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Development of alpha bungaro toxin receptors in cultured chick ciliary ganglion neurons



Development of alpha bungaro toxin receptors in cultured chick ciliary ganglion neurons



Brain Research 208(2): 479-486



Embryonic chick ciliary ganglion neurons were maintained in dissociated cell culture and the progressive appearance of surface receptors for [125I].alpha.-bungarotoxin was studied. Cultures were established from 8 day old embryos and fed a medium supplemented with 180 .mu.g/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 2 wk in culture. Binding of [125I].alpha.-bungarotoxin was determined at room temperature on intact cells still attached to their coverslips. Non-sepcific 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 1st wk in culture from 1.8[femto] per 104 neurons at 1 day in vitro (DIV) to 8.6 f[femto]mol per 104 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. This culture system should be useful for the study of the regulation, surface distribution and intracellular pathways of neuronal .alpha.-bungarotoxin receptors.

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Accession: 005132291

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PMID: 7214156



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