(−)-Nicotine hydrogen tartrate (Sigma-Aldich Corp., St Louis, MO) was dissolved in 0.9 % physiological saline. F344 and HIV-1Tg rats were injected subcutaneously (s.c.) with saline or nicotine (0.4 mg/kg/day) once per day for 27 days before tissue collection (Nesil et al. 2015 (link); Song et al. 2016 (link)). The concentration of nicotine was calculated as a nicotine-free base. This dose of nicotine was determined based on Matta et al. (2007) and based on our previous studies (Nesil et al. 2015 (link); Song et al. 2016 (link)), which showed that 0.04-mg/kg chronic injections of nicotine significantly induced alterations at the transcriptional level in the brain regions of HIV-1Tg rats. Using a rat brain matrix (Kent Scientific, Torrinton, CT), 1-mm slices were taken from each brain. Slices that contained the VTA, NAc, PFC, HIP, and hypothalamus (HYP) were identified based on a rat brain atlas (Paxinos and Watson 1998 ). Tissue from the brain regions of interest was collected from each rat using a 1.5-mm brain punch (Stoelting, Wood Dale, IL) and stored at −80 ° C until use.