Fig. 1: Nicotine and ethanol induce activation and inhibition of VTA DA subpopulations.

a In vivo juxtacellular recordings of VTA dopamine (DA) neurons following intravenous (i.v.) injection of ethanol (EtOH; 250 mg/kg) or nicotine (Nic; 30 µg/kg). Recorded neurons were labeled with neurobiotin (NB) and identified via tyrosine hydroxylase (TH) immunofluorescence. Scale bar: 20 µm. b Examples of normalized firing frequency change after paired Nic and EtOH injections for activated (top) and inhibited (bottom) DA neuron. c Distribution of firing rate responses (% change) to nicotine (black, n = 72) or ethanol (gray, n = 72); Kolmogorov-Smirnov test, D = 0.14, p = 0.49. d Left: Mean time course of normalized firing frequency after nicotine or saline injection in activated (Nic+, red, n = 41) and inhibited (Nic-, blue, n = 28) neurons (Maximum % variation, paired Wilcoxon: Nic+ vs Sal, V = 854, ***p = 1.7e⁻¹¹; Nic- vs Sal, V = 3, ***p = 3.7e⁻⁰⁸). Right: individual firing rate variation from baseline (Nic+ vs Baseline, V = 861, ***p = 2.5e^-08; Nic- vs Baseline, V = 0, ***p = 7.4e^-09). e Same as (d) for ethanol. Ethanol-induced activation (EtOH+, red, n = 52) and inhibition (EtOH-, blue, n = 15) differed significantly from saline (paired Wilcoxon: EtOH+ vs Sal, V = 1290, ***p = 4.5e^-08; EtOH- vs Sal, V = 3, ***p = 0.0003) and from baseline (EtOH+ vs Bas, V = 1378, ***p = 3.6e^-10; EtOH- vs Bas, V = 0, ***p = 6.1e^-05). f Individual neuron responses to ethanol (left) and nicotine (right), ranked from most inhibited (pink) to most activated (white/red). Neurons grouped into EtOH-/Nic-, EtOH+/Nic+, and EtOH+/Nic-. g Correlation between EtOH- and Nic-induced responses (Pearson’s r = 0.28, t₆₉ = 2.45, *p = 0.02). Correlated responses (black dots, n = 57); uncorrelated (white dots, n = 14). Inset: experimental (red line) vs surrogate correlation distributions. h Left: Anatomical distribution of EtOH+/Nic+ (red, n = 39), EtOH-/Nic- (blue, n = 16), and EtOH+/Nic- (orange, n = 14) neurons. Right: EtOH-/Nic- neurons were more medially located than EtOH+/Nic+ neurons (Wilcoxon, V = 472, **p = 0.0028); EtOH+/Nic- locations were not significantly different (V = 147, p = 0.14; V = 312, p = 0.44). Data are presented as mean ± SEM. All statistical tests are two-sided.