Saccharin sodium

Nicotine and control drinking solutions were prepared from nicotine hydrogen tartrate or L-tartaric acid (Sigma-Aldrich), which were dissolved in tap water with the concentrations of 200 µg ml⁻¹ and 300 µg ml⁻¹, respectively. Saccharin sodium (Fisher Scientific) was added at a concentration of 3 mg ml⁻¹ to each solution to sweeten the taste. Nicotine doses are reported as nicotine free base. For brain infusion, nicotine (50 ng µl⁻¹), mecamylamine hydrochloride (2 µg µl⁻¹), AP5 (0.2 µg µl⁻¹), CRF (0.1 µg µl⁻¹, Sigma-Aldrich), and antisauvagine-30 (1 µg µl⁻¹, Tocris Bioscience) were dissolved in sterile saline. Antalarmin hydrochloride (Tocris Bioscience) was dissolved in 5 % DMSO and 95 % sterile saline (1 µg µl⁻¹). Infusion concentrations and amounts for nicotine^{51 (link)}, mecamylamine^{11 (link)}, AP5^{52 (link),53 (link)}, CRF^{54 (link)}, antalarmin^{55 (link)} and antisauvagine-30^{56 (link)} were chosen based on previously published studies. On the test day, the CRF1 antagonist antalarmin or the CRF2 antagonist antisauvagine-30 was administered 40 min before behavioral testing while mecamylamine was administered 25 min before behavioral testing. CRF and nicotine were administered 10 min before behavioral testing. Lipophilic Tracer DiI (D282, Molecular Probe, Invitrogen, 5 mg ml⁻¹ in ethanol) was used to locate the site of brain infusion.