Pulsed laser imaging of Ca2+ influx in a neuroendocrine terminal
The surge of Ca2+ that triggers vesicle fusion is shaped by the distribution of Ca2+ channels and the physical relationship between those channels and the exocytotic apparatus. Although channels and the release apparatus are thought to be tightly associated at fast synapses, the arrangement at neuroendocrine cells is less clear. The distribution of Ca2+ influx near release sites is difficult to determine because of spatial and temporal limitations on Ca2+ imaging techniques. We now present spatially resolved images of Ca2+ influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca2+ influx into neurohypophysial terminals were captured after excitation of Ca2+-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca2+ increases were detected during the first millisecond of an evoked Ca2+ tail current. Steep gradients of Ca2+ were evident, with concentrations near the membrane reaching above 1 mM during a 30 msec depolarization. Ca2+ influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca2+ chelator. During longer depolarizations, mean and peak Ca2+ concentrations reached an asymptote in parallel, suggesting that Ca2+ binding proteins near the membrane rapidly buffer Ca2+ and do not become saturated during prolonged influx. These data support the hypothesis that exocytosis is activated in these terminals by the summation of influx through multiple, randomly spaced Ca2+ channels.