Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) interact with the brain’s endogenous ligand, acetylcholine, and the exogenous ligand, nicotine. Neuronal nAChRs regulate mood, memory, cognition, and reward. A subset of nAChRs contains the alpha(α)6 subunit (encoded by the CHRNA6 gene). α6-containing nAChRs have restricted expression patterns within the central nervous system and make up pentameric nAChR populations of varying compositions (α4α6β2*, α6β2β3*, and α4α6β2β3*, *denotes possible other subunits) (Gotti et al. 2009). α6* nicotinic receptors are expressed on the terminals of dopaminergic neurons in the striatum and reach peak mRNA expression in dopaminergic cell bodies in the ventral tegmental area (VTA) and substantia nigra during adolescence (Azam et al. 2007; Champtiaux et al. 2002; Le Novere et al. 1996; Quik et al. 2011; Yang et al. 2009). Localization of the α6 nAChR subunit to the mesocorticolimibic and nigrostriatal pathways implicates a role of α6* nAChRs in nicotine reinforcement and reward as well as motor control (Bruijnzeel andMarkou 2004; Exley et al. 2008; Gotti et al. 2010; Jackson et al. 2009; Laviolette and van der Kooy 2003; Pons et al. 2008). As research begins to further elucidate α6* nAChR pharmacology, function, and its behavioral role in learning, memory, and addiction, we are limited in our tools to study the receptor complexes at a protein level. α6 nAChR expression has previously been detected via in situ hybridization (mRNA), genetic approaches, or by labeled selective antagonists, αconotoxinMII (α-CtxMII) and PIA (Drenan et al. 2008; Yang et al. 2009). However, a general, validated antibody, selective for α6 nAChR subunits would be a powerful tool to study receptor expression and function. To date, a commercially available and validatedα6 nAChR subunit antibody has not been reported. Previous studies assessing the specificity of α3, α4, and α7 nAChR subunit antibodies have challenged the specificity of these antibodies. Data highlights that the immunoreactivity for the antibody binding of these nAChR subunits is equivalent in wild type and nAChR subunit knock-out (KO) animals (Moser et al. 2007). Thus, the purpose of this study is to validate the specificity of the commercially available polyclonal α6 nAChR subunit antibody from Alomone Labs (cat. # ANC-006, Jerusalem, Israel). In order to detect whether we can quantify α6 nAChR subunit protein expression in wild type (WT) Sprague Dawley rats and C57BL/6J mice, we used quantitative western blot. The α6 nAChR subunit antibody used in this study is from a rabbit source, with rat and mouse reactivity, and was shown to bind toα6 nAChR subunit protein in rat PC12 pheochromocytoma cells as well as rodent brain lysates (Alomone 2019). Although a control antigen (Alomone 2019) blocks the α6 nAChR subunit antibody from binding in the PC12 pheochromocytoma cells, a more standard form of validation is necessary (Uhlen et al. 2016). Thus, the aim of our current studies is to use a genetic approach, to assess α6 nAChR subunit protein expression with the α6 nAChR subunit antibody in wildtype versus α6 KO C57BL/6J mice. As a first approach, we initially set out to develop a protocol to quantify α6 nAChR subunits within the VTA of male Sprague Dawley rats. The VTA is an important structure within the mesolimbic pathway that plays a role in mediating reward, motivation, and attention (Spanagel andWeiss 1999). This pathway is composed of dopaminergic neurons that originate in the VTA and innervate the limbic system including the nucleus accumbens (Di Chiara and Imperato 1988). Although the VTA Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00213-019-05413-x) contains supplementary material, which is available to authorized users.