This project is focused on the structure and function of the node of Ranvier of myelinated nerve. The nodal complex contains axonal specializations with adjacent paranodal terminations of the myelin sheath, and glial processes that invest the nodal gap and the gap substance. Our goal is to extend our work on establishing a better understanding of how the node functions and to continue to identify the locations of macromolecules in the complex that are important to maintain the structural integrity and in conduction and coordination of the nerve impulse. In SPECIFIC AIM 1, we will extend the current knowledge of specific membrane, cytoskeletal and extracellular matrix structures of both CNS and PNS nodes by building high resolution 3D volumes using the improved methodology for higher voltage electron tomographic structure determination. We will explore the relationship between different nodal compartments and their interconnectivity from nerves and fiber tracts with differing conduction properties to test if the number and frequency of action potentials have an effect on the morphology of the nodal compartments. In SPECIFIC AIM 2, we will use novel new methods such as the photoconversion of novel fluorescent labels, double tilt tomography and high resolution electron microscopic imaging for obtaining accurate and high resolution information (50 A) in situ about the arrangements and locations of specific molecules using intermediate high voltage electron tomography. Specifically, we are interested in the relationships between ion channels found in the membranes, cytoskeletal anchoring proteins and cell-cell junctional elements and the relationships between. The data we obtain from these new structures will then be used to construct highly accurate models of the node of Ranvier complex. In SPECIFIC AIM 3, we will deposit this structural data into a recently developed cell-level microanatomical database system that provides a framework for the integration of systems, cellular, subcellular and molecular data from distributed data sources as well as use a computer simulator program of cellular physiology that will use our 3D structural models obtained from tomography to conduct in silico tests of physiological function. The results of these studies will provide a better understanding of the relationship between structure and function at the node of Ranvier and the underlying macromolecular basis for impaired conduction in demyelinating disease and nerve repair.