Acetylcholine is the endogenous agonist for the neuronal nicotinic acetylcholine receptor (nAChR) system, which is involved in attention, memory, affective behaviours and substance use disorders. Brain nAChRs are highly diverse with 11 different subunits that can form multiple receptor subtypes, each with distinct receptor and pharmacological properties. Different neuronal cell types can also express different nAChR subtypes, resulting in highly complex cholinergic signalling. Identifying which nAChR subunit transcripts are expressed in cell types can provide an indication of which nAChR combinations are possible and which receptor subtypes may be most pharmacologically relevant to target. In addition to differences in expression across cell types, nAChRs also undergo changes in expression levels from adolescence to adulthood. In this study, we used fluorescent in situ hybridization to identify and quantify the expression of α4, α5, α6, β2 and β3 nAChR subunit transcripts in dopaminergic, GABAergic, glutamatergic and noradrenergic neurons and astrocytes in the ventral tegmental area (VTA) and locus coeruleus (LC) in adult and adolescent, male and female C57BL/6J mice. There were distinct differences in the pattern of nAChR subunit transcript expression between the two brain regions. LC noradrenergic neurons had high prevalence of α6, β2 and β3 expression, with very low expression of α4, suggesting the α6(non‐α4)β2β3 receptor as a main subtype in these neurons. VTA astrocytes from adult mice showed greater prevalence of α5, α6, β2 and β3 transcript compared with adolescent mice. These data highlight the complex nAChR expression patterns across brain region and cell type.

Background and Purpose Drugs of abuse, including alcohol, increase dopamine in the mesocorticolimbic system via actions on dopamine neurons in the ventral tegmental area (VTA). Increased dopamine transmission can activate inhibitory G protein signalling pathways in VTA dopamine neurons, including those controlled by GABAB and D2 receptors. Members of the R7 subfamily of regulator of G protein signalling (RGS) proteins can regulate inhibitory G protein signalling, but their influence on VTA dopamine neurons is unclear. Here, we investigated the influence of RGS6, an R7 RGS family memberthat has been implicated in the regulation of alcohol consumption in mice, on inhibitory G protein signalling in VTA dopamine neurons. Experimental Approach We used molecular, electrophysiological and genetic approaches to probe the impact of RGS6 on inhibitory G protein signalling in VTA dopamine neurons and on binge‐like alcohol consumption in mice. Key Results RGS6 is expressed in adult mouse VTA dopamine neurons and it modulates inhibitory G protein signalling in a receptor‐dependent manner, tempering D2 receptor‐induced somatodendritic currents and accelerating deactivation of synaptically evoked GABAB receptor‐dependent responses. RGS6−/− mice exhibit diminished binge‐like alcohol consumption, a phenotype replicated in female (but not male) mice lacking RGS6 selectively in VTA dopamine neurons. Conclusions and Implications RGS6 negatively regulates GABAB‐ and D2 receptor‐dependent inhibitory G protein signalling pathways in mouse VTA dopamine neurons and exerts a sex‐dependent positive influence on binge‐like alcohol consumption in adult mice. As such, RGS6 may represent a new diagnostic and/or therapeutic target for alcohol use disorder.

Background: Chronic intermittent ethanol (CIE) vapor inhalation is a widely used model of alcohol dependence, but the impact of CIE on cue-elicited alcohol seeking is not well understood. Here, we assessed the effects of CIE on alcohol-seeking elicited by previously learned cues, and on acquisition of new cue-alcohol associations. Methods:In Experiment 1, male and female Long Evans rats were first trained in a discriminative stimulus (DS) task, in which one auditory cue (the DS) predicts the availability of 15% ethanol and a control cue (the NS) predicts nothing. Rats then underwent CIE or served as controls. Subsets of each group received access to oral ethanol twice a week during acute withdrawal. After CIE, rats were presented with the DS and NS cues under extinction and retraining conditions to determine whether they would alter their responses to these cues. In Experiment 2, rats underwent CIE prior to training in the DS task. We also assessed alcohol consumption, aversion-resistant drinking, somatic withdrawal symptoms, and behavior in an open field. Results:We found that CIE enhanced behavioral responses to previously learned alcohol cues, but only in rats that received access to alcohol during acute withdrawal. CIE disrupted cue responses in rats that did not. When CIE occurred before cue learning, male rats were slower to develop cue responses and less likely to enter the alcohol port, even though they had received alcohol during acute withdrawal. We also found that CIE increased alcohol consumption and aversion-resistant drinking in male but not female rats.Conclusions: These results suggest that CIE alone does not potentiate the motivational value of alcohol cues, but that an increase in cue responses requires the potentiation of the value of alcohol during acute withdrawal. Further, under some conditions CIE may disrupt responses to previously learned and subsequently acquired alcohol cues.

Background: Chronic intermittent ethanol (CIE) vapor inhalation is a widely used model of alcohol dependence, but the impact of CIE on cue-elicited alcohol seeking is not well understood. Here, we assessed the effects of CIE on alcohol-seeking elicited by previously learned cues, and on acquisition of new cue-alcohol associations. Methods: In Experiment 1, male and female Long Evans rats were first trained in a discriminative stimulus (DS) task, in which one auditory cue (the DS) predicts the availability of 15% ethanol and a control cue (the NS) predicts nothing. Rats then underwent CIE or served as controls. Subsets of each group received access to oral ethanol twice a week during acute withdrawal. After CIE, rats were presented with the DS and NS cues under extinction and retraining conditions to determine whether they would alter their responses to these cues. In Experiment 2, rats underwent CIE prior to training in the DS task. We also assessed alcohol consumption, aversion resistant drinking, somatic withdrawal symptoms, and behavior in an open field. Results: We found that CIE enhanced behavioral responses to previously learned alcohol cues, but only in rats that received access to alcohol during acute withdrawal. CIE disrupted cue responses in rats that did not. When CIE occurred before cue learning, male rats were slower to develop cue responses and less likely to enter the alcohol port, even though they had received alcohol during acute withdrawal. We also found that CIE increased alcohol consumption and aversion-resistant drinking in male but not female rats. Conclusions: These results suggest that CIE alone does not potentiate the motivational value of alcohol cues, but that an increase in cue responses requires the potentiation of the value of alcohol during acute withdrawal. Further, under some conditions CIE may disrupt responses to previously learned and subsequently acquired alcohol cues.

Acetylcholine is the endogenous agonist for the neuronal nicotinic acetylcholine receptor (nAChR) system, which is involved in attention, memory, affective behaviors, and substance use disorders. Brain nAChRs are highly diverse with 11 different subunits that can form multiple receptor subtypes, each with distinct receptor and pharmacological properties. Different neuronal cell types can also express different nAChR subtypes, resulting in highly complex cholinergic signaling. Identifying which nAChR subunit transcripts are expressed in cell types can provide an indication of which nAChR combinations are possible and which receptor subtypes may be most pharmacologically relevant to target. In addition to differences in expression across cell types, nAChRs also undergo changes in expression levels from adolescence to adulthood. In this study, we used fluorescent in situ hybridization to identify and quantify the expression of α4, α5, α6, β2 and β3 nAChR subunit transcripts in dopaminergic, GABAergic, glutamatergic, and noradrenergic neurons and astrocytes in the ventral tegmental area (VTA) and locus coeruleus (LC) in adult and adolescent, male and female C57BL/6J mice. There were distinct differences in the pattern of nAChR subunit transcript expression between the two brain regions. LC noradrenergic neurons had high prevalence of α6, β2 and β3 expression, with very low expression of α4, suggesting the α6(non-α4)β2β3 receptor as a main subtype in these neurons. VTA astrocytes from adult mice showed greater prevalence of α5, α6, β2 and β3 transcript compared with adolescent mice. These data highlight the complex nAChR expression patterns across brain region and cell type.