Although nervous system function depends upon synaptic communication, relatively little is known about the regulation of neuronal synapse formation. Neurons both receive afferent and transmit efferent synaptic information. in addition, several different classes of neurotransmitter receptors, with distinct spatial localizations, function within a single neuron. The major goal of the proposed studies is identification of in vivo cellular and molecular mechanisms that regulate the formation of functionally specialized synaptic domains on developing neurons. The chick ciliary ganglion (CG) will be used as a model system because of the well-defined time course of synapse formation and the feasibility of surgical manipulations that prevent innervation or target tissue interactions in situ. CG neurons express two distinct classes of nicotinic cholinergic receptors: acetylcholine receptors (AChRs), and alpha-bungarotoxin receptors (alpha-Bgt-Rs). AChRs and alpha-Bgt-Rs have different functions and distributions, being localized in synaptic and perisynaptic regions, respectively. Three aspects of synaptic regulation will be addressed. First, the role of cell-cell interactions in regulating alpha-Bgt-R expression will be determined. Alpha-Bgt transcript and protein levels will be measured in normal and cell-cell interaction- deprived CGs using the quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays with radiolabeled alpha-Bgt or subunit specific monoclonal antibodies. Second, a similar approach will be used to study two candidate synapse organizing proteins: the chick homolog of gephyrin and a novel utrophin-like protein. In addition, antisera to gephyrin and the utrophin-like protein will be generated and used to determine their distribution in relation to that of AChRs and alpha-Bgt-Rs on the neuronal surface. Third, studies will identify domains of AChR and alpha-Bgt-R subunits responsible for directing their localization to discrete synaptic and perisynaptic regions. Epitope-tagged chimeric receptor subunits will be overexpressed in developing neurons in situ using replication competent avian specific retrovirus vectors. The subcellular distribution of the recombinant subunits will be assessed by ultrastructural analysis. The proposed studies present a unique opportunity for establishing the regulatory mechanisms influencing synapse differentiation on neurons in situ. Detailed understanding of neuronal synapse formation and function is essential in light of their central role in information processing and cell-cell communication in the nervous system. The importance of this knowledge is further underscored by the dire consequences of synaptic pathology, one of the causes of Alzheimer's disease, other dementias, and mental retardation.
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