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. New frozen sectioning techniques, in combination with scanning transmission electron microscopy (STEM), have permitted studies of coordinated changes in cytoplasmic total calcium which accompany regulation of free intracellular calcium by endoplasmic reticulum (ER). The results show that the (previously described) characteristic calcium concentration states of the ER in dendritic spines are associated with specific cytoplasmic calcium levels. This leads to three distinct states of calcium mobilization, which likely reflect the sequential processes of release from intracellular stores, followed by calcium uptake through membrane channels. In spine-bearing dendrites, there is a class of calcium-rich ER which is not present in spines, suggesting more complex patterns of calcium regulation in dendrites. A new method, based on darkfield mass mapping in the STEM, has been developed for determining the in situ molecular mass of organelles within neuronal processes. This capability has proven valuable for uncovering changes in protein binding and water content that reflect synaptic activity. Structural analysis of directly frozen preparation of various organotypic cultures of hippocampus continues to define the organization of dendrites and spines in the pyramidal cells of this tissue. In Part Two, formation of specialized membranes is studied in the context of myelin assembly. Confocal light microscopy has shown that Schwann cells depend on microtubule based intracellular transport and assembly of myelin-specific proteins. Moreover,the various myelin proteins synthesized in the perinuclear region are sorted in the trans-Golgi network into distinct transport vesicles, and this process also appears to be directed by microtubules. There are strong and important associations between microtubules and other cytoskeletal components within cytoplasmic channels of the internode; the organization of the Schwann cell ER appears to depend on these interactions.
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