The DID protocol was also employed as it achieves pharmacologically significant levels of ethanol [31]. For this treatment, animals (approximately 2 months old at the beginning of experiments) were maintained on a 12∶12 reverse light/dark cycle (lights on at 07∶00 PM.) Starting 3 hr after lights off, water bottles were replaced with bottles containing a 20% ethanol solution. The ethanol bottles remained in place for either 2 (first 3 days) or 4 hr (day 4) and then were replaced with water bottles. Except for this short period of time of ethanol drinking, mice had unlimited access to water. This procedure was repeated for 36 consecutive days. Control animals received only water.
