Nicotinic receptor binding sites in rat primary neuronal cells in culture: characterization and their regulation by chronic nicotine

Abstract

We have characterized high affinity neuronal nicotinic acetylcholine receptors labeled by [ 3 H ]cytisine in primary neuronal cell cultures from fetal rat brains. After 15 days in culture, the highest density of [ 3 H ]cytisine binding sites ( B max≈57 fmol/mg protein) was found in cells from the brainstem, which includes the following subcortical brain areas: the septum, thalamus, hypothalamus, midbrain, pons and medulla. A lower density of sites was found in cells from the cerebral cortex, hippocampus, and caudate nucleus. [ 3 H ]Cytisine binds to receptors in primary cells from the brainstem and cerebral cortex with a K d of ≈0.5 nM, and the binding is inhibited by the agonists nicotine, acetylcholine, and epibatidine with IC 50 values of 1 to 20 nM, and by carbachol and the antagonist dihydro-β-erythroidine with IC 50 values of 0.5 to 1.5 μM. Chronic treatment of neuronal cultures with nicotine for 7 days differentially affected the number of nicotinic receptors in cells from different brain areas; it significantly increased the number of nicotinic binding sites in cells from the cerebral cortex, hippocampus, and caudate, but not in cells from the brainstem. The nicotine-induced increase of receptors in cerebral cortical cultures was not blocked by either mecamylamine or dihydro-β-erythroidine. These results indicate that primary cultures of rat neuronal cells provide a good model system in which to study and compare the properties and regulation of native neuronal nicotinic acetylcholine receptors.