(A) Left, localization of the neurons recorded in patch-clamp experiments from mice implanted with saline (MPSal, n=22 neurons from 3 mice) or nicotine minipumps (MPNic, n=30 neurons from 4 mice). The images of two MPSal and four MPNic neurons were missing and thus could not be placed on this map. Right, anatomical sampling was identical between the two groups (t-test, ML: p=0.86; DV: p=0.97). (B) Nicotine-induced current (puff application, 30 µM, 200ms) recorded in voltage-clamp mode (–60 mV) from IPN neurons of naive mice kept in their home-cage (grey, n=56 neurons from 8 mice, I = –384±43 pA) and of mice that underwent the two-bottle choice task (2BC, red, n=207 neurons from 26 mice, I = –227±15 pA). Nicotine-induced currents were of smaller amplitude in 2BC mice than in naive mice (Mann-Whitney, p=0.0002). (C) Left, anatomical position of the neurons recorded in vivo (MPNic: n=20 neurons from 13 mice; MPSal: n=12 neurons from 6 mice). Right: The medio-lateral (ML) and dorsoventral (DV) distributions of the neurons were identical in both groups (Mann-Whitney, ML: p=0.52, DV: p=0.87). (D) Correlation between the mediolateral (but not the dorsoventral) coordinates of the neurons and their response to nicotine, in MPSal (ML: R2=0.43, F1,9 = 6.9, p=0.027; DV: R2=0.008, F1,9 = 0.075, p=0.79) and MPNic mice (ML: R2=0.33, F1,16 = 8.14, p0.011; DV: R2=0.06, F1,16 = 1.13, p=0.3). The correlations are not statistically different between the two groups (LM: F1,25 = 0.61, p=0.44; DV: F1,25 = 0.02, p=0.87). (E) Absence of correlation between the mediolateral or dorsoventral coordinates of the neurons and their basal firing frequency in both MPSal (ML: R2=0.04, F1,9 = 0.44, p=0.52; DV: R2=0.001, F1,9 = 0.015, p=0.9) and MPNic mice (ML: R2=0.12, F1,16 = 2.2, p=0.15; DV: R2=0.07, F1,16 = 1.27, p=0.27). The correlations are not statistically different between the two groups (ML: F1,25 = 0.46, p=0.5; DV: F1,25 = 0.39, p=0.53). (F) Cumulative distribution of the spontaneous firing frequencies of IPN neurons from nicotine- and saline-treated mice (Kolmogorov-Smirnov, p=0.56). Inset: average spontaneous frequency of IPN neurons from nicotine- and saline-treated mice (Mann-Whitney, p=0.7). (G) Positive correlation between the spontaneous activity of IPN neurons and their response to nicotine, for nicotine- (R2=0.48, F1,16 = 14.84, p=0.0014) and saline-treated mice (R2=0.38, F1,9 = 5.64, p=0.04). The correlations are not statistically different between the two groups (F1,25 = 2.65, p=0.11). (H) In vivo juxtacellular recordings of nicotine-evoked responses in nicotine-inhibited IPN neurons of saline- and nicotine-treated animals. Top left, representative electrophysiological recording of an IPN neuron, during an i.v. injection of nicotine (30 µg/kg). Bottom left, average time course and amplitude of the change in firing frequency from baseline after an i.v. injection of saline and nicotine (30 µg/kg), for IPN neurons of saline- (n=12 neurons from 7 mice) and nicotine-treated animals (n=13 neurons from 10 mice). Right, responses were on average lower after chronic exposure to nicotine (p<0.05). (I) Daily percent nicotine consumption for saline- and nicotine-treated mice, for each day in the two-bottle choice task. In saline-treated mice, avoidance to nicotine appears from the second nicotine exposition day onward. In all panels, mice treated with nicotine (MPNic) are shown in red, and control mice treated with saline (MPSal) in grey. *** p<0.001, ** p<0.01, * p<0.05.