9% sterile saline (all concentrations of nicotine refer to the free base form). The dose consumed was calculated as the milligrams of nicotine consumed per day considering the body weight of the mouse (mg/kg/d). Voluntary nicotine intake was assessed in adult male WT (n = 7) and Tabac (n = 6) mice, using the two-bottle assay selleck chemicals as described before (Butt et al., 2005). Naive mice were presented

with two bottles of water in the home cage for acclimatization to the new conditions for the first 3 days of testing. After this period, one of the bottles was filled with a nicotine solution (1 μg/ml) diluted in water. The intake of fluid from each bottle was measured daily Buparlisib manufacturer for 3 days. The concentration of the nicotine solution was then increased and tested for another 3 days. In total, six different concentrations were tested consecutively (1, 5, 12.5, 25, 50, and 100 μg/ml). Percent of nicotine consumption was expressed as a ratio of the volume of nicotine solution consumed divided by the total fluid intake ([ml nicotine × 100%]/ml total). The CPA apparatus used was a rectangular box composed of three distinct compartments

separated by removable doors. The center compartment (10 × 20 × 10 cm) is gray with a polycarbonate smooth floor. The choice compartments (20 × 40 × 20 cm) have different visual and tactile cues. One choice compartment has black walls with a 0.75 cm stainless steel mesh floor. The other compartment has white walls with a 0.25 cm stainless steel mesh floor. Behavior of animals was videotaped and scored by a blind observer. In the preconditioning phase, on day 1, mice (8–12 weeks old) were allowed to explore the three compartments freely for 15 min. This preconditioning session was used to separate mice into groups with approximately equal biases for each side. None of the mice exhibited a strong preference for one side over the other. In the conditioning phase, during the following 3 days, two pairings per day were given at 4–5 hr apart. The doors between the compartments were Liothyronine Sodium closed so that animals

were confined to one side or the other of the conditioning box for 15 min. In the morning the animals were given an i.p. saline injection prior to the placement in the chamber. In the afternoon, animals received a nicotine injection (i.p., 0.5 mg/kg) prior to the placement in the opposing chamber. In the preference test, on day 5, the doors between the compartments were opened again. Mice were placed in the central chamber and were allowed to move freely in the three chambers for 15 min. Time spent on each side was recorded. Recombinant lentiviral vectors were prepared using transient transfection of HEK293T cells. Briefly, 5 × 106 HEK293T cells were seeded on 24 × 10 cm cell-culture dishes precoated with poly-l-lysine (Sigma-Aldrich).