The bottle-brush test was used to test irritability-like behavior during nicotine withdrawal, based on the methods of Kimbrough et al. (25). This test was performed after 48 hours of nicotine withdrawal and measured aggressive and defensive responses. Testing consisted of 10 trials per rat in plastic cages (26.67 cm by 48.26 cm by 20.32 cm; Ancare, Bellmore, NY, USA) with fresh bedding. Three observers who were blinded to treatment scored behavior, and the average scores of the three observers for each behavior were calculated: aggressive responses (smelling, biting, boxing, following, and exploring the target) and defensive responses (escaping, burying, jumping, climbing, grooming, and vocalization). The test was performed in the pre-escalation and post-escalation phases.

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