The aim of this work is to better understand the role of target derived constituents that effect nerve terminal maintenance, elimination and growth. The newly developed techniques of transfer of exogenous genes and other molecules into living cells in vivo and the imaging of the responses of the modified cells over time will be used to study the neuromuscular junction. The role of molecules believed to play a role in motor terminal sprouting will be explored by transfecting muscles, by injection, with genes encoding CNTF, BDNF, NT3, NGF and IGF-2. The response of sympathetic, sensory and motor axons will be explored as will the effect on synapse formation. Protein synthesis will be focally inhibited in muscle by injecting ricin-60 near the synaptic site. Both pre- and postsynaptic specializations will be followed. At issue here is whether synapse loss is due to the removal of some factor initially supplied by the muscle or whether synapse survival depends on a muscle response that requires new protein synthesis. The role of the acetylcholine receptor (AChR) in synapse maintenance will be explored by injecting into the synaptic region a number of agents designed to decrease postsynaptic AChR synthesis. These include antisense oligonucleotides, cDNAs in the antisense direction and cDNAs encoding dominant negative truncations for subunits of the AChR and the 43k protein. In addition, efforts will be made to develop new adenovirus vectors for genes of interest and reporter genes as a means of increasing the yield of transfected muscle fibers to study in situ. Some in situ monitoring studies will also be conducted on transgenic mice lacking s-laminin or other basal lamina proteins as a way of investigating the role of basal lamina in synapse elimination, growth and maturation.
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