Molecular and cellular characterization of nicotinic acetylcholine receptor subtypes in the arcuate nucleus of the mouse hypothalamus

Nicotine, acting through nicotinic acetylcholine receptors (nAChRs), increases the firing rate of both orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC), yet nicotine and other nAChR agonists decrease food intake in mice. Viral-mediated knockdown of the 4 nAChR subunit in all neuronal cell types in the ARC prevents the nicotinic agonist cytisine from decreasing food intake, but it is not known whether the 4 subunit is selectively expressed in anorexigenic neurons or how other nAChR subtypes are distributed in this nucleus. Using translating ribosome affinity purification (TRAP) on ARC tissue from mice with ribosomes tagged in either AgRP or POMC cells, we examined nAChR subunit mRNA levels using real-time PCR. Both AgRP and POMC cells express a comparable panel of nAChR subunits with differences in 7 mRNA levels and a trend for difference in 4 levels, but no differences in 4 expression. Immunoprecipitation of assembled nAChRs revealed that the 4 subunit forms assembled channels with 3, 2 and 4, but not other subunits found in the ARC. Finally, using cell type-selective, virally delivered small hairpin RNAs targeting either the 4 or 7 subunit, we examined the contribution of each subunit in either AgRP or POMC cells to the behavioural response to nicotine, refining the understanding of nicotinic regulation of this feeding circuit. These experiments identify a more complex set of nAChRs expressed in ARC than in other hypothalamic regions. Thus, the ARC appears to be a particular target of nicotinic modulation.