3.3.2. Freezing

A repeated measures ANOVA (shock × virus × SR; 2 × 2 × 6) on freezing behavior during SRs (Figure 4a) indicated significant main effects of drug [F(1,30) = 27.535, *p* < 0.001], shock [F(1,30) = 56.779, *p* < 0.001], and SR [F(5,150) = 14.616, *p* < 0.001]. We also detected the following significant interactions: shock × virus [F(1,30) = 23.092, *p* < 0.001], shock × SR [F(5,150) = 7.636, *p* < 0.05], virus × SR [F(5,150) = 20.135, *p* < 0.001], and shock × virus × SR [F(5,150) = 12.028, *p* < 0.01]. Post hoc analysis revealed that on SR1– SR5, the Shock/GFP group demonstrated a significant increase in freezing levels compared with the NoShock/GFP group (SR1, *p* < 0.01; SR2–SR5, *p* < 0.001) and Shock/OE group (SR1, *p* < 0.05; SR2, *p* < 0.01, SR3–SR5: *p* < 0.001). Moreover, the Shock/OE group showed increased freezing levels compared with the NoShock/OE group (SR1, *p* < 0.01; SR2, SR3, SR5: *p* < 0.05). Taken together, the findings sugges<sup>t</sup> that β-catenin OE ameliorated the shock- and reminders-induced increase in freezing behavior.

Next, 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). In this experiment, the brains were taken for analysis 26 days after virus delivery and following behavioral testing.

## 3.3.3. Saccharin Preference

A mixed design three-way ANOVA on saccharin preference (shock × virus × days; 2 × 2 × 4) indicated significant main effects of shock [F(1,30) = 8.739, *p* < 0.01] and days [F(3,30) = 35.374, *p* < 0.001], with the following significant interactions: shock × virus [F(1,30) = 7.196, *p* < 0.05]; shock × days [F(1,30) = 7.90, *p* < 0.001]; and shock × virus × days [F(1,30) = 2.577, *p* = 0.05] (Figure 4b). Post hoc comparisons revealed that the Shock/GFP group demonstrated decreased saccharin preference compared with the NoShock/GFP group (day 2: *p* < 0.001) and the Shock/OE group (day 2: *p* < 0.001; day 14: *p* < 0.05). This suggests that NAc β-catenin overexpression prevented the shock- and reminders-induced decrease in saccharin preference.

#### 3.3.4. Acoustic Startle Response

A repeated measures ANOVA [shock × virus × time] on startle amplitude in the ASR indicated a significant main effect of virus [F(1,30) = 12.943, *p* < 0.01], and a significant shock × virus interaction [F(1,30) = 6.737, *p* < 0.05] (Figure 4c). Post hoc analysis revealed that the Shock/GFP group demonstrated a significant increase in startle amplitude compared with the Shock/OE and NoShock/GFP groups (both *p* < 0.001). Hence, upregulation of β-catenin in the NAc restored startle amplitude in rats exposed to shock and reminders.

**Figure 4.** The effects of nucleus accumbens (NAc) β-catenin overexpression on behavior in rats exposed to shock and reminders. Compared with shocked rats with β-catenin overexpression in the nucleus accumbens (NAc) (Shock/OE) and non-shocked rats treated with green fluorescent protein (GFP) (NoShock/GFP), shocked rats treated with GFP (Shock/GFP) demonstrated the following: increased freezing on SR1 to SR5 (SR—situational reminder) (**a**); decreased saccharin preference on days 2 and 7 (only the NoShock/GFP group) (**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**); and increased immobility in the forced swim test (FST) (**f**) (data is shown as mean ± sem; \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001).

## 3.3.5. Social Tests

A two-way ANOVA for the social tests revealed significant main effects of shock [preference: F(1,30) = 17.652, *p* < 0.001; recognition: F(1,30) = 4.751, *p* < 0.05] and virus [preference: F(1,30) = 16.185, *p* < 0.001], with a significant shock × virus interaction [preference: F(1,30) = 8.402; recognition: F(1,30) = 8.727, both *p* < 0.01] (Figure 4d). Post hoc analysis

revealed a lower exploration ratio in both tasks in the Shock/GFP group compared with the Shock/OE group (preference, *p* < 0.01; recognition, *p* < 0.001) and the NoShock/GFP group (preference, *p* < 0.001; recognition, *p* < 0.01). This suggests that NAc β-catenin overexpression prevented the effects of shock and reminders on social behaviors. For total exploration time, see Supplementary File, Figure S5.
