This experiment consisted of different stages.

Self-administration: The nicotine self-administration procedure was performed as described previously (7). Drug self-administration occurred under an FR1 schedule of reinforcement, with a 20-s TO period after each drug infusion, for 21 hours/day. Every drug delivery was associated with illumination of the cue light, which lasted for the duration of the TO period. During the TO period, responses were recorded but did not lead to drug delivery. Self-administration sessions (21 hours/day, 7 days/week) were performed in operant chambers that were equipped with two levers and two nosepoke holes. Responses on one of the levers (active lever) resulted in drug delivery, whereas responses on the other lever (inactive lever, a measure of nonspecific activity) were recorded but not reinforced. Food and water delivery occurred after each nosepoke.

Escalation: The escalation phase was identical to the self-administration phase, with the exception that the rats were introduced to the operant chambers every 48 hours (Monday, Thursday, Sunday, Wednesday, and so on).

Extinction: This phase was similar to the self-administration procedure, with the exception that presses on the active lever resulted in saline instead of nicotine infusions.

Prime-induced reinstatement: After the extinction criterion of nicotine-taking behavior was reached, the effects of priming injections of nicotine (0.03 mg/kg per injection; one infusion of nicotine solution at the same concentration as the one that was used during self-administration) or saline on the reinstatement of drug seeking were determined in separate daily 1-hour sessions. The rats were tested after intravenous drug priming injections. During the tests for reinstatement, lever presses resulted in saline infusions. Priming injections were administered immediately before the test sessions began. The experiment was performed using a Latin-square design. The extinction sessions were continued between priming sessions.

Yohimbine-induced reinstatement: After completion of the test of prime-induced reinstatement, the rats were left undisturbed in their home cages for 2 days. They then underwent the extinction phase again for three consecutive days. Once the extinction criterion was met, the rats received an intraperitoneal injection of yohimbine (2 mg/kg) 1 hour before the reinstatement test. Responses on the active and inactive levers were recorded. During the test, lever presses resulted in saline infusions for the duration of the session. Data were recorded using a computer that controlled the operant chambers. For the reinstatement experiments, only responses during the first hour of extinction and reinstatement were analyzed.

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