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An autoradiographic survey of mouse brain nicotinic acetylcholine receptors defined by null mutants.

ABSTRACT: Nine nicotinic receptor subunits are expressed in the central nervous system indicating that a variety of nicotinic acetylcholine receptors (nAChR) may be assembled. A useful method with which to identify putative nAChR is radioligand binding. In the current study the binding of [(125)I]?-bungarotoxin, [(125)I]?-conotoxinMII, 5[(125)I]-3-((2S)-azetidinylmethoxy)pyridine (A-85380), and [(125)I]epibatidine has been measured autoradiographically to provide data on many nAChR binding sites. Each binding site was evaluated semi-quantitatively for samples prepared from wild-type and ?2, ?4, ?6, ?7, ?2, ?4, ?5 and ?3 null mutant mice. Deletion of the ?7 subunit completely and selectively eliminated [(125)I]?-bungarotoxin binding. The binding of [(125)I]?-conotoxinMII was eliminated in most brain regions by deletion of either the ?6 or ?2 subunit and is reduced by deletion of either the ?4 or ?3 subunit. The binding of 5[(125)I]A-85380 was completely eliminated by deletion of the ?2 subunit and significantly reduced by deletion of the ?4 subunit. Most, but not all, ?4-independent sites require expression of the ?6 subunit. The effect of gene deletion on total [(125)I]epibatidine binding was very similar to that on [(125)I]A-85380 binding. [(125)I]Epibatidine also labels ?4* nAChR, which was readily apparent for incubations conducted in the presence of 100nM cytisine. The effects of ?3 gene deletion could not be evaluated, but persistence of residual sites implies the expression of ?3* nAChR. Taken together these results confirm and extend previously published evaluations of the effect of nAChR gene deletion and help to define the nAChR subtypes measurable by ligand binding.

SUBMITTER: Baddick CG

PROVIDER: S-EPMC3162045 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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An autoradiographic survey of mouse brain nicotinic acetylcholine receptors defined by null mutants.

Baddick Christopher G CG Marks Michael J MJ

Biochemical pharmacology 20110510 8

Nine nicotinic receptor subunits are expressed in the central nervous system indicating that a variety of nicotinic acetylcholine receptors (nAChR) may be assembled. A useful method with which to identify putative nAChR is radioligand binding. In the current study the binding of [(125)I]α-bungarotoxin, [(125)I]α-conotoxinMII, 5[(125)I]-3-((2S)-azetidinylmethoxy)pyridine (A-85380), and [(125)I]epibatidine has been measured autoradiographically to provide data on many nAChR binding sites. Each bin ...[more]

PMID: 21575611

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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.

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An autoradiographic survey of mouse brain nicotinic acetylcholine receptors defined by null mutants.

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An autoradiographic survey of mouse brain nicotinic acetylcholine receptors defined by null mutants.

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