Binge drinking is the most common pattern of excessive alcohol intake and is the leading cause of death and disability globally among people between the ages of 15–49 (Lim et al., 2012). Alcohol (ethanol) in relevant concentrations functionally interferes with the release of several brain neurotransmitters, including dopamine (Bainton et al., 2000; Doyon et al., 2003), and acetylcholine, as well as their respective receptors (Larsson et al., 2005), on both activation and desensitisation. Although extensive research has examined the role of nicotinic receptors in nicotine addiction, alcohol-related changes in neuronal nAChRs involved in AUDs remain unclear. These nAChRs belong to a super-family of ligand-gated ion channel receptors, which when activated, allow for the flux of several cations. They are localised both pre- and post-synaptically and cause calcium dependent signalling cascades in many neuronal cell types (Albuquerque et al., 2009). Indeed, identification of changes in nAChRs brought about by alcohol may help to improve pharmacotherapies for alcohol dependence.

The limbic system includes several pathways between the hypothalamic-pituitary-adrenal (HPA) axis and dopaminergic systems, encompassing the PFC, NAc, VTA and the amygdala (Amg). This includes the mesocorticolimbic dopaminergic or ‘reward’ pathway, which is involved in the addiction to many drugs of abuse (Koob and Volkow, 2016). These regions are intimately connected. The NAc receives inputs from the BLA, sending outputs to the basal ganglia and ventral pallidum (Yager et al., 2015). Both receive dopaminergic innervation from the VTA. In the reward pathway (humans and rodents alike), the PFC is thought to be involved in monitoring information regarding choice, and the adjustment of behaviour in response to consequences (St Onge and Floresco, 2010), in turn, the amygdala and NAc play a role in representing the value of the reward (Mendez et al., 2013). Determining how substances of abuse such as ethanol affect nAChRs in these regions is essential to understanding how these drugs regulate reward processes and modulate decision-making and risky behaviour that further contribute to the development of alcohol dependence.

Few studies have investigated ethanol-induced changes in nicotinic receptor expression at the mRNA or protein level, and it remains an area of significance in understanding alcohol dependence. The impact of ethanol on the expression of specific nAChR subunits has mostly been investigated using heterologous systems, such as cell lines expressing nAChR subtypes. However, the mechanisms of ethanol-induced changes in nAChRs in vivo may differ from those in heterologous expression systems. Despite this, genetic analysis and manipulations point to the involvement of α4^∗ nAChRs (where α4^∗ denotes that other subunits are present in the heteropentameric nAChR protein) in alcohol addiction (Butt et al., 2003; Coon et al., 2014). The α4 subunit is the most widely expressed α subunit in the mammalian brain, including dopaminergic projection areas such as the NAc and amygdala, representing principal relay stations in the extended amygdala circuit (Exley and Cragg, 2008). Most studies agree that in the case of nicotine, alterations in presynaptic α4^∗ nAChRs on dopaminergic neurons mediates key pathways involved in reward (Lena et al., 1999; Zhou et al., 2001; McGranahan et al., 2011), and in line with this, the partial α4β2 agonist varenicline enhances dopamine (DA) release in the NAc (Feduccia et al., 2014). Little is known about the response of this receptor subtype to ethanol, however, as in contrast to nicotine, ethanol acts primarily to potentiate the response of nAChRs to acetylcholine (ACh) rather than as a direct agonist (Zuo et al., 2002).

Although available evidence shows that transcription and upregulated protein synthesis is not directly involved in nicotine-induced increases in α4^∗ nAChRs (Pauly et al., 1996; Miao et al., 1998; Huang and Winzer-Serhan, 2006), ethanol regulation of heteromeric nAChRs is still widely unresolved. Until recently, in vivo studies concerning nAChR subunits have been hindered by the lack of selectivity of receptor-specific antibodies. Indeed, several studies have concluded that under commonly used conditions, nAChR antibodies are not suitable for immunocytochemistry (Herber et al., 2004; Jones and Wonnacott, 2005; Moser et al., 2007). An advance has been the generation of transgenic knock-in mice in which the nAChR subunit is fused with YFP (Nashmi et al., 2007), and these have previously been used to successfully quantitate α4 subunit expression in the brain (Chatterjee et al., 2013; Renda and Nashmi, 2014).

Using these knock-in mice, we examine the effect of acute ethanol exposure on nAChRs containing the α4 subunit by western blot and on the gene encoding for the subunit (Chrna4) by real-time PCR (qPCR) in brain regions involved in the mesocortical limbic reward pathway, in particular the PFC, NAc, VTA and amygdala. We further adapted a quantitative immunohistochemistry method to determine the micron-scale localisation of any changes. Given the prominent role of dopaminergic signalling within the NAc and amygdala in the effect of alcohol, we investigated whether α4 subunit expression within the presynaptic boutons of dopaminergic fibres was modulated by binge ethanol exposure. Our results indicate that a single exposure to a sedating dose of ethanol upregulates the expression of synaptic α4^∗-receptors in dopaminergic inputs to the NAc and BLA, directly linking alcohol to the functional dysregulations of dopaminergic signalling observed during the development of alcohol dependence.

We examined the effect of an acute large dose of ethanol (3.6 g/kg, i.p.) or saline (20 ml/kg, i.p) on the animal’s LORR and RORR in order to measure the sedative effect of ethanol in each animal. Latency to and duration of LORR was analogous to that of similarly treated C57BL/6 mice (Figures 2B,C). In agreement with previous studies, the α4-YFP mice, showed no physiological alterations compared to their WT littermates (Nashmi et al., 2003, 2007; Santos et al., 2013). BECs were also measured to confirm treatment (Figure 2A).

In the past, studies examining changes in nAChRs have used receptor-specific antibodies; however, such analyses raise questions due to lack of selectivity of the antibodies for the desired subunit. In this study, we have used α4-YFP knock-in mice to evaluate whether ethanol exposure affects the levels of α4 nAChR subunit protein and/or the Chrna4 mRNA that encodes this subunit. This mouse line displays normal subcellular localisation, ACh-induced currents and ACh-induced Ca²⁺ fluxes (Nashmi et al., 2007). We have also previously used these mice to identify reduced α4^∗ nAChR levels in α5 knock-out mice using western blot (Chatterjee et al., 2013), with Renda and Nashmi (2014) only recently using the same strain to characterise an upregulation of α4^∗ nAChRs after chronic nicotine pre-treatment using qualitative measurements of fluorescence intensity. In the present study, we have adapted a quantitative approach (Belmer et al., 2017) to demonstrate that synaptic α4^∗ nAChRs, in specifically dopaminergic fibres within the NAc and BLA, are upregulated just hours after a single ‘binge’ episode of alcohol administration. For this, we colocalised the α4^∗ nAChRs with the presynaptic bouton marker synaptophysin in dopaminergic axons using a high-resolution confocal laser scanning microscopy approach. Although synaptophysin is a one of the most widely used protein markers of functional synapses and synaptic plasticity in the brain, this approach is not resolutive enough to conclude on the subsynaptic localisation of α4^∗ nAChRs and whether they are located at the synaptic membrane (functional), or located within the submembrane cytoskeleton (inactive). Pharmacology and physiology experiments will therefore be needed to determine whether the alcohol-upregulated α4^∗ nAChRs are pharmacology active and functional.

ACh influences numerous physiological and pathological processes in the CNS via activation of nAChRs (Albuquerque et al., 2009) that modulate neurotransmitter release (MacDermott et al., 1999). The activation of DA receptors can increase acetylcholine release, (DeBoer et al., 1996; Nicola et al., 2000), and nAChRs on dopamine terminals play a key role in facilitating endogenous acetylcholine’s ability to trigger synaptic dopamine release (Marubio et al., 2003; Mao et al., 2011; Cachope et al., 2012; Threlfell et al., 2012). This circuit is potentiated by ethanol, and reduced in mice lacking the α4 subunit (Liu et al., 2013). Interestingly, the increases in α4^∗ nAChRs seen in the amygdala were comprised solely of changes within the BLA. St Onge et al. (2012) previously revealed that disruption in communication between the BLA and NAc biassed choice toward larger, uncertain rewards on a probabilistic discounting task in rodents. Importantly, release of dopamine into the BLA may be distinctly involved in the response to both rewarding and threatening stimuli (Phillips et al., 2003), and changes in synaptic nAChRs on dopaminergic terminals may influence further responses, with damaged dopamine signalling also linked to several neuropsychiatric disorders (Ledonne and Mercuri, 2017). Many previous studies show that direct interaction of BLA activity and NAc dopamine is essential to conditioned reward associations (Ambroggi et al., 2008; Jones et al., 2010) and likely confers the motivational value of alcohol-related stimuli (Ghods-Sharifi et al., 2009). Interestingly, while most of the increase occurred in dopaminergic neurons, a small subset of receptors was upregulated on non-TH⁺ fibres. These may comprise nAChRs on GABAergic interneurons or serotonergic terminals, with conflicting reports of presynaptic nAChR modulation of ACh release from cholinergic axons (Schwartz et al., 1984; Pradhan et al., 2002; Quik and Wonnacott, 2011).

Current research indicates that nAChR subunit genes are potential contributors to the development of both alcoholism and tobacco abuse (Schlaepfer et al., 2008; Hoft et al., 2009a,b; Zuo et al., 2016). Also, SNP analysis of CHRNA4 by Tritto et al. (2001) and Feng et al. (2004), demonstrated that two SNPs are associated with a protective effect against nicotine and alcohol addiction in humans and mice (Tritto et al., 2001; Feng et al., 2004) when exposed chronically. Combined with the results reported here, it is likely that while polymorphisms of the CHRNA4 gene play a role in altering the sensitivity of the subunit to ethanol (Butt et al., 2003; Coon et al., 2014), acute doses of ethanol do not influence transcription.

Previous studies in this area are few and have produced mixed results. An early study on chronic ethanol exposure showed an increase in α4 mRNA levels of 22% after 4 days of direct application of ethanol to neuronal cell culture (Gorbounova et al., 1998). A recent study, and the only found addressing acute ethanol exposure reported no change in α4 mRNA expression after 24 h of application in foetal neural progenitor cells (Balaraman et al., 2012). Interestingly, the same study found a decrease in nAChR subunit mRNAs in a longer term of application (5 days). An in vivo study on nicotine reported a drop in the level of CHRNA4 gene expression in the VTA and an increased expression in the NAc in rat pups after 4 weeks of gestational exposure (Chen et al., 2005). It could be suggested that α4 mRNA levels may be altered only in long-term ethanol consumption, and possibly be limited to distinct developmental periods such as prenatal. As for protein expression, a previous ligand binding study reported an increase in α4β2 nAChRs in M10 cells exposed to ethanol after 48 h, which remained elevated for as long as 6 days after removal of the drug (Dohrman and Reiter, 2003). While not only being a study using cell culture, the cells specifically expressed only α4β2 nAChRs and was a purely in vitro model. In the mammalian brain, α4 nAChR subunits may also form receptors with the α5 nAChR subunit, which plays a distinct role in driving the expression of α4^∗ receptors (Chatterjee et al., 2013), making the in vivo study of ethanol on α4^∗ receptors valuable.

Studies that have focused on the function of nAChRs have demonstrated that deleting the α4 nAChR subunit caused increased sensitivity to nicotine-induced locomotor depression and decreased sensitivity to anxiolytic effects (McGranahan et al., 2011), suggesting an involvement of the amygdala. In addition, other groups showed that α4-knockout (KO) mice did not systemically self-administer nicotine and displayed a lack of nicotine-induced dopamine level increase seen in WT mice exposed to nicotine (Marubio et al., 2003; Pons et al., 2008). Similar observations were made in relation to alcohol addiction, as ethanol-induced place preference was absent in α4KO mice and was increased in mice with a single point mutation producing hypersensitive α4^∗ nAChRs (Liu et al., 2013). This suggests that the α4 subunit is necessary for the development of alcohol and nicotine dependence and increased levels of the subunit will affect susceptibility to addiction.

As discussed previously, we do not know the subcellular mechanism underlying the ethanol-induced increase in α4^∗ nAChR levels. Because we did not see a change in local mRNA expression, ethanol-induced upregulation, like nicotine-induced upregulation, is a post-translational mechanism. Amongst the possible mechanisms, we believe that two particular post-translational mechanisms are unlikely to occur. First, although an increase in NAc dopamine could augment acetylcholine (ACh) levels, and this could, in turn, lead to desensitisation of α4β2 nAChRs, desensitisation does not appear to cause upregulation (Henderson and Lester, 2015). Second, in neurons, nicotine does not affect the rate of nAChR endocytosis from the plasma membrane (Henderson and Lester, 2015).

Upregulation of nAChRs by chronic exposure to nicotine, at smoking-relevant concentrations, is selective at every level explored to date. Some evidence supports the hypothesis that this selective upregulation is necessary and sufficient for the early stages of nicotine dependence (Miwa et al., 2011; Henderson and Lester, 2015; Henderson et al., 2017). Here we describe these tiers of selectivity. At the level of brain regions, nicotine upregulates nAChRs in hippocampus, cortex, amygdala (Le Foll et al., 2016) and midbrain; but not in the thalamus. Among cell types within brain regions, nicotine upregulates nAChRs strongly in the somata of VTA GABAergic neurons, but only modestly in the somata of VTA dopaminergic neurons. There is also selectivity at the level of somatodendritic vs. axon terminal regions: in dopaminergic neurons, upregulation is modest in the somatodendritic region, but strong in the axon terminals, as also found for binge ethanol in the present study. At the level of nAChR composition, selectivity is also observed: the most consistently upregulated receptors are α4β2^∗ (where the asterisk denotes the possible presence of other subunits). In some regions, α6β2^∗ nAChRs are also upregulated. Other nAChR subtypes are upregulated only by much higher, non-pharmacologically relevant nicotine doses. The combinations thought to be resistant to upregulation include α4β4, α4β3, and α7. There is also selectivity at the level of subunit stoichiometry within a nAChR pentamer: (α4)₂(β2)₃, but not (α4)₃(β2)₂, nAChRs are upregulated by chronic exposure to nicotine. Although no single molecular or molecular level mechanism accounts for these tiers of selectivity, in most cases the upregulated nAChRs are (α4)₂(β2)₃. These (α4)₂(β2)₃ nAChRs are constitutively retained to some extent in the endoplasmic reticulum (ER) and cis-Golgi (Kuryatov et al., 2005; Sallette et al., 2005) and also bind nicotine rather strongly at smoking-relevant doses. Nicotine enters the ER, acts as a stabilising pharmacological chaperone, protects nAChRs against ER-associated degradation (ERAD), and enhances exit of nAChRs from the ER, thus enhancing the number which eventually reach the plasma membrane. This post-translational pharmacological chaperoning is the dominant mechanism of nicotine-mediated upregulation (Henderson and Lester, 2015).

In contrast, a more likely mechanism for ethanol-induced upregulation derives from recent experiments showing that >24 hr exposure to menthol upregulates α4β2^∗ nAChRs, both in the absence and presence of nicotine. This upregulation may be an example of chemical chaperoning: the menthol binds to as-yet undetermined non-agonist sites, either on the nAChR or on other components of the pathway taken by nAChRs to exit the ER (Henderson et al., 2016, 2017). We suggest that a binge of ethanol acts via a similar mechanism(s). Ethanol increases the open channel state of nAChRs (Wu and Miller, 1994; Forman and Zhou, 1999; Zuo et al., 2004), and this increased stability, if it also occurs in the ER, may protect nAChRs against ERAD. The rather general mechanism of chemical chaperoning should be distinguished from specific pharmacological chaperoning (Kuryatov et al., 2005; Henderson and Lester, 2015), which also occurs within the ER but involves the binding of nicotine or other agonists to nAChRs themselves, at or very near the ACh binding site within the interface between the α4 and β2 subunits.

An identifiable aspect of alcohol use is its strong correlation with nicotine use, and α4^∗ nAChRs in reward areas of the brain are likely involved in the co-morbidity of these two drugs (Liu et al., 2013), for a comprehensive review see (Tarren and Bartlett, 2017). In line with this, advances in pharmacotherapy for AUDs have indicated the potential of various α4β2^∗ nAChR agonists such as the smoking cessation drugs cytisine and varenicline in reducing ethanol self-administration (Steensland et al., 2007; Hendrickson et al., 2010; Bito-Onon et al., 2011; Mitchell et al., 2012). Increases in α4^∗ nAChRs seen after just a single dose of ethanol may then both increase propensity for nicotine use, as well as increase the efficacy of α4^∗ nAChR targeted therapies for reducing co-morbid alcohol and nicotine use.

The work presented here not only identifies acute changes to a major population of nAChRs caused by an intoxicating dose of alcohol, but also for the first time visualises specific neuronal populations involved in α4^∗ nAChR-mediated alcohol behaviours. Whether similar changes occur following a moderate dose of alcohol and/or after chronic exposure, however, was not elucidated in the present work.

If upregulation of α4 containing nAChRs by ethanol does not involve new protein synthesis, it is believed this process may be contingent on post-translational events, and likely has far-reaching implications (Henderson and Lester, 2015). In addition, whether this process impacts long-term alcohol consumption and seeking remains to be investigated.

HL provided the α4YFP mice and revised the manuscript. JT performed the WB and qPCR experiments and analysis, and drafted the manuscript. AB performed the IHC experiments and analysis. JT, AB, and SB analyzed and interpreted the results. JT, AB, and SB designed the experiments and revised the manuscript. All authors approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.