Pulsed laser imaging of Ca(2+) influx in a neuroendocrine terminal

The surge of Ca(2+) that triggers vesicle fusion is shaped by the distribution of Ca(2+) 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 Ca(2+) influx near release sites is difficult to determine because of spatial and temporal limitations on Ca(2+) imaging techniques. We now present spatially resolved images of Ca(2+) influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca(2+) influx into neurohypophysial terminals were captured after excitation of Ca(2+)-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca(2+) increases were detected during the first millisecond of an evoked Ca(2+) tail current. Steep gradients of Ca(2+) were evident, with concentrations near the membrane reaching above 1 microM during a 30 msec depolarization. Ca(2+) influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca(2+) chelator. During longer depolarizations, mean and peak Ca(2+) concentrations reached an asymptote in parallel, suggesting that Ca(2+) binding proteins near the membrane rapidly buffer Ca(2+) 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 Ca(2+) channels.