Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption

Abstract

The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and β4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in α3β4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that β4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of β4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that β4A90I and β4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant β4D447Y, significantly increased nicotine-evoked current amplitudes, while β4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express β4 or β4 variants in the MHb. Immunoprecipitation studies confirmed that β4 lentiviral-mediated expression leads to specific upregulation of α3β4 but not β2 nAChRs in the Mhb. Mice injected with the β4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the β4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the β4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular β4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.

Role of β4* Nicotinic Acetylcholine Receptors in the Habenulo–Interpeduncular Pathway in Nicotine Reinforcement in Mice

“…The role of β4* nAChRs has been investigated in certain nicotine-mediated behaviors, including nicotine-elicited seizures (Salas et al, 2004), aversion (Frahm et al, 2013;Slimak et al, 2014), anxiety (Salas et al, 2003), and withdrawal (Salas et al, 2004;Zhao-Shea et al, 2013), supporting the involvement of this subunit in regulating the aversive properties of nicotine (Fowler and Kenny, 2014). However, recent findings provide evidence for their role in the habenulo-interpeduncular pathway in nicotine reinforcement.…”

“…Furthermore, variants in the β4 regulatory domain reduce the age of tobacco initiation, while gain-offunction variants in CHRNB4 reduce the risk of nicotine dependence (Haller et al, 2012;Saccone et al, 2009;Schlaepfer et al, 2008). Although cholinergic responses are unaffected in CHRNB4 coding variants, nicotine-elicited currents are increased (Haller et al, 2012), which is reflected as aberrant nicotine aversion in mice expressing these variants (Slimak et al, 2014), and as fewer cigarettes smoked per day in humans (Haller et al, 2012). These studies have shown that the β4-containing (β4*) subpopulation of nAChRs merits further investigation.…”

Diversity of native nicotinic receptor subtypes in mammalian brain

“…A number of SNPs have been found in the b4 subunit gene associated with cigarette consumption in human and the injection of lentiviruses carrying the wild-type b4 subunit or these b4 variants in the MHb of mice leads to aversion or preference for nicotine depending on whether the variants increase or decrease nicotine currents (Slimak et al, 2014).…”

Mechanisms of Action and Persistent Neuroplasticity by Drugs of Abuse

“…In mice chronically treated with nicotine, null mutations of the a2 and a5 subunits highly expressed in the IPN reduced mecamylamine-induced withdrawal, and in wild-type mice microinfusion of mecamylamine into the Hb or IPN precipitated withdrawal (Salas et al, 2009). Moreover, lentiviral expression in the MHb of a gain-of-function Thr374Ile variant of the b4 nAChR subunit associated with reduced risk of smoking resulted in strong nicotine aversion in mice, whereas expression of a loss-of-function Arg348Cys variant failed to induce nicotine aversion (Slimak et al, 2014). Nicotine-induced excitation of the MHb neurons in mice was mediated by tachykinin NK 1 and NK 3 receptors (Dao et al, 2014).…”