Developments of alpha-bungarotoxin receptors in cultured chick ciliary ganglion neurons

We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [125I]alpha-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 micrograms/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [125I]alpha-bungarotoxin was determined at room temperature in intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [125I]alpha-bungarotoxin was saturable with respect to both time of incubation (20-30 min) and concentration of toxin (5-10 nM), with an apparent Kd = 1.0 nM. Binding sites for [125I]alpha-bungarotoxin increased during the first week in culture from 1.8 fmol per 10(4) neurons at 1 day in vitro (DIV) to 8.6 fmol per 10(4) neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of alpha-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels, than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal alpha-bungarotoxin receptors.