Origin sites of calcium release and calcium oscillations in frog sympathetic neurons

In many neurons, Ca2+ signaling depends on efflux of Ca2+ from intracellula r stores into the cytoplasm via caffeine-sensitive ryanodine receptors (RyR s) of the endoplasmic reticulum. We have used high-speed confocal microscop y to image depolarization- and caffeine-evoked increases in cytoplasmic Ca2 + levels in individual cultured frog sympathetic neurons. Although caffeine -evoked Ca2+ wave fronts propagated throughout the cell, in most cells the initial Ca2+ release was from one or more discrete sites that were several micrometers wide and located at the cell edge, even in Ca2+-free external s olution. During cell-wide cytoplasmic [Ca2+] oscillations triggered by cont inual caffeine application, the initial Ca2+ release that began each Ca2+ p eak was from the same subcellular site or sites. The Ca2+ wave fronts propa gated with constant amplitude; the spread was mostly via calcium-induced ca lcium release. Propagation was faster around the cell periphery than radial ly inward. Local Ca2+ levels within the cell body could increase or decreas e independently of neighboring regions, suggesting independent action of sp atially separate Ca2+ stores. Confocal imaging of fluorescent analogs of ry anodine and thapsigargin, and of MitoTracker, showed potential structural c orrelates to the patterns of Ca2+ release and propagation. High densities o f RyRs were found in a ring around the cell periphery, mitochondria in a br oader ring just inside the RyRs, and sarco-endoplasmic reticulum Ca2+ ATPas e pumps in hot spots at the cell edge. Discrete sites at the cell edge prim ed to release Ca2+ from intracellular stores might preferentially convert C a2+ influx through a local area of plasma membrane into a cell-wide Ca2+ in crease.