Modulation of ethanol drinking-in-the-dark by mecamylamine and nicotinic acetylcholine receptor agonists in C57BL/6J mice

Abstract

Rationale: Recent reports describe a restricted access ethanol consumption paradigm where C57Bl/6J mice drink until intoxicated. Termed "drinking in the dark" (DID), this paradigm has been used as a model of binge drinking. Although neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated in alcohol drinking in rats pre-trained to self-administer ethanol, their role in binge-like ethanol consumption is unknown.

Objectives: To determine if nAChRs are involved in binge drinking as measured by the DID assay in C57Bl/6J mice.

Materials and methods: Adult male C57Bl/6J mice were injected i.p. with nicotinic receptor antagonists including mecamylamine, hexamethonium, dihydro-beta-erythroidine, and methyllycaconitine. Immediately following injection, mice were presented with 20% ethanol for 2 h in the DID assay to measure ethanol consumption. Nicotinic agonists including cytisine and nicotine were also evaluated. The effects of mecamylamine and nicotine on ethanol-induced dopaminergic neuronal activation in the VTA were evaluated via immunohistochemistry.

Results: Mecamylamine dose dependently reduced ethanol consumption; whereas, the peripheral antagonist hexamethonium had no significant effect. Nicotinic agonists, cytisine and nicotine, reduced ethanol consumption. None of the effective nicotinic receptor drugs reduced sucrose drinking. Mecamylamine blocked ethanol activation of dopaminergic neurons while nicotine alone activated them without additional activation by ethanol.

Conclusions: Neuronal nAChRs are involved in ethanol consumption in the DID paradigm. The effects of mecamylamine, nicotine, and cytisine on ethanol intake appear to be specific because they do not reduce sucrose drinking. Mecamylamine reduces alcohol consumption by blocking activation of dopaminergic neurons; whereas, nicotinic agonists may activate the same reward pathway as alcohol.

Figure 1

Mecamylamine dose dependently reduces ethanol DID. a) Total ethanol drinking volume (mls ethanol solution) over the course of two hours starting two hours after lights off. Immediately prior to introduction of the ethanol solution into each individual cage, mice were injected i.p. with either 0 (saline), 0.5, 1.0, or 3.0, mg/kg mecamylamine. One group of animals was used for the 0.5 mg/kg dose; whereas a second group of animals was used for the 1 and 3 mg/kg doses (see methods). b) Bar graph representation of total ethanol intake over the two hour DID assay (g/kg) for the three mecamylamine doses. c) Total 10 % sucrose volume intake (mls) after an i.p. injection of 0 (saline), 1 or 6 mg/kg mecamylamine. Mice had access to 10 % sucrose for two hours during the dark cycle starting two hours after lights out. d) Blood ethanol concentration (mM) in mice given an i.p. preinjection of saline (n = 5) or 1 mg/kg mecamylamine (n = 6) immediately prior to an alcohol bottle. Blood was isolated immediately after the two hour drinking session. Data presented as mean +/− SEM. * p < 0.05, *** p < 0.001 compared to same group saline controls, One-Way or Repeated Measure ANOVA, Tukey post-hoc (see Methods and Results section for details).

Figure 2

Nicotine reduces ethanol DID. a) The effect of a preinjection of nicotine on ethanol drinking volume is shown. One group of mice were used for both drug concentrations (n = 7) b) Ethanol intake (g/kg) from a). c) Average effect of a preinjection of nicotine on sucrose intake. Data are presented as mean +/− SEM. * p < 0.05, ** p < 0.01 compared to same group saline controls, Repeated Measures ANOVA, Tukey post-hoc.

Figure 3

Cytisine reduces ethanol DID. a) Total volume of ethanol intake after saline, 1, or 3 mg/kg cytisine pre-injection. Separate groups of animals were used for each dose. b) Ethanol intake (g/kg) from a). Asterisk indicates significance compared to within group intake after a saline pre-injection. c) Effect of saline, 1, or 3 mg/kg cytisine on sucrose intake. * p < 0.05 compared to same group saline controls, One-Way ANOVA, Tukey post hoc.

Figure 4

Mecamylamine and nicotine differentially effect DID ethanol drinking pattern. a) Normalized drinking bouts in two separate groups of mice that received saline/3 mg/kg mecamylamine or saline/0.5 mg/kg nicotine. Dotted line represents the normalized saline value for each group. b) Average 15 minute bout during the first and second hour of the DID assay in the two groups of animals. ** p < 0.01, *** p < 0.001 compared to same group saline controls, One-Way ANOVA, Tukey post hoc.

Figure 5

Mecamylamine and nicotine exhibit distinct effects on ethanol-induced VTA DAergic neuron activation. a) Representative images depicting VTA slices from mice receiving two saline injections (left), saline followed by a 2.0 g/kg ethanol injection (middle), or 3.0 mg/kg mecamylamine followed by a 2.0 g/kg ethanol injection (right). Slices are fluorescently double labeled with anti-tyrosine hydroxylase (red) and anti-c-Fos (green). b) Representative images depicting VTA slices from mice receiving saline injections (left), 0.5 mg/kg nicotine followed by saline (middle), or 0.5 mg/kg nicotine followed by 2.0 g/kg ethanol (right). c) Average number of c-Fos positive, TH positive cells per slice from mice treated as in a). d) Average number of c-Fos positive, TH positive cells per slice from mice treated as in b). Baseline c-Fos positive, TH positive cells from saline injected control mice were subtracted from each value. Cells were counted from 23–33 VTA slices per mouse. Three mice per treatment were used for analysis. Asterisks directly above each bar indicate significance from saline treated control mice. *p < 0.05, **p < 0.01.