Nicotine enhances alcohol intake and dopaminergic responses through β2* and β4* nicotinic acetylcholine receptors

Abstract

Alcohol and nicotine are the most widely co-abused drugs. Both modify the activity of dopaminergic (DA) neurons of the Ventral Tegmental Area (VTA) and lead to an increase in DA release in the Nucleus Accumbens, thereby affecting the reward system. Evidences support the hypothesis that distinct nicotinic acetylcholine receptors (nAChRs), the molecular target of acetylcholine (ACh) and exogenous nicotine, are also in addition implicated in the response to alcohol. The precise molecular and neuronal substrates of this interaction are however not well understood. Here we used in vivo electrophysiology in the VTA to characterise acute and chronic interactions between nicotine and alcohol. Simultaneous injections of the two drugs enhanced their responses on VTA DA neuron firing and chronic exposure to nicotine increased alcohol-induced DA responses and alcohol intake. Then, we assessed the role of β4 * nAChRs, but not β2 * nAChRs, in mediating acute responses to alcohol using nAChR subtypes knockout mice (β2-/- and β4-/- mice). Finally, we showed that nicotine-induced modifications of alcohol responses were absent in β2-/- and β4-/- mice, suggesting that nicotine triggers β2* and β4 * nAChR-dependent neuroadaptations that subsequently modify the responses to alcohol and thus indicating these receptors as key mediators in the complex interactions between these two drugs.

Figure 1. Acute and chronic nicotine enhance alcohol responses of VTA DA neurons.

(a) (top) Typical electrophysiological recording showing the increase in firing rate of VTA DA cell induced by 500 mg/kg i.v. alcohol injection in WT mice. (bottom) Example responses of a VTA DA neuron to consecutive doses of alcohol. Arrowheads indicate the time of the injection. (b) (left) Mean ± SEM DA cell firing frequency modification after injection of saline and the indicated ethanol dose (saline, n = 28; 125 mg/kg, n = 13; 250 mg/kg, n = 14; 500 mg/kg, n = 14; 750 mg/kg, n = 7). Arrowheads indicate the time of the injection. (right) Dose-response curve of ethanol-elicited responses for the same groups of neurons. ***p < 0.001, **p < 0.01, Wilcoxon paired test from baseline. Horizontal lines indicate significant dose effect (one-way ANOVA). (c) (top) Example responses of a VTA DA neuron to consecutive injections of alcohol, nicotine and combined alcohol + nicotine. Arrowheads indicate the time of the injections. (bottom) Barplot of the maximum of firing frequency variation from baseline (mean ± SEM) after alcohol (black), nicotine (white), or combined alcohol + nicotine injections (gray). ***p < 0.001, Wilcoxon paired test from baseline is indicated within each vertical bar. Difference between groups is indicated above the horizontal lines. ***p < 0.001, Wilcoxon paired test between groups. (d) (left) Examples of electrophysiological recordings of the basal activity of a DA neuron of a nic− (black) and a nic+ (gray) mouse. Insets: Enlarged views of two or more action potentials. (right) Barplot of the mean frequency and %SWB for nic− (black, n = 92) and nic+ mice (gray, n = 65). *p < 0.05, Wilcoxon test. (e) Dose-response curve of ethanol-elicited DA cell responses for nic− (black) and nic+ (gray) mice. Mean ± SEM of variation from baseline in firing frequency. Horizontal lines indicate significant dose effect and vertical lines indicate treatment effect (***p < 0.001, *p < 0.5, two-way ANOVA. Nic−: saline: n = 36; 125 mg/kg: n = 19; 250 mg/kg: n = 22; 500 mg/kg: n = 17; 750 mg/kg: n = 9; nic+: saline: n = 19; 125 mg/kg: n = 3; 250 mg/kg: n = 9; 500 mg/kg: n = 9; 750 mg/kg: n = 12).

Figure 2. Chronic nicotine modifies alcohol intake.

(a) Mean ± SEM of ethanol consumption (gr/kg) in nic− (black, n = 18) and nic+ (gray, n = 15) mice during the two bottle choice procedure. Horizontal lines indicate significant dose effect and vertical lines indicate treatment effect. ***p < 0.001, **p < 0.01, repeated measures two-way ANOVA. (b) Mean ± SEM of the preference ratio for alcohol over total fluid intake for the same groups of mice. ***p < 0.001, **p < 0.01, repeated measures two-way ANOVA; (c) Mean ± SEM of the total fluids intake for the same groups of mice. (d) Barplot of the total alcohol intake (within the 18 days procedure) for the same groups. **p < 0.01, Wilcoxon test.

Figure 3. Alcohol-induced responses and alcohol reward are modified in β4−/−, but not in β2−/− mice.

(a) Typical electrophysiological recordings showing the increase in firing rate of VTA DA cells induced by 500 mg/kg i.v. alcohol injection in β2−/− (top) and β4−/− mice (bottom). (b) Dose- response curves of ethanol-elicited DA cell responses for WT (black), β2−/− (red) and β4−/− mice (blue). Horizontal lines indicate significant dose effect and vertical lines indicate strain effect. ***p < 0.001, *p < 0.5, two-way ANOVA (see Results section for statistical details). WT: saline: n = 28; 125 mg/kg: n = 13; 250 mg/kg: n = 14; 500 mg/kg: n = 14; 750 mg/kg: n = 7; β2−/−: saline: n = 15; 125 mg/kg: n = 5; 250 mg/kg: n = 10; 500 mg/kg: n = 11; 750 mg/kg: n = 8; β4−/−: saline: n = 23; 125 mg/kg: n = 9; 250 mg/kg: n = 10; 500 mg/kg: n = 14; 750 mg/kg: n = 7. (c) Mean ± SEM of ethanol intake (gr/kg) in WT (black, n = 18), β2−/− (red, n = 9) and β4−/− mice (blue, n = 6) during the two bottle choice procedure. ***p < 0.001, repeated measures two-way ANOVA; ^##p < 0.01, ^#p < 0.05, multiple comparisons with Bonferroni correction. (d) Mean ± SEM of the preference ratio for alcohol over total fluid intake for the same groups of mice. ***p < 0.001; *p < 0.05, repeated measures two-way ANOVA (see Results section for statistical details).

Figure 4. Alcohol-elicited responses and alcohol intake of β2−/− and β4−/− mice are not modified by chronic nicotine.

(a) Barplot of the mean frequency and %SWB for β2nic− (red, n = 47) and β2nic+ mice (pink, n = 33). **p < 0.01, t-test; *p < 0.05, Wilcoxon test. (b) Barplot of the mean frequency and % SWB for β4nic− (blue, n = 41) and β4nic+ mice (light blue, n = 42). *p < 0.05, t-test. (c) Dose- response curves of ethanol-elicited DA cell responses for β2nic− (red) and β2nic+ (pink). β2nic−: saline: n = 15; 125 mg/kg: n = 5; 250 mg/kg: n = 10; 500 mg/kg: n = 11; 750 mg/kg: n = 8; β2nic+: saline: n = 12; 125 mg/kg: n = 6; 250 mg/kg: n = 7; 500 mg/kg: n = 10; 750 mg/kg: n = 5. (d) Dose- response curves of ethanol-elicited DA cell responses for β4nic− (blue) and β4nic+ (light blue). β4nic−: saline: n = 23; 125 mg/kg: n = 9; 250 mg/kg: n = 10; 500 mg/kg: n = 14; 750 mg/kg: n = 7; β4nic+: saline: n = 15; 125 mg/kg: n = 7; 250 mg/kg: n = 10; 500 mg/kg: n = 8; 750 mg/kg: n = 6. (e) Mean ± SEM of ethanol consumption (gr/kg) in β2nic− (red, n = 9) and β2nic+ (pink, n = 8) mice during the two bottle choice procedure. (f) Mean ± SEM of ethanol consumption (gr/kg) in β4nic− (blue, n = 6) and β4nic+ (light blue, n = 6) mice during the two bottle choice procedure.