The major goal of the proposed studies is to identify molecular mechanisms relevant to the formation of specialized synaptic and perisynaptic regions on developing chick ciliary ganglion (CG) neurons. CG neurons express two classes of nicotinic acetylcholine receptors: nAChRs composed of a3, a5, b4 and occasionally b2 subunits which are concentrated in the postsynaptic membrane, and Bgt-nAChRs which contain a7 subunits, and are localized perisynaptically. The investigator's recent findings show that exogenous, chimeric Bgt-nAChRs engineered to contain the long cytoplasmic loop of the a3 subunit, but not the corresponding loop from the a5 or b4 subunit, become localized in the postsynaptic membrane. These results are interpreted to suggest that endogenous nAChR complexes may also use the a3 loop for appropriate synaptic localization. In addition to AChRs, CG neurons express inhibitory glycine receptors (GlyRs). GlyRs and nAChRs are clustered in separate, but proximate, postsynaptic membrane regions on mature CG neurons. These distinct clusters coexist on the neuron surface under one calyx-type presynaptic terminal. The planned studies will logically extend the findings in 4 aims: (1) identify the essential targeting subdomain in the a3 loop; (2) determine whether this subdomain localizes endogenous nAChRs to the synapse; (3) define the extent of postsynaptic membrane microheterogeneity and the spatial organization of the separate nAChR and GlyR clusters; and (4) identify the protein that interacts with the a3 subdomain and may function in targeting nAChRs to the synapse. The studies will involve molecular and morphological analyses, including retroviral mediated gene transfer in situ, confocal & electron microscopy, and yeast two-hybrid assays.
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