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

ABSTRACT: 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.

SUBMITTER: Calarco CA

PROVIDER: S-EPMC6251769 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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

Calarco Cali A CA Li Zhiying Z Taylor Seth R SR Lee Somin S Zhou Wenliang W Friedman Jeffrey M JM Mineur Yann S YS Gotti Cecilia C Picciotto Marina R MR

The European journal of neuroscience 20180523

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 β4 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 t ...[more]

PMID: 29791746

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Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.

Dataset Information

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

Publications

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

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