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The Journal of Neuroscience, September 1, 1999, 19(17):7450-7457

Pulsed Laser Imaging of Ca²⁺ Influx in a Neuroendocrine Terminal

Thomas E. Fisher and Julio M. Fernandez

Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905

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 µM 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.

Key words: calcium channel; calcium current; neurohypophysis; calcium imaging; secretion; neuroendocrine; neuropeptide

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Copyright © 1999 Society for Neuroscience  0270-6474/99/19177450-08$05.00/0

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