Nicotinic cholinergic receptors in the rat retina and their transport down the optic nerve
Neuronal nicotinic receptors (nAChRs) were measured in the rat retina and optic nerve to determine the specific subtypes and possible transport down the optic nerve. We detected seven nicotinic receptor subunit mRNA transcripts in the rat retina, alpha2-alpha4, alpha6, and beta2-beta4, with RNase protection assays. The density of the sites in the retina measured with [3H]epibatidine ([3H]EB) is ∼30% higher than with [125I]A-85380 and more than twice that measured with [3H]cytisine or [3H](-) nicotine. These data suggest that the retina expresses multiple subtypes of nAChRs, including a large fraction of receptors containing the beta2 subunit. Consistent with this, in binding competition studies nicotinic ligands fit a model for two affinity classes of binding sites. To determine the specific subtypes of nAChRs in the rat retina, we used subunit specific antibodies in immunoprecipitation assays. Immunoprecipitation of [3H]EB-labeled nAChRs with antibodies specific to the beta2 and beta4 subunits indicated that ∼80% of the receptors contained beta2 subunits and ∼25% contained beta4 receptors. Sequential immunoprecipitation assays indicated that the rat retina contains multiple subtypes of nAChRs. The majority of the receptors measured appeared to be simple heteromeric subtypes, composed of a single type of alpha and a single type of beta subunit; but a significant fraction are mixed heteromeric subtypes, composed of two or more alpha and/or beta subunits.
[3H]EB binding in the rat optic nerve suggest a large number of nAChRs and more than one subtype when compared to [3H]cytisine binding. Single immunoprecipitation studies in the rat optic nerve found the subunits alpha2-alpha4, alpha6, beta2, and beta3, but no beta4 nAChR subunit. Sequential immunoprecipitation assays showed at least three beta2 containing simple heteromeric subtypes. Autoradiography studies with unilaterally enucleated rats showed the transport of more than one nAChR subtype down the optic nerve, suggesting presynaptic nAChRs in the superior colliculus and lateral geniculate nucleus.
[125I]alpha-bungarotoxin labeled alpha7 homomeric nAChRs in both the rat retina and optic nerve. RNase protection assays showed alpha7 mRNA in the optic nerve, but not in the rat retina. Autoradiography studies using unilaterally enucleated rats found a small percentage of the alpha7 nAChR transported down the optic nerve.