Localization of alpha-bungarotoxin binding sites in the ciliary ganglion of the embryonic chick: an autoradiographic study at the light and electron microscopic level.

Binding sites for [125I]alpha-bungarotoxin were localized in the chick ciliary ganglion by light and electron microscopic autoradiography. Groups of four ganglia were incubated for 4 h with 20 nM [125I]alpha-bungarotoxin alone or with radioactive alpha-bungarotoxin plus either 1 microM unlabeled alpha-bungarotoxin or 100 microM d-tubocurarine. Specific binding to various morphological regions was determined by subtracting the densities of autoradiographic grains in the presence of competing nonradioactive ligands from the densities in the absence of those ligands. Most of the specific binding in the ganglion (91%) was associated with neurons. Seventy-four per cent was found within 1.2 micron of neuronal plasma membranes and 17% was found overlying neuronal cell bodies. Analysis of the specific binding associated with neurons, but not with the neuronal plasma membranes, revealed that lysosomes and multivesicular bodies were 9- and 32-fold more heavily labeled than other cellular organelles. The grain density over choroid cell bodies was significantly higher than that over ciliary neurons. Most (greater than 75%) of the autoradiographic grains within 0.25 micron of neuronal plasma membranes were found in "complex" contact regions of the membranes, which are characterized by extensive membrane evaginations. However, after correcting for the amount of plasma membrane present in the various regions of the membrane studied, alpha-bungarotoxin binding was found to be uniform. Few (less than 10%) of the specialized membranes between pre- and postsynaptic neurons were found in these "complex" contact regions suggesting that the bulk of alpha-bungarotoxin binding to neuronal membranes is located at some distance from the sites of transmitter release. The density of alpha-bungarotoxin binding sites in the neuronal plasma membrane was low (approximately 100-200 sites/micron 2 of neuronal membrane) compared to published values of the density of binding sites at the neuromuscular junction. Since alpha-bungarotoxin does not bind preferentially to specialized synaptic membranes, it seems unlikely that the binding sites for this toxin are the neuronal nicotinic receptors.