Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChR) on GABAergic interneurons. Single cell PCR-analysis had confirmed that several nAChR subunit mRNAs are coexpressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the coexpression of GAD67 with different nAChR subunits using double in situ hybridization with a digoxigenin-labeled GAD67 probe and 35S-labeled probes for nAChR subunits (α2, α3, α4, α5, α6, α7, β2, β3, and β4). The results revealed that most GAD67-positive interneurons expressed β2, and 67 % also expressed α7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons coexpressed α4, and less than 10% expressed transcripts for α2, α3, α5 or β4. Most GAD67/α2 coexpression was located in CA1/CA3 stratum oriens, and GAD67/α5 coexpression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of α6 and β3 mRNAs was rarely detected in the hippocampus, and mRNAs were not coexpressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric α7 or heteromeric α7*-containing nAChRs. Other possible combinations such as α2β2*, α4β2*, or α5β2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of α and β subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of α3, α5, α7, and β4 subunit mRNAs and α7 binding sites during hippocampal and cortical development. Here, we examined the expression of α2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. α2 subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of α2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of α2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, α2 mRNA expression was located in nonpyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, α2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing α2 subunits could regulate GABAergic activity during a critical period of network formation.
In the enteric nervous system (ENS) excitatory nicotinic cholinergic transmission is mediated by neuronal nicotinic acetylcholine receptors (nAChR) and is critical for the regulation of gastric motility. nAChRs are ligand-gated pentameric ion channels found in the central and peripheral nervous systems. The expression of heteromeric nAChR and receptor subunit mRNAs was investigated in the neonatal rat ENS using receptor autoradiography with the radiolabeled ligand 125I-Epibatidine, and in situ hybridization with subtype specific probes for ligand binding alpha (α2, α3, α4, α5, α6) and structural beta (β2, β3, β4) subunits. The results showed strong nicotine sensitive binding of 125I-Epibatidine around the stomach, and small and large intestines. The binding was partially displaced by A85380, a nicotinic ligand which differentiates between different heteromeric nAChR subtypes, suggesting a mixed receptor population. Radioactive in situ hybridization detected expression of α3, α5, α7, β2 and β4 mRNA in the myenteric plexus of the stomach, and small and large intestines. In the submucosal plexus of the small and large intestines expression of α3, α5 and β4 was found in some ganglia. There was no signal for α4, α6 and β3 in the ENS but positive hybridization signal for α2 transcripts was seen in some areas of the small intestines. However, the signal was not associated with any ganglion cells. The results confirm the presence of heteromeric nAChRs in the ENS similar to those found in the peripheral nervous system, with the majority being composed of α3(α5)β4, and a few α3β2 nAChRs. In addition, homomeric α7 nAChRs could be present.
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