Activation of alpha4* nAChRs is necessary and sufficient for varenicline-induced reduction of alcohol consumption.

Recently, the smoking cessation therapeutic varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist, has been shown to reduce alcohol consumption. However, the mechanism and nAChR subtype(s) involved are unknown. Here we demonstrate that varenicline and alcohol exposure, either alone or in combination, selectively activates dopaminergic (DAergic) neurons within the posterior, but not the anterior, ventral tegmental area (VTA). To gain insight into which nAChR subtypes may be involved in the response to alcohol, we analyzed nAChR subunit gene expression in posterior VTA DAergic neurons. Ethanol-activated DAergic neurons expressed higher levels of alpha4, alpha6, and beta3 subunit genes compared with nonactivated neurons. To examine the role of nicotinic receptors containing the alpha4 subunit (alpha4* nAChRs) in varenicline-induced reduction of alcohol consumption, we examined the effect of the drug in two complementary mouse models, a knock-out line that does not express the alpha4 subunit (alpha4 KO) and another line that expresses alpha4* nAChRs hypersensitive to agonist (Leu9'Ala). While varenicline (0.1-0.3 mg/kg, i.p.) reduced 2% and 20% alcohol consumption in wild-type (WT) mice, the drug did not significantly reduce consumption in alpha4 KO animals. Conversely, low doses of varenicline (0.0125-0.05 mg/kg, i.p.) that had little effect in WT mice dramatically reduced ethanol intake in Leu9'Ala mice. Infusion of varenicline into the posterior, but not the anterior VTA was sufficient to reduce alcohol consumption. Together, our data indicate that activation of alpha4* nAChRs is necessary and sufficient for varenicline reduction of alcohol consumption.

Identification of a BK channel auxiliary protein controlling molecular and behavioral tolerance to alcohol.

Tolerance, described as the loss of drug effectiveness over time, is an important component of addiction. The degree of acute behavioral tolerance to alcohol exhibited by a naïve subject can predict the likelihood of alcohol abuse. Thus, the determinants of acute tolerance are important to understand. Calcium- and voltage-gated (BK) potassium channels, consisting of pore forming alpha and modulatory beta subunits, are targets of ethanol (EtOH) action. Here, we examine the role, at the molecular, cellular, and behavioral levels, of the BK beta4 subunit in acute tolerance. Single channel recordings in HEK-293 cells show that, in the absence of beta4, EtOH potentiation of activity exhibits acute tolerance, which is blocked by coexpressing the beta4 subunit. BK channels in acutely isolated medium spiny neurons from WT mice (in which the beta4 subunit is well-represented) exhibit little tolerance. In contrast, neuronal BK channels from beta4 knockout (KO) mice do display acute tolerance. Brain slice recordings showed tolerance to EtOH's effects on spike patterning in KO but not in WT mice. In addition, beta4 KO mice develop rapid tolerance to EtOH's locomotor effects, whereas WT mice do not. Finally, in a restricted access ethanol self-administration assay, beta4 KO mice drink more than their WT counterparts. Taken together, these data indicate that the beta4 subunit controls ethanol tolerance at the molecular, cellular, and behavioral levels, and could determine individual differences in alcohol abuse and alcoholism, as well as represent a therapeutic target for alcoholism.

Junk food diet-induced obesity increases D2 receptor autoinhibition in the ventral tegmental area and reduces ethanol drinking.

Similar to drugs of abuse, the hedonic value of food is mediated, at least in part, by the mesostriatal dopamine (DA) system. Prolonged intake of either high calorie diets or drugs of abuse both lead to a blunting of the DA system. Most studies have focused on DAergic alterations in the striatum, but little is known about the effects of high calorie diets on ventral tegmental area (VTA) DA neurons. Since high calorie diets produce addictive-like DAergic adaptations, it is possible these diets may increase addiction susceptibility. However, high calorie diets consistently reduce psychostimulant intake and conditioned place preference in rodents. In contrast, high calorie diets can increase or decrease ethanol drinking, but it is not known how a junk food diet (cafeteria diet) affects ethanol drinking. In the current study, we administered a cafeteria diet consisting of bacon, potato chips, cheesecake, cookies, breakfast cereals, marshmallows, and chocolate candies to male Wistar rats for 3-4 weeks, producing an obese phenotype. Prior cafeteria diet feeding reduced homecage ethanol drinking over 2 weeks of testing, and transiently reduced sucrose and chow intake. Importantly, cafeteria diet had no effect on ethanol metabolism rate or blood ethanol concentrations following 2g/kg ethanol administration. In midbrain slices, we showed that cafeteria diet feeding enhances DA D2 receptor (D2R) autoinhibition in VTA DA neurons. These results show that junk food diet-induced obesity reduces ethanol drinking, and suggest that increased D2R autoinhibition in the VTA may contribute to deficits in DAergic signaling and reward hypofunction observed with obesity.

Neuronal nicotinic acetylcholine receptors: common molecular substrates of nicotine and alcohol dependence.

Alcohol and nicotine are often co-abused. As many as 80-95% of alcoholics are also smokers, suggesting that ethanol and nicotine, the primary addictive component of tobacco smoke, may functionally interact in the central nervous system and/or share a common mechanism of action. While nicotine initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels normally activated by endogenous acetylcholine (ACh), ethanol is much less specific with the ability to modulate multiple gene products including those encoding voltage-gated ion channels, and excitatory/inhibitory neurotransmitter receptors. However, emerging data indicate that ethanol interacts with nAChRs, both directly and indirectly, in the mesocorticolimbic dopaminergic (DAergic) reward circuitry to affect brain reward systems. Like nicotine, ethanol activates DAergic neurons of the ventral tegmental area (VTA) which project to the nucleus accumbens (NAc). Blockade of VTA nAChRs reduces ethanol-mediated activation of DAergic neurons, NAc DA release, consumption, and operant responding for ethanol in rodents. Thus, ethanol may increase ACh release into the VTA driving activation of DAergic neurons through nAChRs. In addition, ethanol potentiates distinct nAChR subtype responses to ACh and nicotine in vitro and in DAergic neurons. The smoking cessation therapeutic and nAChR partial agonist, varenicline, reduces alcohol consumption in heavy drinking smokers and rodent models of alcohol consumption. Finally, single nucleotide polymorphisms in nAChR subunit genes are associated with alcohol dependence phenotypes and smoking behaviors in human populations. Together, results from pre-clinical, clinical, and genetic studies indicate that nAChRs may have an inherent role in the abusive properties of ethanol, as well as in nicotine and alcohol co-dependence.

Nicotinic acetylcholine receptors containing the α4 subunit modulate alcohol reward.

BACKGROUND: Nicotine and alcohol are the two most co-abused drugs in the world, suggesting a common mechanism of action might underlie their rewarding properties. Although nicotine elicits reward by activating ventral tegmental area dopaminergic (DAergic) neurons via high-affinity neuronal nicotinic acetylcholine receptors (nAChRs), the mechanism by which alcohol activates these neurons is unclear. METHODS: Because most high-affinity nAChRs expressed in ventral tegmental area DAergic neurons contain the α4 subunit, we measured ethanol-induced activation of DAergic neurons in midbrain slices from two complementary mouse models, an α4 knock-out (KO) mouse line and a knock-in line (Leu9'Ala) expressing α4 subunit-containing nAChRs hypersensitive to agonist compared with wild-type (WT). Activation of DAergic neurons by ethanol was analyzed with both biophysical and immunohistochemical approaches in midbrain slices. The ability of alcohol to condition a place preference in each mouse model was also measured. RESULTS: At intoxicating concentrations, ethanol activation of DAergic neurons was significantly reduced in α4 KO mice compared with WT. Conversely, in Leu9'Ala mice, DAergic neurons were activated by low ethanol concentrations that did not increase activity of WT neurons. In addition, alcohol potentiated the response to ACh in DAergic neurons, an effect reduced in α4 KO mice. Rewarding alcohol doses failed to condition a place preference in α4 KO mice, paralleling alcohol effects on DAergic neuron activity, whereas a sub-rewarding alcohol dose was sufficient to condition a place preference in Leu9'Ala mice. CONCLUSIONS: Together, these data indicate that nAChRs containing the α4 subunit modulate alcohol reward.

Nicotinic acetylcholine receptors containing the α4 subunit are critical for the nicotine-induced reduction of acute voluntary ethanol consumption.

Recently, we investigated the molecular mechanisms of the smoking cessation drug varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist, in its ability to decrease voluntary ethanol intake in mice. Previous to our study, other labs had shown that this drug can decrease ethanol consumption and seeking in rat models of ethanol intake. Although varenicline was designed to be a high affinity partial agonist of nAChRs containing the α4 and ß2 subunits (designated as α4ß2*), at higher concentrations it can also act upon α3ß2*, α6*, α3ß4* and α7 nAChRs. Therefore, to further elucidate the nAChR subtype responsible for varenicline-induced reduction of ethanol consumption, we utilized a pharmacological approach in combination with two complimentary nAChR genetic mouse models, a knock-out line that does not express the α4 subunit (α4 KO) and another line that expresses α4* nAChRs hypersensitive to agonist (the Leu9'Ala line). We found that activation of α4* nAChRs was necessary and sufficient for varenicline-induced reduction of alcohol consumption. Consistent with this result, here we show that a more efficacious nAChR agonist, nicotine, also decreased voluntary ethanol intake, and that α4* nAChRs are critical for this reduction.

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

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.