This project aims to determine the intracellular distribution of diffusible and structural components within axons, dendrites, glia, and synapses. This work is important because of the relationship between the localization and movement of cellular constituents and their role in synaptic transmission. This project depends on several recent technological advances, including direct freezing, cryosectioning, immunocytochemistry in ultrathin cryosections, and quantitative x-ray microanalysis and element-specific x-ray imaging. Quantitative studies of the intracellular calcium distribution in parallel fiber/Purkinje cell cerebellar synapses indicate that total calcium is below 0.7 mmol/kg wet weight in all resting pre-and postsynaptic organelles; thus, high-level calcium stores appear unnecessary for the activity of these synapses. Membrane depolarization, however, elicits a fivefold increase in (extracellularly derived) calcium in the smooth endoplasmic reticulum of presynaptic terminals and dendritic spines, while that in synaptic vesicles is unchanged, thereby identifying the endoplasmic reticulum as a site of calcium sequestration. Immunocytochemical studies using frozen sections of actively myelinating nerve have implicated cytoskeletal and transport proteins, e.g., actin, spectrin and kinesin, in the synthesis and insertion of the myelin-specific proteins PO and myelin-associated glycoprotein. Similarly, a new approach using in situ hybridization methods in frozen sections has revealed the differential localization of m-RNAs encoding for the central nervous system myelin-specific proteins myelin basic protein and proteolipid protein. Thus, this project continues to provide important new information on the detailed relationship between the diffusible and structural components of neurons and glia, and how these regulate neuronal activity.