Alcohol consumption was evaluated using the DID method. The protocol calls for 4 consecutive days of testing. Each day, starting on a Tuesday 3 h after lights were turned off, the water bottles were removed from the cages and replaced with 15 ml centrifuge tubes filled with 20% (v/v) ethanol from 95% USP ethanol. On days 1–3 (Tuesday through Thursday) the length of exposure was 2 h and on the 4th day (Friday) the exposure was 4 h. No alcohol was offered in the intervening period (Saturday through Monday). This protocol was repeated weekly for 16 weeks (Table). Tubes were weighed immediately before and after the exposure period. Volume consumed was converted to g/kg body weight of ethanol.
+ +During a period of 7 weeks, mice received 2 disturbances per day (see Figure). These consisted of being exposed to wet bedding for 1 hour, 1 cm of water in the bottom of the cage with no bedding for 1 hour, no bedding for 1 hour, confinement in a 3 × 3 × 3 cm plastic box for 15 minutes, tilted cage at 45º for 1 hour, and exposure to foreign mouse or fox urine odor for 1 hour. The other stressor was a complete phase shift in the light:dark cycle over each weekend. Disruption of the light cycle (lights constantly on) occurred over the weekend (starting Friday 11:00 am) with the light cycle resuming at 11 am on Monday (lights off). The schedule was repeated weekly over the duration of the study.
+ +Treatment Periods
+ +The entire protocol took 16 weeks to complete and was subdivided into 3 periods: baseline (initial 5 weeks), CMS (next 7 weeks), and postâ€CMS (final 4 weeks). Alcohol was offered as described above for all periods and experimental groups. During the intervening 7â€week CMS period, control animals were subjected to normal housing and experimental (stress) animals were subjected to CMS.
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