This two~part project studies the organization and function of specialized membranes in neurons and glia. The first part aims to characterize calcium regulation during synaptic activity in parallel fiber~Purkinje cell synapses of the cerebellar cortex and in synapses of CA3 hippocampal pyramidal cells. New frozen sectioning techniques, combined with scanning transmission electron microscopy (STEM), have permitted studies of coordinated changes in cytoplasmic total calcium which accompany the regulation of free intracellular calcium by endoplasmic reticulum. A new method, based on darkfield mass mapping in the STEM, has been used to determine the in situ molecular mass of organelles within neuronal processes. Such measurements have provided fundamental new information on the effects of beam~induced mass loss, and have led to new approaches to correcting concentration measurements for such effects. Structural analysis of chemically fixed and directly frozen preparations of a new kind of organotypic culture of hippocampus has identified culture conditions which provide excellent organ~ization of CA3 mossy fiber synapses. These synapses are close enough to the surface to be suitable for direct freezing studies. In the second part, the assembly of specialized myelin membranes is studied. Confocal light microscopy had previously shown that Schwann cells depend on microtubules for intracellular transport and assembly of myelin~specific proteins. Now, we have found that the organization of the Schwann cell microtubule network and therefore also of the organelles and filaments of cytoplasmic channels which depend on microtubules for their organization is obligatorily depend on axonal contact. Thus, the characteristic polarization of Schwann cell surface membranes does not occur in the absence of axons, and the proper sorting and targeting of myelin proteins consequently cannot take place.
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