3.2.1. Freezing

Freezing behavior was monitored during the 1 min exposure to the SRs. A repeated measures ANOVA (shock × drug × SR; 2 × 3 × 6) revealed significant main effects of shock [F(1,42) = 551.682, *p* < 0.001] and SR [F(5,210) = 33.124, *p* < 0.001]; with a shock × SR interaction [F(5,210) = 32.356, *p* < 0.001] (Figure 2a). Post hoc analysis revealed that on SR1 to SR5, the shock groups (Veh; am; am + URB) demonstrated a significant increase in freezing levels compared with the NoShock groups (Veh; am; am + URB; *p* < 0.001). Hence, the preventive effect of URB597 on freezing behavior was blocked by AM251.

## 3.2.2. Saccharin Preference

Saccharin preference was tested on day 2 pre-shock and on days 2, 7, and 14 postshock. A repeated measures ANOVA (2 × 2 × 4) revealed a significant effect of shock [F(1,54) = 99.552, *p* < 0.001] and drug [F(2,54) = 6.626, *p* < 0.01], with a shock × drug interaction [F(1,54) = 4.021, *p* < 0.05] (Figure 2b). Post hoc analysis revealed that the shock groups (Veh, am, am + URB) demonstrated a decrease in saccharin preference compared with their corresponding NoShock groups: NoShock/Veh (days 2, 7, and 14: all *p* < 0.001), NoShock/am (day 2: *p* < 0.05; day 7: *p* < 0.01), NoShock/am + URB (day 2: *p* < 0.01; day 7: *p* < 0.001; days 14 and 21: *p*< 0.05). Moreover, compared with the NoShock/Veh group, we found that the NoShock/am group (days 2 and 14: *p* < 0.01; day 7: *p* < 0.001), and the NoShock/am + URB group (days 2 and 14: *p* < 0.05; day 7: *p* < 0.01) demonstrated decreased saccharin preference. Hence, co-administration of URB597 + AM251 shows that the restoring effect of URB597 on saccharin preference was blocked by AM251 treatment.

The shock-induced decrease in saccharin preference was short-termed (observed only one or two weeks after shock exposure). We have previously shown that in rats exposed to severe shock and to SRs 7, 14, and 21 days after shock exposure, the shock-induced decrease lasted till day 14 post-shock but not till day 28 post-shock (Burstein et al., 2018). In a previous study, we found that the chronic stress-induced decrease in sucrose consumption only lasted 1 week after the stress ended (Segev et al., 2014). Hence, there are fluctuations in saccharin preference at different times after stress exposure.

#### 3.2.3. Acoustic Startle Response

We tested ASR on days 1 and 26, and a repeated measures ANOVA on startle amplitude indicated significant main effects of shock [F(1,54) = 4.060, *p* < 0.001] and time [F(1,54) = 17.854, *p* < 0.001], as well as a shock × time interaction [F(1,54) = 17.854, *p* < 0.001] (Figure 2c). Post hoc analysis revealed that on ASR2, a significant increase in amplitude was found in the shocked groups treated with vehicle (Shock/Veh), AM251 alone (Shock/am), and AM251 and URB597 (Shock/am + URB597) compared with the non-shocked groups (NoShock/Veh: *p* < 0.01, NoShock/am: *p* < 0.01, NoShock/am + URB597: *p* < 0.001). Hence, AM251 had no effect on ASR by itself; the co-administration of URB597 and AM251 in rats

exposed to shock and reminders increased their startle response amplitude, suggesting that the effects of URB597 on startle response were blocked by AM251 treatment.

**Figure 2.** The effects of URB597 on behavior in rats exposed to shock and reminders are mediated by CB1 receptors. The shocked groups (Shock/Veh, Shock/am, Shock/am + URB597) compared with the non-shocked groups (NoShock/Veh, NoShock/am, NoShock/am + URB597), demonstrated the following: increased freezing levels (**a**); a decrease in saccharin preference on days 2, 7, 14, and 21 (**b**); increased acoustic startle response (ASR) amplitude 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 (**f**) (data is shown as mean ± sem; \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001).
