*2.4. Behavioral Testing*

All rats were tested in all behavioral procedures in the same order. The most aversive test (forced swim test—FST) was last. Tests were separated by a 24 h period. All behavioral tests were conducted under dim lighting (15–20 lx) and took place between 12:00 and 16:00. Behavioral testing was taken after SR5 in order to examine the long-term effects of the traumatic event. In a previous study, we found that shocked rats exposed to SRs persistently avoided the dark chamber, whereas, shocked rats not exposed to SRs demonstrated increased avoidance on the first extinction trial, but their extinction kinetic was intact [7].

#### *2.5. Acoustic Startle Response (ASR)*

A soundproof chamber (25 × 25 × 25 cm) containing an acrylic animal holder (8 × 8 × 16 cm; Coulbourn Instruments, Whitehall, PA, USA) that is connected to a piezoelectric accelerometer. For consistency between chambers and experiments, calibration of sensitivity to the movement and sound levels was applied. A single rat was placed in the holder for a 5 min acclimatization period; 30 acoustic startle trials (98 or 120 dB; 50 ms duration, 20–40 s intertrial interval) were presented over the 68 dB white noise background. We analyzed the mean startle amplitude which indicates the averaged response to the 98 and 120 dB in mV [40].

#### *2.6. Social Preference and Social Recognition*

This task assesses sociability and short-term social memory. Habituation for 1 h to the transparent corrals in the home cages of the juvenile and experimental rats preceded the training. The object and the juvenile rat were confined to the corrals that were placed 10 cm from the two opposite corners of the arena. The corrals (9 cm in diameter) allowed physical contact with the experimental rat [3]. The objects were children's Lego blocks.

For the preference and recognition phases, the experimental rat was given 5 min exploration with an intertrial interval of 30 min in a holding cage. For preference, the rat explored the novel object and the unfamiliar juvenile. The ratio was calculated as the time spent exploring the juvenile rat divided by the total exploration time (juvenile rat + novel object). For recognition, the rat explored a novel juvenile and the familiarized juvenile. The ratio was calculated as the time spent exploring the novel juvenile divided by total exploration time (familiar + novel juveniles). The trials were videotaped (Logiteck, C922 Pro Stream, Newark, CA, USA) and recorded (NCH Software, Greenwood Village, CO, USA) and the time spent in corral exploration was analyzed using EthoVision XT9 software.

#### *2.7. Water T-Maze (WTM)*

The WTM is a black Plexiglas maze (length of stem, 64 cm; length of arms, 43 cm; width, 13 cm; height of walls, 42 cm). A transparent plastic escape platform (12 × 12 cm) is hidden at the end of one of the two arms that are filled with water (23 ± 1 ◦C). The test has two phases: acquisition and reversal.

For the first acquisition phase, a rat needs to choose the right arm out of two. If it chooses the right arm, it remains on the platform for 10 more seconds. If it chooses the wrong arm, the rat is confined to the arm for 20 s.

In the second reversal phase (performed 24 h after the acquisition phase), each rat was first tested in a probe trial without the platform. Once the rat entered an arm, it was removed from the maze. If the non-reinforced arm was chosen, the rat was retrained on the discrimination of the previous day and tested again 24 h later. If the reinforced arm was chosen, the rat was trained on the reversal of that discrimination, i.e., the platform was moved to the opposite arm [41]. In both phases, the number of correct trials was recorded until the rat reached five consecutive correct trials.

#### *2.8. Forced Swim Test (FST)*

This test is based on the assumption that when a rat is placed in a container filled with water, it will at first make efforts to escape but eventually will exhibit immobility that may reflect despair [42]. An acrylic cylindrical container (62 cm diameter, 40 cm height) was filled with water at a temperature of 24 ◦C. The water level (34 cm) was such that a rat could not touch the bottom with its hind paws. A rat was forced to swim for 15 min inside the container. Then, after 24 h in the home cage, the rat was put back in the container and forced to swim for 5 min. The time spent immobile was recorded. Immobility was defined as the lack of motion, except for movements necessary to keep the rat's head above water [43,44].

#### *2.9. Saccharin Preference Test*

Water bottles were removed before the dark part of the cycle and replaced with two bottles, one filled with water and the other with saccharin (0.01 mg/L, dissolved in water, Sigma-Aldrich, Rehovot, Israel). Saccharin consumption was measured during the 12 dark hours of the cycle. Following one night of habituation to saccharin, the saccharin preference ratio was calculated as the saccharin consumption divided by the total consumption (consumption of saccharin/water + saccharin).

#### *2.10. Western Blotting (WB)*

Rats were euthanized and brain tissues of the mPFC (prelimbic and infralimbic punched together), NAc shell, basolateral amygdala (BLA), and CA1 area of the hippocampus were removed by cryostat using a 0.5 mm puncher (coordinates relative to Bregma in mm: mPFC: anteroposterior (AP), +3.72; medial–lateral (ML), ±0.4; ventral (V), −4.8; NAc shell: AP, +1.6; ML, ±1; V, −5.5; BLA: AP, −1.596; ML, ±4.2; V, 8.45; CA1: AP, −4.2; ML, ±2.5; V: −2.5). Protein levels were determined by the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The samples were then diluted in an SDS sample buffer, boiled for 5 min at 100 ◦C, and then stored at −80 ◦C. On running day, wells were loaded with 30 μL of samples. Each gel contained at least one sample from each group. Aliquots were subjected to SDS–PAGE (7.5% polyacrylamide; Sigma-Aldrich, Rehovot, Israel) and immunoblot analysis.

Blots were incubated with CB1r (1:1000; abcam, Cambridge, UK; ab25932 [ERP23934- 20]), β–catenin (1:5000; abcam, Cambridge, UK; ab32572 [E247]), or mGluR5 (1:5000; abcam, Cambridge, UK; ab76316 [ERP2425Y]) antibodies overnight at 4 ◦C. This was followed by a 1 h incubation with an HRP-linked secondary antibody at room temperature (1:10,000; goa<sup>t</sup> anti-rabbit IgG; Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 111-035-144). Blots were visualized with ECL (Bio-Lab, Jerusalem, Israel) and an XRS charge-coupled device camera (BioRad Laboratories, Hercules, CA, USA). We used the Quantity One

software (BioRad Laboratories, Hercules, CA, USA) to assess blot density. All protein samples were standardized with β-actin (1:1000; Cell Signaling, Danvers, MA, USA; #5125 [13E5]). For antibody specificity, see the Supplementary File, Figure S1.

#### *2.11. Viral-Mediated Gene Transfer*

The replication-deficient *herpes simplex virus* (HSV) p1005 vector is a "short-term" vector, derived from herpes simplex virus-1 with a high titer range (3–5 × 10<sup>8</sup> transduction unit, TU/mL; an illustration of a modified HSV amplicon plasmid is presented in the Supplementary File, Figure S2). Stereotactic surgery was performed on rats under anesthesia of Domitor (2%, 10 mg/kg, i.p.) and ketamine (10%, 100 mg/kg, s.c.) (Vetmarket, Modiin, Israel). A total of 1μL of the HSV viral vector or green fluorescent protein (GFP) was infused bilaterally into the NAc (Stoelting, Wood Dale, IL, USA) at a rate of 0.1 μL/min (coordinates relative to Bregma: AP, +1.6 mm; LM, ±1 mm; V, −5.5 mm). Vectors were used to overexpress (OE) or downregulate (DR) the expression of β-catenin compared to a GFP control five days before shock day; the vector is expressed in vivo within 2–3 h, with maximal expression from 3–5 days post-injection that lasts only 8 days in vivo [23,45–47]. The viral dose was determined by rendering the >90% cell infection rate in brain tissue, diluted in 60% PBS.

The needle was held in place for 5 additional minutes before being slowly withdrawn. Animals were allowed 5 days of recovery before behavioral experiments began.

#### *2.12. Perfusion and Immunohistochemistry (IHC)*
