*2.13. Experimental Design*

a. Study design for experiments 1 and 2: on day 0, male rats were exposed to a single, severe foot shock (1.5 mA, 10 s) in an inhibitory avoidance apparatus, followed by exposure to contextual 1 min situational reminders (SRs) of the shock on days 5, 10, 15, 20, and 25 (Scheme 1a). Freezing was measured during exposure to SR. Drugs (Vehicle, URB597, AM251, AM251 + URB597) were administered i.p. 1 h after shock exposure. Following habituation to saccharin on day −3 (sacc' hab; pre-shock), preference was tested on day −2 pre-shock and on days 2, 7, and 14 post-shock. Acoustic startle response (ASR) testing was administered twice: one day before the shock ( −1; ASR1) and one day after the last reminder (day 26; ASR2). On day 27, rats were exposed to the social preference and recognition tests, on days 28–29 to the water T-maze (WTM), and on days 30–31 to the forced swim test (FST). In experiment 1, four groups (n = 10 for all groups) were administered with vehicle or URB597, and brains were removed on day 32 for western blotting (WB). In experiment 2, six groups (n = 8 for all groups) were administered with vehicle, AM251, or AM 251 + URB597. No rats were excluded from the experiments.


**Scheme 1.** (**a**) Study design for experiments 1 and 2. Rats were exposed to a severe foot shock (1.5 mA, 10 s) followed by exposure to contextual 1 min situational reminders (SRs). Drugs were administered i.p. 1 h after shock exposure followed by a battery of behavioral tests and brain extraction. (**b**) Study design for experiments 3 and 4. On day −5, the herpes simplex virus (HSV) vector was injected bilaterally into the nucleus accumbens (NAc) to overexpress (OE) or downregulate (DR) β-catenin. On day 0, rats were exposed to a single severe foot shock (1.5 mA, 10 s) followed by exposure to contextual 1 min situational reminders (SRs). In experiment 4, drugs (Vehicle, URB59751) were administered i.p. 1 h after shock exposure. All rats were exposed to a battery of behavioral tests. For experiment 3, the brains were removed following the tests.

b. Study design for experiments 3 and 4: on day −5, the herpes simplex virus (HSV) vector was injected bilaterally into the nucleus accumbens (NAc) to overexpress (OE) or downregulate (DR) β-catenin (Scheme 1b). On day 0, male rats were exposed to a single severe foot shock (1.5 mA, 10 s) in an inhibitory avoidance apparatus followed by exposure to contextual 1 min situational reminders (SRs) of the shock on days: 2, 5, 8, 11, and 14. Following habituation to saccharin on day −3 (sacc' hab; pre-shock), preference was tested on days −2 pre-shock and on days 2, 7, and 14 post-shock. Acoustic startle response (ASR) testing was administered before the shock (day 1; ASR1) and one day after the last reminder (day 15; ASR2). In experiment 4, drugs (Vehicle, URB59751) were administered i.p. 1 h after shock exposure. On day 16 rats were exposed to the social preference and recognition tests, on days 17–18 to the water T-maze (WTM), and on days 19–20 to the forced swim test (FST). For experiment 3, the brains were removed on day 21, and β-catenin, mGluR5, and CB1r expression were measured using western blotting (WB). In experiment 3, GFP or OE was delivered to four groups (n = 7–10 for all groups). In experiment 4, GFP or DR was delivered to eight groups (n = 8 for all groups). No rats were excluded from the experiments.
