Slimak_2014_Front.Hum.Neurosci_8_12

The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the alpha5, alpha3, and beta4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in alpha3beta4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that beta4 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 beta4 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 alpha3. We found that beta4A90I and beta4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant beta4D447Y, significantly increased nicotine-evoked current amplitudes, while beta4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express beta4 or beta4 variants in the MHb. Immunoprecipitation studies confirmed that beta4 lentiviral-mediated expression leads to specific upregulation of alpha3beta4 but not beta2 nAChRs in the Mhb. Mice injected with the beta4-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 beta4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the beta4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular beta4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.