Two components have been identified in extracts prepared from embryonic eye tissue that influence the long-term fate of chick ciliary ganglion neurons in dissociated cell culture. One of the components, with an apparent molecular weight of about 2x104, stimulates overall growth of the neurons but does not stimulate development of choline acetyltransferase activity. The other component, with an apparent molecular weight of about 5x104, stimulates development of choline acetyltransferase levels but has no effect on neuronal growth. The components will be purified from eye extract and examined both in culture and in vivo to determine whether they serve as target-derived trophic factors for ciliary ganglion neurons. To aid in these studies, monoclonal antibodies will be prepared against the partially purified components and used to purify the components further, manipulate component levels in vivo, and construct quantitative radioimmune assays for the components. Conditions will also be sought which induce the expression of adrenergic properties by the normally cholinergic ciliary ganglion neurons in culture as a way of identifying components which influence the differentiation of neural crest derivatives. Studies will be carried out to determine whether the neurons form chemical and electrical synapses on each other in cell culture, and how such connections are affected by input from neurons of the accessory oculomotor nucleus, the normal preganglionic input to ciliary ganglion neurons. Bgt 3.1, a protein neurotoxin present as a minor component in B. multicinctus venom, reversibly blocks acetylcholine (ACh) sensitivity on ciliary ganglion neurons and, in a separate action, induces the internalization of Alpha-bungarotoxin bound to the neurons. Alpha-Bungarotoxin does neither of these. Bgt 3.1 will be radiolabeled and examined as a possible ligand for the neuronal ACh receptor. If suitable, it will be used to study regulation of the neuronal receptor. Also, ultrastructural studies will be carried out to determine the distribution of Alpha-bungarotoxin binding sites during development since they do not appear to be associated with synaptic regions at early times, and to follow the path of bound Alpha-bungarotoxin that is internalized by Bgt 3.1 treatment.
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