SUPPLEMENTARY METHODS Animals 126 Male Wistar rats (Charles River Co., Wilmington, MA) weighing 250-275 g at the beginning of the experiment and 450-550g at the time of testing were used. Rats were pairhoused and maintained on a 12h/12h reverse light-dark cycle in a temperature and humidity controlled vivarium. All training and testing were conducted during the dark phase of the cycle. Standard laboratory rat chow and water were available ad libitum throughout the experiment. All procedures were conducted in accordance to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Scripps Research Institute. Behavioral testing apparatus Self-administration training and testing were conducted inside sound-attenuated operant conditioning chambers (Med Associates, St. Albans, VT) located inside sound-attenuating cubicles equipped with exhaust fans as previously described (Weiss et al, 1993). Each chamber was equipped with a 0.15 ml drinking reservoir positioned 4 cm above the grid floor in the center of the front panel of the chamber, and two retractable levers located 4.5 cm on either side of the drinking reservoir. Responses on the right (active) lever resulted in activation of the infusion pump for 0.5 s, resulting in delivery of 0.1 ml liquid reinforcer into the drinking reservoir, while responses on the left (inactive) lever were recorded but had no other scheduled consequences. Each lever required a 0.01 N force to register a response. Auditory stimuli were presented via a speaker mounted on the rear wall. A microcomputer controlled the delivery of fluids, presentation of auditory stimuli and recording of behavioral data. Locomotor activity was measured in 16 identical metal wire hanging cages, each measuring 36 cm (L) × 25 cm (W) × 20 cm (H). Each cage contained two sets of infrared emitter-detector photocells positioned along the long axis, 1 cm above the grid floor and 8 cm from the front and back of the cage. Movement within the cages produced photocell beam breaks that were automatically recorded by computer. Pre-training procedure for operant self-administration Rats (N = 96) were trained to self-administer ethanol in daily 30 min sessions on a schedule of continuous reinforcement, using the “supersac” sweet solution fading procedure to (Ji et al, 2008; Walker et al, 2008) to establish operant responding for ethanol reinforcement without using food or water deprivation. During the first five days of self-administration training, lever responses resulted in delivery of a solution containing 3% glucose and 0.125% saccharin (“supersac”). Starting on day six, training continued with 10% (w/v) ethanol added to the supersac solution for four days. During the following four training days, glucose was removed from the solution, followed by removal of saccharin after completion of 14 total training days. Ethanol withdrawal ratings After every 3-day intoxication cycle (RW rats) or at the end of the 12-day intoxication period (SW rats), withdrawal from ethanol was evaluated in the rats using five well-characterized behavioral signs and a rating scale adapted from Macey et al. (Macey et al, 1996): ventromedial limb retraction, impaired gait, vocalization, tail rigidity and tremors were examined 12h after removal from the ethanol vapor chambers. Each sign was assigned a score of 0 to 2, based on the following severity scale: 0 = no apparent symptom, 1 = moderate, 2 = severe. The sum of the five observation scores (0 to 10) was used as a quantitative measure of withdrawal severity. For these behavioral observations, rats were individually transferred from their home cages to a quiet observation room. Rats in the SW group were evaluated for withdrawal once before ethanol vapor exposure and 12h following the last exposure period. Rats in the RW group were measured 12h after each 3-day intoxication cycle. Randomly selected rats of the CTRL group were evaluated during the corresponding time periods for comparison. Calculation of fractional inhibition indices For all LY379268-pretreated rats in each intoxication group, responses recorded during the S+ reinstatement tests were subtracted from, and normalized to, the corresponding responses of vehicle-treated rats in the same intoxication group. The resulting indices of fractional inhibition were then fit to an ordinary pharmacokinetic model y = 1 / (1 − 10A − x), where the parameter A estimated the logarithm of EC50, the effective dose for half of the population. Significant differences were assessed by t-tests between rats from different intoxication groups pretreated with the same dose of LY379268. Membrane preparation for the [35S]GTPγS binding assay Brain punches were homogenized and then centrifuged at 3000 rpm for 10 min in lysis buffer [0.32 M sucrose, 2 mM EGTA, 0.002% SDS, protease inhibitor (Roche Diagnostics, Rahway, NJ), pH 7.4]. The tissue pellet was re-suspended in 2mM EGTA, protease inhibitor, pH 7.4 and incubated for 15 minutes at 37 °C. The homogenate was centrifuged at 1400 rpm for 20 min. Membranes were re-suspended in storage buffer at −80 °C (20mM HEPES, 0.1mM EGTA, protease inhibitor, pH 7.4). All steps were processed at 4 °C unless noted differently. Protein concentrations were determined using a bicinchoninic acid assay (Thermo Scientific, Rockford, IL). Calculation of binding parameters in [35S]GTPγS binding data The specific [35S]GTPγS binding rate data were fit to a one-site compartment kinetic model for specific binding y = BMAX x / (KD + x), where the parameter BMAX estimated the maximum specific binding and KD estimated the equilibrium binding constant. KD was also the estimated radioligand concentration needed to achieve a half-maximum binding at equilibrium. Significant differences were assessed by t-tests between parameters associated with rats from different intoxication groups.