Cell autonomy, receptor autonomy, and thermodynamics in nicotine receptor up-regulation

Chronic nicotine exposure, in smokers or in experimental rodents administered nicotine, produces elevated levels of nicotinic acetylcholine receptors in several brain regions. However, there are few data on up-regulation of receptors in specific neuronal subtypes. We tested whether functional up-regulation of nicotinic responses occurs in cultured GABAergic neurons of the ventral midbrain. Fura-2 measurements of nicotinic responses were made on ventral midbrain neurons from knock-in mice heterozygous for the α4-M2 domain Leu9′Ala mutation, which confers nicotine hypersensitivity. Chronic nicotine exposure at a concentration (10 nM for 3 days) that activates only the hypersensitive α4* (Leu9′Ala) receptors, but not wild-type receptors, resulted in significant potentiation of ACh (100 μM)-elicited responses. Experiments were also performed on midbrain neuronal cultures heterozygous for the α4* (Leu9′Ala) mutation as well as for a GFP protein fused to a GABA transporter that reliably reveals GABAergic neurons. In cultures chronically treated with 10 nM nicotine, there was significantly increased α4* nicotinic-induced Ca2+ influx elicited by low concentration of ACh (3 μM). Furthermore, chronic exposure to the competitive antagonist dihydro-β-erythroidine, but not to the noncompetitive antagonist mecamylamine, induced up-regulation of ACh elicited nicotinic responses. These results suggest that occupation of α4* nicotinic receptor binding site(s), at the interface between two subunits, is sufficient to promote assembly and/or up-regulation of functional receptors in GABAergic neurons. Up-regulation in neurons is both “cell-autonomous”, occurring at the cell itself, and “receptor autonomous”, occurring at the receptor itself, and may be a thermodynamic necessity of ligand–protein interactions.