5,180) = 10.960, *p* < 0.001], drug × SR [F(5,180) = 4.910, *p* < 0.001], and shock × drug × SR [F(5,180) = 4.823, *p* < 0.001]. Post hoc analysis revealed that on SR1 to SR5 the Shock/Veh group demonstrated a significant increase in freezing levels compared with the NoShock/Veh group (*p* < 0.001) and the Shock/URB group (SR1; SR2; SR4; SR5: *p* < 0.01; SR3: *p* < 0.001). This suggests that URB597 prevented the shock-induced increase in freezing behavior. In addition, the Shock/URB group showed increased freezing levels compared with the NoShock/URB group (SR1: *p* < 0.001; SR2; SR3: *p* < 0.01; SR4: *p* < 0.05).

## 3.1.2. Saccharin Preference

Saccharin preference was tested on day 2 pre-shock and on days 2, 7, and 14 postshock (Figure 1b). Repeated measures ANOVA (shock × drug × day; 2 × 2 × 4) revealed significant effects of drug [F(1,36) = 16.109, *p* < 0.001] and day [F(3,108) = 5.633, *p* < 0.01]; with the following interactions: shock × drug [F(1,36) = 8.970, *p* < 0.01], shock × day [F(3,108) = 2.896, *p* < 0.05], drug × day [F(3,108) = 5.723, *p* < 0.01], shock × drug × day [F(3,108) = 6.052, *p* < 0.01]. Post hoc analysis revealed that on days 2 and 7, the Shock/Veh group demonstrated decreased saccharin preference compared with the NoShock/Veh (day 2: *p* < 0.001; day 7: *p* < 0.01) and Shock/URB (*p* < 0.001) groups. Hence, URB597 prevented the shock and reminders induced a decrease in saccharin preference (i.e., anhedonia).

#### 3.1.3. Acoustic Startle Response

The ASR test was performed on days 1 (pre-shock) and 26 (Figure 1c). We performed a repeated measures ANOVA (shock × drug × time; 2 × 2 × 2) on mean amplitude, and identified significant effects of shock [F(1,36) = 4.060, *p* < 0.05] and time [F(1,36) = 7.799, *p* < 0.01], with the following significant interactions: shock × time [F(1,36) = 5.3, *p* < 0.05], drug × time [F(1,36) = 6.779, *p* < 0.05], and shock × drug × time [F(1,36) = 8.646, *p* < 0.01]. Post hoc analysis on ASR2 revealed a significant increase in amplitude in Shock/Veh compared with Shock/URB and NoShock/Veh (*p* < 0.05 in both cases). This suggests that URB597 restored the shock/reminders-induced increase in startle response.

## 3.1.4. Social Tests

We performed the social tests on day 27 (Figure 1d). We ran a two-way ANOVA (shock × drug; 2 × 2) and found significant effects of shock [preference: F(1,36) = 4.537, *p* < 0.05; recognition: F(1,36) = 23.199, *p* < 0.001] and drug [preference: F(1,36) = 22.928, *p* < 0.001; recognition: F(1,36) = 6.619, *p* < 0.05], with a significant drug × shock interaction [preference: F(1,36) = 6.259, *p* < 0.05; recognition: F(1,36) = 5.905, *p* < 0.05]. Post hoc analysis revealed a significant decrease in the exploration ratio in both tasks in the Shock/Veh group compared with the NoShock/Veh group (preference: *p* < 0.01; recognition: *p* < 0.001) and Shock/URB597 group (*p* < 0.001 for both preference and recognition). Hence, URB597 restored the impairing effects of shock and reminders on social behavior. For total exploration time, see the Supplementary File, Figure S3.

**Figure 1.** The effects of URB597 on behavior and β-catenin expression in rats exposed to shock and reminders. Compared with non-shocked rats treated with vehicle (NoShock/Veh) and shocked rats treated with URB597 (Shock/URB597), rats that were exposed to shock and treated with vehicle (Shock/Veh) demonstrated the following: increased freezing on SR1 to SR5 (SR—situational reminder) (**a**), decreased saccharin preference on days 2 and 7 (**b**), increased acoustic startle amplitude (ASR) on ASR2 (**c**); decreased social preference and social recognition (**d**); impaired performance in the acquisition and reversal phases in the water T-maze (WTM) (**e**), increased immobility in the forced swim test (FST) (**f**); downregulation in β-catenin protein levels in the medial prefrontal cortex (mPFC) (**g**),

and in the nucleus accumbens (NAc) (**h**); in the CA1, the Shock/Veh group and the NoShock/URB597 group demonstrated downregulation in β-catenin levels compared with the Shock/URB597 group (**i**); in the basolateral amygdala (BLA), no significant differences between the groups were observed (**j**) rat brain atlas illustration indicating punch location are also shown (data is shown as mean ± sem; \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001).
