To test the hypothesis that proteins composing the synaptic apparatus at the neuromuscular junction have a specific and orderly arrangements, the PI and his colleagues have begun developing ways of using EM tomography for making accurate 3D reconstructions of tissue sections. Data obtained from such reconstructions furnish a rich new source of quantitative information about the structural composition and organization of the synaptic apparatus to less than 4nm resolution in all three axes. The position of immunogold labels for synaptic proteins can also be accurately determined.
The specific aims of the experiments outlines in this proposal are: 1. To assess different methods of tissue preparation, data collection, reconstruction and representation for examining fine structure of the neuromuscular synaptic apparatus in 3D. 2. To characterize the 3D fine structure of the apparatus. 3. To map in 3D the position of immunogold labels for specific synaptic proteins to specific structural components of the apparatus. A detailed knowledge of the molecular architecture of the neuromuscular junction is essential for understanding the mechanisms involved in synaptic transmission and maintenance. It is also necessary for determining how the neuromuscular junction forms during ontogenesis and regenerates after trauma and for characterizing mechanisms of pathogenesis. EM tomography offers the opportunity of obtaining such information at a significantly higher level of resolution than that provided by any other method currently available or on the immediate horizon. Moreover, since it can also be used to study the 3D fine structure of any tissue in the body, a systematic approach to its application at the neuromuscular junction, as proposed here, should provide insights that will expedite the entry of others into this nascent field of research methodology.
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