CALCIUM-CONTAINING ORGANELLES DISPLAY UNIQUE REACTIVITY TO CHEMICAL-STIMULATION IN CULTURED HIPPOCAMPAL-NEURONS

Cultured rat hippocampal neurons grown on glass coverslips for 1-3 wee ks were loaded with the calcium-sensitive fluorescent dye Fluo-3 and v iewed with a confocal laser scanning microscope. Large pyramidal-shape d neurons were found to contain dye-accumulating organelles in their s omata, primarily around nuclei and near the base of their primary dend rites. These organelles varied in size and increased in density over w eeks in culture, and were not colocalized with the endoplasmic reticul um or with mitochondria. The Fluo-3 fluorescence in these calcium-cont aining organelles (CCOs) was transiently quenched by exposure to Mn2+, indicating that the dye is a genuine [Ca2+] reporter and is not just a site of accumulating Fluo-3 dye. Recovery of fluorescence in the CCO s after washout of Mn2+ involved activation of a thapsigargin-sensitiv e process. CCOs responded to stimuli that evoke a rise of cytosolic [C a2+] ([Ca](i)) in a unique manner; perfusion of caffeine caused a prol onged rise of [Ca] in the CCOs ([Ca](C)), whereas it caused only a tra nsient rise of [Ca](i). Pulse application of caffeine also caused a fa ster effect on [Ca](C) than on [Ca](i). Glutamate caused a transient r ise of both [Ca](i) and [Ca](C), followed by a prolonged fall of only [Ca](C) to below rest level. This fall was blocked by preincubation wi th thapsigargin. Ryanodine blocked the cytosolic effects of caffeine b ut not its effect on [C](C). A clear distinction between CCOs and the known calcium stores was seen in digitonin-permeabilized cells; in the se, remaining Fluo-3 reported changes in store calcium, i.e., caffeine caused a reduction in Fluo-3 fluorescence in permeabilized cells, whe reas it still caused an increase in [Ca](C). A possible role of CCOs i n regulation of release of calcium from ryanodine-sensitive stores was indicated by the observation that CCO-containing cells exhibited a la rger and faster response to caffeine than cells that did not have them . We propose that CCOs constitute a unique functional compartment invo lved in release of calcium from calcium-sensitive stores.