Sweetened EtOH was prepared by combining 95% ethanol and sucrose in tap water to obtain either a 2% sucrose–15% EtOH (w/v; Experiment 1) or a 2% sucrose–20%

EtOH (w/v; Experiments 2 and 3). Alcohol exposure in the home cage Rats were initially acclimated to the taste and pharmacological effects of EtOH in the home cage. This procedure was the same for Experiments 2 and 3, but differed for Experiment 1. In Experiment 1, rats (n = 25) first received a 24-h session in which only 15% EtOH was available in the home cage, followed by a 24-h session in which only water was available. Subsequently, they received 15% EtOH for 1 h/day (during the light phase) and water Inhibitors,research,lifescience,medical for 23 h/day for 18 consecutive days. EtOH was restricted to 1 h to encourage consumption within a time frame that corresponded to the VE-822 cost length of subsequent behavioral sessions. Experiment 2 (n = 32) and Experiment 3 (n = 28) utilized an intermittent, 24-h access, two-bottle

choice procedure that produces high EtOH intakes in outbred rats (Wise 1973; Simms et al. 2008; Sparks et al. 2013). On Monday, Wednesday Inhibitors,research,lifescience,medical and Friday rats received concurrent access to one bottle containing water and a second bottle containing 20% EtOH for 24-h sessions across Inhibitors,research,lifescience,medical 5–6 weeks. On Tuesday, Thursday, Saturday and Sunday only water was available. In all experiments, the left/right positions of water and EtOH bottles were alternated daily to mitigate the impact of side preferences. Rat weights and volume of ethanol consumed was obtained for each session and used to calculate EtOH intake in Inhibitors,research,lifescience,medical terms of g/kg (grams of EtOH consumed divided by rat weight in kilograms). Spillage was accounted for by subtracting the volume of fluid lost from bottles on an empty cage. Rats that consumed less than 1.0 g/kg by session 7 were given sweetened EtOH for 2–3 sessions to entice drinking.

Rats with the highest EtOH intakes averaged across the last 2 days (Table 1) were selected for behavioral testing. Table 1 Ethanol intake averaged over the last two sessions (mean ± SEM) of exposure in the home cage or Pavlovian discrimination training. Pavlovian discrimination mafosfamide Inhibitors,research,lifescience,medical training Pavlovian discrimination training (PDT) was conducted in daily, 60-min sessions, Monday–Friday. At 5 min after placement into the operant conditioning chamber the house light was illuminated to indicate the start of the session. In each session, rats received 16 presentations each of two different 10-sec auditory conditional stimuli (CS), a continuous white noise and clicker (2 Hz), controlled by a variable-time 67-sec schedule. Presentations of one stimulus (CS+) were paired with EtOH (concentration as per experiment), whereas presentations of the second stimulus (CS−) were not. EtOH (0.2 mL/CS+; 3.2 mL/session) was delivered into the fluid port for oral consumption over the last 6 sec of each CS+. Ports were checked at the end of each session to ensure that all the EtOH had been consumed.