Abstract
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.
Highlights
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)
An advance has been the generation of transgenic knock-in mice in which the nicotinic acetylcholine receptor (nAChR) subunit is fused with yellow fluorescent protein (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 in brain regions involved in the mesocortical limbic reward pathway, in particular the prefrontal cortex (PFC), nucleus accumbens (NAc), ventral tegmental area (VTA) and amygdala
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 basolateral amygdala (BLA), directly linking alcohol to the functional dysregulations of dopaminergic signalling observed during the development of alcohol dependence
Summary
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). 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). Identification of changes in nAChRs brought about by alcohol may help to improve pharmacotherapies for alcohol dependence
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