Identification, pharmacology and anatomical localization of heteromeric neuronal nicotinic acetylcholine receptors in the rat hippocampus Effects of nicotine on nicotinic receptors expressed in rat primary cultured neurons

The first part of the present work is focused on the identification and quantification of the heteromeric neuronal nicotinic receptors (nAChRs) in the rat hippocampus. The density of nAChR subtypes was assessed by immunoprecipitation with subunit-selective antibodies in presence of [3H]epibatidine. Our results show that the predominant hippocampal heteromeric nAChRs are the α4β2 and α4β2α5 subtypes which account for ~40% and ~35%, respectively, of the total [3H]epibatidine-labeled receptors. An additional heteromeric subtype with the subunit composition of α4β2α3 represented ~10% of the total nAChRs, and another 10% of the immunoprecipitated receptors contained α4 and β4 subunits, with or without the α3 subunit. To determine if α4β2 and α4beta;2α5 nAChR subtypes differ in their ligand binding affinities, competition studies with various ligands against [3H]epibatidine were carried out and results were analyzed for one and two sites. Results suggested these subtypes have comparable binding affinities for the nicotinic ligands used here, except in presence of the positive allosteric modulator, galanthamine. Immunohistochemical analyses showed distribution of the α5 subunit similar to the α4 and β2 subunits with prominent expression in specific cell layers of the cornu ammonis 1 and dentate gyrus regions.;The second major focus of this study was to investigate the effects of chronic nicotine treatment on nAChRs expressed in primary neurons isolated from embryonic rat brain. Chronic nicotine treatment produced ~ a two-fold up-regulation of nAChRs, as measured with [125I]epibatidine radioligand binding assay. Biotinylation studies indicate that this up-regulation resulted from an increase in the number of receptors on the cell surface as well as inside the cell. Most of the cortical and hippocampal nAChR subtypes contain α4 and β2 subunits whereas α2, α3, α5, α6 and β4 subunits were detected at lower amounts. Nicotine exposure yielded a two-fold increase only in the β2-containing receptors and a less pronounced up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover of surface receptor is extremely slow in neurons and that chronic nicotine exposure seems to not affect it.