Immunolabeling demonstrates the interdependence of mouse brain alpha4 and beta2 nicotinic acetylcholine receptor subunit expression.

Immunolabeling of beta2 and alpha4 subunits was quantitated in brain sections (14 mum) using [(125)I]mAb 270 and [(125)I]mAb 299, respectively. Specificity was demonstrated by signal loss in beta2(-/-) and alpha4(-/-) brain sections, respectively. Even mild paraformaldehyde fixation severely affected immunolabeling, so this study used unfixed sections. Immunolabeling autoradiography was used to map and quantitate the effects of beta2 and alpha4 subunit-null mutations on their putative partner subunits' protein expression. [(125)I]mAb 299 labeling was nearly eliminated in beta2(-/-) sections, although dorsal interpeduncular nucleus (IPN) retained a faint signal. Therefore, alpha4 subunit expression is almost universally beta2-dependent. In contrast, alpha4-null mutation effects on [(125)I]mAb 270 immunolabeling varied widely among brain regions. In corticothalamic regions, [(125)I]mAb 270 labeling was eliminated. However, in habenulopeduncular regions, alpha4 genotype had no effect. Other (predominantly dopaminergic and optic tract) nuclei also retained reduced [(125)I]mAb 270 labeling in alpha4(-/-) sections. Thus, although most beta2 subunit protein expression is alpha4-dependent, this dependence is not universal. Presumably, residual beta2 subunits are found in non-alpha4* subtypes. Together, these results show that immunolabeling is applicable to reliable, quantitative investigations of neuronal nAChRs, and that subunit-null mutants can be appropriate controls for such experiments. In situ mRNA hybridization was also performed to determine if altered mRNA transcription mediated the interdependence of alpha4 and beta2 subunit expression. alpha4-Null mutation did not affect beta2 mRNA expression, nor did beta2 genotype affect alpha4 mRNA expression. Consequently, it seems that the two subunits' effects on each other's expression are mediated at the protein, rather than gene expression level.