The ultrastructural distribution of putative nicotinic receptors on cultured neurons from the rat superior cervical ganglion

Abstract

The distribution of putative nicotinic receptors on cultured neurons from the rat superior cervical ganglion was determined by electron microscopic autoradiography using a radioactively labeled snake venom neurotoxin, toxin F. In a previous study, we demonstrated that toxin F blocks nicotinic transmission in these cultures of sympathetic neurons and in intact superior cervical ganglia. [ 125I]toxin F bound to two sites in these cultures: one site that was also recognized by the neuromuscular blocker, α-bungarotoxin, and a second site that was not. Since α-bungarotoxin neither blocks nicotinic transmission nor prevents the blocking effects of toxin F, the site specific to the binding of toxin F most probably represents neuronal nicotinic receptors. The total number of each of the toxin F binding sites was unaffected by culture conditions that are known to influence the extent to which these sympathetic neurons synthesize norepinephrine or acetylcholine. Autoradiographic analysis performed under saturating binding conditions (80 nM [ 125I]toxin F) revealed that the density of [ 125I]toxin F binding at synaptic membranes was about 5000 sites/μm 2, either in the absence of any competing ligand or in the presence of 2 μM α-bungarotoxin. In the presence of 2 μM unlabeled toxin F, there was no detectable binding at synapses. The density of these toxin F-specific sites was at least 80-fold higher at synaptic membranes than elsewhere. On the other hand, the data suggest that the toxin F binding site shared with α-bungarotoxin is exclusively extrasynaptic. Two micromolar α-bungarotoxin decreased the density of [ 125I]toxin F binding at non-synaptic sites by approximately two-thirds. These experiments support the hypothesis that toxin F blocks cholinergic transmission in cultures of sympathetic neurons by binding to nicotinic receptors and suggests that these receptors are highly clustered at synaptic membranes.