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A FR3 protocol (three active lever presses required for reward) was then introduced and sessions were reduced to 30?min. After approximately two months the rats were trained only three times per week (Monday, Wednesday and Friday) but instead the sessions were increased to 60?min. An inactive lever (no reinforcer, tone, light or olfactory stimuli) was present throughout the training GSK1349572 ic50 sessions as a measure of nonspecific behavioral activity. The rats were kept on the FR3 schedule for approximately five months and thereafter subjected to extinction. To minimize the number of animals used, in line with the recommendations in the Swedish Animal Welfare Act, the rats participating in the progressive ratio (PR) test in the present study underwent testing evaluating the effects of the monoamine stabilizer (-)-OSU6162 on cue-induced reinstatement and ethanol seeking under PR schedule (Steensland et al, Biological Psychiatry 2012) before subjected to the Ex4 PR test. To prevent carry over effects between the experiments, we allowed a wash out period of two weeks on regular FR1 sessions three times a week between the (-)-OSU6162 and the Ex4 tests. The rats returned to baseline FR1 responding for 20% alcohol between the tests (22.9?��?2.7 and 24.8?��?2.7 active lever presses (mean of three consecutive sessions) before (-)-OSU6162 and Ex4 PR-testing, respectively). When the effect of Ex4 on alcohol seeking behavior under PR schedule was evaluated, the rats had been consuming alcohol for approximately nine months. Seven out of the 24 rats failed to meet the criteria of SAR1B excluded from the PR test. The PR method was adapted from (Bowers et al., 2008?and?Steensland et al., 2010). In brief, during the PR test, the response requirement for each subsequent reward increases according to an exponential function; PR?=?5?��?e(0.1?��?number?of?reinforcement?previously?earned)???5. The PR session started BMS 354825 with the cup filled with alcohol and then the PR schedule proceeded with a schedule of 1, 2, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 15, 17, 20, 22, 25, 38, 32, 36, 40, 45, and 50. If the rat did not emit any lever presses for 3?min, there were 20?s timeout (houselights off and levers retracted) followed by a compound cue consisting of tone and light during 3?s. If the rat continued not to emit any lever presses after the timeout, the re-cues continued with 3?min apart for up to 5 iterations. The maximum time each rat had on the PR test were 60?min but if the rat did nothing during a period of 15?min or after five consecutive re-cues, the program was turned off automatically. The breakpoint was defined as the total number of lever presses established in the last successfully completed ratio during the session. Injections of Ex4 (1.2?��g/kg) or vehicle were given 10?min prior the start of the PR test session.