Chronic nicotine activates stress/reward-related brain regions and facilitates the transition to compulsive alcohol drinking

Abstract

Alcohol and nicotine are the two most co-abused drugs in the world. Previous studies have shown that nicotine can increase alcohol drinking in nondependent rats, yet it is unknown whether nicotine facilitates the transition to alcohol dependence. We tested the hypothesis that chronic nicotine will speed up the escalation of alcohol drinking in rats and that this effect will be accompanied by activation of sparsely distributed neurons (neuronal ensembles) throughout the brain that are specifically recruited by the combination of nicotine and alcohol. Rats were trained to respond for alcohol and made dependent using chronic, intermittent exposure to alcohol vapor, while receiving daily nicotine (0.8 mg/kg) injections. Identification of neuronal ensembles was performed after the last operant session, using immunohistochemistry. Nicotine produced an early escalation of alcohol drinking associated with compulsive alcohol drinking in dependent, but not in nondependent rats (air exposed), as measured by increased progressive-ratio responding and increased responding despite adverse consequences. The combination of nicotine and alcohol produced the recruitment of discrete and phenotype-specific neuronal ensembles (∼4-13% of total neuronal population) in the nucleus accumbens core, dorsomedial prefrontal cortex, central nucleus of the amygdala, bed nucleus of stria terminalis, and posterior ventral tegmental area. Blockade of nicotinic receptors using mecamylamine (1 mg/kg) prevented both the behavioral and neuronal effects of nicotine in dependent rats. These results demonstrate that nicotine and activation of nicotinic receptors are critical factors in the development of alcohol dependence through the dysregulation of a set of interconnected neuronal ensembles throughout the brain.

Keywords: Fos; addiction; alcohol; compulsivity; neuronal ensembles; tobacco.

Figure 1.

Escalation of alcohol self-administration. A, Timeline of the experiment. Dependent and nondependent rats were injected with saline or nicotine (0.8 mg/kg, s.c.) once per day, 15 min before each operant session after 6–8 h of withdrawal from alcohol vapor. The data represent the mean ± SEM number of active lever presses for 10% w/v alcohol in dependent (B) and nondependent (C) rats treated with saline (n = 8–9; black circles) or nicotine (n = 8–9; black squares) and dependent (n = 9; black squares) and nondependent (n = 9; black circles) rats treated with nicotine (0.8 mg/kg, s.c.; D). *p < 0.05, compared with saline group; **p < 0.001, compared with saline group; ^#p < 0.05, compared with baseline.

Figure 2.

Mecamylamine blocks the escalation of alcohol self-administration. Dependent rats were injected with mecamylamine (1 mg/kg, i.p.) or saline 45 min before each nicotine (0.8 mg/kg, s.c.) injection once per day, 15 min before each operant session after 6–8 h of withdrawal from alcohol vapor. The data represent the mean ± SEM number of active lever presses for 10% w/v alcohol in dependent rats treated with nicotine + saline (n = 7; black circles) or nicotine + mecamylamine (n = 7; black squares). *p < 0.05, compared with nicotine + saline group; ^#p < 0.05, compared with baseline.

Figure 3.

Mean (±SEM) reinforcement or last ratio achieved for alcohol during the PR sessions. Alcohol-dependent and nondependent Wistar rats responded for alcohol at 6–8 h of withdrawal from alcohol vapor and 15 min after an injection of saline (n = 8–9; white bars) or nicotine (n = 8–9; black bars). ^#p < 0.05, significant difference between air and vapor in nicotine-treated rats; *p < 0.05, significant difference from respective saline group.

Figure 4.

Compulsive-like drinking (i.e., persistent alcohol drinking despite the aversive bitter taste of quinine added to the alcohol solution). The data represent the percentage change from baseline (i.e., lever presses for alcohol alone before adulteration with quinine) in vapor-exposed (A) and air-control (B) rats that were treated with saline (n = 7–9; white bars) or nicotine (n = 7–9; black bars). *p < 0.05, significant difference between saline and nicotine.

Figure 5.

Nicotine accelerates the escalation of alcohol self-administration, associated with Fos induction in the dmPFC. Top, Number of Fos-immunoreactive nuclei per millimeter squared in the dmPFC. Middle, Number of Fos-immunoreactive nuclei per millimeter squared in the vmPFC. Bottom, Number of Fos-immunoreactive nuclei per millimeter squared in the OFC. The data are represented as mean ± SEM; *p < 0.05, different from the other groups; n = 6–8 per group.

Figure 6.

Nicotine speeds up the escalation of alcohol self-administration, associated with Fos induction in the nucleus accumbens core. Top, Number of Fos-immunoreactive nuclei per millimeter squared in the NAc-Core. Bottom, Number of Fos-immunoreactive nuclei per millimeter squared in the NAc-Shell. *p < 0.05, different from the other groups; n = 6–8 per group.

Figure 7.

Nicotine accelerates the escalation of alcohol self-administration, associated with Fos induction in the central amygdala, and nicotine treatment induces Fos in the basolateral amygdala. Top, Number of Fos-immunoreactive nuclei per millimeter squared in the CeA. Bottom, Number of Fos-immunoreactive nuclei per millimeter squared in the BLA. *p < 0.05, different from the other groups; n = 6–8 per group.

Figure 8.

Nicotine accelerates the escalation of alcohol self-administration, associated with Fos induction in the BNST. Top, Number of Fos-immunoreactive nuclei per millimeter squared in the juxtacapsular bed nucleus of the stria terminalis (jcBNST). Middle, Number of Fos-immunoreactive nuclei per millimeter squared in the posterolateral bed nucleus of the stria terminalis (plBNST). Bottom, Number of Fos-immunoreactive nuclei per millimeter squared in the ventrolateral bed nucleus of the stria terminalis (vlBNST). *p < 0.05, different from the other groups; n = 6–8 per group.

Figure 9.

Nicotine accelerates the escalation of alcohol self-administration, associated with Fos induction in the pVTA. Top, Number of Fos-immunoreactive nuclei per millimeter squared in the aVTA. Bottom, Number of Fos-immunoreactive nuclei per millimeter squared in the pVTA. *p < 0.05, different from the other groups; n = 6–8 per group.

Figure 10.

Mecamylamine blocks the facilitating effect of nicotine on the escalation of alcohol self-administration, associated with a reduction of Fos expression in the dorsal medial prefrontal cortex. *p < 0.05, different from the other groups; n = 7 per group.

Figure 11.

Representative image of double-labeled immunohistochemistry detecting Fos and NeuN associated with nicotine-induced acceleration of the escalation of alcohol self-administration. A, Red-labeled nuclei indicate expression of the general neuronal nuclei marker NeuN. B, Green-labeled nuclei indicate Fos expression. C, Merged image indicating Fos + NeuN double-labeled nuclei in yellow. Scale bar, 50 μm. n = 4 per group.

Figure 12.

Representative image of double-labeled immunohistochemistry detecting Fos and CaMKII (A), Fos and TH (B), and Fos and GAD67 (C) associated with nicotine-induced acceleration of the escalation of alcohol self-administration. A, Red-labeled nuclei indicate expression of the general neuronal nuclei marker NeuN. B, Green-labeled nuclei indicate Fos expression. Merged images indicate nuclei in green. Scale bar, 100 μm. n = 4 per group.