Acute Ethanol Administration Upregulates Synaptic α4-Subunit of Neuronal Nicotinic Acetylcholine Receptors within the Nucleus Accumbens and Amygdala

Alcohol and nicotine are two of the most frequently abused drugs, with their comorbidity well described. Previous data show that chronic exposure to nicotine upregulates high-affinity nicotinic acetylcholine receptors (nAChRs) in several brain areas. Effects of ethanol on specific brain nAChR subtypes within the mesolimbic dopaminergic (DA) pathway may be a key element in the comorbidity of ethanol and nicotine. However, it is unknown how alcohol affects the abundance of these receptor proteins. In the present study, we measured the effect of acute binge ethanol on nAChR α4 subunit levels in the prefrontal cortex (PFC), nucleus accumbens (NAc), ventral tegmental area (VTA), and amygdala (Amg) by western blot analysis using a knock-in mouse line, generated with a normally functioning α4 nAChR subunit tagged with yellow fluorescent protein (YFP). We observed a robust increase in α4-YFP subunit levels in the NAc and the Amg following acute ethanol, with no changes in the PFC and VTA. To further investigate whether this upregulation was mediated by increased local mRNA transcription, we quantified mRNA levels of the Chrna4 gene using qRT-PCR. We found no effect of ethanol on α4 mRNA expression, suggesting that the upregulation of α4 protein rather occurs post-translationally. The quantitative counting of YFP immunoreactive puncta further revealed that α4-YFP protein is upregulated in presynaptic boutons of the dopaminergic axons projecting to the shell and the core regions of the NAc as well as to the basolateral amygdala (BLA), but not to the central or lateral Amg. Together, our results demonstrate that a single exposure to binge ethanol upregulates level of synaptic α4∗ nAChRs in dopaminergic inputs to the NAc and BLA. This upregulation could be linked to the functional dysregulation of dopaminergic signalling observed during the development of alcohol dependence

Investigating the role of neuronal nicotinic acetylcholine receptor subtypes in the acquisition and maintenance of alcohol use disorders

Owing to the substantial comorbidity of nicotine and alcohol, this project was designed to explore the role that neuronal nicotinic acetylcholine receptors play in the development of alcohol use disorders. This study utilised well-established animal models, documenting the impact of 'binge-like' ethanol consumption on the expression of these receptors in key brain areas governing stress and reward. Only by continuing to uncover the neurological changes attributed to alcohol consumption, can we further elucidate treatment options for those affected

Alcohol and nicotine interactions: Pre-clinical models of dependence

While the co-morbidity of alcohol (ethanol) and tobacco (nicotine) dependence is well described, the processes that underpin this strong connection are still under debate. With the increasing popularity of electronic cigarettes (e-cigarettes), it is now becoming more important to look to the neurobiological mechanisms involving alcohol and nicotine interactions to effectively treat a new generation of co-dependent individuals. Researchers have already recognized that the neuropathology produced by the combination of nicotine and ethanol is likely to produce an addictive nature very different to that of either one alone, and are employing a mixture of pre-clinical techniques to establish and investigate every stage in the development of both nicotine and ethanol-seeking behaviors. While it is agreed that multiple pathways orchestrate the complex reward profile of alcohol and nicotine co-addiction, several lines of evidence suggest the convergent site of action is within the mesolimbic dopaminergic system, at neuronal nicotinic acetylcholine receptors (nAChRs). A whole host of strategies are currently being employed to discover and unravel previously unknown or ill understood neurobiological processes in the brain, contributing greatly toward the development of novel pharmacotherapies with the aim of improving patient outcomes. This review intends to shed some light on the most influential and most recent pre-clinical work that is leading the charge in modeling this complicated relationship

Acute alcohol exposure alters the distribution Of alpha4 nicotinic receptors in the mouse brain

A growing body of evidence suggests that nicotinic acetylcholine receptors are important mediators of the effects of alcohol and represent significant pharmacotherapeutic targets for the treatment of alcohol use disorders (AUDs). In particular, the nicotinic alpha4 subunit has been shown to control the brain reward pathway and modulate its response to alcohol. Here we present evidence that a single exposure to alcohol is sufficient to elicit a profound redistribution of alpha4 subunits within the mouse brain reward circuit, including prefrontal-subcortical circuits. Using intraperitoneal bolus injection of a sedative dose of ethanol (3.6 g/kg) in alpha4-YFP transgenic mice, we have quantified the changes in the expression of nicotinic alpha4 subunits by using a combination of western blots and immunohistological techniques combined with 3D reconstruction. Our results show that 24 hr after a single ethanol injection, the expression of alpha4-containing nicotinic receptors is significantly upregulated in the nucleus accumbens (217%) and the amygdala (175%) but not in the prefrontal cortex. This data suggests that acute alcohol exposure alters alpha4 nicotinic receptor signalling principally in the mesolimbic pathway which in turn, might modulate the activity of several neuronal pathways in this brain region

Targeting glucocorticoid receptors that promote resilience in the treatment of addiction

There is strong evidence to suggest that the combination of alcohol and chronic repetitive stress leads to long-lasting effects on brain function, specifically areas associated with stress, motivation and decision-making such as the amygdala, nucleus accumbens and prefrontal cortex. Alcohol and stress together facilitate the imprinting of long-lasting memories. The molecular mechanisms and circuits involved are being studied but are not fully understood. Current evidence suggests that corticosterone (animals) or cortisol (humans), in addition to direct transcriptional effects on the genome, can directly regulate pre- and postsynaptic synaptic transmission through membrane bound glucocorticoid receptors (GR). Indeed, corticosterone-sensitive synaptic receptors may be critical sites for stress regulation of synaptic responses. Direct modulation of synaptic transmission by corticosterone may contribute to the regulation of synaptic plasticity and memory during stress (Johnson et al., 2005; Prager et al., 2010). Specifically, previous data has shown that long term alcohol (1) increases the expression of NR2Bcontaining NMDA receptors at glutamate synapses, (2) changes receptor density, and (3) changes morphology of dendritic spines (Prendergast and Mulholland; 2012). During alcohol withdrawal these changes are associated with increased glucocorticoid signalling and increased neuronal excitability. It has therefore been proposed that these synapse changes lead to the anxiety and alcohol craving associated with withdrawal (Prendergast and Mulholland; 2012). My lab is targeting this receptor system and the amygdala in order to understand the effect of combining alcohol and stress on these pathways. Lastly, we are testing GR specific compounds as potential new medications to promote the development of resilience to developing addiction

Acute Ethanol Administration Upregulates Synaptic α4-Subunit of Neuronal Nicotinic Acetylcholine Receptors within the Nucleus Accumbens and Amygdala

Alcohol and nicotine are two of the most frequently abused drugs, with their comorbidity well described. Previous data show that chronic exposure to nicotine upregulates high-affinity nicotinic acetylcholine receptors (nAChRs) in several brain areas. Effects of ethanol on specific brain nAChR subtypes within the mesolimbic dopaminergic (DA) pathway may be a key element in the comorbidity of ethanol and nicotine. However, it is unknown how alcohol affects the abundance of these receptor proteins. In the present study, we measured the effect of acute binge ethanol on nAChR α4 subunit levels in the prefrontal cortex (PFC), nucleus accumbens (NAc), ventral tegmental area (VTA), and amygdala (Amg) by western blot analysis using a knock-in mouse line, generated with a normally functioning α4 nAChR subunit tagged with yellow fluorescent protein (YFP). We observed a robust increase in α4-YFP subunit levels in the NAc and the Amg following acute ethanol, with no changes in the PFC and VTA. To further investigate whether this upregulation was mediated by increased local mRNA transcription, we quantified mRNA levels of the Chrna4 gene using qRT-PCR. We found no effect of ethanol on α4 mRNA expression, suggesting that the upregulation of α4 protein rather occurs post-translationally. The quantitative counting of YFP immunoreactive puncta further revealed that α4-YFP protein is upregulated in presynaptic boutons of the dopaminergic axons projecting to the shell and the core regions of the NAc as well as to the basolateral amygdala (BLA), but not to the central or lateral Amg. Together, our results demonstrate that a single exposure to binge ethanol upregulates level of synaptic α4∗ nAChRs in dopaminergic inputs to the NAc and BLA. This upregulation could be linked to the functional dysregulation of dopaminergic signalling observed during the development of alcohol dependence

The effect of varenicline on binge-like ethanol consumption in mice is beta4 nicotinic acetylcholine receptor-independent

BACKGROUND: \ud \ud Our laboratory has previously shown that the smoking-cessation agent varenicline, an agonist/partial agonist of alpha4beta2*, alpha3beta4*, alpha3beta2*, alpha6beta2* (* indicates the possibility of additional subunits) and alpha7 subunits of nicotinic acetylcholine receptors (nAChRs), reduces ethanol consumption in rats only after long-term exposure (12 weeks). As compounds having partial agonistic activity on alpha3beta4* nAChRs were shown to decrease ethanol consumption in rodents, we assessed here the involvement of the beta4 subunit in the effect of varenicline in the reduction of short- and long-term binge-like ethanol drinking in mice. \ud \ud METHODS: \ud \ud We used the well-validated drinking-in-the-dark (DID) paradigm to model chronic binge-like ethanol drinking in beta4-/- and beta4+/+ littermate mice and compare the effect of intraperitoneal injection of varenicline (2mg/kg) on ethanol intake following short- (4 weeks) or long-term (12 weeks) exposure. \ud \ud RESULTS: \ud \ud Drinking pattern and amounts of ethanol intake were similar in beta4-/- and beta4+/+ mice. Interestingly, our results showed that varenicline reduces ethanol consumption following short- and long-term ethanol exposure in the DID. Although the effect of varenicline on the reduction of ethanol consumption was slightly more pronounced in beta4-/- mice than their beta4+/+ littermates no significant differences were observed between genotypes. \ud \ud CONCLUSION: \ud \ud In mice, varenicline reduces binge-like ethanol consumption both after short- and long-term exposure in the DID and this effect is independent of beta4 nAChR subunit

The antihypertensive drug pindolol attenuates long-term but not short-term binge-like ethanol consumption in mice

Alcohol dependence is a debilitating disorder with current therapies displaying limited efficacy and/or compliance. Consequently, there is a critical need for improved pharmacotherapeutic strategies to manage alcohol use disorders (AUDs). Previous studies have shown that the development of alcohol dependence involves repeated cycles of binge-like ethanol intake and abstinence. Therefore, we used a model of binge-ethanol consumption (drinking-in-the-dark) in mice to test the effects of compounds known to modify the activity of neurotransmitters implicated in alcohol addiction. From this, we have identified the FDA-approved antihypertensive drug pindolol, as a potential candidate for the management of AUDs. We show that the efficacy of pindolol to reduce ethanol consumption is enhanced following long-term (12weeks) binge-ethanol intake, compared with short-term (4weeks) intake. Furthermore, pindolol had no effect on locomotor activity or consumption of the natural reward sucrose. Because pindolol acts as a dual beta-adrenergic antagonist and 5-HT1A/1B partial agonist, we examined its effect on spontaneous synaptic activity in the basolateral amygdala (BLA), a brain region densely innervated by serotonin and norepinephrine-containing fibres. Pindolol increased spontaneous excitatory post-synaptic current frequency of BLA principal neurons from long-term ethanol-consuming mice but not naive mice. Additionally, this effect was blocked by the 5-HT1A/1B receptor antagonist methiothepin, suggesting that altered serotonergic activity in the BLA may contribute to the efficacy of pindolol to reduce ethanol intake following long-term exposure. Although further mechanistic investigations are required, this study demonstrates the potential of pindolol as a new treatment option for AUDs that can be fast-tracked into human clinical studies