The proposed projects concern the organization of calcium regulatory proteins within the neuronal endomembrane system. Transient rises in intracellular calcium serve as important signals regulating a host of cellular behaviors such as neurotransmitter release, activation of key enzymes, gene transcription and cell movement How neurons regulate intracellular calcium levels and produce calcium responses that are both temporally and spatially limited is a major research area in neurobiology. This information is critical not only for understanding normal neuronal function, but also for understanding various pathological processes, since abnormalities in intracellular calcium handling may be involved in several degenerative disorders such as Alzheimer's and Huntington's disease. A focus of this laboratory has been on defining the subcellular localization of proteins involved in the regulation of intracellular calcium pools. Neurons, like muscle cells, possess intracellular stores of calcium located within the endoplasmic reticulum that are capable of calcium release, storage and uptake. These stores, along with calcium fluxes across the plasma membrane, are believed to contribute to temporal and spatial regulation of calcium dynamics within neurons, although their precise function is unknown. These studies will employ single- and double- labeling immunolocalization techniques at both the light and electron microscopic levels to investigate the structure and distribution of these intracellular calcium pools. Structural aspects of the endoplasmic reticulum, such as the relative volume and surface area of the smooth endoplasmic reticulum within different portions of the Purkinje cell, will also be examined using three-dimensional reconstruction techniques and intermediate high voltage electron microscopy. Concurrently, cultured neurons will be used as model systems to investigate some functional aspects of intracellular calcium regulation.