5 KCl, 1.3 MgCl2, 2 CaCl2, 1.25 KH2PO4, 11 glucose, and 26 NaHCO3 (pH 7.4, osmolarity 310) and allowed to recover for at least 1 hr in oxygenated ACSF at RT. The recording chamber was gravity fed with the same buffer. Hb neurons were visually identified with a microscope (Axioskop 2 FS plus) equipped with a digital camera (SPOT Insight). Patch electrodes were made from borosilicate glass (1B150F-4, World Precision Instruments, Inc.) with a microelectrode puller (P-97, Sutter Instrument,

CO). The internal pipette solution contained (in mM) 130 KCl, 2 MgCl2, 0.5 CaCl2, 5 EGTA, and 10 HEPES (pH 7.3, osmolarity 280; resistance, 5–7 MΩ). Typical series resistance was 15–30 MΩ. Nicotine was locally applied (50 ms, 8–10 psi) at different concentrations (1–600 μM) with a pressure device (PR-10, ALA Scientific Instruments) connected to a focal perfusion system (VM4, ALA Scientific GDC-0068 selleck kinase inhibitor Instruments) controlled with a trigger interface (TIB 14S, HEKA). The pipette was moved within 15–20 μm of the recorded cell with a motorized micromanipulator (LN mini 25, control system SM-5, Luigs & Neumann) for drug application and retracted after the end of the puff to minimize desensitization. In current clamp, the pipette with nicotine was positioned 100 μm from the cell and the drug was applied for 3 s. Neurons showing spontaneous oscillations

were not tested. Currents were recorded with a HEKA amplifier (EPC 10) using PatchMaster software (V2.20, HEKA), and were analyzed with FitMaster software (V2.3, HEKA). Membrane potential was held at −70 mV. Dose-response curves were calculated relative to the maximal response to nicotine (n = 3 cells per genotype). Adult brains were dissected and immediately embedded in O.C.T.

compound (Sakura). Frozen tissues were cut at the cryostat (20 μm coronal sections), thaw mounted much on ultrafrost microscope slides (Menzel Gläser), and stored at −80°C. For total [125I]-epibatidine binding sites, sections of WT and transgenic littermates (n = 3 per genotype) were incubated with 200 pM [125I]-epibatidine (NEN Perkin Elmer, Boston; specific activity 2200 Ci/mmole) in Tris 50 mM (pH 7.4) for 1 hr. For cytisine-resistant [125I]-epibatidine binding sites, sections were first incubated with 25 mM Cytisine (Sigma, St Louis) in Tris 50 mM (pH 7.4) for 30 min, as described previously (Zoli et al., 1995). Quantification of binding was done with ImageJ (NIH). WT (n = 5) and Tabac (n = 5) male mice were single housed in their home cages. Mice were provided with either nicotine or saccharin solutions as their sole source of fluid and bottles were weighed daily to measure nicotine intake. The volume of the drinking solution consumed per day was averaged for the period of consumption (3 days). Drinking solutions were: water, 2% saccharine in water (sweet water), 5 mM quinine (bitter water), or 100 μM nicotine in sweet water.