Video freeze

Behavior was recorded digitally from a camera mounted above each test chamber at 30 FPS. Following the completion of experiments, each recorded EPM video was analyzed using ANY-maze for positional tracking of the animal along the apparatus. For EPM mapping we measured open arm entries, time spent in open arms, and closed arm. VideoFreeze (MedAssociates) was used to measure animal freezing in QC testing from recorded videos. Freezing was determined as an absence of all visible movement except that required for respiration; in VideoFreeze, we set the motion threshold to 18 au with a minimum freeze duration of 1 s (30 frames). Contextual fear was assessed by total freezing time over the first 3 min of the test. Cued fear was assessed by total freezing time in response to the presentation of the first, second, and third CS during ITI. One exception was with freezing measurements during genetic mapping with BXD lines. Due to the variable coat color across lines, we used ANY-maze for animal tracking and freezing measurements as it performed better than VideoFreeze.

Associative fear memory was tested for three consecutive days using VideoFreeze (Med-Associates Inc., Georgia, VT, USA). On training day animals were placed in a chamber with white light for 3 min, then a tone is played paired with a foot shock (0.05mA) 3 times over the next 3 min. This tone/shock pairing occurs once every minute, once the test is complete the animal is returned to their home cage. 24 h later, context day, the animal is placed back in the chamber for 5 min with the white light but no tone or shock and is returned to their home cage after. For cued testing, the chamber is altered to appear as a “new environment”: the white light remains present, but the walls and floor are covered with plastic and vanilla extract is placed underneath the chamber floor. Animals are placed in the new environment for 3 min, then the tone from training day is played for the duration of the next 3 min. The amount of time the animal spends freezing is recorded. Animals were tested between 10 and 13 weeks of age.

Animals were given a behavioral battery to assess various stereotyped behaviors that have been associated with other preclinical models of AUD. Briefly, animals were allowed to acclimate to the room in their home cages for 30 min following room transfer and sessions started at 10AM. First, animals were videotaped in a clean, bare Plexiglass cage for 15 min. Grooming, stereotyped rostro caudal grooming transitions, and rearing were assessed by a blinded scorer (see Supplemental Methods (Kalueff et al., 2007 (link))). Three days later, animals were given a 10-min open field test, digitally scored by EthoVision (Noldus, Leesburg, VA) software. The subsequent day, animals were exposed to 1 uncued foot-shock (1 mA, 1s) during an 8-min session with 2% Simple Green as an olfactory cue, blue polka dots as a visual cue, and metal bar flooring as a tactile cue. Animals were then reintroduced to the same context the next day to evaluate context-dependent fear during a 15-min session. Sessions were scored using Video Freeze (Med Associates Fairfax, VT) software. Finally, animals were given an intermittent access 2-bottle choice between potable water and 10% w/w EtOH, (see Supplemental Methods (Marty et al., 2020a (link); Meyer et al., 2013 (link); Simms et al., 2008 (link))).